From the wound to the bench:exoproteome interplay between wound-colonizing Staphylococcus aureus strains and co-existing bacteria

Wound-colonizing microorganisms can form complex and dynamic polymicrobial communities where pathogens and commensals may co-exist, cooperate or compete with each other. The present study was aimed at identifying possible interactions between different bacteria isolated from the same chronic wound of a patient with the genetic blistering disease epidermolysis bullosa (EB). Specifically, this involved two different isolates of the human pathogen Staphylococcus aureus, and isolates of Bacillus thuringiensis and Klebsiella oxytoca. Particular focus was attributed to interactions of S. aureus with the two other species, because of the high staphylococcal prevalence among chronic wounds. Intriguingly, upon co-cultivation, none of the wound isolates inhibited each other's growth. Since the extracellular proteome of bacterial pathogens is a reservoir of virulence factors, the exoproteomes of the staphylococcal isolates in monoculture and co-culture with B. thuringiensis and K. oxytoca were characterized by Mass Spectrometry to explore the inherent relationships between these co-exisiting bacteria. This revealed a massive reduction in the number of staphylococcal exoproteins upon co-culturing with K. oxytoca or B. thuringiensis. Interestingly, this decrease was particularly evident for extracellular proteins with a predicted cytoplasmic localization, which were recently implicated in staphylococcal virulence and epidemiology. Furthermore, our exoproteome analysis uncovered potential cooperativity between the two different S. aureus isolates. Altogether, the observed exoproteome variations upon co-culturing are indicative of unprecedented adaptive mechanisms that set limits to the production of secreted staphylococcal virulence factors

Extracellular Proteome and Citrullinome of the Oral Pathogen Porphyromonas gingivalis

Porphyromonas gingivalis is an oral pathogen associated with the inflammatory disease periodontitis. Periodontitis and P. gingivalis have been associated with rheumatoid arthritis. One of the hallmarks of rheumatoid arthritis is the loss of tolerance against citrullinated proteins. Citrullination is a post translational modification of arginine residues, leading to a change in structure and function of the respective protein. This modification, which is catalyzed by peptidylarginine deiminases (PADs), plays a role in several physiological processes in the human body. Interestingly, P. gingivalis secretes a citrullinating enzyme, known as P. gingivalis PAD (PPAD), which targets bacterial and human proteins. Because the extent of P. gingivalis protein citrullination by PPAD was not yet known, the present study was aimed at identifying the extracellular proteome and citrullinome of P. gingivalis. To this end, extracellulai proteins of two reference strains, two PPAD-deficient mutants, and three clinical isolates of P. gingivalis were analyzed by mass spectrometry. The results uncovered substantial heterogeneity in the extracellular proteome and citrullinome of P. gingivalis, especially in relation to the extracellular detection of typical cytoplasmic proteins. In contrast, the major virulence factors of P. gingivalis were identified in all investigated isolates, although their citrullination was shown to vary. This may be related to post-translational processing of the PPAD enzyme. Altogether, our findings focus attention on the possible roles of 6 to 25 potentially citrullinated proteins, especially the gingipain RgpA, in periodontitis and rheumatoid arthritis.</p

Protein Arginine Phosphorylation in Staphylococcus aureus COL

Reversible posttranslationelle Modifikationen spielen eine wichtige Rolle in der Regulation zentraler Prozesse in bakteriellen Zellen. Insbesondere die Phosphorylierung von Proteinen kann beispielsweise Signaltransduktionsprozesse beeinflussen und eine differenzierte Reaktion der Zelle auf Stress und Umweltbedingungen ermöglichen. So ist zum Beispiel der humanpathogene Organismus Staphylococcus aureus in der Lage, sich an die veränderten Bedingungen während der Besiedlung des menschlichen Wirtes anzupassen. Aus diesem Grund ermöglicht die Untersuchung von Phosphorylierungen in S. aureus ein besseres Verständnis der Pathophysiologie und Virulenz dieses Organismus. Neben dem Wissen über relativ stabile Phosphorylierungen an den Aminosäuren Serin, Threonin und Tyrosin gewinnen dabei vor allem Erkenntnisse über energiereichere Phosphorylierungen, beispielsweise an Argininen, eine immer größere wissenschaftliche Aufmerksamkeit. Ein Ziel dieser Arbeit war es daher, Vorkommen und biologische Relevanz dieser Proteinmodifikation im globalen Maßstab zu untersuchen. Es gelang in einem ersten Schritt, die Analyse dieser Modifikation methodisch zu optimieren und daraufhin acht Argininphosphorylierungen im Wildtyp S. aureus COL zu identifizieren. In einem zweiten Schritt wurde eine Deletionsmutante analysiert, deren fehlendes Gen ptpB für eine Argininphosphatase codiert. Die Charakterisierung dieses Enzyms in vitro bewies dessen Aktivität und Spezifität in Bezug auf Phosphorylierungen an Argininresten und ermöglichte im weiteren Verlauf die globale Analyse des Phosphoproteoms mit dem Fokus auf Argininphosphorylierungen. Neben der Optimierung der Phosphopeptidanreicherung als Teil der Probenvorbereitung wurde im Zuge dieser Analyse auch die Datenauswertung an die Herausforderungen der energiereichen Phosphorylierungen angepasst. Hierzu wurde die klassische Datenbanksuche um eine Analyse mittels Spektrenbibliotheken erweitert. Mittels synthetischer Peptide konnten qualitativ hochwertige Massenspektren generiert sowie die datenbankgestützten Auswerteverfahren zur Sicherstellung der Datenqualität der Spektrenbibliothek verifiziert werden. In einem weiteren Schritt wurde S. aureus COL in einer Vielzahl von Bedingungen kultiviert und die Analyse mehrerer subzellulärer Fraktionen dazu genutzt, eine möglichst große Abdeckung des Proteoms zu erreichen. Die Kombination aus den Spektren der oben genannten synthetischen Peptide, den Spektren der unphosphorylierten Peptide aus den umfangreichen Kultivierungsansätzen sowie den Spektren der angereicherten Phosphopeptide ermöglichte so schlussendlich die Konstruktion einer Spektrenbibliothek mit 2.270 Proteinen, von denen 392 an mindestens einem Peptid phosphoryliert sind. Ein Vergleich der datenbankgestützten Analyse mit der Analyse durch die erstellten Spektrenbibliotheken zeigte im weiteren Verlauf, dass für die Analyse von Argininphosphorylierungen die spektrenbibliotheksbasierte Analyse eindeutige Vorteile in Bezug auf die Reproduzierbarkeit innerhalb biologischer Replikate aufweist und somit eine zentrale Herausforderung in der Phosphoproteomik untersucht. Deswegen wurden diese Spektrenbibliotheken für eine Analyse des Phosphoproteoms von S. aureus unter Kontroll- und Stressbedingungen genutzt. So konnten in der Mutante 215 an einem Arginin phosphorylierte Peptide unter Kontrollbedingungen identifiziert werden und 117 nach oxidativem Stress. Der oxidative Stress wurde hierbei nach Wachstumsstudien zur phänotypischen Charakterisierung der Mutante ausgewählt, da diese Bedingung nach oxidativem Stress die deutlichsten Veränderungen im Vergleich zum Wildtyp aufwies. Diese phänotypischen Veränderungen konnten im letzten Teil dieser Arbeit auch quantitativ adressiert werden. Im Zuge einer Gesamtproteomquantifizierung von Wildtyp und Mutante unter Kontroll- und Stressbedingungen kristallisierte sich so ein Einfluss der ptpB Deletion auf den Aminosäurestoffwechsel, die oxidative Stressantwort und die Virulenz heraus. Die Quantifizierung von Phosphopeptiden mittels einer Kombination der spektrenbibliotheksbasierten Analyse mit einer Census-basierten Auswertung ermöglichte schließlich die Bestätigung der im Gesamtproteom gemachten Beobachtungen.Reversible posttranslational modifications play an important role during the regulation of many central processes in bacterial cells. Protein phosphorylation, in particular, can influence signal transduction processes and thus enables a distinct reaction of the cell to different stress and environmental conditions. In the case of the human pathogen Staphylococcus aureus, protein phosphorylation is involved in the adaptation to changing conditions during colonisation of human hosts. For this reason, the investigation of phosphorylations in S. aureus allows a better understanding of pathophysiology and virulence of this organism. Apart from stable phosphorylations at the amino acids serine, threonine and tyrosine, insights into energy-rich phosphorylations, for instance at arginine residues, gain more and more scientific attention. For this reason, one purpose of this study was the investigation of incidence and physiological relevance of this protein modification at a global scale. Firstly, the analysis of this modification was methodically optimised resulting in the identification of eight arginine phosphorylations in wild type cells of S. aureus COL. Secondly, the deletion mutant ΔptpB missing the gene that codes for an arginine phosphatase, was analysed. The characterisation of PtpB in vitro proved its activity and specificity towards arginine phosphorylations. This enabled the global analysis of the phosphoproteome with a focus on arginine phosphorylations. In addition to the optimisation of the phosphopeptide enrichment as part of the sample preparation, the data analysis process was adapted to the special challenges of energy-rich phosphorylations. Here, classical database search was extended by spectral library based analyses. In addition, synthetic peptides allow the generation of high quality mass spectra and the verification of database based evaluation strategies to ensure the quality of the spectral library. Next, S. aureus COL was cultivated under various conditions and several subcellular fractions were analysed with the aim to cover a broad part of the proteome. The combination of the spectra of synthetic peptides, the spectra of non-phosphorylated peptides from extensive cultivation experiments and the spectra of enriched phosphopeptides rendered the construction of a spectral library possible. This contained 2,270 proteins out of which 392 were found to be phosphorylated. A comparison of the database based analysis with spectral library based analysis showed the advantages of the latter when comparing the reproducibility of biological replicates. Thereby a permanent issue in phosphoproteomics was investigated. Hence, spectral libraries were used for the analysis of the phosphoproteome of S. aureus under control and stress conditions. 215 arginine phosphosites were identified within the mutant under control conditions and 117 under oxidative stress conditions. Oxidative stress was chosen because phenotypic characterisation of the mutant revealed that the most distinct growth changes in comparison with the wild type occurred after oxidative stress. These phenotypic changes were quantitatively approached in the last part of this work. Total proteome quantification of the wild type and mutant under control and stress conditions revealed an influence of the ptpB deletion on amino acid metabolism, oxidative stress response and virulence. The quantification of phosphopeptides by means of a combination of spectral library with Census based analysis finally confirmed the observations made during total proteome quantification

From the wound to the bench: exoproteome interplay between wound-colonizing Staphylococcus aureus strains and co-existing bacteria

Wound-colonizing microorganisms can form complex and dynamic polymicrobial communities where pathogens and commensals may co-exist, cooperate or compete with each other. The present study was aimed at identifying possible interactions between different bacteria isolated from the same chronic wound of a patient with the genetic blistering disease epidermolysis bullosa (EB). Specifically, this involved two different isolates of the human pathogen Staphylococcus aureus, and isolates of Bacillus thuringiensis and Klebsiella oxytoca. Particular focus was attributed to interactions of S. aureus with the two other species, because of the high staphylococcal prevalence among chronic wounds. Intriguingly, upon co-cultivation, none of the wound isolates inhibited each other's growth. Since the extracellular proteome of bacterial pathogens is a reservoir of virulence factors, the exoproteomes of the staphylococcal isolates in monoculture and co-culture with B. thuringiensis and K. oxytoca were characterized by Mass Spectrometry to explore the inherent relationships between these co-exisiting bacteria. This revealed a massive reduction in the number of staphylococcal exoproteins upon co-culturing with K. oxytoca or B. thuringiensis. Interestingly, this decrease was particularly evident for extracellular proteins with a predicted cytoplasmic localization, which were recently implicated in staphylococcal virulence and epidemiology. Furthermore, our exoproteome analysis uncovered potential cooperativity between the two different S. aureus isolates. Altogether, the observed exoproteome variations upon co-culturing are indicative of unprecedented adaptive mechanisms that set limits to the production of secreted staphylococcal virulence factors

Clp-targeting BacPROTACs impair mycobacterial proteostasis and survival

The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics

Homo-BacPROTAC-induced degradation of ClpC1 as a strategy against drug-resistant mycobacteria

Antimicrobial resistance is a global health threat that requires development of new treatment concepts. These should not only overcome existing resistance, but be designed to slow down emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We developed proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs were derived from cyclomarins, natural products known to bind to ClpC1, which were dimerized resulting in compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduced levels of endogenous ClpC1 in Mycobacterium smegmatis, as well as displayed minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug resistant Mtb isolates. The compounds also killed Mtb resident in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance

Homo-BacPROTAC-induced degradation of ClpC1 as a strategy against drug-resistant mycobacteria

Abstract Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance