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

The Low-Molecular Weight Protein Arginine Phosphatase PtpB Affects Nuclease Production, Cell Wall Integrity, and Uptake Rates of Staphylococcus aureus by Polymorphonuclear Leukocytes

The epidemiological success of Staphylococcus aureus as a versatile pathogen in mammals is largely attributed to its virulence factor repertoire and the sophisticated regulatory network controlling this virulon. Here we demonstrate that the low-molecular-weight protein arginine phosphatase PtpB contributes to this regulatory network by affecting the growth phase-dependent transcription of the virulence factor encoding genes/operons aur, nuc, and psmα, and that of the small regulatory RNA RNAIII. Inactivation of ptpB in S. aureus SA564 also significantly decreased the capacity of the mutant to degrade extracellular DNA, to hydrolyze proteins in the extracellular milieu, and to withstand Triton X-100 induced autolysis. SA564 ∆ptpB mutant cells were additionally ingested faster by polymorphonuclear leukocytes in a whole blood phagocytosis assay, suggesting that PtpB contributes by several ways positively to the ability of S. aureus to evade host innate immunity

The Phosphoarginine Phosphatase PtpB from Staphylococcus aureus Is Involved in Bacterial Stress Adaptation during Infection

Staphylococcus aureus continues to be a public health threat, especially in hospital settings. Studies aimed at deciphering the molecular and cellular mechanisms that underlie pathogenesis, host adaptation, and virulence are required to develop effective treatment strategies. Numerous host-pathogen interactions were found to be dependent on phosphatases-mediated regulation. This study focused on the analysis of the role of the low-molecular weight phosphatase PtpB, in particular, during infection. Deletion of ptpB in S. aureus strain SA564 significantly reduced the capacity of the mutant to withstand intracellular killing by THP-1 macrophages. When injected into normoglycemic C57BL/6 mice, the SA564 ∆ptpB mutant displayed markedly reduced bacterial loads in liver and kidney tissues in a murine S. aureus abscess model when compared to the wild type. We also observed that PtpB phosphatase-activity was sensitive to oxidative stress. Our quantitative transcript analyses revealed that PtpB affects the transcription of various genes involved in oxidative stress adaptation and infectivity. Thus, this study disclosed first insights into the physiological role of PtpB during host interaction allowing us to link phosphatase-dependent regulation to oxidative bacterial stress adaptation during infection

The Transcription Factor SpoVG Is of Major Importance for Biofilm Formation of Staphylococcus epidermidis under In Vitro Conditions, but Dispensable for In Vivo Biofilm Formation

Staphylococcus epidermidis is a common cause of device related infections on which pathogens form biofilms (i.e., multilayered cell populations embedded in an extracellular matrix). Here, we report that the transcription factor SpoVG is essential for the capacity of S. epidermidis to form such biofilms on artificial surfaces under in vitro conditions. Inactivation of spoVG in the polysaccharide intercellular adhesin (PIA) producing S. epidermidis strain 1457 yielded a mutant that, unlike its parental strain, failed to produce a clear biofilm in a microtiter plate-based static biofilm assay. A decreased biofilm formation capacity was also observed when 1457 ∆spoVG cells were co-cultured with polyurethane-based peripheral venous catheter fragments under dynamic conditions, while the cis-complemented 1457 ∆spoVG::spoVG derivative formed biofilms comparable to the levels seen with the wild-type. Transcriptional studies demonstrated that the deletion of spoVG significantly altered the expression of the intercellular adhesion (ica) locus by upregulating the transcription of the ica operon repressor icaR and down-regulating the transcription of icaADBC. Electrophoretic mobility shift assays (EMSA) revealed an interaction between SpoVG and the icaA-icaR intergenic region, suggesting SpoVG to promote biofilm formation of S. epidermidis by modulating ica expression. However, when mice were challenged with the 1457 ∆spoVG mutant in a foreign body infection model, only marginal differences in biomasses produced on the infected catheter fragments between the mutant and the parental strain were observed. These findings suggest that SpoVG is critical for the PIA-dependent biofilm formation of S. epidermis under in vitro conditions, but is largely dispensable for biofilm formation of this skin commensal under in vivo conditions

Characterization of the Secreted Acid Phosphatase SapS Reveals a Novel Virulence Factor of Staphylococcus aureus That Contributes to Survival and Virulence in Mice

Staphylococcus aureus possesses a large arsenal of immune-modulating factors, enabling it to bypass the immune system’s response. Here, we demonstrate that the acid phosphatase SapS is secreted during macrophage infection and promotes its intracellular survival in this type of immune cell. In animal models, the SA564 sapS mutant demonstrated a significantly lower bacterial burden in liver and renal tissues of mice at four days post infection in comparison to the wild type, along with lower pathogenicity in a zebrafish infection model. The SA564 sapS mutant elicits a lower inflammatory response in mice than the wild-type strain, while S. aureus cells harbouring a functional sapS induce a chemokine response that favours the recruitment of neutrophils to the infection site. Our in vitro and quantitative transcript analysis show that SapS has an effect on S. aureus capacity to adapt to oxidative stress during growth. SapS is also involved in S. aureus biofilm formation. Thus, this study shows for the first time that SapS plays a significant role during infection, most likely through inhibiting a variety of the host’s defence mechanisms

The Low-Molecular Weight Protein Arginine Phosphatase PtpB Affects Nuclease Production, Cell Wall Integrity, and Uptake Rates of Staphylococcus aureus by Polymorphonuclear Leukocytes

The epidemiological success of Staphylococcus aureus as a versatile pathogen in mammals is largely attributed to its virulence factor repertoire and the sophisticated regulatory network controlling this virulon. Here we demonstrate that the low-molecular-weight protein arginine phosphatase PtpB contributes to this regulatory network by affecting the growth phase-dependent transcription of the virulence factor encoding genes/operons aur, nuc, and psmα, and that of the small regulatory RNA RNAIII. Inactivation of ptpB in S. aureus SA564 also significantly decreased the capacity of the mutant to degrade extracellular DNA, to hydrolyze proteins in the extracellular milieu, and to withstand Triton X-100 induced autolysis. SA564 ΔptpB mutant cells were additionally ingested faster by polymorphonuclear leukocytes in a whole blood phagocytosis assay, suggesting that PtpB contributes by several ways positively to the ability of S. aureus to evade host innate immunity

Vaginal misoprostol versus combined intracervical foley’s catheter and oxytocin infusion for second trimester pregnancy termination in women with previous caesarean sections: a randomised control trial

Second trimester pregnancy termination has been reported to be associated with 3–5 times higher maternal morbidity and mortality risks more than first trimester termination. Medical methods had been thoroughly assessed and it is considered the anchor of the safe abortion care. Howevere, there is no global agreement regarding the ideal method for induction of the second trimester abortion in a scarred uterus. The aim of this study was to achieve vaginal expulsion in an expeditious manner with less maternal complications. A total of 158 women having, a history of previous caesarean section undergoing second trimester pregnancy termination, were randomly allocated into group (1) vaginal misoprostol group (n = 79) or group (2) combined intracervical foley’s catheter plus intravenous oxytocin infusion group (n = 79). The primary endpoint was complete fetal expulsion. Meanwhile, secondary outcomes were the rates of maternal complications, in terms of the need for surgical evacuation of retained product of conception (ERPOC), severe haemorrhage, uterine rupture, blood transfusion, cervical laceration, diarrhoea, vomiting and fever. The registration number of this trial https://clinicaltrials.gov/ct2/show/NCT04501809. This study showed that the combined use of intracervical foley’s catheter and oxytocin is an effective, and safe alternative to vaginal misoprostol for termination of the second trimester pregnancy in women having a previous caesarean delivery.Impact statement Why was this study conducted? The purpose of this study is to achieve an expeditious delivery for second trimester pregnancy termination in a scarred uterus by combined mechanical and pharmacological methods without significant morbidity. Physicians should balance the benefit of achieving vaginal exlpusion in an expeditious manner versus the risk of uterine rupture or any other maternal complications. What does this add to what is known? This study showed that a combined mechanical and pharmacolohical approach for second trimester termination of pregnancy has comparable efficacy and safety to vaginal misoprostol which is more affordable to the low resources countries where termination of pregnancy in a scarred uterus still has an increased maternal morbidity. What is the implication of this study on clinical practice? The combined use of intracervical foley’s catheter with intravenous oxytocin infusion is a safer option than vaginal misoprostol with less serious maternal complications

The Phosphoarginine Phosphatase PtpB from Staphylococcus aureus Is Involved in Bacterial Stress Adaptation during Infection

Staphylococcus aureus continues to be a public health threat, especially in hospital settings. Studies aimed at deciphering the molecular and cellular mechanisms that underlie pathogenesis, host adaptation, and virulence are required to develop effective treatment strategies. Numerous host-pathogen interactions were found to be dependent on phosphatases-mediated regulation. This study focused on the analysis of the role of the low-molecular weight phosphatase PtpB, in particular, during infection. Deletion of ptpB in S. aureus strain SA564 significantly reduced the capacity of the mutant to withstand intracellular killing by THP-1 macrophages. When injected into normoglycemic C57BL/6 mice, the SA564 ΔptpB mutant displayed markedly reduced bacterial loads in liver and kidney tissues in a murine S. aureus abscess model when compared to the wild type. We also observed that PtpB phosphatase-activity was sensitive to oxidative stress. Our quantitative transcript analyses revealed that PtpB affects the transcription of various genes involved in oxidative stress adaptation and infectivity. Thus, this study disclosed first insights into the physiological role of PtpB during host interaction allowing us to link phosphatase-dependent regulation to oxidative bacterial stress adaptation during infection

Comparison of in vitro assays to study the effectiveness of antiparasitics against Acanthamoeba castellani trophozoites and cysts

We aimed to compare LDH release assay, trypan blue and fluorescent stainings, and non-nutrient Escherichia coli plate assay in determining treatment efficacy of antiamoebic agents against Acanthamoeba castellanii trophozoites/cysts, in vitro. 1BU trophozoites/cysts were challenged with 0.02% polyhexamethylene biguanid (PHMB), 0.1% propamidine isethionate (PD), and 0.0065% miltefosine (MF). Efficacies of the drugs were determined by LDH release and trypan blue assays, by Hoechst 33343, calcein-AM, and ethidium homodimer-1 fluorescent dyes, and by a non-nutrient agar E. coli plate assay. All three antiamoebic agents induced a significant LDH release from trophozoites, compared to controls (p < 0.0001). Fluorescent-dye staining in untreated 1BU trophozoites/cysts was negligible, but using antiamoebic agents, there was 59.3%-100% trypan blue, 100% Hoechst 33342, 0%-75.3% calcein-AM, and 100% ethidium homodimer-1 positivity. On E. coli plates, in controls and MF-treated 1BU trophozoites/cysts, new trophozoites appeared within 24 h, encystment occurred after 5 weeks. In PHMB- and PD-treated 1BU throphozoites/cysts, irregularly shaped, smaller trophozoites appeared after 72 h, which failed to form new cysts within 5 weeks. None of the enzymatic- and dye-based viability assays tested here generated survival rates for trophozoites/cysts that were comparable with those yielded with the non-nutrient agar E. coli plate assay, suggesting that the culture-based assay is the best method to study the treatment efficacy of drugs against Acanthamoeba

The Transcription Factor SpoVG Is of Major Importance for Biofilm Formation of Staphylococcus epidermidis under In Vitro Conditions, but Dispensable for In Vivo Biofilm Formation

Staphylococcus epidermidis is a common cause of device related infections on which pathogens form biofilms (i.e., multilayered cell populations embedded in an extracellular matrix). Here, we report that the transcription factor SpoVG is essential for the capacity of S. epidermidis to form such biofilms on artificial surfaces under in vitro conditions. Inactivation of spoVG in the polysaccharide intercellular adhesin (PIA) producing S. epidermidis strain 1457 yielded a mutant that, unlike its parental strain, failed to produce a clear biofilm in a microtiter plate-based static biofilm assay. A decreased biofilm formation capacity was also observed when 1457 &Delta;spoVG cells were co-cultured with polyurethane-based peripheral venous catheter fragments under dynamic conditions, while the cis-complemented 1457 &Delta;spoVG::spoVG derivative formed biofilms comparable to the levels seen with the wild-type. Transcriptional studies demonstrated that the deletion of spoVG significantly altered the expression of the intercellular adhesion (ica) locus by upregulating the transcription of the ica operon repressor icaR and down-regulating the transcription of icaADBC. Electrophoretic mobility shift assays (EMSA) revealed an interaction between SpoVG and the icaA-icaR intergenic region, suggesting SpoVG to promote biofilm formation of S. epidermidis by modulating ica expression. However, when mice were challenged with the 1457 &Delta;spoVG mutant in a foreign body infection model, only marginal differences in biomasses produced on the infected catheter fragments between the mutant and the parental strain were observed. These findings suggest that SpoVG is critical for the PIA-dependent biofilm formation of S. epidermis under in vitro conditions, but is largely dispensable for biofilm formation of this skin commensal under in vivo conditions