Biology subjects

Colautti, J.

Publications and source records attributed to Colautti, J..

4 recordsLinked to original sources

bioRxiv2026

Oral Actinobacteria sense and defend against parasitic epibionts

Bacteria contend with a wide range of antagonistic interactions from neighboring microbes. One widespread but poorly understood threat is posed by Patescibacteria, which proliferate by colonizing host bacteria surfaces and extracting cellular resources. Despite the ubiquity of Patescibacteria in the environment, how hosts defend against these epibionts remains unknown. Here we show that Actinomycetota fend off Patescibacteria using the Esx secretion system. We identify a secreted effector protein that mediates this defensive behavior and demonstrate that Esx induction is part of a multifaceted, epibiont-specific response that includes multiple predicted cell surface modifications. Finally, we show that this response occurs at the transcriptional level and is mediated by a previously undescribed threat sensing pathway whose activation leads to threonine phosphorylation of an FHA domain-containing output protein. Together with bioinformatic evidence linking this threat sensing pathway to Esx across Actinomycetales, our experimental findings establish a functional role for the elusive and broadly distributed Esx pathway in defense against epibionts. Furthermore, they help resolve the ecological relationship between Patescibacteria and their hosts as one of antagonism, not mutualism.

microbiology↗

bioRxiv2025

Cryo-EM structure of a type VI secretion system delivered membrane-depolarizing toxin involved in bacterial antagonism

Many Gram-negative bacteria use type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighboring competitor cells. Members of the VasX protein family, such as VasX from Vibrio cholerae and Tke5 from Pseudomonas putida, disrupt the inner membrane of target cells by forming ion-permeable channels that dissipate the proton motive force, thereby interfering with essential physiological processes. However, the molecular structure of any VasX family effector has remained unknown. Here, we present a cryo-EM structure of Ptx2, a recently identified VasX family effector exported by a T6SS of Pseudomonas aeruginosa. Our structure reveals that Ptx2 is an elongated, multi-domain protein that bears little resemblance to proteins of known function. Notably, the apparent flexibility of its domains suggests that Ptx2, like other membrane-depolarizing toxins, undergoes substantial conformational changes to facilitate membrane insertion. Guided by these predicted structural rearrangements, we used mutagenesis coupled with phenotypic assays to identify key features required for its toxic activity. Together, these findings provide the first molecular level insights into the structure and mechanism of VasX family effectors and expand our understanding of how these proteins contribute to interbacterial antagonism.

microbiology↗

bioRxiv2025

Proteolytically activated antibacterial toxins inhibit the growth of diverse Gram-positive bacteria

Many species of bacteria produce small-molecule antibiotics that enter and kill a wide range of competitor microbes. However, diffusible antibacterial proteins that share this broad-spectrum activity are not known to exist. Here, we report a family of proteins widespread in Gram-positive bacteria that display potent antibacterial activity against a diverse range of target organisms. Upon entering susceptible cells, these antibacterial proteins (ABPs) enzymatically degrade essential cellular components including DNA, tRNA, and rRNA. Unlike previously characterized bactericidal proteins, which require a specific cell surface receptor and therefore display a narrow spectrum of activity, we find that ABPs act in a receptor-independent manner and consequently kill bacteria spanning multiple bacterial phyla. Target cell entry by ABPs requires proteolytic activation by a cognate, co-exported serine protease and the liberated toxin component of the cleaved ABP is driven across the target cell membrane by the proton motive force. By examining representative ABPs from diverse pathogenic, commensal, and environmental bacteria, we show that broad-spectrum antibacterial activity is a conserved property of this protein family. Collectively, our work demonstrates that secreted proteins can act as broad-spectrum antibiotics, suggesting that ABPs represent one of potentially many such families produced in nature. Significance StatementMany bacteria produce proteins with antibacterial properties. However, owing to their reliance on a specific surface receptor for target cell entry, all known antibacterial proteins are only active against a narrow range of organisms. Using biochemical and genetic approaches, this study reports the discovery of a new family of antibacterial proteins secreted by many Gram-positive bacteria that enter and kill a broad spectrum of bacteria. Entry of these proteins into susceptible bacteria does not require a receptor and instead relies on cleavage by a co-secreted protease and the proton motive force of the target cell. Overall, our findings reveal a new family of antibacterial proteins and provides insight into how these proteins enter and kill a broad range of bacteria.

microbiology↗

bioRxiv2025

Biophysical characterization of Eag chaperones suggests the mechanism of effector transmembrane domain release

The type VI secretion system (T6SS) is a dynamic protein nanomachine found in Gram- negative bacteria that secretes toxic effectors into prey-cells. For secretion, effectors require chaperones or adaptors for proper loading onto the T6SS. Effector associated genes (Eags) are a family of T6SS chaperones that stabilize N-terminal transmembrane domains (TMDs) found in thousands of effectors. Eags are essential for secretion and inhibit effector TMDs from prematurely adopting a membrane-penetrative conformation. However, the mechanism of TMD release from its cognate Eag chaperone is unknown. Here, we take a biochemical and biophysical approach to probe the mechanism of TMD binding and dissociation from Eag chaperones. Using steady-state fluorescence, stopped-flow measurements, and bacterial competition assays, we compare the thermodynamics, kinetics, and in vivo chaperone function of wild-type and point-variant Eag-TMD complexes. Additionally, we solve an X-ray crystal structure of an Eag-TMD point-variant complex that captures an intermediate state of TMD release. Our data reveals the molecular features and specific residue contacts necessary for TMD binding and demonstrates the Eag conformational change required to initiate rapid release of the TMD. Overall, our work details the stability of Eag-TMD complexes and the energetic pathway for the dissociation of effector TMDs from their Eag chaperones.

biochemistry↗