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Comparison of protein patterns of Listeria monocytogenes grown in biofilm or in planktonic mode by proteomic analysis
FEMS Microbiology Letters 210 (2002) 25^31
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Comparison of protein patterns of Listeria monocytogenes grown in bio¢lm or in planktonic mode by proteomic analysis F. Tre¤moulet a , O. Duche¤ a , A. Namane b , B. Martinie c , The European Listeria Genome Consortium 1 , J.C. Labadie a; a
c
Station de Recherches sur la Viande, Laboratoire de Microbiologie, Institut National de la Recherche Agronomique, Theix, 63122 Saint Gene's Champanelle, France b PT Prote¤omique, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France Laboratoire de Microbiologie, Institut National de la Recherche Agronomique, Theix, 63122 Saint Gene's Champanelle, France Received 5 November 2001 ; received in revised form 5 February 2002 ; accepted 13 February 2002 First published online 21 March 2002
Abstract The proteome of a Listeria monocytogenes strain isolated from a food plant was investigated to study the differential protein pattern expressed by biofilms and planktonic bacteria. The approach used in this study was a combination of two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight and database searches for the protein identification. Thirty-one proteins varied significantly between the two growth conditions. Twenty-two and nine proteins were up- and down-regulated respectively and nine proteins were successfully identified. The variations of the protein patterns indicated that the biofilm development is probably controlled by specific regulation of protein expression involved at various levels of cellular physiology. 2 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Bio¢lm; Proteome ; Two-dimensional electrophoresis; Matrix-assisted laser desorption ionization-time of £ight; Listeria monocytogenes
1. Introduction Listeria monocytogenes is a Gram-positive bacterium widely distributed in the food-processing environment and frequently involved in food-borne disease outbreaks [1]. This microorganism is often isolated from the surfaces of processing lines in food plants [2], where it is able to stay alive for several months. For bacteria, the ability to * Corresponding author. Tel. : +33 4 73 62 41 57; Fax : +33 4 73 62 42 68. E-mail address : [email protected] (J.C. Labadie). 1
The European Listeria Genome Consortium is composed of Philippe Glaser, Alexandra Amend, Fernando Baquero-Mochales, Patrick Berche, Helmut Bloecker, Petra Brandt, Carmen Buchrieser, Trinad Chakraborty, Alain Charbit, Elisabeth Couve¤, Antoine de Daruvar, Pierre Dehoux, Eugen Domann, Gustavo Dominguez-Bernal, Lionel Durant, Karl-Dieter Entian, Lionel Frangeul, Ha¢da Fsihi, Francisco Garcia del Portillo, Patricia Garrido, Werner Goebel, Nuria Gomez-Lopez, Torsten Hain, Joerg Hauf, David Jackson, Jurgen Kreft, Frank Kunst, Jorge MataVicente, Eva Ng, Gabriele Nordsiek, Jose Claudio Perez-Diaz, Bettina Remmel, Matthias Rose, Christophe Rusniok, Thomas Schlueter, JoseAntonio Vazquez-Boland, Hartmut Voss, Jurgen Wehland and Pascale Cossart.
grow on surfaces as bio¢lms is the consequence of a survival strategy adopted by many species including L. monocytogenes [3]. Generally, bacterial bio¢lms are composed of microbial communities attached to surfaces and embedded in an extracellular polymeric matrix [4]. One of the most striking properties of these communities is their increased resistance to antimicrobial agents [5]. This property is certainly the result of additive e¡ects of several speci¢c regulatory mechanisms inside the bio¢lms leading to long-term survival. Prigent-Combaret et al. [6] reported that the expression of speci¢c genes was modi¢ed in Escherichia coli growing attached to a surface. For most bacteria including L. monocytogenes, limited information is available about the speci¢c properties that are expressed in bio¢lms. A growing number of works are published on the change in gene expression during the initial phase of bio¢lm development, but information concerning the properties of mature bio¢lms is lacking. In the present work, mature bio¢lms of L. monocytogenes and planktonic culture of the same age were studied by a proteomic analysis of their protein content in order to investigate the induction or repression of individual proteins which could characterize the bio¢lm phenotype.
0378-1097 / 02 / $22.00 2 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 5 7 1 - 2
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Two-dimensional (2-D) electrophoresis has been used in combination with matrix-assisted laser desorption ionization-time of £ight mass spectrometry (MALDI-TOF-MS) and database searching obtained from the DNA genome of the L. monocytogenes EGD-E strain.
2. Materials and methods 2.1. Bacterial strain and culture conditions All experiments throughout this study were performed using a L. monocytogenes strain (serotype 1/2a) isolated several times during 3 years from the facilities of a meatprocessing plant. L. monocytogenes was grown in tryptic soy broth (TSB, Difco Laboratories, Detroit, MI, USA) at 20‡C under agitation (150 rpm) for 24 h. Cultures were harvested by centrifugation (7500Ug, 10 min, 20‡C) and washed twice in saline to obtain the bacterial suspensions used for planktonic and bio¢lm cultures. For planktonic growth, TSB medium was inoculated with bacterial suspension (OD600nm = 0.1) and cultivated for 7 days at 20‡C to obtain planktonic L. monocytogenes in advanced stationary phase. 2.2. Bio¢lm formation Bio¢lms were cultivated on a pre¢ltration membrane disk Glass Fibre Filter Extra Thick (GFF, Pall Bio Pharma, Ann Arbor, MI, USA) according to a new approach recently set up (F. Tre¤moulet, E. Cle¤ment, B. Martinie, J. Labadie, submitted for publication). A sterile GFF was inoculated with 500 Wl of the bacterial suspension and adhesion of cells to ¢bres was completed in 5 min. After washing, the GFF was placed on a sterile GFF deposited in the centre of a Petri plate (90 mm diameter) containing 20 ml of TSB agar medium (TSB, 15 g l31 agar). The bio¢lms obtained were incubated at 20‡C for 7 days. After that time, bacterial bio¢lms were resuspended by vortexing the ¢lters for 5 min. The number of viable cells (planktonic or bio¢lm cells) was estimated by serial decimal dilution, plating onto tryptic soy agar (TSA, Difco) and counting colonies after 24 h at 37‡C. 2.3. Protein extraction Planktonic or sessile bacteria were washed three times by centrifugation at 7000Ug for 10 min at 4‡C with 20 mM Tris bu¡er, pH 7.5, containing 5 mM EDTA and 5 mM MgCl2 . Cells were sonicated (Vibra cell, Bioblock, Illkirch, France) three times for 2 min at 4‡C using a microtip setting at power 5 and 50% pulse duration. The suspension was centrifuged at 14 000Ug for 10 min at 4‡C to remove unbroken cells and cell debris. Protein concen-
tration was determined according to Bradford [7] with bovine serum albumin as standard. Cell-free extracts containing protein were precipitated with ice-cold acetone, incubated for 2 h at 320‡C and centrifuged at 14 000Ug for 25 min. Following precipitation, the protein pellet was resuspended in isoelectric focusing bu¡er containing 8 M urea, 2% CHAPS and traces of bromophenol blue. The protein samples were stored at 320‡C until analysis. This protocol allowed the extraction of total soluble proteins but was not adapted for the extraction of hydrophobic proteins such as membrane proteins. 2.4. 2-D gel electrophoresis 2-D electrophoresis was performed essentially according to O’Farrell [8] with the following modi¢cations. Precast Immobiline DryStrips with a non-linear gradient from pH 3 to 10 (Pharmacia-Biotech, Orsay, France) were rehydrated overnight with 80 Wg protein in 8 M urea, 2 mM tributyl phosphine, 2% ampholytes pH 3^10, 2% CHAPS and traces of bromophenol blue. The ¢rst dimension was carried out with the Multiphor II system (Pharmacia Biotech) for a total of 63.7 kVh according to the manufacturer’s instructions. After isoelectric focusing, the strips were ¢rst equilibrated in equilibration bu¡er (50 mM Tris^HCl, 2% SDS, 30% glycerol, 2 mM TBP, 6 M urea, pH 6.8). The same solution without TBP but with 2.5% iodoacetamide and traces of bromophenol blue was used for another 15 min equilibration period. The second dimensional separation was a vertical sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) with 12.5% acrylamide resolving gels and 1 cm stacking gel (4%). Separation was performed at 19‡C in two steps: 12.5 mA per gels were applied during the stacking period and 25 mA were used for the separation period. Gels were stained either with Coomassie brilliant blue (Brilliant blue R 250, Sigma, St. Louis, MO, USA) or with silver staining according to the method described by Rabilloud [9]. Gels were scanned using a GS-700 Imaging densitometer (BioRad, Yvry sur Seine, France). 2.5. Analysis of protein pattern Gels were analysed with Melanie 3 software (release 3.03, Bio-Rad) for qualitative and quantitative analysis of protein spots visualized on 2-D gels. For each condition (planktonic versus bio¢lm), six gels resulting from three independent protein extractions were analysed and compared. A statistical analysis was performed with Student’s t-test (95% con¢dence interval) which ensured that only signi¢cant changes in the value of protein spots were taken into consideration. Calibration of gels with pI and molecular masses was performed with internal protein standards (2-D SDS^PAGE standards, Bio-Rad).
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2.6. Protein identi¢cation by MALDI-TOF-MS and N-terminal sequencing
for MALDI-TOF-MS and N-terminal sequencing identi¢cation.
Protein spots from 2-D gels stained with Coomassie blue were digested in the gel by trypsin (Promega). Peptides were mixed with a 10 mg l31 solution of K-cyano-4hydroxycinnamic acid and deposited on the sample plate. Samples were allowed to dry and analysed by MALDITOF-MS (Voyager DE-STR, Perkin-Elmer, Norwalk, CT, USA). Monoisotopic peptide masses obtained from mass spectra were considered for protein identi¢cation using the MS-¢t program and searching in a personal L. monocytogenes database. Peptide masses were used to identify the open reading frames encoding the proteins obtained from the L. monocytogenes EGD-E genome database sequenced by ‘The European L. monocytogenes Genome Consortium’. The following parameters were used in the searches: protein molecular mass range from 1000 to 100 000 Da, trypsin digest with one missing cleavage, fragment ion mass tolerance of T 50 ppm and possible oxidation of methionine. To obtain N-terminal amino acid sequences, selected proteins were transferred to polyvinylidene di£uoride membranes (Sequi-Blot PVDF Membrane, BioRad) and microsequenced using an automatic Beckman/ Porton LF3000 protein sequencer. Searches for sequence homology were performed with the BLAST program [10]
2.7. Scanning electron microscopy Bio¢lm bacteria were ¢xed with 6% glutaraldehyde, 0.2 M cacodylate bu¡er (pH 7.4) for 1 h at 4‡C. GFF were rinsed three times in 0.2 M cacodylate bu¡er for 10 min and post-¢xed for 1 h with osmic vapors. GFF samples were dehydrated in a graded ethanol series of 70, 95 and 100% (three times) for 10 min, then in a graded acetone series of 30, 50 and 100% (three times) for 10 min. Samples were mounted on aluminium stubs then coated with gold (Emscope SC500) and observed with a Philips SEM 505 scanning electron microscope.
3. Results 3.1. Growth of planktonic cultures and bio¢lms of L. monocytogenes The growth curves obtained at 20‡C both in planktonic and bio¢lm mode are shown in the Fig. 1. In broth, the initial population of L. monocytogenes was 106 cfu ml31 . The number of cells increased during the ¢rst 24 h to reach
Fig. 1. Growth curves of L. monocytogenes cultivating in TSB medium at 20‡C (a) in liquid medium, (b) in bio¢lms on GFF with an initial inoculation of either 106 or 108 cfu per ¢lter.
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compaction of glass ¢bre (Fig. 2a). Microcolonies were observed within 24 h (Fig. 2b) and a dense bio¢lm of L. monocytogenes after 7 days (Fig. 2c). Seven-day-old bio¢lms were characterized by a variable morphological aspect of the cells, which appeared more coccoid than cells aged 24 h. 3.2. Di¡erential pattern proteins extracted from bio¢lms and free cells To establish di¡erential protein expression in bio¢lms of L. monocytogenes versus their free-living counterparts, statistical analysis of 2-D gels was performed with Melanie 3 software. From more than 550 proteins spots revealed by silver staining (Fig. 3), the expressions of 31 proteins were signi¢cantly (P 6 0.05) a¡ected by the growth in bio¢lms (Table 1). All proteins observed in 2-D gels were synthe-
Table 1 Proteins induced or repressed in L. monocytogenes bio¢lms versus their planktonic counterparts
Fig. 2. Photomicrographs showing the development of L. monocytogenes bio¢lms on GFF cultivating at 20‡C on agar medium. (a) GFF before the inoculation, (b) 24 h after inoculation and (c) 7 days after inoculation.
109 cfu ml31 , a number that remained stable for 7 days. For the bio¢lms, two levels were tested (106 or 108 cfu per ¢lter). For both concentrations, L. monocytogenes grew slower than in planktonic mode. The maximal population was reached within 24 h. After that time, L. monocytogenes bio¢lms remained stable for 7 days, that is close to 109 cfu per GFF. Microscopic observations of FFVs revealed a signi¢cant
Number of spotsa
pIb
Molecular mass (kDa)b
Protein spots induced (+) or repressed (3) by bio¢lm growthc
9 17 24 30 33 50 65 98 101 123 134 149 176 185 188 194 197 207 215 246 252 264 295 296 299 307 312 321 325 328 359
4.8 6 5.3 6.1 5.7 4.9 5.7 5.8 5.7 5.8 4.6 5.4 4.9 5.7 5.3 5.1 5 4.7 5.8 5.9 5.2 5.8 4.9 4.7 5.7 4.9 4.6 4.9 4.6 5.3 5.9
66.7 63.5 62.2 60.3 59.8 57.6 56.4 49.5 48.5 45.9 43.1 40.9 36.7 35.2 35.1 33.4 33.4 31.6 32 29.1 28.6 27.8 25.9 25.9 25.3 25.1 23.9 22.4 20.8 19.9 14.9
+ 3 3 3 3 + 3 + + + + 3 + + + + + 3 + + + + + + 3 + + + 3 + +
a
Spot number indicated on the gel of Fig. 3. pI and molecular mass are experimentally determined with protein standards on 2-D gel. c +, protein spot signi¢cantly (P 6 0.05) more important in L. monocytogenes grown in bio¢lm mode ; 3, protein spot signi¢cantly (P 6 0.05) less important in L. monocytogenes grown in bio¢lm. b
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Fig. 3. 2-D patterns of proteins of L. monocytogenes cultivating 7 days (a) in liquid medium and (b) in bio¢lm on GFF incubated at 20‡C. 2-D gels for electrophoresis were stained with the silver staining method. Circles indicate spots increased signi¢cantly more than 2-fold by the growth condition.
sized in both planktonic and bio¢lm conditions. Among the separated proteins, the comparison of the two conditions indicated that respectively 22 and nine of them were up- or down-regulated in bio¢lms. 3.3. Identi¢cation of the proteins a¡ected by the growth of L. monocytogenes in bio¢lms Actually, a poor number of identi¢ed proteins of L. monocytogenes is available in databases. This lack of
information limits the identi¢cation of proteins by MALDI-TOF. So, a database of peptide masses was created from the L. monocytogenes (strain EGD-E) genome sequence project. Among the 31 proteins modi¢ed by growth conditions, eight were identi¢ed by MALDITOF-MS (Table 2) and one by N-terminal sequencing for the spot 194. The sequence of the ¢rst seven amino acids of the spot 194 was TIANSIT. According to the Listeria genome database, this protein was identi¢ed as CysK protein. The estimated molecular mass and pI ob-
Table 2 Summary of di¡erentially expressed proteins of L. monocytogenes identi¢ed by MALDI-TOF-MS Total number of peptidesa
Protein identi¢ed
% of the protein coveredb
Estimated pI/molecular mass (kDa)
50
6
17
4.9/57.6
185 188
8 9
28 22
5.7/35.2 5.3/35.1
207 215 295 307 312
8 4 4 4 5
Unknown, similar to subunit E3 of pyruvate dehydrogenase (pdhD) 30S ribosomal protein S2 (rpsB) Unknown, highly similar to 6-phosphofructokinase (pfk) Flagellin protein (£aA) Unknown, similar to B. subtilis RecO Superoxide dismutase (sod) Unknown, similar to YvyD of B. subtilis Unknown, similar to cell division initiation protein (divIVA)
40 16 31 35 34
4.7/31.6 5.8/32 4.9/25.9 4.9/25.1 4.6/23.9
Number of spots
a b
Total number of peptides used for comparison with the database. Sequence of amino acids matching the identi¢ed protein.
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tained by 2-D electrophoresis were in agreement with the identi¢cation of these nine proteins.
4. Discussion In this work, we studied the di¡erential protein patterns of L. monocytogenes cultivated in bio¢lm versus planktonic mode. As a ¢rst step, it was decided to determine the growth conditions necessary to obtain protein patterns representative of a 7-day-old L. monocytogenes bio¢lm and of planktonic cells of the same age. The method used to set up bio¢lms using GFFs allowed the formation of a true bio¢lm attested by SEM observations. Microcolonies and single adherent bacteria were observed within 24 h at 20‡C and mature bio¢lm within 7 days. The development of L. monocytogenes during bio¢lm maturation was responsible for changes in the cell morphology which changed from a classical rod shape (24-h-old bio¢lms) to coccoid forms (7-day-old bio¢lms). These changes were certainly the result of physiological modi¢cations related to the maturation of the bio¢lms. The obvious increase in cell numbers in bio¢lms between day 1 and day 7, clearly visible in the SEM photographs, was not re£ected in the cfu ml31 reported in Fig. 1. This observation could be notably explained by the presence of non-cultivable or dead L. monocytogenes in 7-day-old bio¢lms. The GFFs used for cultivating bio¢lms in this work a¡orded a surface for adhesion and colonization allowing the production of a su⁄cient quantity of bio¢lm for 2-D electrophoresis analysis of proteins. As 2-D electrophoresis allows the separation of several hundred proteins in a single gel, this technique has become an important tool for proteome studies investigating cellular physiology. So, we have used this approach to study the di¡erential protein patterns of L. monocytogenes bio¢lms versus planktonic counterparts. Among the 550 proteins separated and detected on 2-D gels, 31 showed signi¢cant variations (P 6 0.05). Twenty-two and nine proteins were respectively up- and down-expressed in bio¢lms and appeared characteristic of a bio¢lm phenotype. The only protein identi¢ed which decreased as a result of bio¢lm growth was £agellin. It is well known that £agella are directly implicated in the initial adhesion of L. monocytogenes [11] at the beginning of bio¢lm formation. As the synthesis of this protein is inhibited during the bio¢lm development of many motile bacteria [6], this property could be one of the most important to phenotypically characterize bio¢lms. Among the proteins whose level increased as a result of bio¢lm development, two key enzymes involved in global carbon metabolism, pyruvate dehydrogenase (PdhD) and 6-phosphofructokinase, have been identi¢ed. PdhD is a component of the pyruvate dehydrogenase complex, which catalyses the overall conversion of pyruvate to acetyl-CoA and CO2 . The second protein, 6-phosphofructokinase, is the key control step of glycolysis and is principally regu-
lated by the intracellular ATP levels. This ¢nding indicated that central metabolism of L. monocytogenes is affected by bio¢lm development. It is also interesting to note that two proteins increased in bio¢lms were identi¢ed as 30S ribosomal proteins. The ¢rst one, YvyD is known to be induced under various stress conditions, including starvation, heat, ethanol and salt stress in Bacillus subtilis [12^ 14]. Drzewiecki et al. [13] showed that, in B. subtilis, YvyD is a very important protein of the bacterial stress response under the control of the general stress transcription factor cB of B. subtilis. The ribosomal proteins YvyD and rpsB, which increased in bio¢lms, may act as sensors to detect physical or chemical changes in the environment of bio¢lms. Two proteins known to be increased in oxidative stress conditions, SOD and CysK, were also up-regulated in bio¢lm conditions [13]. The CysK protein, which encodes O-acetylserine lyase A, is implicated in cysteine biosynthesis and was also induced upon oxidative stresses in B. subtilis [12]. Additionally, the signalling molecules of quorum sensing control the expression of numerous genes, including CysK in E. coli [15], as well as bio¢lm formation [16]. These data suggested that the high density of bacterial cells in bio¢lms possibly led to regulation on quorum sensing, including CysK overexpression. L. monocytogenes bio¢lms overexpressed the DNA repair and protection RecO that could be involved in a speci¢c mechanism of bio¢lm resistance. Finally, the factor DivIVA which controls cellular division was increased in bio¢lm, probably re£ecting speci¢c needs for cell multiplication on surfaces. This study showed that the proteome of L. monocytogenes was greatly in£uenced by bio¢lm development on a surface and proved that speci¢c genes may be induced under such conditions. Thirty-one proteins were di¡erentially synthesized in L. monocytogenes bio¢lms versus planktonic cells and nine of these were identi¢ed. The proteins identi¢ed are implicated at various levels of cellular physiology, indicating that the bio¢lm phenotype results in complex patterns of gene regulation. Understanding the role of these speci¢c proteins during the bio¢lm development should permit a better understanding of the mechanisms sustaining the proliferation and the resistance of bacteria on abiotic surfaces.
References [1] Low, J.C. and Donachie, W. (1997) A review of Listeria monocytogenes and listeriosis. Vet. J. 153, 9^29. [2] Jeong, D.K. and Frank, J.F. (1994) Growth of Listeria monocytogenes at 21‡C in bio¢lms with micro-organisms isolated from meat and dairy processing environments. Lebensm. Wiss. Technol. 27, 415^424. [3] Carpentier, B. and Cerf, O. (1993) Bio¢lms and their consequences, with particular reference to hygiene in the food industry. J. Appl. Bacteriol. 75, 499^511. [4] Costerton, J.W., Caldwell, D.E., Korber, D.R. and Lappin-Scott, H.M. (1995) Microbial bio¢lms. Annu. Rev. Microbiol. 49, 711^745.
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F. Tre¤moulet et al. / FEMS Microbiology Letters 210 (2002) 25^31 [5] Lewis, K. (2001) Riddle of bio¢lm resistance. Antimicrob. Agents Chemother. 45, 999^1007. [6] Prigent-Combaret, C., Vidal, O., Dorel, C. and Lejeune, P. (1999) Abiotic surface sensing and bio¢lm-dependent regulation of gene expression in Escherichia coli. J. Bacteriol. 181, 5993^6002. [7] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. [8] O’Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007^4021. [9] Rabilloud, T. (1992) A comparison between low background silver diammine and silver nitrate protein stains. Electrophoresis 13, 429^ 439. [10] Altschul, S.F., Madden, T.L., Scha¡er, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389^3402. [11] Vatanyoopaisarn, S., Nazli, A., Dodd, C.E., Rees, C.E. and Waites,
[12]
[13]
[14]
[15]
[16]
31
W.M. (2000) E¡ect of £agella on initial attachment of Listeria monocytogenes to stainless steel. Appl. Environ. Microbiol. 66, 860^863. Antelmann, H., Scharf, C. and Hecker, M. (2000) Phosphate starvation-inducible proteins of Bacillus subtilis: proteomics and transcriptional analysis. J. Bacteriol. 182, 4478^4490. Drzewiecki, K., Eymann, C., Mittenhuber, G. and Hecker, M. (1998) The yvyD gene of Bacillus subtilis is under dual control of sigmaB and sigmaH. J. Bacteriol. 180, 6674^6680. Petersohn, A., Brigulla, M., Haas, S., Hoheisel, J.D., Volker, U. and Hecker, M. (2001) Global analysis of the general stress response of Bacillus subtilis. J. Bacteriol. 183, 5617^5631. Baca-DeLancey, R.R., South, M.M., Ding, X. and Rather, P.N. (1999) Escherichia coli genes regulated by cell-to-cell signaling. Proc. Natl. Acad. Sci. USA 96, 4610^4614. De Kievit, T.R., Gillis, R., Marx, S., Brown, C. and Iglewski, B.H. (2001) Quorum-sensing genes in Pseudomonas aeruginosa bio¢lms: their role and expression patterns. Appl. Environ. Microbiol. 67, 1865^1873.
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Comparison of protein patterns of Listeria monocytogenes grown in bio¢lm or in planktonic mode by proteomic analysis F. Tre¤moulet a , O. Duche¤ a , A. Namane b , B. Martinie c , The European Listeria Genome Consortium 1 , J.C. Labadie a; a
c
Station de Recherches sur la Viande, Laboratoire de Microbiologie, Institut National de la Recherche Agronomique, Theix, 63122 Saint Gene's Champanelle, France b PT Prote¤omique, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France Laboratoire de Microbiologie, Institut National de la Recherche Agronomique, Theix, 63122 Saint Gene's Champanelle, France Received 5 November 2001 ; received in revised form 5 February 2002 ; accepted 13 February 2002 First published online 21 March 2002
Abstract The proteome of a Listeria monocytogenes strain isolated from a food plant was investigated to study the differential protein pattern expressed by biofilms and planktonic bacteria. The approach used in this study was a combination of two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight and database searches for the protein identification. Thirty-one proteins varied significantly between the two growth conditions. Twenty-two and nine proteins were up- and down-regulated respectively and nine proteins were successfully identified. The variations of the protein patterns indicated that the biofilm development is probably controlled by specific regulation of protein expression involved at various levels of cellular physiology. 2 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Bio¢lm; Proteome ; Two-dimensional electrophoresis; Matrix-assisted laser desorption ionization-time of £ight; Listeria monocytogenes
1. Introduction Listeria monocytogenes is a Gram-positive bacterium widely distributed in the food-processing environment and frequently involved in food-borne disease outbreaks [1]. This microorganism is often isolated from the surfaces of processing lines in food plants [2], where it is able to stay alive for several months. For bacteria, the ability to * Corresponding author. Tel. : +33 4 73 62 41 57; Fax : +33 4 73 62 42 68. E-mail address : [email protected] (J.C. Labadie). 1
The European Listeria Genome Consortium is composed of Philippe Glaser, Alexandra Amend, Fernando Baquero-Mochales, Patrick Berche, Helmut Bloecker, Petra Brandt, Carmen Buchrieser, Trinad Chakraborty, Alain Charbit, Elisabeth Couve¤, Antoine de Daruvar, Pierre Dehoux, Eugen Domann, Gustavo Dominguez-Bernal, Lionel Durant, Karl-Dieter Entian, Lionel Frangeul, Ha¢da Fsihi, Francisco Garcia del Portillo, Patricia Garrido, Werner Goebel, Nuria Gomez-Lopez, Torsten Hain, Joerg Hauf, David Jackson, Jurgen Kreft, Frank Kunst, Jorge MataVicente, Eva Ng, Gabriele Nordsiek, Jose Claudio Perez-Diaz, Bettina Remmel, Matthias Rose, Christophe Rusniok, Thomas Schlueter, JoseAntonio Vazquez-Boland, Hartmut Voss, Jurgen Wehland and Pascale Cossart.
grow on surfaces as bio¢lms is the consequence of a survival strategy adopted by many species including L. monocytogenes [3]. Generally, bacterial bio¢lms are composed of microbial communities attached to surfaces and embedded in an extracellular polymeric matrix [4]. One of the most striking properties of these communities is their increased resistance to antimicrobial agents [5]. This property is certainly the result of additive e¡ects of several speci¢c regulatory mechanisms inside the bio¢lms leading to long-term survival. Prigent-Combaret et al. [6] reported that the expression of speci¢c genes was modi¢ed in Escherichia coli growing attached to a surface. For most bacteria including L. monocytogenes, limited information is available about the speci¢c properties that are expressed in bio¢lms. A growing number of works are published on the change in gene expression during the initial phase of bio¢lm development, but information concerning the properties of mature bio¢lms is lacking. In the present work, mature bio¢lms of L. monocytogenes and planktonic culture of the same age were studied by a proteomic analysis of their protein content in order to investigate the induction or repression of individual proteins which could characterize the bio¢lm phenotype.
0378-1097 / 02 / $22.00 2 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 5 7 1 - 2
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Two-dimensional (2-D) electrophoresis has been used in combination with matrix-assisted laser desorption ionization-time of £ight mass spectrometry (MALDI-TOF-MS) and database searching obtained from the DNA genome of the L. monocytogenes EGD-E strain.
2. Materials and methods 2.1. Bacterial strain and culture conditions All experiments throughout this study were performed using a L. monocytogenes strain (serotype 1/2a) isolated several times during 3 years from the facilities of a meatprocessing plant. L. monocytogenes was grown in tryptic soy broth (TSB, Difco Laboratories, Detroit, MI, USA) at 20‡C under agitation (150 rpm) for 24 h. Cultures were harvested by centrifugation (7500Ug, 10 min, 20‡C) and washed twice in saline to obtain the bacterial suspensions used for planktonic and bio¢lm cultures. For planktonic growth, TSB medium was inoculated with bacterial suspension (OD600nm = 0.1) and cultivated for 7 days at 20‡C to obtain planktonic L. monocytogenes in advanced stationary phase. 2.2. Bio¢lm formation Bio¢lms were cultivated on a pre¢ltration membrane disk Glass Fibre Filter Extra Thick (GFF, Pall Bio Pharma, Ann Arbor, MI, USA) according to a new approach recently set up (F. Tre¤moulet, E. Cle¤ment, B. Martinie, J. Labadie, submitted for publication). A sterile GFF was inoculated with 500 Wl of the bacterial suspension and adhesion of cells to ¢bres was completed in 5 min. After washing, the GFF was placed on a sterile GFF deposited in the centre of a Petri plate (90 mm diameter) containing 20 ml of TSB agar medium (TSB, 15 g l31 agar). The bio¢lms obtained were incubated at 20‡C for 7 days. After that time, bacterial bio¢lms were resuspended by vortexing the ¢lters for 5 min. The number of viable cells (planktonic or bio¢lm cells) was estimated by serial decimal dilution, plating onto tryptic soy agar (TSA, Difco) and counting colonies after 24 h at 37‡C. 2.3. Protein extraction Planktonic or sessile bacteria were washed three times by centrifugation at 7000Ug for 10 min at 4‡C with 20 mM Tris bu¡er, pH 7.5, containing 5 mM EDTA and 5 mM MgCl2 . Cells were sonicated (Vibra cell, Bioblock, Illkirch, France) three times for 2 min at 4‡C using a microtip setting at power 5 and 50% pulse duration. The suspension was centrifuged at 14 000Ug for 10 min at 4‡C to remove unbroken cells and cell debris. Protein concen-
tration was determined according to Bradford [7] with bovine serum albumin as standard. Cell-free extracts containing protein were precipitated with ice-cold acetone, incubated for 2 h at 320‡C and centrifuged at 14 000Ug for 25 min. Following precipitation, the protein pellet was resuspended in isoelectric focusing bu¡er containing 8 M urea, 2% CHAPS and traces of bromophenol blue. The protein samples were stored at 320‡C until analysis. This protocol allowed the extraction of total soluble proteins but was not adapted for the extraction of hydrophobic proteins such as membrane proteins. 2.4. 2-D gel electrophoresis 2-D electrophoresis was performed essentially according to O’Farrell [8] with the following modi¢cations. Precast Immobiline DryStrips with a non-linear gradient from pH 3 to 10 (Pharmacia-Biotech, Orsay, France) were rehydrated overnight with 80 Wg protein in 8 M urea, 2 mM tributyl phosphine, 2% ampholytes pH 3^10, 2% CHAPS and traces of bromophenol blue. The ¢rst dimension was carried out with the Multiphor II system (Pharmacia Biotech) for a total of 63.7 kVh according to the manufacturer’s instructions. After isoelectric focusing, the strips were ¢rst equilibrated in equilibration bu¡er (50 mM Tris^HCl, 2% SDS, 30% glycerol, 2 mM TBP, 6 M urea, pH 6.8). The same solution without TBP but with 2.5% iodoacetamide and traces of bromophenol blue was used for another 15 min equilibration period. The second dimensional separation was a vertical sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) with 12.5% acrylamide resolving gels and 1 cm stacking gel (4%). Separation was performed at 19‡C in two steps: 12.5 mA per gels were applied during the stacking period and 25 mA were used for the separation period. Gels were stained either with Coomassie brilliant blue (Brilliant blue R 250, Sigma, St. Louis, MO, USA) or with silver staining according to the method described by Rabilloud [9]. Gels were scanned using a GS-700 Imaging densitometer (BioRad, Yvry sur Seine, France). 2.5. Analysis of protein pattern Gels were analysed with Melanie 3 software (release 3.03, Bio-Rad) for qualitative and quantitative analysis of protein spots visualized on 2-D gels. For each condition (planktonic versus bio¢lm), six gels resulting from three independent protein extractions were analysed and compared. A statistical analysis was performed with Student’s t-test (95% con¢dence interval) which ensured that only signi¢cant changes in the value of protein spots were taken into consideration. Calibration of gels with pI and molecular masses was performed with internal protein standards (2-D SDS^PAGE standards, Bio-Rad).
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27
2.6. Protein identi¢cation by MALDI-TOF-MS and N-terminal sequencing
for MALDI-TOF-MS and N-terminal sequencing identi¢cation.
Protein spots from 2-D gels stained with Coomassie blue were digested in the gel by trypsin (Promega). Peptides were mixed with a 10 mg l31 solution of K-cyano-4hydroxycinnamic acid and deposited on the sample plate. Samples were allowed to dry and analysed by MALDITOF-MS (Voyager DE-STR, Perkin-Elmer, Norwalk, CT, USA). Monoisotopic peptide masses obtained from mass spectra were considered for protein identi¢cation using the MS-¢t program and searching in a personal L. monocytogenes database. Peptide masses were used to identify the open reading frames encoding the proteins obtained from the L. monocytogenes EGD-E genome database sequenced by ‘The European L. monocytogenes Genome Consortium’. The following parameters were used in the searches: protein molecular mass range from 1000 to 100 000 Da, trypsin digest with one missing cleavage, fragment ion mass tolerance of T 50 ppm and possible oxidation of methionine. To obtain N-terminal amino acid sequences, selected proteins were transferred to polyvinylidene di£uoride membranes (Sequi-Blot PVDF Membrane, BioRad) and microsequenced using an automatic Beckman/ Porton LF3000 protein sequencer. Searches for sequence homology were performed with the BLAST program [10]
2.7. Scanning electron microscopy Bio¢lm bacteria were ¢xed with 6% glutaraldehyde, 0.2 M cacodylate bu¡er (pH 7.4) for 1 h at 4‡C. GFF were rinsed three times in 0.2 M cacodylate bu¡er for 10 min and post-¢xed for 1 h with osmic vapors. GFF samples were dehydrated in a graded ethanol series of 70, 95 and 100% (three times) for 10 min, then in a graded acetone series of 30, 50 and 100% (three times) for 10 min. Samples were mounted on aluminium stubs then coated with gold (Emscope SC500) and observed with a Philips SEM 505 scanning electron microscope.
3. Results 3.1. Growth of planktonic cultures and bio¢lms of L. monocytogenes The growth curves obtained at 20‡C both in planktonic and bio¢lm mode are shown in the Fig. 1. In broth, the initial population of L. monocytogenes was 106 cfu ml31 . The number of cells increased during the ¢rst 24 h to reach
Fig. 1. Growth curves of L. monocytogenes cultivating in TSB medium at 20‡C (a) in liquid medium, (b) in bio¢lms on GFF with an initial inoculation of either 106 or 108 cfu per ¢lter.
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compaction of glass ¢bre (Fig. 2a). Microcolonies were observed within 24 h (Fig. 2b) and a dense bio¢lm of L. monocytogenes after 7 days (Fig. 2c). Seven-day-old bio¢lms were characterized by a variable morphological aspect of the cells, which appeared more coccoid than cells aged 24 h. 3.2. Di¡erential pattern proteins extracted from bio¢lms and free cells To establish di¡erential protein expression in bio¢lms of L. monocytogenes versus their free-living counterparts, statistical analysis of 2-D gels was performed with Melanie 3 software. From more than 550 proteins spots revealed by silver staining (Fig. 3), the expressions of 31 proteins were signi¢cantly (P 6 0.05) a¡ected by the growth in bio¢lms (Table 1). All proteins observed in 2-D gels were synthe-
Table 1 Proteins induced or repressed in L. monocytogenes bio¢lms versus their planktonic counterparts
Fig. 2. Photomicrographs showing the development of L. monocytogenes bio¢lms on GFF cultivating at 20‡C on agar medium. (a) GFF before the inoculation, (b) 24 h after inoculation and (c) 7 days after inoculation.
109 cfu ml31 , a number that remained stable for 7 days. For the bio¢lms, two levels were tested (106 or 108 cfu per ¢lter). For both concentrations, L. monocytogenes grew slower than in planktonic mode. The maximal population was reached within 24 h. After that time, L. monocytogenes bio¢lms remained stable for 7 days, that is close to 109 cfu per GFF. Microscopic observations of FFVs revealed a signi¢cant
Number of spotsa
pIb
Molecular mass (kDa)b
Protein spots induced (+) or repressed (3) by bio¢lm growthc
9 17 24 30 33 50 65 98 101 123 134 149 176 185 188 194 197 207 215 246 252 264 295 296 299 307 312 321 325 328 359
4.8 6 5.3 6.1 5.7 4.9 5.7 5.8 5.7 5.8 4.6 5.4 4.9 5.7 5.3 5.1 5 4.7 5.8 5.9 5.2 5.8 4.9 4.7 5.7 4.9 4.6 4.9 4.6 5.3 5.9
66.7 63.5 62.2 60.3 59.8 57.6 56.4 49.5 48.5 45.9 43.1 40.9 36.7 35.2 35.1 33.4 33.4 31.6 32 29.1 28.6 27.8 25.9 25.9 25.3 25.1 23.9 22.4 20.8 19.9 14.9
+ 3 3 3 3 + 3 + + + + 3 + + + + + 3 + + + + + + 3 + + + 3 + +
a
Spot number indicated on the gel of Fig. 3. pI and molecular mass are experimentally determined with protein standards on 2-D gel. c +, protein spot signi¢cantly (P 6 0.05) more important in L. monocytogenes grown in bio¢lm mode ; 3, protein spot signi¢cantly (P 6 0.05) less important in L. monocytogenes grown in bio¢lm. b
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Fig. 3. 2-D patterns of proteins of L. monocytogenes cultivating 7 days (a) in liquid medium and (b) in bio¢lm on GFF incubated at 20‡C. 2-D gels for electrophoresis were stained with the silver staining method. Circles indicate spots increased signi¢cantly more than 2-fold by the growth condition.
sized in both planktonic and bio¢lm conditions. Among the separated proteins, the comparison of the two conditions indicated that respectively 22 and nine of them were up- or down-regulated in bio¢lms. 3.3. Identi¢cation of the proteins a¡ected by the growth of L. monocytogenes in bio¢lms Actually, a poor number of identi¢ed proteins of L. monocytogenes is available in databases. This lack of
information limits the identi¢cation of proteins by MALDI-TOF. So, a database of peptide masses was created from the L. monocytogenes (strain EGD-E) genome sequence project. Among the 31 proteins modi¢ed by growth conditions, eight were identi¢ed by MALDITOF-MS (Table 2) and one by N-terminal sequencing for the spot 194. The sequence of the ¢rst seven amino acids of the spot 194 was TIANSIT. According to the Listeria genome database, this protein was identi¢ed as CysK protein. The estimated molecular mass and pI ob-
Table 2 Summary of di¡erentially expressed proteins of L. monocytogenes identi¢ed by MALDI-TOF-MS Total number of peptidesa
Protein identi¢ed
% of the protein coveredb
Estimated pI/molecular mass (kDa)
50
6
17
4.9/57.6
185 188
8 9
28 22
5.7/35.2 5.3/35.1
207 215 295 307 312
8 4 4 4 5
Unknown, similar to subunit E3 of pyruvate dehydrogenase (pdhD) 30S ribosomal protein S2 (rpsB) Unknown, highly similar to 6-phosphofructokinase (pfk) Flagellin protein (£aA) Unknown, similar to B. subtilis RecO Superoxide dismutase (sod) Unknown, similar to YvyD of B. subtilis Unknown, similar to cell division initiation protein (divIVA)
40 16 31 35 34
4.7/31.6 5.8/32 4.9/25.9 4.9/25.1 4.6/23.9
Number of spots
a b
Total number of peptides used for comparison with the database. Sequence of amino acids matching the identi¢ed protein.
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tained by 2-D electrophoresis were in agreement with the identi¢cation of these nine proteins.
4. Discussion In this work, we studied the di¡erential protein patterns of L. monocytogenes cultivated in bio¢lm versus planktonic mode. As a ¢rst step, it was decided to determine the growth conditions necessary to obtain protein patterns representative of a 7-day-old L. monocytogenes bio¢lm and of planktonic cells of the same age. The method used to set up bio¢lms using GFFs allowed the formation of a true bio¢lm attested by SEM observations. Microcolonies and single adherent bacteria were observed within 24 h at 20‡C and mature bio¢lm within 7 days. The development of L. monocytogenes during bio¢lm maturation was responsible for changes in the cell morphology which changed from a classical rod shape (24-h-old bio¢lms) to coccoid forms (7-day-old bio¢lms). These changes were certainly the result of physiological modi¢cations related to the maturation of the bio¢lms. The obvious increase in cell numbers in bio¢lms between day 1 and day 7, clearly visible in the SEM photographs, was not re£ected in the cfu ml31 reported in Fig. 1. This observation could be notably explained by the presence of non-cultivable or dead L. monocytogenes in 7-day-old bio¢lms. The GFFs used for cultivating bio¢lms in this work a¡orded a surface for adhesion and colonization allowing the production of a su⁄cient quantity of bio¢lm for 2-D electrophoresis analysis of proteins. As 2-D electrophoresis allows the separation of several hundred proteins in a single gel, this technique has become an important tool for proteome studies investigating cellular physiology. So, we have used this approach to study the di¡erential protein patterns of L. monocytogenes bio¢lms versus planktonic counterparts. Among the 550 proteins separated and detected on 2-D gels, 31 showed signi¢cant variations (P 6 0.05). Twenty-two and nine proteins were respectively up- and down-expressed in bio¢lms and appeared characteristic of a bio¢lm phenotype. The only protein identi¢ed which decreased as a result of bio¢lm growth was £agellin. It is well known that £agella are directly implicated in the initial adhesion of L. monocytogenes [11] at the beginning of bio¢lm formation. As the synthesis of this protein is inhibited during the bio¢lm development of many motile bacteria [6], this property could be one of the most important to phenotypically characterize bio¢lms. Among the proteins whose level increased as a result of bio¢lm development, two key enzymes involved in global carbon metabolism, pyruvate dehydrogenase (PdhD) and 6-phosphofructokinase, have been identi¢ed. PdhD is a component of the pyruvate dehydrogenase complex, which catalyses the overall conversion of pyruvate to acetyl-CoA and CO2 . The second protein, 6-phosphofructokinase, is the key control step of glycolysis and is principally regu-
lated by the intracellular ATP levels. This ¢nding indicated that central metabolism of L. monocytogenes is affected by bio¢lm development. It is also interesting to note that two proteins increased in bio¢lms were identi¢ed as 30S ribosomal proteins. The ¢rst one, YvyD is known to be induced under various stress conditions, including starvation, heat, ethanol and salt stress in Bacillus subtilis [12^ 14]. Drzewiecki et al. [13] showed that, in B. subtilis, YvyD is a very important protein of the bacterial stress response under the control of the general stress transcription factor cB of B. subtilis. The ribosomal proteins YvyD and rpsB, which increased in bio¢lms, may act as sensors to detect physical or chemical changes in the environment of bio¢lms. Two proteins known to be increased in oxidative stress conditions, SOD and CysK, were also up-regulated in bio¢lm conditions [13]. The CysK protein, which encodes O-acetylserine lyase A, is implicated in cysteine biosynthesis and was also induced upon oxidative stresses in B. subtilis [12]. Additionally, the signalling molecules of quorum sensing control the expression of numerous genes, including CysK in E. coli [15], as well as bio¢lm formation [16]. These data suggested that the high density of bacterial cells in bio¢lms possibly led to regulation on quorum sensing, including CysK overexpression. L. monocytogenes bio¢lms overexpressed the DNA repair and protection RecO that could be involved in a speci¢c mechanism of bio¢lm resistance. Finally, the factor DivIVA which controls cellular division was increased in bio¢lm, probably re£ecting speci¢c needs for cell multiplication on surfaces. This study showed that the proteome of L. monocytogenes was greatly in£uenced by bio¢lm development on a surface and proved that speci¢c genes may be induced under such conditions. Thirty-one proteins were di¡erentially synthesized in L. monocytogenes bio¢lms versus planktonic cells and nine of these were identi¢ed. The proteins identi¢ed are implicated at various levels of cellular physiology, indicating that the bio¢lm phenotype results in complex patterns of gene regulation. Understanding the role of these speci¢c proteins during the bio¢lm development should permit a better understanding of the mechanisms sustaining the proliferation and the resistance of bacteria on abiotic surfaces.
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F. Tre¤moulet et al. / FEMS Microbiology Letters 210 (2002) 25^31 [5] Lewis, K. (2001) Riddle of bio¢lm resistance. Antimicrob. Agents Chemother. 45, 999^1007. [6] Prigent-Combaret, C., Vidal, O., Dorel, C. and Lejeune, P. (1999) Abiotic surface sensing and bio¢lm-dependent regulation of gene expression in Escherichia coli. J. Bacteriol. 181, 5993^6002. [7] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. [8] O’Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007^4021. [9] Rabilloud, T. (1992) A comparison between low background silver diammine and silver nitrate protein stains. Electrophoresis 13, 429^ 439. [10] Altschul, S.F., Madden, T.L., Scha¡er, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389^3402. [11] Vatanyoopaisarn, S., Nazli, A., Dodd, C.E., Rees, C.E. and Waites,
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W.M. (2000) E¡ect of £agella on initial attachment of Listeria monocytogenes to stainless steel. Appl. Environ. Microbiol. 66, 860^863. Antelmann, H., Scharf, C. and Hecker, M. (2000) Phosphate starvation-inducible proteins of Bacillus subtilis: proteomics and transcriptional analysis. J. Bacteriol. 182, 4478^4490. Drzewiecki, K., Eymann, C., Mittenhuber, G. and Hecker, M. (1998) The yvyD gene of Bacillus subtilis is under dual control of sigmaB and sigmaH. J. Bacteriol. 180, 6674^6680. Petersohn, A., Brigulla, M., Haas, S., Hoheisel, J.D., Volker, U. and Hecker, M. (2001) Global analysis of the general stress response of Bacillus subtilis. J. Bacteriol. 183, 5617^5631. Baca-DeLancey, R.R., South, M.M., Ding, X. and Rather, P.N. (1999) Escherichia coli genes regulated by cell-to-cell signaling. Proc. Natl. Acad. Sci. USA 96, 4610^4614. De Kievit, T.R., Gillis, R., Marx, S., Brown, C. and Iglewski, B.H. (2001) Quorum-sensing genes in Pseudomonas aeruginosa bio¢lms: their role and expression patterns. Appl. Environ. Microbiol. 67, 1865^1873.
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