Analysis of the Streptococcus agalactiae exoproteome

J O U RN A L OF P ROTE O M IC S 8 9 ( 2 01 3 ) 1 5 4 –16 4

Available online at www.sciencedirect.com

ScienceDirect www.elsevier.com/locate/jprot

Analysis of the Streptococcus agalactiae exoproteome Salvatore Papasergia , Roberta Galbob,⁎, Veronica Lanza-Cariccioa , Maria Dominaa , Giacomo Signorinoa , Carmelo Biondoa , Ida Perniceb , Claire Poyartc , Patrick Trieu-Cuotd , Giuseppe Tetia , Concetta Beninatia a

Metchnikoff Laboratory, University of Messina, Messina I-98125, Italy Dipartimento di Scienze Biologiche e Ambientali, University of Messina, Messina I-98125, Italy c Institut Cochin, Université Paris Descartes Faculté de Médecine, CNRS, 75014 Paris, France d Institut Pasteur, Unité de Biologie des Bactéries Pathogènes à Gram Positif, CNRS ERL3526, 75015 Paris, France b

AR TIC LE I N FO

ABS TR ACT

Article history:

The two-component regulatory system CovRS is the main regulator of virulence gene

Received 16 April 2013

expression in Group B Streptococcus (GBS), the leading cause of invasive infections in

Accepted 2 June 2013

neonates. In this study we analyzed by mass spectrometry the GBS extracellular protein

Available online 14 June 2013

complex (i.e. the exoproteome) of NEM316 wild-type (WT) strain and its isogenic covRS deletion mutant (ΔcovRS). A total of 53 proteins, 49 of which had classical secretion signals,

Keywords:

were identified: 12 were released by both strains while 21 and 20 were released exclusively

Bacterial infections

by WT and ΔcovRS strains, respectively. In addition to known surface proteins, we detected

Proteomics

here unstudied cell-wall associated proteins and/or orthologs of putative virulence factors

Mass spectrometry

present in other pathogenic streptococci. While the functional role of these proteins

Virulence factors

remains to be elucidated, our data suggest that the analysis of the exoproteome of bacterial pathogens under different gene expression conditions may be a powerful tool for the rapid identification of novel virulence factors and vaccine candidates. Biological significance We believe that this manuscript will be of interest to Journal of Proteomics readers since the paper describes the identification of several putative virulence factors and vaccine candidates of the group B streptococcus, an important pathogen, using a simple proteomics strategy involving LC–MS analysis of culture supernatants obtained from two strains with divergent gene expression patterns. This technique provided the most comprehensive inventory of extracellular proteins obtained from a single streptococcal species thus far. The approach described has the added benefit of being easily applicable to a large number of different strains, making it ideal for the identification of conserved vaccine candidates. © 2013 Elsevier B.V. All rights reserved.

1.

Introduction

Group B Streptococcus (GBS), or Streptococcus agalactiae, is a common colonizer of the lower gastrointestinal tract and vaginal genital mucosa of humans [1]. However, under certain circumstances, GBS can invade mucosal barriers and cause

systemic infections, including sepsis and meningitis. This is exemplified by the frequent occurrence of GBS disease in human newborns (approximately 0.8 cases per 1000 live births) [2] and in elderly adults (approximately 25.4 cases per 100,000 population) [3,4]. Moreover, the increasing emergence of antibiotic-resistant isolates raises additional concern.

⁎ Corresponding author at: Torre Biologica IIp, Policlinico, Via Consolare Valeria, 1, 98125 Messina, Italy. Tel.: +39 090 221 3310; fax: +39 090 221 3312. E-mail address: [email protected] (R. Galbo). 1874-3919/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jprot.2013.06.003

J O U RN A L OF P ROT EO M IC S 8 9 ( 2 01 3 ) 1 5 4 –1 64

Thus, GBS is as a Janus-faced organism and a major health problem for which additional prophylactic and therapeutic strategies are needed. Well-characterized GBS virulence factors include the polysialic acid capsule, β-hemolysin/cytolysin (β-H/C), pili and other surface proteins that mediate binding to host cells, extracellular matrix, and blood components [5]. In order to cause infection, GBS must adapt to the changing microenvironments associated with different host niches. Indeed, as clearly shown in the case of Salmonella [6], inappropriate or constitutive expression of virulence factors can impair the ability of a pathogen to persist in the host. Therefore, GBS survival is highly dependent on fine-tuning gene regulation during infection to correctly express virulence factors in response to distinct host microenvironmental conditions [7]. Gene expression in GBS NEM316 is controlled by 6 stand-alone regulators and by 20 two-component regulatory systems (TCS), which are frequently involved in the sensing of environmental stimuli. The TCS CovRS (also known as CsrRS) is shared by several streptococcal species (including Group A Streptococcus or GAS) and is considered as the master regulator of virulence gene expression in GBS [8,9]. Accordingly, the CovRS system regulates up to 7% of the GBS genes, most of which encode putative secreted or surface components, including several small molecule transport systems, cytolysins, and adhesins [7,10]. However, whereas CsrRS mediates the conversion of GAS from a colonizing to an invasive phenotype in response to signaling by host LL-37 [11], the GBS CovRS controls the response to acidic stress and is required for intracellular bacterial survival in macrophages [12]. Similar to surface antigens, extracellular released proteins are important in pathogenesis, due to their early interaction with host cells and their ability to stimulate immune responses. Studies have focused on the identification of GBS surface antigens by proteomics [13,14], but a comprehensive analysis of GBS extracellular protein complex (i.e. the exoproteome) has not been performed thus far. In the present study, we compared the exoproteomes of GBS NEM316 WT and its covRS deletion mutant (ΔcovRS). Our results revealed that deletion of this TCS results in dramatic qualitative changes in the protein secretion pattern, particularly in the release of known or putative virulence factors. These data may be of interest to better understand the complex mechanism by which GBS adapt its physiology to host microenvironments and for the identification of novel candidate vaccines.

2.

Materials and methods

2.1.

Strains and culture conditions

GBS strain NEM316, capsular serotype III (ST 23), originally isolated from a neonatal blood culture, and its derivative mutant ΔcovRS [8], were grown in Todd Hewitt Broth (THB) or in Carey's chemically defined medium (CCDM; [15]) at 37 °C with 5% CO2.

2.2.

Protein isolation and electrophoresis

To collect proteins present in culture supernatants, NEM316 WT and ΔcovRS mutant strains were grown in CCDM (200 ml) until the late exponential phase and cells were separated from

155

the medium by centrifugation at 10,000 ×g for 5 min at 4 °C. The supernatants were then filtered using 0.22-μm pore size filters (Millipore) to remove residual bacterial cells and kept frozen (−80 °C) until use. A sodium deoxycholate-trichloro-acetic acid (DOC–TCA) precipitation method was used for precipitating proteins from bacterial culture supernatants. Briefly, DOC was added to reach a final concentration of 0.03%, followed by incubation at room temperature for 5 min. Subsequently, TCA was added to a final concentration of 7.5% and, after 1 h, the pellets were collected by centrifugation at 10,000 ×g at 4 °C for 30 min, washed twice with ice-cold acetone, air dried and kept frozen (−80 °C) until use. Protein yield was determined on solubilized samples by the Bradford method using bovine serum albumin as a standard (Protein Assay, Biorad). For sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, protein pellets were resuspended to the desired concentration with solubilization buffer [16] prior to PAGE using 12% acrylamide gels. Proteins in gels were silver stained with Silver Stain Plus (Biorad), following manufacturer's instructions.

2.3.

Protein identification by nano-LC/MS/MS

MS was used to analyze bacterial culture supernatants obtained as described above. After DOC–TCA precipitation, protein pellets obtained from culture supernatants were solubilized with 0.1% RapiGest SF (Waters) and reduced with 5 mM dithiothreitol for 10 min at 100 °C. The pH was adjusted to 8.0 using ammonium bicarbonate (50 mM, pH 8.5) before digestion with trypsin for 20 h at 37 °C at a trypsin-to-protein ratio of 1/20 (wt/wt). The digestion reaction was stopped with 0.1% formic acid. Before analysis, peptide mixtures were desalted with OASIS HLB cartridges (Waters) following the manufacturer's protocol. Desalted peptides were concentrated with a vacuum concentrator (Eppendorf) and kept at −20 °C until further analysis. In selected experiments, culture supernatant proteins were identified by MS after excising bands from SDS-PAGE gels. Major bands were sliced from the gel, minced into small pieces and treated as described by Shevchenko et al. [17]. Briefly, destained gel pieces were treated with digestion buffer (20 mM ammonium bicarbonate pH 8.5, containing 12.5 μg/ml trypsin for 60 min in ice), and incubated at 37 °C for 16 h. The generated peptides were extracted twice with 1% formic acid/acetonitrile (1:2) at 37 °C for 30 min, concentrated with a vacuum concentrator (Eppendorf) and kept at −20 °C until further analysis. Products obtained by tryptic digestion of extract from gel slices or from whole culture supernatant precipitates were separated by nano-LC on a NanoAcquity UPLC System (Waters) connected to an Electron Spray Ionization (ESI) mass spectrometer equipped with a nanospray source (Q-ToF Micro, Waters). Samples were loaded onto a Nano-Acquity 1.7 μm BEH130 C18 column (100 μm i.d. × 100 mm; Waters) through a NanoAcquity 5-μm Symmetry C18 trap column (180 μm i.d. × 20 mm; Waters). Peptides were eluted with a 60-min gradient of 2–50% acetonitrile in 0.1% formic acid at a flow rate of 400 nl/min. The eluted peptides were subjected to automated data-dependent acquisition using the MassLynx software, version 4.1 (Waters), while an MS survey scan was used to automatically select multicharged peptides over the m/z ratio range of 350–1500 for further MS/MS fragmentation. Up to three different

156

J O U RN A L OF P ROTE O M IC S 8 9 ( 2 01 3 ) 1 5 4 –16 4

components were individually subjected to MS/MS fragmentation following each MS survey scan. After data acquisition, individual MS/MS spectra were combined, smoothed, and centroided using ProteinLynx version 3.5 (Waters) to obtain the peak list file. Search and identification of peptides were performed in batch mode with Mascot (http://www. matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH= PMF) using the NCBI database (http://www.ncbi.nlm.nih.gov/). The Mascot search parameters were set as follows: (i) 2 as the number of allowed missed cleavages, (ii) methionine oxidation and glutamine–asparagine deamination as the variable modifications, (iii) 50 ppm as the peptide tolerance, (iv) 0.3 Da as the MS/MS tolerance and (v) +2, and +3 as the peptide charges. Only significant hits, as defined by the Mascot probability analysis, were considered.

2.4.

Bioinformatics analysis

The identified proteins were analyzed using different on line servers. LocateP (http://www.cmbi.ru.nl/locatep-db/cgi-bin/ locatepdb.py) and SMART (http://smart.embl-heidelberg.de/) were employed for the prediction of, respectively, cellular localization and domain architecture. The identification of leader peptides was performed using signalP (http://www.cbs. dtu.dk/services/SignalP/) and comparative sequence analysis using the BLAST engine of the NCBI server. The presence of repeats and conserved domains was verified with, respectively, SMART (http://smart.embl-heidelberg.de/) and Pfam (http:// pfam.sanger.ac.uk/) softwares.

2.5.

Western blot analysis

Cloning, expression and purification of recombinant gbs0428 and gbs0791 proteins were performed as previously described [18]. Briefly, chromosomal DNA of GBS strain NEM316 was used as a template for PCR amplification of gbs0428 and gbs0791 sequences. Primers (Table S1) were selected as to avoid amplification of secretory leader peptide and the cell-wall anchor sequences. Amplicons were cloned into the bacterial expression vector pGEX-SN that allows the expression of recombinant proteins as fusions to glutathione S-transferase (GST) [19]. GST fusion proteins were expressed in Escherichia coli strain AD202

Fig. 1 – Growth curves of the WT NEM316 strain and covRS deletion mutant in Carey's chemically defined medium (CCDM). Samples for electrophoresis and LC/MS/MS analysis were collected at the time points shown by arrows.

and purified by affinity chromatography [18]. Recombinant GST protein, used as a control, was also produced using the same procedures. Sera were collected from mice immunized as previously described [20]. Recombinant BibA, PilB and Bsp and the corresponding specific rabbit antisera were obtained as previously described [21]. Western blots were performed, as previously described [20], by reacting specific murine or rabbit antisera against SDS-PAGE-separated culture supernatant proteins.

3.

Results and discussion

3.1. Deletion of the CovRS system results in major changes in the exoproteome To gain a comprehensive view of proteins secreted by GBS under varying gene expression conditions, we compared the exoproteome of the WT strain NEM316 with that of its isogenic mutant derivative ΔcovRS bearing a deletion encompassing both the covR and covS genes [8] which constitute the master regulation system of virulence gene expression. In agreement with previous studies [8], we found that the covRS deletion abrogated GBS ability to grow in RPMI, a minimal chemically defined growth medium, but did not affect its ability to grow in THB (data not shown). This complex infusion medium, however, was considered less-than-ideal for LC–MS/MS analysis of supernatants, since it was anticipated that the heterologous proteins present at high concentrations in THB might interfere with the detection of GBS products. For this reason, the mutant was tentatively cultivated in CCDM, an enriched, chemically defined and dialyzable medium [15]. Fig. 1 shows that the ΔcovRS strain grew well in CCDM, although the final OD value was lower relative to that obtained with the WT strain. Supernatants collected during late exponential phase of growth (Fig. 1) contained similar amounts of proteins (1.1 and 1.3 μg/ml in non-precipitated supernatants for WT and ΔcovRS strains, respectively), but exhibited divergent banding patterns by SDS-PAGE analysis (Fig. 2). After excising selected bands from these gels, LC–MS/MS analysis of eluted material confirmed the presence of different protein species in ΔcovRS vs WT supernatants (Fig. 2). Therefore, in further experiments whole supernatants were directly subjected to LC–MS/MS analysis after precipitation and trypsin treatment without previous electrophoretic separation. A total of 53 proteins were detected in three separate experiments, each conducted using different supernatant samples. Cumulative results from these experiments are reported in Tables 1–3. Peptide identification is reported for each experiment in Tables S2–S4. Twelve proteins were detected in the supernatants of both NEM316 WT and ΔcovRS strains (Table 1), while 21 and 20 were detected exclusively in the supernatants of WT (Table 2) and ΔcovRS strains (Table 3), respectively. Based on sequence prediction analysis, proteins were grouped in four cellular compartment categories: 1) extracellularly released proteins, indicated henceforth as “secreted” and comprising those possessing an N-terminus secretory signal peptide with no anchor sequence; 2) cell wall-bound proteins possessing a signal peptide and an LPXTG motif or a LysM anchor domain; 3) membrane-bound proteins possessing a

J O U RN A L OF P ROT EO M IC S 8 9 ( 2 01 3 ) 1 5 4 –1 64

157

3.3. Proteins present exclusively in the WT strain exoproteome

Fig. 2 – Comparative SDS-PAGE analysis of culture supernatant proteins from the WT NEM316 strain and its covRS deletion mutant. Precipitates from culture supernatants were analyzed by SDS-PAGE followed by silver stain. In equivalent gels, bands were excised as indicated and subjected to trypsin treatment followed by protein identification by LC/MS/MS.

leader peptide and a transmembrane or lipid anchor domain; and 4) cytoplasmic or moonlighting proteins devoid of signal peptides and of surface anchor domains. The latter group also included non-canonical surface antigens [22], which are separately listed in Tables 2 and 3. All identified proteins are shown in Fig. 3 as percentages relative to the total number of proteins predicted to be encoded by NEM316 genome.

3.2.

Proteins present in the exoproteome of both strains

Three predicted secreted, 6 cell wall-anchored, 2 membranebound, and 1 cytoplasmic proteins were detected in this group (Table 1). Predicted secreted products included proteins likely to be involved in cell surface physiology, such as PcsB, which is required for correct cell wall separation [23]. CAMP factor was also found in the supernatants of both strains, although a considerably higher number of spectra matching CAMP factor peptides was found in WT supernatants (61 versus 3 spectra; Tables S2–S4), suggesting that this protein was more abundantly expressed by the WT strain, as compared to the ΔcovRS mutant. This is in agreement with the notion that CAMP factor gene expression is downregulated in the absence of CovRS [8]. The cell wall-associated antigens found in this group comprised 2 proteins with the LysM peptidoglycan interaction domain (including Sip, a well-characterized protective antigen [24]) and 4 proteins with an LPXTG peptidoglycan binding motif. Two of these were represented by the backbone and ancillary protein 1 subunits of the type PI-2a pilus [25]. The other 2 LPXTG antigens were a hypothetical nucleotidase [26] and a Rib-like protein [27,28]. Predicted membrane associated proteins included gbs1838, displaying an ABC amino-acid transporter domain [29], and gbs1279, displaying similarity with members of the C39A subfamily of peptidases.

Five predicted secreted proteins with unknown function were found in this group (Table 2). Three cell wall-anchored proteins with an LPXTG motif were also identified: 1) PI-2a pilus ancillary protein PilC (gbs1474); 2) gbs1929, a putative bifunctional cyclic phosphodiesterase–nucleotidase precursor whose gene transcripts are upregulated during growth at 40 °C [30] or in amniotic fluid [31]; and 3) gbs1539, a hypothetical protein with unknown function. Twelve proteins of this group displayed the characteristics of membrane anchored/associated antigens, including hyaluronate lyase [32] and two penicillin-binding proteins [33,34]. The cytoplasmic glyceraldehyde 3-phosphate dehydrogenases (GAPDH) were found exclusively in WT supernatants. This moonlighting enzyme is released upon cell lysis, can induce apoptosis in murine macrophages [35] and is involved in binding to the host extracellular matrix [36,37]. GAPDH expression is upregulated during growth in human blood [38].

3.4. Proteins present exclusively in the ΔcovRS mutant exoproteome Twenty out of the 53 identified proteins were detected exclusively in the exoproteome of the ΔcovRS mutant (Table 3). Proteins predicted to be extracellularly released by bioinformatics analysis comprised 6 antigens, 2 of which (gbs0419 and gbs0661) were orthologs of virulence factors identified in other streptococcal species. In fact, gbs0661 was found to share partial homology with endA, a surface nuclease of pneumococci that promotes bacterial spreading from the lungs into the bloodstream after escape from extracellular neutrophil DNA traps [39]. Moreover, gbs0419 was found to belong to the GDXG family of lipolytic enzymes and to share more than the 90% homology with GAS secreted esterase (Sse), an important virulence factor in this species [40,41]. The five cell wall-anchored proteins found exclusively in ΔcovRS supernatants all displayed an LPXTG motif and included: 1) BibA (gbs2018), an adherence and anti-phagocytic factor with immuno-protective activity [42]; 2) C5a peptidase (gbs0451), which inactivates the C5a complement factor and may also act as an adhesin; 3) FbsA (gbs1087), the major fibrinogen binding protein of GBS and an important virulence factor [43,44]; 4) gbs0428, a hypothetical protein displaying two SSURE domains homologous with those of the recently characterized plasminogen and fibronectin binding protein B (PfbB) of Streptococcus pneumoniae [45]; and 5) gbs0791, a hypothetical protein with two tandem repeats. Six predicted membrane proteins were found exclusively in ΔcovRS supernatants, among which gbs0155, the penicillin-binding protein 1B, involved in peptidoglycan biosynthesis. The only non canonical surface protein detected in this group was the 30S Ribosomal protein S8 (gbs0072) [46].

3.5. Western blot validation of LC–MS/MS protein identification In further experiments we sought to further validate the above-described LC–MS/MS method for protein identification in bacterial culture supernatants. To this end, 5 out of the 53

158

J O U RN A L OF P ROTE O M IC S 8 9 ( 2 01 3 ) 1 5 4 –16 4

Table 1 – Proteins detected in both WT and ΔcovRS supernatants. Cell localization and NEM316 locus

Secreted gbs2000

gbs0016 gbs1727

Gene product name or function

CAMP factor

Cell wall separation protein PcsB Hypothetical protein

Cell-wall anchored gbs0031 Group B streptococcal surface immunogenic protein (Sip) gbs2107 LysM domain hypothetical protein gbs1478 PilA

ΔcovRS

WT

Comments

No. of unique peptides identified

Coverage %

No. of unique peptides identified

Coverage %

9

54

3

18

13

70

8

44

15

52

13

45

17

86

15

76

Protective antigen and vaccine candidate with a LysM domain [24]

6

50

6

50

5

7

5

7

9

20

9

20

Highly conserved among Streptococcus species Adhesin subunit of PI-2a pilus [25]. Upregulated in ΔcovRS mutant relative to the WT parental strain NEM316 [55] Major subunit of PI-2a pilus [25]. Upregulated in ΔcovRS mutant relative to the WT parental strain NEM316 [55] Belongs to the Rib/alpha group of immune-protective GBS proteins [27,28] Ortholog of Staphylococcus aureus adenosine synthetase (Adsa) [53]

gbs1477

PilB

gbs0470

Hypothetical protein

15

18

4

5

gbs1403

YhcR-like metallophosphatase domain, hypothetical protein

4

10

5

13

Membrane gbs1838

2

5

1

3

gbs1279

8

21

10

27

2

3

1

5

Cytoplasmic gbs0293

Hypothetical protein

proteins identified were recombinantly expressed and used to immunize mice or rabbits. Specific antisera were then used to detect the presence of the corresponding proteins in culture supernatants by Western blot analysis (Fig. 4). Sera raised against proteins gbs0428, gbs0791 or BibA produced reactive bands only in the ΔcovRS, but not in the WT supernatant lane, in agreement with LC–MS/MS results. Moreover, serum raised

Pore-forming extracellular hemolysin that synergizes with staphylococcal beta-lysin Required for correct cell wall separation and cell wall homeostasis [23] Hypothetical protein with a C-terminal CHAP domain (cysteine, histidinedependent amido-hydrolases/peptidases) frequently found in N-acetylmuramoyl-L-alanine amidases involved in cell wall metabolism [58]. Annotated as “immunogenic secreted protein” in the NEM316 genome because of homology with the homonymous GAS protein [59]

Protein with an ABC amino-acid transporter domain [29] Protein with clostridial hydrophobic tryptophan (ChW) repeats and a C-terminal C39 peptidase domain, which is common in the C39A subfamily of peptidases. Displays a Bsp-like repeat [60]

No match with characterized proteins in databases

against PilB (which was detected by LC–MS/MS analysis in both WT and ΔcovRS supernatants) produced bands in both lanes. Anti-Bsp serum reacted more intensely against WT, compared with ΔcovRS, supernatants in agreement with LC–MS/MS identification of Bsp in WT supernatants only. Therefore, LC– MS/MS findings were confirmed by Western blot analysis even in the case of proteins, such as gbs0428 or gbs0791, for which

159

J O U RN A L OF P ROT EO M IC S 8 9 ( 2 01 3 ) 1 5 4 –1 64

Table 2 – Proteins detected exclusively in WT supernatants. Cell localization and NEM316 locus Secreted gbs1556

Gene product name or function

No. total Coverage unique % peptides identified

gbs1420

Transglutaminase-like domain hypothetical protein Bsp-like hypothetical protein

21

70

10

24

gbs1659

Putative amino acid ABC transporter

3

14

gbs1586

3

14

gbs1061

Putative peptydyl-prolyl cistransisomerase cyclophilin type chaperone protein Hypothetical protein

1

17

Cell-wall anchored gbs1474

PilC

3

14

gbs1929

2′-Phosphodiesterase/3′-nucleotidase

3

7

gbs1539

Hypothetical protein

1

6

Membrane gbs1270

Hyaluronate lyase

1

2

gbs1073

Putative phage infection protein

13

23

gbs1299

1

6

gbs1431

Phage superinfection exclusion family hypothetical protein Putative RND export transporter

7

27

gbs0785

Penicillin-binding protein 2B

2

6

gbs0277

Penicillin-binding protein 2×

1

2

gbs1829

Conserved hypothetical protein

1

10

gbs0903

Hypothetical YbbR-like protein

2

10

gbs1194

Hypothetical protein

2

10

gbs2022 gbs0942 gbs0778

Hypothetical protein Lipoprotein Hypothetical lipoprotein

2 1 1

13 3 14

1

6

Non canonical surface gbs1811 Glyceraldehyde-3-phoshate dehydrogenase

one peptide only was detected by LC–MS/MS (confront Fig. 4 and Table 3). To colonize host surfaces or to disseminate inside the body, a pathogen must physically associate with host tissues, obtain nutrients essential for growth and evade the immune system. To accomplish these tasks, bacteria should export proteins through the cell membrane that subsequently localize to the membrane itself, to the cell wall or are directly released in the extracellular milieu. In order to identify proteins exported under different gene expression conditions, we optimized here a method (i.e. liquid chromatography-ESI-Q-TOF mass spectrometry analysis of a dialyzable medium used for bacterial growth)

Comments

Hypothetical coiled coil protein displaying a transglutaminase domain Contains 4 bacterial SH3 (src homology-3) domains [61] and one Bsp-like domain [60] Homologous to polar amino acid ABC transporters of the PBPb superfamily [62] Members of this chaperone family catalyze correct folding of various proteins types [63] No match with characterized proteins in databases

Subunit of PI-2a pilus. Found to be upregulated in ΔcovRS relative to the WT parental strain NEM316 [55] Gene transcripts are upregulated during growth at 40 °C [30] or in amniotic fluid [31] Protein with unknown function

Virulence factor involved in extracellular matrix degradation [32] Hypothetical protein homologous to phage infection protein family members Hypothetical lipoprotein, homologous to proteins affecting phage replication Protein with a 350 aa long region showing homology with the membrane fusion subunits of the resistance–nodulation–division (RDN) export transporter superfamily proteins Penicillin-binding protein, probably implicated in cell wall polymerization [33,34] Penicillin-binding protein, probably implicated in cell wall polymerization [33,34] Displays a domain conserved among Streptococcus and Listeria spp. Displays a domain conserved among Streptococcus and Listeria spp. Hypothetical protein homologous with Bacillus subtilis Ybbr protein [64] No match with characterized proteins in databases No match with characterized proteins in databases No match with characterized proteins in databases

Involved in binding to the host extracellular matrix [36,37]. Gene transcripts are upregulated during growth in human blood [38]

that allowed fast and sensitive characterization of the GBS exoproteome. We believe this method is advantageous over classical proteomics approaches based on two-dimensional gel electrophoresis, which have been recently applied to bacterial exoproteome analysis [47–52]. The use of high-resolution nanoLC avoids the complexities, labor and long analysis time inherent in gel fractionation and subsequent steps. These features make the method described here, and similar methods, ideal to rapidly compare different strains for exoproteome expression or to examine the same strain under different physiological conditions. Moreover, LC–MS is apparently more sensitive than traditional two-dimensional gel fractionation

160

J O U RN A L OF P ROTE O M IC S 8 9 ( 2 01 3 ) 1 5 4 –16 4

Table 3 – Proteins detected exclusively in ΔcovRS supernatants. Cell localization and NEM316 locus Secreted gbs0827 gbs1895 gbs0358 gbs0661 gbs0419 gbs0951

Gene product name or function

Foldase protein PrsA Transglutaminase domain hypothetical protein Hypothetical protein Putative DNA-entry nuclease GDXG lipolytic enzyme family protein Hypothetical protein, conserved among streptococci

No. of unique peptides identified

Coverage %

Comments

1

3

24

66

6 2

25 16

2

8

5

29

Homologous to chaperone-like peptidyl-prolyl-cistrans-isomerases [65] Displays a CYKS motif, in addition to a transglutaminase/protease-like domain No match with characterized proteins in databases Shows partial homology with pneumococcal surface nuclease endA [39] Putative esterase, homologous to group A streptococcal Sse [40,41] Uncharacterized protein that is conserved among Streptococcus ssp.

22

51

Cell-wall anchored gbs2018

BibA

gbs0451

C5a peptidase

3

4

gbs1087

FbsA

4

10

gbs0791

Hypothetical protein

1

2

gbs0428

Hypothetical protein with SSURE domains

1

4

Membrane gbs0851

Hypothetical protein

1

13

gbs1606 gbs0155 gbs2106 gbs0687

Hypothetical protein Penicillin binding protein 1B Hypothetical protein Putative metallopeptidase

1 1 1 1

2 2 17 7

gbs0255

Hypothetical protein

1

8

Non canonical surface gbs0072 30S ribosomal protein S8

1

Cytoplasmic gbs0839

Phosphocarrier protein HPr

1

12

gbs0332

Acyl carrier protein (ACP)

1

14

proteomic profiling. Thus far, LC/MS/MS analysis of gel-free supernatants from gram positive bacteria has been performed only for Clostridium spp. [48]. We provide in the present study a comprehensive profile of the GBS exoproteome, defined here as including two sets of components: 1) proteins containing peptide sequences that signal for active exportation through the membrane and are secreted directly in the extracellular milieu or remain exposed on the bacterial surface before being eventually released; 2) proteins that are devoid of secretion signals and are released by noncanonical pathways. The vast majority (92.6%) of the proteins identified here belonged to the first category. This suggests that GBS mostly use classical secretion pathways for protein export. Moreover, this data suggest that our extracellular preparations had minimal or no cytoplasmic contamination, a factor that

Adherence and anti-phagocytic factor with immuno-protective activity [42] Peptidase, which inactivates the C5a complement factor and may also act as an adhesin Major human fibrinogen binding protein and virulence factor [43,44] Displays two tandem bacterial immunoglobulin-like repeats Displays two SSURE domains homologous with those of pneumococcal PfbB [45]; gene transcripts are upregulated upon exposure to human blood [30]

No match with characterized proteins in databases; it is encoded by the gene immediately adjacent to FbsB No match with characterized proteins in databases Probably involved in peptidoglycan biosynthesis Protein displaying a lysozyme-like domain Protein displaying two consecutive PepSY (peptidase propeptide and YPEB) domains [66] No match with characterized proteins in databases

Protein proposed to have dual (cytoplasmic and surface) localization in bacterial cells [46]

Phosphocarrier protein (HPr) may be involved in the internalization and the phosphorylation of an array of carbohydrates ACP participates in fatty acid biosynthesis

often prevents accurate qualitative and quantitative proteomic characterization. To our knowledge, our data present the most comprehensive inventory of experimentally confirmed extracellular proteins from any streptococcal species. At variance with extracellular proteins, surface proteins of Group A Streptococci and GBS have received considerable attention. The “surfome” of GBS has been essentially analyzed by: 1) “brute force” confirmation of surface localization of all predicted surface proteins using immunofluorescence [24]; and 2) protease “shaving” of whole cells followed by MS/MS analysis of proteolytic peptides [13]. Our data indicate that exoproteome analysis may usefully complement the above techniques in the identification of novel virulence factors and vaccine candidates. Notably, the GBS exoproteome comprised, in addition to proteins known or

J O U RN A L OF P ROT EO M IC S 8 9 ( 2 01 3 ) 1 5 4 –1 64

161

Fig. 3 – Overview of proteins identified by LC/MS/MS in the present study. Identified (light gray) and unidentified (dark gray) proteins were grouped into topological groups using locateP database. Numbers and percentages refer to proteins predicted to be encoded in the NEM316 genome. expected to be extracellularly secreted, a large proportion of predicted surface-associated proteins that were likely released by GBS in the culture medium after remaining anchored for variable periods of time to the membrane or the cell wall. The vast majority of previously described immunoprotective antigens and/or virulence factors of GBS were detected in the exoproteome. These included three different pilus components, protein Sip, C5a peptidase, Rib, hyaluronate lyase, β-hemolysin, CAMP factor, fibrinogen binding protein FbsA, BibA and three penicillin binding proteins. Since exoproteome analysis of a single strain requires little time and labor, this method seems ideally suited to analyze the degree of expression and conservation of vaccine candidates in a large number of clinical isolates. This may allow the rapid screening and prioritization of candidates to select those with the highest probability of being immunoprotective against a wide number of pathogenic strains. Importantly, in addition to known antigens, we were able to experimentally validate in the present study a number of hypothetical culture supernatant proteins that were predicted to translocate across the cell membrane. Some of these putative proteins were orthologs of virulence factors described in other streptococci. In addition, gbs1403 might be the ortholog of Staphylococcus aureus adenosine synthetase (Adsa), also an important virulence factor [53]. Other putative proteins validated here showed no homology with sequences present in database. It will be of interest to ascertain the functional role of these proteins as virulence factors and immunoprotective agents. In this study, we also examined the effects of the deletion of the CovRS TCS, a major regulatory system activated by environmental changes, on the GBS exoproteome. Although the effects of this system on gene expression have been previously examined in depth through “transcriptome” analysis, we considered it important to experimentally verify the

Fig. 4 – Western blot analysis of culture supernatants from WT and ΔcovRS strains. Proteins were separated by SDS-PAGE and blots were overlaid with the indicated mouse (A) or rabbit (B) polyclonal antibodies. The arrows indicate the position of the respective proteins.

presence of the expected gene products at the protein level and/or in their predicted subcellular location. Our data confirm and extend those of previous transcriptome studies [8,54]. Firstly, we observed dramatic qualitative changes in protein secretion in the absence of the CovRS TCS, as 77% of all detected proteins were present exclusively in the WT or in the ΔcovRS mutant. Our data suggest that covRS selectively regulates the expression of secreted proteins, since only 7% of NEM316 genes are regulated by this system at the transcriptional level. Interestingly, we found that, in addition to gene products previously known to be regulated by CovRS, this system may indirectly affect the expression of a number of hypothetical proteins whose genes are not considered as belonging to the covRS regulon. Among these, we found, for example, the putative cell wall-anchored cyclic phosphodiesterase gbs1929 and the putative secreted Bps-like protein gbs1420, which were detected in WT but not in ΔcovRS supernatants. Conversely, the putative secreted proteins gbs0661, gbs0419, and gbs0951 were detected exclusively in ΔcovRS supernatants. PI-2a pilus subunits were recently found to be under the positive control of the Rga transcriptional regulator, which, in

162

J O U RN A L OF P ROTE O M IC S 8 9 ( 2 01 3 ) 1 5 4 –16 4

turn, is downregulated by the CovRS TCS [55]. Accordingly, gbs1478, gbs1477, and gbs1474 encoding the PI-2a subunits PilA, PilB, and PilC, respectively, were found to be upregulated in ΔcovRS relative to the WT parental strain NEM316 [55]. However, in the present study, PilA and PilB were detected at a similar level in both strains. This suggests that the release of pilus subunit fragments in culture supernatants may be affected by factors other than pilus gene expression levels, including SrtA-mediated cell wall anchoring versus SrtCmediated polymerization. In addition, the release in the growth medium of peptides from pilus subunits, which are known to be particularly protease-resistant [56,57], may be more affected by variations in surface or extracellular protease activity than by other factors.

4.

Conclusions

We provide here a comprehensive profile of GBS exoproteome. It was found that the deletion of the CovRS regulation system results in dramatic qualitative changes in protein secretion, particularly in the release or known or putative virulence factors. Because of its simplicity, the technique used here, involving nanoLC/MS/MS analysis of culture supernatants, seems ideally suited to test a large number of bacterial strains under similar or different physiological conditions. This may be useful for the rapid identification of effective vaccine candidates and to better understand the complex mechanism by which bacterial pathogens adapt their physiology to different host microenvironments. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2013.06.003.

Acknowledgments This study was supported by grants Progetti di Ricerca di Rilevante Interesse nazionale 2005 (DM n° 287/2005) and 2008 (DM n° 1407/2008) from the Ministero dell'Istruzione, dell'Università e della Ricerca of Italy.

REFERENCES

[1] Hansen SM, Uldbjerg N, Kilian M, Sorensen UB. Dynamics of Streptococcus agalactiae colonization in women during and after pregnancy and in their infants. J Clin Microbiol 2004;42: 83–9. [2] Schrag SJ, Stoll BJ. Early-onset neonatal sepsis in the era of widespread intrapartum chemoprophylaxis. Pediatr Infect Dis J 2006;25:939–40. [3] Edwards MS, Baker CJ. Group B streptococcal infections in elderly adults. Clin Infect Dis 2005;41:839–47. [4] Dermer P, Lee C, Eggert J, Few B. A history of neonatal group B Streptococcus with its related morbidity and mortality rates in the United States. J Pediatr Nurs 2004;19:357–63. [5] Rajagopal L. Understanding the regulation of Group B streptococcal virulence factors. Future Microbiol 2009;4:201–21. [6] Mouslim C, Delgado M, Groisman EA. Activation of the RcsC/YojN/RcsB phosphorelay system attenuates Salmonella virulence. Mol Microbiol 2004;54:386–95.

[7] Maisey HC, Doran KS, Nizet V. Recent advances in understanding the molecular basis of group B Streptococcus virulence. Expert Rev Mol Med 2008;10:e27. [8] Lamy MC, Zouine M, Fert J, Vergassola M, Couve E, Pellegrini E, et al. CovS/CovR of group B streptococcus: a two-component global regulatory system involved in virulence. Mol Microbiol 2004;54:1250–68. [9] Santi I, Grifantini R, Jiang SM, Brettoni C, Grandi G, Wessels MR, et al. CsrRS regulates group B Streptococcus virulence gene expression in response to environmental pH: a new perspective on vaccine development. J Bacteriol 2009;191:5387–97. [10] Jiang SM, Ishmael N, Dunning Hotopp J, Puliti M, Tissi L, Kumar N, et al. Variation in the group B Streptococcus CsrRS regulon and effects on pathogenicity. J Bacteriol 2008;190: 1956–65. [11] Tran-Winkler HJ, Love JF, Gryllos I, Wessels MR. Signal transduction through CsrRS confers an invasive phenotype in group A Streptococcus. PLoS Pathog 2011;7:e1002361. [12] Cumley NJ, Smith LM, Anthony M, May RC. The CovS/CovR acid response regulator is required for intracellular survival of group B Streptococcus in macrophages. Infect Immun 2012;80:1650–61. [13] Doro F, Liberatori S, Rodriguez-Ortega MJ, Rinaudo CD, Rosini R, Mora M, et al. Surfome analysis as a fast track to vaccine discovery: identification of a novel protective antigen for Group B Streptococcus hypervirulent strain COH1. Mol Cell Proteomics 2009;8:1728–37. [14] Johri AK, Margarit I, Broenstrup M, Brettoni C, Hua L, Gygi SP, et al. Transcriptional and proteomic profiles of group B Streptococcus type V reveal potential adherence proteins associated with high-level invasion. Infect Immun 2007;75: 1473–83. [15] Carey RB, Eisenstein TK, Shockman GD, Greber TF, Swenson RM. Soluble group- and type-specific antigens from type III group B Streptococcus. Infect Immun 1980;28:195–203. [16] Sambrook J, Russell DW. The condensed protocols from molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; 2006 [17] Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 2006;1:2856–60. [18] Cardaci A, Papasergi S, Midiri A, Mancuso G, Domina M, Cariccio VL, et al. Protective activity of Streptococcus pneumoniae Spr 1875 protein fragments identified using a phage displayed genomic library. PLoS One 2012;7:e36588. [19] Beghetto E, Gargano N, Ricci S, Garufi G, Peppoloni S, Montagnani F, et al. Discovery of novel Streptococcus pneumoniae antigens by screening a whole-genome lambda-display library. FEMS Microbiol Lett 2006;262: 14–21. [20] Mandanici F, Gomez-Gascon L, Garibaldi M, Olaya-Abril A, Luque I, Tarradas C, et al. A surface protein of Streptococcus suis serotype 2 identified by proteomics protects mice against infection. J Proteomics 2010;73:2365–9. [21] Lalioui L, Pellegrini E, Dramsi S, Baptista M, Bourgeois N, Doucet-Populaire F, et al. The SrtA Sortase of Streptococcus agalactiae is required for cell wall anchoring of proteins containing the LPXTG motif, for adhesion to epithelial cells, and for colonization of the mouse intestine. Infect Immun 2005;73:3342–50. [22] Hughes MJ, Moore JC, Lane JD, Wilson R, Pribul PK, Younes ZN, et al. Identification of major outer surface proteins of Streptococcus agalactiae. Infect Immun 2002;70:1254–9. [23] Reinscheid DJ, Gottschalk B, Schubert A, Eikmanns BJ, Chhatwal GS. Identification and molecular analysis of PcsB, a protein required for cell wall separation of group B Streptococcus. J Bacteriol 2001;183:1175–83. [24] Maione D, Margarit I, Rinaudo CD, Masignani V, Mora M, Scarselli M, et al. Identification of a universal Group B

J O U RN A L OF P ROT EO M IC S 8 9 ( 2 01 3 ) 1 5 4 –1 64

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

Streptococcus vaccine by multiple genome screen. Science 2005;309:148–50. Dramsi S, Caliot E, Bonne I, Guadagnini S, Prevost MC, Kojadinovic M, et al. Assembly and role of pili in group B streptococci. Mol Microbiol 2006;60:1401–13. Oussenko IA, Sanchez R, Bechhofer DH. Bacillus subtilis YhcR, a high-molecular-weight, nonspecific endonuclease with a unique domain structure. J Bacteriol 2004;186:5376–83. Stalhammar-Carlemalm M, Stenberg L, Lindahl G. Protein rib: a novel group B streptococcal cell surface protein that confers protective immunity and is expressed by most strains causing invasive infections. J Exp Med 1993;177:1593–603. Larsson C, Lindroth M, Nordin P, Stalhammar-Carlemalm M, Lindahl G, Krantz I. Association between low concentrations of antibodies to protein alpha and Rib and invasive neonatal group B streptococcal infection. Arch Dis Child Fetal Neonatal Ed 2006;91:F403–8. Davidson AL, Dassa E, Orelle C, Chen J. Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol Mol Biol Rev 2008;72:317–64 [table of contents]. Mereghetti L, Sitkiewicz I, Green NM, Musser JM. Identification of an unusual pattern of global gene expression in group B Streptococcus grown in human blood. PLoS One 2009;4:e7145. Sitkiewicz I, Green NM, Guo N, Bongiovanni AM, Witkin SS, Musser JM. Transcriptome adaptation of group B Streptococcus to growth in human amniotic fluid. PLoS One 2009;4:e6114. Lin B, Hollingshead SK, Coligan JE, Egan ML, Baker JR, Pritchard DG. Cloning and expression of the gene for group B streptococcal hyaluronate lyase. J Biol Chem 1994;269: 30113–6. Pares S, Mouz N, Petillot Y, Hakenbeck R, Dideberg O. X-ray structure of Streptococcus pneumoniae PBP2x, a primary penicillin target enzyme. Nat Struct Biol 1996;3:284–9. Sauvage E, Kerff F, Terrak M, Ayala JA, Charlier P. The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev 2008;32:234–58. Oliveira L, Madureira P, Andrade EB, Bouaboud A, Morello E, Ferreira P, et al. Group B Streptococcus GAPDH is released upon cell lysis, associates with bacterial surface, and induces apoptosis in murine macrophages. PLoS One 2012;7:e29963. Magalhaes V, Veiga-Malta I, Almeida MR, Baptista M, Ribeiro A, Trieu-Cuot P, et al. Interaction with human plasminogen system turns on proteolytic activity in Streptococcus agalactiae and enhances its virulence in a mouse model. Microbes Infect 2007;9:1276–84. Bergmann S, Rohde M, Hammerschmidt S. Glyceraldehyde-3-phosphate dehydrogenase of Streptococcus pneumoniae is a surface-displayed plasminogen-binding protein. Infect Immun 2004;72:2416–9. Mereghetti L, Sitkiewicz I, Green NM, Musser JM. Extensive adaptive changes occur in the transcriptome of Streptococcus agalactiae (group B Streptococcus) in response to incubation with human blood. PLoS One 2008;3:e3143. Beiter K, Wartha F, Albiger B, Normark S, Zychlinsky A, Henriques-Normark B. An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. Curr Biol 2006;16:401–7. Zhu H, Liu M, Sumby P, Lei B. The secreted esterase of group a Streptococcus is important for invasive skin infection and dissemination in mice. Infect Immun 2009;77:5225–32. Liu M, Zhu H, Zhang J, Lei B. Active and passive immunizations with the streptococcal esterase Sse protect mice against subcutaneous infection with group A streptococci. Infect Immun 2007;75:3651–7. Santi I, Scarselli M, Mariani M, Pezzicoli A, Masignani V, Taddei A, et al. BibA: a novel immunogenic bacterial adhesin contributing to group B Streptococcus survival in human blood. Mol Microbiol 2007;63:754–67.

163

[43] Schubert A, Zakikhany K, Pietrocola G, Meinke A, Speziale P, Eikmanns BJ, et al. The fibrinogen receptor FbsA promotes adherence of Streptococcus agalactiae to human epithelial cells. Infect Immun 2004;72:6197–205. [44] Pietrocola G, Visai L, Valtulina V, Vignati E, Rindi S, Arciola CR, et al. Multiple interactions of FbsA, a surface protein from Streptococcus agalactiae, with fibrinogen: affinity, stoichiometry, and structural characterization. Biochemistry 2006;45: 12840–52. [45] Papasergi S, Garibaldi M, Tuscano G, Signorino G, Ricci S, Peppoloni S, et al. Plasminogen- and fibronectin-binding protein B is involved in the adherence of Streptococcus pneumoniae to human epithelial cells. J Biol Chem 2010;285:7517–24. [46] Severin A, Nickbarg E, Wooters J, Quazi SA, Matsuka YV, Murphy E, et al. Proteomic analysis and identification of Streptococcus pyogenes surface-associated proteins. J Bacteriol 2007;189:1514–22. [47] Desvaux M, Dumas E, Chafsey I, Chambon C, Hebraud M. Comprehensive appraisal of the extracellular proteins from a monoderm bacterium: theoretical and empirical exoproteomes of Listeria monocytogenes EGD-e by secretomics. J Proteome Res 2010;9:5076–92. [48] Sengupta N, Alam SI, Kumar B, Kumar RB, Gautam V, Kumar S, et al. Comparative proteomic analysis of extracellular proteins of Clostridium perfringens type A and type C strains. Infect Immun 2010;78:3957–68. [49] Wolf C, Kusch H, Monecke S, Albrecht D, Holtfreter S, von Eiff C, et al. Genomic and proteomic characterization of Staphylococcus aureus mastitis isolates of bovine origin. Proteomics 2011;11: 2491–502. [50] Muthukrishnan G, Quinn GA, Lamers RP, Diaz C, Cole AL, Chen S, et al. Exoproteome of Staphylococcus aureus reveals putative determinants of nasal carriage. J Proteome Res 2011;10:2064–78. [51] Sibbald MJ, Winter T, van der Kooi-Pol MM, Buist G, Tsompanidou E, Bosma T, et al. Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in Staphylococcus aureus. J Bacteriol 2010;192:3788–800. [52] Ziebandt AK, Kusch H, Degner M, Jaglitz S, Sibbald MJ, Arends JP, et al. Proteomics uncovers extreme heterogeneity in the Staphylococcus aureus exoproteome due to genomic plasticity and variant gene regulation. Proteomics 2010;10: 1634–44. [53] Thammavongsa V, Kern JW, Missiakas DM, Schneewind O. Staphylococcus aureus synthesizes adenosine to escape host immune responses. J Exp Med 2009;206:2417–27. [54] Lembo A, Gurney MA, Burnside K, Banerjee A, de los Reyes M, Connelly JE, et al. Regulation of CovR expression in Group B Streptococcus impacts blood–brain barrier penetration. Mol Microbiol 2010;77:431–43. [55] Dramsi S, Dubrac S, Konto-Ghiorghi Y, Da Cunha V, Couve E, Glaser P, et al. Rga, a RofA-like regulator, is the major transcriptional activator of the PI-2a pilus in Streptococcus agalactiae. Microb Drug Resist 2012;18:286–97. [56] Mora M, Bensi G, Capo S, Falugi F, Zingaretti C, Manetti AG, et al. Group A Streptococcus produce pilus-like structures containing protective antigens and Lancefield T antigens. Proc Natl Acad Sci U S A 2005;102:15641–6. [57] Rodriguez-Ortega MJ, Norais N, Bensi G, Liberatori S, Capo S, Mora M, et al. Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome. Nat Biotechnol 2006;24:191–7. [58] Bateman A, Rawlings ND. The CHAP domain: a large family of amidases including GSP amidase and peptidoglycan hydrolases. Trends Biochem Sci 2003;28:234–7. [59] McIver KS, Subbarao S, Kellner EM, Heath AS, Scott JR. Identification of isp, a locus encoding an immunogenic secreted protein conserved among group A streptococci. Infect Immun 1996;64:2548–55.

164

J O U RN A L OF P ROTE O M IC S 8 9 ( 2 01 3 ) 1 5 4 –16 4

[60] Reinscheid DJ, Stosser C, Ehlert K, Jack RW, Moller K, Eikmanns BJ, et al. Influence of proteins Bsp and FemH on cell shape and peptidoglycan composition in group B Streptococcus. Microbiology 2002;148:3245–54. [61] Ponting CP, Aravind L, Schultz J, Bork P, Koonin EV. Eukaryotic signalling domain homologues in archaea and bacteria. Ancient ancestry and horizontal gene transfer. J Mol Biol 1999;289:729–45. [62] Higgins CF, Ames GF. Two periplasmic transport proteins which interact with a common membrane receptor show extensive homology: complete nucleotide sequences. Proc Natl Acad Sci U S A 1981;78:6038–42. [63] Galat A. Peptidylprolyl cis/trans isomerases (immunophilins): biological diversity – targets – functions. Curr Top Med Chem 2003;3:1315–47.

[64] Barb AW, Cort JR, Seetharaman J, Lew S, Lee HW, Acton T, et al. Structures of domains I and IV from YbbR are representative of a widely distributed protein family. Protein Sci 2011;20:396–405. [65] Heikkinen O, Seppala R, Tossavainen H, Heikkinen S, Koskela H, Permi P, et al. Solution structure of the parvulin-type PPIase domain of Staphylococcus aureus PrsA — implications for the catalytic mechanism of parvulins. BMC Struct Biol 2009;9:17. [66] Yeats C, Rawlings ND, Bateman A. The PepSY domain: a regulator of peptidase activity in the microbial environment? Trends Biochem Sci 2004;29:169–72.