Coenzyme A precursors flow from mother to zygote and from microbiome to host.

Coenzyme A (CoA) is essential for metabolism and protein acetylation. Current knowledge holds that each cell obtains CoA exclusively through biosynthesis via the canonical five-step pathway, starting with pantothenate uptake. However, recent studies have suggested the presence of additional CoA-generating mechanisms, indicating a more complex system for CoA homeostasis. Here, we uncovered pathways for CoA generation through inter-organismal flows of CoA precursors. Using traceable compounds and fruit flies with a genetic block in CoA biosynthesis, we demonstrate that progeny survive embryonal and early larval development by obtaining CoA precursors from maternal sources. Later in life, the microbiome can provide the essential CoA building blocks to the host, enabling continuation of normal development. A flow of stable, long-lasting CoA precursors between living organisms is revealed. This indicates the presence of complex strategies to maintain CoA homeostasis.

Tryptic Shaving of Staphylococcus aureus Unveils Immunodominant Epitopes on the Bacterial Cell Surface.

The opportunistic pathogen Staphylococcus aureus has become a major threat for human health and well-being by developing resistance to antibiotics and by fast evolution into new lineages that rapidly spread within the healthy human population. This calls for development of active or passive immunization strategies to prevent or treat acute phase infections. Since no such anti-staphylococcal immunization approaches are available for clinical implementation, the present studies were aimed at identifying new leads for their development. For this purpose, we profiled the cell-surface-exposed staphylococcal proteome under infection-mimicking conditions by combining two approaches for "bacterial shaving" with immobilized or soluble trypsin and subsequent mass spectrometry analysis of liberated peptides. In parallel, non-covalently cell-wall-bound proteins extracted with potassium thiocyanate and the exoproteome fraction were analyzed by gel-free proteomics. All data are available through ProteomeXchange accession PXD000156. To pinpoint immunodominant bacterial-surface-exposed epitopes, we screened selected cell-wall-attached proteins of S. aureus for binding of immunoglobulin G from patients who have been challenged by different types of S. aureus due to chronic wound colonization. The combined results of these analyses highlight particular cell-surface-exposed S. aureus proteins with highly immunogenic exposed epitopes as potential targets for development of protective anti-staphylococcal immunization strategies.

Exoproteome Heterogeneity among Closely Related Staphylococcus aureus t437 Isolates and Possible Implications for Virulence.

Staphylococcus aureus with spa-type t437 has been identified as a predominant community-associated methicillin-resistant S. aureus clone from Asia, which is also encountered in Europe. Molecular typing has previously shown that t437 isolates are highly similar regardless of geographical regions or host environments. The present study was aimed at assessing to what extent this high similarity is actually reflected in the production of secreted virulence factors. We therefore profiled the extracellular proteome, representing the main reservoir of virulence factors, of 20 representative clinical isolates by mass spectrometry. The results show that these isolates can be divided into three groups and nine subgroups based on exoproteome abundance signatures. This implies that S. aureus t437 isolates show substantial exoproteome heterogeneity. Nonetheless, 30 highly conserved extracellular proteins, of which about 50% have a predicted role in pathogenesis, were dominantly identified. To approximate the virulence of the 20 investigated isolates, we employed infection models based on Galleria mellonella and HeLa cells. The results show that the grouping of clinical isolates based on their exoproteome profile can be related to virulence. We consider this outcome important as our approach provides a tool to pinpoint differences in virulence among seemingly highly similar clinical isolates of S. aureus.

A Lactococcus lactis expression vector set with multiple affinity tags to facilitate isolation and direct labeling of heterologous secreted proteins.

The gram-positive bacterium Lactococcus lactis is a useful host for extracellular protein production. A main advantage of L. lactis over other bacterial expression systems is that lactococcal cells display low levels of autolysis and proteolysis. Previously, we developed a set of vectors for nisin-inducible extracellular production of N- or C-terminally hexa-histidine (His6)-tagged proteins. The present study was aimed at expanding our portfolio of L. lactis expression vectors for protein purification and site-specific labeling. Specifically, we present two new groups of vectors allowing N- or C-terminal provision of proteins with a Strep-tag II or AVI-tag. Vectors for AVI-tagging encode an additional His6-tag for protein purification. Another set of vectors allows removal of N-terminal Strep- or His6-tags from expressed proteins with the tobacco etch virus protease. Two possible applications of the developed vectors are presented. First, we show that Strep-tagged LytM of Staphylococcus aureus in the growth medium of L. lactis can be directly bound to microtiter plates coated with an affinity reagent and used for enzyme-linked immunosorbent assays. Second, we show that the AVI-tagged Sle1 protein from S. aureus produced in L. lactis can be directly biotinylated and fluorescently labeled. The fluorescently labeled Sle1 was successfully applied for S. aureus re-binding studies, allowing subcellular localization by fluorescence microscopy. In conclusion, we have developed a set of expression vectors that enhances the versatility of L. lactis as a system for production of proteins with tags that can be used for affinity purification and site-specific protein labeling.

Expression of prophage-encoded endolysins contributes to autolysis of Lactococcus lactis.

Analysis of autolysis of derivatives of Lactococcus lactis subsp. cremoris MG1363 and subsp. lactis IL1403, both lacking the major autolysin AcmA, showed that L. lactis IL1403 still lysed during growth while L. lactis MG1363 did not. Zymographic analysis revealed that a peptidoglycan hydrolase activity of around 30 kDa is present in cell extracts of L. lactis IL1403 that could not be detected in strain MG1363. A comparison of all genes encoding putative peptidoglycan hydrolases of IL1403 and MG1363 led to the assumption that one or more of the 99 % homologous 27.9-kDa endolysins encoded by the prophages bIL285, bIL286 and bIL309 could account for the autolysis phenotype of IL1403. Induced expression of the endolysins from bIL285, bIL286 or bIL309 in L. lactis MG1363 resulted in detectable lysis or lytic activity. Prophage deletion and insertion derivatives of L. lactis IL1403 had a reduced cell lysis phenotype. RT-qPCR and zymogram analysis showed that each of these strains still expressed one or more of the three phage lysins. A homologous gene and an endolysin activity were also identified in the natural starter culture L. lactis subsp. cremoris strains E8, Wg2 and HP, and the lytic activity could be detected under growth conditions that were identical as those used for IL1403. The results presented here show that these endolysins of L. lactis are expressed during normal growth and contribute to autolysis without production of (lytic) phages. Screening for natural strains expressing homologous endolysins could help in the selection of strains with enhanced autolysis and, thus, cheese ripening properties.

A human monoclonal antibody targeting the conserved staphylococcal antigen IsaA protects mice against Staphylococcus aureus bacteremia.

Due to substantial therapy failure and the emergence of antibiotic-resistant Staphylococcus aureus strains, alternatives for antibiotic treatment of S. aureus infections are urgently needed. Passive immunization using S. aureus-specific monoclonal antibodies (mAb) could be such an alternative to prevent and treat severe S. aureus infections. The invariantly expressed immunodominant staphylococcal antigen A (IsaA) is a promising target for passive immunization. Here we report the development of the human anti-IsaA IgG1 mAb 1D9, which was shown to bind to all 26 S. aureus isolates tested. These included both methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA, respectively). Immune complexes consisting of IsaA and 1D9 stimulated human as well as murine neutrophils to generate an oxidative burst. In a murine bacteremia model, the prophylactic treatment with a single dose of 5 mg/kg 1D9 improved the survival of mice challenged with S. aureus isolate P (MSSA) significantly, while therapeutic treatment with the same dose did not influence animal survival. Neither prophylactic nor therapeutic treatment with 5 mg/kg 1D9 resulted in improved survival of mice with S. aureus USA300 (MRSA) bacteremia. Importantly, our studies show that healthy S. aureus carriers elicit an immune response which is sufficient to generate protective mAbs against invariant staphylococcal surface antigens. Human mAb 1D9, possibly conjugated to for example another antibody, antibiotics, cytokines or chemokines, may be valuable to fight S. aureus infections in patients.

Versatile vector suite for the extracytoplasmic production and purification of heterologous His-tagged proteins in Lactococcus lactis.

Recent studies have shown that the Gram-positive bacterium Lactococcus lactis can be exploited for the expression of heterologous proteins; however, a versatile set of vectors suitable for inducible extracellular protein production and subsequent purification of the expressed proteins by immobilized metal affinity chromatography was so far lacking. Here we describe three novel vectors that, respectively, facilitate the nisin-inducible production of N- or C-terminally hexa-histidine (His6)-tagged proteins in L. lactis. One of these vectors also encodes a tobacco etch virus (TEV) protease cleavage site allowing removal of the N-terminal His6-tag from expressed proteins. Successful application of the developed vectors for protein expression, purification and/or functional studies is exemplified with six different cell wall-bound or secreted proteins from Staphylococcus aureus. The results show that secretory production of S. aureus proteins is affected by the position, N- or C-terminal, of the His6-tag. This seems to be due to an influence of the His6-tag on protein stability. Intriguingly, the S. aureus IsdB protein, which is phosphorylated in S. aureus, was also found to be phosphorylated when heterologously produced in L. lactis, albeit not on the same Tyr residue. This implies that this particular post-translational protein modification is to some extent conserved in S. aureus and L. lactis. Altogether, we are confident that the present vector set combined with the L. lactis expression host has the potential to become a very useful tool in optimization of the expression, purification and functional analysis of extracytoplasmic bacterial proteins.

The Staphylococcal Cassette Chromosome mec type V from Staphylococcus aureus ST398 is packaged into bacteriophage capsids.

The Staphylococcal Cassette Chromosome mec (SCCmec) confers methicillin resistance to Staphylococcus aureus. While SCCmec is generally regarded as a mobile genetic element, the precise mechanisms by which large SCCmec elements are exchanged between staphylococci have remained enigmatic. In the present studies, we observed that the clinical methicillin-resistant S. aureus (MRSA) isolate UMCG-M4 with the sequence type 398 contains four prophages belonging to the serological groups A, B and Fa. Previous studies have shown that certain serological group B bacteriophages of S. aureus are capable of generalized transduction. We therefore assessed the transducing capabilities of the phages from strain UMCG-M4. The results show that some of these phages can indeed transduce plasmid pT181 to the recipient S. aureus strain RN4220. Therefore, we also investigated the possible involvement of these transducing phages in the transmission of the large SCCmec type V (5C2&5) element of S. aureus UMCG-M4. While no transduction of the complete SCCmec element was observed, we were able to demonstrate that purified phage particles did contain large parts of the SCCmec element of the donor strain, including the methicillin resistance gene mecA. This shows that staphylococcal phages can encapsulate the resistance determinant mecA of a large SCCmec type V (5C2&5) element, which may lead to its transfer to other staphylococci.

Exploiting the peptidoglycan-binding motif, LysM, for medical and industrial applications.

The lysin motif (LysM) was first identified by Garvey et al. in 1986 and, in subsequent studies, has been shown to bind noncovalently to peptidoglycan and chitin by interacting with N-acetylglucosamine moieties. The LysM sequence is present singly or repeatedly in a large number of proteins of prokaryotes and eukaryotes. Since the mid-1990s, domains containing one or more of these LysM sequences originating from different LysM-containing proteins have been examined for purely scientific reasons as well as for their possible use in various medical and industrial applications. These studies range from detecting localized binding of LysM-containing proteins onto bacteria to actual bacterial cell surface analysis. On a more applied level, the possibilities of employing the LysM domains for cell immobilization, for the display of peptides, proteins, or enzymes on (bacterial) surfaces as well as their utility in the development of novel vaccines have been scrutinized. To serve these purposes, the chimeric proteins containing one or more of the LysM sequences have been produced and isolated from various prokaryotic and eukaryotic expression hosts. This review gives a succinct overview of the characteristics of the LysM domain and of current developments in its application potential.

Efficient production of secreted staphylococcal antigens in a non-lysing and proteolytically reduced Lactococcus lactis strain.

Cell surface-exposed and secreted proteins are attractive targets for vaccination against pathogenic gram-positive bacteria. To obtain sufficient amounts of such antigens, efficient protein production platforms are needed. In this study, a pipeline for the production and purification of surface-exposed and secreted antigens of the gram-positive bacterial pathogen Staphylococcus aureus is presented. Cytoplasmic or extracellular production of S. aureus antigens was achieved using the Lactococcus lactis strain PA1001, which lacks the major extracellular protease HtrA and the autolysin AcmA to minimize proteolysis and cell lysis, respectively. For most tested S. aureus antigens, secretory production directed by the signal peptide of the major secreted protein Usp45 of L. lactis resulted in higher yields than intracellular production without a signal peptide. Additionally, secretory production of His-tagged antigens allowed their facile one-step purification from the growth medium by metal affinity chromatography. For three of the purified antigens, biological activity was confirmed through enzyme activity assays. We, furthermore, show that the present pipeline can be used to produce staphylococcal antigens with an N-terminal AVI-tag for site-specific labeling with biotin or a C-terminal cell wall-binding domain for cell surface display. We conclude that our L. lactis-based pipeline allows the efficient production of S. aureus antigens and their subsequent purification in one step.

Distinction of Staphylococcal Cassette Chromosome mec type V elements from Staphylococcus aureus ST398.

Methicillin resistant S. aureus (MRSA) is a major threat for human health and well-being. In recent years, it has become clear that livestock is a potential reservoir for MRSA, most livestock-associated isolates belonging to the ST398 lineage. Importantly, ST398 strains were also reported as causative agents of severe invasive infections in humans with no evidence for livestock associations. Here we document the sequence of the J1 region of the type V (5C2&5) SCCmec element and its right chromosomal junction in the clinical PVL-positive ST398 MRSA isolate UMCG-M4. Sequence comparisons show that this SCCmec element and related type V elements from other S. aureus isolates share a common core structure, but differ substantially in the so-called J1 region. Additional PCR analyses and typing studies indicate that the J1 region of strain UMCG-M4 is specific for SCCmec elements of PVL-positive ST398 isolates. Lastly, we show that the sequenced right chromosomal junction is invariant in strains of the ST398 lineage.

Distinct roles of phenol-soluble modulins in spreading of Staphylococcus aureus on wet surfaces.

The human pathogen Staphylococcus aureus is renowned for the rapid colonization of contaminated wounds, medical implants, and food products. Nevertheless, little is known about the mechanisms that allow S. aureus to colonize the respective wet surfaces. The present studies were therefore aimed at identifying factors used by S. aureus cells to spread over wet surfaces, starting either from planktonic or biofilm-associated states. Through proteomics analyses we pinpoint phenol-soluble modulins (PSMs) as prime facilitators of the spreading process. To dissect the roles of the eight PSMs produced by S. aureus, these peptides were chemically synthesized and tested in spreading assays with different psm mutant strains. The results show that PSMα3 and PSMγ are the strongest facilitators of spreading both for planktonic cells and cells in catheter-associated biofilms. Compared to the six other PSMs of S. aureus, PSMα3 and PSMγ combine strong surfactant activities with a relatively low overall hydropathicity. Importantly, we show that PSM-mediated motility of S. aureus facilitates the rapid colonization of wet surfaces next to catheters and the colonization of fresh meat.

Differential Virulence of Aggregatibacter actinomycetemcomitans Serotypes Explained by Exoproteome Heterogeneity.

Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative bacterial pathogen associated with periodontitis and nonoral diseases like rheumatoid arthritis and Alzheimer´s disease. Aa isolates with the serotypes a, b, and c are globally most prevalent. Importantly, isolates displaying these serotypes have different clinical presentations. While serotype b isolates are predominant in severe periodontitis, serotypes a and c are generally encountered in mild periodontitis or healthy individuals. It is currently unknown how these differences are reflected in the overall secretion of virulence factors. Therefore, this study was aimed at a comparative analysis of exoproteomes from different clinical Aa isolates with serotypes a, b, or c by mass spectrometry, and a subsequent correlation of the recorded exoproteome profiles with virulence. Overall, we identified 425 extracellular proteins. Significant differences in the exoproteome composition of isolates with different serotypes were observed in terms of protein identification and abundance. In particular, serotype a isolates presented more extracellular proteins than serotype b or c isolates. These differences are mirrored in their virulence in infection models based on human salivary gland epithelial cells and neutrophils. Remarkably, serotype a isolates displayed stronger adhesive capabilities and induced more lysis of epithelial cells and neutrophils than serotype b or c isolates. Conversely, serotype c isolates showed relatively low leukotoxicity, while provoking NETosis to similar extents as serotype a and b isolates. Altogether, we conclude that the differential virulence presentation by Aa isolates with the dominant serotypes a, b, or c can be explained by their exoproteome heterogeneity. IMPORTANCE Periodontitis is an inflammatory disease that causes progressive destruction of alveolar bone and supporting tissues around the teeth, ultimately resulting in tooth loss. The bacterium Aggregatibacter actinomycetemcomitans (Aa) is a prevalent causative agent of periodontitis, but this oral pathogen is also associated with serious extraoral diseases like rheumatoid arthritis and Alzheimer's disease. Clinical Aa isolates are usually distinguished by serotyping, because of known serotype-specific differences in virulence. Aa with serotype b is associated with aggressive forms of periodontitis, while isolates with serotypes a or c are usually encountered in cases of mild periodontitis or healthy individuals. The molecular basis for these differences in virulence was so far unknown. In the present study, we pinpoint serotype-specific differences in virulence factor production by clinical Aa isolates. We consider these findings important, because they provide new leads for future preventive or therapeutic approaches to fight periodontitis and associated morbidities.

Partially overlapping substrate specificities of staphylococcal group A sortases.

Sortases catalyze the covalent attachment of proteins with a C-terminal LPxTG motif to the cell walls of Gram-positive bacteria. Here, we show that deletion of the srtA genes of Staphylococcus aureus and Staphylococcus epidermidis resulted in the dislocation of several LPxTG proteins from the cell wall to the growth medium. Nevertheless, proteomics and Western blotting analyses revealed that substantial amounts of the identified proteins remained cell wall bound through noncovalent interactions. The protein dislocation phenotypes of srtA mutants of S. aureus and S. epidermidis were reverted by ectopic expression of srtA genes of either species. Interestingly, S. epidermidis contains a second sortase A, which was previously annotated as ``SrtC.'' Ectopic expression of this SrtC in srtA mutant cells reverted the dislocation of some, but not all, cell wall associated proteins. Similarly, defects in biofilm formation were reverted by ectopic expression of SrtC in some, but not all, tested srtA mutant strains. Finally, overexpression of SrtA resulted in increased levels of biofilm formation in some tested strains. Taken together, these findings show that the substrate specificities of SrtA and SrtC overlap partially, and that sortase levels may be limiting for biofilm formation in some staphylococci.

Requirement of signal peptidase ComC and thiol-disulfide oxidoreductase DsbA for optimal cell surface display of pseudopilin ComGC in Staphylococcus aureus.

Staphylococcus aureus is an important Gram-positive bacterial pathogen producing many secreted and cell surface-localized virulence factors. Here we report that the staphylococcal thiol-disulfide oxidoreductase DsbA is essential for stable biogenesis of the ComGC pseudopilin. The signal peptidase ComC is indispensable for ComGC maturation and optimal cell surface exposure.

Staphylococcus aureus cell wall maintenance - the multifaceted roles of peptidoglycan hydrolases in bacterial growth, fitness, and virulence.

Staphylococcus aureus is an important human and livestock pathogen that is well-protected against environmental insults by a thick cell wall. Accordingly, the wall is a major target of present-day antimicrobial therapy. Unfortunately, S. aureus has mastered the art of antimicrobial resistance, as underscored by the global spread of methicillin-resistant S. aureus (MRSA). The major cell wall component is peptidoglycan. Importantly, the peptidoglycan network is not only vital for cell wall function, but it also represents a bacterial Achilles' heel. In particular, this network is continuously opened by no less than 18 different peptidoglycan hydrolases (PGHs) encoded by the S. aureus core genome, which facilitate bacterial growth and division. This focuses attention on the specific functions executed by these enzymes, their subcellular localization, their control at the transcriptional and post-transcriptional levels, their contributions to staphylococcal virulence and their overall importance in bacterial homeostasis. As highlighted in the present review, our understanding of the different aspects of PGH function in S. aureus has been substantially increased over recent years. This is important because it opens up new possibilities to exploit PGHs as innovative targets for next-generation antimicrobials, passive or active immunization strategies, or even to engineer them into effective antimicrobial agents.

Connections between Exoproteome Heterogeneity and Virulence in the Oral Pathogen Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterial pathogen associated with severe periodontitis and nonoral diseases. Clinical isolates of A. actinomycetemcomitans display a rough (R) colony phenotype with strong adherent properties. Upon prolonged culturing, nonadherent strains with a smooth (S) colony phenotype emerge. To date, most virulence studies on A. actinomycetemcomitans have been performed with S strains of A. actinomycetemcomitans, whereas the virulence of clinical R isolates has received relatively little attention. Since the extracellular proteome is the main bacterial reservoir of virulence factors, the present study was aimed at a comparative analysis of this subproteome fraction for a collection of R isolates and derivative S strains, in order to link particular proteins to the virulence of A. actinomycetemcomitans with serotype b. To assess the bacterial virulence, we applied different infection models based on larvae of the greater wax moth Galleria mellonella, a human salivary gland-derived epithelial cell line, and freshly isolated neutrophils from healthy human volunteers. A total number of 351 extracellular A. actinomycetemcomitans proteins was identified by mass spectrometry, with the S strains consistently showing more extracellular proteins than their parental R isolates. A total of 50 known extracellular virulence factors was identified, of which 15 were expressed by all investigated bacteria. Importantly, the comparison of differences in exoproteome composition and virulence highlights critical roles of 10 extracellular proteins in the different infection models. Together, our findings provide novel clues for understanding the virulence of A. actinomycetemcomitans and for development of potential preventive or therapeutic avenues to neutralize this important oral pathogen. IMPORTANCE Periodontitis is one of the most common inflammatory diseases worldwide, causing high morbidity and decreasing the quality of life of millions of people. The bacterial pathogen Aggregatibacter actinomycetemcomitans is strongly associated with aggressive forms of periodontitis. Moreover, it has been implicated in serious nonoral infections, including endocarditis and brain abscesses. Therefore, it is important to investigate how A. actinomycetemcomitans can cause disease. In the present study, we applied a mass spectrometry approach to make an inventory of the virulence factors secreted by different clinical A. actinomycetemcomitans isolates and derivative strains that emerged upon culturing. We subsequently correlated the secreted virulence factors to the pathogenicity of the investigated bacteria in different infection models. The results show that a limited number of extracellular virulence factors of A. actinomycetemcomitans have central roles in pathogenesis, indicating that they could be druggable targets to prevent or treat oral disease.

Staphylococcus aureus populations from the gut and the blood are not distinguished by virulence traits-a critical role of host barrier integrity.

BACKGROUND: The opportunistic pathogen Staphylococcus aureus is an asymptomatically carried member of the microbiome of about one third of the human population at any given point in time. Body sites known to harbor S. aureus are the skin, nasopharynx, and gut. In particular, the mechanisms allowing S. aureus to pass the gut epithelial barrier and to invade the bloodstream were so far poorly understood. Therefore, the objective of our present study was to investigate the extent to which genetic differences between enteric S. aureus isolates and isolates that caused serious bloodstream infections contribute to the likelihood of invasive disease. RESULTS: Here, we present genome-wide association studies (GWAS) that compare the genome sequences of 69 S. aureus isolates from enteric carriage by healthy volunteers and 95 isolates from bloodstream infections. We complement our GWAS results with a detailed characterization of the cellular and extracellular proteomes of the representative gut and bloodstream isolates, and by assaying the virulence of these isolates with infection models based on human gut epithelial cells, human blood cells, and a small animal infection model. Intriguingly, our results show that enteric and bloodstream isolates with the same sequence type (ST1 or ST5) are very similar to each other at the genomic and proteomic levels. Nonetheless, bloodstream isolates are not necessarily associated with an invasive profile. Furthermore, we show that the main decisive factor preventing infection of gut epithelial cells in vitro is the presence of a tight barrier. CONCLUSIONS: Our data show that virulence is a highly variable trait, even within a single clone. Importantly, however, there is no evidence that blood stream isolates possess a higher virulence potential than those from the enteric carriage. In fact, some gut isolates from healthy carriers were more virulent than bloodstream isolates. Based on our present observations, we propose that the integrity of the gut epithelial layer, rather than the pathogenic potential of the investigated enteric S. aureus isolates, determines whether staphylococci from the gut microbiome will become invasive pathogens. Video Abstract.

Requirement of the agr locus for colony spreading of Staphylococcus aureus.

The important human pathogen Staphylococcus aureus is known to spread on soft agar plates. Here, we show that colony spreading of S. aureus involves the agr quorum-sensing system. This finding can be related to the agr-dependent expression of biosurfactants, such as phenol-soluble modulins, suggesting a connection between spreading motility and virulence.

The cell surface proteome of Staphylococcus aureus.

The Gram-positive bacterium Staphylococcus aureus is a wide spread opportunistic pathogen that can cause a range of life-threatening diseases. To obtain a better understanding of the global mechanisms for pathogenesis and to identify novel targets for therapeutic interventions, the S. aureus proteome has been recently 'dissected' in several studies. Proteins that are exposed on the cell surface - collectively referred to as the 'surfacome' - have received particular attention, because they can directly interact with extracellular molecules, including drugs and antibodies. Accordingly, these proteins represent interesting candidate targets for active or passive immunization against S. aureus. Here, we review the proteomics strategies used, and we compare the results that were so far obtained. Since the surfacome is part of the cell wall proteome, we first present an overview of general properties of the S. aureus cell envelope, cell wall-associated proteins and mechanisms for protein attachment to the cell wall. Then we zoom in on the surfacome, and discuss the pro's and con's of the specific strategies that have been applied for surfacome profiling. The insights thus obtained may serve as leads for future studies on the S. aureus surfacome and possible applications.