C KEBBI-BEGHDADI¹, L PILLOUX¹, G GREUB<sup>1

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

Waddlia chondrophila is a strict intracellular bacterium causing adverse pregnancy outcomes in humans and abortion in ruminants. Similarly to classical Chlamydiae responsible for some severe human and animal diseases, W. chondrophila belongs to the Chlamydiales order and harbors a biphasic life cycle. Like for every obligate intracellular bacteria, it is crucial for W. chondrophila to modulate its environment to establish a replicative niche, acquire nutrients and escape the host defense mechanisms. These bacteria-host interactions are triggered by bacterial effector proteins that are actively secreted, mainly by the Type 3 Secretion System (T3SS), in the membrane of the bacteria-containing vacuole or in the host cytoplasm.
The hypothetical protein WcT3SS_001 was previously identified as an immunological protein (Kebbi-Beghdadi et al, 2012) and is predicted by several in silico algorithms to be a T3SS effector. We demonstrated by immunofluorescence and confocal microscopy that this putative T3SS effector is secreted in the host cell cytoplasm. Analysis of the mRNA and protein expression levels during the bacterial replication cycle by RT-qPCR and western blot indicated that the WcT3SS_001 gene is transcribed during the early phase of the cycle and that the protein is produced and secreted 16 to 24 hours post infection. This time point corresponds to the beginning of the replication phase, which could suggest that this effector plays a role in the establishment of favorable conditions to sustain an exponential multiplication of the bacteria.
To further demonstrate that WcT3SS_001 is indeed secreted by the T3SS and since Chlamydiales bacteria are only poorly amenable to genetic modifications, we expressed the protein in a heterologous system (Yersinia enterocolitica) where type three secretion can be selectively blocked or induced. In addition, we will also express this protein, fused to GFP, in human epithelial cells and identify its eukaryotic target(s) by co-immunoprecipitation. These results will provide insights into the function of this hypothetical protein and potentially open novel perspectives for the development of new drugs active against chlamydiae.