Anne-Kathrin Ziebandt

Mapping the Pathways to Staphylococcal Pathogenesis by Comparative Secretomics

SUMMARY The gram-positive bacterium Staphylococcus aureus is a frequent component of the human microbial flora that can turn into a dangerous pathogen. As such, this organism is capable of infecting almost every tissue and organ system in the human body. It does so by actively exporting a variety of virulence factors to the cell surface and extracellular milieu. Upon reaching their respective destinations, these virulence factors have pivotal roles in the colonization and subversion of the human host. It is therefore of major importance to obtain a clear understanding of the protein transport pathways that are active in S. aureus. The present review aims to provide a state-of-the-art roadmap of staphylococcal secretomes, which include both protein transport pathways and the extracytoplasmic proteins of these organisms. Specifically, an overview is presented of the exported virulence factors, pathways for protein transport, signals for cellular protein retention or secretion, and the exoproteomes of different S. aureus isolates. The focus is on S. aureus, but comparisons with Staphylococcus epidermidis and other gram-positive bacteria, such as Bacillus subtilis, are included where appropriate. Importantly, the results of genomic and proteomic studies on S. aureus secretomes are integrated through a comparative “secretomics” approach, resulting in the first definition of the core and variant secretomes of this bacterium. While the core secretome seems to be largely employed for general housekeeping functions which are necessary to thrive in particular niches provided by the human host, the variant secretome seems to contain the “gadgets” that S. aureus needs to conquer these well-protected niches.

Repair of Global Regulators in Staphylococcus aureus 8325 and Comparative Analysis with Other Clinical Isolates

ABSTRACT The pathogenicity of Staphylococcus aureus strains varies tremendously (as seen with animals). It is largely dependent on global regulators, which control the production of toxins, virulence, and fitness factors. Despite the vast knowledge of staphylococcal molecular genetics, there is still widespread dispute over what factors must come together to make a strain highly virulent. S. aureus NCTC8325 (RN1 and derivatives) is a widely used model strain for which an incomparable wealth of knowledge has accumulated in the almost 50 years since its isolation. Although RN1 has functional agr, sarA, and sae global regulators, it is defective in two regulatory genes, rsbU (a positive activator of SigB) and tcaR (an activator of protein A transcription), and is therefore considered by many to be a poor model for studies of regulation and virulence. Here, we repaired these genes and compared the resulting RN1 derivatives with other widely used strains, Newman, USA300, UAMS-1, and COL, plus the parental RN1, with respect to growth, extracellular protein pattern, hemolytic activity, protein A production, pigmentation, biofilm formation, and mouse lethality. The tcaR-repaired strain, showed little alteration in these properties. However, the rsbU-repaired strain was profoundly altered. Hemolytic activity was largely decreased, the exoprotein pattern became much more similar to that of typical wild-type (wt) S. aureus, and there was a surprising increase in mouse lethality. We note that each of the strains tested has a mutational alteration in one or more other regulatory functions, and we conclude that the repaired RN1 is a good model strain for studies of staphylococcal regulation and pathobiology; although strain Newman has been used extensively for such studies in recent years, it has a missense mutation in saeS, the histidine kinase component of the sae signaling module, which profoundly alters its regulatory phenotype. If this mutation were repaired, Newman would be considerably improved as a model strain.

Extracellular proteins of Staphylococcus aureus and the role of SarA and sigma B.

Staphlococcus aureus synthesizes a large number of extracellular proteins that have been postulated to play a role in bacterial virulence. The proteomic approach was used to analyse the pattern of extracellular proteins of two different S. aureus strains, RN6390 and COL. Thirty‐nine protein spots were identified by N‐terminal sequencing or MALDI‐TOF‐MS. The differences of the extracellular protein patterns between both strains are striking. Among the 18 proteins identified in S. aureus COL there are nine proteins not yet discovered in S. aureus RN6390. These are enterotoxin B, leukotoxin D, enterotoxin, serin proteases (SplA and SplC), thermonuclease, an IgG binding protein and two so far unknown proteins in S. aureus with similarities to SceD precursor in Staphylococcus carnosus and to synergohymenotropic toxin precursor in Streptococcus intermedius. In contrast, lipase as well as staphylokinase identified in S. aureus RN6390 were not detectable in S. aureus COL under the same conditions. By using a regulatory mutant of sarA (ALC136) isogenic to strain RN6390 we identified five proteins positively regulated by SarA and 12 proteins negatively regulated by SarA. Besides V8 protease (StsP) and Hlb already described to be regulated by the sar locus new putatively sarA‐dependent proteins were identified, e.g. glycerolester hydrolase and autolysin both down‐regulated in the sarA mutant, and aureolysin, staphylokinase, staphopain and format tetrahydrofolate lyase up‐regulated in the mutant. Moreover, the role of σB in expression of extracellular proteins was studied. Interestingly, we found 11 proteins at an enhanced level in a sigB mutant of S. aureus COL, among them enterotoxin B, α and β hemolysin, serine proteases SplA and SplB, leukotoxin D, and staphopain homologues. The σB‐dependent repression of gene expression occurs at the transcriptional level. Only one protein, SceD, was identified whose synthesis was down‐regulated in the mutant indicating that its gene belongs to the σB‐dependent general stress regulon.

Characterization of the sigma(B) regulon in Staphylococcus aureus.

The sigma(B)-dependent stress regulon in gram-positive bacteria might fulfill a physiological role in stress response and virulence similar to that of the sigma(S) regulon in Escherichia coli and other gram-negative bacteria. In order to obtain evidence for the function of the sigma(B) regulon of Staphylococcus aureus, especially in virulence control, sigma(B)-dependent stress genes were identified. The two-dimensional protein pattern of wild-type cells of S. aureus COL was compared with that of an isogenic sigB mutant. By this approach, we found that the synthesis of about 27 cytoplasmic proteins seemed to be under the positive control of sigma(B). N-terminal sequencing of 18 proteins allowed the identification of their genes on the almost finished genome sequence of S. aureus COL and the analysis of the promoter structure. Transcriptional analyses of 11 of these genes confirmed their sigma(B) dependency, and moreover, about 7 additional sigma(B)-dependent genes were found which are cotranscribed with the newly detected genes, forming operons. Altogether, we identified 23 sigma(B)-dependent genes and their corresponding proteins. Among them are proteins probably involved in the generation of NADH or in membrane transport mechanisms. Furthermore, at least one clpC-homologous gene was localized on the S. aureus sequence solely transcribed by sigma(B). In contrast, a second clpC-homologous gene in S. aureus forming an operon with ctsR, yacH, and yacI was sigma(B) independently expressed.

Proteomics uncovers extreme heterogeneity in the Staphylococcus aureus exoproteome due to genomic plasticity and variant gene regulation

Sequencing of at least 13 Staphylococcus aureus isolates has shown that genomic plasticity impacts significantly on the repertoire of virulence factors. However, genome sequencing does not reveal which genes are expressed by individual isolates. Here, we have therefore performed a comprehensive survey of the composition and variability of the S. aureus exoproteome. This involved multilocus sequence typing, virulence gene, and prophage profiling by multiplex PCR, and proteomic analyses of secreted proteins using 2‐DE. Dissection of the exoproteomes of 25 clinical isolates revealed that only seven out of 63 identified secreted proteins were produced by all isolates, indicating a remarkably high exoproteome heterogeneity within one bacterial species. Most interesting, the observed variations were caused not only by genome plasticity, but also by an unprecedented variation in secretory protein production due to differences in transcriptional and post‐transcriptional regulation. Our data imply that genomic studies on virulence gene conservation patterns need to be complemented by analyses of the extracellular protein pattern to assess the full virulence potential of bacterial pathogens like S. aureus. Importantly, the extensive variability of secreted virulence factors in S. aureus also suggests that development of protective vaccines against this pathogen requires a carefully selected combination of invariably produced antigens.

Staphylococcal Major Autolysin (Atl) Is Involved in Excretion of Cytoplasmic Proteins

Many microorganisms excrete typical cytoplasmic proteins into the culture supernatant. As none of the classical secretion systems appears to be involved, this type of secretion was referred to as “nonclassical protein secretion.” Here, we demonstrate that in Staphylococcus aureus the major autolysin plays a crucial role in release of cytoplasmic proteins. Comparative secretome analysis revealed that in the wild type S. aureus strain, 22 typical cytoplasmic proteins were excreted into the culture supernatant, although in the atl mutant they were significantly decreased. The presence or absence of prophages had little influence on the secretome pattern. In the atl mutant, secondary peptidoglycan hydrolases were increased in the secretome; the corresponding genes were transcriptionally up-regulated suggesting a compensatory mechanism for the atl mutation. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic indicator enzyme, we showed that all clinical isolates tested excreted this protein. In the wall teichoic acid-deficient tagO mutant with its increased autolysis activity, GAPDH was excreted in even higher amounts than in the WT, confirming the importance of autolysis in excretion of cytoplasmic proteins. To answer the question of how discriminatory the excretion of cytoplasmic proteins is, we performed a two-dimensional PAGE of cytoplasmic proteins isolated from WT. Surprisingly, the most abundant proteins in the cytoplasm were not found in the secretome of the WT, suggesting that there exists a selection mechanism in the excretion of cytoplasmic proteins. As the major autolysin binds at the septum site, we assume that the proteins are preferentially released at and during septum formation.

Role of the Twin-Arginine Translocation Pathway in Staphylococcus

In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (DeltatatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of DeltatatAC and Deltatat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB.