Myo1C knockdown and pharmacology inhibition disturb Chlamydia trachomatis development and bacterial exit by extrusion in an actin-dependent manner.

CUERVO BUSTAMANTE ME; DIEGO DEL BALZO; DAMIANI MT; CAPMANY A

Mendoza

Congreso; SAIB 2022; 2022

Sociedad Argentina de Investigación en Bioquimica y Boligía Molecular

Chlamydia trachomatis is an obligate intracellular Gram-negative bacterium and the most frequent bacterial agent of sexually transmitted infections. This bacterium modifies the actin cytoskeleton to ensure its entry, development, and exit. An actin belt surrounds chlamydial inclusion during bacterial growth. The myosin protein superfamily plays a central role in the modulation of the actin cytoskeleton. However, the involvement of myosin in chlamydial infection has not been explored deeply. We previously described the participation of Myo1C in actin cytoskeleton stability. In this context, we hypothesize that Myo1C could be involved in actin cytoskeleton modulation during C. trachomatis infection. Myo1C is a single-headed class I myosin strongly present in dynamic regions of the plasma membrane, where it modulates the actin network. Working with our model of C. trachomatis infection in Hela cells, we determined, by confocal microscopy, that endogenous and over-express Myo1C was recruited to the chlamydial inclusion. The knockdown of Myo1C impaired the C. trachomatis invasion and development, causing destabilization of the actin belt that surrounds the inclusion. Moreover, this phenotype was rescued in HeLa cells overexpressing mouse GFP-Myo1C transfecting with siRNA- human Myo1C. Likewise, the over-expression of the negative mutant Myo1C ΔABL, which does not bind to actin, induces a significantly higher percentage of incomplete or absent actin belt surrounding the inclusion. Besides, it’s known that an actin belt is necessary for C. trachomatis to exit by extrusion. So, our objective was to determine if Myo1C is involved in this process. We purified extrusions by differential centrifugation at 48 hours of infection. We infected a monolayer of Hela with extrusion obtained from cells that overexpressed GFP, GFP-Myo1Cwt, or GFP-Myo1CΔABL cells to quantify chlamydial extrusion levels per condition. We determined a significant decrease in the extrusions recovered after over-expression of GFP-Myo1CΔABL. In line with these results, we detected significantly lowers extrusion levels after Myo1C depletion. Likewise, extrusions recovered at 48 h pi from cells incubated for 6 hours with pentachloropseudilin (PClP), a Myo1-specific inhibitor, were significantly lower than the control condition. Altogether these results indicate that MYO1C would stabilize the surrounding actin ring and that the knockdown and pharmacological Myo1C inhibition disrupts C. trachomatis development and exit by extrusion.