human cecum content modulates production of extracellular proteins by food and probiotic bacteria

R E S EA RCH L E T T E R

Human cecum content modulates production of extracellularproteins by food and probiotic bacteria

Borja Sanchez1, Lorena Ruiz1, Adolfo Suarez2, Clara G. de los Reyes-Gavilan1 & Abelardo Margolles1

1Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lacteos de Asturias, Consejo Superior de Investigaciones

Cientıficas (IPLA-CSIC), Villaviciosa, Asturias, Spain; and 2Seccion de Aparato Digestivo, Hospital de Cabuenes, Gijon, Asturias, Spain

Correspondence: Borja Sanchez, Instituto de

Productos Lacteos de Asturias, Consejo

Superior de Investigaciones Cientıficas (IPLA-

CSIC), Ctra. Infiesto s/n, 33300 Villaviciosa,

Asturias, Spain. Tel.: +34 985 89 21 31;

fax: +34 985 89 22 33;

e-mail: [email protected]

Received 2 August 2011; revised 26 August

2011; accepted 27 August 2011.

Final version published online 3 October

2011.

DOI: 10.1111/j.1574-6968.2011.02408.x

Editor: Marco Soria

Keywords

extracellular proteins; food bacteria;

probiotics.

Abstract

Lactic acid bacteria (LAB) are responsible for different types of food fermenta-

tions that provide humans with many different classes of fermented products.

During the 20th century, some LAB strains as well as several members of the

genus Bifidobacterium started to be extensively used in human nutrition as pro-

biotics because of their health-promoting effects. Nowadays, the subset of

extracellular proteins is being investigated as potential mediators of the process

known as bacteria–host molecular crosstalk. Inclusion of human cecum extracts

in laboratory culture medium modified the production of extracellular proteins

by food and probiotic microorganisms. By proteomic and genetic means, the

specific overproduction of two proteins was revealed to occur at transcriptional

level. This work sheds light on the potential molecular effectors that food bac-

teria could use for interacting with the human gut and revealed that they may

be produced under very specific environmental conditions.

Introduction

Lactic acid bacteria (LAB) have been part of human

nutrition since ancient times, being involved in the pro-

duction of an endless number of fermented products.

These fermented foods play important roles in human

customs. It is generally accepted that LAB were initially

responsible for spontaneous food fermentations, some

strains being selected by humans with the aim of control-

ling these spontaneous processes. During the 20th century,

the initial work of microbiologists such as Metchnikov,

Jensen, Cheplin, and Rettger provided the first evidence

that some LAB and bifidobacteria strains could exert

beneficial effects on human health. These microorganisms

were subsequently denominated as probiotics (Araya

et al., 2002).

A growing interest regarding the inclusion of probiotic

strains within the formulation of foods and supplements

has emerged in recent times, and an increasing variety of

commercial products containing them can be found

worldwide (Sanchez et al., 2009a). Probiotics can exert

several beneficial effects on human health including

favorable balance of intestinal microbiota (Salminen &

Gueimonde, 2004). Indeed, in certain autoimmune

diseases, an imbalance has been demonstrated between

beneficial and detrimental commensal microorganisms,

termed dysbiosis (Sartor, 2008; Qin et al., 2010).

Probiotics ingested with foods exert their health bene-

fits through production of beneficial compounds, modu-

lation of other intestinal microbial populations, and

interactions with eukaryotic cells (intestinal epithelium

and immune system). The molecular mechanisms respon-

sible for the interaction of food bacteria with eukaryotic

cells of the intestine remain unclear. Some of these inter-

actions have been proposed mediated by extracellular and

cell surface-associated proteins (Sanchez et al., 2010).

Production of extracellular proteins by food bacteria may

be affected by environmental conditions; thus, these pro-

teins might go unnoticed in our controlled laboratory

conditions as compared with the in vivo situation in the

gastrointestinal tract (GIT).

In this work, we aimed to analyze possible changes that

could occur in production levels of extracellular proteins

synthesized by a set of food and probiotic bacteria in

FEMS Microbiol Lett 324 (2011) 189–194 ª 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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simulated environmental conditions of the colon, using

cecum samples of healthy adults as compared with stan-

dard culture conditions.

Materials and methods

Human donors and sampling conditions

Cecum content was obtained from endoscopic explora-

tion of the colon of four individuals complaining of non-

specific slight digestive pains. In all cases, the exploration

did not reveal any pathology; thus, the four patients were

considered healthy donors. The four donors were submit-

ted to a diet free from residues during the 48 h prior to

exploration, supplemented with oral intake of the laxative

Fosfosoda® (Labs. Casen-Fleet, Zaragoza, Spain). All

patients provided written informed consent for their sam-

ples to be used for research purposes. Ethical approval for

this study was obtained from the Regional Ethics Com-

mittee for Clinical Investigation. This allowed the endo-

scopic exploration of the cecum.

Colonoscopies were performed with the introduction

of an Olympus video-colonoscope (Olympus America,

Inc., Center Valley, PA). The liquid present in the cecum

was aspired through the instrument. The first 5 mL was

discarded, and the remainder of the content placed in a

sterile recipient and stored at �20 °C until processing.

Prior to their use, cecum contents were centrifuged three

times (12 000 g, 4 °C, 10 min) and the supernatants

recovered and sterilized by filtration (0.45 lm). This

large filter would allow, theoretically, the passage of

certain host molecules involved in signaling, whereas

bacteria, which are usually in the range of micrometers,

would stay retained in the filter. The different cecum

contents were pooled (cecum extract) and used to study

their effect on the different bacterial strains throughout

this work.

Bacterial strains and growth conditions

Bifidobacterium animalis ssp. lactis IPLA4549, B. animalis

ssp. lactis IPLAR2, Bifidobacterium bifidum LMG11041T,

Bifidobacterium longum ssp. longum NCIMB8809, Lacto-

bacillus acidophilus DSM20079T, Lactobacillus casei ssp.

rhamnosus GG (ATCC53103), Lactobacillus delbrueckii

ssp. delbrueckii IPLAlb101, and Lactobacillus reuteri

DSM20016T were routinely grown at 37 °C in MRS broth

(Difco®; Becton Dickinson, Franklin Lakes, NJ) supple-

mented with 0.05% (w/v) L-cysteine (MRSC) (Sigma

Chemical Co., St. Louis, MO). Lactococcus lactis ssp.

cremoris MG1363 and Streptococcus thermophilus

LMG18311 were propagated on M17 broth (Difco®;

Becton Dickinson) supplemented with 1% (w/v) glucose

(GM17) at 30 °C. All cultures were incubated in anaero-

bic jars (Anaerocult A System; Merck KGaA, Darmstadt,

Germany).

Bacterial growth in simulated gut conditions

The environmental conditions of the large intestine were

simulated by supplementing the growth media with 0.1%

or 1.0% (v/v) cecum extract. Overnight cultures of the

different bacterial strains were used to inoculate (1% v/v)

50 mL of fresh media containing 0%, 0.1%, or 1.0% (v/v)

sterilized cecum extract. Cultures were made in triplicate

from three independent precultures; cells were harvested

at different phases of the growth curve, depending on the

experiment. With this setup, bacteria enter stationary

phase of growth after 7–10 h of growth, depending on

the strain. No apparent inhibitory effect on growth was

observed after addition of 1.0% (v/v) cecum extract.

Precipitation and identification of secreted

proteins

Precipitation of extracellular proteins was performed as

described previously (Sanchez et al., 2009b). Fifty millili-

ter aliquots of fresh MRSC or GM17 broth containing

0%, 0.1%, or 1.0% (v/v) cecum extract were inoculated

(1% v/v) from an overnight culture of the different bacte-

rial strains. Cultures were allowed to enter stationary

phase of growth; cells were harvested by centrifugation

(9300 g, 4 °C, 10 min). Supernatants were then filtered

(0.45 lm). Sodium deoxycholate 10 mg (Sigma) was

added and mixed, and the resulting solution was incu-

bated at 4 °C for 30 min. Chilled trichloroacetic acid

(TCA; Sigma) was added at a final concentration of 6%

(w/v), and proteins were allowed to precipitate at 4 °Cfor 2 h. Proteins were recovered by centrifugation

(9300 g, 4 °C, 10 min); pellets were washed twice with

2 mL of chilled acetone (Sigma). Pellets were allowed to

dry at room temperature, and proteins were resolubilized

by ultrasonication (Ultrasonic bath; Deltasonic, Meaux,

France) in 200 lL of 19 Laemmli buffer for 10 min

(Laemmli, 1970).

Protein loadings were standardized on a volume-for-

volume basis; usually the extracellular protein amount

present in 50 mL of supernatant ranged from 200 to

500 lg. The total protein amounts contained in 50 mL of

control samples [MRSC, GM17 supplemented or not with

0.1% or 1% (v/v)], or 40 lg of extracellular protein

extracts were resolved by SDS-PAGE using a final poly-

acrylamide concentration of 12.5% (w/v) (Laemmli,

1970). Proteins whose electrophoretic bands showed

changes in intensity with the presence of cecum extract

were submitted to MALDI-MS/MS analysis and identified

ª 2011 Federation of European Microbiological Societies FEMS Microbiol Lett 324 (2011) 189–194Published by Blackwell Publishing Ltd. All rights reserved

190 B. Sanchez et al.

at the Proteomics Core Facility of CNIC (Madrid, Spain)

using standard protocols.

Quantitative PCR

Relative expression of the genes coding for Imp11 and

Imp23 was determined by quantitative PCR (qPCR). Ten

milliliters of MRS containing 0% or 1.0% (v/v) cecum

extract was left in the anaerobic chamber MG500 (Don

Whitley Scientific, West Yorkshire, UK) under 10% (v/v)

H2, 10% CO2, and 80% N2 at 37 °C overnight. These

aliquots were inoculated (1% v/v) with overnight bacterial

cultures made in MRSC; samples were taken after 90 min

(early exponential phase), 3 h (middle exponential

phase), and 12 h (early stationary phase). Cells were col-

lected by centrifugation (9300 g, 5 min), and the proto-

cols for cell lysis, RNA isolation, and cDNA synthesis

were performed as previously described (Gueimonde

et al., 2007). The qPCR experiments were run in an ABI

Prism 7500 Fast real-time PCR system (Applied Biosys-

tems, Foster City, CA). Specific primers were designed for

imp11 (SABLF, 5′-CGTACGTGTGATCAAGCCCGCA-3′;SABLR, 5′-GGAATAGGTGTCTGCCTGGGCA-3′) and for

imp23 psacid (PSACIDF, 5′-TCAGCAGCCACTAATAGCGACTCA-3′; PSACIDR, 5′-CACCTGGTACACCTCCAGGAGCT-3′). Their specificity was verified before the quanti-

tative analysis. At least three independent qPCR runs

were performed for each cDNA. Relative expression of

stated genes under the experimental conditions was esti-

mated according to DDCt method using an intergenic

spacer region between the 16s and 23s rRNAs as an

endogenous control, employing previously described

primers (Gueimonde et al., 2004; Haarman & Knol,

2006). Expression rate was related to that of the corre-

sponding genes in the absence of cecum extract, which

was given the arbitrary value 1.

Results and discussion

Research studies focusing on characterization of food and

probiotic bacterial strains generally involve the use of syn-

thetic, defined, or complex culture media that do not

reproduce adequately the conditions of the GIT, which is

the natural habitat or the site of action of most of these

bacteria. As a consequence, expression of some cellular

and extracellular proteins may change with respect to the

in vivo situation. Key proteins that might be potentially

involved in interactions with the human host could be

found by trying to mimic the environmental conditions

that those bacteria face in the human intestine. Once

released from the bacterial cell to the surrounding media,

extracellular proteins would be able to interact directly

with mucosal cells including epithelial and immune cells

(Sanchez et al., 2008), thus constituting potential media-

tors of bacteria–host crosstalk (Lebeer et al., 2010). In the

present work, we explored the subset of extracellular

proteins produced by a panel of LAB and bifidobacteria

frequently found in foods or that are normal inhabitants

of the human GIT. We aimed to detect changes in the

production of extracellular proteins as affected by the

presence of cecum extract in the culture medium.

A panel of food/probiotic bacteria was used, among

which representative strains for dairy starters, adjunct

dairy cultures, commensal species inhabiting the human

GIT, and probiotic strains were chosen. In addition, the

strain B. animalis ssp. lactis R2, a strain producing a ropy

exopolysaccharide that may be relevant for the food

industry (Ruas-Madiedo & de los Reyes-Gavilan, 2005),

was also included (Table 1) (Gasson, 1983).

In our experimental design, different subinhibitory

concentrations of cecum extract obtained from the pooled

cecum contents of four healthy donors were added to the

growth culture media. The highest amount of extracellu-

lar proteins was recovered from the supernatants of bac-

teria cultured to stationary phase of growth. Therefore,

we used extracellular proteins isolated in this phase for

obtaining preliminary electrophoretic profiles.

In general, the extracellular protein profiles of the cul-

tures of selected bacteria were affected qualitatively by the

presence and concentration of cecum extract initially

added to the growth medium. Many of the new bands

were identified as components of the cecum extract

(Fig. 1, see Supporting Information, Table S1), but two of

them were shown to be highly upregulated bacterial

proteins: surface antigen (Imp11; accession number

Table 1. Strains used in this study

Strain

Source of isolation/

reference

Bifidobacterium animalis ssp. lactis IPLA4549 Fermented milk

Bifidobacterium animalis ssp. lactis R2 Spontaneous ropy

mutant

Bifidobacterium bifidum LMG11041T BCCM/LMG

Bifidobacterium longum ssp. longum

NCIMB8809

NCIMB

Lactobacillus acidophilus DSM20079T DSMZ

Lactobacillus casei ssp. rhamnosus GG

(ATCC53103)

ATCC

Lactobacillus delbrueckii ssp. delbrueckii

IPLAlb101

Fermented milk

Lactobacillus reuteri DSM20016T DSMZ

Lactococcus lactis ssp. cremoris MG1363 Gasson (1983)

Streptococcus thermophilus LMG18311 BCCM/LMG

ATCC, American Type Culture Collection; BCCM/LMG, Belgium Coor-

dinated Collection of Microorganisms; DSMZ, Deutsche Sammlung

von Mikroorganismen und Zellkulturen; IPLA, Instituto de Productos

Lacteos de Asturias – CSIC; T, type strain.


Modulation of extracellular proteins by cecum content 191

ZP_00121020) from Bifidobacterium longum and a small

extracellular protein of unknown function (Imp23; acces-

sion number YP_193019) produced by L. acidophilus

(Fig. 2a). After their identification, we could further

demonstrate the induction of the corresponding genes: the

expression level of imp11 remained at a twofold higher

level in the presence of cecal content along the growth

curve, whereas imp23 was considerably more induced in

exponential than in stationary phase (Fig. 2b).

It is known that intestinal bacteria are able to react to

the GIT environment by activating certain genes, nor-

mally under the control of inducible promoters (Guei-

monde et al., 2009; Rivera-Amill et al., 2001; Sleator

et al., 2005). Our results suggest that the expression of

certain genes, whose products could be relevant for the

physiology of the bacterium in the GIT, may be up- or

down-regulated in conditions used in the laboratory, thus

escaping analysis. In contrast, the actual relevance regard-

ing bacteria–host interaction of proteins produced at

higher amounts in nonconditioned media with respect to

simulated GIT conditions should be carefully addressed.

For instance, S-layer protein A from L. acidophilus NCFM

(ATCC 700396) has been shown to modulate immune

functions of dendritic cells through direct interaction

with the surface lectin DC-SIGN (Konstantinov et al.,

2008), whereas cell wall hydrolase from GG strain has

been suggested an important factor for GIT homeostasis,

being involved in maintenance of the mucosal barrier

(Seth et al., 2008) and, recently, in the attenuation of

inflammatory processes (Yan et al., 2011).

In conclusion, bacteria present in food and probiotic

products change their extracellular protein profiles when

grown in media simulating the conditions of the large

intestine. Thus, genes and proteins only expressed under

intestinal stimuli can pass unnoticed in laboratory experi-

mental conditions. Further experimentation is ongoing in

1 2 3 4 5 6 7 8 9[caecum extract]

0 0 1 1 0 0 1 1

[caecum extract]

Imp01

97

66

45

kDa

97

66

45

kDa

0 0. 0 .0 0.1 1

p

Imp02 Imp03

Imp04

30

20.1

30

20.1

14.4 14.4

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27[caecum extract]

0 0.1 1 0 1

[caecum extract]

0 0.1 1

Imp06

Imp07

Imp15

Imp16

Imp17Imp18

Imp19Imp20

Imp21

Imp30 Imp32

97

66

45

kDa

97

66

45

kDaImp31

Imp34

0 0 0.1 1 0.1 0 0.1 1 0 0.1 1

Imp05

Imp06

Imp12

Imp13

Imp14

Imp15Imp22

Imp23

Imp24Imp29

Imp33

30

20.1

14 4

30

20.1

14.4Imp08 Imp09 Imp10 Imp11Imp25 Imp26 Imp27

Imp29Imp28

14.

Fig. 1. Representative polyacrylamide gels showing the changes in the extracellular proteome as affected by the presence of the two

concentrations (0.1 or 1.0% v/v) of cecum content. Lanes 1–3: Bifidobacterium animalis IPLA4549, lanes 4–6: B. animalis R2, lanes 7–9:

Lactococcus lactis MG1363, lanes 10–12: Lactobacillus rhamnosus GG, lanes 13–15: Lactobacillus delbrueckii IPLA lb101, lanes 16–18:

Lactobacillus acidophilus DSM 20079, lanes 19–21: Lactobacillus reuteri DSM 20016, lanes 22–24: Bifidobacterium longum NCIMB 8809, and

lanes 25–27: Bifidobacterium bifidum LMG11041. Cecum extract concentration (% v/v) is indicated under the lane numbers. The most

outstanding bands showing reproducible differences in their production were labeled as Imp#, and were submitted to MS analysis. Bands Imp11

and Imp23 were chosen for further experimentation.



our laboratory to elucidate the precise mechanism of

action of those two proteins.

Acknowledgements

BS was the recipient of a Juan de la Cierva postdoctoral

contract from the Spanish Ministerio de Ciencia e Inno-

vacion. LR was the recipient of an I3P predoctoral grant

from CSIC. Research in our group is supported by Grants

AGL2007-61805 and RM2010-00012-00-00 from the

Spanish Ministerio de Ciencia e Innovacion.

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0 0.1 10 0.1 1

L. acidophilus DSM20079TB. longum NCIMB8809

kD

Cecum extract (% v/v)

kD

(a)

9766

45

30

kDa9766

45

30

kDa

Imp23

Imp1130

20.1

14.4

20.1

14.4

5

6

uct

ion

Imp11

Imp23

(b)

2

3

4

pre

ssio

n fo

ld in

d

1

2

Early exponential Middle exponential Early stationary

Rel

ativ

e ex

p

Phase of growth

Fig. 2. (a) SDS-PAGE gels showing increases in production of Imp11

and Imp23 in the presence of 0.1% or 1.0% (v/v) cecum content in

growth medium at stationary phase. (b) qPCR analysis of genes

coding for Imp11 and Imp23 in early exponential, middle exponential,

and early stationary phase of growth.


Modulation of extracellular proteins by cecum content 193

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Table S1. Information concerning the identification of

the bands marked by arrows in the supplementary figure.

Please note: Wiley-Blackwell is not responsible for the

content or functionality of any supporting materials sup-

plied by the authors. Any queries (other than missing

material) should be directed to the corresponding author

for the article.



human cecum content modulates production of extracellular proteins by food and probiotic bacteria

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