Proteomics of Bacillus pumilus

Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on secretome analysis, Bacillus pumilus strain Jo2, possessing high secretion capability, was chosen for an omics based investigation. The physiology of Bacillus pumilus cells growing either in minimal or complex medium was analyzed by a combination of proteomic and metabolomic methods. Master gels of the cytosolic and the secreted proteome covering major parts of the main metabolic pathways were created by means of 2D gel electrophoresis. Quantification of 2D gels allowed displaying the most abundant proteins in these sub-proteomes. Application of the GeLC-MS/MS technique tripled the number of identified proteins and enabled detection of many intrinsic membrane proteins. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43 % of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC-MS, IP-LC/MS and H-NMR methods numerous metabolites were analyzed and assigned to the reconstructed metabolic pathways. Our data indicate that applying a combination of proteomic and metabolomic techniques a comprehensive view of the physiology of growing B. pumilus cells can be gained. In addition, selected production-relevant genome features such as the restriction modification system, NRPS clusters and the secretory system of B. pumilus Jo2 are discussed. In their natural habitat, the soil, B. pumilus cells are often exposed to growth limiting conditions due to the lack of sufficient amounts of nutrients. Such limitations can also occur during fermentation conditions and will negatively influence the efficiency of the process. Glucose is the main carbon and energy source of B. pumilus. Thus, a deficiency of glucose has an enormous impact on cell growth. A 1D LC-MS/MS approach was performed to quantify the proteins using an N14/N15 labeling and to analyze the changes in the protein equipment when B. pumilus cells stop their exponential growth and become stationary due to limitation of glucose. 1033 proteins in the cytosolic fraction of B. pumilus cells were quantified and 272 of them appeared to be upregulated when the cells experience glucose starvation. 2D-PAGE was used to analyze the exoproteome of those cells. Glucose starving B. pumilus cells seemed to focus on usage of proteins and peptides as alternative carbon and energy sources instead of other carbohydrates. Especially the exoproteome of glucose starving cells is dominated by proteases and peptidases. Furthermore, cells used fatty acids as carbon source indicated by upregulation of enzymes involved in β-oxidation and the methylcitrate pathway. Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus. Using the physiological knowledge gained during our studies, we analyzed samples taken during an industrial fermentation process. Five samples were taken during the processes using a protease overexpressing B. pumilus strain and a non-overexpressing B. pumilus reference strain. 2D-PAGE was employed to analyze the samples. 448 proteins could be identified in the samples from the protease overexpressing stain as well as 453 proteins in the reference strain. The proteins were quantified relatively comparing the different growth phases of each strain as well as comparing the strains to each other. The physiological knowledge gained from the shake flask studies enabled us to interpret the findings. Both strains showed an induction of proteins involved in acquisition of alternative carbon sources and of proteins involved in degradation and usage of fatty acids, e.g. the methylcitrate pathway, when they stop exponential growth. This is comparable to the results gained from the analysis of B. pumilus cells under glucose limitation, indicating similar conditions during the processes. Especially in the late phases of the fermentation processes the cells were obviously exposed to severe stress conditions. Our results demonstrated that overexpressing cells showed a significantly stronger oxidative stress response at the end of the fermentation process compared to non-overexpressing cells, which indicated that not only the high cell densities but also the overproduction of the target protein might be responsible for these conditions.In den letzten Jahren gelangten Bacillus pumilis-Stämme als mögliche alternative Produktionsstämme mehr und mehr in den Fokus biotechnologischer Unternehmen. Basierend auf Sekretomanalysen, in denen er hohe Sekretionskapazitäten zeigte, wurde B. pumilus Jo2 für omics-basierte weiterführende Untersuchungen ausgewählt. Mittels einer Kombination aus Proteom- und Metabolomanalyse wurde die Physiologie von in einem Minimalmedium sowie in einem komplexen Medium wachsenden B. pumilus-Zellen analysiert. Mastergele aus 2D-Gelelektrophoresen cytosolischer sowie sekretierte Proteine decken den größten Teil der zentralen Stoffwechselpozesse ab. Ebenso zeigten die 2D-Gele, welche Proteine zu den höchstabuntantesten der jeweiligen Subproteome gehörten. Mittels GeLC-MS/MS konnte die Zahl der identifizierten Proteine verdreifacht und zahlreiche intrinsische Membranproteine identifiziert werden. Insgesamt wurden in wachsenden B. pumilus-Zellen 1542 Proteine identifiziert werden. Darunter waren 1182 cytosolische, 297 Membran- und Lipoproteine sowie 63 sekretierte Proteine. Somit konnten 43% der 3616 im Genom codierten Proteine abgedeckt werden. Mittels GC-MS-, IP-LC/MS- und H-NMR-Techniken wurde eine Vielzahl an Metaboliten identifiziert und den rekonstruierten Stoffwechselprozessen zugeordnet. Zusätzlich wurden einige produktionsrelevandte Features wie das restriktions-Modifikations-System, NRPS-Cluster und das Sekretionssystem im Genom identifiziert und genauer beschrieben. In seinem natürlichen Umfeld im Boden ist B. pumilus oft limitierenden Bedingungen ausgesetzt. Solche Mangelsituationen können auch im Verlauf von industriellen Fermentationsprozessen auftreten und die Enzymproduktion negativ beeinflussen. Glukose ist die bevorzugte C- und Energiequelle für B. pumilus. Daher hat ein Mangel an Glukose einen enormen Einfluss auf die Zellen. Mittels quantitativer 1D LC-MS/MS-Analyse N14/N15-markierter Proteine wurden Veränderungen in der Proteinausstattung von zellen untersucht, die aufgrund von Glukosemangel in die stationäre Phase übergehen. 1033 Proteine wurden dabei im Cytosol von B. pumilus-Zellen quantifiziert. 272 dieser Proteine zeigten unter Glukosemangel eine deutlich gesteigerte Akkumulation. 2D-PAGE wurde genutzt, um das Exoproteom unter diesen Bedingungen zu analysieren. Die Verwertung von Proteinen und Peptiden hat für B. pumilus-Zellen unter Glukoemangel eine besondere Bedeutung. Vor allem im Exoproteom dominierten Proteasen und Peptidasen. Darüber hinaus nutzen die Zellen Fettsäuren als alternative Quellen, was an einer erhöhten Akkumulation von Enzymen der β-Oxidation sowie dem Methylcitrat-Weg zu erkennen war. Es ist bereits beschrieben, dass B. pumilus-Stämme eine deutlich höhere Resistenz gegenüber oxidativen Stress aufweisen als andere industriell relevante Bacilli. In dieser Studie wurde die Reaktion von B. pumilus-Zellen auf eine Behandlung mit eienr hohen Wasserstoffperoxid-Konzentration auf Proteom- sowie auf Transkriptomebene analysiert. Es wurden Gene/Proteine induziert, die in verwandten Bacilli zu Regulons gehören, die bei der oxidativen Stressantwort dieser Organismen wichtige Funktionen erfüllen. Dazu gehören Fur, Spx, SOS oder CtsR. Bemerkenswert ist die Tatsache, dass Teile des PerR-Regulons aus B. subtilis im Genom von B. pumilus nicht codiert sind. So fehlt im genom die Katalase KatA ebenso wie das DNA-Schutzprotein MrgA oder die Alkylhydroperoxid-Reductase AhpCF. Daten dieser Studie deuten darauf hin, dass die Katalase KatX2 die Funktion der KatA bei der oxidativen Stressantwort von B. pumilus erfüllt. Die genomweite Expressionsanalyse zeigte eine Induktion von bacillithiol-relevandten Genen. Die Analyse der intrazellulären Metabolite stellte eine hohe intrazelluläre Konzentration dieses schützenden Metabolites fest, was darauf hin deutet, dass Bacillithiol an der Peroxid-Stressantwort von B. pumilus beteiligt ist. Basierend auf den bisherigen physiologischen Erkenntnissen wurden Proben aus dem einem industriellen Fermentationsprozess analysiert. Fünf Proben wurden im Verlauf des Prozesses untersucht, sowohl für einen Protease-überexprimierenden Stamm als auch einen nicht-überexprimierenden Referenzstamm. Mittels 2D-PAGE wurden 228 Proteine in überexprimierenden Zellen und 453 Proteine in nicht-überexprimierenden Zellen identifiziert. Diese Proteine wurden relativ quantifiziert und die verschiendenen probenzeitpunkte miteinander verglichen. Die Erfahrungen aus den Schüttelkolben-Versuchen ermöglichten eine Interpretation der Ergebnisse. Beide Stämme zeigten eine Induktion von Proteinen, die an der Verwertung alternativer Kohlenstoff-Quellen beteiligt sind und solcher, die an der Verwertung von Fettsäuren beteiligt sind wie dem Methylcitrat-Weg. das ist vergleichbar mit den Ergebnissen, die bei der Analyse der reaktion auf Glukoselimitation gewonnen wurden und deutet auf ähnliche Bedingungen während der Fermentation hin. Besonders in den späteren Phasen der Prozesse waren die Zellen offensichtlich diversen Stressbedingungen ausgesetzt. Die Ergebnisse zeigten, dass gegen Ende des Prozesses in überexprimierende Zellen eine deultlich stärkere oxidative Stressantwort zu sehen war als in nicht-überexprimierenden Zellen. das deutet darauf hin, dass nicht nur hohe Zelldichten für diese Stresssituation verantwortlich sind, sondern auch die Überexpression eines Proteins einen Einfluss darauf hat

Toward the Relevance of Platelet Subpopulations for Transfusion Medicine

Circulating platelets consist of subpopulations with different age, maturation state and size. In this review, we address the association between platelet size and platelet function and summarize the current knowledge on platelet subpopulations including reticulated platelets, procoagulant platelets and platelets exposing signals to mediate their clearance. Thereby, we emphasize the impact of platelet turnover as an important condition for platelet production in vivo. Understanding of the features that characterize platelet subpopulations is very relevant for the methods of platelet concentrate production, which may enrich or deplete particular platelet subpopulations. Moreover, the concept of platelet size being associated with platelet function may be attractive for transfusion medicine as it holds the perspective to separate platelet subpopulations with specific functional capabilities

Bacillus pumilus KatX2 confers enhanced hydrogen peroxide resistance to a Bacillus subtilis PkatA::katX2 mutant strain

Abstract Background Bacillus pumilus cells exhibit a significantly higher resistance to hydrogen peroxide compared to closely related Bacilli like Bacillus subtilis. Results In this study we analyzed features of the catalase KatX2 of B. pumilus as one of the most important parts of the cellular response to hydrogen peroxide. KatX2, the vegetative catalase expressed in B. pumilus, was compared to the vegetative catalase KatA of B. subtilis. Data of our study demonstrate that B. pumilus can degrade toxic concentrations of hydrogen peroxide faster than B. subtilis. By replacing B. subtilis katA gene by katX2 we could significantly enhance its resistance to H2O2 and its potential to eliminate this toxic compound. Mutant cells showed a 1.5- to 2-fold higher survival to toxic concentrations of hydrogen peroxide compared to wild type cells. Furthermore, we found reversible but also irreversible oxidations of the KatX2 protein which, in contrast to KatA, contains several cysteine residues. Conclusions Our study indicates that the catalase KatX2 plays a major role in the increased resistance of B. pumilus to oxidative stress caused by hydrogen peroxide. Resistance to hydrogen peroxide of other Bacilli can be enhanced by exchanging the native catalase in the cells with katX2

Activated platelets kill Staphylococcus aureus, but not Streptococcus pneumoniae—The role of FcγRIIa and platelet factor 4/heparinantibodies

Abstract Background Heparin induced thrombocytopenia (HIT) is likely a misdirected bacterial host defense mechanism. Platelet factor 4 (PF4) binds to polyanions on bacterial surfaces exposing neo‐epitopes to which HIT antibodies bind. Platelets are activated by the resulting immune complexes via FcγRIIA, release bactericidal substances, and kill Gram‐negative Escherichia coli. Objectives To assess the role of PF4, anti‐PF4/H antibodies and FcγRIIa in killing of Gram‐positive bacteria by platelets. Methods Binding of PF4 to protein‐A deficient Staphylococcus aureus (SA113Δspa) and non‐encapsulated Streptococcus pneumoniae (D39Δcps) and its conformational change were assessed by flow cytometry using monoclonal (KKO,5B9) and patient derived anti‐PF4/H antibodies. Killing of bacteria was quantified by counting colony forming units (cfu) after incubation with platelets or platelet releasate. Using flow cytometry, platelet activation (CD62P‐expression, PAC‐1 binding) and phosphatidylserine (PS)‐exposure were analyzed. Results Monoclonal and patient‐derived anti‐PF4/H antibodies bound in the presence of PF4 to both S. aureus and S. pneumoniae (1.6‐fold increased fluorescence signal for human anti‐PF4/H antibodies to 24.0‐fold increase for KKO). Staphylococcus aureus (5.5 × 104cfu/mL) was efficiently killed by platelets (2.7 × 104cfu/mL) or their releasate (2.9 × 104cfu/mL). Killing was not further enhanced by PF4 or anti‐PF4/H antibodies. Blocking FcγRIIa had no impact on killing of S. aureus by platelets. In contrast, S. pneumoniae was not killed by platelets or releasate. Instead, after incubation with pneumococci platelets were unresponsive to TRAP‐6 stimulation and exposed high levels of PS. Conclusions Anti‐PF4/H antibodies seem to have only a minor role for direct killing of Gram‐positive bacteria by platelets. Staphylococcus aureus is killed by platelets or platelet releasate. In contrast, S. pneumoniae affects platelet viability

α‐hemolysin of Staphylococcus aureus impairs thrombus formation

Abstract Background Toxins are key virulence determinants of pathogens and can impair the function of host immune cells, including platelets. Insights into pathogen toxin interference with platelets will be pivotal to improve treatment of patients with bacterial bloodstream infections. Materials and Methods In this study, we deciphered the effects of Staphylococcus aureus toxins α‐hemolysin, LukAB, LukDE, and LukSF on human platelets and compared the effects with the pore forming toxin pneumolysin of Streptococcus pneumoniae. Activation of platelets and loss of platelet function were investigated by flow cytometry, aggregometry, platelet viability, fluorescence microscopy, and intracellular calcium release. Thrombus formation was assessed in whole blood. Results α‐hemolysin (Hla) is known to be a pore‐forming toxin. Hla‐induced calcium influx initially activates platelets as indicated by CD62P and αIIbβ3 integrin activation, but also induces finally alterations in the phenotype of platelets. In contrast to Hla and pneumolysin, S. aureus bicomponent pore‐forming leukocidins LukAB, LukED, and LukSF do not bind to platelets and had no significant effect on platelet activation and viability. The presence of small amounts of Hla (0.2 µg/ml) in whole blood abrogates thrombus formation indicating that in systemic infections with S. aureus the stability of formed thrombi is impaired. Damage of platelets by Hla was not neutralized by intravenous immune globulins. Conclusion Our findings might be of clinical relevance for S. aureus induced endocarditis. Stabilizing the aortic‐valve thrombi by inhibiting Hla‐induced impairment of platelets might reduce the risk for septic (micro‐)embolization

GPVI expression is linked to platelet size, age, and reactivity

Platelets within one individual display heterogeneity in reactivity, size, age, and expression of surface receptors. To investigate the combined intra-individual contribution of platelet size, platelet age, and receptor expression levels on the reactivity of platelets, we studied fractions of large and small platelets from healthy donors separated by differential centrifugation. Size-separated platelet fractions were perfused over a collagen-coated surface to assess thrombus formation. Multicolour flow cytometry was used to characterise resting and stimulated platelet subpopulations. Platelet age was determined based on RNA and HLA-I labelling. Signal transduction was analysed by measuring consecutive phosphorylation of serine/threonine-protein kinase Akt. Large platelets adhered faster to collagen under flow and formed larger thrombi, compared to small platelets. Among the large platelets a highly reactive juvenile platelet subpopulation was identified with high GPVI expression. Elevated GPVI expression correlated with high HLA-I expression, RNA content and increased platelet reactivity. Akt phosphorylation and activation upon collagen stimulation differed stronger between juvenile and older platelets than between large and small platelets. GPVI expression and platelet reactivity decreased throughout platelet storage at 22°C and was better maintained throughout cold storage at 4°C. We further detected a higher GPVI expression in platelets of patients with immune thrombocytopenia. Our findings show that high GPVI expression is a feature of highly reactive juvenile platelets, which are predominantly found among the large platelet population explaining the better performance of large platelets during thrombus formation. These data are important for studies of thrombus formation, platelet storage and ITP

Pneumolysin induces platelet destruction, not platelet activation, which can be prevented by immunoglobulin preparations in vitro.

Community-acquired pneumonia by primary or superinfections with Streptococcus pneumoniae can lead to acute respiratory distress requiring mechanical ventilation. The pore-forming toxin pneumolysin alters the alveolar-capillary barrier and causes extravasation of protein-rich fluid into the interstitial pulmonary tissue, which impairs gas exchange. Platelets usually prevent endothelial leakage in inflamed pulmonary tissue by sealing inflammation-induced endothelial gaps. We not only confirm that S pneumoniae induces CD62P expression in platelets, but we also show that, in the presence of pneumolysin, CD62P expression is not associated with platelet activation. Pneumolysin induces pores in the platelet membrane, which allow anti-CD62P antibodies to stain the intracellular CD62P without platelet activation. Pneumolysin treatment also results in calcium efflux, increase in light transmission by platelet lysis (not aggregation), loss of platelet thrombus formation in the flow chamber, and loss of pore-sealing capacity of platelets in the Boyden chamber. Specific anti-pneumolysin monoclonal and polyclonal antibodies inhibit these effects of pneumolysin on platelets as do polyvalent human immunoglobulins. In a post hoc analysis of the prospective randomized phase 2 CIGMA trial, we show that administration of a polyvalent immunoglobulin preparation was associated with a nominally higher platelet count and nominally improved survival in patients with severe S pneumoniae-related community-acquired pneumonia. Although, due to the low number of patients, no definitive conclusion can be made, our findings provide a rationale for investigation of pharmacologic immunoglobulin preparations to target pneumolysin by polyvalent immunoglobulin preparations in severe community-acquired pneumococcal pneumonia, to counteract the risk of these patients becoming ventilation dependent. This trial was registered at www.clinicaltrials.gov as #NCT01420744

Concentration of thiol compounds in <i>B. pumilus</i> cells.

<p>Cytosolic concentration of bacillithiol (BSH), CoA and cysteine (Cys) per mg cell dry weight (CDW) during the exponential growth phase (OD_{500 nm} 0.6 at 0 min) and 10, 30 and 60 min after H₂O₂ treatment.</p

Selected induced genes and proteins.

<p>Selected genes and proteins that are induced in H₂O₂ treated <i>B. pumilus</i> cells.</p><p>Genes and proteins are listed, which could be assigned to putative regulons known from other <i>Bacilli</i>. Complete lists of upregulated as well as downregulated genes/proteins is given in supporting information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085625#pone.0085625.s002" target="_blank">Tables S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085625#pone.0085625.s003" target="_blank">S3</a>. For transcriptome, selected genes are shown for 3 and 8 minutes after stress compared to the control conditions (0 min). For a complete list of induced and repressed genes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085625#pone.0085625.s003" target="_blank">Table S3</a>. Differential regulation was determined from the biological triplicate measurements by false-discovery rate (FDR) from the Cyber-T p-values <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085625#pone.0085625-Baldi1" target="_blank">[27]</a> by means of multiple testing correction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085625#pone.0085625-vanHijum1" target="_blank">[26]</a>. Differential regulation was defined as a two-fold or higher differential expression with a FDR cut-off value of 0.05 or lower. Protein quantification was performed by the Delta 2D software (Decodon) from 3 biological replicates with a FDR cut-off value of 0.05 or lower.</p