Biology subjects

Alcock, F.

Publications and source records attributed to Alcock, F..

6 recordsLinked to original sources

bioRxiv2025

The Staphylococcus aureus LXG-domain toxins EsxX and SAR0287 do not promote virulence in a zebrafish larval infection model

The Staphylococcus aureus type VIIb secretion system (T7SSb) is a multiprotein secretion system that secretes toxins with antibacterial activity, but which is also required for full virulence in animal models of infection. S. aureus strains carry one of four T7SSb locus types, named essC1 to essC4, each of which encodes a characteristic LXG-family substrate at the T7SS locus. In essC2 strains this LXG-domain protein is EsxX, which has a glycine zipper sequence in its C-terminus and has potent antibacterial membrane-depolarising activity. In this work we recognise conserved features of the essC2 and essC3 systems, identifying the LXG protein SAR0287 as structurally and functionally similar to EsxX. Using a zebrafish larval hindbrain ventricle infection model we demonstrate that the T7SSb of essC2 and essC3 representative strains contribute to bacterial replication and zebrafish mortality. However, there is no significant loss of virulence in the model system if EsxX or SAR0287 are absent. These findings indicate there is no discernible role for either toxin in this virulence model.

microbiology↗

bioRxiv2025

Distinct immunity protein families mediate compartment-specificneutralisation of a bacterial toxin

Staphylococcus aureus utilises a type VII secretion system (T7SS) to secrete antibacterial toxins that target competitor bacteria. EsxX is a T7SS substrate protein secreted by ST398 strains that has an LXG domain at its N-terminus. Here we show that the EsxX C-terminus is a membrane-depolarising toxin with a glycine zipper motif. EsxX is profoundly toxic to bacteria, displaying toxicity from both cytoplasmic and extracellular compartments. A pair of polytopic membrane proteins, ExiCD, protect cells from intoxication by extracellular EsxX. By contrast, a distinct soluble heterodimer, ExiAB, neutralises cytoplasmic EsxX by sequestration of its glycine zipper motif in a binding groove on ExiB. The exiA-exiB gene pair co-occur in staphylococcal genomes with esxX, invoking a model whereby ExiAB protects EsxX-producing cells from self-toxicity prior to EsxX secretion. By contrast ExiCD is encoded by both EsxX producers and in antitoxin islands of competitor strains that do not encode EsxX, consistent with providing immunity against the secreted form of the toxin. This work defines a new class of antibacterial toxin requiring two distinct types of immunity protein which follow different phylogenetic distributions.

microbiology↗

bioRxiv2023

A high throughput assay to measure Type VII secretion in Staphylococcus aureus

Successful colonisation by the opportunistic pathogen Staphylococcus aureus depends on its ability to interact with other microorganisms. S. aureus strains harbour a T7b-subtype type VII secretion system (T7SSb), a protein secretion system found in a wide variety of Bacillota which functions in bacterial antagonism and virulence. Assessment of T7SSb activity in S. aureus has been hampered by low secretion activity under laboratory conditions, and the lack of a sensitive assay to measure secretion. Here we have utilised NanoLuc Binary Technology to develop a simple assay to monitor protein secretion via detection of bioluminescence. Fusion of the 11 amino acid NanoLuc fragment to the conserved substrate EsxA permits its extracellular detection upon supplementation with the large NanoLuc fragment and luciferase substrate. Following miniaturisation of the assay to 384 well format, we use high-throughput analysis to demonstrate that T7SSb-dependent protein secretion differs across strains and growth temperature. We further show that the same assay can be used to monitor secretion of the surface-associated toxin substrate TspA. Using this approach we identify three conserved accessory proteins required to mediate TspA secretion. Co-purification experiments confirm that all three proteins form a complex with TspA.

microbiology↗

bioRxiv2023

EsxB, EsxC and EsxD facilitate export of the antibacterialnuclease EsaD by the Staphylococcus aureus Type VII secretionsystem

The type VIIb protein secretion system (T7SSb) plays a role in interbacterial competition in Gram-positive Firmicute bacteria and secretes various toxic effector proteins. The mechanism of secretion and the roles of numerous conserved genes within T7SSb gene clusters remain unknown. EsaD is a nuclease toxin secreted by the Staphylococcus aureus T7SSb, which forms a complex with its cognate immunity protein, EsaG, and chaperone EsaE. Encoded upstream of EsaD are three small secreted proteins, EsxB, EsxC and EsxD. Here we show that EsxBCD bind to the transport domain of EsaD and function as EsaD export factors. We report the first structural information for a complete T7SSb substrate pre-secretion complex. Cryo-EM of the EsaDEG trimer and the EsaDEG-EsxBCD hexamer shows that incorporation of EsxBCD confers a conformation comprising a flexible globular cargo domain attached to a long narrow shaft that is likely to be crucial for efficient toxin export.

microbiology↗

bioRxiv2020

Structure of a proton-powered molecular motor that drives protein transport and gliding motility

Ion-driven motors are rare in biology. The archetypes of the three classes identified to date are ATP synthase, the bacterial flagellar motor, and a proton-driven motor that powers gliding motility and protein secretion in Bacteroidetes bacteria. Whilst the molecular mechanism of ATP synthase is now well understood, structural information is lacking for the other two classes of motor. Here we present the structure of the Bacteroidetes gliding motility motor determined by cryo-electron microscopy. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM proteins are positioned within a ring of five GldL proteins. Combining mutagenesis and single-molecule tracking, we identify protonatable amino acid residues within the transmembrane domain of the complex that are important for motor function. Our data imply a mechanism in which proton flow leads the periplasm-spanning GldM dimer to rotate with respect to the intra-membrane GldL ring to drive processes at the bacterial outer membrane. This work provides a molecular basis for understanding how the gliding motility motor is able to transduce the energy of the inner membrane protonmotive force across the bacterial cell envelope.

biochemistry↗

bioRxiv2019

Ciprofloxacin facilitates the transfer of XDR plasmids from commensal E. coli into epidemic fluoroquinolone-resistant Shigella sonnei

The global dissemination of a ciprofloxacin-resistant (cipR) S. sonnei clone outlines the mobility of this important agent of diarrheal disease, and threatens the utility of ciprofloxacin as a first-line antimicrobial for shigellosis. Here, we aimed to track the emergence of cipR S. sonnei in Vietnam to understand how novel antimicrobial resistant (AMR) Shigella clones become established in new locations. From 2014 to 2016, we isolated and genome sequenced 79 S. sonnei from children hospitalized with dysenteric diarrhea in southern Vietnam. The novel cipR S. sonnei clone displaced the resident ciprofloxacin-susceptible lineage while acquiring resistance against third-generation cephalosporins, macrolides, and aminoglycosides. This process was not the result of a single clonal expansion, as we identified at least thirteen independent acquisitions of ESBL-encoding plasmids. The frequency and diversity of the variable AMR repertoire in an expanding clonal background of S. sonnei is unprecedented and we speculated that it was facilitated by horizontal gene transfer from commensal organisms in the human gut. Consequently, we characterized non-Shigella Enterobacteriaceae from Shigella-infected and healthy children by shotgun metagenomics. We identified a wide array of AMR genes and plasmids in the commensal Enterobacteriaceae, including an E. coli isolated from a Shigella-infected child with an identical ESBL plasmid to that characterized in the infecting S. sonnei. We confirmed that these AMR plasmids could be exchanged between commensal E. coli and S. sonnei and found that supplementation of ciprofloxacin into the conjugation media significantly increased the conjugation frequency of IncI/blaCTX-M-15, IncB/O/blaCTX-M-27 and IncF/blaCTX-M-27 plasmids. In a setting with high antimicrobial use and a high prevalence of AMR commensals, cipR S. sonnei may be propelled towards pan-resistance by adherence to outdated international treatment guidelines. Our work highlights the role of the gut microbiota in transferring resistance plasmids into enteric pathogens and provides essential data to restrict the use of ciprofloxacin globally.

microbiology↗