Patologia ed epidemiologia molecolare delle infezioni associate all'impianto

Staphylococcus aureus and Staphylococcus epidermidis are leading pathogens of implant-related infections. This study aimed at investigating the diverse distribution of different bacterial pathogen factors in most prevalent S. aureus and S. epidermidis strain types causing orthopaedic implant infections. In this study the presence both of the ica genes, encoding for biofilm exopolysaccharide production, and the insertion sequence IS256, a mobile element frequently associated to transposons, was investigated in relationship with the prevalence of antibiotic resistance among Staphylococcus epidermidis strains. The investigation was conducted on 70 clinical isolates derived from orthopaedic implant infections. Among the clinical isolates investigated a dramatic high level of association was found between the presence of ica genes as well as of IS256 and multiple resistance to all the antibiotics tested. Noteworthy, a striking full association between the presence of IS256 and resistance to gentamicin was found, being none of the IS256-negative strain resistant to this antibiotic. This association is probably because of the link of the corresponding aminoglycoside-resistance genes, and IS256, often co-existing within the same staphylococcal transposon. Moreover we investigated the prevalence of aac(6’)-Ie-aph(2’’), aph (3’) IIIa, and ant(4’) genes, encoding for the three forms of aminoglycoside-modifying enzymes (AME), responsible for resistance to aminoglycoside antibiotics. All isolates were characterized by automated ribotyping, so that the presence of antibiotic resistance determinants was investigated in strains exhibiting different ribopatterns. Interestingly, combinations of coexisting AME genes appeared to be typical of specific ribopatterns. 200 S. aureus isolates, categorized into ribogroups by automated ribotyping, i.e. rDNA restriction fragment length polymorphism analysis, were screened for the presence of a panel of adhesins genes, accessory gene regulatory (agr) polymorphisms and toxins. For many ribogroups, characteristic tandem genes arrangements could be identified. Surprisingly, the isolates of the most prevalent cluster, enlisting 27 isolates, were susceptible to almost all antibiotics and never possessed the lukD/lukE gene, thus suggesting the role of factors other than antibiotic resistance and the here investigated toxins in driving the major epidemic clone to the larger success. Afterwards, .in the predominant S. aureus cluster, the bbp gene encoding bone sialoprotein-binding protein appeared a typical virulence trait, found in 93% of the isolates. Conversely, the bbp gene was identified in just 10% of the remaining isolates of the collection. In this cluster, co-presence of bbp with the cna gene encoding collagen adhesin was a pattern consistently observed. These findings indicate a crucial role of both these adhesins, able to bind the most abundant bone proteins, in the pathogenesis of orthopaedic implant infections, there where biomaterials interface bone tissues. Moreover a PCR screening for the ebpS gene, conducted on over two hundred S. aureus clinical isolates from implant related infections revealed the detection of six strains exhibiting an altered amplicon size, shorter than expected. In order to elucidate the sequence changes present in these gene variants, the trait comprised between the primers was analyzed in all six isolates bearing the modification and in four isolates exhibiting the regular amplicon size. From nucleotide translation, the corresponding encoded protein was found to lack an entire peptide segment of 60 amino acids. These variants, missing an entire hydrophobic region, could actually facilitate current structural studies, helping to assess whether the absent domain is strictly necessary for a functional adhesin conformation and its contribution to the topology of the protein. This study suggests that epidemic clones appear to pursue different survival strategies, where adhesins, when present, exhibit diverse importance as virulence factors. A practical message arising from the present study is that strategies for the prevention and treatment of implant orthopaedic infections should target adhesins conjointly present in epidemic clones. Furthermore, the choice of reference strains for testing the anti-infective properties of biomaterials should focus on a selection of the most prevalent clones as they exhibit distinct profiles of adhesins.

Functional characterization of Streptococcus pyogenes pili

Group A Streptococcus is a Gram-positive human pathogen able to colonize both upper respiratory tract and skin. GAS is responsible for several acute diseases and autoimmune sequelae that account for half a million deaths worldwide every year (Cunningham et al., 2000). As other bacteria, GAS infections requires the capacity of the pathogen to adhere to host tissues and to form cell aggregates. The ability to persist in distinct host niches like the throat and the skin and to trigger infections is associated with the expression of different GAS virulence factors. GAS pili has been described as important virulence factors encoded by different FCT-operon regions. Based on this information, we decided to study the possible effect of environmental conditions that could regulate the pili expression. In this study we reported the influence of pH environment variations in biofilm formation for strains pertaining to a panel of different GAS FCT-types. The biofilm formation was promoted, excepted in the FCT-1 strains, by a changing in pH from physiological to acidic condition of growth in in vitro biofilm assay. By analyzing the possible association between biofilm formation and pH dependence, we have found that in FCT-2 and FCT-3 strains, the biofilm is promoted by pH reduction leading to an increase of pili expression. These data confirmed a direct link between pH dependent pilus expression and biofilm formation in GAS. As pili are a multi component structure we decided to investigate the functional role of one of its subunits, the AP-1 protein. AP-1 is highly conserved through the different FCT-types and suggests a possible essential role for the pili function. We focused our attention on the AP-1 protein encoded by the FCT-1 strains (M6). In particular this AP-1 protein contains the von Willebrand Factor A (VWFA) domain, which share an homology with the human VWFA domain that has been reported to be involved in adhesion process. We have demonstrated that the AP-1 protein binds to human epithelial cells by its VWFA domain, whereas the biofilm formation is mediated by the N-terminal region of AP-1 protein. Moreover, analyzing the importance of AP-1 in in vivo experiments we found a major capacity of tissue dissemination for the wild-type strain compared to the isogenic AP-1 deletion mutant. Pili have been also reported as potential vaccine candidates against Gram positive bacteria. For these reason we decided to investigate the relationship between cross reaction of sera raised against different GAS and GBS pilin subunits and the presence of a conserved Cna_B domain, in different pilin components. Our idea was to investigate if, using pilus conserved domains, a broad coverage vaccine against streptococcal infection could be possible.

LrhA als Regulator der Flagellen, Motilität, Chemotaxis und Typ 1 Fimbrien in Escherichia coli

Das lrhA-Gen von E. coli kodiert für einen Transkriptionsregulator der LysR-Familie. Die Funktion von LrhA war ungeklärt und sollte durch Vergleich der Gesamt-mRNA aus einem E. coli-Wildtyp und einer isogenen lrhA-Mutante mit Hilfe von Genomanalysen untersucht werden. In der lrhA-Mutante war der mRNA-Gehalt vieler Gene um den Faktor 3 bis 80 erhöht. Es handelt sich um Flagellen-, Motilitäts- und Chemotaxisgene, bzw. um Gene der Typ 1 Fimbrien. Diese Ergebnisse wurden in Expressionsmessungen bestätigt. LrhA war in der Lage an den Promotor von flhDC zu binden, aber nicht an die Promotoren der übrigen Gene für Motilität und Chemotaxis. FlhDC kodiert für den übergeordneten Regulator FlhD2C2 der Fagellensynthese.LrhA war außerdem in der Lage an die Promotoren der Gene für Typ 1 Fimbrien fimA und fimE zu binden. Typ 1 Fimbrien stellen in E. coli Virulenzfaktoren dar. Eine Regulation weiterer Virulenzfaktoren durch LrhA konnte in DNA-Pathoarrays ausgeschlossen werden.LrhA ist damit ein wichtiger Transkriptionsregulator, der die Expression der Gene für Flagellen, Motilität, Chemotaxis und Typ 1 Fimbrien reguliert. FlhDC, fimA und fimE stellen dabei direkte Zielgene von LrhA dar. Außerdem konnte eine positive Autoregulation von LrhA nachgewiesen werden.

Structural and functional characterization of Group B Streptococcus class C sortases

In Group B Streptococcus (GBS) three structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies and in vivo mutagenesis we performed a broad characterization of GBS sortase C. The high resolution X-ray structure of the enzymes revealed that the active site, located into the β-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the “lid”. We show that the catalytic triad and the predicted N- and C-terminal trans-membrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild type protein.

Trascriptome analysis and functional genomics of Neisseria meningitidis in human blood

Neisseria meningitidis (Nm) is the major cause of septicemia and meningococcal meningitis. During the course of infection, it must adapt to different host environments as a crucial factor for survival. Despite the severity of meningococcal sepsis, little is known about how Nm adapts to permit survival and growth in human blood. A previous time-course transcriptome analysis, using an ex vivo model of human whole blood infection, showed that Nm alters the expression of nearly 30% of ORFs of the genome: major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. Starting from these data, mutagenesis studies of a subset of up-regulated genes were performed and the mutants were tested for the ability to survive in human whole blood; Nm mutant strains lacking the genes encoding NMB1483, NalP, Mip, NspA, Fur, TbpB, and LctP were sensitive to killing by human blood. Then, the analysis was extended to the whole Nm transcriptome in human blood, using a customized 60-mer oligonucleotide tiling microarray. The application of specifically developed software combined with this new tiling array allowed the identification of different types of regulated transcripts: small intergenic RNAs, antisense RNAs, 5’ and 3’ untranslated regions and operons. The expression of these RNA molecules was confirmed by 5’-3’RACE protocol and specific RT-PCR. Here we describe the complete transcriptome of Nm during incubation in human blood; we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. In addition the tiling array analysis demonstrated that Nm expresses a set of new transcripts, not previously identified, and suggests the presence of a circuit of regulatory RNA elements used by Nm to adapt to proliferate in human blood.

Clostridium difficile: Entdeckung und Charakterisierung der autokatalytischen Prozessierung von Toxin B

Clostridium difficile, der Auslöser der nosokomialen Antibiotika-assoziierten Durchfälle und der Pseudomembranösen Kolitis, besitzt zwei Hauptvirulenzfaktoren: die Toxine A und B. In vorangegangenen Veröffentlichungen wurde gezeigt, dass Toxin B durch einen zytosolischen Faktor der eukaryotischen Zielzelle während des Aufnahmeweges in die Zelle gespalten wird. Nur die N-terminale katalytische Domäne erreicht das Zytosol. Hierbei wurde davon ausgegangen, dass eine Protease der Zielzelle die Spaltung katalysiert. In dieser Arbeit konnte gezeigt werden, dass die Spaltung von Toxin B ein intramolekularer Prozess ist, der zytosolisches Inositolphosphat der Zielzelle als Kofaktor zur Aktivierung der intrinsischen Protease benötigt. Die Freisetzung der katalytischen Domäne durch Inositolphosphat-induzierte Spaltung ist nicht nur das Prinzip des Clostridium difficile Toxin B sondern auch des Toxin A, als auch des alpha Toxin von Clostridium novyi und das Letale Toxin von Clostridium sordellii. Der kovalente Inhibitor von Aspartatproteasen 1,2-epoxy-3-(p-nitrophenoxy)propan (EPNP), wurde dazu verwendet die intrinsische Protease von Toxin B zu blockieren und ermöglichte die Identifikation des katalytischen Zentrums. EPNP modifiziertes Toxin B verliert die intrinsische Proteaseaktivität und Zytotoxizität, aber wenn es direkt in das Zytosol der Wirtszelle injiziert ist, bleibt die Toxizität erhalten. Diese ist damit der erste Bericht eines bakteriellen Toxins, das eukaryotische Signale zur induzierten Autoproteolyse nutzt, um seine katalytisch-toxische Domäne in das Zytosol der Zielzelle freizusetzen. Durch diese Ergebnisse kann das Modell der Toxin-Prozessierung nun um einen weiteren entscheidenden Schritt vervollständigt werden.

Biofilm formation by the anaerobic pathogen Clostridium difficile

Clostridium difficile is an obligate anaerobic, Gram-positive, endospore-forming bacterium. Although an opportunistic pathogen, it is one of the important causes of healthcare-associated infections. While toxins TcdA and TcdB are the main virulence factors of C. difficile, the factors or processes involved in gut colonization during infection remain unclear. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Little is known about biofilm formation by anaerobic gut species. Biofilm formation by C. difficile could play a role in virulence and persistence of C. difficile, as seen for other intestinal pathogens. We demonstrate that C. difficile clinical strains, 630, and the strain isolated in the outbreak, R20291, form structured biofilms in vitro. Biofilm matrix is made of proteins, DNA and polysaccharide. Strain R20291 accumulates substantially more biofilm. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella and a putative quorum sensing regulator, LuxS, Spo0A, are required for maximal biofilm formation by C. difficile. Moreover we demonstrate that bacteria in C. difficile biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI, and that inhibitory and sub-inhibitory concentrations of the same antibiotic induce biofilm formation. Surprisingly, clinical C. difficile strains from the same out-break, but from different origin, show differences in biofilm formation. Genome sequence analysis of these strains showed presence of a single nucleoide polymorphism (SNP) in the anti-σ factor RsbW, which regulates the stress-induced alternative sigma factor B (σB). We further demonstrate that RsbW, a negative regulator of alternative sigma factor B, has a role in biofilm formation and sporulation of C. difficile. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.

Drosophila melanogaster as a model to study host-parasitoid interactions: the case of the polydnaviral protein TnBVANK1

Parasitic wasps attack a number of insect species on which they feed, either externally or internally. This requires very effective strategies for suppressing the immune response and a finely tuned interference with the host physiology that is co-opted for the developing parasitoid progeny. The wealth of physiological host alterations is mediated by virulence factors encoded by the wasp or, in some cases, by polydnaviruses (PDVs), unique viral symbionts injected into the host at oviposition along with the egg, venom and ovarian secretions. PDVs are among the most powerful immunosuppressors in nature, targeting insect defense barriers at different levels. During my PhD research program I have used Drosophila melanogaster as a model to expand the functional analysis of virulence factors encoded by PDV focusing on the molecular processes underlying the disruption of the host endocrine system. I focused my research on a member of the ankyrin (ank) gene family, an immunosuppressant found in bracovirus, which associates with the parasitic wasp Toxoneuron nigriceps. I found that ankyrin disrupts ecdysone biosynthesis by impairing the vesicular traffic of ecdysteroid precursors in the cells of the prothoracic gland and results in developmental arrest.

Polymerizing activity and regulation of group B Streptococcus pilus 2a sortase C1

Group B Streptococcus [GBS; Streptococcus agalactiae] is the leading cause of life-threatening diseases in newborn and is also becoming a common cause of invasive diseases in non-pregnant, elderly and immune-compromised adults. Pili, long filamentous fibers protruding from the bacterial surface, have been discovered in GBS, as important virulence factors and vaccine candidates. Gram-positive bacteria build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates. Despite the availability of several crystal structures, pilus-related C sortases remain poorly characterized to date and their mechanisms of transpeptidation and regulation need to be further investigated. The available three-dimensional structures of these enzymes reveal a typical sortase fold except for the presence of a unique feature represented by an N-terminal highly flexible loop, known as the “lid”. This region interacts with the residues composing the catalytic triad and covers the active site, thus maintaining the enzyme in an auto-inhibited state and preventing the accessibility to the substrate. It is believed that enzyme activation may occur only after lid displacement from the catalytic domain. In this work we provide the first direct evidence of the regulatory role of the lid, demonstrating that it is possible to obtain in vitro an efficient polymerization of pilin subunits using an active C sortase lid mutant carrying a single residue mutation in the lid region. Moreover, biochemical analyses of this recombinant mutant reveal that the lid confers thermodynamic and proteolytic stability to the enzyme. A further characterization of this sortase active mutant showed promiscuity in the substrate recognition, as it is able to polymerize different LPXTG-proteins in vitro.

Molecular variability of Meningococcal antigens in carriage and disease strains and in other Neisseria species

This PhD thesis is focused on the study of the molecular variability of some specific proteins, part of the outer membrane of the pathogen Neisseria meningitidis, and described as protective antigens and important virulence factors. These antigens have been employed as components of the vaccine developed by Novartis Vaccines against N. meningitidis of serogroup B, and their variability in the meningococcal population is a key aspect when the effect of the vaccine is evaluated. The PhD project has led to complete three major studies described in three different manuscritps, of which two have been published and the third is in preparation. The thesis is structured in three main chapters, each of them dedicated to the three studies. The first, described in Chapter 1, is specifically dedicated to the analysis of the molecular conservation of meningococcal antigens in the genomes of all species classified in the genus Neisseria (Conservation of Meningococcal Antigens in the Genus Neisseria. A. Muzzi et al.. 2013. mBio 4 (3)). The second study, described in Chapter 2, focuses on the analysis of the presence and conservation of the antigens in a panel of bacterial isolates obtained from cases of the disease and from healthy individuals, and collected in the same year and in the same geographical area (Conservation of fHbp, NadA, and NHBA in carrier and pathogenic isolates of Neisseria meningitidis collected in the Czech Republic in 1993. A. Muzzi et al.. Manuscript in preparation). Finally, Chapter 3 describes the molecular features of the antigens in a panel of bacterial isolates collected over a period of 50 years, and representatives of the epidemiological history of meningococcal disease in the Netherlands (An Analysis of the Sequence Variability of Meningococcal fHbp, NadA and NHBA over a 50-Year Period in the Netherlands. S. Bambini et al.. 2013. PloS one e65043).

Structural and functional characterization of Group B Streptococcus pilus 2b

Group B Streptococcus (GBS) is a Gram-positive human pathogen representing one of the most common causes of life-threatening bacterial infections such as sepsis and meningitis in neonates. Covalently polymerized pilus-like structures have been discovered in GBS as important virulence factors as well as vaccine candidates. Pili are protein polymers forming long and thin filamentous structures protruding from bacterial cells, mediating adhesion and colonization to host cells. Gram-positive bacteria, including GBS, build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates that are the backbone protein forming the pilus shaft and two ancillary proteins. Also the cell-wall anchoring of the pilus polymers made of covalently linked pilin subunits is mediated by a sortase enzyme. GBS expresses three structurally distinct pilus types (type 1, 2a and 2b). Although the mechanisms of assembly and cell wall anchoring of GBS types 1 and 2a pili have been investigated, those of pilus 2b are not understood until now. Pilus 2b is frequently found in ST-17 strains that are mostly associated with meningitis and high mortality rate especially in infants. In this work the assembly mechanism of GBS pilus type 2b has been elucidated by dissecting through genetic, biochemical and structural studies the role of the two pilus-associated sortases. The most significant findings show that pilus 2b assembly appears “non-canonical”, differing significantly from current pilus assembly models in Gram-positive pathogens. Only sortase-C1 is involved in pilin polymerization, while the sortase-C2 does not act as a pilin polymerase, but it is involved in cell-wall pilus anchoring. Our findings provide new insights into pili biogenesis in Gram-positive bacteria. Moreover, the role of this pilus type during host infection has been investigated. By using a mouse model of meningitis we demonstrated that type 2b pilus contributes to pathogenesis of meningitis in vivo.

Molecular epidemiology of the nasal colonization by methicillin-susceptible Staphylococcus aureus in Swiss children

Nasal carriage of Staphylococcus aureus contributes to an increased risk of developing an infection with the same bacterial strain. Genetic regulatory elements and toxin-expressing genes are virulence factors associated with the pathogenic potential of S. aureus. We undertook an extensive molecular characterization of methicillin-susceptible S. aureus (MSSA) carried by children. MSSA were recovered from the nostrils of children. The presence of Panton-Valentine leukocidin (PVL), exfoliatins A and B (exfoA and exfoB), and the toxic-shock staphylococcal toxin (TSST-1) and agr group typing were determined by quantitative PCR. A multiple-locus variable-number of tandem repeat analysis (MLVA) assay was also performed for genotyping. Five hundred and seventy-two strains of MSSA were analysed. Overall, 30% were positive for toxin-expressing genes: 29% contained one toxin and 1.6% two toxins. The most commonly detected toxin gene was tst, which was present in 145 (25%) strains. The TSST-1 gene was significantly associated with the agr group 3 (OR 56.8, 95% CI 32.0-100.8). MLVA analysis revealed a large diversity of genetic content and no clonal relationship was demonstrated among the analysed MSSA strains. Multilocus sequence typing confirmed this observation of diversity and identified ST45 as a frequent colonizer. This broad diversity in MSSA carriage strains suggests a limited selection pressure in our geographical area.

Monomeric and dimeric IgG fractions show differential reactivity against pathogen-derived antigens

Polyvalent Ig preparations, derived from the pooled plasma of thousands of healthy donors, contain a complex mix of both 'acquired' and natural antibodies directed against pathogens as well as foreign and self/auto antigens (Ag). Depending on their formulation, donor pool size, etc., liquid Ig preparations contain monomeric and dimeric IgG. The dimeric IgG fraction is thought to represent mainly idiotype-antiidiotype Ab pairs. Treatment of all IgG fractions at pH 4 effectively monomerizes the IgG dimers resulting in separated idiotype-antiidiotype Ab pairs and thus in a comparable F(ab')(2) binding site availability of the different IgG fractions. Previously, we identified an increased anti-self-reactivity within the monomerized dimer fraction. This study addressed if, among the different IgG fractions, an analogous preferential reactivity was evident in the response against different pathogen-derived protein and carbohydrate antigens. Therefore, we assessed the activity of total unseparated IgG, the monomeric and dimeric IgG fractions against antigenic structures of bacterial and viral antigens/virulence factors. All fractions showed similar reactivity to protein antigens except for exotoxin A of Pseudomonas aeruginosa, where the dimeric fraction, especially when monomerized, showed a marked increase in reactivity. This suggests that the production of antiidiotypic IgG antibodies contributes to controlling the immune response to certain categories of pathogens. In contrast, the monomeric IgG fractions showed increased reactivity towards pathogen-associated polysaccharides, classically regarded as T-independent antigens. Taken together, the differential reactivity of the IgG fractions seems to indicate a preferential segregation of antibody reactivities according to the nature of the antigen.

Moraxella catarrhalis - pathogen or commensal?

Moraxella catarrhalis is an exclusively human commensal and mucosal pathogen. Its role as a disease-causing organism has long been questioned. Today, it is recognized as one of the major causes of acute otitis media in children, and its relative frequency of isolation from both the nasopharynx and the middle ear cavity has increased since the introduction of the heptavalent pneumococcal conjugate vaccine, which is associated with a shift in the composition of the nasopharyngeal flora in infants and young children. Although otitis media caused by M. catarrhalis is generally believed to be mild in comparison with pneumococcal disease, numerous putative virulence factors have now been identified and it has been shown that several surface components of M. catarrhalis induce mucosal inflammation. In adults with chronic obstructive pulmonary disease (COPD), M. catarrhalis is now a well-established trigger of approximately 10% of acute inflammatory exacerbations.Although the so-called cold shock response is a well-described bacterial stress response in species such as Escherichia coli, Bacillus subtilis or - more recently - Staphylococcus aureus, M. catarrhalis is the only typical nasopharyngeal pathogen in which this response has been investigated. Indeed, a 3-h 26°C cold shock, which may occur physiologically, when humans inspire cold air for prolonged periods of time, increases epithelial cell adherence and enhances proinflammatory host responses and may thus contribute to the symptoms referred to as common cold, which typically are attributed to viral infections.

Prevalence of genes encoding extracellular proteases in Staphylococcus aureus - important targets triggering immune response in vivo

Proteases of Staphylococcus aureus have long been considered to function as important virulence factors, although direct evidence of the role of particular enzymes remains incomplete and elusive. Here, we sought to provide a collective view of the prevalence of extracellular protease genes in genomes of commensal and pathogenic strains of S. aureus and their expression in the course of human and mouse infection. Data on V8 protease, staphopains A and B, aureolysin, and the recently described and poorly characterized group of six Spl proteases are provided. A phylogenetically diverse collection of 167 clinical isolates was analyzed, resulting in the comprehensive genetic survey of the prevalence of protease-encoding genes. No correlation between identified gene patterns with specific infections was established. Humoral response against the proteases of interest was examined in the sera derived from human patients and from a model mouse infection. The analysis suggests that at least some, if not all, tested proteases are expressed and secreted during the course of infection. Overall, the results presented in this study support the hypothesis that the secretory proteases as a group may contribute to the virulence of S. aureus.