Proof of concept: targeting Chlamydia trachomatis- host interplay as a novel therapeutic approach

DEL BALZO D; GAMBARTE J; ALONSO BIVOU M; DAMIANI MT

Mendoza

Congreso; IV Reunión Conjunta de soc. de biología; 2020

SBCUYO

The Chlamydia trachomatis (CT) gram-negative bacterial species can be subdivided by tissue tropism and virulence into three biovars, namely genital, lymphogranuloma venereum, and trachoma biovars. Although all of them are primary causative agents of disease in humans, the genital biovar is responsible for the most frequent bacterial sexually transmitted infection. As a species, CT develops only within human cells in a unique biphasic cycle and, upon certain harmful stimuli, it can progress to a state of latency called aberrant or persistent state. These particular characteristics make CT able to develop difficult to eradicate recurrent chronic infections in an alarmingly increasing portion of the reproductive-age population with the therapeutic tools available to date. To tackle this problem new pharmacological solutions need to be evaluated. In our lab, we have previously reported that CT activates and usurps the eukaryotic AKT/AS160/RAB14 pathway to ensure the provision of nutrients from the Golgi apparatus that are essential for its survival within infected cells. In this work we evaluate the anti-chlamydial efficacy of four AKT kinase inhibitors that are now at different stages of clinical trials, mostly for oncological purposes, but could potentially be repositioned. To this aim, we first verified the abrogation of AKT activity in our cellular model of infection by evaluating the phosphorylation state of AKT and its substrate, AS160, by western blot. Two of the inhibitors present an allosteric inhibition of the kinase activity impeding also its own phosphorylation (S473), whereas, the other two are orthosteric inhibitors whose effect produces increased AKT phosphorylation (S473) but reduced AS160 phosphorylation (S318). A significant defect in chlamydial development upon AKT inhibition was observed by a decrease in CT inclusion area and a reduction of infectious progeny by Inclusion Forming Unit analysis. To rule out the cytotoxic effect of the inhibitors on the host, cellular death after treatment at the concentration assayed was quantified by trypan blue staining. Based on our previous studies, we evaluated the transport of sphingomyelin from the Golgi apparatus to the inclusion using a Bodipy-ceramide probe. Upon AKT inhibition, we constatated a retention of the lipid probe in the Golgi and a reduced delivery to the inclusion by confocal microscopy, as expected by the decrease in chlamydial inclusion growth. The present results provide proof of principle that targeting host-pathogen interactions can be a worthy source of new pharmacological approaches to control bacterial infections. Further studies on in vivo models should be performed to assess the suitability of the use of these inhibitors as an anti-chlamydial specific treatment.