Biology subjects

Doyle, M. T.

Publications and source records attributed to Doyle, M. T..

4 recordsLinked to original sources

bioRxiv2025

A Protein Antibiotic Inhibits the BAM Complex to Kill Without Cell Entry

Many antibiotics are ineffective against Gram-negative pathogens such as Pseudomonas aeruginosa because they cannot penetrate the bacterial outer membrane. Here, we show that protein antibiotics called L-type pyocins kill P. aeruginosa by inhibiting the {beta}-barrel assembly machinery (BAM) complex at the cell surface, halting outer-membrane protein assembly. Using single-particle cryo-electron microscopy, we show that L-type pyocins bind a surface-exposed region of BamA and deploy a C-terminal peptide that competitively inhibits the BAM complex, demonstrating that cell entry is not required for antibiotic activity. We combine genetics, multi-omics and cryo-electron tomography to show that BAM complex inhibition by L-type pyocins or the peptide antibiotic darobactin triggers a multifaceted transcriptomic, proteomic and morphological response. Despite this, BAM inhibition ultimately leads to a catastrophic loss of membrane integrity and cell death. These results validate BAM as a target for antibiotics that do not enter the cell and define an engineerable system for their development.

microbiology↗

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

The patatin-like protein PlpD forms novel structurally dynamic homodimers in the Pseudomonas aeruginosa outer membrane

Members of the Omp85 superfamily of outer membrane proteins (OMPs) found in Gram-negative bacteria, mitochondria and chloroplasts are characterized by a distinctive 16-stranded {beta}-barrel transmembrane domain and at least one periplasmic POTRA domain. All previously studied Omp85 proteins promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of an Omp85 protein family that contains an N-terminal patatin-like (PL) domain that is thought to be translocated across the OM by a C-terminal {beta}-barrel domain. Challenging the current dogma, we found that the PlpD PL-domain resides exclusively in the periplasm and, unlike previously studied Omp85 proteins, PlpD forms a homodimer. Remarkably, the PL-domain contains a segment that exhibits unprecedented dynamicity by undergoing transient strand-swapping with the neighboring {beta}-barrel domain. Our results show that the Omp85 superfamily is more structurally diverse than currently believed and suggest that the Omp85 scaffold was utilized during evolution to generate novel functions.

microbiology↗

bioRxiv2021

Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding

Transmembrane {beta}-barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the {beta}-barrel assembly machine (BAM) via an unexplained process that occurs without known external energy sources. Here we used single-particle cryo-EM to visualize the folding dynamics of a model {beta}-barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "{beta}-signal" motif of EspP to correctly orient {beta}-strands in the OM during folding. We also found that the folding of EspP proceeds via remarkable "hybrid-barrel" intermediates in which membrane integrated {beta}-sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that {beta}-sheets progressively fold towards BamA to form a {beta}-barrel. Along with in vivo experiments that tracked {beta}-barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate {beta}-barrel folding.

biochemistry↗