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학 사 학 논문

Molecular structure and immunological function of lipoteichoic acid purified

from Enterococcus faecalis

Enterococcus faecalis lipoteichoic acid

분 조 면역학적 기능 연

2015년 8월

울 학 학원

치 학과 면역 분 미생물 전공

정

Molecular structure and immunological

function of lipoteichoic acid purified from Enterococcus faecalis

by

Jung Eun Baik

Under the supervision of

Professor Seung Hyun Han, Ph. D.

A Thesis Submitted in Partial Fulfillment of

the Requirements for the Degree of

Doctor of Philosophy

August 2015

School of Dentistry

The Graduate School

Seoul National University

ABSTRACT

Molecular structure and immunological function of

lipoteichoic acid purified from Enterococcus faecalis

Jung Eun Baik

Immunology and Molecular Microbiology Major

Department of Dental Science

The Graduate School

Seoul National University

Enterococcus faecalis is a Gram-positive commensal bacterium that is

frequently found in mucosal tissues of the oral cavity, gastrointestinal tract,

and genital tract in humans. E. faecalis is one of the major opportunistic

pathogens causing inflammatory diseases such as bacteremia and refractory

apical periodontitis. Unlike Gram-negative bacteria that express

lipopolysaccharide (LPS), a major etiologic component that causes

inflammatory responses, Gram-positive bacteria express lipoteichoic acid

(LTA) that is considered as the counterpart of LPS. Despite the importance of

LTA, its molecular structure and immunological functions have been poorly

characterized because the earlier LTA preparations were often contaminated

and/or structurally damaged due to improper purification processes. Recently,

a novel method has been introduced for purification of LTA using the

sequential application of mild organic solvent extraction,

hydrophobic-interaction column chromatography and ion-exchange column

chromatography. The aims of the present study were (1) to purify highly pure

and structurally intact LTA from E. faecalis, (2) to investigate its structural

and functional relationship in the modulation of innate immune responses, and

(3) to elucidate the action mechanism of endodontic medicament against E.

faecalis and its LTA.

LTAs from E. faecalis, Staphylococcus aureus, Bacillus anthracis, and

Lactobacillus plantarum were purified by butanol extraction followed by

chromatography with the hydrophobic-interaction and ion-exchange columns

using octyl-sepharose and diethlyaminoethyl (DEAE)-sepharose, respectively.

Endotoxin level in the LTA preparations was determined by Limulus

amebocyte lysate (LAL) assay. The contamination of LTA preparations with

nucleic acid was examined by using spectrophotometry. The contamination of

LTA preparations with proteins was determined via coomassie blue and silver

stainings. A murine macrophage cell-line (RAW 264.7) and bone

marrow-derived macrophage (BMM) from wild-type or Toll-like receptor

(TLR) 2-deficient C57/BL6 mice were used to determine the abilities of the

purified LTAs to induce the production of tumor necrosis factor-alpha

(TNF-α), interleukin (IL)-6, interferon gamma-inducible protein 10 (IP-10),

macrophage inflammatory protein-1 alpha (MIP-1α), and monocyte

chemoattractant protein-1 (MCP-1). To determine the production of

proinflammatory mediators in macrophages, RAW 264.7 cells and BMMs

were stimulated with purified LTAs in the presence or absence of recombinant

mouse interferon-gamma (IFN-γ) and the production of TNF-α, IL-6, IP-10,

MIP-1α, and MCP-1 in the culture supernatant was measured by

enzyme-linked immunosorbent assay (ELISA). The nitric oxide (NO)

production in LTA-stimulated macrophages was determined by reacting with

an equal volume of Griess reagent. The ability to activate TLR2 or TLR4 by

purified LTAs was determined by flow cytometry with nuclear factor

kappa-light-chain-enhancer of activated B cells (NF-κB) reporter cell lines,

CHO/CD14/TLR2 and CHO/CD14/TLR4, respectively. DNA-binding activity

of NF-κB was determined by electrophoretic mobility shift assay (EMSA).

Backbone structure of LTAs was determined by Western blot analysis. The

production of TNF-α in macrophages upon stimulation with heat-killed or

calcium hydroxide-killed E. faecalis was determined by ELISA. The

immunostimulating abilities of intact E. faecalis LTA or calcium

hydroxide-treated E. faecalis LTA to induce the production of NO, IP-10, and

MIP-1α were determined by ELISA. TLR2-stimulating activity of intact E.

faecalis LTA or calcium hydroxide-treated E. faecalis LTA was determined

by flow cytometry using CHO/CD14/TLR2. The structure of glycolipid in E.

faecalis LTA or in calcium hydroxide-treated E. faecalis LTA was analyzed

by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)

mass spectrometry and thin layer chromatography (TLC).

Upon exposure to E. faecalis LTA, RAW 264.7 cells significantly produced

TNF-α in a concentration-dependent manner. Although E. faecalis LTA

weakly induced the production of NO in RAW 264.7 cells, its ability to induce

NO was remarkably increased in the presence of IFN-γ under the same

condition. E. faecalis LTA preferentially activated TLR2 cells rather than

TLR4. Concomitantly, E. faecalis LTA enhanced the DNA-binding activity of

NF-κB, which plays an important role in the transcriptional activation of

genes encoding inflammatory mediators. E. faecalis LTA was not able to

induce TNF-α in macrophages lacking TLR2 while significantly induced

TNF-α in wild-type macrophages. Compared with LTAs purified from other

Gram-positive bacteria such as S. aureus, L. plantarum, and B. anthracis, E.

faecalis LTA showed a moderate potential in the activation of TLR2 and the

induction of inflammatory mediators. E. faecalis LTA weakly interacted with

anti-polyglycerophosphate antibody compared with other

polyglycerophosphate-type LTAs such as S. aureus LTA, L. plantarum LTA,

and B. anthracis LTA, implying that E. faecalis LTA might have a subtle

structural difference in its polyglycerophosphate backbone compared with

other polyglycerophosphate-type LTAs. The structural analysis using

MALDI-TOF demonstrated that the glycolipids from E. faecalis LTA consist

of dihexosyl diacylglycerol harboring fatty acids ranging from C16 to C22.

Interestingly, unlike most LTAs which likely possess saturated and

mono-unsaturated acyl chains, E. faecalis LTA contained mono- and

di-unsaturated fatty acids. Heat-killed E. faecalis potently induced the

production of TNF-α in RAW 264.7 cells while such induction was

significantly dampened by calcium hydroxide-killed E. faecalis under the

same condition. Treatment of E. faecalis LTA with calcium hydroxide

remarkably abrogated its ability to induce the production of proinflammatory

mediators such as NO, TNF-α, IP-10, and MIP-1α in RAW 264.7 cells.

Furthermore, calcium hydroxide-treated E. faecalis LTA was not able to

activate TLR2 signaling pathway. Structural analysis using MALDI-TOF and

TLC showed that calcium hydroxide deacylated the glycolipid moiety of E.

faecalis LTA, implying that the lipid moiety of E. faecalis LTA is crucial for

its inflammatory potential.

A novel purification method using mild organic solvent extraction,

hydrophobic-interaction and ion-exchange chromatography provided a

highly-pure and structurally-intact E. faecalis LTA preparation. E. faecalis

LTA is one of the major immunostimulating components in the Gram-positive

bacterial cell wall capable of activating the TLR2 signaling pathway. LTAs of

E. faecalis have moderate inflammatory potential compared to LTAs purified

from different bacterial species. E. faecalis LTA is a

polyglycerophosphate-type LTA with subtle structural differences of

substituent in repeating units and fatty acid composition. The lipid moiety of

E. faecalis LTA plays a crucial role in determining its ability to induce the

production of TNF-α, IL-6, IP-10, MIP-1α, and MCP-1 in macrophages by

affecting TLR2 activation. Treatment with calcium hydroxide abrogates the

inflammatory potential of E. faecalis LTA by inducing deacylation of the lipid

moiety of LTA. Thus, development of medicament targeting either the lipid

moiety of LTA or TLR2 signaling would be a good therapeutic candidate.

Collectively, E. faecalis LTA is an important virulence factor of E. faecalis for

inducing inflammatory responses through TLR2 signaling pathway and its

lipid moiety is essential for its inflammatory activity.

Keywords: Lipoteichoic acid, Enterococcus faecalis, Gram-positive bacteria,

Innate immune response, Toll-like receptor 2, Endodontic infection

Student number: 2007-22098

CONTENTS

Abstract

Contents

List of figures

List of tables

Abbreviations

Chapter I. Introduction 1

1.1. Enterococcus faecalis 1

1.1.1 General characteristics of E. faecalis 1

1.1.2. Virulence factors of E. faecalis 2

1.1.2.1. Secreted proteins 2

1.1.2.2. Surface proteins 3

1.1.2.3. Bacterial cell wall polysaccharide 4

1.2. LTA 6

1.2.1. Role of LTA in bacterial physiology 6

1.2.2. Structure and biosynthesis of LTA 9

1.2.3. Recognition of LTA in host innate immunity 13

1.2.4. Limitation of earlier studies for LTA 20

1.3. Endodontic infection and endodontic treatment 22

1.3.1. Primary root canal infection 22

1.3.2. Secondary/persistent root canal infection 23

1.3.3. Endodontic treatment and calcium hydroxide 23

1.4. Aim of the present study 25

Chapter II. Materials and Methods 26

2.1. Preparation of highly-pure and structurally-intact LTA 26

2.1.1. Reagents and chemicals 26

2.1.2. Bacterial strains and culture condition 27

2.1.3. Purification of LTA from Gram-positive bacteria 27

2.1.4. Phosphate assay 28

2.1.5. Limulus amebocyte lysate assay 29

2.1.6. Visualization of LTAs on SDS-PAGE gel using coomassie

blue staining, silver staining, and Western blotting 29

2.2. Structural analysis of E. faecalis LTA 30

2.2.1. Preparation of glycolipid anchor from E. faecalis LTA 30

2.2.2. Structural analysis of E. faecalis LTA using MALDI-TOF

and TLC 31

2.3. Functional analysis of LTA 32

2.3.1. Culture of RAW 264.7 32

2.3.2. Determination of nitric oxide 32

2.3.3. Enzyme-linked immunosorbent assay (ELISA) 32

2.3.4. Flow cytometric analysis 33

2.3.5. Electrophoretic mobility shift assay (EMSA) 33

2.3.6. Stimulation of bone marrow–derived macrophages 35

2.3.7. Preparation of killed whole cells by heat or calcium

hydroxide 35

2.3.8. Treatment of E. faecalis LTA with calcium hydroxide 36

2.3.9. Statistical Analysis 36

Chapter III. Results 37

3.1. Purification of highly-pure LTA from E. faecalis 37

3.2. Determination of immunological function of E. faecalis LTA 41

3.2.1. LTA from E. faecalis has inflammatory potential 41

3.2.2. TLR2 is crucial for activation of macrophage by E. faecalis

LTA 45

3.2.3. TLR2 is important for the inflammatory potential of E.

faecalis LTA in macrophage 47

3.2.4. E. faecalis LTA induces the activation of transcription

factor NF-κB 49

3.3. Structural and functional relationship of E. faecalis LTA 51

3.3.1. Highly-pure LTA was prepared from various Gram-positive

bacteria 51

3.3.2. E. faecalis LTA exhibits a modest ability to induce the

production of NO, IL-6, MCP-1 in macrophages

comparable to those of LTA purified from other bacterial

species

57

3.3.3. E. faecalis LTA has modest TLR2-stimulating activity

comparable to other bacterial

LTAs

59

3.3.4. E. faecalis LTA more weakly interacts with

anti-polyglycerophosphate antibody compared to other

bacterial LTA

61

3.3.5. Differential bacterial LTA exhibit subtle difference in their

backbone structure 64

3.4. Molecular mechanism of endodontic medicament 68

3.4.1. Calcium hydroxide inhibits the ability of E. faecalis to

induce TNF-α production in RAW 264.7 cells 68

3.4.2. Calcium hydroxide inhibits the ability of E. faecalis LTA to

induce the production of inflammatory mediators in murine

macrophages

70

3.4.3. Attenuated induction of inflammatory mediators by

calcium hydroxide-treated E. faecalis LTA is due to the loss

of TLR2-stimulating activity required for NF-κB activation

74

3.4.4. Calcium hydroxide deacylates the glycolipid moiety of

LTA from E. faecalis 76

Chapter IV. Discussion 81

Chapter V. References 94

List of publications 123

문초록

List of Figures

Figure 1. Structural classification of LTA 11

Figure 2. Biosynthesis of LTA 12

Figure 3. Bacterial cell wall structure 18

Figure 4. Elution profile of E. faecalis LTA from an

octyl-sepharose hydrophobic-interaction column

followed by elution on a DEAE-sepharose

anion-exchange column

39

Figure 5. Visualization of E. faecalis LTA using silver

staining

40

Figure 6. Induction of TNF-α and NO productions by RAW

264.7 cells stimulated with E. faecalis LTA

43

Figure 7. Induction of TNF-α and NO productions by RAW

264.7 cells stimulated with E. faecalis LTA in the

presence of polymyxin B

44

Figure 8. Activation of TLR2 by E. faecalis LTA 46

Figure 9. Activation of TLR2 on macrophage by E. faecalis

LTA

48

Figure 10. Enhanced DNA-binding activity of NF-κB by E.

faecalis LTA

50

Figure 11. Elution profiles of LTAs from octyl-sepharose

hydrophobic-interaction column

53

Figure 12. Elution profiles of LTAs from DEAE-sepharose

anion-exchange column

54

Figure 13. Visualization of LTA preparations using

coomassie blue staining and silver staining

55

Figure 14. Comparison of immunostimulating abilities of

LTAs purified from various Gram-positive

bacterial species.

58

Figure 15. Comparison of TLR2-activating ability among

LTAs purified from various Gram-positive

bacterial species

60

Figure 16. Characterization of backbone structure of LTAs 63

Figure 17. Experimental scheme for isolation of glycolipid

from E. faecalis LTA

65

Figure 18. Mass spectra obtained from the glycolipid anchor

of E. faecalis LTA

66

Figure 19. Calcium hydroxide attenuates the ability of E.

faecalis to induce TNF-α production in RAW

264.7 cells

69

Figure 20. Calcium hydroxide inactivates the ability of LTA

from E. faecalis to induce TNF-α production in

RAW 264.7 cells

72

Figure 21. Calcium hydroxide abolishes the ability of E.

faecalis LTA to stimulate the expression of

inflammatory mediators

73

Figure 22. Calcium hydroxide eliminates the ability of E.

faecalis LTA to stimulate TLR2

75

Figure 23. Experimental scheme for isolation of glycolipid

from calcium hydroxide-treated E. faecalis LTA

78

Figure 24. Calcium hydroxide delipidates LTA of E. faecalis 79

Figure 25. Identifications of free fatty acids generated from

the glycolipids after calcium hydroxide treatment

80

Figure 26. Proposed structure and immunological functions

of E. faecalis LTA

93

List of tables

Table 1. Known virulence factors of E. faecalis 5

Table 2. Known functions of LTA 8

Table 3. TLRs in human and mice 17

Table 4. Comparison of immunological properties

between LTA and LPS

19

Table 5. Determination of impurities in LTA

preparations

56

Table 6. Predictive structure of the glycolipid moiety of

E. faecalis LTA

67

Table 7. Structural characterization of LTAs 88

Abbreviations

Ace adhesin of collagen from E. faecalis

AS aggregation substance protein

BM bone marrow cell

BMM bone marrow-derived macrophage

Ca(OH)2 calcium hydroxide

CD14 cluster of differentiation 14

CD25 cluster of differentiation 25

CD36 cluster of differentiation 36

CFU colony forming uint

CpG cytosine-phosphate-guanine

DAG diacylglycerol

ELISA enzyme-linked immunosorbent assay

ElrA enterococcal leucine-rich-repeat-containing protein A

EMSA electrophoretic mobility shift assay

Epa enterococcal polysaccharide antigen

FA fatty acid

NAG α-D-N-acetylglucosamine

GU guanine-uracil

IFN-γ interferon-gamma

IL-6 interleukin-6

IP-10 interferon gamma-induced protein 10

LBP lipopolysaccharide binding protein

LPS lipopolysaccharide

LTA lipoteichoic acid

NF-κB nuclear factor kappa-light-chain-enhancer of activated

B cells

NO nitric oxide

MALDI-TOF matrix assisted laser desorption/ionization

time-of-flight mass spectrometry

MBL mannose-binding lectin

M-CSF macrophage colony-stimulating factor

MCP-1 monocyte chemoattractant protein-1

MD-2 differentiation factor 2

MIP-1α macrophage inflammatory protein-1 alpha

MW molecular weight

MyD88 myeloid differentiation primary response gene 88

PAFR platelet activating factor receptor

PGN peptidoglycan

PMB polymyxin B

SrA scavenger receptor class A

TAG triacylglycerol

TCL thin layer chromatography

TIR Toll/interleukin-1 receptor

TLR toll-like receptor

TNF-α tumor necrosis factor

TRIF TIR-domain-containing adapter-inducing interferon-β

WT wild-type

1

Chapter I. Introduction

1.1. Enterococcus faecalis

1.1.1. General characteristics of E. faecalis

Enterococcus faecalis is a Gram-positive, nonmotile, and facultative anaerobe that

is a commensal in the mucus area of the oral cavity, the gastrointestinal tract, and

the genital tract in humans [1]. Enterococci are one of the major opportunistic

pathogens causing a variety of diseases including bacteremia, endocarditis, and

refractory apical periodontitis. In recent years, these bacterial species have been

ranked in the top three nosocomial bacterial pathogens frequently isolated from

patients with bacteremia [2] and they also have been known as the most prevalent

species isolated from failed endodontic root canals [3]. Moreover, enterococcal

infections usually pose therapeutic difficulties because of their ability to persist in

harsh environmental conditions and their resistance to multiple antibiotics.

According to previous reports, enterococci are able to grow in the range of 10°C

and 45°C, in 6.5% NaCl broth, at pH 9.6, and they can survive at 60°C for 30 min

[4]. In addition, they have resistance to lethal levels of bile salts, sodium dodecyl

sulfate, detergents, hyperosmolarity, ethanol, azide, hydrogen peroxide, UV,

irradiation heat, sodium hypochlorite, acidity and alkalinity [5-10]. Over the past

two decades, enterococci have gained great attention due to the emergence of

multi-drug resistant strain such as vancomycin-resistant enterococci (VRE) [11].

2

Moreover, enterococci have substantial attention because they have also served as

donors of vancomycin resistance genes to other pathogens such as

methicillin-resistant Staphylococcus aureus (MRSA) [12]. Currently, the molecular

mechanism of enterococcal antibiotic resistance has been revealed that enterococci

intrinsically have resistance to a number of antibiotics and also readily acquire

additional resistance by uptaking exogenous genes that confer another antibiotic

resistance [13]. To date, 12 enterococci species have been identified, and

approximately 90% of the Enterococcus clinical isolates are Enterococcus faecalis.

1.1.2. Virulence factors of E. faecalis

Various etiologic virulence factors have been identified in E. faecalis. These

virulence factors are either released from bacteria or expressed on the surface and

they are related to bacterial adhesion, colonization, resistance to host defense, tissue

destruction, and induction of inflammation (Table 1).

1.1.2.1. Secreted proteins

E. faecalis secrete several proteins into the extracellular space to destruct host

tissues or to lyse target cells or molecules. Gelatinase is an extracellular protease

involved in tissue destruction by hydrolyzing components of the extracellular

3

matrix such as gelatin, collagen, and fibrinogen [14]. Cytolysin is a secreted toxin

produced by ~ 30% of E. faecalis strains, which can lyse not only host cells

including red blood cells [15], polymorphonuclear leukocytes (PMNs), and

macrophages [16], but also a broad range of Gram-positive, but not Gram-negative

bacteria [17, 18]. Sex pheromones, small secreted hydrophobic peptides comprised

of 7 - 8 amino acids, function as signaling peptides capable of regulating the

expression of virulence factors such as cytolysin [19]. Some of the sex pheromones

released from E. faecalis have been reported to induce abnormal production of

superoxides and lysosomal enzymes in neutrophils resulting in tissue destruction

[20, 21].

1.1.2.2. Surface proteins

E. faecalis expresses various virulence factors on its surface, which are likely to be

involved in the transfer of plasmid DNA and bacterial adherence. Aggregation

substance protein (AS) is a bacterial clumping factor that mediates the interaction

between donor and recipient bacteria, facilitating plasmid exchange [22].

Enterococcal microbial surface component recognizing adhesive matrix molecule

(MSCRAMM) is a family of bacterial adhesin that is involved in bacterial binding

to host extracellular matrix [23]. Adhesin of collagen from E. faecalis (Ace) is a cell

wall-anchored adhesin interacting with collagen and laminin [23, 24]. Another

4

enterococcal protein, enterococcal leucine-rich-repeat-containing protein (ElrA) has

been reported to affect the adhesion of E. faecalis to macrophages [25].

1.1.2.3. Bacterial cell wall polysaccharides

Polysaccharides of bacterial cell walls are crucial for bacterial pathogenesis,

mediating evasion of phagocytosis and inducing inflammatory responses.

Enterococcal polysaccharide antigen (Epa) is a rhamnose-containing polysaccharide

facilitating biofilm formation and decreasing susceptibility to PMN-mediated

killing [26-28]. Lipoteichoic acid (LTA), a Gram-positive bacterial membrane

bound polysaccharide, seems to be a major etiologic factor that has a broad

spectrum of pathogenic potentials: (i) LTA from Gram-positive bacteria triggers

inflammatory responses and results in tissue damage [29], (ii) E. faecalis LTA plays

an important role in biofilm formation, providing bacterial resistance to

antimicrobial peptides, antibiotics, or disinfectants [30], and (iii) LTA acts as a

representative target for generating opsonic antibodies during an infection with E.

faecalis [31].

5

Table 1. Known virulence factors of E. faecalis

Function Factor Reference

Bacterial adhesion and colonization

AS [32, 33]

Adhesins (Ace and ElrA) [23, 25]

Epa [27, 28]

LTA [34]

Resistance to host defense AS [35, 36]

Tissue destruction

LTA [37, 38]

Gelatinase [39, 40]

Cytolysin [41]

Induction of inflammation LTA [42, 43]

Sex pheromones [20, 21]

AS denotes aggregation substance protein

ElrA denotes enterococcal leucine-rich-repeat-containing protein A

Epa denotes enterococcal polysaccharide antigen

Ace denotes adhesin of collagen from E. faecalis

6

1.2. LTA

1.2.1. Role of LTA in bacterial physiology

Although the exact role of LTA in bacterial physiology is still unclear, it is

assumed that LTA is crucial for the (i) control of bacterial division and growth, (ii)

protection of bacteria against environmental stress, (iii) interaction with host

receptors or biomaterials (Table 2). According to several studies using

LTA-deficient strains of Gram-positive bacteria including S. aureus, Bacillus

subtilis, Bacillus anthracis, and Listeria monocytogenes, the strains lacking LTA

result in growth defects such as aberrant cell division and septum formation,

decreased autolysis, and reduced levels of peptidoglycan (PGN) hydrolases [44-46]

suggesting that LTA may be required for proper positioning of proteins related with

the cell-division machinery. Indeed, LTA of S. aureus has a high affinity to

autolysins, enzymes required for cleaving PGN during cell division [47].

LTA-deficiency in B. subtilis causes FtsZ, a key protein in bacterial division, to

become incapable of assembling at the division site [48]. In addition to bacterial

division, LTA is also responsible for protecting of bacteria from environmental

stress such as osmotic pressure since S. aureus lacking LTA can survive under

high-osmolality medium such as high concentration of NaCl or sucrose [49-51].

Moreover, LTA is important for the modulation of bacterial susceptibility to cationic

antimicrobial substances such as antimicrobial peptides or enzymes including

defensins and lysozyme as well as cationic antibiotics such as vancomycin by

modifying D-alanine contents which results in decrease of negative net charge in the

7

cell envelops [52-56]. More recently, several regulatory systems such as sensor

kinase ApsS have been shown to recognize defensins or other antimicrobial peptides

and to modulate alanylation in the presence of antimicrobial challenge [57, 58]. On

the other hand, LTA is also involved in bacterial interaction with host cells or

biomaterials. For example, mutants of S. aureus, E. faecalis, L. monocytogenes, and

Streptococcus pyrogenes deficient in dlt operon have shown decreased bacterial

adherence and biofilm formation [59-61]. Indeed, passive immunization with

monoclonal antibodies targeting the polyglycerophosphate moiety of LTA exhibited

significant increases of mortality in a murine S. aureus peritonitis model. In addition,

numerous studies have reported that LTA can activate host innate immune systems

via Toll-like receptors (TLR) 2 [62-64]. However, the proinflammatory potency of

LTA is still controversial because previously commercially available LTA have been

reported to be contaminated with other bacterial cell wall components such as

lipopolysaccharide (LPS), bacterial lipoprotein, and PGN.

8

Table 2. Known functions of LTA

State Function Reference

Growth of bacteria Bacterial division [50, 51]

Autolysin activity [61, 65, 66]

Protection of bacteria

Resistance to antimicrobial peptides [52, 54, 55]

Resistance to antibiotics [53]

Resistance to low osmolarity [51]

Interaction with host or abiotic surface

Binding to scavenger receptors (e.g. SrA)

[67, 68]

Activation of the complement system (e.g. MBL and L-ficolin)

[69-71]

Induction of inflammation (e.g. TLR2, CD36, CD14, and LBP)

[62-64]

Adhesion and biofilm formation [59-61]

SrA denotes scavenger receptor class A

MBL denotes mannose-binding lectin

TLR2 denotes Toll-like receptor 2

CD36 denotes cluster of differentiation 36

CD14 denotes cluster of differentiation 14

LBP denotes lipopolysaccharide binding protein

9

1.2.2. Structure and biosynthesis of LTA

LTA is an alditolphosphate-containing polymer attached to cytoplasmic

membranes of Gram-positive bacteria via glycolipid anchors. LTA is classified into

two groups based on its chemical structures of the repetitive backbone (i.e., type I

and II). Type I LTA, expressed in most Gram-positive bacteria including S. aureus,

B. subtilis, E. faecalis, and Lactobacillus plantarum, is usually comprised of

disaccharide-containing glycolipids linked to repeating polyglycerophosphate units,

whose sn-2 position is esterified with hydroxyl, D-alanyl, or glycosyl substituent

[64] (Figure 1A). Type II LTA, which is expressed by a small number of bacteria

such as Streptococcus pneumoniae and Streptococcus oralis, is comprised of

polyribitolphosphate repeating units linked to a positively charged glycolipid [72]

(Figure 1B). In addition to differences of backbone structure, type II LTA uniquely

contains phosphocholine residue in its repeating units which plays an important

roles in bacterial growth and virulence by mediating the interaction with

choline-binding proteins (CBP) [73]. Recently, most enzymes involved in LTA

synthesis have been identified including YpfP, LtaA, and LtaS [74] (Figure 2). The

YpfP enzyme produces the glycolipid anchor in the bacterial cytoplasm by adding

two glucose residues from UDP-glucose to diacylglycerol. Subsequently, the

flippase, LtaA, translocates the glycolipid from the inner to the outer leaflet of the

cytoplasmic membrane followed by extension of the glycerophosphate polymer on

the glycolipid by LTA synthase, LtaS. In addition, further studies using mutagenesis

10

have also revealed that dltABCD gene cluster is responsible for modification of

D-alanine after LTA or TA biosynthesis. The D-alanylation of LTA have reported

to be crucial roles in the regulation of autolysis [75, 76], divalent cationic ions

homeostasis through the interaction with Mg2+ [77, 78], biofilm formation [30], and

bacterial resistance to cationic antimicrobial peptides by affecting their net charge

[79].

11

Figure 1. Structural classification of LTA (A) Polyglycerophosphate-type LTA as

found in S. aureus and (B) polyribitolphosphate-type LTA from S. pneumoniae.

Chemical structures of S. aureus LTA and S. pneumoniae LTA proposed by Morath.,

et al [80] and Gisch., et al [81], respectively. The repeating unit of S. pneumococcal

LTA consists of the

pseudopentasaccharide-2-acetamido-4-amino-2,4,6-trideoxygalactose (AATGal),

glucose (D-Glu), and ribitol-phosphate (Ribitol-P) followed by two

N-acetylgalactosamine (NAcGal) moieties, both substituted with phosphocholine in

position O-6. R denotes backbone substitutions. n denotes number of repeating units,

12

NAG denotes N-acetylglocosamine.

Figure 2. Biosynthesis of LTA. Polyglycerophosphate-type LTA synthesis

machinery of S. aureus. The glycolipid anchor dihexose-DAG is produced by YpfP

and moved to the outside of the membrane by LtaA. The LtaS is an enzyme

required for the producing the polyglycerophosphate chain. Coincidently, DltA

ligates D-alanines onto DltC. The D-alanines are subsequently transported across

the membrane by DltB. Then, DltD aids in the transfer of the D-alanines to

glycerophosphate (GroP) of LTA. DAG denotes diacylglycerol. D-Ala denotes

D-alanine.

13

1.2.3. Recognition of LTA in host innate immunity

The human body is constantly challenged by diverse bacteria, which not only have

co-evolved with the host by affecting various physiological processes including

nutrient production/absorption, gut homeostasis, tissue development, and host

defense [82, 83], but also cause infectious diseases that are leading causes of death

worldwide including sepsis and pneumonia. The Gram-positive bacteria lactobacilli,

commonly found in the gastrointestinal tract, are representative commensal bacteria

beneficial to the host as they enhance intestinal barrier function and produce

antimicrobial substances [84, 85]. On the other hand, S. aureus is a versatile

Gram-positive pathogen that causes a range of illnesses from minor skin infections

to life-threatening infections such as sepsis, pneumonia, and endocarditis [86]. For

maintaining homeostasis with bacteria, the host immune system has developed

under the dual pressure of defending against pathogen and coexisting with

commensal bacteria. Upon the exposure to bacteria, the host triggers both innate

and adaptive immune systems. While adaptive immunity provides highly effective

protection against particular pathogens by recognizing specific antigens during the

late phase of infection, innate immunity is responsible for the protection of host at

the early phase of infection by using germ line-encoded receptors that recognize a

wide range of highly conserved microbial moieties, termed microbial-associated

molecular patterns (MAMPs) from both commensal and pathogenic bacteria [87].

Among these germ line-encoded receptors, TLRs, members of interleukine-1 (IL-1)

14

superfamily of transmembrane receptors, are one of the key MAMP receptors

capable of inducing innate immune responses against a broad range of

microorganism including bacteria, yeast, and viruses [88]. To date, a total of 10 and

13 TLRs have been identified in human and mice, respectively. Each TLR forms a

homo- or heterodimer complex and recognizes various bacterial MAMPs such as

LTA, lipoprotein, LPS, flagellin, and bacterial DNA (Table 3).

Bacteria can be classified into Gram-negative and Gram-positive according to their

cell wall structures (Figure 3). Gram-negative bacteria are comprised of thin PGNs

with two bilayer membranes including inner- and outer membranes which are

primarily composed of phospholipids and LPS, respectively. In contrast,

Gram-positive bacteria are surrounded by thick PGNs with a single cytoplasmic

membrane and express LTA instead of LPS. Among various bacterial components,

LPS, an amphiphilic molecule composed of hydrophobic lipid A and two distinct

carbohydrate including highly conserved core polysaccharide and heterogeneous

O-polysaccharide, is considered to be a major etiologic component of

Gram-negative bacteria because it can stimulate host cells leading to cell death and

the production of inflammatory mediators including cytokines, chemokines, and

lipid metabolites [89]. Moreover, LPS alone is sufficient to cause septic shock when

administered to mice [90]. Upon entering the blood stream, LPS forms a complex

with LPS-binding protein (LBP) and soluble cluster of differentiation (CD) 14 [91,

15

92]. Then, the LPS in the complex is transferred to the myeloid differentiation

factor 2 (MD-2) followed by interaction with TLR4 on target cells such as

macrophages [93]. These interactions subsequently activate innate immune

responses by triggering both myeloid differentiation primary response gene 88

(MyD88)-dependent and MyD88-independent signaling pathways which lead to the

activation of nuclear factor-kappa B (NF-κB) transcription factor and the production

of interferon (IFN)-β, respectively [94-96].

LTA of Gram-positive bacteria is considered to be a counterpart of the LPS of

Gram-negative bacteria. LTA is an amphiphile consisted of hydrophobic glycolipids

and hydrophilic polysaccharides that can induce various proinflammatory cytokines

and chemokines [29, 62-64, 97]. Although both LTA and LPS share structural and

immunological characteristics, they have distinctive properties (Table 4). For

example, unlike LPS, LTA is actively released from the bacterial cell wall during

bacterial division or bacteriolysis induced by external factors such as lysozymes,

cationic peptides, and beta-lactam antibiotics [29, 98, 99]. In addition, the released

LTA in the host bloodstream is recognized by not only LBP and CD14 but also

mannose-binding protein (MBP), L-ficolin, CD36, and soluble TLR2 which are able

to either facilitate LTA-mediated immune responses or neutralize the LTA as a

scavenger [70, 71, 100-102]. Moreover, LTA is recognized by TLR2 and triggers a

cell signaling cascade through the MyD88-dependent but not the

16

MyD88-independent pathway [103]. Furthermore, LPS by itself is a powerful agent

that can provoke inflammatory responses, whereas LTA exhibits relatively weak

induction of inflammatory responses that can be amplified in the presence of other

bacterial components such as PGN [104]. Given these distinctions, host immune

responses to LPS may not be directly applicable to those of LTA and Gram-positive

bacteria.

17

Table 3. TLRs in human and mice

TLR Localization Species Ligands Recognized

pathogen

TLR1 Extracellular Human and mice Triacyl lipopeptide Bacteria

TLR2 Extracellular Human and mice

Lipoproteins, PGN, LTA,

zymosan, and mannan

Bacteria

TLR3 Endolysosomal Human and mice dsRNA dsRNA

TLR4 Extracellular

and Endolysomal

Human and mice LPS Gram-negative

bacteria

TLR5 Extracellular Human and mice Flagellin Bacteria

TLR6 Extracellular Human and mice Diacylipopeptide, LTA, and zymosan

Bacteria

TLR7 Endolysosomal Human and mice GU-rich ssRNA

and short dsRNA Viruses and

bacteria

TLR8 Endolysosomal Human and mice GU-rich ssRNA,

short dsRNA Viruses and

bacteria

TLR9 Endolysosomal Human and mice CpG DNA

Bacteria, viruses and protozoan parasites

TLR10 Extracellular Human N.D. N.D.

TLR11 Endolysosomal Mice Profilin and

flagellin

Apicomplexan parasites and

bacteria

TLR12 Endolysosomal Mice Profilin Apicomplexan

parasites

TLR13 Endolysosomal Mice

Bacterial 23S rRNA with

CGGAAAGACC motif

Gram-negative and

Gram-positive bacteria

GU denotes guanine-uracil

CpG denotes cytosine-phosphate-guanine

N.D. denotes not determined

Referenced from Broz et al., [88]

18

Figure 3. Bacterial cell wall structure. (A) The Gram-positive bacterial cell wall

is composed of thick PGN with a single cytoplasmic membrane. Teichoic acids (TA)

are expressed as major polysaccharides of Gram-positive bacteria. These polymers

are covalently anchored to PGN (wall teichoic acids; WTAs) or the cytoplasmic

membrane (LTAs). Both WTAs and LTAs are often substituted with D-alanyl esters.

Gram-positive bacteria usually have diacylated lipoproteins in their cell wall surface.

(B) The Gram-negative bacterial cell wall is composed of a thin PGN with an inner

and an outer membrane. They express LPS in the outer membrane as representative

polysaccharide. Gram-negative bacterial usually have triacylated lipoproteins.

19

Table 4. Comparison of immunological properties between LTA and LPS

PAFR denotes platelet activating factor receptor

TRIF denotes TIR-domain-containing adapter-inducing interferon-β

LTA LPS

Expression Gram-positive bacteria Gram-negative bacteria

Structure Amphipathic

(glycolipid + polysaccharide) Amphipathic

(lipid A + polysaccharide)

Secretion Yes No

Cytotoxicity No Yes

Induction of inflammatory

response Yes Yes

Induction of sepsis No

(Yes in the presence of PGN) Yes

Membrane receptors

TLR2, CD14, CD36, PAFR TLR4, CD14

Soluble binding proteins

LBP, sCD14, MBP, L-ficolin LBP, sCD14, MD2

Signaling pathway MyD88-dependent Both MyD88- and TRIF-dependent

20

1.2.4. Limitation of earlier studies for LTA

Even though numerous studies have been reported to elucidate the role of LTA in

host inflammatory responses during Gram-positive bacterial infection [29, 62-64, 97,

105], the biological properties of LTA and its inflammatory potency have been

controversial because commercial LTA preparations used in early studies had been

contaminated with endotoxins and/or they had been structurally damaged [106, 107].

Recent studies have reported that the non-hydrophobic phosphate containing

fraction separated from commercially available S. aureus LTA using reverse-phase

chromatography showed potent inflammatory activity [108, 109]. Moreover,

re-purified LTA from commercially available S. aureus LTA had no inflammatory

potential. More recent studies using nuclear magnetic resonance (NMR) have

demonstrated that the commercial phenol-extracted S. aureus LTA showed dramatic

loss of glycerophosphate repeats (approximately 48% of loss) and D-alanine

residues (approximately 71% of loss), which are crucial for biological activity of

LTA [80, 107]. In addition, the commercial S. aureus LTA was contaminated with

considerable impurities including non-LTA (no phosphate, LPS), non-LPS

(non-reactive to Limulus amebocyte lysate, bacterial DNA) [80]. This has led to

contradictory immunological properties of LTA being reported due to the erroneous

reflections of LTA-mediated immune responses. Recently, an upgraded purification

method capable of extracting highly pure and structurally intact LTA was introduced

[80, 107]. The method uses butanol, a mild organic solvent, to replace phenol and

21

serial applications of two different chromatographic columns such as a

hydrophobic-interaction column and a subsequent ion-exchange column. Hence, the

need for pure LTA has become a prerequisite to elucidate the role of LTA in

bacterial pathogenesis and host immunity.

22

1.3. Endodontic infection and endodontic treatment

Endodontic infection is the infection of root canal and is a major etiological cause

of apical periodontitis. Although certain chemical and physical factors are able to

induce periradicular inflammation, microorganisms are essential for the endodontic

infection and progression of apical periodontitis [110-112]. According to clinical

conditions, endodontic infections can be divide into (1) primary root canal infection

caused by initially infected microorganisms and (2) secondary/persistent root canal

infection caused by very limited microorganisms that were members of the primary

infection with resistance to intracanal antimicrobial procedure or with the ability to

survive nutrient deprivation during endodontic treatment.

1.3.1. Primary root canal infection

Primary root canal infection is caused by microorganisms that initially colonize in

the necrotic pulp tissue, which are generally polymicrobial infection with anaerobic

bacteria and are composed of 10 to 30 species per canal [113]. Although several

bacterial species belong to the genera Bacteroides, Porphyromonas, Prevotella,

Fusobacterium, Treponema, Peptostreptococcus, Eubacterium, and Campylobacter

usually isolated in the infected root canal, the bacterial profiles vary from individual

to individual [114]. It indicates that primary root canal infection has heterogeneous

etiology and multiple bacteria play a role in the progress of the disease.

23

1.3.2. Secondary/persistent root canal infection

Secondary/persistent root canal infection is caused by microorganisms that

survived after endodontic treatment. It frequently leads to the progress of

endodontic failure and apical periodontitis. The microbial profiles associated with

secondary/persistent root canal infection are usually composed of a single species or

relatively lower number of species compared with primary root canal infection [113,

115]. Bacterial species that were identified in secondary/persistent root canal belong

to the genera Enterococcus, Streptococcus, Actinomyces, Propionibacterium,

Staphylococcus, and Pseudomonas [114]. Among various microbial species

identified in the secondary/persistent root canal infection, E. faecalis has gained

considerable attention by its frequent recovery in root-filled teeth with apical

periodontitis after endodontic treatment although they occupy only a small

proportion of the flora in untreated canals [116-118]. E. faecalis can survive in

harsh environmental conditions including intracanal irrigants and medications [119]

and can invade the dentinal tubules in variable depths [120] followed by

colonization of the root canal [121, 122]. These abilities of E. faecalis may explain

its ability to survive after a root canal treatment and to induce persistent apical

periodontitis.

1.3.3. Endodontic treatment and calcium hydroxide

Since endodontic infection followed by apical periodontitis is an infectious

24

inflammatory disease, the major objectives of endodontic treatment are not only to

eliminate viable bacteria in the root canal system but also to inactivate bacterial

virulence factors that could potentially influence the progress of apical periodontitis.

Calcium hydroxide has been widely used as an endodontic medicament since the

1920s because of its highly effective bactericidal activity [123]. Calcium hydroxide

releases hydroxide ions in aqueous solutions, giving rise to a high alkaline

environment (approximately pH 12.5-12.8) in which most bacteria cannot survive

[124]. In addition, accumulating reports suggest that calcium hydroxide can

inactivate LPS [125], and that calcium hydroxide-treated LPS does not induce

either the release of TNF-α from monocytes [126] or the stimulation of osteoclast

formation, which is a major cell type responsible for alveolar bone loss [127]. Mass

spectrometry demonstrated that calcium hydroxide detoxifies LPS by hydrolyzing

fatty acids in the lipid A moiety [128]. Although the inactivation of LPS from

Gram-negative bacteria by calcium hydroxide and other endodontic irrigants has

been extensively investigated, the effects on LTA of Gram-positive bacteria have

been poorly studied.

25

1.4. Aim of the present study

The aim of the present study is to investigate the structure and immunological

functions of E. faecalis LTAs by (1) preparing highly-pure and structurally intact

LTA from E. faecalis, (2) by studying its structural and functional relationship in

innate immune responses, and (3) by elucidating the molecular mechanism of

endodontic medicament against E. faecalis and its LTA.

26

Chapter II. Materials and Methods

2.1. Preparation of highly-pure and structurally-intact LTA

2.1.1. Reagents and chemicals

E. faecalis ATCC29212, S. aureus ATCC29213, and B. anthracis ATCC14185

were purchased from the American Type Culture Collection (Manassas, VA, USA).

L. plantarum KCTC 10887BP was obtained from Korean Collection for Type

Culture (Daejon, Korea). Luria-bertani (LB), tryptic soy broth (TSB), brain heart

infusion (BHI) and Mann, Rogosa, and Sharpe (MRS) media were purchased from

BD Biosciences (Franklin, NJ, USA). octyl-sepharose CL-4B, DEAE-sepharose,

Salmonella typhosa LPS, E. coli LPS, and polymyxin B were purchased from

Sigma-Aldrich (St. Louis, MO, USA). Highly pure LPS from E. coli O111:B4, a

synthetic lipopeptide stimulating TLR2, Pam2CSK4 and Pam3CSK4 were obtained

from InVivoGen (San Diego, CA, USA). Spectra/Por 6 semi-permeable dialysis

membrane was purchased from Spectrum® Laboratories Inc. (Ranch Dominquez,

CA, USA). Endotoxin-free distilled water was obtained from Dai Han Pharm Co.

Ltd. (Seoul, Korea). Mouse recombinant interferon-gamma (mrIFN-γ) was obtained

from R&D Systems (Minneapolis, MN, USA). All other reagents were purchased

from Sigma-Aldrich unless otherwise indicated.

27

2.1.2. Bacterial strains and culture condition

E. faecalis ATCC29212 and L. plantarum KCTC 10887BP were grown in BHI

medium and in MRS medium at 37oC without shaking overnight, respectively. S.

aureus ATCC29213 was cultured in TSB medium, and B. anthracis ATCC14185 in

BHI medium supplemented with 0.7% bicarbonate at 37oC with shaking at 200 rpm

overnight. After culturing these bacteria, they were centrifuged at 11,068 × g at 4oC

for 10 min, washed with PBS three times and the pellets was kept until used.

2.1.3. Purification of LTA from Gram-positive bacteria

To purify LTAs from E. faecalis, S. aureus, L. plantarum, or B. anthracis, each

bacterial pellet with approximately 80 to 100 g (wet weight) were resuspended in

0.1 M sodium citrate buffer (pH 4.7) and disrupted by ultrasonication (Labsonic P,

B. Braun Biotech, Allentown, PA, USA) followed by mild organic solvent

extraction with an equal volume of n-butanol for 30 min. After phase separation by

using centrifugation, the aqueous phase was collected and subjected to dialysis in a

semi-permeable dialysis membrane against endotoxin-free distilled water. The

extract in 0.1M sodium acetate buffer (pH 4.7) containing 15% n-propanol was

subjected to hydrophobic-interaction chromatography using octyl-sepharose CL-4B.

After removing the unbound materials with 15% n-propanol in 0.1 M sodium

acetate buffer (pH 4.7), the bound materials were eluted with 35% n-propanol in 0.1

28

M of the sodium acetate buffer. After determining the column fractions containing

LTA by using phosphate assay, the fractions were pooled and dialyzed against

endotoxin-free distilled water. The LTA-containing fractions were further subjected

to ion-exchange chromatography using DEAE-sepharose. After washing out the

unbound materials with 30% n-propanol in 0.1 M sodium acetate buffer, the bound

materials were eluted with a linear salt gradient (0-1 M NaCl in 0.1 M sodium

acetate buffer containing 30% n-propanol). The fractions containing LTA were

determined using the phosphate assay and further subjected to dialysis against

endotoxin-free distilled water. After measuring the dry weight of the purified LTA,

the LTA was dissolved in endotoxin-free water and kept at -80oC until use.

2.1.4. Phosphate assay

The phosphate contents of the eluted fractions or purified LTAs were determined

by measuring inorganic phosphorus. Briefly, each sample was subjected to strong

acidic digestion by boiling with a nitric acid-sulfuric acid mixture. The quantity of

the phosphate in each sample was determined by measuring the optical density at

600 nm using a microtiter plate reader (VERSAmax; Molecular Devices, Sunnyvale,

CA) after treatment of molybdate and stannous chloride. Sodium phosphate was

used as a standard.

29

2.1.5. Limulus amebocyte lysate assay

The quantity of endotoxin was determined using a commercially available Limulus

amebocyte lysate (LAL) test kit (QCL-1000; Cambrex Bio Science, Walkersville,

MD, USA).

2.1.6. Visualization of LTAs on SDS-PAGE gel using coomassie blue

staining, silver staining, and Western blotting

LTAs from E. faecalis, S. aureus, L. plantarum, or B. anthracis were mixed with 4

× sample buffer (8% SDS, 10% 2-mercaptoethanol, 30% glycerol, 0.02%

bromophenol blue in 0.25 M Tris-HCl, pH 6.8) and incubated at 100oC for 15 min.

The solution was then loaded into each well and run at 120 V for 1.5 h in 15%

SDS–PAGE gels. For coomassie blue staining, the SDS-PAGE gel was treated with

commassie blue solution (0.1% coomassie blue R-250/10% acetic acid/45%

methanol) at room temperature for 1 h with gentle shaking. Then, the gel was

incubated with de-staining solution (10% methanol/10% acetic acid) at room

temperature with gentle agitation overnight. For silver staining, the SDS–PAGE gel

was treated with fixation solution (50% methanol/12% acetic acid/0.0185% CH2O)

for 1 h. Then, the gel was washed twice with 50% ethanol for 20 min each, after

which the gel was treated with sensitizing solution (0.02% Na2S2O3·5H2O) for 1

min. After washing three times with distilled water for 20 sec each, the gel was

30

incubated with silver reaction solution (0.2% AgNO3/0.027% CH2O) for 1 h. After

washing twice with non-pyrogenic water for 20 sec each, the gel was incubated with

developing solution (6% Na2CO3/0.0004% Na2S2O3·5H2O/0.0185% CH2O) for 5

-10 min. After obtaining the desired band intensity, the gel was washed and

incubated with stopping solution (50% methanol/12% acetic acid) to halt the silver

reaction. For Western blot, the gel was electro-transferred to a PVDF membrane

(Milipore, Bedford, MA, USA) and the membrane was blocked with 5% skim milk

in TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.6) at room temperature for 1 h. After

washing three times with TBST (50 mM Tris-HCl, 150 mM NaCl, 0.05% tween 20,

pH 7.6) for 10 min at room temperature, the membrane was kept at 4°C overnight

with mouse monoclonal anti-LTA antibody (clone: 55; Hycult biotech, Uden,

Netherlands) in the blocking buffer. Subsequently, the membrane was washed three

times with TBST for 10 min at room temperature, and incubated with horseradish

peroxidase-conjugated goat anti-mouse IgG polyclonal antibody (West Grove, PA,

USA) in the blocking buffer at room temperature for 1 h. The immunoreactive

bands were detected with ECL Western blotting reagents (Neuronex Co.,Pohang,

Korea).

2.2. Structural analysis of LTA

2.2.1. Preparation of glycolipid anchor from E. faecalis LTA

31

The lyophilized E. faecalis LTA (1 mg) were exposed to 98% acetic acid (1 ml) to

cleave the phosphodiester bond on the LTA, then sonicated for 30 min, boiled at

100oC for 3 h, and lyophilized. Then, a solvent comprised of chloroform, methanol,

and water (1:1:0.9, v/v/v) was added to the lyophilized products, and the chloroform

phase containing the glycolipid anchors of E. faecalis LTA was obtained.

2.2.2. Structural analysis of E. faecalis LTA using matrix-assisted laser

desorption/ionization-time of flight mass spectrometer (MALDI-TOF)

and thin layer chromatography (TLC)

For MALDI-TOF analysis, the glycolipids of E. faecalis LTA were mixed with 30

mg/ml 2,5-dihydroxybenzoic acid (Sigma-Aldrich) in 70% acetonitrile, deposited

onto a metallic sample holder and subsequently subjected to MALDI-TOF mass

spectrometry using Biflex IV systems (Bruker Daltonics). For TLC, glycolipid from

E. faecalis LTA (300 μg) treated with 25 mg/ml calcium hydroxide, 5.25% sodium

hypochlorite or 0.2 N sodium hydroxide at 37oC for 60 min were deposited onto a

silica gel TLC plate (Silica gel 60; Merck, Whitehouse Station, NJ) and developed

with a chloroform/methanol/water (65:25:4, v/v/v). Then the glycolipids were

visualized by spraying with 5% phosphomolybdic acid in ethanol followed by

heating at 120°C.

32

2.3. Functional analysis of LTA

2.3.1. Culture of RAW 264.7 cells

A murine macrophage cell line, RAW 264.7 (TIB-71), was purchased from the

American Type Culture Collection. The cells were cultured in Dulbecco modified

Eagle’s medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal

bovine serum (FBS) (HyClone, Logan, UT), 100 U/ml of penicillin, and 100 μg/ml

streptomycin at 37°C in a humidified incubator with 5% CO2.

2.3.2. Determination of nitric oxide

The production of nitric oxide (NO) in macrophages after stimulation with various

stimuli was determined by reacting with equal volumes of the Griess reagent (1%

sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2% phosphoric

acid). After incubation for 5 min at room temperature, the absorbance was measured

at 540 nm with the microtiter-plate reader. NaNO2 was used as a standard for nitrite

concentrations.

2.3.3. Enzyme-linked immunosorbent assay (ELISA)

33

The amounts of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, interferon

gamma-induced protein 10 (IP-10), macrophage inflammatory protein-1 alpha

(MIP-1α), and monocyte chemoattractant protein-1 (MCP-1) in culture supernatants

of macrophages after stimulation with various stimuli for 24 h were determined by

using a commercially available enzyme-linked immunosorbent assay (ELISA) kit

(R&D Systems).

2.3.4. Flow cytometry

CHO/CD14/TLR2 and CHO/CD14/TLR4, two NF-κB reporter cell lines which are

coexpressing CD14 and TLR2 or TLR4, respectively, were kindly provided by Dr.

Douglas Golenbock (Boston Medical Center, Boston medical center, Boston, MA,

USA) and grown in Ham’s F-12 media containing 10% heat-inactivated FBS, 1

mg/ml G418, 400 μg/ml hygromycin, 100 U/ml penicillin, and 100 μg/ml

streptomycin at 37ºC in a 5% CO2 humidified incubator. After stimulation of the

cell lines with purified LTA or LPS, the ability to activate TLR2 or TLR4 was

determined by measuring the expression of CD25 using a FACS Calibur with

CellQuest software (BD Biosciences).

2.3.5. Electrophoretic mobility shift assay (EMSA)

34

RAW 264.7 cells cultured on a 60-mm culture dish were stimulated with 0.5 μg/ml

of E. coli LPS or E. faecalis LTAs at 0, 3, 10, or 30 μg/ml for 90 min. To prepare

nuclear extracts, the cells were lysed with hypotonic buffer [10 mM HEPES (pH

7.9), 1.5 mM MgCl2] on ice for 15 min and the nuclei were precipitated using

centrifugation at 3000 × g for 5 min. After lysing the nuclear by treating with a

hypertonic buffer (30 mM HEPES, 1.5 mM MgCl2, 450 mM KCl, 0.3 mM EDTA,

10% glycerol, 1 mM DTT, 1 mM PMSF, and 1 μg/ml each of aprotinin and

leupeptin) on ice for 40 min, the nuclear extracts were collected by centrifugation

and stored at -80oC until use. Double-stranded deoxyoligonucleotide probe

containing the consensus recognition sites (in italics) of NF-κB was

5’-GATCTCAGAGGGGACTTTCCGAGAGA-3’ and synthesized oligonucleotides

were end-labeled with [γ-32P]-dATP. Nuclear extracts were incubated with the

32P-labeled DNA probes in binding buffer (100 mM KCl, 30 mM HEPES, 1.5 mM

MgCl2, 0.3 mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF, and 1 μg/ml each

of aprotinin and leupeptin) containing 1 μg of poly(dI-dC) for 20 min at room

temperature. Then, the mixtures were electrophoresed on a 4.8% polyacrylamide gel

in 0.5 × TBE buffer (44.5 mM Tris, 44.5 mM boric acid, and 1 mM EDTA), after

which the gel was dried and subjected to autoradiography. To confirm specific

binding, one picomole of unlabeled probe was used as a “cold competitor.” The

relative NF-κB-binding activity was quantitated by using a densitometer,

BIO-PROFIL X-press Zoom 2000 (Vilber-Lourmat, Marnela-Vallee, France).

35

2.3.6. Stimulation of bone marrow-derived macrophages

TLR2-deficient mouse was kindly provided by Dr. Sizuo Akira (Osaka University,

Osaka, Japan), and the wild-type C57BL/6 was purchased from Orient Bio Inc

(Seoul, Korea). Bone marrow cells were isolated from the femur and the tibia of 6-

to 8-week-old mice, adjusted to 1×106 cells/ml, and differentiated to macrophages

by incubation with 10% L929-cultured media in DMEM supplemented with 10%

FBS, 50 μM 2-mercaptoethanol, and antibiotics for 3 days. The cells were washed

once with PBS, and the adherent cells were cultured in a fresh DMEM medium

containing 10% FBS and antibiotics without L929-cultured media for 12 h. Then,

the cells were stimulated with E. faecalis LTA (3 or 30 μg/ml), Pam3CSK4 (10

μg/ml), or S. typhosa LPS (0.5 μg/ml) for 16 h. At the end of the stimulation, the

culture supernatants were used for the analysis of TNF-α production by the ELISA.

2.3.7. Preparation of killed whole cells by heat or calcium hydroxide

E. faecalis was cultured in BHI media at 37°C to mid-log phase (A600, 0.6).

Bacterial cells were precipitated by centrifugation and washed three times with PBS.

Colony-forming units (CFU)/ml were re-determined by spectrophotometry based on

a standard curve (A600 vs CFU). For the preparation of heat-killed E. faecalis, 1.0 ×

36

1010 CFU/ml was heated at 60°C for 60 min. For the preparation of calcium

hydroxide-killed E. faecalis, 70 μl of 250 mg/ml of calcium hydroxide was added to

630 μl of 1.7 × 1010 CFU/ml of E. faecalis followed by incubation at 37°C for 24 h.

The mixture was neutralized to pH 7 by slowly adding 1 N HCl, and the final

bacterial cell concentration was adjusted to 1.0×1010 CFU/ml. To ensure the bacteria

were completely killed, the killed bacteria were plated on BHI media containing 1.5%

agar and cultured overnight at 37°C. No bacterial colonies were observed.

2.3.8. Treatment of E. faecalis LTA with calcium hydroxide

The E. faecalis LTA or endotoxin-free water, a control, was incubated with

various concentrations of calcium hydroxide (0.25, 2.5, or 25 mg/ml) for 60 min or

at 25 mg/ml for various time periods (short-term periods: 5, 10, 20, 40, or 60 min;

long-term periods: 1, 3, 6, 12, or 24 h). At the end of the incubation period, the

mixture was neutralized to pH 7 by adding 1 N HCl. E. faecalis LTA was not lost

during the treatment as confirmed by the measurement of phosphate contents in the

E. faecalis LTA.

2.3.9. Statistical Analysis

All experiments including mass spectrometric analysis were performed at least two

or three times. To analyze the statistics of in vitro experiments including the

production of proinflammatory mediators in RAW 264.7 cells, comparisons

37

between experimental groups and control groups were compared using a Student’s

t-test. Differences were considered significant when P < 0.05.

38

Chapter III. Results

3.1. Purification of highly-pure LTA from E. faecalis

LTA is one of the most important cell-wall virulence factors of Gram-positive

bacteria, determining innate immunity. To examine the ability of LTA from E.

faecalis to induce inflammatory responses, LTA from E. faecalis was purified by

using butanol extraction and octyl-sepharose chromatography, followed by

DEAE-sepharose chromatography [80]. Column fractions were analyzed for

phosphate contents, which have a linear correlation with LTA contents [107]. After

purification by octyl-sepharose chromatography, fractions 9 to 19 were shown to

contain abundant phosphate (Figure 4A) and were therefore pooled and further

subjected to DEAE-sepharose column chromatography. As shown in Figure 4B,

fractions 7 to 10, containing high amounts of phosphate, were pooled, lyophilized,

quantified, and used for subsequent experiments. The purified LTAs were further

examined for endotoxin contamination using the Limulus amebocyte lysate test. The

level of endotoxin was less than 20.29 pg/mg of the purified LTA preparation,

indicating that the amount of endotoxin in the LTA preparation was biologically

irrelevant. To further determine protein contamination in LTA preparation, LTA

purified from E. faecalis was subjected to silver staining. As shown in Figure 5,

LTAs with heterogeneous size ranging from 7 to 25 kDa were detected in the

purified LTA whereas no detectable protein band was observed. Furthermore,

nucleic acid were not detected in one mg of the purified LTAs as determined by

39

spectrophotometry. These results indicate that E. faecalis LTAs purified by a novel

purification method using mild organic solvent extraction, hydrophobic-interaction

and ion-exchange chromatography were not contaminated with biologically active

molecules such as nucleic acid, protein, and endotoxin.

40

Figure 4. Elution profile of LTA from an octyl-sepharose

hydrophobic-interaction column followed by elution on a DEAE-sepharose

anion-exchange column. LTA was prepared from E. faecalis by sequential

application of butanol extraction, hydrophobic-interaction column chromatography,

and ion-exchange column chromatography. (A) A phosphate assay was performed

to measure the quantity of LTA after octyl-sepharose column chromatography.

Fractions 9 to 19 were pooled and subjected to DEAE-sepharose column

chromatography. (B) A phosphate assay was performed to quantify of LTA after

DEAE-sepharose column chromatography. The octyl-sepharose column and

DEAE-sepharose column were eluted with 35% n-propanol in 0.1 M of the sodium

acetate buffer and 0.1 M sodium acetate buffer containing 30% n-propanol with a

linear salt gradient, respectively (dotted line).

41

Figure 5. Visualization of E. faecalis LTA using silver staining. E. faecalis LTA

(Ef.LTA; 0.1, 5, 10, or 20 μg) was separated using SDS-PAGE and visualized by

silver staining to examine residual proteins in LTA preparation. Bovine serum

albumin (BSA; 0.1, 1, 10, or 100 ng) was used as a standard. SM, size marker.

42

3.2. Determination of immunological function of E. faecalis

LTA

3.2.1. LTA from E. faecalis has inflammatory potential

Since E. faecalis is a pathogenic Gram-positive bacterium closely related to

refractory apical periodontitis and its LTA is considered as a major virulence factor

of Gram-positive bacteria, the ability of E. faecalis LTA to activate innate immune

responses was examined by determining the production of proinflammatory

mediators, TNF-α and NO, in the murine macrophage cell-line, RAW 264.7 cells.

The cells were stimulated with E. faecalis LTA for 18 h for the analysis of TNF-α

production. E. faecalis LTA significantly induced (P < 0.05) the production of

TNF-α in a time- and concentration-dependent manner in RAW 264.7 cells (Figure

6A and B). In contrast to TNF-α, the production of NO in RAW cells stimulated

with E. faecalis LTA was not observed even at concentrations up to 10 μg/ml

(Figure 6C). Previously, it had been reported that highly pure LTA weakly induced

NO production in RAW 264.7 cells in the absence of IFN-γ [106]. More recently, as

certain Gram-positive bacterial LTA such as L. plantarum LTA alone had no

inflammatory potential but induced NO production in the presence of IFN-γ [129,

130], the ability to induce the production of NO in RAW 264.7 cells by E. faecalis

LTA in the presence of IFN-γ was further examined. When RAW 264.7 cells were

stimulated with E. faecalis LTA for 24 h in the presence of IFN-γ (0.1 ng/ml), E.

faecalis LTA showed a dose-dependent induction of NO in RAW 264.7 cells

43

(Figure 6D). These findings suggest that LTA derived from E. faecalis is an

inflammatory agent capable of activating host innate immunity, whose

inflammatory potential gets significantly potentiated by IFN-γ. To further confirm

that the activation of macrophages by E. faecalis LTA was not caused by endotoxin

contamination during LTA preparation, RAW 264.7 cells were stimulated with E.

faecalis LTA under the same condition but in the presence of polymyxin B (25

μg/ml), an inhibitor of endotoxin. Polymyxin B did not suppress the production of

TNF-α or NO production induced by E. faecalis LTA (Figure 7A and C), whereas it

substantially (P < 0.05) inhibited the production of both inflammatory mediators

induced by LPS (Figure 7B and D). These results imply that the ability of E.

faecalis LTA to activate macrophages is not caused by endotoxin contamination in

the LTA preparation.

44

Figure 6. Induction of TNF-α and NO productions by RAW 264.7 cells

stimulated with E. faecalis LTA. RAW 264.7 cells (1 × 106 cells/ml) were (A)

stimulated with 30 μg/ml of E. faecalis LTA (Ef.LTA) for various times (0, 1, 3, 6,

12, 18, or 24 h), or the cells were (B) stimulated with various concentrations of E.

faecalis LTA (Ef.LTA; 1, 3, 10, or 30 μg/ml) for 18 h. At the end of the incubation

period, culture media were harvested and analyzed for TNF-α by using an ELISA.

RAW 264.7 cells (1 × 106 cells/ml) were stimulated with E. faecalis LTA (0, 0.1, 1,

or 10 μg/ml) in the (C) absence or in the (D) presence of recombinant mouse IFN-γ

(0.1 ng/ml) for 24 h. Nitrite accumulation was used as an indicator of NO

production in the cultured media. Error bars indicate SD, and an asterisk represents

significance at P < 0.05 compared with the nontreatment control.

45

Figure 7. Induction of TNF-α and NO productions by RAW 264.7 cells

stimulated with E. faecalis LTA in the presence of polymyxin B. RAW 264.7

cells (1 × 106 cells/ml) were stimulated with E. faecalis LTA (Ef.LTA; 1, 3, 10, or

30 μg/ml) or E. coli LPS (Ec.LPS; 0.5 μg/ml) for 24 h in the (A) absence or (B)

presence of polymyxin B (25 μg/ml) for 18 h. At the end of the incubation period,

culture media were harvested and analyzed for TNF-α by ELISA. RAW 264.7 cells

(1 × 105 cells/ml) were stimulated with (C) 30 μg/ml of E. faecalis LTA or (D) 0.1

μg/ml of E. coli LPS together with recombinant mouse IFN-γ (0.1 ng/ml) in the

absence or presence of polymyxin B (25 μg/ml) for 48 h. Nitrite accumulation was

used as an indicator of NO production in the culture media. Error bars indicate SD,

and an asterisk represents significance at P < 0.05 compared with the nontreatment

46

control.

47

3.2.2. TLR2 is crucial for activation of macrophage by E. faecalis LTA

TLR2 appears to be the primary mediator of the innate immune response to LTAs

from various Gram-positive bacteria [102]. To examine whether the E. faecalis

LTA is able to stimulate TLR2 or TLR4, CHO/CD14/TLR2 and CHO/CD14/TLR4

cells, which express human TLR2 and TLR4, respectively, were treated with E.

faecalis LTA (0, 1, 3, 10, or 30 μg/ml) for 16 h. Then, TLR2- or TLR4-dependent

NF-κB activation was determined using flow cytometry by analyzing CD25

expression. As shown in Figure 8, CD25 expression on the CHO/CD14/TLR2

increased with E. faecalis LTA treatment in a concentration-dependent manner, as

observed when using a representative TLR2 ligand, Pam3CSK4. In contrast, no

expression of CD25 was observed in CHO/CD14/TLR4 cells under the same

conditions, whereas E. coli LPS, a representative TLR4 ligand, substantially

stimulated CD25 expression (Figure 8).

48

Figure 8. Activation of TLR2 by E. faecalis LTA. (A) CHO/CD14/TLR2 or

CHO/CD14/TLR4 cells were treated with E. faecalis LTA (Ef.LTA; 0, 1, 3, 10, or

30 μg/ml) for 16 h. Pam3CSK4 (10 μg/m) and E. coli LPS (Ec.LPS; 0.5 μg/ml) were

used as positive controls for TLR2- and TLR4 stimulations, respectively. At the end

of the incubation period, TLR2- or TLR4-dependent NF-κB activation was

determined by flow cytometric analysis of CD25. The percentage of CD25-positive

cells was shown in the upper right in each histogram. One of three similar results is

49

shown.

50

3.2.3. TLR2 is important for the inflammatory potential of E. faecalis LTA in macrophage

To confirm whether TLR2, but not TLR4 signaling is responsible for the

inflammatory responses in macrophages by E. faecalis LTA, bone marrow-derived

macrophages were prepared from a TLR2-defi cient mouse. Then, the macrophages

were stimulated with 3 or 30 μg/ml of E. faecalis LTA, 10 μg/ml of Pam3CSK4 (a

TLR2-specific ligand), or 0.5 μg/ml of S. typhosa LPS (a TLR4-specific ligand) for

16 h, and the production of TNF-α was measured. As shown in Figure 9, like

Pam3CSK4, E. faecalis LTA was not able to induce TNF-α expression in

TLR2-deficient macrophages, whereas TNF-α was induced in wild-type

macrophages under the same condition. In contrast, the S. typhosa LPS significantly

(P < 0.05) induced the production of TNF-α (P < 0.05) to a similar extent in both

TLR2-deficient macrophages and wild-type macrophages. These results indicate

that the inflammatory responses in macrophages stimulated with E. faecalis LTA

are mediated via TLR2 signaling pathway. In addition, the results further imply that

purified LTA from E. faecalis does not contain a biologically relevant amount of

endotoxin that could stimulate TLR4.

51

Figure 9. Activation of TLR2 on macrophages by E. faecalis LTA. After

preparing bone marrow-derived macrophages from TLR2-deficient mouse (TLR2

KO) and C57BL/6 wild-type mouse (wild-type), the cells were stimulated with 3 or

30 μg/ml of E. faecalis LTA (Ef.LTA), 10 μg/ml of Pam3CSK4, or 0.5 μg/ml of S.

typhosa LPS (St.LPS) for 16 h and the production of TNF-α was measured by

ELISA. Error bars indicate SD, and an asterisk represents significance at P < 0.05

compared with the nontreatment control.

52

3.2.4. E. faecalis LTA induces the activation of transcription factor

NF-κB

NF-κB is an important transcription factor that initiates the transcription of genes

encoding a number of inflammatory mediators [131]. To examine whether E.

faecalis LTA induces activation of NF-κB, the DNA-binding activity of NF-κB was

measured. As shown in Figure 10, E. faecalis LTA significantly enhanced (P < 0.05)

the DNA-binding activity of NF-κB. These findings indicate that E. faecalis LTA

induces the NF-κB signal transduction cascade.

53

Figure 10. Enhanced DNA-binding activity of NF-κB by E. faecalis LTA. (A)

RAW 264.7 cells (3 × 105 cells/ml) were stimulated with E. faecalis LTA (Ef.LTA;

0, 3, 10, or 30 μg/ml) or E. coli LPS (Ec.LPS; 0.5 μg/ml) for 90 min. Nuclear

extracts were prepared and incubated with 32P-labeled oligonucleotides containing

consensus sequences recognizing NF-κB. One picomole of the unlabeled probe was

used as a control for a competition assay to confirm specific binding and marked as

“Cold.” Reaction products were electrophoresed on a 4.8% polyacrylamide gel,

after which the gel was dried and autoradiographed. One of three similar results is

shown. (B) Relative NF-κB-binding activity was determined by densitometric

analysis of the corresponding bands from three independent electrophoretic mobility

shift assay results. Error bars indicate SD, and an asterisk represents significance at

P < 0.05 compared with the nontreatment control.

54

3.3. Structural and functional relationship of E. faecalis LTA

3.3.1. Highly pure LTAs were prepared from various Gram-positive

bacteria

A previous study has reported that LTAs from various Gram-positive bacteria have

differential inflammatory potentials [129]. Prior to comparing the

immunostimulating potential of E. faecalis LTA to other bacterial LTAs, various

Gram-positive bacterial LTAs were purified by performing sequential application of

butanol extraction, octyl-sepharose chromatography, and DEAE-sepharose

chromatography. As shown in Figure 11, the abundant phosphates were detected in

the relatively different fractions among LTA preparations of each bacterial species

following by octyl-sepharose chromatography (fractions from 8 to 19 in E. faecalis,

from 8 to 13 in S. aureus, from 7 to 15 in L. plantarum, and from 10 to 14 in B.

anthracis, respectively). After pooling the phosphate-containing fractions and

subjecting them to DEAE-sepharose chromatograpy, phosphate contents in each

fraction were further analyzed. As shown in Figure 12, the profiles of

phosphate-containing fractions were exhibited with subtle differences (fraction from

6 to 11 and 16 in E. faecalis, from 5 to 13 in S. aureus, from 7 to 21 in L. plantarum,

and from 7 to 12 in B. anthracis, respectively). The purified LTAs were further

examined to measure endotoxin contents using the Limulus amebocyte lysate test.

The level of endotoxin was less than 20 pg/mg in every purified LTA (20 pg of

endotoxin in 1 mg of E. faecalis LTA, 17 pg of endotoxin in 1 mg of S. aureus LTA,

55

and no endotoxin was detected in both L. plantarum LTA and B. anthracis LTA,

respectively), suggesting that LTA preparations did not contain a biologically

relevant amount of endotoxin. Furthermore, contaminations with nucleic acids and

proteins were negligible in the purified LTAs as determined by spectrophotometry

(Table 5). To further determine contamination in LTA preparations, purified LTAs

were separated in 15% SDS-PAGE gel and were subjected to coomassie blue

staining and silver staining. As shown in Figure 13, no protein bands detected in

LTA preparation in the gel stained with coomassie blue solution whereas bands with

heterogeneous size ranged from 7 to 25 kDa in S. aureus LTA, E. faecalis LTA and

B. anthracis LTA or from 7 to 240 kDa in L. plantarum LTA were detected by silver

staining. These results indicate that the purified LTAs were not contaminated with

biologically active molecules such as nucleic acid, protein, and endotoxin.

56

Figure 11. Elution profiles of LTAs from octyl-sepharose

hydrophobic-interaction column. LTAs were prepared from E. faecalis, S. aureus,

L. plantarum, or B. anthracis through the sequential application of butanol

extraction, hydrophobic-interaction column chromatography. Phosphate assays were

performed to determine LTA-containing fractions after octyl-sepharose column

chromatography. The octyl-sepharose column was eluted with 35% n-propanol in

0.1 M of the sodium acetate buffer (dotted line).

57

Figure 12. Elution profiles of LTAs from DEAE-sepharose anion-exchange

column. Phosphate assays were performed to determine LTA-containing fractions

after DEAE-sepharose column chromatography. The DEAE-sepharose column was

eluted with 0.1 M sodium acetate buffer containing 30% n-propanol with a linear

salt gradient, respectively (dotted line).

58

Figure 13. Visualization of LTA preparations using coomassie blue staining and

silver staining. Five or ten micrograms of LTAs from S. aureus (Sa.LTA), E.

faecalis (Ef.LTA), L. plantarum (Lp.LTA), or B. anthracis (Ba.LTA) were separated

using SDS-PAGE and visualized by (A) coomassie blue staining and (B) silver

staining. Bovine serum albumin (BSA; 0.1, 1, 10, or 100 ng) was used as positive

control. SM; size marker

59

Table 5. Determination of impurities in LTA preparations

S. aureus LTA

E. faecalis LTA

L. plantarum LTA

B. anthracis LTA

Nucleic acid

(ng/mg of LTA) 3.8 N.D. N.D. N.D.

Endotoxin

(pg/mg of LTA) 17 20 N.D. N.D.

Protein

(ng/mg of LTA) < 100 < 100 < 100 < 100

N.D. denotes not detected

60

3.3.2. E. faecalis LTA exhibits a modest ability to induce the production

of NO, IL-6, MCP-1 in macrophages comparable to those of LTA

purified from other bacterial species

To further examine whether E. faecalis LTA exhibits differential ability to activate

host immune responses compared to other LTAs purified from different bacterial

species, RAW 264.7 cells were stimulated with LTA purified from E. faecalis, S.

aureus, L. plantarum, or B. anthracis for 24 h and subsequently studied for the

production of proinflammatory mediators such as NO, IL-6, and MCP-1. As shown

in Figure 14. S. aureus LTA significantly induced the production of NO, IL-6, and

MCP-1 in RAW 264.7 cells but other LTAs purified from E. faecalis, L. plantarum,

or B. anthracis did not induce NO production but weakly induced IL-6 and MCP-1

compared to those by S. aureus LTA. To determine whether these abilities of each

LTA altered under inflammatory conditions, the inflammatory potential of each LTA

in the presence of IFN-γ was further examined. When the RAW 264.7 cells were

stimulated with purified LTAs for 24 h in the presence of IFN-γ, the LTAs

including E. faecalis LTA, L. plantarum LTA, and B. anthracis LTA significantly

augmented the production of NO, IL-6, and MCP-1 although S. aureus LTA

exhibited the highest inflammatory potentials (Figure 14). These findings suggest

that inflammatory potential of E. faecalis LTA is modest compared to those of other

LTAs purified from differential species.

61

Figure 14. Comparison of immunostimulating abilities of LTAs purified from

various Gram-positive bacterial species. RAW 264.7 cells (5 × 105 cells/ml) were

stimulated with LTAs (5 μg/ml) from S. aureus (Sa.LTA), E. faecalis (Ef.LTA), L.

plantarum (Lp.LTA), or B. anthracis (Ba.LTA) in the absence or presence of

recombinant mouse IFN-γ (0.5 ng/ml) for 24 h. At the end of the incubation period,

culture media were harvested and analyzed for (A) NO, (B) IL-6, and (C) MCP-1.

Nitrite accumulation was used as an indicator of NO production in the culture media

as described in the materials and methods. The production of IL-6 and MCP-1 were

determined by using ELISA. Error bars indicate SD and an asterisk represents

significance at P < 0.05 compared with the nontreatment control.

62

3.3.3. E. faecalis LTA has modest TLR2-stimulating activity comparable to other bacteria LTAs

To examine whether the modest ability of E. faecalis LTA to induce NO, IL-6,

and MCP-1 is due to their modest TLR2-stimulating activity compared to other

LTAs, CHO/CD14/TLR2, a NF-κB reporter cell lines capable of inducing

expression of membrane-bound CD25 in proportion to the activation of TLR2, were

treated with E. faecalis LTA, S. aureus LTA, L. plantarum LTA, or B. anthracis

LTA for 16 h. Then TLR2-dependent NF-κB activation was determined using flow

cytometry. As shown in Figure 15A, CD25 expression on the CHO/CD14/TLR2

was increased with all of LTA treatment groups as observed when using a

representative TLR2 ligand, Pam2CSK4 and these expressions were not altered in

the presence of an endotoxin inhibitor, polymyxin B. As expected, the highest

TLR2-activating potential was observed when the cells were exposed to S. aureus

LTA whereas E. faecalis LTA moderately or L. plantarum LTA and B. anthracis

LTA weakly activated TLR2 under the same condition. In contrast, CD25

expression was not observed in CHO/CD14/TLR4 cells under the same conditions,

whereas E. coli LPS, a representative TLR4 ligand, substantially stimulated CD25

expression (Figure 15B). These results indicate that all of purified LTAs

preferentially stimulate TLR2 rather than TLR4 and the TLR2-activating potentials

of purified LTAs are coincident with their inflammatory potentials.

63

Figure 15. Comparison of TLR2-activating ability among LTAs purified from

various Gram-positive bacterial species. CHO/CD14/TLR2 or CHO/CD14/TLR4

cells were treated with 5 μg/ml of LTAs purified from S. aureus (Sa.LTA), E.

faecalis (Ef.LTA), L. plantarum (Lp.LTA), or B. anthracis (Ba.LTA) in the absence

or presence of polymyxin B (PMB; 25 μg/ml) for 16 h . Pam2CSK4 (0.1 μg/ml) and

E. coli LPS (Ec.LPS; 0.1 μg/ml) were used as positive controls for TLR2- and

TLR4 stimulation, respectively. At the end of the incubation period, TLR2- or

TLR4-dependent NF-κB activation was determined by flow cytometric analysis of

CD25. The percentage of CD25-positive cells was shown in the upper right in each

histogram.

64

3.3.4. E. faecalis LTA more weakly interacts with

anti-polyglycerophosphate antibody compared to other bacterial LTAs

The differential TLR2-stimulating activity of LTAs from different bacterial species

can be deducted by their structural differences such as molecular size, backbone

structure, and fatty acid composition [73]. Since LTA has been classified into two

major types based on its backbone structure including polyglycerophosphate-type

and polyribitolphosphate-type, the backbone structure of each purified LTA was

determined by Western blot analysis using anti-polyglecerolphosphates antibody.

Like S. aureus LTA, which is a representative of polyglycerophosphate-type LTAs,

LTAs purified from L. plantarum, or B. anthracis could interact with

anti-polyglycerolphosphates antibody but such an interaction appeared to be

weakened in E. faecalis LTA although previously it had been known as

polyglycerophosphate-type LTA. S. pneumoniae LTA, which has been known as

polyribitolphosphate-type LTA [81], did not show such an interaction (Figure 16).

Interestingly, most LTAs including S. aureus LTA (from 7 to 22 kDa, theological

number of repeating unit: 26 to 96), E. faecalis LTA (from 7 to 25 kDa, theological

number of repeating unit: 26 to 109), and B. anthracis LTA (from 7 to 20 kDa,

theological number of repeating unit: 26 to 87) exhibited relatively similar

molecular sizes whereas L. plantarum LTA (7 to 240 kDa, theological number of

repeating unit: 26 to 1096) was larger in size than other LTAs, indicating that LTA

from L. plantarum was composed of higher number of repeating units compared to

65

LTAs from S. aureus, E. faecalis, and B. anthracis. These results indicate that E.

faecalis LTA exhibits subtle differences in its backbone structure such as number of

repeating units and composition of substituents compared to other bacterial LTAs.

66

Figure 16. Characterization of backbone structure of LTAs. LTAs (5 or 10 μg)

from S. aureus (Sa.LTA), E. faecalis (Ef.LTA), L. plantarum (Lp.LTA), or B.

anthracis (Ba.LTA) were separated on SDS-PAGE gel and subsequently subjected

to Western blot using anti-polyglycerophosphates antibody. LTA (5 or 10 μg) from S.

aureus RN4220 (SaRN.LTA) and S. pneumoniae (Sp.LTA) were used as positive

and negative controls, respectively.

67

3.3.5. Differential bacterial LTAs exhibit subtle differences in their backbone structure

Glycolipids are pivotal for the immunostimulating potential of LTA [80, 129].

Despite the importance of glycolipids, the molecular structure in relation to E.

faecalis LTA has not been identified yet. To determine the glycolipid molecular

structure, the glycolipid of E. faecalis LTA was obtained using acidic hydrolysis

followed by MALDI-TOF mass spectrometry (Figure 17). Mass spectra showed that

peaks were mainly observed from 930 to 1,070 mass units (m/z) (Figure 18).

Among the distinct peaks, 12 representative peaks were analyzed with BioTools

software for molecular weight–based predictions of their structures. The glycolipids

were shown to be dihexosyl diacylglycerol, in which the acyl chain lengths varied

from C16 to C22. Interestingly, not only saturated and monounsaturated fatty acids,

which are general compositions of fatty acid in most of LTAs including S. aureus

LTA [64], but also diunsaturated fatty acids were observed (Table 6). These results

indicate that the modest TLR2-stimulating potentials of E. faecalis LTAs might be

due to subtle differences in their glycolipid structures compared to those of S.

aureus LTA.

68

Figure 17. Experimental scheme for isolation of glycolipid from E. faecalis LTA.

In order to disintegrate polyglycerophosphates of LTA, E. faecalis LTA (450 mg)

was solubilized and boiled in 98% acetic acid at 100oC for 3 h followed by

lyophilization. Then, a solvent comprised of chloroform, methanol, and water

(1:1:0.9, v/v/v) was added to the lyophilized products and mixed vigorously. After

phase separation, the organic phase containing the glycolipids of LTA was collected

and concentrated and followed by subjecting MALDI-TOF analysis.

69

Figure 18. Mass spectra obtained from the glycolipid anchor of E. faecalis LTA.

For structure and nomenclature of residues see Table 6. The numbers with arrows

indicate peak numbers in Table 6.

70

Table 6. Predictive structure of the glycolipid moiety of E. faecalis LTA

Peak

number

aObserved molecular mass (Da)

Calculated molecular mass (Da)

Mass difference

(Da)

Expected chemical formula

Fatty acids in the glycolipids

Carbon number

bPossible fatty acid

chains

Number of double

bonds

1 939.833 939.602 0.231 C49H88NaO15 34 C16/C18 2

2 941.828 941.617 0.211 C49H90NaO15 34 C16/C18 1

3 955.804 955.633 0.171 C50H92NaO15 35 C17/C18 1

4 967.857 967.633 0.224 C51H92NaO15 36 C18/C18 2

5 981.836 981.649 0.187 C52H94NaO15 34 C18/C19 2

6 983.849 983.665 0.184 C52H96NaO15 37 C18/C19 1

7 997.835 997.68 0.155 C53H98NaO15 38 C19/C19 1

8 1009.905 1009.68 0.225 C54H98NaO15 39 C19/C20 2

9 1023.827 1023.696 0.131 C55H100NaO15 40 C20/C20 2

10 1025.855 1025.712 0.143 C55H102NaO15 40 C20/C20 1

11 1051.902 1051.727 0.175 C57H104NaO15 42 C20/C22 2

12 1067.844 1067.759 0.085 C58H108NaO15 43 C21/C22 1

a Sodium ion was detected as a counter ion in all peaks.

b One of the possible combinations is shown.

71

3.4. Molecular mechanism of endodontic medicament

3.4.1. Calcium hydroxide inhibits the ability of E. faecalis to induce

TNF-α production in RAW 264.7 cells

Calcium hydroxide is a widely used endodontic medicament for eliminating viable

bacteria and inactivating virulence factors. To examine whether calcium hydroxide

is able to attenuate the inflammatory ability of E. faecalis, RAW 264.7 cells were

treated with heat-killed E. faecalis or calcium hydroxide-killed E. faecalis at 105 to

108 CFU/ml for 18 h. In RAW 264.7 cells, TNF-α expression induced by the

calcium hydroxide-killed E. faecalis was less than that induced by the heat-killed E.

faecalis (Figure 19), suggesting that calcium hydroxide can attenuate the capacity of

E. faecalis to induce expression of a proinflammatory cytokine, TNF-α.

72

Figure 19. Calcium hydroxide attenuates the ability of E. faecalis to induce

TNF-α production in RAW 264.7 cells. RAW 264.7 cells (1 × 106 cells/ml) were

stimulated with heat-killed or calcium hydroxide-killed E. faecalis [Ca(OH)2-killed

E. faecalis] at 105, 106, 107, or 108 CFU/ml for 18 h. At the end of the incubation

period, culture media were harvested to determine TNF-α production by using

ELISA. Error bars indicate SD and an asterisk represents significance at P < 0.05

compared with the nontreatment control.

73

3.4.2. Calcium hydroxide inhibits the ability of E. faecalis LTA to induce

the production of inflammatory mediators in murine macrophages

To examine whether calcium hydroxide attenuates the immunostimulating

potential of E. faecalis LTA, the expression of TNF-α in macrophages by calcium

hydroxide-treated E. faecalis LTA was determined. When E. faecalis LTA was

pretreated with 25 mg/ml of calcium hydroxide for 1, 3, 6, 12, or 24 h before

stimulating RAW 264.7 cells, TNF-α expression was not observed in any treatment

groups (Figure 20A). Thus, E. faecalis LTA was exposed to calcium hydroxide for

shorter time periods, 5-60 min. Treatment of E. faecalis LTA with 25 mg/ml of

calcium hydroxide even for 5 min completely abrogated the capacity of E. faecalis

LTA to stimulate RAW 264.7 (Figure 20B), suggesting that inactivation of E.

faecalis LTA by calcium hydroxide at 25 mg/ml is an immediate response. To

identify the minimum concentration of calcium hydroxide needed to inactivate LTA,

E. faecalis LTA was pretreated with various concentrations (0.25, 2.5, or 25 mg/ml)

of calcium hydroxide for 60 min before the stimulation of RAW 264.7 cells.

Expression of TNF-α was significantly attenuated at 2.5 mg/ml of calcium

hydroxide (P < 0.05), whereas no effect was observed at 0.25 mg/ml (P < 0.05)

(Figure 20C). These results indicate that concentrations of calcium hydroxide higher

than 2.5 mg/ml are necessary for the significant inactivation of E. faecalis LTA.

Furthermore, to validate and generalize whether calcium hydroxide is able to

attenuate the inflammatory ability of E. faecalis LTA, RAW 264.7 cells were

74

stimulated with intact or calcium hydroxide–treated E. faecalis LTA for 24 h, and

the culture media were analyzed for the productions of NO, IP-10, and MIP-1α. As

shown in Figure 21, the intact E. faecalis LTA augmented the expressions of

inflammatory mediators, but pretreatment of E. faecalis LTA with calcium

hydroxide abolished their expressions. These results suggest that calcium hydroxide

can attenuate the capacity of E. faecalis LTA to induce the production of

proinflammatory mediators.

75

Figure 20. Calcium hydroxide inactivates the ability of LTA from E. faecalis to

induce TNF-α production in RAW 264.7 cells. E. faecalis LTA was either treated

with 25 mg/ml of calcium hydroxide for long-term (1 to 24 h) (A) or short-term (5

to 60 min) (B) periods or treated with various calcium hydroxide concentrations

(0.25, 2.5, or 25 mg/ml) for 60 min (C). After neutralization to pH 7, the calcium

hydroxide-treated E. faecalis LTAs were used to stimulate RAW 264.7 cells (1 × 106

cells/ml) for 18 h. At the end of the stimulation, the culture media were collected for

the analysis of TNF-α by using ELISA. Error bars indicate SD and an asterisk

represents significance at P < 0.05 compared with the nontreatment control.

76

Figure 21. Calcium hydroxide abolishes the ability of E. faecalis LTA to

stimulate the expression of inflammatory mediators. E. faecalis LTA was treated

with or without calcium hydroxide (25 mg/ml) at 37oC for 60 min. After pH

neutralization, the calcium hydroxide-treated E. faecalis LTA was used for the

stimulation of RAW 264.7 cells (1 × 106 cells/ml) for 24 h. At the end of the

incubation, culture media were collected and analyzed for the production of (A) NO,

(B) IP-10, and (C) MIP-1α. Error bars indicate SD and an asterisk represents

significance at P < 0.05 compared with the nontreatment control.

77

78

3.4.3. Attenuated induction of inflammatory mediators by calcium

hydroxide-treated LTA is due to the loss of TLR2-stimulating activity

required for NF-κB activation

Since E. faecalis LTA preferentially recognized by TLR2, to further address the

mechanism for the inactivation of E. faecalis LTA by calcium hydroxide, E. faecalis

LTA was pre-treated with calcium hydroxide at 25 mg/ml for 60 min and its

TLR2-stimulating activity was determined using CHO/CD14/TLR2 cells. Flow

cytometric analysis demonstrated that CD25 expression on the CHO/CD14/TLR2

cells increased with E. faecalis LTA treatment in a concentration-dependent manner.

In contrast, no expression of CD25 was observed upon exposure to the calcium

hydroxide-treated E. faecalis LTA (Figure 22). These results indicate that calcium

hydroxide abrogates the ability of E. faecalis LTA to stimulate TLR2, resulting in

the attenuated induction of inflammatory mediators.

79

Figure 22. Calcium hydroxide eliminates the ability of E. faecalis LTA to

stimulate TLR2. E. faecalis LTA was pre-treated with calcium hydroxide at 25

mg/ml for 60 min and used to stimulate CHO/CD14/TLR2 for 16 h. At the end of

the incubation period, TLR2-dependent NF-kB activation was determined by flow

cytometric analysis of CD25. The percentage of CD25-positive cells is shown in the

upper right of each histogram.

80

3.4.4. Calcium hydroxide deacylates the glycolipid moiety of LTA from E.

faecalis

Since calcium hydroxide elicits high alkalinity through the release of hydroxide

ions, as sodium hydroxide can also delipidate the LTA structure [124, 132], it was

further examined whether calcium hydroxide could deacylate the glycolipid anchor

of LTA from E. faecalis using MALDI-TOF mass spectrometry (Figure 23). As

shown in Figure 24B, peaks for glycolipid anchors from E. faecalis LTA were

detected on the mass spectra in an expected manner. However, such peaks were not

observed in the glycolipid fractions of calcium hydroxide-treated (Figure 24D) or

sodium hydroxide-treated E. faecalis LTAs (Figure 24E). Notably, the glycolipids

undergoing deacylation cannot be detected within the m/z ranges at 930 – 1,070

because they fragment into small pieces that are free fatty acids, probably with a

minimum size of 250.230 Da (for C16:2) to a maximum size of 338.355 Da (for C22)

and a remaining dihexose with a glycerol backbone at 384.090 Da. In order to

confirm the calcium hydroxide-mediated deacylation of LTA, the release of fatty

acids from the glycolipids in the E. faecalis LTA treated with calcium hydroxide

was examined using TLC analysis. As shown in Figure 25, various glycolipids from

the intact E. faecalis LTA were detected in ladders via TLC due to differences in

lengths and saturation degrees of the fatty acid chains. However, free fatty acids

released from the calcium hydroxide-treated E. faecalis LTA were detected in

identical patterns as those of E. faecalis LTA deacylated by sodium hydroxide

81

treatment. Release of fatty acid with different retention factor also were observed in

preparation of glycolipid from E. faecalis LTA treated with sodium hypochlorite,

representative endodontic irrigation solution. Collectively, these results suggest that

calcium hydroxide deacylates the LTA structure, leading to abolishment of the

inflammatory activity of E. faecalis LTA.

82

Figure 23. Experimental scheme for isolation of glycolipid from calcium

hydroxide-treated E. faecalis LTA. In order to prepare glycolipids of E. faecalis

LTA treated with calcium hydroxide [Ca(OH)2] or sodium hydroxide (NaOH), E.

faecalis LTA (450 mg) was treated with 25 mg/ml of calcium hydroxide or 0.2 N

sodium hydroxide for 1 h followed by adjusting pH at 7 by adding HCl. After

lyophilizing the samples, they were solubilized and boiled in 98% acetic acid at

100oC for 3 h followed by lyophilization. Then, a solvent comprised of chloroform,

methanol, and water (1:1:0.9, v/v/v) was added to the lyophilized products then

mixed vigorously. After phase separation, the organic phase containing the

glycolipids of LTA was collected, concentrated and subjected to MALDI-TOF or

TLC.

83

Figure 24. Calcium hydroxide delipidates LTA of E. faecalis. E. faecalis LTA

(450 mg) was incubated in 25 mg/ml calcium hydroxide or 0.2 N sodium hydroxide

at 37oC for 60 min, and their glycolipids were analyzed using MALDI-TOF mass

spectrometry. The panels show MALDI-TOF mass spectra for (A) proteomics-grade

water, (B) the glycolipid fraction from E. faecalis LTA treated with

proteomics-grade water, (C) calcium hydroxide at 25 mg/ml, (D) the glycolipid

fraction from E. faecalis LTA treated with 25 mg/ml calcium hydroxide, (E) sodium

hydroxide at 0.2 N, and (F) the glycolipid fraction from E. faecalis LTA treated with

0.2 N sodium hydroxide. One of three representative results is shown.

84

Figure 25. Identifications of free fatty acids generated from the glycolipids

after calcium hydroxide treatment. E. faecalis LTA (300 μg) was incubated in 25

mg/ml calcium hydroxide, 5.25% sodium hypochlorite or 0.2 N sodium hydroxide

at 37oC for 60 min, and their glycolipids were prepared as described in Figure 23.

TLC was performed in a solvent containing chloroform, methanol, and water

(66:25:4, v/v/v), and fatty acids were visualized with 5% ethanolic

phosphomolybdic acid. Lane 1, glycolipid from E. faecalis LTA (Ef.LTA); Lane 2,

glycolipid from calcium hydroxide-treated E. faecalis LTA [Ca(OH)2]; Lane 3,

glycolipid from sodium hypochlorite–treated E. faecalis LTA (NaOCl). Lane 4,

glycolipid from sodium hydroxide–treated E. faecalis LTA (NaOH). The positions

of the origin and TLC solvent front are indicated by arrows. One of three similar

results is shown. Rf, retention factor.

85

86

Chapter IV. Discussion

4.1. Structure and Immunological function of E. faecalis LTA

In the present study, the structure and immunological functions of E. faecalis LTA

were investigated using highly-pure and structurally intact LTA purified by a novel

purification method. E. faecalis LTA has an immunostimulating ability to induce

the inflammatory mediators in macrophages through the activation of TLR2

signaling pathway. Furthermore, the lipid moiety of E. faecalis LTA plays a crucial

role for determining its immunostimulating potency by affecting TLR2 activation.

Thus, these results provide important clues for understanding the pathogenesis and

immunological properties of E. faecalis, a bacterium that has been getting increased

attention from biomedical scientists for its clinical importance as a problematic

nosocomial pathogen with high resistance to antibiotics and disinfectants.

LTA appears to be one of the major immunostimulating components of

Gram-positive bacteria responsible for innate immune responses. Like E. faecalis

LTA, LTAs from other Gram-positive bacteria have been reported to induce various

proinflammatory cytokines and NO production. Indeed, the capacity of LTA to

induce proinflammatory cytokines (eg, TNF-α and IL-1β) and chemokines (e.g.

IL-8) has been shown in various experimental models [133]. Moreover, a previous

study also reported that the production of TNF-α in macrophages by Gram-positive

87

bacterial culture supernatants (GPCS) was abrogated when treated with LTA

hydrolyzing agents or anti-LTA antibodies [105]. Furthermore, LTA also plays a key

role in serious inflammatory responses or septic shock caused by Gram-positive

bacteria, as shown by the study in mice in which LTA from S. aureus mimicked

Gram-positive bacterial sepsis when coadministered with PGN. Thus, E. faecalis

LTA is expected to play a key role in E. faecalis-mediated inflammatory responses.

It would be intriguing to speculate what essential moieties in the LTA structure are

responsible for its inflammatory potential. Among various structural moieties in the

LTA structure, the glycolipid moiety plays a pivotal role in the immunostimulating

capacity of E. faecalis LTA. This hypothesis is further supported by previous

studies as follows. First, a recent study using x-ray crystallography showed that acyl

chains were directly involved in the binding of LTA to its recognition receptor,

TLR2 [134]. Second, upon exposure to alkaline conditions capable of deacylating

LTA, LTA from S. aureus, B. subtilis, and L. plantarum could not stimulate TLR2

and lost their abilities to induce TNF-α and NO production in macrophages [129,

135]. Third, a study using synthetic LTA showed that the glycolipid was critical for

the immunostimulatory activity of LTA [62]. Finally, Enterococcus hirae ATCC

9790 expressed two kinds of LTA, diacylated and tetraacylated, the latter of which

is more potent than the former with regard to immunostimulatory potential [136].

88

The structure of E. faecalis LTA has been relatively well characterized except for

its composition of fatty acids. Its hydrophilic chains are repeating

1,3-polyglycerophosphates; the C-2 position of the glycerol residues are

nonstoichiometrically substituted with hydroxy, D-alanine, kojibiose, or

[D-Ala→6]-α-D-Glcp-(1→2-[D-Ala→6]-α-D-Glcp-1→) [31]. The structure of the

glycolipid anchor is Glc-α-(1→2)-Glc-α-(1→3)-diacylglycerol, and the fatty acids

are saturated straight chains and cis-monounsaturated chains [64, 137]. The

MALDI-TOF mass spectra of the present study also suggest that the glycolipids

from E. faecalis LTA consist of dihexosyl diacylglycerol harboring fatty acids

ranging from C16 to C22. Interestingly, the fatty acid structures are likely to be not

only saturated and monounsaturated chains but also diunsaturated chains. One

possible explanation for the detection of the diunsaturated LTA in this study is that

LTA with diunsaturated fatty acids may have been in a negligible amount in a

previous study [64] because most LTAs probably possessed saturated and

monounsaturated acyl chains. Coincidently, the mass spectra in the present study

also showed that the diunsaturated LTAs were detected at mass peaks with low

intensity as shown in Figure 18. Another possible explanation is the difference in E.

faecalis strains that were used in the present study (ATCC 29212) and the previous

study (DSM 20371 and 20478) [64]. Remarkably, E. faecalis can produce LTA with

two or four fatty acids depending on the strains [64]. It should be further

investigated if both acyl chains possess one of each double bond or if one is

89

saturated and the other acyl chain has two double bonds.

4.2. Differential inflammatory and TLR2-activating potential

of LTA among bacterial species

In this study, it was observed that the inflammatory and TLR2-stimulating

activities of LTAs from E. faecalis, L. plantarum, or B. anthracis appear to be less

potent than that of S. aureus LTA. These different potentials of LTAs might be due

to subtle differences of their molecular structures capable of affecting the interaction

with host molecules such as composition of substituents, number of repeating units,

and profiles of glycolipids (Table 7).

First, D-alanine could be important in the determination of pathogenic potential to

LTAs. Previous studies using a deletion mutant lacking the dltA gene, which is

missing D-alanine in the LTA structure, showed not only reduced production of

proinflammatory mediators production from peripheral blood mononuclear cells

(PBMCs) but also diminished biofilm formation [30], lower binding of opsonic

antibodies [31], and increased sensitivity to antimicrobial peptides compared with

wild-type bacteria [57, 58, 138]. Since the exposed amine group (-NH2+) of

D-alanine has been known to be responsible for the net charge of LTA, the deletion

of D-alanine might lead to a change of the electrostatic force resulting in the

decreased activity of LTA. Similar results were observed in LTAs from other

90

Gram-positive bacteria. For example, S. aureus, B. subtilis, and Group B

streptococcus (GBS) have different contents of D-alanine in their LTA, 70%, 25%,

and 46%, respectively and S. aureus LTA appeared to have the most powerful

inflammatory potential [80]. Similarly, LTAs from S. aureus and Lactobacillus

rhamnosus are better inducers of NO in macrophages than LTA from B. subtilis,

which exhibits a low percentage of alanylated LTA [139, 140]. In addition, a

previous study using synthetic LTA also demonstrated that the replacement of

D-alanine substituents with L-alanine leads to the reduction of the inflammatory

activity of the LTA by at least 10-fold [62]. Although the determination of the

D-alanine contents in LTA from E. faecalis, L. plantarum, and B. anthracis will be

needed to understand the differential potential of LTAs, the differential contents of

D-alanine in LTA might be one of the possible explanations.

Second, the composition of substituents in the repeating units and the structure of

hexoses in the glycolipids of LTAs also might affect the distinctive immunological

activity of LTAs. Previously, it had been demonstrated that S. aureus LTA composed

of glycerolphosphate polymers is esterified with D-alanine or

α-D-N-acetylglucosamine, whereas those of E. faecalis LTA are

non-stoichiometrically substituted with D-alanine, kojibiose, or

[D-Ala→6]-a-D-Glcp-(1→2-[D-Ala→6]-a-D-Glcp-1→) [31]. Another structural

analysis using nuclear magnetic resonance (NMR) revealed that L. plantarum LTA

91

has a-linked glucose and galactose on the glycerophosphate backbone instead of

α-D-N-acetylglucosamine substituents. Moreover, the Western blot analysis using

a-glycerophosphate antibody in this study also demonstrated that L. plantarum LTA

was composed of higher number of repeating units (approximately 11-fold higher)

compared to LTAs from S. aureus, E. faecalis, and B. anthracis. Since the

composition of carbohydrate seems to be closely associated with the recognition of

LTAs by host resulting in determination of the inflammatory potential [141], the

different number of repeating units and distinctive substituents in structures of LTAs

can act as another structural determinant for their activity.

Third, the differential compositions of fatty acids also appear to be critical for

conferring immunostimulating potentials of LTAs. According to previous

accumulating results suggest that LTAs with unsaturated fatty acids tend to be less

potent than LTAs with saturated fatty acids. For example, pneumococcal LTA

comprised of unsaturated fatty acids is 100-fold weaker than staphylococcal LTA

comprised of all saturated fatty acids [135]. LTA from L. plantarum hardly induces

the expression of inflammatory mediators in macrophages [129], and the structural

analysis using mass spectrometry suggests that its glycolipid also contains

unsaturated fatty acids [142]. E. faecalis LTA was also less potent than

staphylococcal LTA based on the fact that staphylococcal LTA alone sufficiently

induced NO production [143], while LTA from E. faecalis or L. plantarum required

92

IFN-g for substantial induction of NO production [144]. Indeed, another previous

study has reported that, unlike saturated fatty acids such as palmitic acid and lauric

acid, unsaturated fatty acids inhibit NF-κB activation in macrophages induced by

LPS or lipopeptide, a synthetic TLR2 agonist [145]. The preferred induction of

negative regulators by unsaturated fatty acids compared to saturated fatty acids

might be one possible explanation. This hypothesis is in accord with a previous

study showed that L. plantarum LTA, which possess unsaturated fatty acids,

inhibited E. coli LPS-induced TNF-α production in THP-1 cells through the

induction of interleukin-1 receptor-associated kinase M (IRAK-M). More recently,

it has been reported that the replacement of fatty acids in lipopeptide from saturated

form (palmitic acid) to unsaturated form (linoleic acid) can attenuate the recruitment

of neutrophils in vivo by activating peroxisome proliferator-activated receptor γ,

which is an intracellular receptor for unsaturated fatty acids [146, 147] that has an

anti-inflammatory activity. Another explanation is different affinities to lipid rafts,

which are highly ordered phospholipid microdomains enriched with saturated

phospholipid and cholesterol [148]. The formation of lipid rafts is required for the

stimulation of immune cells by various virulence factors, including LPS [149, 150].

Indeed, polyunsaturated fatty acids disturb immunostimulatory signals by altering

the structures of lipid rafts [151, 152]. Thus, LTA with unsaturated fatty acids might

have a lower affinity for lipid rafts, possibly resulting in lower immunostimulatory

potential.

93

Table 7. Structural characterization of LTAs

Structure of LTA

Ef.LTA Sa.LTA Lp.LTA Ba.LTA

Type of backbone

Glycero

phosphate

Glycero

phosphate

Glycero

phosphate

Glycero

phosphate

Variation of MW

(kDa)

7 ~ 25 7 ~ 22 7 ~ 240 7 ~ 20

R-groups

D-Ala, Kojibiose, [D-Ala→6]-α-D-Glcp-(1

→2)-[D-ala → 6]- α –D-Glcp-(1 →

D-Ala, NAG D-Ala, Glu/Gal Not

defined

Composition

of glycolipid Dihexosyl-DAG Dihexosyl-DAG

Trihexosyl-DAG

Trihexosyl-TAG

Not defined

Type of fatty acid

(most abundant FA)

Unsatureated

(C18/C19:1)

Saturated

(C17/C18)

Unsatureated

(C20/C21:2)

Not defined

MW denotes molecular weight

NAG denotes α-D-N-acetylglucosamine

DAG denotes diacylglycerol

TAG denotes triacylglycerol

FA denotes fatty acid

94

4.3. Molecular mechanism of endodontic medicament against

E. faecalis and its LTA

Calcium hydroxide is widely used as an intracanal medicament in endodontic

treatments for the elimination of viable bacteria and inactivation of bacterial

virulence factors. In the present study, it was demonstrated that treating E. faecalis

with calcium hydroxide, thus killing the cells, alleviated the induction of TNF-a in

RAW 264.7 cells by E. faecalis. Subsequent experiments using purified LTA from E.

faecalis demonstrated that calcium hydroxide could deacylate the LTA from E.

faecalis, resulting in the impairment of LTA immunostimulating activity.

Furthermore, inactivation of E. faecalis LTA by calcium hydroxide appears to occur

immediately (within 5 min) at a relatively low calcium hydroxide concentration (2.5

mg/ml). Previous studies had shown that the minimal inhibitory concentration of

calcium hydroxide against E. faecalis was 16 mg/ml [153] and that the effective

treatment time to kill E. faecalis was within 100 min [154], supporting this

suggestion that proper endodontic treatment with calcium hydroxide could

effectively detoxify E. faecalis and its LTA. Thus, the results provide an important

insight for understanding the molecular mechanisms by which the endodontic

medicament calcium hydroxide inactivates LTA of E. faecalis.

95

Calcium hydroxide can also inactivate LPS in Gram-negative bacteria [125],

which is considered to be a counterpart of LTA because of structural and functional

similarities. Indeed, calcium hydroxide-treated LPS failed to induce the production

of TNF-α from monocytes [126] and stimulate osteoclast formation [127].

Concomitant with the mechanism for the inactivation of LTA, hydroxide ions of

calcium hydroxide also detoxifies LPS via hydrolysis of fatty acids in the lipid A

moiety [128]. In contrast to hydroxide ions, calcium ions does not seem to be

involved in the inactivation of E. faecalis LTA because previously it has been

reported to promote bacterial adhesion by interacting with LTA [165]. In contrast,

chlorhexidine, a widely used endodontic medicament like calcium hydroxide,

inactivates both LPS and LTA but via a neutralizing attachment [155, 156].

Therefore, damaging the lipid moieties of bacterial virulence factors composed of

glycolipids might be a unique detoxification mechanism of calcium hydroxide.

Although the present study has shown that calcium hydroxide can detoxify LTA

from E. faecalis and reduce the induction of TNF-a in RAW 264.7 cells by E.

faecalis, there are obstacles that must be resolved in order to more efficiently use

calcium hydroxide as endodontic treatments. Firstly, calcium hydroxide is known to

be less effective in the presence of dentine with buffering capacity [119]. Secondly,

it is not certain at the present time whether calcium hydroxide is able to detoxify E.

faecalis and its LTA in biofilms as it is in planktonic conditions, because E. faecalis

is more resistant to alkaline stress in biofilms than in planktonic states [154].

96

Thirdly, it is unknown how to efficiently deliver calcium hydroxide in effective

amounts for an adequate time into root canals [157].

E. faecalis is more frequently found in obturated root canals exhibiting signs of

chronic apical periodontitis [3] although it is also detected in root-filled teeth with

or without periradicular lesions as determined by species-specific 16S ribosomal

RNA gene-based polymerase chain reaction [158]. Moreover, E. faecalis can be

cultured from periapical lesions refractory to endodontic treatment because of the

following unique characteristics of E. faecalis: (1) E. faecalis can invade dentinal

tubules, whereas not all bacteria have this ability [119]; (2) unlike other pathogens,

E. faecalis can colonize the root canal and survive without the support of other

bacteria [159]; and (3) E. faecalis is resistant to the antimicrobial effects of calcium

hydroxide [160], probably because of an effective proton pump mechanism, which

maintains optimal cytoplasmic pH levels.

It has been an important issue to identify an efficient endodontic treatment method

[161, 162]. A recent report has recommended 2% chlorhexidine with full-strength

sodium hypochlorite for the complete elimination of E. faecalis [163], although the

use of this method requires caution because this combination forms a precipitate

[164]. Thus, for a more effective endodontic treatment, it would be worthy of

testing whether an endodontic treatment is suitable not only for the elimination of E.

97

faecalis but also for the inactivation of its residual LTA even in the condition of

periodontal inflammation using periodontal ligament cells and oral epithelial cells.

In this context, present studies investigating the capacity of calcium hydroxide to

inactivate the LTA of E. faecalis might provide an experimental model for use in

developing effective endodontic medicaments and therapeutics targeting E. faecalis

LTA.

4.4. Conclusion

The identification of virulence factors and the determination of their

structure-function relationships in host immunity are crucial for understanding

bacterial pathogenesis and host immunity. Therefore, it is important to investigate

the structure and functional analysis of LTA, which has been regarded as a major

virulence factor of Gram-positive bacteria as a counterparts to LPS of

Gram-negative bacteria. In the present study, the structure and immunological

functions of E. faecalis LTA in host immune responses were demonstrated using

highly pure LTA that was prepared by the improved purification method (Figure

26). Conclusively, these results could provide useful information to enhance our

understanding of the pathogenesis of E. faecalis and host immunity.

98

Figure 26. Proposed structure and immunological functions of E. faecalis LTA.

E. faecalis LTA exhibits subtle differences in its glycolipid and backbone structure

compared to S. aureus LTA (Red square). The glycolipid plays an important role for

recognition and activation of TLR2. The glycolipid of E. faecalis preferentially has

unsaturated fatty acid and its composition of di-hexose also differs from those of S.

aureus. In addition, the substituents at the sn-2 position of the glycerophosphate

backbone of E. faecalis LTA, which is responsible for host humoral factors such as

antimicrobial peptides, LBP, CD14, C-type lectin, and immunoglobulins, also differ

from those of S. aureus LTA. Calcium hydroxide can inactivate E. faecalis LTA by

hydrolyzing acyl chains in glycolipid, which results in abrogation of TLR2

activation by E. faecalis LTA. The structure of substituents and di-hexose are

referenced by Theilacker C., et al [31, 137].

99

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List of publications

(* indicates the 1st author, # indicates publications related with this

thesis)

1. Jung Eun Baik*, #, Young Hee Ryu, Ji Young Han, Jintaek Im, Kee-Yeon

Kum, Cheol-Heui Yun, Kangseok Lee, Seung Hyun Han. (2008.08)

Lipoteichoic acid partially contributes to the inflammatory responses to

Enterococcus faecalis. Journal of Endodontics 34(8):975-982

2. Jung Eun Baik*, #, Kee-Yeon Kum, Cheol-Heui Yun, Jin-Kyung Lee,

Kang seok Lee, Kack Kyun Kim, Seung Hyun Han. (2008.11) Calcium

hydroxide inactivates lipoteichoic acid from Enterococcus faecalis. Journal

of Endodontics 34 (11):1355-1359

129

3. Young Hee Ryu, Jung Eun Baik, Jae Seung Yang, Seok-Seong Kang,

Jintaek Im, Cheol-Heui Yun, Dong Wook Kim, Kangseok Lee, Dae Kyun

Chung, Hyang Ran Ju, Seung Hyun Han . (2009.01) Differential

immunostimulatory effects of Gram-positive bacteria due to their

lipoteichoic acids. International Immunopharmacology 9 (1):127-133

4. Jin-Kyung Lee*, Jung Eun Baik*, #, Cheol-Heui Yun, Kangseok Lee ,

Seung Hyun Han, Woo Cheol Lee, Yoon Lee, Won-Jun Son, Seung-Ho

Baek, Kwang-Shik Bae, Kee-Yeon Kum (2009.02). Chlorhexidine gluconate

attenuates the ability of lipoteichoic acid from Enterococcus faecalis to

stimulate Toll-like receptor 2. Journal of Endodontics 35 (2):212-215

(Co-first author)

5. Raghu P. Kataru, Keehoon Jung, Cholsoon Jang, Hanseul Yang, Reto A.

Schwendener, Jung Eun Baik, Seung Hyun Han, Kari Alitalo, Gou Young

Koh. (2009.05) Critical role of CD11b+ macrophages and VEGF in

inflammatory lymphangiogenesis, antigen clearance, and inflammation

resolution. Blood 113 (22):5650-9

6. Jun Ho Jeon, Sun Kyung Kim, Jintaek Im, Ki Bum Ahn, Jung Eun Baik,

Ok-Jin Park, Cheol-Heui Yun, Seung Hyun Han. (2011.02) Trp-P-1, a

carcinogenic heterocyclic amine, inhibits lipopolysaccharide-induced

maturation and activation of human dendritic cells. Cancer Letters 301

(1):63-74

7. Jung Eun Baik*, #, Kyoung-Soon Jang, Seok-Seong Kang, Cheol-Heui

130

Yun, Kangseok Lee, Byung-Gee Kim, Kee-Yeon Kum, Seung Hyun Han.

(2011.02) Calcium hydroxide inactivates lipoteichoic acid from

Enterococcus faecalis through deacylation of the lipid moiety. Journal of

Endodontics 37 (2):191-196

8. Kyoung-Soon Jang, Jung Eun Baik, Seung Hyun Han, Dae Kyun Chung,

Byung-Gee Kim. (2011.04) Multi-spectrometric analyses of lipoteichoic

acids isolated from Lactobacillus plantarum. Biochemical and Biophysical

Research Communications 407 (4):823-830

9. Seulggie Choi*, Jung Eun Baik*, Jun Ho Jeon, Kun Cho, Deog-Gyu Seo,

Kee-Yeon Kum, Cheol-Heui Yun, Seung Hyun Han. (2011.09) Identification

of Porphyromonas gingivalis lipopolysaccharide-binding proteins in human

saliva. Molecular Immunology 48 (15-16):2207-2213 (Co-first author)

10. Seok-Seong Kang, Young Hee Ryu, Jung Eun Baik, Cheol-Heui Yun,

Kangseok Lee, Dae Kyun Chung, Seung Hyun Han. (2011.08) Lipoteichoic

acid from Lactobacillus plantarum induces nitric oxide production in the

presence of interferon-γ in murine macrophages. Molecular Immunology

48 (15-16): 2170-2177

11. Kyoung-Soon Jang*, Jung Eun Baik*, Seok-Seong Kang, Jun Ho Jeon,

Seulggie Choi, Yung-Hun Yang, Byung-Gee Kim, Cheol-Heui Yun, Seung

Hyun Han. (2012.03) Identification of staphylococcal lipoteichoic

acid-binding proteins in human serum by high resolution LTQ-Orbitrap mass

spectrometry. Molecular Immunology 50 (3):177-183 (Co-first author)

131

12. Jun Ho Jeon, Sun Kyung Kim, Jung Eun Baik, Seok-Seong Kang,

Cheol-Heui Yun, Dae Kyun Chung, Seung Hyun Han. (2012.06)

Lipoteichoic acid of Staphylococcus aureus enhances IL-6 expression in

activated human basophils Comparative Immunology, Microbiology &

Infectious Diseases 35: 363-374

13. Seok-Seong Kang, Hye Jin Kim, Mi Seon Jang, Sunjin Moon, Sang In

Lee, Jun Ho Jeon, Jung Eun Baik, Ok-Jin Park, Young Min Son, Gi Rak

Kim, Donghyun Joo, Heebal Kim, Jae Yong Han, Cheol-Heui Yun, Seung

Hyun Han. (2012.06) Gene expression profile of human peripheral blood

mononuclear cells induced by Staphylococcus aureus lipoteichoic acid.

International Immunopharmacology 13: 454-460

14. Jung Eun Baik*, Sun Woong Hong, Seulggie Choi, Jun Ho Jeon, Ok-Jin

Park, Kun Cho, Deog-Gyu Seo, Kee-Yeon Kum, Cheol-Heui Yun, Seung

Hyun Han. (2013.04) Alpha-amylase is a human salivary protein with

affinity to lipopolysaccharide of Aggregatibacter actinomycetemcomitans.

Molecular Oral Microbiology 28:142-153

15. Ok-Jin Park, Ji Young Han, Jung Eun Baik, Jun Ho Jeon, Seok-Seong

Kang, Cheol-Heui Yun, Jong-Won Oh, Seung Hyun Han. (2013.12)

Lipoteichoic acid of Enterococcus faecalis induces the expression of

chemokines via TLR2 and PAFR signaling pathways. Journal of Leukocyte

Biology 94 (6):1275-1284

16. Sun Woong Hong, Jung Eun Baik, Cheol-Heui Yun, Deog-Gyu Seo,

132

Seung Hyun Han. (2014.02) Lipoteichoic acid of Streptococcus mutans

interacts with Toll-like receptor 2 through the lipid moiety for induction of

inflammatory mediators in murine macrophages. Molecular Immunology

57 (2):284-291

17. Ki Bum Ahn, Jun Ho Jeon, Jung Eun Baik, Ok-Jin Park, Seok-Seong

Kang, Cheol-Heui Yun, Jong-Hwan Park, Seung Hyun Han. (2014.02)

Muramyl dipeptide potentiates staphylococcal lipoteichoic acid induction of

cyclooxygenase-2 expression in murine macrophages. Microbes and

Infection 16 (2): 153-160

18. Jintaek Im, Taeheon Lee, Jun Ho Jeon, Jung Eun Baik, Kyoung Whun

Kim, Seok-Seong Kang, Cheol-Heui Yun, Heebal Kim, Seung Hyun Han.

(2014.07) Gene expression profiling of bovine mammary gland epithelial

cells stimulated with lipoteichoic acid plus peptidoglycan from

Staphylococcus aureus. International Immunopharmacology 21

(1):231-240

19. Sun Woong Hong, Deog-Gyu Seo, Jung Eun Baik, Kun Cho,

Cheol-Heui Yun, Seung Hyun Han. (2014.10) Differential profiles of

salivary proteins with affinity to Streptococcus mutans lipoteichoic acid in

caries-free and caries-positive human subjects. Molecular Oral

Microbiology 99 (5):208-218

20. Jung Eun Baik*, Young-Oh Jang, Seok-Seong Kang, Kun Cho,

Cheol-Heui Yun, Seung, Hyun Han. (2015.05) Differential profiles of

133

gastrointestinal proteins interacting with peptidoglycans from Lactobacillus

plantarum and Staphylococcus aureus. Molecular Immunology 65

(1):77-85

21. Seok-Seong Kang, Jung Eun Baik, Jae Seung Yang, Kun Cho,

Cheol-Heui Yun, Seung Hyun Han. (2015.09) Protein profiles in mucosal

and systemic compartments in response to Vibrio cholerae in a mouse

pulmonary infection model. Microbial Pathogenesis 86:10-17

22. Jintaek Im, Jung Eun Baik, Kyoung Whun Kim, Jun Ho Jeon,

Kee-Yeon Kum, Cheol-Heui Yun, Seung Hyun Han. (2015.04) Enterococcus

faecalis lipoteichoic acid suppresses Aggregatibacter

actinomycetemcomitans lipopolysaccharide-induced IL-8 expression in

human periodontal ligament cells. International Immunology (in press)

23. Seok-Seong Kang, Sun Kyung Kim, Sung-Moo Park, Jung Eun Baik,

Sun Woong Hong, Jae Seung Yang, Cheol-Heui Yun, Seung Hyun Han.

Staphylococcal LTA antagonizing the B-cell mitogenic potential of LPS is a

new type of T-independent polysaccharide antigen. Journal of Immunology

(in revision)

24. Jung Eun Baik*, Deog-Gyu Seo, Sun Woong Hong, Seok-Seong Kang,

Kun Cho, PhD, Cheol-Heui Yun, Seung Hyun Han. Identification of

Porphyromonas gingivalis lipopolysaccharide-binding proteins in saliva

from patients with periodontitis. (in preparation)

134

25. Jung Eun Baik*, Hyuk-Il Choe, Sun Woong Hong, Seok-Seong Kang,

Kun Cho, Cheol-Heui Yun, Seung Hyun Han. Human salivary proteins with

affinity to lipoteichoic acid of Enterococcus faecalis. (in preparation)

26. Jung Eun Baik*, Jae Seung Yang, Seok-Seong Kang, Seung Hyun Han.

Lipoteichoic acid is important for the induction of protective immunity

against staphylococcal pneumonia. (in preparation)

문초록

Enterococcus faecalis는 반 강, 장 , 생식 같 체 내

막 직에 상 균 재하는 통 그람양 균 알 지만,

근 병원 내에 감염 통하여 균 증과 난 근단 주염과 같

염증질 발하는 원 균 보고 고 다. 균 포벽 에

라 그람 균과 그람양 균 할 , 그람 균 포벽

질 lipopolysaccharide (LPS)는 체 내 염증 발하는 핵심독

들에 한 체 역반 에 역할 상 규 어 는 반 ,

들과 능 사하다고 알 진 그람양 균 포벽 질

lipoteichoic acid (LTA) 경우, 그람양 균에 한 감염 및 염증 발에

핵심 역할 할 것 라고 상 에도 하고 에 사용하 한

리/ 방법에 염과 상 발 하여 재 지 들

천 역반 에 역할 하게 규 지 않았다. 본 연 에 는 도

높고 훼 없는 E. faecalis LTA 하고, 들

역학 능연 통하여 주 역 에 여하는 핵심작용 규 하는

한편, 상에 사용하는 근 독 산 슘과

상 작용 연 통하여 E. faecalis LTA 역할 검증하는 연

진행하 다.

LTA 하 하여, E. faecalis 비 한 다양한 그람양 균

[ 색포도상 균 (Staphylococcus aureus), 탄 균 (Bacillus anthracis), 그리고

산균(Lactobacillus plantarum)] 동반한 틸알코 추출법

행하여 포벽 쇄 후, octyl-sepharose 용한 상 작용

크 마토그래피, Diethylaminoethyl (DEAE)-sepharose 용한

크 마토그래피 차 행하 다. LTA 내 핵산 염여

법, 내독 염여 게 액 용한 내독 시험법(Limulus

Amebocyte lysate, LAL), 그리고 단백질 염여 coomassie blue 염색법과

염색법 통하여 각각 하 다. LTA 천 역 능

평가하 해 마우스 식 포 (마우스 식 포주 RAW 264.7 포

C57BL/6 상마우스 또는 TLR-결 마우스 골 래 식 포) LTA

극 후 생 산 질 [Nitric oxide (NO)]과 염증 싸 토 [Tumor

necrosis factor-alpha (TNF-α), interleukin (IL)-6, interferon gamma-induced

protein 10 (IP-10), macrophage inflammatory protein-1 alpha (MIP-1α), and

monocyte chemoattractant protein-1 (MCP-1)] 양 Griess시약 용한

아질산염 법과 효 역 법(Enzyme-linked immunosorbent assay,

ELISA) 통하여 각각 하 다. 톨 사 용체(Toll-like receptor, TLR) 2

신 달 사하 해, TLR2 또는 TLR4 사 에 라

포막에 항원(Cluster of differentiation, CD) 25 발 하도

작 햄스 난 (Chinse hamster ovary cell, CHO)/CD14/TLR2 포주

CHO/CD14/TLR4 포주에 LTA 처리 후 포 용하여

하 다. 염증매개 발 에 여하는 사 NF-κB DNA결합

electrophoretic mobility shift assay (EMSA)법 용하여 하 다.

LTA 에 재하는 반복단 골격 하 하여

polyglycerophosphate에 한 마우스 래 단클 항체 용한

웨스 블랏(Western blot)법 행하 다. E. faecalis LTA 에

재하는 당지질 하여 E. faecalis LTA에 아 트산 처리 통한 산

가 해 후 용매 용한 상 리법(Phase separation) 용하여 당지질

리 한 후 매트릭스 보 탈착/ 질량 법(Matrix-assisted laser

desorption/ionization-time of flight mass spectrometer, MALDI-TOF)과 얇

막 크 마토그래피(Thin layer chromatography, TLC) 하 다.

근 독 에 한 근단 주염 발 균 E. faecalis 및 들

LTA 사하 하여, 산 슘 시간별, 도별 처리한 E.

faecalis 및 들 LTA RAW 264.7 포 CHO/CD14/TLR2 포 극 후

염증매개 발 양상과 TLR2신 달양상 변 각각 ELISA법과 포

통하여 사하 다.

본 연 에 행한 LTA 리/ 법 용하여 E. faecalis 및 다양한

그람양 균 LTA 보할 었 , LTA 내에는 생 학

나타내는 핵산, 단백질, 내독 염 검출 지 않았다. E. faecalis

LTA 식 포 극 시 통계 는 높 NO, IL-6,

MCP-1생 찰 할 었다. TLR 신 달능 비 해본 결과, E. faecalis

LTA TLR2 시키는 반 , TLR4는 시키지 못하 다. E. faecalis

LTA 상마우스 골 래 식 포 극 시 통계

TNF-α 생 도한 반 , TLR2-결 골 래 식 포에 는 러한

상 찰 할 없었다. 추가 식 포에 E. faecalis LTA 처리 시

NF-κB 사 DNA결합 증가함 찰 할 었다. E. faecalis

LTA 염증매개 발 능 및 TLR2 능 다양한 그람양 균 래

LTA과 비 한 결과, S. aureus LTA 경우 높 염증매개 발 및

TLR2 도하는 반 , E. faecalis LTA 간 B. anthracis

LTA L. plantarum LTA 경우 낮 보 할 었다.

균 에 LTA 각 다 천 역 능 들

차 에 한 것 지 알아보 해 골격

당지질 특 해본 결과, 균 래 LTA는

공통 들 에 polyglycerophosphate 골격 가지고

나, 들 경우 지방산 및 포 도에 차

보 할 었다. 다 상에 사용하는

근 독 산 슘 E. faecalis 사 뿐만 아니라 사 후 잔 하는

들 LTA에 한 염증 발능 어할 는지 해본 결과, 열처리

사 E. faecalis 용하여 식 포 극 시 높 염증매개 발

도 는 반 , 산 슘 사 E. faecalis 경우 동 한 건에

염증매개 발 격 낮아짐 찰 할 었고, 러한 상

산 슘 들 LTA에 처리하 도 동 하게 나타났다.

CHO/CD14/TLR2 포 용한 TLR2 비 실험 통하여, 산 슘에 한

E. faecalis LTA 는 LTA에 한 TLR2신 달

시킴 어나는 상 할 었고, MALDI-TOF 및

TLC실험 통한 E. faecalis LTA 당지질 통하여 산 슘처리 시

LTA 당지질 내 지방산 해리 함 , E. faecalis 천 역

에 어 당지질 핵심 역할 하는 것 규 할 었다.

상 연 결과들 다 과 같 결 얻 었다. 틸알코

추출법, 상 작용 크 마토그래피법, 크 마토그래피법 차

행하여 다양한 그람양 균 도 높 LTA 하 다. E. faecalis

LTA 천 역 포 TLR2신 달 시킴 염증매개

발 발하는 균 병독 역할 한다. E. faecalis LTA는 다

그람양 균 LTA과 비 하 , 간 염증매개 발 발능 및

TLR2 능 보 , 러한 차 는 들 상 차 에 한

것 사료 다. 근 독 산 슘 근 감염 균 E. faecalis

LTA 당지질에 재하는 지방산 해리 도 통하여 천 역 포 내

TLR2 및 에 염증매개 발 도 시킨다. 결

E. faecalis LTA 천 역 포 내 TLR2 통하여 염증매개

발 시키는 독 작용하여, LTA 당지질 들 TLR2 도에

필 핵심작용 작용한다.

주요어: Lipoteichoic acid, Enterococcus faecalis, 그람양 균, 천 역,

톨 사 용체2, 근 감염

학 : 2007-22098