Biology subjects

Bleves, S.

Publications and source records attributed to Bleves, S..

7 recordsLinked to original sources

bioRxiv2026

The Type VI secretion effector TpeX expands the pore-forming virulence arsenal of Pseudomonas aeruginosa

The type VI secretion system (T6SS) is a major weapon used by Pseudomonas aeruginosa to antagonize competing bacteria through the delivery of a diverse repertoire of toxic effectors. Although several P. aeruginosa T6SS effectors target bacterial membranes, the mechanisms underlying membrane disruption remain poorly characterized. Here, we study TpeX (PA5265), an accessory T6SS effector from P. aeruginosa PAO1 with structural similarity to the VasX pore-forming effector of Vibrio cholerae. We show that membrane-targeted TpeX exerts a bactericidal activity in Escherichia coli, resulting in dissipation of the membrane potential and loss of membrane integrity. The TpeX C-terminal region containing the predicted colicin-like transmembrane domain is sufficient to confer toxicity, although with reduced activity, supporting its role as the membrane-disrupting module. TpeX also oligomerizes upon membrane targeting, forming at least dimers, and structural modelling predicts a membrane-embedded pore compatible with the observed permeabilization phenotype. We further identify TpiX (PA5264), the protein encoded by the downstream gene, as the cognate immunity protein, which partially protects cells from TpeX toxicity and interacts with TpeX. Finally, AlphaFold 3 modelling predicts an interaction between TpeX and the HcpB-VgrG6 T6SS spike, suggesting a possible mechanism for effector recruitment and delivery. Together, our results identify TpeX as a bactericidal, colicin-like pore-forming T6SS effector whose membrane activity is controlled by a cognate immunity protein, thereby expanding the repertoire of membrane-targeting weapons used by P. aeruginosa in interbacterial competition.

microbiology↗

bioRxiv2025

Molecular Insights into the bactericidal Toxin Tle1 of Pseudomonas aeruginosa: Interaction with VgrG, its adaptor, and immunity protein

The Type VI secretion system (T6SS) delivers a wide range of antibacterial effectors, including phospholipases of the Tle family. Here, we characterize Tle1 from Pseudomonas aeruginosa and demonstrate that it functions as a bactericidal toxin with moderate activity when associated to the membranes in the periplasm. Bacterial two-hybrid assays revealed specific protein-protein interactions within the tle1 locus, involving the immunity protein Tli1a, the chaperone/adaptor Tla1, and the spike protein VgrG4a. These interactions were independently validated by co-purification assays. Structural modeling with AlphaFold 3 produced a high-confidence ternary complex in which a VgrG4a trimer accommodates one Tle1 monomer and one Tla1 monomer. The three predicted interfaces (Tle1-Tla1, Tla1-VgrG4a, and Tle1-VgrG4a) were confirmed experimentally in vivo and important charged residues mediating these interfaces were identified. Furthermore, modeling of the Tle1-Tli1a complex suggests an inhibition mechanism that does not occlude the catalytic pocket. Consistently, Tli1a was localized to the outer membrane of P. aeruginosa, supporting in silico predictions of an outer membrane lipoprotein and positioning it ideally to neutralize periplasmic Tle1 activity. We did not observe the second candidate immunity protein, Tli1b, either in P. aeruginosa or in E. coli. Together, these findings elucidate the molecular interactions underlying Tle1 delivery and inhibition and highlight the role of Tli1a as a dedicated immunity protein that protects P. aeruginosa from self-intoxication.

microbiology↗

bioRxiv2025

Bacterial Hsp90 promotes virulence factor production through maintenance of NRPS megaenzymes

Pathogenic bacteria produce virulence factors critical to host infection. Here, we demonstrate the crucial role of the bacterial Hsp90 chaperone in the production of two major virulence factors, the colibactin genotoxin in Escherichia coli and the pyoverdine siderophore in Pseudomonas aeruginosa. Colibactin, a hybrid polyketide/non-ribosomal peptide (PK-NRP), and pyoverdine, a non-ribosomal peptide (NRP), are metabolites produced by complex biosynthetic pathways involving large cytoplasmic enzymes called megasynthases. Using comparative proteomics, we found that megasynthase abundance was markedly reduced in hsp90 deletion mutants of E. coli and P. aeruginosa compared to wild-type strains. This reduction was independent of transcriptional or translational regulation. We further revealed an interplay between Hsp90 and the HslUV protease in controlling megasynthase levels. Remarkably, we found that Hsp90 stabilizes additional NRP and PK-NRP megasynthases, suggesting a general role for Hsp90 as a chaperone of these enzymes. These findings open new avenues for enhancing the biosynthesis of complex metabolites for biotechnological applications through proteostasis modulation, and may also have implications for combating bacterial infections.

microbiology↗

bioRxiv2021

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through loss of the Rieske cytochrome bc1 sub-unit

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of 2-heptyl-3-hydroxy-4-quinolone (PQS) dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat secretion substrate responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance of P. aeruginosa biofilms. Author SummaryPseudomonas aeruginosa is a highly adaptable human pathogen responsible for causing chronic biofilm-associated infections. Biofilms are highly refractory to host defences and antibiotics and thus difficult to eradicate. The biofilm extracellular matrix incorporates extracellular DNA (eDNA). This stabilizes biofilm architecture and helps confer tolerance to antibiotics. Since mechanisms that control eDNA release are not well understood, we screened a P. aeruginosa mutant bank for strains with defects in eDNA release and discovered a role for the twin-arginine translocation (Tat) pathway that exports folded proteins across the cytoplasmic membrane. Perturbation of the Tat pathway resulted in defective biofilms susceptible to antibiotic treatment as a consequence of perturbed pseudomonas quinolone (PQS) signalling. This resulted in the failure to produce or release biofilm components including eDNA, phenazines and rhamnolipids as well as microvesicles. Furthermore, we discovered that perturbation of PQS signalling was a consequence of the inability of tat mutants to translocate the Rieske subunit of the cytochrome bc1 complex involved in electron transfer and energy transduction. Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation in P. aeruginosa, our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.

microbiology↗

bioRxiv2019

A T6SS trans-kingdom effector is required for the delivery of a novel antibacterial toxin in Pseudomonas aeruginosa

Pseudomonas aeruginosa has evolved multiple strategies to disarm and take advantage of its host. For this purpose this opportunist pathogen has particularly developed protein secretion in the surrounding medium or injection into host cells. Among this, the Type VI Secretion System (T6SS) is utilized to deliver effectors into eukaryotic host as well as target bacteria. It assembles into a contractile bacteriophage tail-like structure that functions like a crossbow, injecting an arrow loaded with effectors into the target cell. The repertoire of T6SS antibacterial effectors of P. aeruginosa is remarkably broad to promote environmental adaptation and survival in various bacterial communities, and presumably in the eukaryotic host too. Here we report the discovery a novel pair of antibacterial effector and immunity of P. aeruginosa, Tle3 and Tli3. Tli3 neutralizes the toxicity of Tle3 in the periplasm to protect from fratricide intoxication. The characterization of the secretion mechanism of Tle3 indicates that it requires a cytoplasmic adaptor, Tla3, to be targeted and loaded onto the VgrG2b spike and thus delivered by the H2-T6SS machinery. Tla3 is different from the other adaptors discovered so far, and defines a novel family among T6SS. Interestingly this led us to discover that VgrG2b that we previously characterized as an anti-eukaryotic effector possesses an antibacterial activity as well, as it is toxic towards Escherichia coli. VgrG2b is thus a novel trans-kingdom effector targeting both bacteria and eukaryotes. VgrG2b represents an interesting target for fighting against P. aeruginosa in the environment and in the context of host infection. HighlightsO_LITle3 and Tli3 are a novel pair of antibacterial toxin and immunity of P. aeruginosa C_LIO_LITla3 recruits Tle3 in the cytoplasm, and targets it to VgrG2b C_LIO_LIVgrG2b is required for Tle3 delivery into target bacteria by the H2-T6SS C_LIO_LITla3 defines a novel type of T6SS adaptor with a DUF2875 C_LIO_LIVgrG2b is a new trans-kingdom effector targeting both bacteria and eukaryotes C_LI

microbiology↗

bioRxiv2018

Killing from the inside: Intracellular role of T3SS in the fate of Pseudomonas aeruginosa within macrophages revealed by mgtC and oprF mutants

While considered solely an extracellular pathogen, increasing evidence indicates that Pseudomonas aeruginosa encounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-type P. aeruginosa PAO1 strain by live and electron microscopy. P. aeruginosa first resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis. Two bacterial factors, MgtC and OprF, recently identified to be important for survival of P. aeruginosa in macrophages, were found to be involved in bacterial escape from the phagosome as well as cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes of P. aeruginosa were down-regulated within macrophages in both mgtC and oprF mutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern of mgtC and oprF mutants, a T3SS mutant ({Delta}pscN) exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellular P. aeruginosa towards macrophages. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life of P. aeruginosa, allowing internalized bacteria to evade macrophages.\n\nAuthor summaryThe ability of professional phagocytes to ingest and kill microorganisms is central to host defense and Pseudomonas aeruginosa has developed mechanisms to avoid being killed by phagocytes. While considered an extracellular pathogen, P. aeruginosa has been reported to be engulfed by macrophages in animal models. Here, we visualized the fate of P. aeruginosa within cultured macrophages, revealing macrophage lysis driven by intracellular P. aeruginosa. Two bacterial factors, MgtC and OprF, recently discovered to be involved in the intramacrophage survival of P. aeruginosa, appeared to play role in this cytotoxicity caused by intracellular bacteria. We provided evidence that type III secretion system (T3SS) gene expression is lowered intracellularly in mgtC and oprF mutants. We further showed that intramacrophage P. aeruginosa uses its T3SS, specifically the ExoS effector, to promote phagosomal escape and cell lysis. We thus describe a transient intramacrophage stage of P. aeruginosa that could contribute to bacterial dissemination.

microbiology↗

bioRxiv2017

Pseudopaline, a staphylopine-like metallophore involved in zinc and nickel uptake in Pseudomonas aeruginosa

Metal uptake is vital for all living organisms. In metal scarce conditions, a common bacterial strategy consists in the biosynthesis of metallophores, their export in the extracellular medium and the recovery of a metal-metallophore complex through dedicated membrane transporters. Staphylopine is a recently described metallophore distantly related to plant nicotianamine that contributes to the broad-spectrum metal uptake capabilities of Staphylococcus aureus. Here, we characterize a four genes operon (PA4837-PA4834) in Pseudomonas aeruginosa involved in the biosynthesis and trafficking of a staphylopine-like metallophore named pseudopaline. Pseudopaline differs from staphylopine with regard to the stereochemistry of its histidine moiety associated to an alpha ketoglutarate moiety instead of pyruvate. In vivo, the pseudopaline operon is regulated by zinc through the Zur repressor. The metal-uptake property of the pseudopaline system appears different from that of staphylopine with a predominant effect on nickel uptake, and on zinc uptake in metal scarce conditions mimicking a chelating environment, thus reconciling the regulation of the cnt operon by zinc with its function as a zinc importer under metal scarce conditions.\n\nAUTHOR SUMMARYZinc is an essential micronutrients for bacteria, particularly important at the host-pathogen interface where the host tends to sequester metals in a so called nutritional immunity framework, and the pathogenic bacterium increases its metal uptake efforts in order to keep up with its metal requirements. Here we reveal a novel metallophore, named pseudopaline, which is synthesized and exported by Pseudomonas aeruginosa and serves for the uptake of nickel in metal poor media, and for the uptake of zinc in metal scarce conditions that mimic the chelating environment that presumably prevails within a host.

microbiology↗