Biology subjects

Pace, H.

Publications and source records attributed to Pace, H..

3 recordsLinked to original sources

bioRxiv2021

Protein-lipid interaction at low pH induces oligomerisation of the MakA cytotoxin from Vibrio cholerae

Many pathogenic bacteria produce protein toxins that target and perturb host cell membranes. The secreted -pore-forming toxins (-PFTs) cause membrane damage via pore formation. This study demonstrates a remarkable, hitherto unknown mechanism by an -PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other -PFTs. In contrast, MakA protein alone induces tube-like structures in the acidic lysosomal host cell compartment. In vitro studies unravel the dynamics of tubular growth, which occur in a pH-, lipid- and concentration-dependent manner. A 3.7-[A] cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. In its active -PFT conformation, MakA embeds its transmembrane helices into a thin annular lipid bilayer and spirals around a central cavity. Our study provides molecular insights into a novel tubulation mechanism of an -PFT protein, revealing a new mode of action by a secreted bacterial toxin.

cell biology↗

bioRxiv2021

A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion

The protein MakA was discovered as a motility-associated secreted toxin from Vibrio cholerae, Here, we show that MakA is part of a gene cluster encoding four additional proteins: MakB, MakC, MakD and MakE. The MakA, MakB and MakE proteins were readily detected in culture supernatants of wild type V. cholerae whereas secretion was very much reduced from a flagellum deficient mutant. Crystal structures of MakA, MakB and MakE revealed structural relationship to a superfamily of bacterial pore-forming proteins. Cloning and expression of MakA/B/E in Escherichia coli resulted in toxicity of the bacteria towards Caenorhabditis elegans used as a predatory organism model. None of these Mak proteins alone or in pairwise combinations were cytolytic but an equimolar mixture of MakA, MakB and MakE acted as a tripartite cytolytic toxin in vitro causing lysis of erythrocytes and cytotoxicity on cultured human colon carcinoma cells. Formation of oligomeric complexes on liposomes was observed by electron microscopy. Oligomer interaction with membranes was initiated by MakA membrane binding followed by MakB and MakE joining in formation of a pore structure. A predicted membrane insertion domain of MakA was shown by site-directed mutagenesis to be essential for toxicity towards C. elegans. Bioinformatic analyses revealed that the makCDBAE gene cluster is present as a novel genomic island in the vast majority of sequenced genomes of V. cholerae and the fish pathogen V. anguillarum. We suggest that the hitherto unrecognized cytolytic MakA/B/E toxin can contribute to Vibrionaceae fitness and virulence potential in different host environments and organisms. Significance StatementVibrio cholerae, responsible for outbreaks and pandemics of cholera disease, is a highly motile organism by virtue of a single flagellum. We describe that the flagellum facilitates the secretion of three V. cholerae proteins encoded by a hitherto unrecognized novel genomic island. The proteins MakA/B/E can form a tripartite cytolytic toxin that lyses erythrocytes and is cytotoxic to cultured human cells. A structural basis for the Mak protein cytolytic activity was obtained by X-ray crystallography. Flagellum-facilitated secretion, remarkably ensuring spatially co-ordinated delivery of Mak proteins, revealed a new role for the V. cholerae flagellum considered of particular significance for the bacterial environmental persistence. Our findings will pave the way for the development of new diagnostics and therapeutic strategies against pathogenic Vibrionaceae.

biochemistry↗

bioRxiv2021

Membrane insertion of Cavin1 facilitates caveolae assembly

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades and lipid sorting. The caveolae coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modelling to show that Cavin1 inserts into membranes. We establish that initial PI(4,5)P2-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of the HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the co-assembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane. Significance statementCaveolae are cholesterol enriched membrane invaginations coupled to severe muscle and lipid disorders. Their formation is dependent on assembly of the protein Cavin1 at the lipid membrane interface driving membrane curvature. In this work, we dissect the mechanism for how Cavin1 binds and inserts into membranes using a combination of biochemical and biophysical characterization as well as computational modelling. The proposed model for membrane assembly potentiates dynamic switching between shielded and exposed hydrophobic helices used for membrane insertion and clarifies how Cavin1 can drive membrane curvature and the formation of caveolae.

cell biology↗