Auswirkungen von metabolischen Veränderungen verursacht durch das Umgebungsmilieu auf die Wirksamkeit von Antibiotika gegen Chlamydia trachomatis
Zusammenfassung der Projektergebnisse
Urogenital tract infection with Chlamydia trachomatis is the most prevalent bacterial sexually transmitted disease worldwide, which can have serious consequences including ectopic pregnancy and secondary sterility in women. Antibiotic treatment with azithromycin or doxycycline usually has a high efficacy but treatment failure has been reported in up to 8% of the cases. The underlying reasons for this are not fully understood. In C. trachomatis phenotypic resistance mechanisms are more likely to occur than genetic resistances. For instance it is known that the eradication of C. trachomatis by chlamydial first-line antibiotics is hampered under different environmental conditions such as hypoxia (2% O2) or in Interferonγ (IFN-γ)-induced persistent C. trachomatis infection. Furthermore, host cellular factors might change the antibiotic efficacy against this obligate intracellular bacterium. In this project we investigated the influence of environmental conditions on the antibiotic efficacy against C. trachomatis and the responsible bacterial and host factors. We analyzed the antibiotic efficacy in a C. trachomatis infection model of HeLa cells under different environmental conditions such as iron-deprivation induced by the iron chelator deferoxamine (DFO). Furthermore, the extra- and intracellular pH was changed by applying different CO2 concentrations or acidifying the cytosol by using cariporide, a specific inhibitor of the cellular pH regulator sodium/hydrogen exchanger (NHE1). As an obligate-intracellular bacterium C. trachomatis depends on the host metabolism which is affected by environmental changes. Therefore, we checked the host metabolic activity with the metabolic analyzer XF24 that allows determining the glycolytic activity under real-time conditions. We could show that not only in IFN-γ-induced C. trachomatis persistence, but also in a model of DFO-induced persistence the antibiotic efficacy is reduced. Host glycolysis was enhanced in DFO-induced persistent C. trachomatis infected cells as well as in active infection under normoxia and hypoxia. Stimulation of glycolysis results in production of lactate and protons with a subsequent decrease in pH. While it has been reported that an altered pH directly affected the antibiotic efficacy against extracellular bacterial uropathogens, it is so far not known how an alteration in the pH impacts the antibiotic efficacy against intracellular C. trachomatis. Therefore, we focused on the influence of the extra- and intracellular pH on the efficacy of azithromycin, doxycycline and rifampicin for the treatment of C. trachomatis infections. Acidification of the extra- and/or intracellular pH leads to a significant reduction in azithromycin efficacy whereas doxycycline and rifampicin were not affected by the changes. Furthermore, the infection with C. trachomatis itself causes an acidification of the cytoplasm of the host cell. Cellular import and export mechanisms can influence the intracellular antibiotic concentration. Thus, the uptake of azithromycin is dependent on the extracellular pH in phagocytic cells. Macrolide antibiotics protonate at lower pH which hinders the drug from crossing the cell membrane. Moreover, the export of drugs is well described for tumor cells known as multidrug resistance. Therefore, we analyzed the best known drug transporter, P-glycoprotein (pgp) to evaluate if the observed decrease of azithromycin efficacy is due to an increased efflux of the drug. Neither the pH nor a C. trachomatis infection changed the abundancy of pgp mRNA. Further analysis, need to clarify the influence of import and export mechanisms on the antibiotic efficacy against C. trachomatis. The abundancy and activation of other members of multidrug resistance proteins need to be analyzed to exclude enhanced export. Furthermore, import mechanism could be analyzed by competitive inhibitors of azithromycin transport such as L-carnitin.
