1 Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland
2 Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK
3 Department of Biochemistry, University of Pavia, 27100 Pavia, Italy
4 Department of Microbiology and Immunobiology, The Queen's University of Belfast, Grosvenor Road, Belfast BT12 6BN, UK
Correspondence
Timothy J. Foster
tfoster{at}tcd.ie
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Cambridge Unit for Medical Research, Addenbrooke's Hospital, Cambridge CB2 2XY, UK.
Present address: Division of Immunity and Infection, Medical School, University of Birmingham, Birmingham B15 2TT, UK.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell-wall-associated surface proteins share several common features that enable them to be covalently anchored to cell-wall peptidoglycan. They possess an N-terminal signal peptide required for Sec-dependent secretion and a conserved C-terminal cell-wall sorting signal which is essential for attachment of a protein to the cell wall by sortase (Mazmanian et al., 2001; Navarre & Schneewind, 1999
). The C-terminal sorting signal comprises a conserved LPXTG motif followed by a hydrophobic stretch of amino acids and positively charged residues at the extreme C terminus. Sortase, encoded by srtA, is a membrane-bound transpeptidase that cleaves polypeptides between the threonine and glycine residues of the LPXTG motif and covalently links them to the nascent pentaglycine crossbridge in peptidoglycan (Ton-That et al., 1999
). Mutants lacking srtA are defective in the display of surface proteins and are attenuated in animal infection models (Mazmanian et al., 2000
). Interrogation of the many microbial genome sequences now available showed that LPXTG proteins and homologues of the srtA gene exist in many Gram-positive bacteria (Janulczyk & Rasmussen, 2001
; Pallen et al., 2001
).
S. aureus is known to express 11 LPXTG proteins including protein A (Spa), the clumping factors ClfA and ClfB, the collagen-binding protein Cna, the serine asparate repeat proteins SdrC, SdrD and SdrE, the fibronectin-binding proteins FnbpA and FnbpB (reviewed by Foster & Höök, 1998), the plasmin-sensitive protein Pls (Savolainen et al., 2001
) and FmtB (Komatsuzawa et al., 2000
). Many share a common domain organization with a similar sized N-terminal A region (ca 500 residues) which contains ligand binding activity. The ClfA and ClfB A regions are composed of independently folded subdomains consisting primarily of
-sheets and coils with a small amount of
-helix (Perkins et al., 2001
; Deivanayagam et al., 2002
). Repeat regions occur as dipeptides in SD repeats of Sdr proteins (Josefsson et al., 1998a
) that act as a stalk to project the N-terminal A region away from the bacterial cell surface (Hartford et al., 1997
) or larger tandem repeated domains which vary in size and identity between proteins. The B repeats of Sdr proteins bind Ca2+ and form rigid rod-like structures (Josefsson et al., 1998b
) while the D repeats of the FnbpA and FnbpB proteins are flexible and bind fibronectin (McGavin et al., 1993
; House-Pompeo et al., 1996
).
The aims of this study were (i) to identify the diversity of LPXTG surface proteins from S. aureus genome sequences, (ii) to study the structural organization of each protein, (iii) to determine if any surface protein genes are associated with invasive disease, and (iv) to measure antibody titres in convalescent patients' sera. This study has identified 10 novel LPXTG proteins from S. aureus genome sequences which are described using the nomenclature Staphylococcus aureus surface (Sas) protein (Mazmanian et al., 2001).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Primary structure analysis.
On-line bioinformatic tools were used to characterize protein sequences identified from the in silico analysis. Signal peptides and repeat domains were identified using the SIGNALP (http://www.cbs.dtu.dk/services/SignalP/) and REPRO (http://mathbio.nimr.mrc.ac.uk/rgeorge/repro/) algorithms. Similarity searches were carried out using NCBI BLAST (Altschul et al., 1997
) and protein sequence alignments used CLUSTAL W (EMBL) (Thompson et al., 1994
). ARTEMIS, a genome visualization program available from the Sanger Centre (http://www.sanger.ac.uk/Software/Artemis/), was used to examine gene localization. Protein secondary structure was predicted from a consensus of algorithms offered at http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sspred.html and http://www.expasy.ch/ sites.
PCR analysis of variable repeat regions.
The entire ORFs of sasG and sdrD were PCR-amplified from strain 8325-4 (Novick, 1967) genomic DNA using primers detailed in Table 1
.
|
Construction and purification of histidine-tagged fusion proteins.
Recombinant N-terminal domains of Sas proteins were expressed with hexahistidine affinity tags at their N termini using the expression vector pQE30 (Qiagen). Each gene fragment was amplified by PCR using 8325-4 genomic DNA as the template, except for sasK. Out of the six genomes studied sasK is only present in Mu50 and N315. In the absence of these strains, a strain of the same clonal type, EMRSA-3 (B. Cookson, personal communication), was used (Enright et al., 2002). The forward and reverse primers are listed in Table 1
. The PCR fragments were cloned into pQE30 at the BamHI and HindIII sites and the resulting plasmids transformed into Escherichia coli TOPP-3 (Stratagene) for protein expression. The recombinant proteins were purified using Ni2+-chelate chromatography (O'Connell et al., 1998
). The SasF40-211 construct was used to immunize a rabbit to generate anti-SasF antibodies.
Isolation of IgG from convalescent patients' sera.
Antisera from 33 individuals with S. aureus infections were obtained from the Ospedale di Circolo di Varese, Italy. The patients were diagnosed with a variety of staphylococcal infections, including sepsis, pneumonia and peritonitis. S. aureus was the only pathogen isolated in the majority of cases. IgG from the sera was purified by chromatography on Vectron-SA (Biovectra). The concentration of the purified antibodies was quantified by absorbance at 280 nm with human IgG as the standard. Control IgGs were obtained from 12 healthy adults representing the same age group as the patients tested.
Statistical analysis.
Patients (n=33) were divided into three groups according to age, group I (2045 years), group II (4565 years) and group III (6590 years). One-way ANOVA was used to compare variability in patient IgG response within each age group to each protein.
ELISA assay.
IgG from sera was tested for reactivity with recombinant SasG50-428, SasA91-575, SasI40-321, SasF40-211, SasK27-175, SasC38-432, SasH39-253, SasJ48-477 and SasE36-292. Microtitre wells were incubated overnight with 100 µl of 50 mM sodium carbonate, pH 9·5, containing 10 µg ml-1 of each recombinant protein. To block additional protein-binding sites, the wells were treated for 1 h at 22 °C with 200 µl PBS containing 2 % (w/v) BSA. The wells were then washed five times with PBST (0·1 % Tween 20 in PBS) and incubated with 2 µg of antibody present in 100 µl of 2 % BSA in PBS at 22 °C. Unbound antibody was removed by washing the wells five times with PBST. Bound antibody was detected by incubation (1 h at 37 °C) of the plates with a rabbit anti-human IgG conjugated to horseradish peroxidase (Dako, Gostrup, Denmark). After washing, binding was quantified using the substrate p-phenylenediamine dihydrochloride (Sigma) and measuring the absorbance at 492 nm in a microplate reader (Bio-Rad). An ELISA reading of greater than two times the mean of the controls was deemed to represent a response to recent exposure to the antigen.
Bacterial strains.
The presence of nine genes encoding putative cell-wall-anchored proteins was examined in 155 isolates recovered from patients with invasive S. aureus disease (94 hospital-acquired and 61 community-acquired) and 179 isolates recovered from healthy blood donors. These isolates were collected within Oxfordshire, UK, between 1997 and 1998 using a prospective case control design (Peacock et al., 2002). They include EMRSA-16 and MSSA strains being sequenced by the Sanger Centre. Invasive isolates were recovered from a normally sterile body site in the Diagnostic Microbiology Laboratory, John Radcliffe Hospital, Oxford. Of these, 86 % were blood culture isolates and 14 % were from cerebrospinal fluid or tissue recovered at operation or biopsy from sites of deep infection such as osteomyelitis and deep abscesses. They include EMRSA-16 and MSSA strains being sequenced by the Sanger Centre.
PCR screening of sas genes in clinical isolates.
The presence of sas genes was analysed by PCR amplification using primers designed to non-variable regions within each gene (Table 2). Genomic DNA from each bacterial strain was isolated using the Wizard Genomic DNA Purification kit (Promega) with the addition of lysostaphin (30 µg ml-1) at the cell lysis step. The PCR mixtures for all nine sets of primers were as follows: 1xreaction buffer, 1·5 mM MgCl2, 100 pmol forward and reverse primers, 100 ng template DNA, 200 µM dNTP mix and 2·5 U Taq polymerase. The PCR cycling conditions were as follows: 94 °C for 3 min, followed by 30 cycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min, which was suitable for amplifying the same size PCR product from each gene. Aliquots of the reaction mixtures were analysed by 1 % agarose gel electrophoresis. Positive controls, EMRSA-16 and MSSA, and a negative control (reaction mixture minus DNA) were included in each PCR run.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A conserved motif in the signal sequences
A feature of proteins belonging to the LPXTG family is the presence of an N-terminal secretory signal sequence. Signal sequences were identified using the SIGNALP prediction algorithm. When the signal sequences from all 21 S. aureus LPXTG proteins were aligned, 15 were found to contain a conserved sequence, (Y/F)SIRK, or variants thereof (data not shown). Subsequent BLAST searches confirmed the observations by Tettelin et al. (2001) that this motif is common among sortase substrates from Gram-positive cocci, although apparently neither exclusive to nor universal among these proteins. SasA is encoded by a gene within an accessory secA2/secY2 cluster. As noted by Bensing & Sullam (2002)
for the GspA LPXTG protein from Streptococcus gordonii, which is encoded in a similar secA2/secY2 cluster, the signal sequence of SasA is predicted by SIGNALP to be unusually long (90 residues). This suggests that the accessory SecA2/SecY2 system might be required for the export of SasA in Staphylococcus aureus, as is the case for GspB in Streptococcus gordonii (Bensing & Sullam, 2002
). All of the other uncharacterized LPXTG proteins had conventional signal peptides.
Primary characterization of the Sas proteins
SasG.
The primary structure of SasG resembles that of proteins Pls of Staphylococcus aureus and Aap of Staphylococcus epidermidis (GenBank accession nos P80544 and CAB77251, respectively; Fig. 1). A distinct subdomain organization of the A regions of these proteins is evident upon primary and secondary structure analysis. Pls and Aap both contain a repeat domain at the extreme N terminus of the A region which is lacking in SasG. All three proteins contain a previously undescribed conserved domain at the C terminus of the A region which is either 212 residues (SasG and Aap) or 217 residues (Pls) in length (Fig. 1
). In SasG this domain lies between residues 208420 and is 52 % identical to that of Pls and 59 % identical to that of Aap. Interestingly, the N-terminal 181 residues of this domain also occur within the A region of SasA (residues 296476; Fig. 1
) and within the N terminus of a non-LPXTG protein of Bacillus thuringiensis (GenBank accession no. P56957, Cry22AA) and one of Lactococcus lactis (GenBank accession no. AAK06279, YwfH), showing 32, 36 and 40 % amino acid identity with the SasG domain, respectively. Fig. 2
shows an alignment of the conserved part of this domain from all six proteins (5660 residues in length). The A regions of SasG, Pls and Aap appear to be composed of subdomains, with those of Pls and Aap comprising a repeat domain and a non-repeat domain followed by a 212/217 residue conserved domain. The A domain of SasG is smaller and appears to comprise two subdomains, a unique N-terminal domain and a domain that is related to the conserved domain of Pls and Aap.
|
|
|
|
The sasG gene resides on a 5 kb fragment which is absent from strain EMRSA-16. This region bears two sar homologues, sarH2 and sarH3, and a gene of unknown function. There is a high level of amino acid sequence identity (8799 %) between the SasG proteins from 8325, COL, N315 and Mu50, whereas SasG from MSSA is only 5569 % identical to these. Residues 50206 of MSSA SasG are more conserved (96 % identical) than residues 207410, which correspond to the domain conserved in Pls and Aap. This is only 64 % identical to SasG from the other strains. The B repeats of MSSA SasG are 96100 % identical but they show only 63 % identity to the highly conserved B repeats of SasG from 8325, COL, N315 and Mu50.
SasA.
This protein has a similar domain organization to proteins of the Sdr family (Josefsson et al., 1998a). It contains a large N-terminal A region which we propose to be composed of three separately folded subdomains, N1 (residues 91244), N2 (residues 245476) and N3 (residues 477575). Residues 91244 of SasA contain a high proportion of hydrophilic residues similar to domain N1 of ClfA and ClfB (28 % serine compared to 9 % serine in the rest of the A region; Perkins et al., 2001
; Deivanayagam et al., 2002
). This is probably responsible for the aberrant migration of recombinant proteins on SDS-PAGE gels. Furthermore, treatment of full-length recombinant SasA A region with purified metalloprotease, a treatment which removes domain N1 of ClfB (McAleese et al., 2001
), resulted in loss of residues 91244 from the full-length molecule (data not shown). Domain N2 carries the 181 residue conserved domain, the C-terminal end of which possibly represents the border between N2 and N3.
Two 88 residue B repeats which are 42 % identical occur after region A. Unlike SasG, there is no variation in the number of these repeats in SasA from all six sequenced strains. These repeats are 4557 % identical to repeats that occur in the Bhp protein from S. epidermidis (GenBank accession no. AAK29746). The A and B domains of SasA are linked to the cell-wall-anchoring domain by SX dipeptide repeats, which resemble the SD dipeptide repeats of the ClfSdr protein family. The length of the SX repeat varies from strain to strain (Table 3).
Other Sas proteins.
Many of the remaining uncharacterized Sas proteins also seem to comprise a modular organization. SasC has a 585 residue N-terminal A region with a similar predicted secondary structure to A regions of ClfSdr proteins. Therefore, it is likely also to have a subunit organization. Region A is followed by two 300 residue repeat domains with 38 % identity (Fig. 1). At the carboxy terminus of the second repeat lies a 12 aa motif [AT(D/T)EEKQ(A/V)A(L/V)NQ] which is repeated twice. The N terminus of the protein (residues 381305) is 49 % identical to the FmtB protein involved in expression of methicillin resistance (Komatsuzawa et al., 2000
).
SasI lacks a typical A region but contains two 140 residue repeat domains at the N terminus which are 38 % identical (Fig. 1). Hydropathy analysis predicts a 60 residue hydrophilic domain in the centre of the protein (residues 480540).
SasF is predicted to be composed of -helices with a potential coiled-coil domain at the N terminus. The LPXTG motif of this protein varies by a single residue from the canonical sequence (LPKAG). To determine if this variation of the classical LPXTG motif enables SasF to be sorted and covalently anchored to the cell-wall peptidoglycan, SasF-specific antisera were used to probe the cell-wall fraction of exponentially grown 8325-4 cells by Western blotting. A single protein band of 65 kDa was detected (Fig. 4
) suggesting that the SasF protein is cell-wall-associated and has undergone sorting despite containing a variation in the LPXTG motif.
|
SasE, SasD, SasH and SasJ do not carry repeat domains and are present in all six genome sequences. The sasE and sasJ genes encode proteins that have been implicated in transferrin binding (Taylor & Heinrichs, 2002). They lie adjacent to and divergent from an iron-regulated operon containing the gene for sortase B (Mazmanian et al., 2002
). SasH has 32 % identity to a 5' nucleotidase from Bacillus halodurans.
ELISA-based screen using convalescent patients' sera
To determine if the newly identified surface proteins are expressed during growth of the bacteria in vivo, recombinant N-terminal truncates from SasG, SasA, SasI, SasF, SasK, SasC, SasH, SasJ and SasE were purified and used in ELISA-based assays to screen for antibodies in sera from patients who had recovered from S. aureus infections. IgG isolated from sera of 33 patients was tested by ELISA for reactivity with the recombinant proteins (Fig. 5). We observed higher titres of antibodies to SasG, SasA, SasI, SasH and SasJ compared to the control IgG, suggesting that these proteins are expressed during S. aureus infections. Sera from individual patients varied substantially in titre towards particular proteins. For example, IgG from patients 3, 12, 16 and 17 did not react with SasG. However, IgG from patients 1, 10, 13 and 30 had higher titres against SasG. Likewise, patients 4, 5, 10, 11 and 13 had high titres against SasA compared to that of IgG purified from patients 20, 23 and 32. Similar variability was observed when the same panel of IgG was tested for the reactivity with fibronectin-binding protein (Casolini et al., 1998
).
|
The sasG and sasH genes are positively associated with disease isolates
To determine if any of the novel surface protein genes are more commonly associated with disease, the presence or absence of the nine genes was determined for 155 isolates associated with invasive disease and 179 carriage isolates obtained from healthy blood donors. Genomic DNA was analysed by PCR with primers specific for each gene. On univariate analysis sasG and sasH were significantly more common in the invasive group (Table 4). To adjust for any effects of clonality, the prevalence of each gene in the disease and carriage groups was compared within but not between clonal complexes using the MantelHaenszel method. The clonal structure of this collection of isolates has been defined by multilocus sequence typing (http://www.mlst.net). The presence of sasG and sasH remained significantly associated with disease isolates (Table 4
). When isolates from nosocomial infections were removed from the analysis and community-acquired disease isolates were compared with carriage strains in a stratified analysis, both sasG and sasH remained associated with disease [Odds ratios 3·1 (P=0·003) and 3·4 (P=0·005), respectively]. We examined the possibility that identification of these virulence-associated genes was actually the result of linkage disequilibrium. Conditional logistic regression modelling of the relationship of the two factors both to disease and to each other suggested that sasG and sasH are both independent of each other and are independently associated with disease.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is well established that proteins bearing an N-terminal signal peptide and C-terminal cell-wall sorting signal with a conserved LPXTG motif are anchored to the cell-wall envelope by a transpeptidation mechanism involving sortase (Mazmanian et al., 2001). Most of the newly characterized LPXTG proteins examined in this study bear the typical features of this family of proteins and hence are predicted to be cell-wall-associated. Exceptions include SasD and SasF, which contain an LPXAG motif within the cell-wall sorting signal. Using SasF-specific antibodies, Western blotting of the cell-wall fraction of 8325-4 cells detected a single 65 kDa protein corresponding to the predicted molecular mass of the SasF protein. Since SasF can be solubilized by lysostaphin during the generation of stable protoplasts, it suggests that SasF is associated with the cell-wall fraction and infers that sorting has occurred. These data therefore indicate that a substitution of threonine with an alanine residue within the LPXTG motif does not affect the sorting reaction. We propose that SasD is also cell-wall-associated.
The domain architecture of proteins is well documented (Doolittle, 1995). In this study, primary structural analysis strongly suggests that proteins of the LPXTG family are modular in architecture and have evolved through the acquisition of distinct domain-sized polypeptides, some of which have expanded by duplication and homologous recombination. Many of these domains are found in other species, such as the B repeats of SasA and SasG, the 212 residue conserved domain of SasG, the SD and SX repeats of the Sdr proteins and the (Y/F)SIRK motif containing signal peptide, suggesting they have been acquired by recent horizontal transfer.
The conserved (Y/F)SIRK motif is present in the signal peptides of many S. aureus and other Gram-positive LPXTG proteins. The possibility that this sequence targets the protein to sortase is unlikely, since it is also found in non-cell-wall-anchored proteins and more importantly, some S. aureus LPXTG proteins (Cna, SasF, SasK, SasH and SasA) do not carry the motif. Therefore, the significance of this motif and its function are not clear. SasA bears an unusually long signal peptide that probably targets this protein for secretion by the accessory SecA2/SecY2 system (Bensing & Sullam, 2002), although why this should require an accessory secretion system is also unclear. Specific amino acid patterns have previously been identified within signal peptides of proteins of similar function or of proteins destined for different cellular compartments (Edman et al., 1999
). Expression of hybrid proteins with different signal peptide types within wild-type and SecA2 mutants could address the effects of the (F/Y)SIRK motif and of the SasA extended signal sequence on efficiency and mechanism of secretion.
Another typical domain arrangement of LPXTG proteins is the modular A region. Most LPXTG proteins of S. aureus contain a 500 residue A region, which has recently been proposed to be composed of three independently folded subdomains in the case of ClfA and ClfB (Perkins et al., 2001
; Deivanayagam et al., 2002
). N1 is extremely hydrophilic with an elongated secondary structure, whereas N2 and N3 are globular proteins bearing the ligand binding activity. As bacterial cells enter stationary phase, domain N1 of both proteins is cleaved from the bacterial cell surface by a staphylococcal metalloprotease and in the case of ClfB, ligand binding activity is lost (McAleese et al., 2001
; J. Higgins & T. J. Foster, unpublished data). The site of cleavage is a predicted
-helix which separates domains N1 and N23. A similar phenomenon occurs with Pls expression on the bacterial cell surface. As cells progress to stationary phase, full-length Pls (230 kDa) is proteolytically cleaved to a 175 kDa truncated form (Savolainen et al., 2001
). The cleavage site (between residues 387 and 388) may represent the border between subdomain N2 (residues 49387) and subdomain N3 (residues 388655) bearing the 217 residue conserved domain (Fig. 1
). The SasA A region is also predicted to have three subdomains. The architecture of this region is very similar to ClfA and ClfB. Like these proteins, SasA has an extremely hydrophilic N1 subdomain that is proteolytically cleaved off from full-length recombinant SasA region A by metalloprotease. The site of cleavage is also a predicted
-helix. It is therefore possible that similar processing occurs in SasA as with ClfA and ClfB.
Repeated sequences are also a common feature of the newly characterized LPXTG proteins. The SX dipeptide repeats of SasA resemble the SD dipeptide repeats of the ClfSdr family. The length of the SX repeats varies considerably from strain to strain, as do the SD repeats (McDevitt & Foster, 1995), and it seems reasonable to think that they have the same function, namely to project the ligand-binding region from the cell surface (Hartford et al., 1997
). Despite the strain-to-strain length variation of the SD and SX repeats, the underlying frequency of recombination between the DNA repeats is actually quite low. The SD-repeat-encoding region of clfA of strain Newman is composed of an array of 18 bp DNA repeats which are saturated with base substitution mutations such that very few are identical (Shields et al., 1995
). PCR analysis of the SD repeat region yielded a single PCR product indicative of a low frequency of recombination (McDevitt & Foster, 1995
). Similar results were obtained by PCR across the SX-repeat-encoding region of SasA (data not shown).
In contrast, the B repeats of SasG are between 98 and 100 % identical at the amino acid and nucleotide levels. Such a high level of DNA sequence identity is likely to result in a high frequency of unequal recombination and a detectable level of length variants in any population. This explains the observation that PCR of sasG from genomic DNA isolated from a bacterial population generated a number of different sized fragments varying by the length of one repeat. The SdrD protein also contains a B repeat region which varies in the number of repeats between different strains (Josefsson et al., 1998a). PCR amplification of the sdrD gene from strain 8325-4, which bears five B repeat domains, yielded only a single PCR product. The DNA encoding B repeats is 6878 % identical so frequency of unequal recombination would be lower than for sasG, explaining the failure to detect variants by PCR. Length variation of sasG is reminiscent of the Bca protein from Group B streptococci, where it is believed to be a mechanism for evading the immune response during infection, a shorter protein being selected in neonates when compared with the corresponding isogenic maternal strain (Madoff et al., 1996
). Similar variants were detected when the B repeat region of pls was analysed by PCR but this was attributed to homology between one of the primers and the repeats (Savolainen et al., 2001
). The Pls B repeats did not vary in length after 200 generations of growth in broth, suggesting that shorter variants, for example, did not have a growth advantage in vitro (Savolainen et al., 2001
).
The success of S. aureus as a human pathogen is likely to involve many virulence determinants. However, it has been shown that certain individual virulence factors are linked to specific diseases. For example, the collagen-binding adhesin (Cna) is present in only 32 % of carriage isolates but is found in 52 % of isolates from invasive disease (Peacock et al., 2002). An earlier study suggested an association between cna and septic arthritis (Switalski et al., 1993
). In this study, the sasG and sasH genes were found to be virulence-associated genes; genes encoding the surface proteins FnbpA, Cna and SdrE were previously found to be virulence-associated in the same panel of invasive strains, indicating the important role played by some of the surface-expressed proteins in disease pathogenesis (Peacock et al., 2002
). These associations help prioritize future studies of LPXTG proteins and indicate that sasG and sasH warrant early, in-depth characterization.
To determine if the new collection of Sas proteins is expressed during growth in infected human patients, the N-terminal region of each was expressed and used in ELISA tests with IgG purified from sera of convalescent patients. Elevated titres occurred in five of nine Sas proteins compared to control IgG, suggesting that exposure to antigens had occurred during previous infection(s). Variability in patients' IgG response to each protein was observed. ANOVA indicated that this variability in antibody response is dependent on the age of the patients, with older patients showing higher variability compared to younger groups. Another factor that may contribute to this variability is the presence or absence of the particular gene from the infecting strain. For example, sasG and sasH occur at low frequency, 53 and 68 %, respectively, in the invasive isolate and only 4/33 and 7/33 patients had elevated titres to the SasG and SasH proteins, respectively. In contrast, sasA, sasI and sasJ are present in 100, 96 and 97 % of the strains, and a greater number of patients had elevated titres to the SasA, SasI and SasJ (11/33, 10/33 and 18/33, respectively).
In the cases of sasK and sasE, which are present in fewer than 44 % of the clinical isolates tested, the low titres might be due to the infecting strains lacking these genes. SasI was also shown to be expressed during infection because sera from convalescent patients reacted with SasI peptides displayed on the surface of E. coli (Etz et al., 2002), while SdrD was identified by serological proteome analysis (Vytvytska et al., 2002
).
It is now apparent that S. aureus can express up to 21 different LPXTG-anchored surface proteins. The domain architecture, repeat regions and common signal peptide motifs suggest that the evolution of LPXTG-anchored surface proteins may have arisen in a modular fashion. Some of these proteins were recognized by sera from infected patients and were also found to be associated with clinically invasive disease but their functions remain to be elucidated. One or more of these proteins could be suitable for development as vaccine candidates, alongside the current contenders ClfA (Josefsson et al., 2001) and Cna (Nilsson et al., 1998
; Visai et al., 2000
).
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bensing, B. A. & Sullam, P. M. (2002). An accessory sec locus of Streptococcus gordonii is required for export of the surface protein GspB and for normal levels of binding to human platelets. Mol Microbiol 44, 10811094.[CrossRef][Medline]
Casolini, F., Visai, L., Joh, D., Conaldi, P. G., Toniolo, A., Höök, M. & Speziale, P. (1998). Antibody response to fibronectin-binding adhesin FnbpA in patients with Staphylococcus aureus infections. Infect Immun 66, 54335442.
Cucarella, C., Solano, C., Valle, J., Amorena, B., Lasa, L. & Penadés, J. R. (2001). Bap, a Staphylococcus aureus surface protein involved in biofilm formation. J Bacteriol 183, 28882896.
Deivanayagam, C. C., Wann, E. R., Chen, W., Carson, M., Rajashankar, K. R., Höök, M. & Narayana, S. V. L. (2002). A novel variant of the immunoglobulin fold in surface adhesins of Staphylococcus aureus: crystal structure of the fibrinogen-binding MSCRAMM, clumping factor A. EMBO J 21, 66606672.
Doolittle, R. F. (1995). The multiplicity of domains in proteins. Annu Rev Biochem 64, 287314.[CrossRef][Medline]
Edman, M., Jarhede, T., Sjöström, M. & Wieslander, A. (1999). Different sequence patterns in signal peptides from mycoplasmas, other gram-positive bacteria, and Escherichia coli: a multivariate data analysis. Proteins 35, 195205.[CrossRef][Medline]
Enright, M. C., Day, N. P. J., Davies, C. E., Peacock, S. J. & Spratt, B. J. (2000). Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol 38, 10081015.
Enright, M. C., Robinson, D. A., Randle, G., Feil, E. J., Grundmann, H. & Spratt, B. G. (2002). The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proc Natl Acad Sci U S A 99, 76877692.
Etz, H., Minh, D. B., Henics, T. & 15 other authors (2002). Identification of in vivo expressed vaccine candidate antigens from Staphylococcus aureus. Proc Natl Acad Sci U S A 99, 65736578.
Foster, T. J. & Höök, M. (1998). Surface protein adhesins of Staphylococcus aureus. Trends Microbiol 6, 484488.[CrossRef][Medline]
Hartford, O., Francois, P., Vaudaux, P. & Foster, T. J. (1997). The dipeptide repeat region of the fibrinogen-binding protein (clumping factor) is required for functional expression of the fibrinogen-binding domain on the Staphylococcus aureus cell surface. Mol Microbiol 25, 10651076.[Medline]
House-Pompeo, K., Xu, Y., Joh, D., Speziale, P. & Höök, M. (1996). Conformational changes in the fibronectin binding MSCRAMMs are induced by ligand binding. J Biol Chem 271, 13791384.
Janulczyk, R. & Rasmussen, M. (2001). Improved pattern for genome-based screening identifies novel cell wall-attached proteins from Gram-positive bacteria. Infect Immun 69, 40194016.
Josefsson, E., McCrea, K. W., Ní Eidhin, D., O'Connell, D., Cox, J., Höök, M. & Foster, T. J. (1998a). Three new members of the serine-aspartate repeat protein multigene family of Staphylococcus aureus. Microbiology 144, 33873395.[Abstract]
Josefsson, E., O'Connell, D., Foster, T. J., Durussel, I. & Cox, J. A. (1998b). The binding of calcium to the B-repeat segment of SdrD, a cell surface protein of Staphylococcus aureus. J Biol Chem 273, 3114531152.
Josefsson, E., Hartford, O., O'Brien, L., Patti, J. M. & Foster, T. J. (2001). Protection against experimental Staphylococcus aureus arthritis by vaccination with clumping factor A, a novel virulence determinant. J Infect Dis 184, 15721580.[CrossRef][Medline]
Komatsuzawa, H., Ohta, K., Sugai, M., Fujiwara, T., Glanzmann, P., Berger-Bachi, B. & Suginaka, H. (2000). Tn551-mediated insertional inactivation of the fmtB gene encoding a cell wall-associated protein abolishes methicillin resistance in Staphylococcus aureus. J Antimicrob Chemother 45, 421431.
Kuroda, M., Ohta, T., Uchiyama, I. & 34 other authors (2001). Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357, 12251240.[CrossRef][Medline]
Madoff, L. C., Michel, J. L., Gong, E. W., Kling, D. E. & Kasper, D. L. (1996). Group B streptococci escape host immunity by deletion of tandem repeat elements of the alpha C protein. Proc Natl Acad Sci U S A 93, 41314136.
Mazmanian, S. K., Lui, G., Jensen, E. R., Lenoy, E. & Schneewind, O. (2000). Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections. Proc Natl Acad Sci U S A 97, 55105515.
Mazmanian, S. K., Ton-That, H. & Schneewind, O. (2001). Sortase-catalysed anchoring of surface proteins to the cell wall of Staphylococcus aureus. Mol Microbiol 40, 10491057.[CrossRef][Medline]
Mazmanian, S. K., Ton-That, H., Su, K. & Schneewind, O. (2002). An iron-regulated sortase anchors a class of surface protein during Staphylococcus aureus pathogenesis. Proc Natl Acad Sci U S A 99, 22932298.
McAleese, F. M., Walsh, E. J., Sieprawska, M., Potempa, J. & Foster, T. J. (2001). Loss of clumping factor B fibrinogen binding activity by Staphylococcus aureus involves cessation of transcription, shedding and cleavage by metalloprotease. J Biol Chem 276, 2996929978.
McDevitt, D. & Foster, T. J. (1995). Variation in the size of the repeat region of the fibrinogen receptor (clumping factor) of Staphylococcus aureus strains. Microbiology 141, 937943.[Abstract]
McGavin, M. J., Gurusiddappa, S., Lingren, P. E., Lindberg, M., Raucci, G. & Höök, M. (1993). Fibronectin receptors from Streptococcus dysgalactiae and Staphylococcus aureus: involvement of conserved residues in ligand binding. J Biol Chem 268, 2394623953.
Navarre, W. W. & Schneewind, O. (1999). Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol Mol Biol Rev 63, 174229.
Nilsson, I. M., Patti, J. M., Bremell, T., Höök, M. & Tarkowski, A. (1998). Vaccination with a recombinant fragment of the collagen adhesin provides protection against Staphylococcus aureus-mediated septic death. J Clin Invest 101, 26402649.
Novick, R. P. (1967). Properties of a cryptic high frequency transducing phage in Staphylococcus aureus. Virology 33, 155166.[Medline]
O'Connell, D., Nanavaty, T., McDevitt, D., Gurusiddappa, S., Höök, M. & Foster, T. J. (1998). The fibrinogen-binding MSCRAMM (clumping factor) of Staphylococcus aureus has a Ca2+-dependent inhibitory site. J Biol Chem 273, 68216829.
Pallen, M. J., Lam, A. C., Antonio, M. & Dunbar, K. (2001). An embarrassment of sortases a richness of substrates? Trends Microbiol 9, 97102.[CrossRef][Medline]
Peacock, S. J., Foster, T. J., Cameron, B. J. & Berendt, A. R. (1999). Bacterial fibronectin-binding proteins and endothelial cell surface fibronectin mediate adherence of Staphylococcus aureus to resting human endothelial cells. Microbiology 145, 34773486.
Peacock, S. J., Moore, C. E., Justice, A., Kantzanou, M., Story, L., Mackie, K., O'Neill, G. & Day, N. P. J. (2002). Virulent combinations of adhesin and toxin genes in natural populations of Staphylococcus aureus. Infect Immun 70, 49874996.
Perkins, S., Walsh, E. J., Deivanayagam, C. C. S., Narayana, S. V. L., Foster, T. J. & Höök, M. (2001). Structural organization of the fibrinogen-binding region of the clumping factor B MSCRAMM of Staphylococcus aureus. J Biol Chem 276, 4472144728.
Savolainen, K., Paulin, L., Westerlund-Wikström, B., Foster, T. J., Korhonen, T. K. & Kuusela, P. (2001). Expression of pls, a gene closely associated with the mecA gene of methicillin-resistant Staphylococcus aureus, prevents bacterial adhesion in vitro. Infect Immun 69, 30133020.
Shields, D. C., McDevitt, D. & Foster, T. J. (1995). Evidence against concerted evolution in a tandem array in the clumping factor gene of Staphylococcus aureus. Mol Biol Evol 12, 963965.
Switalski, L. M., Patti, J. M., Butcher, W., Gristina, A. G., Speziale, P. & Höök, M. (1993). A collagen receptor on Staphylococcus aureus strains isolated from patients with septic arthritis mediates adhesion to cartilage. Mol Microbiol 7, 99107.[Medline]
Taylor, J. M. & Heinrichs, D. E. (2002). Transferrin binding in Staphylococcus aureus: involvement of a cell wall-anchored protein. Mol Microbiol 43, 16031614.[CrossRef][Medline]
Tettelin, H., Nelson, K. E., Paulsen, I. T. & 39 other authors (2001). Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293, 498506.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Ton-That, H., Lui, G., Mazmanian, S. K., Faull, K. F. & Schneewind, O. (1999). Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc Natl Acad Sci U S A 96, 1242412429.
Visai, L., Xu, Y., Casolini, F., Rindi, S., Höök, M. & Speziale, P. (2000). Monoclonal antibodies to CNA, a collagen-binding microbial surface component recognizing adhesive matrix molecules, detach Staphylococcus aureus from a collagen substrate. J Biol Chem 275, 3983739845.
Vytvytska, O., Nagy, E., Bluggel, M., Meyer, H. E., Kurzbauer, R., Huber, L. A. & Klade, C. S. (2002). Identification of vaccine candidate antigens of Staphylococcus aureus by serological proteome analysis. Proteomics 2, 580590.[CrossRef][Medline]
Received 13 September 2002;
revised 15 December 2002;
accepted 24 December 2002.