Biology subjects

Valastyan, J. S.

Publications and source records attributed to Valastyan, J. S..

6 recordsLinked to original sources

bioRxiv2026

Molecular basis of quorum-sensing signal transduction by CqsS and its inhibition by CqsA the autoinducer synthase

Vibrio bacteria possess multiple quorum-sensing systems, each of which conveys information into the cell concerning the cell density and species composition of the vicinal community. Germane to the present work is the CqsA-CqsS quorum-sensing pathway that vibrios use for intra-genus communication. CqsA is the synthase for the autoinducer (S)-3-hydroxytridecan-4-one (CAI-1), and CqsS, a bi-functional two-component kinase-phosphatase, is the CAI-1 receptor. Here, we determine the cryo-EM structures of the full-length Vibrio harveyi CqsS dimer in the apo- and ATP{gamma}S-bound states. Our structures reveal the molecular basis for the initiation of the phosphorylation cascade, which we support through biochemical and genetic analyses. Autophosphorylation of CqsS occurs in cis at the conserved histidine residue (H1), whereas phospho-transfer to the conserved aspartate residue (D1) in the C-terminal receiver domain occurs in trans. By co-expressing CqsS and CqsA to capture CqsS bound to CAI-1, we unexpectedly discover a tight interaction between the CqsS intracellular domain and CqsA. In the complex, the CqsS H1 residues are positioned away from the catalytic cavity, and CqsS kinase activity is inhibited. Cryo-EM analysis, focused on the transmembrane domain of CqsS in this complex, reveals the presence of two CAI-1 molecules. CAI-1 binding induces an obvious movement of transmembrane helix 6, disrupting its extension into the cytosol, providing a structural basis for how CAI-1 binding in the periplasm is coupled to signal transduction into the cytoplasm. Together, our findings advance the understanding of CqsS-CAI-1-mediated quorum-sensing signal transduction, an activity that underpins the coordination of group behaviors in vibrios.

microbiology↗

bioRxiv2026

Discovery of a Human Metabolite that Mimics the Bacterial Quorum-Sensing Autoinducer AI-2

Bacteria use small molecules to orchestrate collective behaviors in a process called quorum sensing (QS), which relies on the production, release, and group-wide detection of extracellular signal molecules referred to as autoinducers. One QS autoinducer, termed AI-2, is broadly used for inter-species bacterial communication, including in the mammalian gut. AI-2 consists of a family of interconverting compounds and adducts originating from 4,5-hydroxy-2,3-pentanedione. This complex speciation, coupled with the inherent instability of AI-2 congeners, have complicated isolation efforts. It has been known that mammalian epithelial cells produce an AI-2 mimic to which bacteria respond. However, the identity of the AI-2 mimic has remained elusive, presumably due to its instability, similar to that of known AI-2 compounds. Here, we developed a reactivity-based metabolomics approach to capture and identify a mammalian AI-2 mimic. Using a chemical strategy targeted at the -diketone moiety of known AI-2s, we identify the unusual sugar L-xylosone, as well as the related metabolite L-xylulose, as AI-2 mimics. While L-xylulose is a common and naturally occurring sugar known in human metabolism, L-xylosone is a rare and highly reactive oxidation product. We established a facile synthetic route to access pure enantiomers of xylosone and confirmed that, like AI-2, the L-configuration is required for recognition by the bacterial AI-2 receptor, LuxP, whereas D-xylosone is inactive. L-xylosone is new to the human metabolome, suggesting that other chemically reactive small molecules that mediate host-microbe interactions await discovery. The identification of L-xylosone expands the AI-2 family of molecules and adds a new word to the lexicon of host-bacterial interactions.

biochemistry↗

bioRxiv2025

A transcription factor-sRNA-mediated double-negative feedback loop confers pathogen-specific control of quorum-sensing genes

The cell-to-cell communication process called quorum sensing enables bacteria to synchronize collective behaviors. Quorum sensing relies on the production, release, and detection of signaling molecules called autoinducers. In Vibrio cholerae, the VqmA transcription factor, following binding of the DPO autoinducer, activates expression of the gene encoding the VqmR small regulatory RNA. VqmR controls traits including biofilm formation. Here, we identify repressors of DPO-VqmA-VqmR signaling. We focus on one identified repressor, the LuxT transcription factor. We show that LuxT represses vqmR transcription. VqmR post-transcriptionally represses luxT translation. This arrangement forms a double-negative feedback loop between the two regulators. Reciprocal control hinges on the N-terminal 8 amino acids of LuxT. The nucleotide sequence encoding this LuxT region serves as the VqmR binding site in the luxT mRNA and the amino acids specified by this same N-terminal region are required for LuxT to bind the vqmR promoter. This same LuxT N-terminal region also expands the DNA motifs to which LuxT can bind. We show this regulatory circuit is unique to V. cholerae and closely related species and absent from other vibrios. We define the set of LuxT-controlled genes in V. cholerae and show that LuxT promotes biofilm formation, a key requirement for successful colonization of eukaryotic hosts. ImportanceBacterial quorum sensing enables control of collective behaviors. In Vibrio cholerae, the DPO-VqmA-VqmR quorum-sensing circuit governs key processes, including biofilm formation. Here, we identify a double-negative feedback loop between the transcription factor LuxT and the small RNA VqmR. This regulatory circuit depends on an eight amino acid N-terminal region that exists only in V. cholerae LuxT and LuxT from its close relatives. This short peptide sequence confers three distinct functions: It enables LuxT to repress vqmR, renders luxT mRNA susceptible to VqmR repression, and governs which DNA motifs LuxT can bind. Our findings reveal a pathogen-specific regulatory module that links small RNA targeting of mRNAs to transcription factor DNA binding specificity. The results show how evolution tailors bacterial regulatory circuits to adapt to different environments.

microbiology↗

bioRxiv2025

Inhibitors of the PqsR Quorum-Sensing Receptor Reveal Differential Roles for PqsE and RhlI in Control of Phenazine Production

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and it is resistant to many current antibiotic therapies, making development of new anti-microbial treatments imperative. The cell-to-cell communication process called quorum sensing controls P. aeruginosa pathogenicity. Quorum sensing relies on the production, release, and group-wide detection of extracellular signal molecules called autoinducers. Quorum sensing enables bacteria to synchronize group behaviors. P. aeruginosa possesses multiple quorum-sensing systems that control overlapping regulons, including those required for virulence and biofilm formation. Interventions that target P. aeruginosa quorum-sensing receptors are considered a fruitful avenue to pursue for new therapeutic advances. Here, we developed a P. aeruginosa strain that carries a bioluminescent reporter fused to a target promoter that is controlled by two P. aeruginosa quorum-sensing receptors. The receptors are PqsR, which binds and responds to the autoinducer called PQS (2-heptyl-3-hydroxy-4(1H)-quinolone) and RhlR, which binds and responds to the autoinducer called C4-HSL (C4-homoserine lactone). We used this reporter strain to screen >100,000 compounds with the aim of identifying inhibitors of either or both the PqsR and RhlR quorum-sensing receptors. We report results for 30 PqsR inhibitors from this screen. All of the identified compounds inhibit PqsR with IC50 values in the nanomolar to low micromolar range and they are readily docked into the autoinducer binding site of the PqsR crystal structure, suggesting they function competitively. The majority of hits identified are not structurally related to previously reported PqsR inhibitors. Recently, RhlR was shown to rely on the accessory protein PqsE for full function. Specifically, RhlR controls different subsets of genes depending on whether it is bound to PqsE or to C4-HSL, however, the consequences of differential regulation on the quorum-sensing output response have not been defined. PqsR regulates pqsE. That feature of the system enabled us to exploit our new set of PqsR inhibitors to show that RhlR requires PqsE to activate the biosynthetic genes for pyocyanin, a key P. aeruginosa virulence factor, while C4-HSL is dispensable. These results highlight the promise of inhibition of PqsR as a possible P. aeruginosa therapeutic to suppress production of factors under RhlR-PqsE control.

microbiology↗

bioRxiv2021

Secreted Proteases Control the Timing of Aggregative Community Formation in Vibrio cholerae

Bacteria orchestrate collective behaviors using the cell-cell communication process called quorum sensing (QS). QS relies on the synthesis, release, and group-wide detection of small molecules called autoinducers. In Vibrio cholerae, a multicellular community aggregation program occurs in liquid, during stationary phase, and in the high-cell-density QS state. Here, we demonstrate that this aggregation program consists of two subprograms. In one subprogram, which we call void formation, structures form that contain few cells but provide a scaffold within which cells can embed. The other subprogram relies on flagellar machinery and enables cells to enter voids. A genetic screen for factors contributing to void formation, coupled with companion molecular analyses, showed that four extracellular proteases, Vca0812, Vca0813, HapA, and PrtV control the onset timing of both void formation and aggregation, and moreover, proteolytic activity is required. These proteases, or their downstream products, can be shared between void-producing and non-void-forming cells and can elicit aggregation in a normally non-aggregating V. cholerae strain. Employing multiple proteases to control void formation and aggregation timing could provide a redundant and irreversible path to commitment to this community lifestyle. ImportanceBacteria can work as collectives to form multicellular communities. Vibrio cholerae, the bacterium that causes the disease cholera in humans, forms aggregated communities in liquid. Aggregate formation relies on a chemical communication process called quorum sensing. Here we show that, beyond overarching control by quorum sensing, there are two aggregation subprograms. One subprogram, which we call void formation, creates a scaffold within which cells can embed. The second subprogram, which allows bacteria to enter the scaffold, requires motility. We discovered that four extracellular proteases control the timing of both void formation and aggregation. We argue that, by using redundant proteases, V. cholerae ensures the reliable execution of this community formation process. These findings may provide insight into how V. cholerae successfully alternates between the marine environment and the human host: transitions that are central to the spread of the disease cholera.

microbiology↗

bioRxiv2020

Saccharomyces cerevisiae Requires CFF1 to Produce 4-hydroxy-5-methylfuran-3(2H)-one, a Mimic of the Bacterial Quorum-Sensing Autoinducer AI-2

Quorum sensing is a process of cell-to-cell communication that bacteria use to orchestrate collective behaviors. Quorum sensing depends on the production, release, and detection of extracellular signal molecules called autoinducers (AIs) that accumulate with increasing cell density. While most AIs are species-specific, the AI called AI-2 is produced and detected by diverse bacterial species and it mediates inter-species communication. We recently reported that mammalian cells produce an AI-2 mimic that can be detected by bacteria through the AI-2 receptor, LuxP, potentially expanding the role of the AI-2 system to inter-domain communication. Here, we describe a second molecule capable of inter-domain signaling through LuxP, 4-hydroxy-5-methylfuran-3(2H)-one (MHF) that is produced by the yeast Saccharomyces cerevisiae. Screening the S. cerevisiae deletion collection revealed Cff1p, a protein with no known role, to be required for MHF production. Cff1p is proposed to be an enzyme, possibly an epimerase or isomerase, and substitution at the putative catalytic residue eliminated MHF production in S. cerevisiae. Sequence analysis uncovered Cff1p homologs in many species, primarily bacterial and fungal, but also viral, archaeal, and higher eukaryotic. Cff1p homologs from organisms from all domains can complement a S. cerevisiae cff1{Delta} mutant and restore MHF production. In all test cases, the identified catalytic residue is conserved and required for MHF to be produced. These findings increase the scope of possibilities for inter-domain interactions via AI-2 and AI-2 mimics, highlighting the breadth of molecules and organisms that could participate in quorum sensing. ImportanceQuorum sensing is a cell-to-cell communication process that bacteria use to monitor local population density. Quorum sensing relies on extracellular signal molecules called autoinducers (AIs). One AI, called AI-2, is broadly made by bacteria and used for inter-species communication. Here, we describe a eukaryotic AI-2 mimic, 5-methylfuran-3(2H)-one, (MHF), that is made by the yeast Saccharomyces cerevisiae, and we identify the Cff1p protein as essential for MHF production. Hundreds of viral, archaeal, bacterial, and eukaryotic organisms possess Cff1p homologs. This finding, combined with our results showing that homologs from all domains can replace S. cerevisiae Cff1p, suggests that like AI-2, MHF is widely produced. Our results expand the breadth of organisms that may participate in quorum-sensing-mediated interactions.

microbiology↗