The discovery of SycO reveals a new function for type three secretion effector chaperones

Abstract

The Type Three Secretion (T3S) system is a device used by many Gram-negative pathogens that allows bacteria to deliver effector proteins straight into the eukaryotic cell cytosol. These effectors interfere with various signaling pathways to subvert the host cell functions. The secretion machinery of the T3S system consist of a basal body spanning the bacterial inner and outer membrane followed by a stiff hollow needle outside the bacterium. The fully assembled secretion apparatus constitute a continuous hollow conduit that connects the bacteria to the eukaryotic target cell. After cell contact, virulence proteins -called effectors- are injected directly into the cytosol of the host cell via the T3S apparatus. Several effectors of the T3S system require the assistance of specific cytosolic chaperones to be efficiently exported. There are three classes of T3S chaperones. Effector proteins are assisted by Class I chaperones. Although Class I chaperones are well characterized, their main function is still a matter of controversy. In this thesis, we demonstrate that orf155 encodes a specific chaperone for the effector YopO that we called SycO. We showed that SycO enhances YopO secretion in vitro and is required for translocation of YopO into infected cells. By pulldown assay we demonstrated that residues 20 to 77 of YopO are required and sufficient for SycO binding. Using crosslinking experiments and size exclusion chromatography analysis, we determined the stoichiometry of purified SycO and YopO-SycO complexes. SycO alone forms dimers in solution and the YopO-SycO complex has a 1:2 stoichiometry. These results suggested that SycO is a typical chaperone of the Class I. YopO is a serine/theronine kinase that interacts with Rho and Rac and disrupts the cytoskeleton of the target cells. YopO has been shown to localize at the cell plasma-membrane. By transfection of YopO-EGFP hybrid proteins into HEK293T cells, we demonstrated that the chaperone-binding domain (CBD) coincides with the membrane localization domain of YopO. Nevertheless, the CBD was not needed for the kinase activity of YopO. By ultracentrifugation, we also showed that the CBD causes YopO aggregation in the bacteria, when SycO does not cover it. Further, we show that the CBD of YopE and YopT also caused aggregation in the bacteria in the absence of SycE and SycT respectively. YopE, YopT and T3S effectors in other systems also act at the membrane of the eukaryotic host cell. We propose a new hypothesis concerning the role of T3S chaperones. The sub-cellular localization domain of effectors is aggregation-prone and creates the need for a chaperone inside bacteria. We propose that masking such aggregation-prone localization domains may be a general function for type III effector chaperones.