Harald Kusch

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.

Proteomic analysis of antioxidant strategies of Staphylococcus aureus: diverse responses to different oxidants.

The high resolution 2‐D protein gel electrophoresis technique combined with MALDI‐TOF MS and a recently developed fluorescence‐based thiol modification assay were used to investigate the cellular response of Staphylococcus aureus to oxidative stress. Addition of hydrogen peroxide, diamide, and the superoxide generating agent paraquat to exponentially growing cells revealed complex changes in the protein expression pattern. In particular, proteins involved in detoxification, repair systems, and intermediary metabolism were found to be up‐regulated. Interestingly, there is only a small overlap of proteins induced by all these stressors. Exposure to hydrogen peroxide mediated a significant increase of DNA repair enzymes, whereas treatment with diamide affected proteins involved in protein repair and degradation. The activity of proteins under oxidative stress conditions can be modulated by oxidation of thiol groups. In growing cells, protein thiols were found to be mainly present in the reduced state. Diamide mediated a strong increase of reversibly oxidized thiols in a variety of metabolic enzymes. By contrast, hydrogen peroxide resulted in the reversible oxidation especially of proteins with active site cysteines. Moreover, high levels of hydrogen peroxide influenced the pI of three proteins containing cysteines within their active sites (GapA1, AhpC, and HchA) indicating the generation of sulfinic or sulfonic acid by irreversible oxidation of thiols.

Human Immune Proteome in Experimental Colonization with Staphylococcus aureus

ABSTRACT More than 20% of adults are persistently colonized with Staphylococcus aureus. When hospitalized, these carriers have increased risks of infection with their own strains. However, a recent study demonstrated a lower incidence of bacteremia-related death among carriers than among noncarriers, raising the question whether the adaptive immune system plays a protective role. In fact, S. aureus carriers mount a highly specific neutralizing antibody response against superantigens of their colonizing strains. We now used 2-dimensional immunoblotting to investigate the profiles of antibodies from healthy individuals against S. aureus extracellular proteins. Moreover, we tested whether symptom-free experimental colonization of these individuals with an S. aureus strain of low virulence, 8325-4, is sufficient to induce an antibody response. Sera obtained before and 4 weeks after colonization were screened for immunoglobulin G (IgG) antibody binding to extracellular staphylococcal proteins. At baseline, most volunteers harbored IgG directed against conserved virulence factors, including alpha-hemolysin (Hla), beta-hemolysin (Hlb), phospholipase C (Plc), staphylococcal serine protease (SspA), and cysteine protease (SspB). However, the variability of spot patterns and intensities was striking and could be important in case of infection. Experimental nasal colonization with S. aureus 8325-4 did not elicit new antibodies or boost the humoral response. Thus, the high antibody prevalence in humans is likely not induced by short-term nasal colonization, and presumably minor infections are required to trigger anti-S. aureus antibody responses.

Verticillium transcription activator of adhesion Vta2 suppresses microsclerotia formation and is required for systemic infection of plant roots

Six transcription regulatory genes of the Verticillium plant pathogen, which reprogrammed nonadherent budding yeasts for adhesion, were isolated by a genetic screen to identify control elements for early plant infection. Verticillium transcription activator of adhesion Vta2 is highly conserved in filamentous fungi but not present in yeasts. The Magnaporthe grisea ortholog conidiation regulator Con7 controls the formation of appressoria which are absent in Verticillium species. Vta2 was analyzed by using genetics, cell biology, transcriptomics, secretome proteomics and plant pathogenicity assays. Nuclear Vta2 activates the expression of the adhesin-encoding yeast flocculin genes FLO1 and FLO11. Vta2 is required for fungal growth of Verticillium where it is a positive regulator of conidiation. Vta2 is mandatory for accurate timing and suppression of microsclerotia as resting structures. Vta2 controls expression of 270 transcripts, including 10 putative genes for adhesins and 57 for secreted proteins. Vta2 controls the level of 125 secreted proteins, including putative adhesins or effector molecules and a secreted catalase-peroxidase. Vta2 is a major regulator of fungal pathogenesis, and controls host-plant root infection and H2 O2 detoxification. Verticillium impaired in Vta2 is unable to colonize plants and induce disease symptoms. Vta2 represents an interesting target for controlling the growth and development of these vascular pathogens.

Genomic and proteomic characterization of Staphylococcus aureus mastitis isolates of bovine origin.

Staphylococcus aureus colonizes and infects humans as well as animals. In the present study, 17 S. aureus strains isolated from cows suffering from mastitis were characterized. The well‐established multilocus sequence typing (MLST) technique and a diagnostic microarray covering 185 S. aureus virulence and resistance genes were used for genetic and epidemiological analyses. Virulence gene expression studies were performed by analyzing the extracellular protein pattern of each isolate on 2‐D gels. By this way, a pronounced heterogeneity of the extracellular proteome between the bovine isolates has been observed which was attributed to genome plasticity and variation of gene expression. Merely 12 proteins were expressed in at least 80% of the isolates, i.e. Atl, Aur, GlpQ, Hla, LtaS, Nuc, PdhB, SAB0846, SAB2176, SAB0566, SspA, and SspB forming the core exoproteome. Fifteen extracellular proteins were highly variably expressed and only present in less than 20% of the isolates. This includes the serine proteases SplB, C, and F, and the superantigens SEC‐bov, SEL and TSST‐1. Compared to human isolates we identified at least six proteins with significantly different expression frequencies. While SAB0846 was expressed more frequently in bovine isolates, LytM, EbpS, Spa, Geh, and LukL1 were seen less frequently in these isolates.

Characterization of the global impact of low temperature gas plasma on vegetative microorganisms.

Plasma medicine and also decontamination of bacteria with physical plasmas is a promising new field of life science with huge interest especially for medical applications. Despite numerous successful applications of low temperature gas plasmas in medicine and decontamination, the fundamental nature of the interactions between plasma and microorganisms is to a large extent unknown. A detailed knowledge of these interactions is essential for the development of new as well as for the enhancement of established plasma‐treatment procedures. In the present work we introduce for the first time a growth chamber system suitable for low temperature gas plasma treatment of bacteria in liquid medium. We have coupled the use of this apparatus to a combined proteomic and transcriptomic analyses to investigate the specific stress response of Bacillus subtilis 168 cells to treatment with argon plasma. The treatment with three different discharge voltages revealed not only effects on growth, but also clear evidence of cellular stress responses. B. subtilis suffered severe cell wall stress, which was made visible also by electron microscopy, DNA damages and oxidative stress as a result of exposure to plasma. These biological findings were supported by the detection of reactive plasma species by OES measurements.

Secrets of the secretome in Staphylococcus aureus.

Secreted proteins constitute a reservoir of virulence factors. Here, an in silico analysis of the secretome of 15 S. aureus reference strains is presented revealing that about 30% of the encoded proteome of this bacterium could be secreted. In total 1354 proteins were predicted to be localized outside the cytoplasm representing the pan-secretome of S. aureus. 41% of these proteins have been identified to be expressed using different approaches. The function of at least 50% of the secreted proteins encoded by S. aureus is not yet clear and a possible role in virulence has to be elucidated.

Proteomic analysis of the bacterial pathogen Bartonella henselae and identification of immunogenic proteins for serodiagnosis.

Bartonella henselae is a slow growing, fastidious and facultative intracellular pathogen causing cat scratch disease and vasculoproliferative disorders. To date, knowledge about the pathogenicity of this human pathogenic bacterium is limited and, additionally, serodiagnosis still needs further improvement. Here, we investigated the proteome of B. henselae using 2‐D SDS‐PAGE and MALDI‐TOF‐MS. We provide a comprehensive 2‐D proteome reference map of the whole cell lysate of B. henselae with 431 identified protein spots representing 191 different proteins of which 16 were formerly assigned as hypothetical proteins. To unravel immunoreactive antigens, we applied 2‐D SDS‐PAGE and subsequent immunoblotting using 33 sera of patients suffering from B. henselae infections. The analysis revealed 79 immunoreactive proteins of which 71 were identified. Setting a threshold of 20% seroreactivity, 11 proteins turned out to be immunodominant antigens potentially useful for an improved Bartonella‐specific serodiagnosis. Therefore, we provide for the first time (i) a comprehensive 2‐D proteome map of B. henselae for further proteome‐based studies focussed on the pathogenicity of B. henselae and (ii) an integrated view into the humoral immune responses targeted against this newly emerged human pathogenic bacterium.

Aureolib — A Proteome Signature Library: Towards an Understanding of Staphylococcus aureus Pathophysiology

Gel-based proteomics is a powerful approach to study the physiology of Staphylococcus aureus under various growth restricting conditions. We analyzed 679 protein spots from a reference 2-dimensional gel of cytosolic proteins of S. aureus COL by mass spectrometry resulting in 521 different proteins. 4,692 time dependent protein synthesis profiles were generated by exposing S. aureus to nine infection-related stress and starvation stimuli (H2O2, diamide, paraquat, NO, fermentation, nitrate respiration, heat shock, puromycin, mupirocin). These expression profiles are stored in an online resource called Aureolib (http://www.aureolib.de). Moreover, information on target genes of 75 regulators and regulatory elements were included in the database. Cross-comparisons of this extensive data collection of protein synthesis profiles using the tools implemented in Aureolib lead to the identification of stress and starvation specific marker proteins. Altogether, 226 protein synthesis profiles showed induction ratios of 2.5-fold or higher under at least one of the tested conditions with 157 protein synthesis profiles specifically induced in response to a single stimulus. The respective proteins might serve as marker proteins for the corresponding stimulus. By contrast, proteins whose synthesis was increased or repressed in response to more than four stimuli are rather exceptional. The only protein that was induced by six stimuli is the universal stress protein SACOL1759. Most strikingly, cluster analyses of synthesis profiles of proteins differentially synthesized under at least one condition revealed only in rare cases a grouping that correlated with known regulon structures. The most prominent examples are the GapR, Rex, and CtsR regulon. In contrast, protein synthesis profiles of proteins belonging to the CodY and σB regulon are widely distributed. In summary, Aureolib is by far the most comprehensive protein expression database for S. aureus and provides an essential tool to decipher more complex adaptation processes in S. aureus during host pathogen interaction.

Protecs, a comprehensive and powerful storage and analysis system for OMICS data, applied for profiling the anaerobiosis response of Staphylococcus aureus COL.

Broad functional genomic studies call for comprehensive and powerful data repositories for storage of genome sequences, experimental information, protein identification data, protein properties and expression values. The better such data repositories can integrate and display complex data in a clear and structured way the more biologically meaningful conclusions or novel hypotheses can be derived from extensive omics data sets. This work presents the web accessible database system Protecs and how it was used to support analysis of 50 samples drawn from four Staphylococcus aureus cultivations under anaerobiosis. Protecs incorporates findings from visualization science, e.g. micro charts and heat maps in the user interface. Its integrated tools for expression data analysis in combination with TIGR Multi Experiment Viewer were used to highlight similar gene expression profiles in single or multiple experiments based on the continuously updated S. aureus master gel. Raw data analysis results are available online at www.protecs.uni‐greifswald.de. Our meta‐study revealed that S. aureus responds in different anaerobiotic experimental setups (growth without oxygen; growth without oxygen but with supplemental pyruvate and uracil; growth without oxygen but with NO  3− ; growth without oxygen but with NO  3− and without functional nreABC genes) with a general anaerobiosis response. Among others, this response is characterized by an induction of fermentation enzymes (PflB, Ldh1, SACOL0135, SACOL0660) as well as the response regulator SrrA. Interestingly, especially genes with a high codon adaptation index highly overlap with anaerobically induced genes.