LP PILLOUX¹, CK KEBBI-BEGHDADI¹, AC CROXATTO¹, GG GREUB<sup>1

1</sup>Center for Research on Intracellular Bacteria (CRIB), Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne, Switzerland

Like other obligate intracellular microorganisms, Chlamydia-related bacteria are able to modulate important functions of their host cells to allow their survival and replication. All bacteria of the Chlamydiales order exhibit a common "core genome" encoding bacterial proteins essential for their intracellular way of life, in particular those forming the Type III secretion system (T3SS). T3SS is a large proteic complex able to inject bacterial effector proteins within the eukaryotic host cells. In Chlamydiales bacteria, effectors are secreted in the inclusion lumen, inserted in the inclusion membrane or translocated into the host cell cytosol. Their functions are multiple, including cytoskeletal rearrangements, subversion of signalling pathways, activation or inhibition of host cell death and modulation of intracellular trafficking. In classical chlamydiae, several proteins have been identified as effectors secreted by the T3SS but nothing is known about those secreted by Chlamydia-related bacteria. Since these bacteria are emerging pathogens for humans and animals, identifying their secreted effector proteins and investigating their mode of action is essential.
We used multiple approaches to identify T3SS effectors of Chlamydia-related bacteria. The first one is based on bioinformatic algorithms that predict T3SS effectors even though no consensus sequence for type III secretion has been identified. We have also developed and optimized two alternative methods to recover bacterial proteins secreted in the host cell cytoplasm. These methods rely on selective permeabilization of the eukaryotic membrane or on fractionation of host cell components; the latter technique also allows the recovery of bacterial proteins localized into the inclusion membrane. After mass spectrometry analysis, we obtained a list of putative effectors, whose secretion by T3SS was confirmed using a heterologous system. These effectors were further expressed in eukaryotic cells to identify their interactants.
This large screening led to the identification of new chlamydial T3SS effectors, increasing our understanding of chlamydial pathogenesis and opening perspectives for new drug targets.