The role of bacterial protein tyrosine phosphatases in the regulation of the biosynthesis of secreted polysaccharides

SIGNIFICANCE: Tyrosine phosphorylation and associated protein tyrosine phosphatases are gaining prominence as critical mechanisms in the regulation of fundamental processes in a wide variety of bacteria. In particular, these phosphatases have been associated with the control of the biosynthesis of capsular polysaccharides and extracellular polysaccharides, critically important virulence factors for bacteria.RECENT ADVANCES: Deletion and over-expression of the phosphatases result in altered polysaccharide biosynthesis in a range of bacteria. The recent structures of associated auto-phosphorylating tyrosine kinases has suggested that the phosphatases may be critical for the cycling of the kinases between monomers and higher order oligomers. CRITICAL ISSUES: Additional substrates of the phosphatases apart from cognate kinases are currently being identified. These are likely to be critical to our understanding of the mechanism by which polysaccharide biosynthesis is regulated. FUTURE DIRECTIONS: Ultimately, these protein tyrosine phosphatases are an attractive target for the development of novel anti-microbials. This is particularly the case for the polymerase and histidinol phosphatase family, which are predominantly found in bacteria. Furthermore, the determination of bacterial tyrosine phosphoproteomes will likely help to uncover the fundamental roles, mechanism and critical importance of these phosphatases in a wide range of bacteria.Alistair James Standish and Renato Moron

Sweet and Sour Ehrlichia: Glycoproteomics and Phosphoproteomics Reveal New Players in Ehrlichia ruminantium Physiology and Pathogenesis

Ehrlichia ruminantium; N-glycoproteins; O-GlcNAcylated proteinsEhrlichia ruminantium; N-glicoproteïnes; Proteïnes O-GlcNAciladesEhrlichia ruminantium; N-glicoproteínas; Proteínas O-GlcNAciladasUnraveling which proteins and post-translational modifications (PTMs) affect bacterial pathogenesis and physiology in diverse environments is a tough challenge. Herein, we used mass spectrometry-based assays to study protein phosphorylation and glycosylation in Ehrlichia ruminantium Gardel virulent (ERGvir) and attenuated (ERGatt) variants and, how they can modulate Ehrlichia biological processes. The characterization of the S/T/Y phosphoproteome revealed that both strains share the same set of phosphoproteins (n = 58), 36% being overexpressed in ERGvir. The percentage of tyrosine phosphorylation is high (23%) and 66% of the identified peptides are multi-phosphorylated. Glycoproteomics revealed a high percentage of glycoproteins (67% in ERGvir) with a subset of glycoproteins being specific to ERGvir (n = 64/371) and ERGatt (n = 36/343). These glycoproteins are involved in key biological processes such as protein, amino-acid and purine biosynthesis, translation, virulence, DNA repair, and replication. Label-free quantitative analysis revealed over-expression in 31 proteins in ERGvir and 8 in ERGatt. While further PNGase digestion confidently localized 2 and 5 N-glycoproteins in ERGvir and ERGatt, respectively, western blotting suggests that many glycoproteins are O-GlcNAcylated. Twenty-three proteins were detected in both the phospho- and glycoproteome, for the two variants. This work represents the first comprehensive assessment of PTMs on Ehrlichia biology, rising interesting questions regarding ER-host interactions. Phosphoproteome characterization demonstrates an increased versatility of ER phosphoproteins to participate in different mechanisms. The high number of glycoproteins and the lack of glycosyltransferases-coding genes highlight ER dependence on the host and/or vector cellular machinery for its own protein glycosylation. Moreover, these glycoproteins could be crucial to interact and respond to changes in ER environment. PTMs crosstalk between of O-GlcNAcylation and phosphorylation could be used as a major cellular signaling mechanism in ER. As little is known about the Ehrlichia proteins/proteome and its signaling biology, the results presented herein provide a useful resource for further hypothesis-driven exploration of Ehrlichia protein regulation by phosphorylation and glycosylation events. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD012589

Sweet and Sour Ehrlichia: Glycoproteomics and Phosphoproteomics Reveal New Players in Ehrlichia ruminantium Physiology and Pathogenesis

Unraveling which proteins and post-translational modifications (PTMs) affect bacterial pathogenesis and physiology in diverse environments is a tough challenge. Herein, we used mass spectrometry-based assays to study protein phosphorylation and glycosylation in Ehrlichia ruminantium Gardel virulent (ERGvir) and attenuated (ERGatt) variants and, how they can modulate Ehrlichia biological processes. The characterization of the S/T/Y phosphoproteome revealed that both strains share the same set of phosphoproteins (n = 58), 36% being overexpressed in ERGvir. The percentage of tyrosine phosphorylation is high (23%) and 66% of the identified peptides are multi-phosphorylated. Glycoproteomics revealed a high percentage of glycoproteins (67% in ERGvir) with a subset of glycoproteins being specific to ERGvir (n = 64/371) and ERGatt (n = 36/343). These glycoproteins are involved in key biological processes such as protein, amino-acid and purine biosynthesis, translation, virulence, DNA repair, and replication. Label-free quantitative analysis revealed over-expression in 31 proteins in ERGvir and 8 in ERGatt. While further PNGase digestion confidently localized 2 and 5 N-glycoproteins in ERGvir and ERGatt, respectively, western blotting suggests that many glycoproteins are O-GlcNAcylated. Twenty-three proteins were detected in both the phospho- and glycoproteome, for the two variants. This work represents the first comprehensive assessment of PTMs on Ehrlichia biology, rising interesting questions regarding ER–host interactions. Phosphoproteome characterization demonstrates an increased versatility of ER phosphoproteins to participate in different mechanisms. The high number of glycoproteins and the lack of glycosyltransferases-coding genes highlight ER dependence on the host and/or vector cellular machinery for its own protein glycosylation. Moreover, these glycoproteins could be crucial to interact and respond to changes in ER environment. PTMs crosstalk between of O-GlcNAcylation and phosphorylation could be used as a major cellular signaling mechanism in ER. As little is known about the Ehrlichia proteins/proteome and its signaling biology, the results presented herein provide a useful resource for further hypothesis-driven exploration of Ehrlichia protein regulation by phosphorylation and glycosylation events. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD012589

Role of the Low Molecular Weight Protein Phosphatases PtpA and PtpB on Infectivity of Staphylococcus aureus

The bacterium, Staphylococcus aureus (S. aureus) is an opportunistic pathogen which can infect a variety of tissues resulting in a wide spectrum of infections ranging from mild cutaneous lesions to serious clinical manifestations such as endocarditis and osteomyelitis. This pathogen is also a common cause of implant-associated infections (IAIs), which are usually difficult to treat. A major characteristic of infections caused by S. aureus is being recurrent and long-standing. This latter characteristic is probably due to the ability of this pathogen to penetrate and survive within different types of cells in the human body including both professional and non-professional phagocytic cells (NPPCs). Many bacterial pathogens that are capable of surviving intracellularly in host immune cells secrete signaling molecules to modulate host cell signaling in order to survive in these cell types. The molecular mechanisms promoting the intracellular survival of S. aureus in professional phagocytes are not fully understood. However, the survival of S. aureus in these immune cells contributes to the dissemination of this pathogen to different body organs during infections using the so-called “trojan-horse delivery system” mechanism. This mechanism is clearly dangerous when macrophages particularly are occupied with viable S. aureus, owing to the mobility and long-living nature of macrophages compared to other immune cells. Across evolution, bacterial pathogens adapt their genomes in order to be able to counteract adverse environmental conditions during infections. Post-translational modifications (PTMs) of proteins are common mechanisms used by bacterial pathogens to modulate their immune evasion strategies. One common PTM mechanism utilized by many bacterial pathogens is phosphorylation/dephosphorylation of bacterial and host proteins. S. aureus is known to use this reversible phosphorylation of proteins to modulate metabolic processes and the activity of diverse global regulators, however its relation to staphylococcal pathogenesis is not fully characterized and asks for further investigation. This thesis focuses on the characterization of potential roles of two low molecular weight protein phosphatases on the infectivity of S. aureus. These two proteins are called PtpA (Protein tyrosine phosphatase A) and PtpB (Protein tyrosine phosphatase B). Both these phosphatases were not fully characterized in S. aureus till the beginning of this study, despite the fact that homologues for both proteins have been already reported to promote infectivity of pathogens such as Mycobacterium tuberculosis (Mtb), Salmonella typhimurium (S. typhimurium), or Yersinia spp. By studying the impact of these proteins on the interactions of S. aureus with host cells, especially macrophages, it became clear that both, PtpA and PtpB, play important roles in pathogenesis of S. aureus by enhancing the bacterium’s ability to survive inside macrophages. Both proteins also promoted the in vivo infectivity of S. aureus in a mouse model of infection. Moreover, a number of intracellular host proteins were identified as putative binding candidates for PtpA after being secreted inside macrophages during infections. Importantly, the protein Coronin-1A was phosphorylated on tyrosine residues when macrophages were infected with S. aureus. This protein is a crucial component of the cytoskeleton of highly motile host cells and is implicated in various immune-mediated responses. Thus, PtpA could be identified as a tyrosine phosphatase secreted by S. aureus to promote the intramacrophage survival capacity of this pathogen during infections, presumably by interacting with intracellular host proteins including Coronin-1A. In the second half of this study, i investigated the impact of a ptpB deletion on the stress response and infectivity of S. aureus. Here, i observed that this protein arginine phosphatase (PAP) is also required for the intracellular survival of S. aureus inside human macrophages. Subsequent analyses revealed that the phosphatase activity of PtpB in S. aureus is modulated by the oxidative status of the bacterial cell. When mimicking different kind of stresses encountered by S. aureus upon engulfment by macrophages, i noticed that the deletion of ptpB reduced the capacity of S. aureus to cope with oxidative-, nitrosative- and acidic stress, suggesting that PtpB enhances the intracellular survival capacity of S. aureus inside macrophages by increasing the bacterial fitness against the major stresses generated inside these immune cells to kill the internalized bacterial cells. Additionally, PtpB also exerted a protective effect in S. aureus against phagocytosis by polymorphonuclear leukocytes (PMNs). In this regard, cells of the ptpB mutant displayed additionally a decreased ability to release nucleases, which are important to degrade the Neutrophil Extracellular Traps (NETs) produced by PMNs upon activation. PtpB is also required for the overall proteolytic activity of S. aureus. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis uncovered a modulatory effect of PtpB on the expression of various virulence factor encoding genes including psmα (endocing phenol-soluble module α), aur (encoding aureolysin), nuc (encoding nuclease) and also RNAIII (a regulator of the agr locus). Finally, i found that PtpB is also involved in maintaining the cell wall integrity of S. aureus, presumably by modulating the activity of selected autolysins/regulators involved in cell wall homeostasis.Der opportunistische Krankheitserreger S. aureus ist dazu imstande, verschiedene Organe/Gewebetypen zu infizieren, und dadurch Infektionen der Haut bis hin zu ernsten klinischen Komplikationen wie Endokarditis, Pneumonie oder Osteomyelitis auszulösen. Dieser Erreger ist zudem ein häufiger Verursacher von Implantat-assoziierten Infektionen, die in der Regel nur schwierig zu behandeln sind. S. aureus-Infektionen sind zudem oft wiederkehrend und chronisch, wobei die letztere Eigenschaft vermutlich auf die Fähigkeit des Pathogens zurückzuführen ist, in verschiedene Wirtszelltypen, wie professionelle und nicht-professionelle Phagozyten inserieren zu können und in ihnen für mehrere Tage zu überleben. Die intrazelluläre Überlebensfähigkeit von S. aureus in diesem Immunzelltyp ist eine wichtige Pathogenese-Eigenschaft dieses Bakteriums, die zur Verbreitung des Bakteriums im Körper des Menschen bis in entfernte anatomische Bereiche beiträgt, ein Mechanismus, der im Englischen als “trojan-horse delivery system” bezeichnet wird. Die molekularen Mechanismen, die die intrazelluläre Überlebensfähigkeit von S. aureus in diesen professionellen Phagozyten erhöhen, sind bisher nicht vollständig aufgeklärt. Für verschiedene andere pathogene Bakterien ist jedoch bekannt, dass sie sich intrazellulär in Immunzellen behaupten können, indem sie dort Signalmoleküle sekretieren, die die Wirtszell-Signalwege so modulieren, dass die internalisierten Bakterienzellen in diesem Wirtszelltyp zu überleben vermögen. Viele dieser Pathogene nutzen dabei post-translationale Modifikationen (PTM) von Proteinen, um ihre Immunevasionsstrategien zu modulieren. Die Phosphorylierung/Dephosphorylierung von Wirtszellproteinen stellt dabei für viele Pathogene eine wichtige Form der PTM dar, die Abwehrmechanismen des Wirtes zu umgehen. Die reversible Phosphorylierung wird auch von S. aureus dazu genutzt, metabolische Prozesse und die Aktivität verschiedener globaler Regulatoren zu steuern. Die Bedeutung dieses PTM Mechanismus für die Infektiosität von S. aureus wurde bisher jedoch nur unzureichend charakterisiert, weshalb weitere Untersuchungen in diesem Bereich wünschenswert sind. Diese Arbeit fokussierte sich daher auf die Charakterisierung zweier Niedermolekulargewichts-Proteinphosphatasen, PtpA und PtpB, hinsichtlich ihrer Rolle während der Pathogenese von S. aureus. Beide Phosphatasen sind in S. aureus schon seit längerem bekannt, wurden bis dato aber noch nicht in Hinblick auf ihre Bedeutung für die Infektiosität von S. aureus untersucht, obwohl für andere pathogene Bakterien wie Mycobacterium tuberculosis (Mtb), Salmonella typhimurium (S. typhimurium) und Yersinia spp. gezeigt werden konnte, dass Homologe der beiden Proteine wichtige Virulenzfaktoren darstellen. Durch meine Untersuchungen konnte ich zeigen, dass sowohl PtpA als auch PtpB wichtige Virulenzfaktoren für S. aureus darstellen, die beide die Überlebenskapazität von S. aureus in Makrophagen steigern und die Virulenz des Pathogens während der Infektion erhöhen. PtpA wird dabei vom Bakterium in das umgebende Milieu sekretiert, um mutmaßlich mit Wirtsfaktoren, wie Coronin-1A zu interagieren. Dieses Protein ist eine wichtige Komponente des Zytoskeletts migrierender Wirtszellen und ebenso für die Immunantwort der Wirtszelle von Bedeutung. Da PtpA auch nach Phagozytose durch Makrophagen innerhalb der Wirtszelle sekretiert wird, liegt die Vermutung nahe, dass auch S. aureus durch die Sekretion dieser Protein-Tyrosin-Phosphatase das intrazelluläre Überleben der Bakterienzelle innerhalb der Immunzelle fördert. In Einklang mit dieser Hypothese konnte in Untersuchungen mit einem S. aureus-basierten Leberabszessmodell der Maus gezeigt werden, dass PtpA für die in vivo Infektiosität von S. aureus von großer Bedeutung ist. Im zweiten Teil meiner Promotion beschäftigte ich mich mit der Protein-Arginin-Phosphatase PtpB. Ebenso wie PtpA fördert PtpB das intrazelluläre Überleben von S. aureus in Makrophagen, anders als PtpA wird diese Phosphatase von S. aureus jedoch nicht in das umgebende Milieu sekretiert, sondern übt ihren regulatorischen Einfluss innerhalb der Bakterienzelle aus. PtpB scheint dabei die Fähigkeit von S. aureus zu fördern, mit Stressbedingungen, wie sie im Phagolysosom von Makrophagen nach Aufnahme von Cargo anzutreffen sind, umzugehen. So verminderte die Deletion von ptpB in S. aureus die Fähigkeit des Bakteriums, mit oxidativem-, nitrosativem- oder Säurestress umzugehen. Weiterführende Untersuchungen zeigten zudem, dass die Protein-Arginin-Phosphatase auch eine protektive Rolle für S. aureus, der Phagozytose durch polymorphkernige Leukozyten (PMNs) zu entgehen, einnimmt. Ebenso förderte PtpB die Sekretion von extrazellulären Nukleasen, einem weiteren wichtigen Immunevasionsmechanismus von S. aureus, den von Neutrophilen gebildeten extrazellulären Netzen zu entgehen. Zusätzlich unterstützte PtpB die proteolytische Aktivität von S. aureus und dessen Widerstandsfähigkeit gegenüber lytischen Agenzien wie Triton X-100 oder Lysostaphin. Darüber hinaus konnte ich zeigen, dass PtpB die Transkription von verschiedenen, für Virulenzfaktoren kodierende Gene beeinflusst, darunter psmα (codiert für die phenollöslichen Moduline α1-4), aur (codiert für die Proteinase Aureolysin), nuc (codiert für die Nuklease 1) und RNAIII (eine regulatorische RNA und Masterregulator des agr Lokus). Last not least zeigte eine ptpB Deletionsmutante in dem S. aureus-basierten Leberabszessmodell der Maus eine deutlich verminderte Fähigkeit, vier Tage nach Infektion eine erhöhte Bakterienlast in der Leber und in den Nieren hervorzurufen, und unterstreicht damit die hohe Bedeutung auch dieser Phosphatase für die Virulenz von S. aureus

Bacterial Phosphoproteomic Analysis Reveals the Correlation Between Protein Phosphorylation and Bacterial Pathogenicity

AbstractIncreasing evidence shows that protein phosphorylation on serine, threonine and tyrosine residues is a major regulatory post-translational modification in the bacteria. This review focuses on the implications of bacterial phosphoproteome in bacterial pathogenicity and highlights recent development of methods in phosphoproteomics and the connectivity of the phosphorylation networks. Recent technical developments in the high accuracy mass spectrometry have dramatically transformed proteomics and made it possible the characterization of a few exhaustive site-specific bacterial phosphoproteomes. The high abundance of tyrosine phosphorylations in a few bacterial phosphoproteomes suggests their roles in the pathogenicity, especially in the case of pathogen–host interactions; the high abundance of multi-phosphorylation sites in bacterial phosphoprotein is a compensation of the relatively small phosphorylation size and an indicator of the delicate regulation of protein functions

Comprehensive definition of Ser/Thr/Tyr phosphorylation in mycobacteria: towards understanding reprogramming of normal macrophage function by pathogenic mycobacteria

Mycobacterium tuberculosis, the causative agent for the disease Tuberculosis, is a serious public health problem that is responsible for 1.6 million deaths each year. The WHO’s recent report on Tuberculosis estimates that a third of the world’s population is latently infected with the bacteria, and, of those, 10% will progress to active disease. M. tuberculosis is a successful pathogen mainly due to its ability to adapt and survive in changing environments. It can survive a dormant state with limited metabolic activity during latent infection, while also being able to escape the macrophage and disseminate into active disease. Efforts to eradicate the disease must be based on understanding the biology of this organism, and the mechanisms it uses to infect, colonize, and evade the immune system. Understanding the behaviour of pathogenic mycobacteria in the macrophage is also important to the discovery of new drug targets. In this thesis, we employed state of the art mass spectrometry techniques, which allowed us to unpack the biology of this bacterium in different growth environments and expand our understanding of the mechanisms it employs to adapt and survive. We investigated protein regulation by the process of phosphorylation, through sensory kinases, which add a phosphate group to a protein of interest, thereby regulating its function. First, we interrogated the phosphoproteomic landscape between M. bovis BCG and M. smegmatis to explain how differential protein regulation results in the differences between slow and fast growth of mycobacteria. Second, we focused on Protein Kinase G (PknG), which plays an important role in bacterial survival by blocking phagosome/lysosome fusion. We identified the in vivo physiological substrates of this kinase in actively growing M.bovis BCG culture. Our results revealed that this kinase is a regulator of protein synthesis. We then examined the mechanisms of survival in murine RAW 246.7 macrophages mediated by PknG, using M. bovis BCG reference strain and PknG knock-out mutant. Our results indicated strong evidence that pathogenic mycobacteria disrupt the macrophagic cytoskeleton, through phosphorylation of proteins that are involved in cytoskeleton rearrangement. These results explain the strategies that pathogenic mycobacteria employ mediated by PknG to block phagosome-lysosome fusion and evade the host immune system and survive for prolonged periods in the macrophages. The findings of this thesis contribute to our understanding of the physiology of pathogenic mycobacteria and their interaction with the host

The novel anti-infective BDM-I : clinical utility and mechanism of action

Since the introduction of antibiotics into clinical use, bacteria have continued to evolve and acquire mechanisms of resistance. In a relatively short time period, this has rapidly escalated into a serious health crisis recognised by major governing/scientific bodies worldwide. The rate at which antibiotic resistance spreads now outpaces our current ability to discover and manufacture new antibiotics. Steps taken to combat antibiotic resistance have had little impact in preventing the spread of relevant pathogens, especially within hospital settings. The current state of antibiotic development has only exacerbated these issues by severely limiting the arsenal of therapeutics available to treat problematic infections. Without sufficient incentives, large pharmaceutical companies have focused on developing financially ‘safe’ drugs to treat non-infectious related conditions, leaving the bulk of antibiotic research to smaller biotechnology companies and academia, often in collaboration together. In this study, we have examined the clinical utility and mechanism of action (MoA) of BDM-I, which is a small synthetic molecule currently being developed by the Australian biotechnology company Opal Biosciences Limited. Importantly, BDM-I (3,4- methylenedioxy-β-nitropropene) appears to be a novel antimicrobial compound and has shown promising activity in vitro against clinically relevant pathogens, such as MRSA and VRE. In this regard, previous studies have shown that BDM-I does not inhibit common antimicrobial targets, and proposed that it binds to bacterial tyrosine phosphatases. While an antibiotic inhibiting a novel cellular target(s) is desirable, in this case the specific MoA (of BDM-I) and its physiological effect on bacterial cells is not known, thus limiting the potential for further development. Therefore, due to this knowledge gap, we attempted to gain insight into the BDM-I MoA (including its binding partner) using an omics approach (i.e. whole genome sequencing and proteomics). Additionally, we also aimed to study the activity of BDM-I against the clinically relevant ESKAPE pathogens