R E S E A R C H L E T T E R

Amethod for the identi¢cationof proteins secreted by lactic acidbacteria grown in complexmediaBorja Sanchez1, Sthephane Chaignepain2, Jean-Marie Schmitter2 & Marıa C. Urdaci1

1Laboratoire de Microbiologie et Biochimie Appliquee, Universite de Bordeaux, UMR 5248 CBMN, UBX1-ENITAB, ENITAB, Gradignan, France; and2Universite de Bordeaux, UMR 5248 CBMN, UBX1-ENITAB, Institut Europeen de Chimie et Biologie 2, Pessac, France

Correspondence: Borja Sanchez,

Laboratoire de Microbiologie et Biochimie

Appliquee, Universite de Bordeaux, UMR

5248 CBMN, UBX1-ENITAB, ENITAB, 1 cours

du General de Gaulle, 33175 Gradignan

Cedex, France. Tel.: 133 5 57 35 59 92; fax:

133 5 57 35 07 39; e-mail:

b-sanchez@enitab.fr

Received 16 October 2008; accepted 16 March

2009.

First published online 15 April 2009.

DOI:10.1111/j.1574-6968.2009.01599.x

Editor: Wolfgang Kneifel

Keywords

secreted proteins; protein precipitation; lactic

acid bacteria.

Abstract

Lactic acid bacteria (LAB) are known for their special nutritional requirements,

being usually cultured in complex media to achieve optimal growth. In this paper,

a protocol based on trichloroacetic acid precipitation of peptides and proteins is

presented. The method has been tested on four probiotic LAB strains grown in De

Man Rogosa Sharpe (MRS) broth, a complex medium that is often used for the

culture of such bacteria. This protocol allowed the detection of 19 proteins after

sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 10 of them being

successfully identified by tandem MS. Thereafter, the 10 were found to be secreted

or surface associated by bioinformatic means. In conclusion, this work supplies a

method for the identification of proteins secreted by LAB, allowing discrimination

between the proteins present in the MRS and those produced by probiotic LAB.

Introduction

Secreted proteins are thought to play essential roles in the

molecular intercommunication between host–bacteria, and

in the monitoring of the bacterial environment (van Pijke-

ren et al., 2006). In commensal and probiotic lactic acid

bacteria (LAB), these proteins could be responsible for

bacterial–intestinal cell intercommunication, and for certain

probiotic traits such as pathogen inhibition and immuno-

modulation (Buck et al., 2005). Thus, identification of

proteins secreted by probiotic LAB is crucial for elucidating

their mechanism of action.

Secreted proteins are transported from the bacterial

cytoplasm to the bacterial environment. This is usually

achieved by the presence of a signal peptide in the N-

terminal part of the protein, which directs the protein

toward the secretion machinery (van Wely et al., 2001).

Included in this group are some surface-associated proteins

that are released into the external medium due to the

physiological turnover of the cell wall (Turner et al., 2004).

Although several scientific papers describe the precipita-

tion of secreted proteins in probiotic bacteria, they usually

start from chemically defined media, in order to avoid the

presence of proteins originating in the protein extracts that

are part of such media (Trost et al., 2005; Sanchez et al.,

2008). In contrast, some LAB strains are not able to grow or

grow deficiently in defined media, making difficult

the identification of the secreted proteins following those

methods.

In the present work, a trichloroacetic acid (TCA)-based

protocol for the precipitation and identification of proteins

secreted by LAB is presented. This method allowed the

identification of the proteins secreted by four probiotic LAB,

which were grown in De Man Rogosa Sharpe (MRS) medium.

Materials and methods

Bacterial strains used and growth conditions

Lactobacillus gasseri B3, Lactobacillus reuteri Protectis and

Lactobacillus rhamnosus R-11 were isolated from the pro-

biotic products Bions3, Stimulobiotic and Biotravel, all

manufactured in France. Lactococcus lactis n35 was isolated

from a sheep artisanal cheese.

FEMS Microbiol Lett 295 (2009) 226–229c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Strains were identified at the species level by partial 16S

rRNA gene sequencing using the universal 20F (50-AG

AGTTTGATCATGGCTCAG-30) and 1500R (50-GGTTACC

TTGTTACGACTT-30) primers (Weisburg et al., 1991).

Sequences were used to query the GenBank database, the

strain identification score being shown in Table 1. Strains

were routinely grown aerobically without shaking at 37 1C in

MRS broth (Becton Dickinson France SAS, Le Pont-De-

Claix, France). Lactococcus lactis n35, able to grow in MRS,

was used as positive control.

Precipitation and identification ofsecreted proteins

Precipitation of secreted proteins was achieved by adding

minor modifications to the method described by Sanchez

et al. (2008). Briefly, 50-mL aliquots of fresh MRS broth

were inoculated (0.1% v/v) from a 24-h culture and were

grown aerobically overnight at 37 1C, at the end of which all

cultures were typically at early stationary phase. Protein

loadings were standardized on a volume-for-volume basis,

which usually corresponded to a protein amount of around

40 mg. Aliquots of 5 mL were harvested by centrifugation

(10 min, 3500 g, 4 1C), and the supernatant was filtered

(0.45mm). Ten milligrams of sodium deoxycholate (Sigma-

Aldrich Chimie, Saint-Quentin Fallavier, France) were

added and mixed – the resulting solution was incubated at

4 1C for 30 min. Chilled TCA (Sigma-Aldrich Chimie) was

added at a final concentration of 6% (w/v) and proteins

were allowed to precipitate for 2 h at 4 1C. Proteins were

recovered by centrifugation (10 min, 9300 g, 4 1C) and

pellets were washed twice with 2 mL of chilled acetone

(Sigma-Aldrich Chimie). Pellets were allowed to dry at

room temperature (RT) and proteins were resolubilized by

ultrasonication (10 min, Ultrasonic bath, Deltasonic,

Meaux, France) in 40mL of 1� Laemmli buffer (Laemmli,

1970). These 40 mL were resolved by sodium dodecyl sulfate-

polyacrylamide gel electrophoresis using a final polyacryl-

amide concentration of 12.5% (w/v) (Laemmli, 1970).

Selected bands were excised from gels and digested with

trypsin using standard protocols, the resulting peptide

mixture being analyzed by tandem MS (MS/MS). Data were

acquired using a MALDI Q-Tof Premier mass spectrometer

(Waters, Manchester, UK), with a-cyano-4-hydroxy-cin-

namic acid (Sigma-Aldrich Chimie) used as a matrix

(3.6 mg mL�1 solution in 50% acetonitrile in 0.1% aqueous

trifluoroacetic acid). Monoisotopic masses were corrected

using the pseudomolecular ion of Glu-Fibrinopeptide as a

lock mass (1570.6774 Da).

Proteins were identified using the MS/MS search module

from the online version of MASCOT software (http://www.ma

trixscience.com) against the nonredundant protein NCBI

database, using the monoisotopic masses derived from

trypsinolysis. The following parameters were used: peptide

Table 1. Secreted proteins identified in the supernatant of several LAB strains

Bands� Putative function Microorganism Accession no.w MM pl

MS/

MSz MWE‰ SPz PSORTBk

S1 Serpin B1 Sus scrofa gi|417185 42.5 6.0 2 127 No Cytoplasmic

S2 Hypothetical protein Usp45 Lactococcus lactis ssp. lactis Il1403 gi|15674211 47.0 8.3 1 82 Yes Extracellular

S3 Cell wall hydrolase Lactobacillus casei ATCC 334 gi|116493849 49.4 4.9 2 68 Yes Extracellular

S4 Peptidoglycan-binding LysM Lactobacillus reuteri 100-23 gi|92089070 24.9 4.8 1 105 Yes Extracellular

S5 Mannosyl-glycoprotein

endo-b-N-

acetylglucosamidase

Lactobacillus reuteri F275 gi|148545058 60.4 9.5 6 150 Yes Extracellular

S6 Hypothetical protein LJ0155 Lactobacillus johnsonii NCC 533 gi|42518241 74.5 9.7 4 103 Yes Extracellular

S7 Hypothetical protein LJ0437 Lactobacillus johnsonii NCC 533 gi|42518532 110.0 6.2 2 70 Yes Cell wall (LPXTG)

S8 Aggregation-promoting

factor

Lactobacillus gasseri gi|1619598 32.0 9.6 1 139 Yes Extracellular

S9 Muramidase (lysozyme

subfamily 2)

Lactobacillus gasseri ATCC 33323 gi|116628837 66.7 9.8 2 143 Yes Extracellular

S10 Peptidoglycan-binding LysM Lactobacillus reuteri 100-23 gi|92090142 21.6 7.8 2 163 Yes Extracellular

S11 Mucus adhesion-promoting

protein

Lactobacillus reuteri gi|9929262 28.6 9.8 1 52 Yes Extracellular

�Codes refer to bands marked by arrows in Fig. 1.wProtein sequence GI number.zFragmented MS/MS peptides allowing the identification of the protein.‰MOWSE score resulting from the ion MS/MS search against the nonredundant NCBI protein database. All scores are statistically significant (Po 0.05).zSignal peptides were predicted using the PSORTB package (Gardy et al., 2005).kFinal subcellular localization was predicted using the PSORTB package (Gardy et al., 2005).

MM, molecular mass.

FEMS Microbiol Lett 295 (2009) 226–229 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

227Proteins secreted by lactic acid bacteria

charge 11, peptide tolerance � 0.1 Da, MS/MS tolerance

� 0.1 Da, and one missed cleavage allowed for trypsin. Gels

were repeated three times from independent cultures.

Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) enzymatic assay

In order to test the presence of small amounts of cytoplas-

mic enzymes in the supernatant medium, GAPDH enzy-

matic assays were conducted as follows: 50mL of

supernatant media were incubated with glyceraldehyde-3-

phosphate 2 mM, 1 mM NAD1 in 950 mL of assay buffer

(triethanolamine 40 mM, Na2HPO4 50 mM, EDTA 5 mM

and dithiothreitol 0.1 mM; pH 8.6) (all reagents purchased

from Sigma-Aldrich Chimie). GAPDH enzymatic activity

was determined spectrophotometrically at RT by monitor-

ing NADH apparition at 340 nm. Cytoplasmic extracts were

used as positive controls. One unit of GAPDH activity was

defined as the amount of protein capable of generating

1 nmol NADH min–1.

Results and discussion

In spite of the interest in the identification of secreted

proteins in LAB, few reports are available in the scientific

literature (Lee et al., 1997; Turner et al., 1997, 2004; Peant &

LaPointe, 2004; Yan et al., 2007; Sanchez et al., 2008).

Complex media might contain several peptides/proteins

and other molecules that interfere with protein detection

techniques, making difficult the identification of proteins

secreted by LAB. The absence of a method for the systematic

identification of proteins secreted by LAB, grown in com-

plex media, prompted us to undertake this study.

The method described in the present paper allowed the

simple precipitation and identification of the proteins

secreted by the four LAB strains used in this study. In total,

19 bands yielded good tryptic profiles after tandem MS

analysis (MS/MS). Ten of them were successfully identified

(Table 1), and nine not identified (labeled with asterisks in

Fig. 1). For unidentified bands, it seems that databases do

not yet contain homologs of such proteins. Bands that did

not yield good tryptic profiles are not indicated in Fig. 1.

Secreted protein profiles were consistent between replicates

and between MRS batches; however, variations might ap-

pear if a different MRS is used or if changes in any

environmental parameters are introduced. In this way, it is

known that L. lactis is able to grow and produce several

peptides in media derived from molasses, soybean and

different source of MRS, the amount of secreted proteins

being affected by pH or the presence of surfactants (Todorov

& Dicks, 2004; Rodrigues et al., 2006; Xiao et al., 2007).

Following our methodology, the strain L. lactis n35 was

shown to mostly produce a single protein of around 55 kDa

(S2, Fig. 1, lane 4) identified as protein Usp45. Lactococcus

lactis species has been shown to predominantly produce this

45-kDa protein, which is a chromosomally encoded protein

of unknown function (van Asseldonk et al., 1990). Remark-

ably, a shift between the theoretical and experimental

molecular masses of Usp45, 45 vs. 55 kDa, respectively, was

observed. The aminoacidic analysis of Usp45 showed that

the central zone of the protein (between residues 263 and

334) was rich in polar amino acids, notably serine and

threonine. For this reason, post-translational modifications

of such residues, like O-glycosylations, may be responsible

for the mass shift, but this point needs further research.

With regard to the proteins secreted by the other strains,

L. rhamnosus R-11 was shown to secrete two proteins,

whereas L. reuteri Protectis and L. gasseri B3 secreted several

(Fig. 1, lanes 3, 5 and 6). All identified proteins were shown

to carry a signal peptide and were identified as extracellular

by the PSORTB 2.0 software (Gardy et al., 2005). The only

exception was S7, which carried a C-terminal LPXTG motif,

a sequence that may allow the covalent binding of the

protein to the cell wall (Siezen et al., 2006). Four proteins

secreted by the strain L. gasseri B3 (labeled S8) were

identified as aggregation-promoting factor, suggesting that

the lower bands might be proteolytic products of the highest

band. The rest of the secreted proteins are listed in Table 1

and included cell wall hydrolase (S3), peptidoglycan-

binding proteins (S4 and S10), mannosyl-glycoprotein

endo-b-N-acetylglucosamidase (S5), muramidase (S9), mu-

cus adhesion-promoting protein (S11) and two hypothetical

proteins (S6 and S7). Again, the finding of only proteins

MM 1 2 3 4 5 6

97

S1 S3S7*

kDa

66

45

S6S5S9

*

**

30

S2

S8S11

*

**

***

20.1 S10

S4

Fig. 1. Representative sodium dodecyl sulfate polyacrylamide gel show-

ing the proteins secreted by the four LAB strains used in this study. Lane

1, Lactobacillus rhamnosus R-11 total extract obtained by sonication,

which evidences the low complexity of secreted cytoplasmic profiles;

lane 2, porcine serpin isolated from fresh MRS; lane 3, Lactobacillus

reuteri Protectis secreted proteins; lane 4, Lactococcus lactis ssp. lactis

n35 secreted proteins showing protein Usp45; lane 5, L. rhamnosus R-11

secreted proteins; and lane 6, Lactobacillus gasseri B3 secreted proteins.�Proteins not identified. MM, molecular mass.

FEMS Microbiol Lett 295 (2009) 226–229c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

228 B. Sanchez et al.

carrying export signals (such as signal peptides) is consistent

with the aim of our protocol.

Interestingly, our method showed that MRS contained

considerable amounts of a protein of about 50 kDa, which

was identified as porcine serpin, a leukocyte elastase inhi-

bitor (band S1, Fig. 1). Serpin was degraded to different

degrees by the four LAB strains, and might have interfered

with the detection of bacterial proteins in this zone of the

molecular mass.

Finally, GAPDH enzymatic assays were performed in

order to determine the presence of lysis in the supernatant

media, and no GAPDH activity was detected (data not

shown). Because secreted proteins were precipitated in the

stationary phase of culture, cell lysis cannot be excluded, but

at least it was minor.

In conclusion, this work provides an efficient, reproduci-

ble and simple protocol for the identification of proteins

secreted by LAB strains. Further analysis of the function of

these proteins, as well as the study of certain environmental

effects on synthesis and secretion, will help to elucidate the

mechanisms of action of probiotic bacteria in their ecologi-

cal niches.

Acknowledgement

B.S. was the recipient of a Cların postdoctoral contract from

the Gobierno del Principado de Asturias funded by the Plan

de Ciencia, Tecnologıa e Innovacion de Asturias 2006–2009.

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229Proteins secreted by lactic acid bacteria