Akt/AS160 Signaling Pathway Inhibition Impairs Infection by Decreasing Rab14-Controlled Sphingolipids Delivery to Chlamydial Inclusions.

Anahí Capmany,María T Damiani,Mariano Alonso Bivou,Julián Gambarte Tudela

doi:10.3389/fmicb.2019.00666

Abstract

Chlamydia trachomatis, an obligate intracellular bacterium, intercepts different trafficking pathways of the host cell to acquire essential lipids for its survival and replication, particularly from the Golgi apparatus via a Rab14-mediated transport. Molecular mechanisms underlying how these bacteria manipulate intracellular transport are a matter of intense study. Here, we show that C. trachomatis utilizes Akt/AS160 signaling pathway to promote sphingolipids delivery to the chlamydial inclusion through Rab14-controlled vesicular transport. C. trachomatis provokes Akt phosphorylation along its entire developmental life cycle and recruits phosphorylated Akt (pAkt) to the inclusion membrane. As a consequence, Akt Substrate of 160 kDa (AS160), also known as TBC1D4, a GTPase Activating Protein (GAP) for Rab14, is phosphorylated and therefore inactivated. Phosphorylated AS160 (pAS160) loses its ability to promote GTP hydrolysis, favoring Rab14 binding to GTP. Akt inhibition by an allosteric isoform-specific Akt inhibitor (iAkt) prevents AS160 phosphorylation and reduces Rab14 recruitment to chlamydial inclusions. iAkt further impairs sphingolipids acquisition by C. trachomatis-inclusion and provokes lipid retention at the Golgi apparatus. Consequently, treatment with iAkt decreases chlamydial inclusion size, bacterial multiplication, and infectivity in a dose-dependent manner. Similar results were found in AS160-depleted cells. By electron microscopy, we observed that iAkt generates abnormal bacterial forms as those reported after sphingolipids deprivation or Rab14 silencing. Taken together, our findings indicate that targeting the Akt/AS160/Rab14 axis could constitute a novel strategy to limit chlamydial infections, mainly for those caused by antibiotic-resistant bacteria.

Highlights

Chlamydia trachomatis (C. trachomatis), a Gram-negative obligate intracellular pathogen, is the most frequent cause of bacterial sexually transmitted diseases worldwide (WHO, 2014)
Akt phosphorylation and consequent activation occurred at every time post-infection, we identified three main peaks of phosphorylation: at the beginning (2 h pi), at the middle (8 h pi), and at later stages (36 h pi) of the bacterial developmental cycle (Figure 1A)
Infection with C. trachomatis caused a dramatic redistribution of endogenous phosphorylated Akt (pAkt) that concentrated at the perinuclear region where these bacteria localized, suggesting pAkt recruitment to chlamydial inclusion membranes (Figure 1C)

Summary

Introduction

Chlamydia trachomatis (C. trachomatis), a Gram-negative obligate intracellular pathogen, is the most frequent cause of bacterial sexually transmitted diseases worldwide (WHO, 2014). Its biphasic developmental cycle involves two bacterial forms: infectious non-replicative elementary bodies (EBs), and non-infectious replicant-competent metabolically active reticulate bodies (RBs) (Bastidas et al, 2013; Vromman et al, 2014). Under stressing conditions, such as sustained treatment with penicillin (Cevenini et al, 1988), gammainterferon (INFγ) (Beatty et al, 1993, 1994a) and nutrients or sphingolipids deprivation (Raulston, 1997; Robertson et al, 2009; Capmany and Damiani, 2010), reticulate bodies acquire abnormal characteristics. Bacteria are released either by host cell lysis or extrusion of inclusions to spread the infection to neighboring cells (Hybiske and Stephens, 2007)

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Conclusion