Biology subjects

Moynihan, P.

Publications and source records attributed to Moynihan, P..

6 recordsLinked to original sources

bioRxiv2026

Discoidins are cytosolic lectins that shape intracellular host defence by sensing virulence-associated mycobacterial glycolipids and glycopeptidolipids

Mycobacteria are surrounded by a dynamic, glycolipid-rich envelope that controls both virulence and immune recognition, yet how cytosolic lectins detect and interpret the organization of surface glycans during intracellular infection remains unclear. Here, we show that Dictyostelium discoideum discoidins act as cytosolic sensors of mycobacterial envelope organization. During Mycobacterium marinum infection, Discoidin A and Discoidin E assemble into foci on intracellular bacilli and bind surface-associated glycan patches. Using transposon mutagenesis, synthetic mycobacterial glycan arrays, biochemical fractionation and targeted envelope mutants, we find that discoidins recognize a restricted set of lipid-linked glycans enriched in methylated L-rhamnose motifs, including structures associated with LOS, PGL and GPL. Binding depends on the H-type lectin {beta}-galactoside-binding pocket and is inhibited by point mutations and TDG, a soluble disaccharide competitor, demonstrating glycan-dependent recognition. Specific assays led to exclusion of major structural carbohydrates, including AG-PG, AM/LAM, -glucan and TDM, while protease treatment of capsular material and fractionation of polar lipids, identified both GPL and LOS-like glycolipids as dominant discoidin ligands. Disruption of LOS biosynthesis or PDIM/PGL-dependent envelope organization reduced discoidin binding to intact bacteria even though some ligands remained detectable by dot blot in various envelope extracts. Thus, discoidins do not simply detect ligand abundance, but binding depends on envelope perturbations and unmasking of glycolipids and glycopeptidolipids. These findings reveal pathogen envelope glycans as direct targets of cytosolic lectin surveillance and establish discoidins as probes of mycobacterial envelope remodelling during intracellular infection. Author SummaryMany disease-causing bacteria are surrounded by a protective outer layer that helps them survive inside host cells and avoid being eliminated. This surface is not static, it can be remodelled during infection, changing which molecules are exposed to the host. How host cells detect these changes is still not fully understood. We studied this question using Dictyostelium discoideum, a single-celled amoeba that shares many cellular defence mechanisms with human immune phagocytes. We focused on discoidins, a family of proteins that bind sugars. We found that discoidins accumulate on intracellular Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, the bacterium that causes tuberculosis, and recognize specific sugar-containing molecules exposed at the bacterial surface. Importantly, discoidins do not simply detect whether these molecules are present. Instead, they respond to how they are displayed and exposed on the bacterial surface. Changes in the organization of the bacterial outer layer strongly affected discoidin binding, masking or revealing specific target molecules. Our findings show that discoidins act as sensors of bacterial surface remodelling during infection. More broadly, they reveal an ancient mechanism by which host cells can monitor pathogens by detecting changes in the sugars exposed on their surface.

microbiology↗

bioRxiv2026

A Type VII-secreted toxin enables inter-mycobacterial competition

Summary ParagraphMost bacteria live in complex environments where resources are scarce and competition is fierce. These organisms have evolved mechanisms to compete with other bacteria, often through the specialised secretion of proteinaceous toxins. Mycobacteria have not previously been reported to engage in this form of competition. The thick and unusual cell wall of mycobacteria, comprised of peptidoglycan, arabinogalactan and mycolic acids, is generally thought to be highly protective to these bacteria. Many enzymes have evolved to maintain this structure, including the GH183 family, which cleaves arabinogalactan. Here, we establish for the first time that some mycobacteria have weaponised a subset of these endo-D-arabinanases to enable inter-bacterial competition. We show that mycobacteria secrete an endo-D-arabinanase effector via the type VII secretion system that specifically targets the arabinogalactan layer of the Mycobacteriales cell envelope. Using structural biology and biochemistry, we identify the molecular basis for this activity and reveal a new protein family that protects the bacterium from the activity of this toxin. Finally, our data uncover widespread T7-secreted toxins in the Mycobacteriales, pointing to extensive inter-mycobacterial competition.

microbiology↗

bioRxiv2026

Chemical genomics screening reveals novel functions for unannotated genes in Vibrio cholerae C6706

Vibrio cholerae, the causative agent of cholera, remains a major global health burden despite ongoing initiatives for vaccination and improved sanitation. Much of the V. cholerae life cycle occurs in aquatic environments, requiring gene functions that enable survival under diverse stresses associated with this niche and during the transition to the human host. Although numerous V. cholerae genome sequences are available, functional annotation across its pan-genome remains incomplete, and many annotated genes lack experimental validation. In this study, we employed a chemical genomics approach to profile fitness phenotypes across 104 diverse stress conditions using a library of 3,026 single-gene mutants of V. cholerae C6706, a widely used research strain. In total, we identified significant growth phenotypes for 1,518 mutants, of which 88 correspond to unannotated (hypothetical or putative) genes. Together, these data provide a comprehensive resource for exploring gene function in V. cholerae, supported by detailed quality metrics and open-access tools to facilitate community-driven discovery. AUTHOR SUMMARYAlthough many bacterial pathogens have been extensively sequenced, we still know relatively little about what many of their genes actually do. In many cases, gene functions are solely predicted by computer algorithms but have not been confirmed in the laboratory, and we can assume that some of those predictions may be incorrect. Chemical genomics -- a method that tests thousands of single-gene mutants under different stresses -- can help reveal what these genes are responsible for. Using this approach, we screened a library of Vibrio cholerae C6706 mutants under 104 different conditions. We found that more than half of the mutants showed measurable effects on growth, including many genes that were previously uncharacterized. Here, we describe our experimental pipeline, present key quality checks, and share how other researchers can use this dataset as a resource to explore V. cholerae mutant phenotypes.

systems biology↗

bioRxiv2025

A new role for lipoproteins LpqZ and FecB in orchestrating mycobacterial cell envelope biogenesis

Accounting for more deaths than any other bacterial species, Mycobacterium tuberculosis (Mtb) represents a critical threat to public health worldwide. A key factor contributing to Mtbs virulence is its unique cell envelope, which acts as a protective barrier. Among the components of this envelope, lipoproteins represent a critical but understudied group of proteins. In this study, we focused on 79 conserved putative lipoproteins, shared between Mtb and the closely related M. marinum. Leveraging the CRISPR1-Cas9 (Sth1Cas9) gene editing system for Mycobacteria, we generated frameshift mutations, targeting one conserved lipoprotein-coding gene at a time. We identified two mutants, lpqZ and fecB, that exhibited increased susceptibility to all tested antibiotics, suggesting essential roles in cell envelope biogenesis. Interestingly, despite having homology to periplasmic substrate binding proteins (SBPs), neither protein is associated with any inner membrane transporter complex. Instead, co-immunoprecipitation experiments revealed that LpqZ interacts with AftA and FecB interacts with AftB. Both these interaction partners are essential enzymes involved in arabinogalactan and lipoarabinomannan synthesis. Accordingly, we observed alterations for both glycoconjugates in lpqZ and fecB mutants. Together, these findings show that orphaned SBP-like proteins have been neofunctionalized in mycobacteria to aid key enzymes involved in cell envelope biosynthesis.

microbiology↗

bioRxiv2019

African Salmonella Typhimurium sequence type 313 lineage 2 evades MAIT cell recognition by overexpressing RibB

Mucosal-associated invariant T (MAIT) cells are a subset of innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defence. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with a selective absence of MAIT cells have not been identified. We report that typhoidal and non-typhoidal S. enterica strains generally activate MAIT cells. However, African invasive disease-associated multidrug-resistant S. Typhimurium sequence type 313 lineage 2 strains escape MAIT cell recognition through overexpression of ribB, a bacterial gene that encodes the 4-dihydroxy-2-butanone 4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. This MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium sequence type 313 lineage 2 in vivo.

immunology↗

bioRxiv2019

A bacterial endosymbiont enables fungal immune evasion during fatal mucormycete infection

Opportunistic infections by environmental fungi are a growing clinical problem, driven by an increasing population of people with immunocompromising conditions. Spores of the Mucorales order are ubiquitious in the environment but can also cause acute invasive infections in humans through germination and evasion of the mammalian host immune system. How they achieve this, and the evolutionary drivers underlying the acquisition of virulence mechanisms, are poorly understood. Here we show that a clinical isolate of Rhizopus microsporus contains a Ralstonia pickettii bacterial endosymbiont required for virulence in both zebrafish and mice, and that this endosymbiosis enables secretion of factors that potently suppress growth of the soil amoeba Dictyostelium discoideum, as well as their ability to engulf and kill other microbes. As amoebae are natural environmental predators of both bacteria and fungi, we propose this tri-kingdom interaction contributes to establishing the endosymbiosis, and acquisition of anti-phagocyte activity. Importantly, we show this activity also protects fungal spores from phagocytosis and clearance by human macrophages, and endosymbiont removal renders the fungal spores avirulent in vivo. Together, these findings describe a novel role for a bacterial endosymbiont in Rhizopus microsporus pathogenesis in animals, and suggest a mechanism of virulence acquisition through environmental interactions with amoebae. In briefHow environmental fungi evolved the mechanisms that enable them to cause opportunistic infections in humans is unclear. Here, we identify a novel tri-kingdom interaction, whereby a bacterial endosymbiont, living within a clinical isolate of the ubiquitous environmental fungus Rhizopus microsporus, causes the generation of a secreted activity that blocks the growth and predatory activity of amoebae. We suggest this provides a new evolutionary driver for the establishment of bacterial/fungal endosymbiosis and demonstrate this is critical for fungal pathogenicity in vivo.

microbiology↗