Biology subjects

Heras, B.

Publications and source records attributed to Heras, B..

6 recordsLinked to original sources

bioRxiv2025

Extracellular loops of the β-barrel domain catalyze rapid folding for function of self-associating autotransporters

Bacterial aggregation is a phenotype associated with disease pathogenesis. Aggregate formation enhances biofilm development, host colonization, and resistance to antibiotics and host defenses. Antigen 43 (Ag43) is a surface-located autotransporter produced by pathogenic Escherichia coli that mediates cell aggregation in biofilms. Two Ag43 molecules, each from neighboring bacterial cells, fold into elongated {beta}-helical passenger domains that associate in a head-to-tail manner while being anchored to the cell surface by their outer membrane-embedded {beta}-barrels. In this study, we conduct mutational analyses on Ag43 to show that the {beta}-hairpin structure of the fourth and fifth extracellular loops of the {beta}-barrel domain have a crucial role for passenger domain folding and subsequent formation of bacterial aggregates. This work provides mechanistic insight into the role of the autotransporter {beta}-barrel domain to nucleate the rapid folding of the passenger domain into the {beta}-helix that enables bacterial interactions during infection.

biochemistry↗

bioRxiv2023

Bacterial suppressor-of-copper-sensitivity (Scs) proteins exhibit diverse thiol-disulfide oxidoreductase cellular functions

Disulfide bond (Dsb) proteins catalyse oxidative protein folding governing bacterial survival and virulence. Dsb systems in Escherichia coli K-12 are well-studied, yet what determines dithiol oxidase or disulfide reductase activity remains unknown. Past studies suggest oligomerisation of periplasmic thiol oxidoreductases dictates the direction of thiol catalytic activity. Here, we studied three suppressor-of-copper-sensitivity C (ScsC) Dsb-like proteins known to exist in the reduced state and bind to copper. These proteins adopt different quaternary structures: Salmonella enterica ScsC (StScsC) is monomeric, while ScsC from Proteus mirabilis (PmScsC) and Caulobacter crescentus (CcScsC) are trimeric. When expressed in the model organism E. coli K-12, we showed that all three ScsC proteins exhibit both dithiol oxidation and disulfide reduction activity, despite structural differences. Interestingly, while ScsC reductase function was supported by the canonical E. coli DsbD reductase, oxidase activity depended on environmental oxidation. However, an engineered monomeric PmScsC synergises with E. coli DsbB to gain dithiol oxidase activity at the expense of reductase function. Thus, oligomerisation could be one mechanism by which ScsC proteins avoid interactions with the periplasmic thiol oxidase pathway. This tightly controls their re-oxidation and maintains ScsC proteins in the reduced state required for binding and sequestering toxic levels of cellular copper.

microbiology↗

bioRxiv2021

Fine tuning the mechanism of Antigen 43 self-association to modulate aggregation levels of Escherichia coli pathogens

Bacterial aggregates and biofilms allow bacteria to colonise a diverse array of surfaces that can ultimately lead to infections, where the protection they afford permits bacteria to resist anti-microbials and host immune factors. Despite these advantages there is a trade-off, whereby bacterial spread is reduced. As such, biofilm development needs to be regulated appropriately to suit the required niche. Here we investigate members from one of largest groups of bacterial adhesins, the autotransporters, for their critical role in the formation of bacterial aggregates and biofilms. We describe the structural and functional characterisation of autotransporter Ag43 homologues from diverse pathogenic Escherichia strains. We reveal a common mode of trans-association that leads to cell clumping and show that subtle variations in these interactions governs their aggregation kinetics. Our in depth investigation reveals an underlying molecular basis for the tuning of bacterial aggregation.

biochemistry↗

bioRxiv2020

High-throughput cell-based assays for the preclinical development of DsbA inhibitors as antivirulence therapeutics.

Antibiotics are failing fast, and the development pipeline is alarmingly dry. New drug research and development is being urged by world health officials, with new antibacterials against multidrug-resistant Gram-negative pathogens as the highest priority. Antivirulence drugs, which are inhibitors of bacterial pathogenicity factors, are a class of promising antibacterials, however, their development is often stifled by lack of standardised preclinical testing akin to what guides antibiotic development. The lack of established target-specific microbiological assays amenable to high-throughput, often means that cell-based testing of virulence inhibitors is absent from the discovery (hit-to-lead) phase, only to be employed at later-stages of lead optimization. Here, we address this by establishing a pipeline of bacterial cell-based assays developed for the identification and early preclinical evaluation of DsbA inhibitors. Inhibitors of DsbA block bacterial oxidative protein folding and were previously identified by biophysical and biochemical assays. Here we use existing Escherichia coli DsbA inhibitors and uropathogenic E. coli (UPEC) as a model pathogen, to demonstrate that a combination of a cell-based AssT sulfotransferase assay and the UPEC motility assay, modified for a higher throughput format, can provide a robust and target-specific platform for the evaluation of DsbA inhibitors. Our pipeline could also be used in fragment and compound screening for the identification of new DsbA inhibitor classes or hits with a broad spectrum of activity. In conclusion, the establishment of accurate, high-throughput microbiological assays for antivirulence drug identification and early preclinical development, is a significant first step towards their translation into effective therapeutics. ImportanceThe safety net of last resort antibiotics is quickly vanishing as bacteria become increasingly resistant to most available drugs. If no action is taken, we will likely enter a post-antibiotic era, where common infections and minor injuries are once again lethal. The paucity in new antibiotic discovery of the past decades has compounded the problem of increasing antibiotic resistance, to the point that it now constitutes a global health crisis that demands global action. There is currently an urgent need for new antibacterial drugs with new targets and modes of action. To address this, research and development efforts into antivirulence drugs, such as DsbA inhibitors, have been ramping up globally. However, the development of microbiological assays as tools for effectively identifying and evaluating antivirulence drugs is lagging behind. Here, we present a high-throughput cell-based screening and evaluation pipeline, which could significantly advance development of DsbA inhibitor as antivirulence therapeutics.

microbiology↗

bioRxiv2019

Sortase-assembled pili promote extracellular electron transfer and iron acquisition in Enterococcus faecalis biofilm

Enterococcus faecalis sortase-assembled endocarditis and biofilm-associated pili (Ebp) are a virulence factor implicated in enterococcal biofilm-associated infections and gastrointestinal colonization. We previously showed that E. faecalis biofilm metabolism is influenced by extracellular electron transfer (EET) under iron-rich conditions, raising the question of whether Ebp pili also play a role in EET. Here, we report a novel role of Ebp pili in E. faecalis adhesion to the iron oxides magnetite, goethite and hematite, where the EbpA tip adhesin contributes to this interaction. Adhesion by Ebp pili is conditionally important for EET to iron oxides, as pilus mutants are attenuated in EET under non-static growth conditions. In alignment with the established role of EET in redox homeostasis, we find that EET to ferricyanide supports E. faecalis anaerobic growth on glycerol. Further, in an antibiotic-treated mouse gastrointestinal colonization model, we show that E. faecalis mutants deficient in EET poorly colonize the intestinal niche. Taken together, our findings suggest that Ebp pili can influence E. faecalis metabolic fitness by promoting EET to iron oxides, raising new questions of how Ebp pili shape E. faecalis interactions with environmental ecosystems. Furthermore, the important role of EET in E. faecalis colonization of the dysbiotic gastrointestinal environment highlights the need for further inquiry into how EET contributes to E. faecalis microbial pathogenesis. ImportanceIn this study, we explored the interplay between extracellular electron transfer (EET) and an Enterococcus faecalis biofilm factor, the endocarditis and biofilm-associated pili (Ebp). We demonstrate that Ebp pili have a novel role in adhesion to iron oxides, which consequently promotes EET to iron oxides under non-static conditions. Along with our findings that E. faecalis EET can be coupled to anaerobic cell growth, our results point to a potential ecological role of Ebp pili in natural environments, outside of its established function in adhesion to host ligands. We provide the first evidence of the contribution of EET to E. faecalis colonization of the antibiotic-treated murine intestinal niche, which adds to the limited experimental evidence linking EET and microbial pathogenesis, as well as highlights the need for further studies of EET in bacterial pathogens.

microbiology↗

bioRxiv2018

The atypical thiol-disulfide exchange protein α-DsbA2 from Wolbachia pipientis is a homotrimeric disulfide isomerase

DiSulfide Bond (DSB) oxidative folding enzymes are master regulators of virulence localized to the periplasm of many Gram-negative bacteria. The archetypal DSB machinery from Escherichia coli K12 has a dithiol oxidizing redox relay pair (DsbA/B), a disulfide isomerizing redox relay pair (DsbC/D) and specialist reducing enzymes DsbE and DsbG that also interact with DsbD. By contrast the Gram-negative bacterium Wolbachia pipientis encodes just three DSB enzymes. Two of these -DsbA1 and -DsbB form a redox relay pair analogous to E. coli DsbA/B. The third enzyme -DsbA2 incorporates a DsbA-like sequence but does not interact with -DsbB. In comparison with other DsbA enzymes, -DsbA2 has [~]50 extra N-terminal residues. The crystal structure of -DsbA2{Delta}N, the N-terminally truncated form in which these residues are removed confirms the DsbA-like nature of this domain. However, -DsbA2 does not have DsbA-like activity: it is structurally and functionally different as a consequence of its N-terminal residues. First, -DsbA2 is a powerful disulfide isomerase and a poor dithiol oxidase - ie its role is to shuffle rather than introduce disulfide bonds. Moreover, small-angle X-ray scattering of -DsbA2 reveals a homotrimeric arrangement. Our results allow us to draw conclusions about the factors required for functionally equivalent enzymatic activity across structurally diverse protein architectures.

biochemistry↗