Biology subjects

Cabanes, D.

Publications and source records attributed to Cabanes, D..

7 recordsLinked to original sources

bioRxiv2026

Structural mechanism of metal-induced DNA release by an ArsR/SmtB transcriptional regulator

Metal-responsive transcriptional regulators must couple metal coordination to DNA release at the DNA-binding interface. However, the pathway connecting these states remains poorly understood. Here, we define this mechanism for the ArsR/SmtB-family repressor CadC from Listeria monocytogenes by combining crystal structures of apo, DNA-bound, and cadmium-bound states with biochemical and in vivo analyses. The DNA-bound structure reveals how CadC dimers bind sequence-specifically to operator DNA, via coordinated major-groove, wing and backbone interactions, with high-affinity binding depending on operator architecture. Cadmium is coordinated at the 3N sensor by an unusual Cys3-Met ligand set and stabilizes a DNA-incompatible conformation through remodeling of DNA-contacting elements and steric incompatibility between the ordered N-terminus and DNA. Mutational analyses show how these structural transitions link to metal-dependent transcriptional control in Listeria cells. We show that CadC binds zinc and supports zinc-responsive transcription in bacterial cells, and that host zinc availability modifies CadC-dependent infection phenotypes. Together, results reveal how metal coordination is converted into DNA release through coupled allosteric remodeling and steric exclusion, establishing a mechanism for signal-dependent transcriptional switching in ArsR/SmtB metalloregulators.

microbiology↗

bioRxiv2026

Wall teichoic acid glycosylation shapes surface and secreted protein distribution in Listeria monocytogenes.

Listeria monocytogenes relies on a tightly controlled set of surface-associated and secreted proteins to mediate host interaction and infection. The correct localization and exposure of these proteins at the bacterial surface are critical for virulence, yet the role of cell wall components in organizing this process remains incompletely understood. In particular, wall teichoic acid (WTA) glycosylation has been implicated in anchoring and function of selected surface proteins, but its global impact on protein distribution across the bacterial cell envelope is unclear. Here, we performed a comprehensive proteomic analysis to investigate how WTA glycosylation influences protein distribution in L. monocytogenes. Using isogenic mutants lacking rhamnose ({Delta}rmlT) or GlcNAc ({Delta}lmo1079) WTA glycosylation, we compared the exoproteome, the surface-accessible proteome and the surface-exposed proteome. Loss of WTA glycosylation did not result in a global disruption of the surface proteome but instead induced a redistribution of proteins across extracellular and surface-associated fractions. This effect was dependent on protein anchoring mechanisms, with limited changes observed for LPXTG-anchored proteins, moderate effects on non-covalently associated proteins, and a marked enrichment of lipoproteins in the surface-exposed proteome, particularly in the {Delta}lmo1079 mutant. In parallel, virulence-associated proteins displayed altered accessibility and exposure, with a progressive shift towards increased surface localization and a combination of shared and mutant-specific responses. This global effect was supported by functional annotation, which revealed that the affected proteins were associated with similar biological processes across fractions, highlighting a broad rather than pathway-specific impact of WTA glycosylation loss Together, these findings indicate that WTA glycosylation plays a key role in organizing the bacterial surface by modulating protein retention, exposure and release. Rather than affecting specific proteins, WTA glycosylation broadly shapes the spatial distribution of proteins across the cell envelope, with potential consequences for host- pathogen interactions.

microbiology↗

bioRxiv2026

Src-dependent tyrosine-phosphorylation of NM2A has a protective role against bacterial pore-forming toxins

Pore-forming toxins (PFTs) are key bacterial virulence factors that disrupt host plasma membrane (PM) integrity, triggering cellular stress and initiating repair mechanisms. The cytolysin Listeriolysin O (LLO), secreted by Listeria monocytogenes, has well established roles in infection, yet the host signaling responses to LLO-induced damage remain poorly understood. Here, we identify a previously unrecognized protective pathway in which LLO triggers rapid activation of the tyrosine kinase Src, leading to phosphorylation of the non-muscle myosin II heavy chain NMHC2A at tyrosine 158. While Src activation and NMHC2A tyrosine phosphorylation have been observed during Listeria infection, we demonstrate here that both responses are directly driven by LLO. This phosphorylation event does not alter NMHC2A motor activity in vitro but is required for cytoskeletal reorganization and efficient responses to PM damage. Using Caenorhabditis elegans, we further show that phosphorylation of the NMHC2A homolog NMY-2 at the conserved tyrosine 163 is required for survival under PFT-induced stress and heat shock, revealing an evolutionarily conserved defense mechanism. Together, our findings establish Src-mediated NMHC2A phosphorylation as a critical link between PFT-induced PM damage sensing and actomyosin remodeling, advancing our understanding of host defense against bacterial toxins. Author SummaryPathogenic bacteria often produce toxins that create pores in the membranes of host cells, compromising cell survival and helping the infection spread. To counter this, host cells must quickly detect and repair the damage. We studied how cells respond to Listeriolysin O (LLO), produced by the human pathogenic bacterium Listeria monocytogenes. We found that LLO activates the host Src kinase, which in turn modifies a key structural protein, non-muscle myosin II (NMII). Although this modification does not affect NMIIs normal motor function, it is crucial for organizing the cellular skeleton and protecting the cells membrane after toxin damage. Importantly, we found that this protective mechanism is conserved in the worm Caenorhabditis elegans, where it also defends against a similar bacterial toxin and even heat stress. Our findings reveal a new way that cells use existing structural proteins to rapidly respond to external threats, providing insight into how organisms protect themselves from harmful microbes and environmental stress.

microbiology↗

bioRxiv2025

Rhamnose biosynthesis is not impaired by the deletion of putative rfbC genes, slr0985 and slr1933, in Synechocystis sp. PCC 6803

Cyanobacterial extracellular polymeric substances (EPS), mainly composed by heteropolysaccharides, can be attached to the cell wall (CPS) or released to the environment (RPS). These polymers have an unusually highly diversified monosaccharidic composition, making them attractive for biotechnological/biomedical applications. However, their production is still poorly understood hindering their optimisation for industrial needs. This work aimed at better understanding the biosynthesis of the 6-deoxysugars fucose and rhamnose in the model cyanobacterium Synechocystis sp. PCC 6803. To that end, genes encoding proteins putatively involved in the biosynthesis of GDP-L-fucose [sll1213 (fucS)] and dTDP-L-rhamnose [slr0985 (rfbC1) and slr1933 (rfbC2)] were deleted. As previously observed, {Delta}fucS had significant growth impairment and its RPS did not contain any fucose or rhamnose. Here, we also showed that both deoxyhexoses pathways are completely impaired in {Delta}fucS. In contrast, both {Delta}rfbC1 and {Delta}rfbC1{Delta}rfbC2 although producing significantly less RPS and more CPS than the wild type, did not show major differences regarding the RPS monosaccharidic composition. These results strongly suggest that their gene products are not essential for rhamnose biosynthesis. Transcriptional analysis revealed that one of the gmd genes (slr1072), putatively encoding a GDP-mannose 4,6-dehydratase, was upregulated in all the knockout strains, and that the three EPS-related genes in the same operon as rfbC1 (slr0982, slr0983 and slr1610) were upregulated in both {Delta}rfbC strains. Altogether, our results reveal that rhamnose biosynthesis in Synechocystis depends on FucS but not on the putative RfbC enzymes, underlining the need to further elucidate the mechanisms involved in the biosynthesis of this deoxyhexose. IMPORTANCEThis study contributes to the overall knowledge of deoxyhexoses biosynthesis in Synechocystis sp. PCC 6803. Here we demonstrated that the {Delta}fucS strain not only produces EPS without fucose and rhamnose but that both pathways are completely impaired. Furthermore, we also showed that the deletion of both putative rfbC genes do not affect rhamnose biosynthesis, despite having an impact on carbohydrates production/export, shifting RPS to CPS production. Altogether, our results suggest that the rfbC genes are not correctly annotated and highlight the intricacies and/or potential crosstalk between the two deoxyhexoses pathways, yet to be completely unravelled in Synechocystis. The understanding of cyanobacterial EPS assembly and export is crucial for the optimisation of their production and tailoring for industrial/commercial applications.

microbiology↗

bioRxiv2024

Molecular properties of RmlT, a wall teichoic acid rhamnosyltransferase that modulates Listeria monocytogenes virulence and antibiotic resistance

Wall teichoic acids (WTAs) from the major Gram-positive foodborne pathogen Listeria monocytogenes are peptidoglycan-associated glycopolymers decorated by monosaccharides that, while not essential for bacterial growth, are required for bacterial virulence and resistance to antimicrobials. Here we report the structure and function of a bacterial WTAs rhamnosyltransferase, RmlT, strictly required for L. monocytogenes WTAs rhamnosylation. In particular, we demonstrated that RmlT transfers rhamnose from dTDP-L-rhamnose to naked WTAs, and that specificity towards TDP-rhamnose is not determined by its binding affinity. Structures of RmlT with and without its substrates showed that this enzyme is a dimer, revealed the residues responsible for interaction with the substrates and that the catalytic residue pre-orients the acceptor substrate towards the nucleophilic attack to the sugar. Additionally, the structures provided indications for two potential interaction pathways for the long WTAs on the surface of RmlT. Finally, we confirmed that WTAs glycosyltransferases are promising targets for next-generation strategies against Gram-positive pathogens by showing that inactivation of the RmlT catalytic activity results in a decreased infection in vivo.

microbiology↗

bioRxiv2021

Stabilin-1 plays a protective role against Listeria monocytogenes infection through the regulation of cytokine and chemokine production and immune cell recruitment

Scavenger receptors are part of a complex surveillance system expressed by host cells to efficiently orchestrate innate immune response against bacterial infections. Stabilin-1 (STAB-1) is a scavenger receptor involved in cell trafficking, inflammation and cancer, however its role in infection remains to be elucidated. Listeria monocytogenes (Lm) is a major intracellular human food-borne pathogen causing severe infections in susceptible hosts. Using a mouse model of infection, we demonstrate here that STAB-1 controls Lm-induced cytokine and chemokine production and immune cell accumulation in Lm-infected organs. We show that STAB-1 also regulates the recruitment of myeloid cells in response to Lm infection and contributes to clear circulating bacteria. In addition, whereas STAB-1 appears to promote bacterial uptake by macrophages, infection by pathogenic Listeria induces the down regulation of STAB-1 expression and its delocalization from the host cell membrane. We propose STAB-1 as a new SR involved in the control of Lm infection through the regulation of host defense mechanisms, a process that would be targeted by bacterial virulence factors to promote infection.

microbiology↗

bioRxiv2020

Src-dependent NM2A tyrosine-phosphorylation regulates actomyosin dynamics

Non-muscle myosin 2A (NM2A) is a key cytoskeletal enzyme that along with actin assembles into actomyosin filaments inside cells. NM2A is fundamental in cellular processes requiring force generation such as cell adhesion, motility and cell division, and plays important functions in different stages of development and during the progression of viral and bacterial infections. We previously identified a novel tyrosine phosphorylation on residue 158 (pTyr158) in the motor domain of NM2A. This phosphorylation is dependent on Src kinase and is promoted by Listeria monocytogenes infection of epithelial cells, however its role is unknown. Here we show that Listeriolysin O (LLO), the pore-forming toxin (PFT) secreted by L. monocytogenes, is sufficient to trigger NM2A pTyr158 by activating Src, an upstream regulator of actomyosin remodeling. We further address the role of NM2A pTyr158 on the organization and dynamics of the actomyosin cytoskeleton and find that, by controlling the activation of the NM2A, the status of the pTyr158 alters cytoskeletal organization, dynamics of focal adhesions and cell motility. In vitro, we observe that non-phosphorylatable and phospho-mimetic versions of NM2A at Tyr158 display motor and ATPase activities similar to the wild-type NM2A, which indicates that the phenotype of these mutants in cells is independent of their ability to translocate actin filaments. Importantly, we find the regulation of this phosphorylation site to be of physiological relevance in Caenorhabditis elegans, in particular in response to intoxication by a PFT and to heat shock. We conclude that the control of the phosphorylation status at NM2A Tyr158 is a conserved trait that contributes to the regulation of actomyosin dynamics and the ability of cells to respond to bacterial infection. We propose Src-dependent NM2A pTyr158 as a novel layer of regulation of the actomyosin cytoskeleton.

cell biology↗