Gierys, A. J. | Search bioRxiv

bioRxiv2025

Biophysical characterization of Eag chaperones suggests the mechanism of effector transmembrane domain release

The type VI secretion system (T6SS) is a dynamic protein nanomachine found in Gram- negative bacteria that secretes toxic effectors into prey-cells. For secretion, effectors require chaperones or adaptors for proper loading onto the T6SS. Effector associated genes (Eags) are a family of T6SS chaperones that stabilize N-terminal transmembrane domains (TMDs) found in thousands of effectors. Eags are essential for secretion and inhibit effector TMDs from prematurely adopting a membrane-penetrative conformation. However, the mechanism of TMD release from its cognate Eag chaperone is unknown. Here, we take a biochemical and biophysical approach to probe the mechanism of TMD binding and dissociation from Eag chaperones. Using steady-state fluorescence, stopped-flow measurements, and bacterial competition assays, we compare the thermodynamics, kinetics, and in vivo chaperone function of wild-type and point-variant Eag-TMD complexes. Additionally, we solve an X-ray crystal structure of an Eag-TMD point-variant complex that captures an intermediate state of TMD release. Our data reveals the molecular features and specific residue contacts necessary for TMD binding and demonstrates the Eag conformational change required to initiate rapid release of the TMD. Overall, our work details the stability of Eag-TMD complexes and the energetic pathway for the dissociation of effector TMDs from their Eag chaperones.

biochemistry↗

bioRxiv2024

Transcription Factors DksA and PsrA are synergistic contributors to L. pneumophila virulence in Acathamoeba castellanii protozoa

The environmental bacterium Legionella pneumophila, an intracellular parasite of free-living freshwater protozoa as well as an opportunistic human pathogen, has a biphasic lifestyle. The switch from the vegetative replicative form to the environmentally resilient transmissive phase form is governed by a complex stringent response-based regulatory network that includes RNA polymerase co-factor DksA. Here we report that, through a dysfunctional DksA mutation (DksA1), a synergistic interplay was discovered between DksA and transcription regulator PsrA using the Acanthamoeba castellanii protozoan infection model. Surprisingly, in trans expression of PsrA partially rescued the growth defect of a dksA1 strain. While in trans expression of DksA expectantly could fully rescued the growth defect of the dksA1 strain, it could also surprisingly rescue the growth defect of a {Delta}psrA strain. Conversely, the severe intracellular growth defect of a {Delta}dksA strain could be rescued by in trans expression of DksA and DksA1, but not PsrA. In vitro phenotypic assays show that either DksA or DksA1 were required for extended culturability of bacterial cells, but normal cell morphology and pigmentation required DksA only. Comparative structural modeling predicts that the DksA1 mutation affects coordination of Mg2+ into the active site of RNAP compromising transcription efficiency. Taken together, we propose that PsrA transcriptionally assists DksA in the expression of select transmissive phase traits. Additionally, in vitro evidence suggests that the long chain fatty chain metabolic response is mediated by PsrA together with DksA inferring a novel regulatory link to the stringent response pathway.

microbiology↗