Untersuchung neuer Therapieoptionen und Wirkstoffoptimierung für S. aureus-induzierte Infektionen in murinen in vivo Modellen

Nosokomiale Infektionen stellen weltweit eine große Herausforderung für das Gesundheitssystem dar und resultieren, neben steigenden Kosten im Gesundheitswesen und verlängerten Aufenthalten der Patienten in der Klinik, in jährlich mehreren zehntausend Todesopfern. Ein prominenter Verursacher nosokomialer Infektionen ist das Gram-positive Bakterium Staphylococcus aureus (S. aureus). Auf Grund diverser Strategien zur Umgehung der Wirtsimmunantwort und des Antibiotikadrucks, gestaltet sich eine Behandlung dieses Pathogens als besonders kompliziert. Krankheitsbilder wie Wundinfektionen, Sepsis und Fremdkörper-assoziierte Infektionen in Zusammenhang mit Biofilmbildung machen S. aureus zu einem gefürchteten Pathogen in Krankenhäusern und Pflegeheimen. Durch die Akkumulation von Resistenzmechanismen gegen gängige Antibiotika in den letzten Jahrzehnten wird eine antimikrobielle Behandlung zunehmend erschwert. Die zunehmende Anzahl Antibiotika-resistenter Bakterien macht eine Entwicklung neuer Behandlungsmethoden und Substanzen, sowie die Optimierung bisheriger Behandlungsoptionen dringend erforderlich. Der Einsatz von Tiermodellen zur Testung vielversprechender Substanzen und Behandlungsschemata unter in vivo Bedingungen in einem dem Menschen nahekommenden Wirtssystem ist dabei unerlässlich. Im Rahmen dieser Arbeit wurden diverse neue und optimierte Behandlungsoptionen in verschiedenen murinen Infektionsmodellen untersucht, um die Wirkstoffentwicklung voranzutreiben. Dabei wurden sowohl das Pathogen S. aureus, als auch der Wirt und die Wirkstoffe selbst in die Betrachtungen einbezogen. Auf Seiten des Pathogens S. aureus wurden neue Zielstrukturen für Wirkstoffe näher beleuchtet. So konnte das, mit dem Kohlenstoffmetabolismus des Bakteriums in Verbindung stehende, Histidin-haltige Phosphatträgerprotein HPr als neue vielversprechende Zielstruktur identifiziert werden. Eine Deletion des für HPr codierenden Gens ptsH resultierte in einer verringerten Biofilmbildung und Infektiosität in vivo (Publikation A). Aus dem Reservoir der Proteinphosphorylierung-vermittelnden Enzyme von S. aureus konnten die beiden niedermolekularen Protein-Phosphatasen PtpA und PtpB als vielversprechende neue Zielstrukturen identifiziert werden. Das Fehlen bereits einer der Phosphatasen resultierte in einer drastischen Reduktion der Infektiosität des Bakteriums in vivo (Publikationen B und C). Im Bereich der Wirkstoffoptimierung wurde zum einen ein neues Rifampicin-Derivat als Inhibitor der bakteriellen RNA-Polymerase untersucht. Auf Grund der enormen Bedeutung von Rifampicin für die Behandlung vieler Infektionskrankheiten und der zunehmenden Resistenzentwicklung ist eine Optimierung des Wirkstoffs dringend notwendig.Zwar konnte im Rahmen der durchgeführten in vivo Experimente kein positiver Behandlungserfolg mit dem in vitro sehr potenten Wirkstoffkandidaten verzeichnet werden, doch es war möglich, ein neues, einfaches murines Wundmodell in haarlosen SKH1 Mäusen zu etablieren und die Grundlage für weiterführende Strukturanalysen zur Wirkstoffoptimierung zu schaffen (Publikation D). Zum anderen wurde die Behandlungsoptimierung des schlecht wasserlöslichen, starke Nebenwirkungen verursachenden und nur schwer zellgängigen Antibiotikums Clarithromycin angestrebt. Durch den Einschluss des Wirkstoffes in Polymer-Nanokapseln wurde die intrazelluläre Bioverfügbarkeit des Stoffes und die Abtötungskinetik intrazellulär persistierender Bakterien enorm gesteigert (Publikation E). Auf Seiten des Wirtes wurde der Einfluss des Interleukins 17C (IL-17C) auf die Heilung und Bakterienbeseitigung in S. aureus-infizierten Wunden untersucht. Das Peptidhormon gilt als ein wichtiger Mediator der Immunantwort im Wirt und kann bei einer Dysfunktion Hauterkrankungen wie Psoriasis verursachen. Eine Behandlung mit Anti-IL-17C Antikörpern könnte dieses Krankheitsbild zwar lindern, gleichzeitig aber auch einen negativen Einfluss auf die Wundheilung haben. Die Untersuchungen zeigten, dass die Expression von IL-17C in S. aureus infiziertem Wundgewebe in Menschen deutlich erhöht ist. In IL-17C defiziente Mäuse konnte eine deutlich verlangsamte Heilungskinetik der infizierten Wunden festgestellt werden, es zeigten sich jedoch keinerlei Unterschiede in der Bakterienlast in den Wunden. Der zu Grunde liegende Mechanismus für die verminderte Wundheilung konnte nicht identifiziert werden (Publikation F). Im Rahmen dieser Arbeit konnten durch den Einsatz muriner Infektionsmodelle neue Angriffspunkte für die antimikrobielle Substanzentwicklung identifiziert und auf ihre Eignung zur Therapie von S. aureus untersucht werden. Zudem konnten bestehende Therapieansätze optimiert werden. Die Untersuchungen liefern somit eine Reihe neuer Ansatzpunkte für die zukünftige Entwicklung antimikrobieller Substanzen gegen das Humanpathogen S. aureus.Nosocomial infections are a major burden on the health care system worldwide and result in tens of thousands of deaths each year in addition to rising costs and extended hospitalization. A prominent cause of nosocomial infections is the gram-positive bacterium Staphylococcus aureus (S. aureus). Due to its various strategies to hide from the host immune response and antibiotic pressure, treatment of this pathogen is complicated. Diseases such as wound infections, sepsis, and foreign body infections, associated with biofilm formation, make S. aureus a challanging pathogen in hospitals and nursing homes. The development of resistance mechanisms against common antibiotics in recent decades has made antimicrobial treatment increasingly difficult. The growing number of antibiotic-resistant bacteria necessitates the development of new treatment methods and substances, as well as the optimization of existing treatment options. The use of animal models to test in vitro promising substances and procedures under realistic and complex conditions is essential. This work investigated new and optimized treatment options in different S. aureus based murine infection models to advance drug development, and included pathogen-, host- and substance-specific traits. On the side of the pathogen, S. aureus, new target structures for drug development were investigated. In connection with the carbon metabolism of the bacterium, the histidine-containing protein HPr (encoded by ptsH) has been identified as a new promising target structure. Deletion of ptsH resulted in reduced biofilm formation and infectivity in vivo (publication A). From the range of protein phosphorylation mediating enzymes of S. aureus, the phosphatases PtpA and PtpB were identified as new promising target structures. The absence of even one of the phosphatases resulted in a drastic reduction of bacterial infectivity in vivo (publications B and C). In the field of drug optimization, a new rifampicin derivative was investigated as an inhibitor of bacterial RNA polymerase. Rifampicin is an important antibiotic in tuberculosis treatment, and it serves as a reserve antibiotic for various bacterial infections. However, because of the increasing development of antibiotic resistance, optimization of this substance is urgently required. Although no positive treatment success was achived with the in vitro highly potent substance under in vivo conditions, it was possible to establish a murine wound healing model in hairless SKH1 mice, and further structure analyses may help to develop a more functional derivate (publication D). In addition, an optimization of the treatment procedure for the antibiotic clarithromycin, which is poorly soluble in water, causes strong side effects, and is barely cell permeable, was sought. The inclusion of the active substance into polymer nano capsules greatly increased the bioavailability of the substance and the elimination of intracellularly surviving bacteria (publication E). On the host side, the influence of interleukin 17C (IL-17C) on wound healing and elimination of bacteria in S. aureus-infected wounds was investigated. The peptide hormone mediates the immune response in the host and may cause skin diseases such as psoriasis in the event of dysfunction. Treatment with anti- IL-17C antibodies may cure this condition but is also considered to have an impact on wound healing and bacterial clearance. This study revealed that the expression of IL-17C in S. aureus infected wound tissue is significantly increased in humans. Although IL-17C-deficient mice had a significant delay in wound healing, a change in bacterial load in the wound could not be detected. The underlying mechanism for the delayed wound healing remained unclear (publication F). In the scope of this work, murine infection models were used to identify new potential targets for antimicrobial substance development and to investigate their suitability. Existing therapeutic approaches were optimized, as well. The findings provide new approaches for the development of antimicrobial substances against the human pathogen S. aureus

Impact of the Histidine‐Containing Phosphocarrier Protein HPr on Carbon Metabolism and Virulence in Staphylococcus aureus

Carbon catabolite repression (CCR) is a common mechanism pathogenic bacteria use to link central metabolism with virulence factor synthesis. In gram‐positive bacteria, catabolite control protein A (CcpA) and the histidine‐containing phosphocarrier protein HPr (encoded by ptsH) are the predominant mediators of CCR. In addition to modulating CcpA activity, HPr is essential for glucose import via the phosphotransferase system. While the regulatory functions of CcpA in Staphylococcus aureus are largely known, little is known about the function of HPr in CCR and infectivity. To address this knowledge gap, ptsH mutants were created in S. aureus that either lack the open reading frame or harbor a ptsH variant carrying a thymidine to guanosine mutation at position 136, and the effects of these mutations on growth and metabolism were assessed. Inactivation of ptsH altered bacterial physiology and decreased the ability of S. aureus to form a biofilm and cause infections in mice. These data demonstrate that HPr affects central metabolism and virulence in S. aureus independent of its influence on CcpA regulation

IL-17C and IL-17RE Promote Wound Closure in a Staphylococcus aureus-Based Murine Wound Infection Model

The epithelial cytokine interleukin-17C (IL-17C) mediates inflammation through the interleukin 17 receptor E (IL-17RE). Prior studies showed a detrimental role of IL-17C in the pathogenesis of immune-mediated skin diseases (e.g., psoriasis). Here, we examined the role of IL-17C/IL-17RE in wound closure in a Staphylococcus aureus wound infection model. We demonstrate that wound closure is significantly delayed in IL-17RE (Il-17re−/−)- and 17C (Il-17c−/−)-deficient mice. There was no significant difference between WT, Il-17re−/−, and Il-17c−/− mice in the absence of infection. Deficiency for IL-17RE and IL-17C did not significantly affect the elimination of bacteria. IL-17C expression was increased in the epidermis of human S. aureus-infected skin. Our results indicate that the IL-17C/IL-17RE axis contributes to the closure of infected wounds but does not contribute to the elimination of S. aureus

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 transiting multi-planet system HD3167: a 5.7 MEarth Super-Earth and a 8.3 MEarth mini-Neptune

HD3167 is a bright (V=8.9 mag) K0V star observed by the NASA's K2 space mission during its Campaign 8. It has been recently found to host two small transiting planets, namely, HD3167b, an ultra short period (0.96 d) super-Earth, and HD3167c, a mini-Neptune on a relatively long-period orbit (29.85 d). Here we present an intensive radial velocity follow-up of HD3167 performed with the FIES@NOT, [email protected], and HARPS-N@TNG spectrographs. We revise the system parameters and determine radii, masses, and densities of the two transiting planets by combining the K2 photometry with our spectroscopic data. With a mass of 5.69+/-0.44 MEarth, radius of 1.574+/-0.054 REarth, and mean density of 8.00(+1.0)(-0.98) g/cm^3, HD3167b joins the small group of ultra-short period planets known to have a rocky terrestrial composition. HD3167c has a mass of 8.33 (+1.79)(-1.85) MEarth and a radius of 2.740(+0.106)(-0.100) REarth, yielding a mean density of 2.21(+0.56)(-0.53) g/cm^3, indicative of a planet with a composition comprising a solid core surrounded by a thick atmospheric envelope. The rather large pressure scale height (about 350 km) and the brightness of the host star make HD3167c an ideal target for atmospheric characterization via transmission spectroscopy across a broad range of wavelengths. We found evidence of additional signals in the radial velocity measurements but the currently available data set does not allow us to draw any firm conclusion on the origin of the observed variation.Comment: 18 pages, 11 figures, 5 table

Impact of the Histidine-Containing Phosphocarrier Protein HPr on Carbon Metabolism and Virulence in Staphylococcus aureus

Carbon catabolite repression (CCR) is a common mechanism pathogenic bacteria use to link central metabolism with virulence factor synthesis. In gram-positive bacteria, catabolite control protein A (CcpA) and the histidine-containing phosphocarrier protein HPr (encoded by ptsH) are the predominant mediators of CCR. In addition to modulating CcpA activity, HPr is essential for glucose import via the phosphotransferase system. While the regulatory functions of CcpA in Staphylococcus aureus are largely known, little is known about the function of HPr in CCR and infectivity. To address this knowledge gap, ptsH mutants were created in S. aureus that either lack the open reading frame or harbor a ptsH variant carrying a thymidine to guanosine mutation at position 136, and the effects of these mutations on growth and metabolism were assessed. Inactivation of ptsH altered bacterial physiology and decreased the ability of S. aureus to form a biofilm and cause infections in mice. These data demonstrate that HPr affects central metabolism and virulence in S. aureus independent of its influence on CcpA regulation

Possible evidence for partial differentiation of asteroid Lutetia from Rosetta

The petrologic diversity of meteorites demonstrates that planetesimals ranged from unmelted, variably metamorphosed aggregates to fully molten, differentiated bodies. However, partially differentiated bodies have not been unambiguously identified in the asteroid belt. New constraints on the density, composition, and morphology of 21 Lutetia from the Rosetta spacecraft indicate that the asteroid's high bulk density exceeds that of most known chondritic meteorite groups, yet its surface properties resemble those of some carbonaceous and enstatite chondrite groups. This indicates that Lutetia likely experienced early compaction processes like metamorphic sintering. It may have also partially differentiated, forming a metallic core overlain by a primitive chondritic crust

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