assessment of pasteurella multocida a...

Research ArticleAssessment of Pasteurella multocidaA Lipopolysaccharide as an Adhesin in an In Vitro Model ofRabbit Respiratory Epithelium

Carolina Gallego12 Stefany Romero3 Paula Esquinas4 Pilar Patintildeo5

Nhora Martiacutenez5 and Carlos Iregui5

1Laboratory of Veterinary Pathology Universidad de Ciencias Aplicadas y Ambientales Calle 222 No 55-37 Bogota Colombia2Faculty of Science Pontificia Universidad Javeriana Carrera 7 No 43-82 Bogota Colombia3Academic Assistant Veterinary Medicine Program Universidad de La Salle Cra 7 No 179-03 Bogota Colombia4Laboratory of Cytogenetics and Genotyping of Domestic Animal UGA Faculty of Veterinary MedicineNational University of Colombia Bogota Colombia5Laboratory of Veterinary Pathology Faculty of Veterinary Medicine National University of Colombia Bogota Colombia

Correspondence should be addressed to Carlos Iregui caireguicunaleduco

Received 25 September 2016 Revised 6 December 2016 Accepted 29 December 2016 Published 29 January 2017

Academic Editor Douglas Morck

Copyright copy 2017 Carolina Gallego et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The role of the P multocida lipopolysaccharide (LPS) as a putative adhesin during the early stages of infection with this bacteriumin the respiratory epithelium of rabbits was investigated By light microscopy and double enzyme labeling of nasal septa tissues theamount of bacteria attached to the respiratory epithelium and the amount of LPS present in goblet cells at different experimentaltimes were estimated Transmission electron microscopy (TEM) and LPS labeling with colloidal gold particles were also used todetermine the exact location of LPS in the cells Septa that were challenged with LPS of P multocida and 30 minutes later with Pmultocida showed more adherent bacteria and more severe lesions than the other treatments Free LPS was observed in the lumenof the nasal septum forming bilamellar structures and adhering to the cilia microvilli cytoplasmic membrane and cytoplasmof epithelial ciliated and goblet cells The above findings suggest that P multocida LPS plays an important role in the process ofbacterial adhesion and that it has the ability of being internalized into host cells

1 Introduction

Pasteurellamultocida is considered themost important causalagent of respiratory diseases in rabbits and in other speciesthis group of microorganisms is responsible for the largesteconomic losses in production farm animals [1] Underproduction conditions rabbits are frequently subjected toovercrowding drastic changes in temperature and humidityor stress all of which contribute to the appearance ofrespiratory pathologies caused by P multocida [2ndash5]

Bacterial adhesion to the mucosal surfaces of a hostresults from physicochemical interactions between thepathogens and the host epithelial cells and the ultimatecolonization of deeper layers of themucosa and the successful

establishment of an infection depends on this interactionTherefore adhesion is considered the first essential step thata pathogen must overcome to cause disease [6ndash9] Suchinteractions occur between molecules located on the surfaceof host epithelial cells as well as molecules on the bacteriaLipopolysaccharide (LPS) is the most abundant superficialconstituent of Gram-negative bacteria This glycolipid mole-cule is fundamental for the survival of these microorganismsand is responsible for causing severe systemic effects in thehosts such as endotoxemia therefore LPS is consideredan important virulence factor of these pathogens [10ndash13]However studies on the involvement of LPS during the firststeps of infection namely during adhesion and colonizationof the surface epithelia before the bacteria causes systemic

HindawiVeterinary Medicine InternationalVolume 2017 Article ID 8967618 13 pageshttpsdoiorg10115520178967618

2 Veterinary Medicine International

effects are rare and most are devoted to other bacterialadhesive structures such as the fimbria [14ndash17]

Belanger et al [18] proposed that the LPS ofActinobacilluspleuropneumoniae would be responsible for the adhesion ofthe bacterium to the respiratory epithelial cells of swine laterstudies of E coli Salmonella enterica subsp enterica serovarTyphimuriumHelicobacter pylori Klebsiella pneumonia andPseudomonas aeruginosa showed that it was possible toblock the adhesion of the corresponding microorganismsto different cell surfaces by suppressing or modifying thestructure of their respective LPSmolecules and by employingmonoclonal antibodies against the O antigen of the molecule[16 17 19ndash23]

Recently Bravo et al [16] showed that the core of Styphimurium LPS plays an important role in the interactionof the bacteria with HeLa epithelial cells as well as BHK andIB3 cells lines The inner core of the molecule has Glc I andGal I residues which are essential for adhesion and entry ofthe microorganism into epithelial cells in vitro The authorsspeculate that a lectin type receptor could be involved in theinteraction of the host cells with the outer core structurewhich is composed of (Gal I 120572 1-3 Glu) [16]

In addition a role for Helicobacter pylori LPS O antigenin adhesion and colonization of the gastric mucosa of micehas also been proposed Moreover it has been shown thatthe core of H pylori LPS is involved in pathogen bindingto laminin an extracellular matrix and basement membraneglycoprotein of the epithelium that is essential for the processof bacterial adhesion [17]

On the other hand few reports document themechanismby which LPS is internalized into the cytoplasm of the hostcells caspases 4 5 and 11 are considered intracytoplasmicreceptors for E coli and S typhimurium LPS in macrophagecell cultures and somatic cells such as mouse enterocytes inwhich they induce pyroptosis and apoptosis [23] In additionLPS can be internalized by the lipopolysaccharide bindingprotein (LBP) into the cytoplasm of monocytes and macro-phages as well as by nonimmune human cells such as theHEK293 cell line These findings have led to the hypothesisthat LPS plays an important role in the regulation of intracel-lular pro- and anti-inflammatory signals [24]

In P multocida different studies reported the significanceof several surface structures such as adhesins includinghighly hydrated polyanionic polysaccharides on the capsulethat are covalently bound to the surface of bacteria throughphospholipids or lipid A Other structures also include outermembrane proteins (OMPs) which may serve as adhesinsor invasins or participate in the formation of biofilms suchas type IV fimbriae the fibronectin binding protein andfilamentous hemagglutinin [25] however no study has beendevoted to exploring the role of P multocida LPS as anadhesion molecule For the first time we show that the LPSof P multocida significantly increases the adhesion of themicroorganism to the apical surface of the respiratory epithe-lium of the rabbit nasal septum and that the increased num-ber of adhered microorganisms causes more severe damageto the host cells We also show that LPS is internalized intothe cytoplasm of respiratory cells and that goblet cells play animportant role during these first steps of infection

2 Materials and Methods

21 Pasteurella multocida Strain Pasteurella multocida Astrain 001 was obtained from turbinates trachea and lungsof rabbits with signs of rhinitis and pneumonia from farmsin the Sabana de Bogota (Colombia) The organism wasgrown in brain heart infusion (BHI) agar gray nonhemolyticcolonies with a round morphology were selected Thecolonies were identified as bipolar-staining Gram-negativecoccobacilli andwere shown to be catalase- oxidase- indole-ornithine decarboxylase- glucose- sucrose- and mannitol-positive using biochemical tests The colonies did not growon MacConkey agar and were urease-negative [1] Molecularidentification of P multocida of capsular serogroup A wasperformed by expanding the sequence of the hyaD cap genelocus with the following primers F 51015840 TGC CAA AAT CGCAGT CAG 31015840 R 51015840 TTG CCA TCA TTG TCA GTG 31015840 [26]

22 Extraction Purification Quantification and BiologicalActivity of P multocida LPS LPS was extracted using thephenolhot water method [30] Pasteurella multocida (58 g)were resuspended in 29mL of ultrapure water The aqueousphase was dialyzed against sterile water and then lyophilizedThe sample was reconstituted in 10mL of 01M Tris buffercontaining 015M NaCl and 1N HCl pH 7 and treatedwith RNase (05mgmL) DNase (005mgmL) and 100 120583L of4mMMgCl

2for 2 hours at 60∘CThe sample was then treated

with proteinase K (005mgmL) and 100120583L of 1mM CaCl2

for 18 hours at 37∘C The extract was frozen at minus80∘C andlyophilized for 48 h A mass of 704mg was obtained and wasthen subjected to gel permeation chromatography brieflythe extract was resuspended in 5mL of 005M Tris buffercontaining 15 sodium deoxycholate and 1mM EDTA pH95 and passed through a GE Healthcare HiPrep 1660Sephacryl S-200HR column (SigmaAldrich St LouisMOUSA) Fractions of 25mL were collected at a flow rate of08mLmin and measured at 220 nm 260 nm and 280 nmusing spectrophotometry At each stage of purification andcharacterization aliquots were sampled and were analyzedwith a spectrophotometer by scanning between 200 nmand 600 nm (NanoDrop 2000 Spectrophotometer ThermoScientific Wilmington DE USA)

The resolving gel for electrophoresis was prepared at 12the stacking gel was prepared at 4 and the electrophoresisconditions were 003A45min and 002A1 h respectivelyThe lyophilized fractions were reconstituted in pyrogen-freewater to a final concentration of 1mgmL prepared in 1xLaemmli buffer and heated at 95∘C for 5 minutes before theywere added to the gel Staining was performed with silvernitrate and Coomassie blue [31]

The amount of polysaccharide in the extract was quanti-fied using the Purpald test a reference curve was constructedwith a standard of 3-deoxy-120572-D-mannooctulosonic acid(Kdo) (Sigma Co St Louis MO USA) in concentrationsranging from 08mM to 0025mM Commercial Escherichiacoli 0111B4 S typhimurium and Rhodobacter sphaeroidesDSM158 LPSswere diluted to two concentrations 04mgmLand 02mgmL Different concentrations of the P multo-cida LPS fractions were used ranging from 1mgmL to

Veterinary Medicine International 3

0125mgmL Duplicates of each sample were placed in a96-well microplate containing 32mM sodium periodate theplates were incubated at room temperature for 25min Sub-sequently 136mM Purpald reagent was added to each welland incubated for 20min at room temperature Immediately64mM of sodium periodate was added and incubated atroom temperature for 20min Then 20120583L of 2-isopropanolwas added Spectrophotometry was performed at 550 nm

The sterility of the LPS was confirmed with cultures inagar BHI in the presence and absence of 5 ovine bloodwhich was incubated at 37∘C and observed daily for six days

The biological activity of LPS was determined in miceby evaluating its pathogenic effects Twenty-five 120583g of LPSdiluted in 100 120583L of PSS was intraperitoneally inoculated infive mice 125 120583L of PSS was also IP injected in 5 additionalmice as a negative control After eight hours the animalswere euthanized and the lungs and liver were evaluated byhistopathology

23 Simultaneous and Sequential Exposure of Rabbit NasalSepta Explants to P multocida and to LPS (HampE) The nasalsepta of rabbit fetuses were cultured ex vivo using themethods reported by [32 33] Briefly fetuses were obtainedby cesarean on gestational day 26 under anesthesia andwere immediately euthanatized by medullar sectioning twosequential approximately 2mm thick cross sections of thenasal cavity were maintained in Dulbeccorsquos minimal essentialmedium (MEM) during the experiment All procedures wereapproved and authorized by the Bioethics Committee of theFaculty of VeterinaryMedicine andAnimal Science NationalUniversity of Colombia (Act 0062010)

Experimental Design The tissues were treated as described inTable 1

The tissues were immersed in 10mL of MEM in 5 cmwide times 2 cm high Petri dishes the tissues were incubated in ahumid chamber at 37∘Cwith a 5CO

2atmosphere for 2 h At

the appropriate times the tissues were immersed in McDow-ell and Trump fixative (commercial 4 formaldehyde and1 glutaraldehyde) diluted in Sorensonrsquos sodium phosphatebuffer

The lesions on the respiratory epithelium were evaluatedwith a 100x objective and were semiquantitatively assessedThe severity and extent of the lesions were graded using thescale reported by Bernet et al [29] (Table 2)

The following changes or lesions were evaluated desqua-mated cells (DC) activity of goblet cells (AGC) cilia loss(CL) the presence of intracytoplasmic vacuoles (IV) anddead cells (DC)

24 Evaluating the Amount of P multocida LPS on theApical Surface of Respiratory Epithelial Cells and within theCytoplasm of Goblet Cells as well as the Number of Pmultocida That Adhered to the Respiratory Epithelium (LH-IIP) P multocida and P multocida LPS were simultaneouslydetected on the respiratory epithelium of nasal septa by adouble labeling technique using indirect immunoperoxidase(IIP) for the bacterium and lectin histochemistry (LH)for LPS Briefly polyclonal ovine antibodies labeled with

horseradish peroxidase (HRP) were employed to detect Pmultocida a specific commercial Limulus polyphemus lectin(LPA) conjugated with alkaline phosphatase was added tothe nasal explants (alkaline phosphatase-conjugated Limuluspolyphemus lectin horseshoe crab EY Laboratories IncSan Mateo CA USA) This lectin specifically binds the 2-keto-3-deoxyoctanate (KDO) sugar of the core of LPS [34]The peroxidase technique was performed first and then thetissues were incubated with the LPA lectin

25 Analysis of the Images of LPS Lectin Histochemistry andIndirect Immunoperoxidase Staining of Pasteurella multocidaThe program ImageJ version 141 GPL (General PublicLicense) which is available for all systems and platformsand is widely used for analyzing biomedical images wasimplemented to evaluate the quantity of LPS on the apicalsurface of epithelial cells and the number of P multocidathat had adhered to the ciliated border of the respiratoryepithelium and within the cytoplasm of goblet cells [35]

26 Transmission Electron Microscopy The tissues weredehydrated in an ascending ethanol series from 50 to90 and were then embedded in LR White resin Ultrathinsections (90 to 100 nm) were cut and mounted on nickelgrids or nickel-covered 200 mesh Formvar grids the sampleswere rehydrated in distilled water Nonspecific sites wereblocked with 10 bovine serum albumin in PBS for 15minutes incubated on a drop of lectin Limulus polyphemusdiluted 1 3 in a buffer solution and conjugated to 5 nm widecolloidal gold particles for 30 minutes at 37∘C The gridswere washed and contrasted with 1 uranyl acetate for 3ndash5 minutes and observed with a Jeol 1400 Plus transmissionelectron microscope at 80 kV [36] No IIP was necessarybecause bacteria are more easily visible with this techniquethan with light microscopy

27 Statistical Analysis For the model assumptions theShapiro-Wilk test was used to determine the normality ofthe errors and Levenersquos test was used to determine thehomogeneity of the variance Analysis of variance (ANOVA)with a confidence interval of 95 and Tukeyrsquos multiplecomparison test were used to determine the differencesbetween treatments [37]

3 Results

31 Pasteurellamultocida Strain According to the taxonomictools of SeqMatch classification from the RDP the compari-son of the BlastN of Greengens and the basic alignment inNCBI the strain of P multocida used in this study shares99 identity with the genus and species Pasteurellamultocidasubspmultocida

32 Extraction Purification Quantification and BiologicalActivity of P multocida LPS A purified smooth chemotypeof P multocida LPS with the presence of KDO and heptosesin its core was obtained It was also protein-free (Figure 1)

33 Simultaneous and Sequential Exposure of Rabbit NasalSepta Explants to P multocida LPS (HampE) Nasal septa that


Table1Ex

perim

entalp

rotocolfor

expo

singfetalrabbitn

asalseptato

Pmultocid

aandLP

SIIP(in

direct

immun

operoxidase)L

H(le

ctin

histo

chem

istry)TE

M(tr

ansm

issionelectro

nmicroscop

y)and

C(minus)(negativ

econ

trol)lowast

[2728]

Treatm

ent

Negativec

ontro

lPmultocid

aLP

S+Pmultocid

asim

ultaneou

slyLP

S+Pmultocid

a30

min

later

Pmultocid

a+LP

S30

min

later

LPS

Treatm

ent1

Treatm

ent2

Treatm

ent3

Treatm

ent4

Treatm

ent5

Treatm

ent6

Techniqu

eIIP-LH

TEM

IIP-LH

TEM

IIP-LH

IIP-LH

TEM

IIP-LH

TEM

Num

bero

fexplants

73

73

77

37

3

Doselowast

MEM

MEM

1times108cfumL

1times108cfumL

10120583gmL+1times

108cfumL

10120583gmL+1times

108cfumL

10120583gmL+1times

108cfumL

1times108cfumL+

10120583gmL

10120583gmL

Expo

sure

time

2h2h

2h2h

2h2h

2h2h

2h


Table 2 Grading the severity of the lesions using HampE adaptedfrom Bernet et al [29]

(a)

Grade Severity Description

minus Absent (i) Cells with normal activity(ii) Normal architecture is readily visible

+ Light(i) Cells with scant activity(ii) Lesions are observed with somedifficulty

++ Moderate(i) Cells with moderate activity(ii) Lesions are easily observed withnormal architecture still visible

+++ Severe(i) Cells are very active(ii) Lesions are easily observed the normalarchitecture is not identified

(b)

Extension Description

Focal F lesion in an area comprising less than 10 ofthe section

Multifocal M lesions in more than two areas eachcomprising less than 10 of the section

Diffuse G multiple areas or an area greater than 50 ofthe section

974KDa

662KDa

45KDa31KDa

215KDa

144KDa

1 2 3 4 5 6 7 8 9 10

Figure 1 Electrophoretic profile of LPS of P multocida A UN0011 low molecular weight marker 2 commercial LPS of Escherichiacoli 0111B4 (InvivoGen San Diego CA USA) (006mgwell)3 commercial lipopolysaccharide of 119878 typhimurium ATCC 7823(Sigma Co St Louis MO USA) (01mgwell) 4 commercial LPSof Rhodobacter sphaeroides DSM 158 (InvivoGen San Diego CAUSA) 5 LPS of P multocida A UN001 (006mgwell) 6 LPS of Pmultocida A UN001 (003mgwell) 7 LPS of P multocida A UN001(006mgwell) 8 LPS of P multocida A UN001 (006 mgwell) 9LPS of P multocidaAUN001 (006mgwell) 10 LPS of P multocidaA UN001 (003mgwell)

were only challenged with P multocida showedmoderate celldesquamation and increased goblet cells activity consisting ofdilatation of the cytoplasm Most of these cells exhibited thischange Slight cilia loss was present in ciliated cells in somethe cytoplasm was vacuolated and others were dead

Table 3 Grading of the lesions after each treatment (HampE)desquamated cells (DQ) increased goblet cells activity (AGC) cilialoss (CL) presence of intracytoplasmic vacuoles (IV) and dead cells(DC)

Treatment DQ AGC CL IV DC(1) Negative control minus minus minus minus minus

(2) Exposure to P multocida ++ ++ + + +(3) Simultaneous exposure to Pmultocida LPS and P multocida +++ +++ ++ ++ ++

(4) Exposure to P multocida LPSfollowed by P multocida 30minlater

+++ +++ ++ ++ ++

(5) Exposure to P multocidafollowed by P multocida LPS30min later

++ ++ ++ ++ ++

(6) Exposure to LPS of Pmultocida +++ +++ + + +

The changes and lesions in the respiratory epithelium oftreatments 3 and 4 that is simultaneous exposure to P mul-tocida LPS and P multocida and exposure to P multocida LPSfollowed byPmultocida 30min later weremore severeThesechanges consisted of desquamated cells and the presence ofdetritus in the septal lumen In addition increased dilatationof the cytoplasm of goblet cells and a notorious extrusionof their secretion were common findings in many othersmoderate loss of cilia cytoplasmic vacuolization and deathof the ciliated cells were more abundant No such changes orlesions were observed in the nasal septa of the control group(treatment 1) that was only incubated with MEM withoutbacteria or LPS (Table 3)

34 Evaluating the Amount of P multocida LPS on theApical Surface of Respiratory Epithelial Cells and within theCytoplasm of Goblet Cells as well as the Number of Pmultocida That Adhered to the Respiratory Epithelium (LH-IIP) Thequantity of Pmultocida LPS on the apical surface ofgoblet cells and ciliated cells and within their cytoplasmand the number of bacteria that adhered to the respiratoryepithelium are recorded in Table 4 The results correspondto the mean of pixels of seven repetitions for each treatmentthe colorimetric information is transformed to numericinformation under the color model RGB (Red-Green-Blue)Based on the previous analysis the intensity and extent of thelabeling for LPS and for P multocida were increased in thetreatment group in which P multocida LPS and P multocidawere administered simultaneously followed by the challengewith P multocida LPS and 30min later with P multocida thechallenge with P multocida and 30min later with its LPS andfinally the tissues that were only exposed to P multocida

Positive P multocida staining was brown and had a gran-ular appearance whereas the LPS staining was reddish and ofdiffuse character in experiments 2 and 3 (Table 1) both colorswere adjacent at the interface (Figure 2) On the other handin tissues that were only exposed toPmultocida (Figure 2(b))the brown color was mainly observed on the ciliated borderof the corresponding cells and free in the lumen of the


(a)

lowast

(b)

lowast

(c)

GC

lowastlowast

(d)

Figure 2 Simultaneous and sequential exposure to P multocida and to its LPS and to P multocida alone in nasal septa explants LH andIIP (a) Tissue cultures that were not exposed to LPS or P multocida ciliated cells and goblet cells were not labeled (b) Tissues that wereonly exposed to P multocida (thick arrow) LPS is visible within the cytoplasm of goblet cells (lowast) faint LPS labeling lines the apical borderof apparently ciliated cells that have lost their cilia an apparent brown color is located intracytoplasmically in a nonidentifiable cell (thinarrow) (c) Simultaneous exposure to P multocida LPS and P multocida (lowast) Most of the LPS labeling is located within the cytoplasm of gobletcells several goblet cells are extruding their content along with LPS (arrow) (d) Tissues that were exposed to P multocida LPS followed by Pmultocida (lowast) 30min later show a similar appearance and signs as in (c) note the clearer interface between LPS and the bacteria (thin arrows)GC goblet cell HampE 100x

Table 4 Mean estimated quantity of LPS on the apical surface ofepithelial cells andwithin the goblet cells and the number of bacteriathat adhered to the ciliated border (mean of 7 repetitions for eachexperiment)

TreatmentEstimated numberof bacteria (pixels)

(mean plusmn SD)

Estimated LPSquantity (pixels)(mean plusmn SD)

(1) Negative control 0 0(2) P multocida 598976 plusmn 1281 1247 plusmn 465(3) LPS and Pmultocidasimultaneously

10240411 plusmn 3241 2080027 plusmn 8950

(4) LPS and 30minlater P multocida 10204817 plusmn 118870 2079876 plusmn 33380

(5) P multocida and30min later its LPS 6340926 plusmn 694 31586 plusmn 480

(6) LPS P multocida 0 1998847 plusmn 55056

tissue Interestingly the characteristic reddish LPS stain-ing was also visible within the goblet cells and apparentlylining the apical border of nonidentifiable epithelial cells

Moreover both colors the brown P multocida and thereddish LPS were also observed in close proximity to eachother in the treatment groups where both the bacterium andits LPS were administered albeit to a lesser extent (Figures2(c) and 2(d))

No positive labeling was observed for either antigen Pmultocida or its LPS in tissue that was not exposed to theantigens (Figure 2(a))

A confidence interval of 95 (120572 = 005) was used for allstatistical analyses

35 Transmission Electron Microscopy (TEM) Tissues fromthe negative control (treatment 1) that were only culturedwith MEM explants that were only exposed to P multocida(treatment 2) explants that were exposed to LPS and then toP multocida 30 minutes later (treatment 4) and explants thatwere only treated with LPS (treatment 6) were processed forTEM and LH (Table 1)

A Pasteurella multocida gatF mutant (AL2116) was alsoused that was unable to assemble the external core of LPSbeyond Glc IV GatF is the galactosyltransferase which addsGal I to the 4th position of the Glc IV [38] in these experi-ments no bacteria were seen to adhere to the respiratory nasal


CE

C

Figure 3 Respiratory epithelium of the nasal septum (treatment1) Negative control (treatment 1) Ciliated epithelial cell (CE) andcilia (C) MET and gold-labeling of LPS using a lectin of Limuluspolyphemus

epithelium moreover LPS staining with Limulus polyphemuscolloidal gold-labeled lectin was very poor or was completelyabsent and no ultrastructural changes in the respiratoryepithelium were observed after challenge with the mutantstrain (kindly donated by Dr Ben Adler Bacterial Pathogen-esis Research Group Department of Microbiology MonashUniversity)

No ultrastructural changes or positive labeling withcolloidal gold was observed in tissues that were not treatedwith P multocida or its LPS (Figure 3)

36 Location of the Ultrastructural Changes in the NasalSepta Caused by P multocida at Two Hours after Incubation(Treatment 2) In nasal septa that were only exposed toP multocida for two hours the bacteria adhered to themicrovilli and cilia of the corresponding cells In bacterialculture the external bacterial membrane and even more sothe capsule were LPS-positive as they reacted with colloidalgold-labeled lectin (Figure 4(a)) In tissue culture LPS islocalized at the interface of the bacterium and the cellmembrane of microvilli of ciliated cells which are also LPS-positive (Figures 4(b) and 4(c)) Moreover in the lumenof the organ thread-like structures resembling a cellularbilayer membrane were also evident and were associatedwith the microvilli and cilia and even within the cytoplasmof epithelial cells (Figures 4(d) 4(e) and 4(f)) many ofthese threads spanned the distance between cilia microvilliand the apical membrane building an apparent scaffold Noor only a few ultrastructural changes were observed in theepithelial cells of these septa

37 Location of the Ultrastructural Changes in Epithelial CellsCaused by Exposure to P multocida LPS and Then to Pmultocida 30 Minutes Later (Treatment 3) Nasal septa thatwere sequentially exposed to P multocida LPS and to Pmultocida 30min later showed increased numbers of bacteriaattached to the cilia of epithelial cells positive labeling for

LPS (colloidal gold nanoparticles of 5 nm in diameter) wasalso visible on the outer membrane of the bacteria as wellas in the cilia and cytoplasm of ciliated cells The bacterialost their capsule Moderate ultrastructural changes such asepithelial cell cytoplasmic vacuolization were more evidentin this experiment (Figure 5)

38 Location of the Ultrastructural Changes in Nasal SeptaInduced by P multocida LPS at Two Hours after Incubation(Treatment 6) Intense labeling in the cilia and within thecytoplasm of goblet cells and ciliated cells was observedin nasal septa that were exposed to P multocida LPS fortwo hours Severe cytoplasmic vacuolization was observed inepithelial cells that were only exposed to LPS (Figure 6)

4 Discussion

The LPS of Gram-negative bacteria is considered one of themain virulence factors of these pathogens causing severesystemic damage in the interaction with their hosts [39ndash41]a similar role has been recognized for the LPS of P multocida[39ndash42] Accordingly most studies with thesemolecules havebeen devoted to understanding the systemic effects caused byLPS once it reaches the circulatory stream [11 41 43 44] Incontrast much less effort has been dedicated to investigatingthe role of LPS in its respective bacteria during the first phasesof an infection and attachment to the mucosal surfaces of thecorresponding hosts [45] In an in vitro model we providemorphological evidence that P multocida A LPS mediatesthe adherence of the bacterium to the apical surface of thenasal respiratory epithelium in rabbits to our knowledge thisfinding has not been reported for this pathogen

It is considered that aGram-negative bacteriumpossessesapproximately 35 times 106 LPS molecules that occupy an area of49 120583m2 on its outer membrane Given that the approximatesurface of one of these microorganisms ranges from 6 to9 120583m2 the LPS would cover approximately 75 of its surfaceconstituting the main component of the outer membrane ofthese agents [11 46 47] Taking into account the amountof LPS on the surface of a Gram-negative bacterium itsprivileged localization on the surface of the microorganismand its rich content of carbohydrates some of which haveadhesive properties and so forth it is surprising that the roleof this molecule during the first steps of infection with thesemicroorganisms has not been more thoroughly investigatedRecently the role of the core oligosaccharide of Helicobacterpylori LPS in mediating the adhesion of the bacterium tolaminin an extracellular matrix glycoprotein in host base-ment membrane that is believed to be essential in the cellularadhesion process was suggested [17] In the same vein Bravoet al [16] showed that the outer core of S typhi LPS which iscomposed of Glc I Gal I andGlc II was required for effectiveadhesion and bacterial entry into HEp-2 cells

Based on these findings and on our own previous results[48] we proposed that the LPS of P multocida could be agood adhesive candidate for mediating pathogen attachmentto the respiratory epithelium of rabbits during the first phasesof infection Accordingly by simultaneously and sequentiallyexposing the nasal septa of fetal rabbits to P multocida LPS


(a)

B

CE

CE

MV

(b)

BB

BB

(c)

(d)

CE

(e)

CE

C

CE

(f)

Figure 4 Respiratory epithelium of the nasal septum that was exposed to P multocidaAUN001 (treatment 2) MET and gold-labeling of LPSusing a lectin of Limulus polyphemus (a) Bacterial structure with positive labeling on the outer membrane (thin arrow) Note the abundantcapsule that surrounds each bacteria Bacterial culture (b) Note the interface of the LPS between the bacterium (B) and microvilli (thinarrow) Some LPS is free in the lumen admixed with amorphous material but is also visible at the surface of some microvilli Microvilli (MV)of a ciliated epithelial cell (CE) and cilia (thick arrows) (c) This particular bacterium has lost its capsule (arrow) compared with three otherbacteria (B) (d) Thread-like bilaminar structure (thick arrow) located in the lumen of the nasal septum that is positively labeled with LPS(thin arrows) (e) Multiple thread-like LPS-positive bilaminar structures are associated with the cytoplasmic membrane and microvilli of anciliated epithelial cell (CE) (black arrows) some gold particles seem to ingress into the cell (white arrow) (f) A bilaminar structure bridgestwo cilia (black arrow) and similar thread-like bilaminar structures bridge the cilia and the cytoplasm of the same cell where the base of acilia was not included (red arrow) large numbers of gold particles are already present within the cytoplasm of the same cell (arrowheads)

and to the bacterium it was possible to show that the LPS ofPmultocida significantly increases the amount ofPmultocidathat attaches to the respiratory epithelial cells of the nasalsepta compared with the septa that were only exposed to thepathogen This result was confirmed by TEM experimentswhich showed that higher numbers of bacteria adhered to thecilia of epithelial cells when the septawere previously exposedto LPS (Figure 5)

Using lectin histochemistry (LH) P multocida LPS wasmore visible within the cytoplasm of goblet and ciliated cellson their apical border surrounding the cilia and in the extra-cellular medium This result was confirmed by TEM On theother hand as shown in Figure 2 larger numbers of bacteriaadhered to goblet cells (119875 lt 005) moreover in the twotreatments where nasal septa cultures were simultaneouslyand sequentially exposed to the LPS and the bacterium theseverity of the lesions in the respiratory epithelial cells wassignificantly increased (119875 lt 0001) compared with the septa

that were only treated with P multocida or were treated withthe microorganism and then its LPS 30min later (Table 3)

However and perhaps more importantly an unexpectedfinding in this study was that the explants that were onlyexposed to P multocida were also positively labeled for itsLPS by LH understandably in a lower amount comparedwith the explants that were exposed to the molecule andthe bacterium This staining included the same structures aswhen the LPS and the bacterium were added simultaneouslyor sequentially that is within the cytoplasm of goblet cellsand ciliated cells as well as a thin film over their respectiveapical membrane Previous findings were corroborated byTEM where in addition to the surface of the bacterium themolecule seems to join the microorganism to the microvilliand cilia of ciliated cells (Figures 4 and 5) More detailedlocation and relationship of the molecule with the apicalmembrane of goblet cells and ciliated cells were shown by thistechnique where it was observed to adhere to the cilia the


CE

Bac

C

VAC

CE

Figure 5 Respiratory epithelium of the nasal septum that wasexposed to P multocida LPS and then to P multocida 30minlater (Treatment 4) MET and gold-labeling of LPS using a lectinof Limulus polyphemus Bacterial structures (Bac) were positivelylabeled on their outer membrane (arrows) and were associated withthe cilia (C) of a ciliated epithelial cell (CE) all microorganismshave lost their capsule Large numbers of colloidal gold particles arevisible at the apical cytoplasm of the cell Cytoplasmic vacuolization(VAC)

CE

GCVAC

Figure 6 Respiratory epithelium of the nasal septum that wasexposed to P multocida AUN001 LPS (treatment 6) MET and gold-labeling of LPS using a lectin of Limulus polyphemus Intense P mul-tocida LPS-positive labeling is associated with the cilia cytoplasmicmembrane and cytoplasm (arrows) of a ciliated epithelial cell (CE)and with a goblet cell severe intracytoplasmic vacuolization (VAC)is observed

microvilli the apicalmembrane or even spanning two neigh-boring cilia (Figure 4) building a mesh-like structure thatmay favor bacterial adherence finally the molecule reachedthe cytoplasm of the epithelial cells One possible explanationfor this finding would be that P multocida spontaneouslyreleases its LPS from its outer membrane from the very firstincubation time [49 50] or from the very first moment ofits interaction with the respiratory epithelium as shown inthis work Interestingly the experiment in which the most

significant number of bacteria adhered was the experimentin which nasal septa cultures were simultaneously exposed tothe LPS and to P multocida This finding suggests that undernatural conditions the adherence of the bacterium to therespiratory epithelium would occur in a matter of minutesThis result fits well with the experimental results of infectionin rabbits which develop clinical signs after two hours In anycase the unexpected release of LPS under the experimentalconditions indirectly favors the suggested adhesive role of theP multocida LPS under natural conditions

Notably in this work the microorganisms display anabundant capsule immediately before they are added to thenasal septa cultures compared to the bacteria adhering tomicrovilli or cilia which have completely lost their capsuleor when only a small amount of the capsule remains (Figures4 and 5) The presence of a capsule on Gram-negative andGram-positive bacteria has repeatedly been reported as animpediment for their ability to adhere to mucosal surfacesOne mechanism proposed for this interference is that thecapsule masks adhesin(s) that might be located deeper on thebacterial surface [10 51 52] thismechanismworks well in thecase of P multocida in this study

Lipopolysaccharide-positive labeling of the same P mul-tocida A within the cytoplasm of goblet cells in the sameexperimental model used in this research has been alreadyreported [32] the only difference was that the previous studylabeled the LPS with polyclonal antibodies instead of withLimulus polyphemus lectin as in this work Using TEMEsquinas et al [53] showed that myelin-like structures werecontained within the vacuoles in the cytoplasm of ciliatedcells and suggested that these structures might correspondto P multocida LPS this study confirms that the LPS of Pmultocida effectively reaches the cytoplasm of these cells andthat cytoplasmic vacuoles are formed in these cells whichmay be caused by this molecule There are some possibilitiesto explain how the molecule reaches the cytoplasm of gobletcells and ciliated cells One is that the LPS of P multocidasimilar to the LPS of other Gram-negative pathogens wouldbe recognized by free mucus- and glycocalyx-located CD14receptors that would be internalized by activation of theTLR4MD2 complex [24 54ndash56] Another soluble receptoris LPS binding protein (LBP) this receptor binds LPS inthe blood plasma and transfers it to cellular receptors thusincreasing the activation of cells that lackmCD14 [57 58]Themechanisms for the internalization and intracellular trans-port of the endotoxin are not clear but some authors proposethat it travels after interacting with CD14 [59] whereas otherssuggest that the internalization involves a clathrin-dependentpathway through which it is transported via lipid rafts to theGolgi apparatus to bind intracellular TLR4 [15 60ndash62]

Recent studies report that LBP catalyzes the intercalationof LPS into the reconstituted phospholipid bilayers providingan additional mechanism of LBP-mediated transport of LPSinto host cell membranes and the cytoplasm of humanmonocytes and macrophages [24] In this regard it is quiteinteresting that the LPS of P multocida adopted a thread-likebilaminar appearance and was observed in direct interactionwith the cell membrane of the ciliated cells and their ciliain this work (Figures 4(d) 4(e) and 4(f)) which could be


evidence of the intercalation described by [24] It should benoted that these bilamellar structures were only observedin the LPS that was spontaneously released by the bacteria(treatment 2 Table 1) and were not observed in experimentsthat used purified LPS (treatments 3 and 4 Table 1) which ismore similar to the natural conditions of the infection

In experiments with purified LPS (treatments 3 and4 Table 1) it was found that LPS induced ultrastructuralchanges of the respiratory epithelium such as cytoplasmicvacuolization and dilated interepithelial spaces of the nasalsepta the changes induced by P multocida LPS alone weremore severe than the changes caused by the bacterium alone(Figures 5 and 6) This finding could be explained by theLPS dose to which the explants were exposed surely theamount of LPS that is spontaneously released by the bacteriais much lower than the amount of purified LPS used in theexperiments Studies exist that show that the effect of LPSis dose-dependent It is possible that in the first instancethe pathogen does not try to activate mechanisms thatlead to tissue damage but induces intracellular signals thatstimulate the immune system Some authors argue that thisupregulationmay occur in the body in response to high dosesof LPS or by a state of hyperreactivity to LPS On the otherhand when there is contact with low levels of LPS beneficialeffects on the host such as the development of resistance toinfections may be triggered [24 63ndash66]

Yan et al [67] proposed a mechanism by which LPSupregulates mucin genes such as MUC5AC and MUC2expression in human epithelial cells they demonstrated thatbacterial LPS utilizes reactive oxygen species (ROS) fortransmitting signals to provoke the host defense responseand upregulate MUC5AC mucin expression Gram-negativebacteria would use some substrates present in glycoproteinsproduced by goblet cells as the first binding site to initiateadhesion It is proposed that themechanismwould start frommetaplasia of mucus-producing cells induced by proinflam-matory cytokines such as TNF alpha released from leukocytesby the action of LPS [32 67ndash71]

More recently the PLUNC (palate lung and nasal epithe-lium clone) protein which is related to the LPS bindingprotein (LBP) family has been demonstrated in epithelialcells goblet cells and glandular airway cells [72 73] and ithas been suggested that it is stored and released from thegranules of neutrophils [74] PLUNC recognizes and bindsto LPS and it is proposed that PLUNC may play a role inthe innate immune response of the upper airways [73 75]Consistent with these findings our results might suggest thatthe positive labeling for P multocida LPS mixed with mucusin the cytoplasm of inflammatory cells that accompany thismucus and inside the glandular and epithelial cells couldbe explained in part by the binding of the endotoxin to thissubstance [45]

It can be concluded that the LPS of P multocidaAUN001plays an important role in the adhesion of bacteria to therabbit respiratory epithelium by increasing the number ofadherent bacteria in the presence of LPS Lipopolysaccharideis spontaneously released by the bacteria and interacts withstructures such as the cilia microvilli and cytoplasmicmembrane to achieve internalization into the cytoplasm of

epithelial ciliated and goblet cells These results should bethe foundation of new research focused on determiningexactly which components of P multocida LPS interact withthe host cell not only to advance the understanding of thepathogenesis of this disease but also to propose mechanismsto inhibit this interaction

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contributions

Carolina Gallego and Stefany Romero are lead authorsbecause they made contributions of equal importance in thisresearch

Acknowledgments

This study was financed by Colciencias the National Uni-versity of Colombia and the University of Applied andEnvironmental Sciences

References

[1] F Dziva A P Muhairwa M Bisgaard and H ChristensenldquoDiagnostic and typing options for investigating diseases asso-ciated with Pasteurella multocidardquo Veterinary Microbiology vol128 no 1-2 pp 1ndash22 2008

[2] E Kawamoto T Sawada and T Maruyama ldquoEvaluation oftransport media for Pasteurella multocida isolates from rabbitnasal specimensrdquo Journal of Clinical Microbiology vol 35 no 8pp 1948ndash1951 1997

[3] H Takashima H Sakai T Yanai and T Masegi ldquoDetectionof antibodies against Pasteurella multocida using immunohis-tochemical staining in an outbreak of rabbit pasteurellosisrdquoTheJournal of Veterinary Medical Science vol 63 no 2 pp 171ndash1742001

[4] Z Jaglic E Jeklova L Leva et al ldquoExperimental study of patho-genicity of Pasteurella multocida serogroup F in rabbitsrdquoVeteri-nary Microbiology vol 126 no 1ndash3 pp 168ndash177 2008

[5] A B J Stahel R K Hoop P Kuhnert and B M KorczakldquoPhenotypic and genetic characterization of Pasteurella multo-cida and related isolates from rabbits in Switzerlandrdquo Journal ofVeterinary Diagnostic Investigation vol 21 no 6 pp 793ndash8022009

[6] M Jacques ldquoRole of lipo-oligosaccharides and lipopolysaccha-rides in bacterial adherencerdquo Trends in Microbiology vol 4 no10 pp 408ndash410 1996

[7] S N Abraham B L Bishop N Sharon and I Ofek ldquoAdhesionof bacteria to mucosal surfacesrdquo Mucosal Immunology Two-Volume Set pp 35ndash48 2005

[8] E Fadda and R J Woods ldquoMolecular simulations of carbohyd-rates and protein-carbohydrate interactions motivation issuesand prospectsrdquoDrugDiscovery Today vol 15 no 15-16 pp 596ndash609 2010

[9] S Sattin and A Bernardi ldquoGlycoconjugates and glycomimeticsas microbial anti-adhesivesrdquo Trends in Biotechnology vol 34no 6 pp 483ndash495 2016


[10] M Jacques M Kobisch M Belanger and F Dugal ldquoVirulenceof capsulated and noncapsulated isolates of Pasteurella multo-cida and their adherence to porcine respiratory tract cells andmucusrdquo Infection and Immunity vol 61 no 11 pp 4785ndash47921993

[11] E T Reitschel T Kirikae FU Shade et al ldquoBacterial endotoxinmolecular relationships of structure to activity and functionrdquoThe FASEB Journal vol 8 no 2 pp 217ndash225 1994

[12] C Erridge E Bennett-Guerrero and I R Poxton ldquoStructureand function of lipopolysaccharidesrdquo Microbes and Infectionvol 4 no 8 pp 837ndash851 2002

[13] L N Lopez-Bojorquez A Z Dehesa and G Reyes-TeranldquoMolecular mechanisms involved in the pathogenesis of septicshockrdquo Archives of Medical Research vol 35 no 6 pp 465ndash4792004

[14] C Risco J L Carrascosa and M A Bosch ldquoUptake and sub-cellular distribution of Escherichia coli lipopolysaccharide byisolated rat Type II pneumocytesrdquo Journal of Histochemistry andCytochemistry vol 39 no 5 pp 607ndash615 1991

[15] M W Hornef B H Normark A Vandewalle and S NormarkldquoIntracellular recognition of lipopolysaccharide by toll-likereceptor 4 in intestinal epithelial cellsrdquo The Journal of Experi-mental Medicine vol 198 no 8 pp 1225ndash1235 2003

[16] D Bravo A Hoare A Silipo et al ldquoDifferent sugar residues ofthe lipopolysaccharide outer core are required for early interac-tions of Salmonella enterica serovars Typhi and Typhimuriumwith epithelial cellsrdquo Microbial Pathogenesis vol 50 no 2 pp70ndash80 2011

[17] P-C Chang C-J Wang C-K You and M-C Kao ldquoEffectsof a HP0859 (rfaD) knockout mutation on lipopolysaccharidestructure of Helicobacter pylori 26695 and the bacterial adhe-sion on AGS cellsrdquo Biochemical and Biophysical Research Com-munications vol 405 no 3 pp 497ndash502 2011

[18] M Belanger S Rioux B Foiry and M Jacques ldquoAffinity forporcine respiratory tract mucus is found in some isolates ofActinobacillus pleuropneumoniaerdquo FEMSMicrobiology Lettersvol 97 no 1-2 pp 119ndash125 1992

[19] E L Fletcher SM J Fleiszig andN A Brennan ldquoLipopolysac-charide in adherence of Pseudomonas aeruginosa to the corneaand contact lensesrdquo Investigative Ophthalmology amp Visual Sci-ence vol 34 no 6 pp 1930ndash1936 1993

[20] S K Gupta R S Berk S Masinick and L D Hazlett ldquoPiliand lipopolysaccharide of Pseudomonas aeruginosa bind to theglycolipid asialo GM1rdquo Infection and Immunity vol 62 no 10pp 4572ndash4579 1994

[21] G B Pier N L Koles G Meluleni K Hatano and M PollackldquoSpecificity and function of murine monoclonal antibodiesand immunization- induced human polyclonal antibodies tolipopolysaccharide subtypes of Pseudomonas aeruginosa sero-group 06rdquo Infection and Immunity vol 62 no 4 pp 1137ndash11431994

[22] A Venable M Mitalipova I Lyons et al ldquoLectin binding pro-files of SSEA-4 enriched pluripotent human embryonic stemcell surfacesrdquo BMC Developmental Biology vol 5 article no 152005

[23] J Yang Y Zhao and F Shao ldquoNon-canonical activation ofinflammatory caspases by cytosolic LPS in innate immunityrdquoCurrent Opinion in Immunology vol 32 pp 78ndash83 2015

[24] F Kopp S Kupsch and A B Schromm ldquoLipopolysaccharide-binding protein is bound and internalized by host cells andcolocalizes with LPS in the cytoplasm implications for a role

of LBP in intracellular LPS-signalingrdquo Biochimica et BiophysicaActaampMolecular Cell Research vol 1863 no 4 pp 660ndash6722016

[25] T Hatfaludi K Al-Hasani J D Boyce and B Adler ldquoOutermembrane proteins of Pasteurella multocidardquoVeterinaryMicro-biology vol 144 no 1-2 pp 1ndash17 2010

[26] K M Townsend J D Boyce J Y Chung A J Frost and BAdler ldquoGenetic organization of Pasteurella multocida cap lociand development of a multiplex capsular PCR typing systemrdquoJournal of ClinicalMicrobiology vol 39 no 3 pp 924ndash929 2001

[27] S L Brockmeier and K B Register ldquoExpression of the der-monecrotic toxin by Bordetella bronchiseptica is not necessaryfor predisposing to infection with toxigenic Pasteurella multo-cidardquo Veterinary Microbiology vol 125 no 3-4 pp 284ndash2892007

[28] D JHalloyNAKirschvink JMainil andPGGustin ldquoSyner-gistic action of E coli endotoxin and Pasteurella multocida typeA for the induction of bronchopneumonia in pigsrdquo VeterinaryJournal vol 169 no 3 pp 417ndash426 2005

[29] D Bernet H Schmidt W Meier P Burkhardt-Holm and TWahli ldquoHistopathology in fish proposal for a protocol to assessaquatic pollutionrdquo Journal of Fish Diseases vol 22 no 1 pp 25ndash34 1999

[30] OWestphal andK Jann ldquoBacterial lipopolysaccharides extrac-tion with phenol-water further applications of this procedurerdquoMethods in Carbohydrate Chemistry vol 5 p 9 1965

[31] D Gualtero J E Castellanos and G I Lafaurie ldquoPorphyro-monas gingivalis libre de polisacaridos utilizando cromatografıade alta resolucion sephacryl S-200rdquoActa Biologica Colombianavol 13 no 3 pp 147ndash160 2008

[32] P Esquinas ldquoComparacion ultraestructural de fosa nasal ynasofaringe de Conejos sanos y enfermos con el sındromede neumonıa enzooticardquo in Abstracts IV Reunion Anual dePatologıa 2004

[33] C Gallego A M Middleton N Martınez S Romero and CIregui ldquoInteraction ofBordetella bronchiseptica and its lipopoly-saccharide with in vitro culture of respiratory nasal epitheliumrdquoVeterinary Medicine International vol 2013 Article ID 3470869 pages 2013

[34] T G Pistole ldquoInteraction of bacteria and fungi with lectins andlectin-like substancesrdquo Annual Review of Microbiology vol 35pp 85ndash112 1981

[35] M D Abramofff P J Magalhaes and S J Ram ldquoImage proc-essing with ImageJ Part IIrdquo Biophotonics International vol 11no 7 pp 36ndash43 2005

[36] S E Wong C E Winbanks C S Samuel and T D HewitsonldquoLectin histochemistry for light and electron microscopyrdquoMethods in Molecular Biology vol 611 pp 103ndash114 2010

[37] RMartınez NMartinez andM VMartinezDiseno de Exper-imentos en Ciencias Agropecuarias y Biologicas con SAS SPSSR Y STATISTIX Fondo Nacional UniversitariomdashInstitucionAuxiliar del Cooperativismo 2011

[38] M Harper F St Michael M John et al ldquoPasteurella mul-tocida heddleston serovar 3 and 4 strains share a commonlipopolysaccharide biosynthesis locus but display both inter-and intrastrain lipopolysaccharide heterogeneityrdquo Journal ofBacteriology vol 195 no 21 pp 4854ndash4864 2013

[39] K Le Blay P Gueirard N Guiso and R Chaby ldquoAntigenic poly-morphism of the lipopolysaccharides from human and animalisolates of Bordetella bronchisepticardquoMicrobiology vol 143 no4 pp 1433ndash1441 1997


[40] E T Harvill A Preston P A Cotter A G Allen D J Maskelland J FMiller ldquoMultiple roles for Bordetella lipopolysaccharidemolecules during respiratory tract infectionrdquo Infection andImmunity vol 68 no 12 pp 6720ndash6728 2000

[41] M Harper F St Michael M John et al ldquoPasteurella multocidaHeddleston serovars 1 and 14 express different lipopolysaccha-ride structures but share the same lipopolysaccharide biosyn-thesis outer core locusrdquo Veterinary Microbiology vol 150 no3-4 pp 289ndash296 2011

[42] M Harper J D Boyce and B Adler ldquoPasteurella multocidapathogenesis 125 years after Pasteurrdquo FEMS Microbiology Let-ters vol 265 no 1 pp 1ndash10 2006

[43] J C Hodgson ldquoEndotoxin and mammalian host responsesduring experimental diseaserdquo Journal of Comparative Pathologyvol 135 no 4 pp 157ndash175 2006

[44] K Singh J W Ritchey and AW Confer ldquoMannheimia haemo-lytica bacterial-host interactions in bovine PneumoniardquoVeteri-nary Pathology vol 48 no 2 pp 338ndash348 2011

[45] P Patino Efectos morfologicos y distribucion del lipopolisacaridode Pasteurella multocida en el tracto respiratorio de conejosexpuestos intranasalmente [MS thesis] Universidad Nacionalde Colombia 2016

[46] R Y Hampton and C R H Raetz ldquoMacrophage catabolism oflipid A is regulated by endotoxin stimulationrdquo Journal of Bio-logical Chemistry vol 266 no 29 pp 19499ndash19509 1991

[47] P G Adams L Lamoureux K L Swingle H Mukundan andG A Montano ldquoLipopolysaccharide-induced dynamic lipidmembrane reorganization tubules perforations and stacksrdquoBiophysical Journal vol 106 no 11 pp 2395ndash2407 2014

[48] M P Carrillo N M Martinez M D P Patino and C AIregui ldquoInhibition of pasteurella multocida adhesion to rab-bit respiratory epithelium using lectinsrdquo Veterinary MedicineInternational vol 2015 Article ID 365428 10 pages 2015

[49] E T Rietschel and H Brade ldquoBacterial endotoxinsrdquo ScientificAmerican vol 267 no 2 pp 54ndash61 1992

[50] I W Devoe and J E Gilchrist ldquoRelease of endotoxin in theform of cell wall blebs during in vitro growth of Neisseriameningitidisrdquo Journal of Experimental Medicine vol 138 no 5pp 1156ndash1167 1973

[51] H Al-Haj Ali T Sawada H Hatakeyama Y Katayama NOhtsuki and O Itoh ldquoInvasion of chicken embryo fibroblastcells by avian Pasteurella multocidardquo Veterinary Microbiologyvol 104 no 1-2 pp 55ndash62 2004

[52] T O Schaffner J Hinds K A Gould et al ldquoA point mutationin cpsE renders Streptococcus pneumoniaenonencapsulated andenhances its growth adherence and competencerdquo BMC Micro-biology vol 14 no 1 article no 210 2014

[53] P Esquinas L Botero M D P Patino C Gallego and C IreguildquoUltrastructural comparison of the nasal epithelia of healthyand naturally affected rabbits with pasteurella multocida ArdquoVeterinary Medicine International vol 2013 Article ID 3213908 pages 2013

[54] N Mukaida Y Ishikawa N Ikeda et al ldquoNovel insight intomolecularmechanismof endotoxin shock biochemical analysisof LPS receptor signaling in a cell-free system targeting NF-120581B and regulation of cytokine productionaction through 1205732integrin in vivordquo Journal of Leukocyte Biology vol 59 no 2 pp145ndash151 1996

[55] M Yarim S Karahan and N Kabakci ldquoImmunohistochemicalinvestigation of CD14 in experimental rabbit pneumonic pas-teurellosisrdquo Revue de Medecine Veterinaire vol 156 no 1 pp13ndash19 2005

[56] M Lu A W Varley S Ohta J Hardwick and R S MunfordldquoHost inactivation of bacterial lipopolysaccharide prevents pro-longed tolerance following gram-negative bacterial infectionrdquoCell Host and Microbe vol 4 no 3 pp 293ndash302 2008

[57] J Pugin C-C Schurer-Maly D Leturcq A Moriarty R JUlevitch and P S Tobias ldquoLipopolysaccharide activation ofhuman endothelial and epithelial cells is mediated by lipopoly-saccharide-binding protein and soluble CD14rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 90 no 7 pp 2744ndash2748 1993

[58] A Haziot E Ferrero F Kontgen et al ldquoResistance to endotoxinshock and reduced dissemination of gram-negative bacteria inCD14-deficientmicerdquo Immunity vol 4 no 4 pp 407ndash414 1996

[59] T Vasselon E Mailman R Thieringer and P A DetmersldquoInternalization of monomeric lipopolysaccharide occurs aftertransfer out of cell surface CD14rdquo Journal of ExperimentalMedicine vol 190 no 4 pp 509ndash521 1999

[60] LGuillott SMedjane K Le-Barillec et al ldquoResponse of humanpulmonary epithelial cells to lipopolysaccharide involves toll-like receptor 4 (TLR4)-dependent signaling pathways evidencefor an intracellular compartmentalization of TLR4rdquo Journal ofBiological Chemistry vol 279 no 4 pp 2712ndash2718 2004

[61] C M Greene and N G McElvaney ldquoToll-like receptor expres-sion and function in airway epithelial cellsrdquo Archivum Immu-nologiae et Therapiae Experimentalis vol 53 no 5 pp 418ndash4272005

[62] SWWongM-J Kwon AM K Choi H-P Kim K NakahiraandDHHwang ldquoFatty acidsmodulate toll-like receptor 4 acti-vation through regulation of receptor dimerization and recruit-ment into lipid rafts in a reactive oxygen species-dependentmannerrdquo Journal of Biological Chemistry vol 284 no 40 pp27384ndash27392 2009

[63] A B Lentsch and P A Ward ldquoRegulation of experimental lunginflammationrdquo Respiration Physiology vol 128 no 1 pp 17ndash222001

[64] C-C Tsai M-T Lin J-J Wang J-F Liao and W-T HuangldquoThe antipyretic effects of baicalin in lipopolysaccharide-evoked fever in rabbitsrdquo Neuropharmacology vol 51 no 4 pp709ndash717 2006

[65] B A Mizock ldquoThe multiple organ dysfunction syndromerdquoDisease-a-Month vol 55 no 8 pp 476ndash526 2009

[66] O S Ali L Adamu F F J Abdullah et al ldquoAlterations in inter-leukin-1120573 and interleukin-6 inmice inoculated through the oralroutes using graded doses of p multocida type b 2 and itslipopolysacchariderdquoAmerican Journal of Animal andVeterinarySciences vol 10 no 1 pp 1ndash8 2015

[67] F Yan W Li H Jono et al ldquoReactive oxygen speciesregulate Pseudomonas aeruginosa lipopolysaccharide-inducedMUC5AC mucin expression via PKC-NADPH oxidase-ROS-TGF-120572 signaling pathways in human airway epithelial cellsrdquoBiochemical andBiophysical ResearchCommunications vol 366no 2 pp 513ndash519 2008

[68] P-R Burgel E Escudier A Coste et al ldquoRelation of epidermalgrowth factor receptor expression to goblet cell hyperplasia innasal polypsrdquo Journal of Allergy and Clinical Immunology vol106 no 4 pp 705ndash712 2000

[69] K Takeyama J V Fahy and J A Nadel ldquoRelationship of epider-mal growth factor receptors to goblet cell production in humanbronchirdquo American Journal of Respiratory and Critical CareMedicine vol 163 no 2 pp 511ndash516 2001

[70] J H Kim S Y Lee S M Bak et al ldquoEffects of matrix metal-loproteinase inhibitor on LPS-induced goblet cell metaplasiardquo


American Journal of PhysiologymdashLung Cellular and MolecularPhysiology vol 287 no 1 pp L127ndashL133 2004

[71] W Li F Yan H Zhou et al ldquoP aeruginosa lipopolysaccharide-induced MUC5AC and CLCA3 expression is partly throughDuox1 in vitro and in vivordquo PLoS ONE vol 8 no 5 Article IDe63945 2013

[72] C D Bingle K Wilson H Lunn et al ldquoHuman LPLUNC1 is asecreted product of goblet cells and minor glands of the respir-atory and upper aerodigestive tractsrdquo Histochemistry and CellBiology vol 133 no 5 pp 505ndash515 2010

[73] L Bingle and C D Bingle ldquoDistribution of human PLUNCBPIfold-containing (BPIF) proteinsrdquo Biochemical Society Transac-tions vol 39 no 4 pp 1023ndash1027 2011

[74] J A Bartlett B J Hicks J M Schlomann S RamachandranWM Nauseef and P B McCray ldquoPLUNC is a secreted product ofneutrophil granulesrdquo Journal of Leukocyte Biology vol 83 no 5pp 1201ndash1206 2008

[75] B Ghafouri E Kihlstrom C Tagesson and M LindahlldquoPLUNC in human nasal lavage fluid multiple isoforms thatbind to lipopolysacchariderdquo Biochimica et Biophysica Acta vol1699 no 1-2 pp 57ndash63 2004

Submit your manuscripts athttpswwwhindawicom

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Case Reports in Veterinary Medicine


effects are rare and most are devoted to other bacterialadhesive structures such as the fimbria [14ndash17]

Belanger et al [18] proposed that the LPS ofActinobacilluspleuropneumoniae would be responsible for the adhesion ofthe bacterium to the respiratory epithelial cells of swine laterstudies of E coli Salmonella enterica subsp enterica serovarTyphimuriumHelicobacter pylori Klebsiella pneumonia andPseudomonas aeruginosa showed that it was possible toblock the adhesion of the corresponding microorganismsto different cell surfaces by suppressing or modifying thestructure of their respective LPSmolecules and by employingmonoclonal antibodies against the O antigen of the molecule[16 17 19ndash23]

Recently Bravo et al [16] showed that the core of Styphimurium LPS plays an important role in the interactionof the bacteria with HeLa epithelial cells as well as BHK andIB3 cells lines The inner core of the molecule has Glc I andGal I residues which are essential for adhesion and entry ofthe microorganism into epithelial cells in vitro The authorsspeculate that a lectin type receptor could be involved in theinteraction of the host cells with the outer core structurewhich is composed of (Gal I 120572 1-3 Glu) [16]

In addition a role for Helicobacter pylori LPS O antigenin adhesion and colonization of the gastric mucosa of micehas also been proposed Moreover it has been shown thatthe core of H pylori LPS is involved in pathogen bindingto laminin an extracellular matrix and basement membraneglycoprotein of the epithelium that is essential for the processof bacterial adhesion [17]

On the other hand few reports document themechanismby which LPS is internalized into the cytoplasm of the hostcells caspases 4 5 and 11 are considered intracytoplasmicreceptors for E coli and S typhimurium LPS in macrophagecell cultures and somatic cells such as mouse enterocytes inwhich they induce pyroptosis and apoptosis [23] In additionLPS can be internalized by the lipopolysaccharide bindingprotein (LBP) into the cytoplasm of monocytes and macro-phages as well as by nonimmune human cells such as theHEK293 cell line These findings have led to the hypothesisthat LPS plays an important role in the regulation of intracel-lular pro- and anti-inflammatory signals [24]

In P multocida different studies reported the significanceof several surface structures such as adhesins includinghighly hydrated polyanionic polysaccharides on the capsulethat are covalently bound to the surface of bacteria throughphospholipids or lipid A Other structures also include outermembrane proteins (OMPs) which may serve as adhesinsor invasins or participate in the formation of biofilms suchas type IV fimbriae the fibronectin binding protein andfilamentous hemagglutinin [25] however no study has beendevoted to exploring the role of P multocida LPS as anadhesion molecule For the first time we show that the LPSof P multocida significantly increases the adhesion of themicroorganism to the apical surface of the respiratory epithe-lium of the rabbit nasal septum and that the increased num-ber of adhered microorganisms causes more severe damageto the host cells We also show that LPS is internalized intothe cytoplasm of respiratory cells and that goblet cells play animportant role during these first steps of infection

2 Materials and Methods

21 Pasteurella multocida Strain Pasteurella multocida Astrain 001 was obtained from turbinates trachea and lungsof rabbits with signs of rhinitis and pneumonia from farmsin the Sabana de Bogota (Colombia) The organism wasgrown in brain heart infusion (BHI) agar gray nonhemolyticcolonies with a round morphology were selected Thecolonies were identified as bipolar-staining Gram-negativecoccobacilli andwere shown to be catalase- oxidase- indole-ornithine decarboxylase- glucose- sucrose- and mannitol-positive using biochemical tests The colonies did not growon MacConkey agar and were urease-negative [1] Molecularidentification of P multocida of capsular serogroup A wasperformed by expanding the sequence of the hyaD cap genelocus with the following primers F 51015840 TGC CAA AAT CGCAGT CAG 31015840 R 51015840 TTG CCA TCA TTG TCA GTG 31015840 [26]

22 Extraction Purification Quantification and BiologicalActivity of P multocida LPS LPS was extracted using thephenolhot water method [30] Pasteurella multocida (58 g)were resuspended in 29mL of ultrapure water The aqueousphase was dialyzed against sterile water and then lyophilizedThe sample was reconstituted in 10mL of 01M Tris buffercontaining 015M NaCl and 1N HCl pH 7 and treatedwith RNase (05mgmL) DNase (005mgmL) and 100 120583L of4mMMgCl

2for 2 hours at 60∘CThe sample was then treated

with proteinase K (005mgmL) and 100120583L of 1mM CaCl2

for 18 hours at 37∘C The extract was frozen at minus80∘C andlyophilized for 48 h A mass of 704mg was obtained and wasthen subjected to gel permeation chromatography brieflythe extract was resuspended in 5mL of 005M Tris buffercontaining 15 sodium deoxycholate and 1mM EDTA pH95 and passed through a GE Healthcare HiPrep 1660Sephacryl S-200HR column (SigmaAldrich St LouisMOUSA) Fractions of 25mL were collected at a flow rate of08mLmin and measured at 220 nm 260 nm and 280 nmusing spectrophotometry At each stage of purification andcharacterization aliquots were sampled and were analyzedwith a spectrophotometer by scanning between 200 nmand 600 nm (NanoDrop 2000 Spectrophotometer ThermoScientific Wilmington DE USA)

The resolving gel for electrophoresis was prepared at 12the stacking gel was prepared at 4 and the electrophoresisconditions were 003A45min and 002A1 h respectivelyThe lyophilized fractions were reconstituted in pyrogen-freewater to a final concentration of 1mgmL prepared in 1xLaemmli buffer and heated at 95∘C for 5 minutes before theywere added to the gel Staining was performed with silvernitrate and Coomassie blue [31]

The amount of polysaccharide in the extract was quanti-fied using the Purpald test a reference curve was constructedwith a standard of 3-deoxy-120572-D-mannooctulosonic acid(Kdo) (Sigma Co St Louis MO USA) in concentrationsranging from 08mM to 0025mM Commercial Escherichiacoli 0111B4 S typhimurium and Rhodobacter sphaeroidesDSM158 LPSswere diluted to two concentrations 04mgmLand 02mgmL Different concentrations of the P multo-cida LPS fractions were used ranging from 1mgmL to

















3 Results





Table1Ex

perim

entalp

rotocolfor

expo

singfetalrabbitn

asalseptato

Pmultocid

aandLP

SIIP(in

direct

immun

operoxidase)L

H(le

ctin

histo

chem

istry)TE

M(tr

ansm

issionelectro

nmicroscop

y)and

C(minus)(negativ

econ

trol)lowast

[2728]

Treatm

ent

Negativec

ontro

lPmultocid

aLP

S+Pmultocid

asim

ultaneou

slyLP

S+Pmultocid

a30

min

later

Pmultocid

a+LP

S30

min

later

LPS

Treatm

ent1

Treatm

ent2

Treatm

ent3

Treatm

ent4

Treatm

ent5

Treatm

ent6

Techniqu

eIIP-LH

TEM

IIP-LH

TEM

IIP-LH

IIP-LH

TEM

IIP-LH

TEM

Num

bero

fexplants

73

73

77

37

3

Doselowast

MEM

MEM

1times108cfumL

1times108cfumL

10120583gmL+1times

108cfumL

10120583gmL+1times

108cfumL

10120583gmL+1times

108cfumL

1times108cfumL+

10120583gmL

10120583gmL

Expo

sure

time

2h2h

2h2h

2h2h

2h2h

2h



(a)






(b)





974KDa

662KDa

45KDa31KDa

215KDa

144KDa

1 2 3 4 5 6 7 8 9 10







+++ +++ ++ ++ ++


++ ++ ++ ++ ++






(a)

lowast

(b)

lowast

(c)

GC

lowastlowast

(d)




(mean plusmn SD)



10240411 plusmn 3241 2080027 plusmn 8950











CE

C








4 Discussion





(a)

B

CE

CE

MV

(b)

BB

BB

(c)

(d)

CE

(e)

CE

C

CE

(f)







CE

Bac

C

VAC

CE


CE

GCVAC














Competing Interests




Acknowledgments


References
























































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






















3 Results





Table1Ex

perim

entalp

rotocolfor

expo

singfetalrabbitn

asalseptato

Pmultocid

aandLP

SIIP(in

direct

immun

operoxidase)L

H(le

ctin

histo

chem

istry)TE

M(tr

ansm

issionelectro

nmicroscop

y)and

C(minus)(negativ

econ

trol)lowast

[2728]

Treatm

ent

Negativec

ontro

lPmultocid

aLP

S+Pmultocid

asim

ultaneou

slyLP

S+Pmultocid

a30

min

later

Pmultocid

a+LP

S30

min

later

LPS

Treatm

ent1

Treatm

ent2

Treatm

ent3

Treatm

ent4

Treatm

ent5

Treatm

ent6

Techniqu

eIIP-LH

TEM

IIP-LH

TEM

IIP-LH

IIP-LH

TEM

IIP-LH

TEM

Num

bero

fexplants

73

73

77

37

3

Doselowast

MEM

MEM

1times108cfumL

1times108cfumL

10120583gmL+1times

108cfumL

10120583gmL+1times

108cfumL

10120583gmL+1times

108cfumL

1times108cfumL+

10120583gmL

10120583gmL

Expo

sure

time

2h2h

2h2h

2h2h

2h2h

2h



(a)






(b)





974KDa

662KDa

45KDa31KDa

215KDa

144KDa

1 2 3 4 5 6 7 8 9 10







+++ +++ ++ ++ ++


++ ++ ++ ++ ++






(a)

lowast

(b)

lowast

(c)

GC

lowastlowast

(d)




(mean plusmn SD)



10240411 plusmn 3241 2080027 plusmn 8950











CE

C








4 Discussion





(a)

B

CE

CE

MV

(b)

BB

BB

(c)

(d)

CE

(e)

CE

C

CE

(f)







CE

Bac

C

VAC

CE


CE

GCVAC














Competing Interests




Acknowledgments


References
























































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







Table1Ex

perim

entalp

rotocolfor

expo

singfetalrabbitn

asalseptato

Pmultocid

aandLP

SIIP(in

direct

immun

operoxidase)L

H(le

ctin

histo

chem

istry)TE

M(tr

ansm

issionelectro

nmicroscop

y)and

C(minus)(negativ

econ

trol)lowast

[2728]

Treatm

ent

Negativec

ontro

lPmultocid

aLP

S+Pmultocid

asim

ultaneou

slyLP

S+Pmultocid

a30

min

later

Pmultocid

a+LP

S30

min

later

LPS

Treatm

ent1

Treatm

ent2

Treatm

ent3

Treatm

ent4

Treatm

ent5

Treatm

ent6

Techniqu

eIIP-LH

TEM

IIP-LH

TEM

IIP-LH

IIP-LH

TEM

IIP-LH

TEM

Num

bero

fexplants

73

73

77

37

3

Doselowast

MEM

MEM

1times108cfumL

1times108cfumL

10120583gmL+1times

108cfumL

10120583gmL+1times

108cfumL

10120583gmL+1times

108cfumL

1times108cfumL+

10120583gmL

10120583gmL

Expo

sure

time

2h2h

2h2h

2h2h

2h2h

2h



(a)






(b)





974KDa

662KDa

45KDa31KDa

215KDa

144KDa

1 2 3 4 5 6 7 8 9 10







+++ +++ ++ ++ ++


++ ++ ++ ++ ++






(a)

lowast

(b)

lowast

(c)

GC

lowastlowast

(d)




(mean plusmn SD)



10240411 plusmn 3241 2080027 plusmn 8950











CE

C








4 Discussion





(a)

B

CE

CE

MV

(b)

BB

BB

(c)

(d)

CE

(e)

CE

C

CE

(f)







CE

Bac

C

VAC

CE


CE

GCVAC














Competing Interests




Acknowledgments


References
























































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of








(a)






(b)





974KDa

662KDa

45KDa31KDa

215KDa

144KDa

1 2 3 4 5 6 7 8 9 10







+++ +++ ++ ++ ++


++ ++ ++ ++ ++






(a)

lowast

(b)

lowast

(c)

GC

lowastlowast

(d)




(mean plusmn SD)



10240411 plusmn 3241 2080027 plusmn 8950











CE

C








4 Discussion





(a)

B

CE

CE

MV

(b)

BB

BB

(c)

(d)

CE

(e)

CE

C

CE

(f)







CE

Bac

C

VAC

CE


CE

GCVAC














Competing Interests




Acknowledgments


References
























































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







(a)

lowast

(b)

lowast

(c)

GC

lowastlowast

(d)




(mean plusmn SD)



10240411 plusmn 3241 2080027 plusmn 8950











CE

C








4 Discussion





(a)

B

CE

CE

MV

(b)

BB

BB

(c)

(d)

CE

(e)

CE

C

CE

(f)







CE

Bac

C

VAC

CE


CE

GCVAC














Competing Interests




Acknowledgments


References
























































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







CE

C








4 Discussion





(a)

B

CE

CE

MV

(b)

BB

BB

(c)

(d)

CE

(e)

CE

C

CE

(f)







CE

Bac

C

VAC

CE


CE

GCVAC














Competing Interests




Acknowledgments


References
























































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







(a)

B

CE

CE

MV

(b)

BB

BB

(c)

(d)

CE

(e)

CE

C

CE

(f)







CE

Bac

C

VAC

CE


CE

GCVAC














Competing Interests




Acknowledgments


References
























































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







CE

Bac

C

VAC

CE


CE

GCVAC














Competing Interests




Acknowledgments


References
























































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of













Competing Interests




Acknowledgments


References
























































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of




















































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






assessment of pasteurella multocida a...

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