A predation assay using amoebae to screen for virulence factors unearthed the first W. chondrophila inclusion membrane protein

Abstract

Waddlia chondrophila is an intracellular bacterium phylogenetically related to the well-studied human and animal pathogens of the Chlamydiaceae family. In the last decade, W. chondrophila was convincingly demonstrated to be associated with adverse pregnancy outcomes in humans and abortions in animals. All members of the phylum Chlamydiae possess a Type Three Secretion System that they use for delivering virulence proteins into the host cell cytosol to modulate their environment and create optimal conditions to complete their life cycle. To identify W. chondrophila virulence proteins, we used an original screening approach that combines a cosmid library with an assay monitoring resistance to predation by phagocytic amoebae. This technique combined with bioinformatic data allowed the identification of 28 candidate virulence proteins, including Wimp1, the first identified inclusion membrane protein of W. chondrophila.

Eukaryotic Cell Permeabilisation to Identify New Putative Chlamydial Type III Secretion System Effectors Secreted within Host Cell Cytoplasm

“…In addition, T3SS-dependent secretion of Wcw_1706 was also convincingly demonstrated using a third Y. enterocolitica strain in which secretion depends on the presence of calcium in the culture medium (Supplementary Figure S4 [33,54]).…”

“…Bacterial pellets were resuspended in SDS loading buffer immediately after centrifugation. Both fractions were normalised according to their OD 600 values and analysed by SDS-PAGE and immunoblotting Type III-dependent secretion of Wcw_1706 was also assessed using the Y. enterocolitica MRS40 (pNG40031) strain as described in [33].…”

“…TepP was detected only in the case of a functional T3SS. To exclude bacterial lysis or fraction contamination, the supernatant fractions were probed against the strictly cytosolic Y. enterocolitica MreB protein ( Supplementary Figures S1-S3) [33].…”

Amoebae as Targets for Toxins or Effectors Secreted by Mammalian Pathogens

“…Chlamydiae such as the abortigenic Waddlia chondrophila , can resist predation by A. castellanii using effectors secreted by the Type 3 Secretion System such as Wimp1 (Wcw_1131). This putative Ras guanine-nucleotide exchange factor, expressed early during the course of a replication cycle, localizes to the inclusion membrane of W. chondrophila [ 43 ]. These Chlamydial Inclusion membrane proteins (Incs) are suggested to induce the formation of the inclusion membrane, important for the chlamydial developmental cycle [ 44 ].…”

The Chlamydia trachomatis IncM Protein Interferes with Host Cell Cytokinesis, Centrosome Positioning, and Golgi Distribution and Contributes to the Stability of the Pathogen-Containing Vacuole

“…Chlamydial T3S effectors include inclusion membrane proteins (Incs), characterized by at least one bilobal hydrophobic motif mediating their insertion in the inclusion membrane (5,(12)(13)(14). Incs are present in all Chlamydiae (13,15,16), but only some Incs are conserved among Chlamydia species (13,17). However, apart from the characteristic hydrophobic motif, Incs from the same Chlamydia species are normally unrelated to each other.…”