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Advances in microscopy hold great promise for allowing quantitative and precise measurement of morphological and molecular phenomena at the single-cell level in bacteria; however, the potential of this approach is ultimately limited by the availability of methods to faithfully segment cells independent of their morphological or optical characteristics. Here, we present Omnipose, a deep neural network image-segmentation algorithm. Unique network outputs such as the gradient of the distance field allow Omnipose to accurately segment cells on which current algorithms, including its predecessor, Cellpose, produce errors. We show that Omnipose achieves unprecedented segmentation performance on mixed bacterial cultures, antibiotic-treated cells and cells of elongated or branched morphology. Furthermore, the benefits of Omnipose extend to non-bacterial subjects, varied imaging modalities and three-dimensional objects. Finally, we demonstrate the utility of Omnipose in the characterization of extreme morphological phenotypes that arise during interbacterial antagonism. Our results distinguish Omnipose as a powerful tool for characterizing diverse and arbitrarily shaped cell types from imaging data. Omnipose is a deep neural network algorithm for image segmentation that improves upon existing approaches by solving the challenging problem of accurately segmenting morphologically diverse cells from images acquired with any modality.

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques 1 , 2 . Because previously described cytidine deaminases operate on single-stranded nucleic acids 3 , their use in base editing requires the unwinding of double-stranded DNA (dsDNA)—for example by a CRISPR–Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria 4 . As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases 9 , 10 .Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders. An interbacterial toxin that catalyses the deamination of cytidines within double-stranded DNA forms part of a CRISPR-free, RNA-free base editing system that enables manipulation of human mitochondrial DNA.

A functionally diverse superfamily of bacterial phospholipase enzymes that mediate antagonisitc interactions as effectors of the type VI secretion system is uncovered; these enzymes degrade the bacterial membrane, representing a novel mechanism of bacterial competition. Antibacterial virulence factors identified Secreted bacterial phospholipases have important roles in bacterial pathogenesis, targeting host cellular membranes and causing tissue destruction, inflammation and the disruption of intracellular trafficking pathways. Here Joseph Mougous and colleagues report the discovery of a diverse superfamily of bacterial phospholipases and show that they do more than simply target eukaryotic host cells. They also have intra- and interspecies antibacterial activity through the degradation of phosphatidylethanolamine in bacterial membranes. This work suggests that interbacterial interactions may be significant factors in the progression of infections, and indicates vulnerabilities that might yield candidate antibacterial targets. Membranes allow the compartmentalization of biochemical processes and are therefore fundamental to life. The conservation of the cellular membrane, combined with its accessibility to secreted proteins, has made it a common target of factors mediating antagonistic interactions between diverse organisms. Here we report the discovery of a diverse superfamily of bacterial phospholipase enzymes. Within this superfamily, we defined enzymes with phospholipase A 1 and A 2 activity, which are common in host-cell-targeting bacterial toxins and the venoms of certain insects and reptiles 1 , 2 . However, we find that the fundamental role of the superfamily is to mediate antagonistic bacterial interactions as effectors of the type VI secretion system (T6SS) translocation apparatus; accordingly, we name these proteins type VI lipase effectors. Our analyses indicate that PldA of Pseudomonas aeruginosa , a eukaryotic-like phospholipase D 3 , is a member of the type VI lipase effector superfamily and the founding substrate of the haemolysin co-regulated protein secretion island II T6SS (H2-T6SS). Although previous studies have specifically implicated PldA and the H2-T6SS in pathogenesis 3 , 4 , 5 , we uncovered a specific role for the effector and its secretory machinery in intra- and interspecies bacterial interactions. Furthermore, we find that this effector achieves its antibacterial activity by degrading phosphatidylethanolamine, the major component of bacterial membranes. The surprising finding that virulence-associated phospholipases can serve as specific antibacterial effectors suggests that interbacterial interactions are a relevant factor driving the continuing evolution of pathogenesis.

The human gut microbiome is a dynamic and densely populated microbial community that can provide important benefits to its host. Cooperation and competition for nutrients among its constituents only partially explain community composition and interpersonal variation. Notably, certain human-associated Bacteroidetes—one of two major phyla in the gut—also encode machinery for contact-dependent interbacterial antagonism, but its impact within gut microbial communities remains unknown. Here we report that prominent human gut symbionts persist in the gut through continuous attack on their immediate neighbors. Our analysis of just one of the hundreds of species in these communities reveals 12 candidate antibacterial effector loci that can exist in 32 combinations. Through the use of secretome studies, in vitro bacterial interaction assays and multiple mouse models, we uncover strain-specific effector/immunity repertoires that can predict interbacterial interactions in vitro and in vivo, and find that some of these strains avoid contact-dependent killing by accumulating immunity genes to effectors that they do not encode. Effector transmission rates in live animals can exceed 1 billion events per minute per gram of colonic contents, and multiphylum communities of human gut commensals can partially protect sensitive strains from these attacks. Together, these results suggest that gut microbes can determine their interactions through direct contact. An understanding of the strategies human gut symbionts have evolved to target other members of this community may provide new approaches for microbiome manipulation.

Type VI secretion systems (T6SSs) translocate effectors into target cells and are made of a contractile sheath and a tube docked onto a multi-protein transmembrane complex via a baseplate. Although some information is available about the mechanisms of tail contraction leading to effector delivery, the detailed architecture and function of the baseplate remain unknown. Here, we report the 3.7 Å resolution cryo-electron microscopy reconstruction of an enteroaggregative Escherichia coli baseplate subcomplex assembled from TssK, TssF and TssG. The structure reveals two TssK trimers interact with a locally pseudo-3-fold symmetrical complex comprising two copies of TssF and one copy of TssG. TssF and TssG are structurally related to each other and to components of the phage T4 baseplate and of the type IV secretion system, strengthening the evolutionary relationships among these macromolecular machines. These results, together with bacterial two-hybrid assays, provide a structural framework to understand the T6SS baseplate architecture. Type VI secretion systems (T6SSs) translocate effector proteins into eukaryotic and bacterial recipient cells and are present in many Gram-negative bacteria. Here the authors present the 3.7 Å cryoEM structure of the E.coli T6SS baseplate wedge comprising TssK–TssF–TssG and propose a model for the T6SS baseplate and needle complex.

The Firmicutes are a phylum of bacteria that dominate numerous polymicrobial habitats of importance to human health and industry. Although these communities are often densely colonized, a broadly distributed contact-dependent mechanism of interbacterial antagonism utilized by Firmicutes has not been elucidated. Here we show that proteins belonging to the LXG polymorphic toxin family present in Streptococcus intermedius mediate cell contact- and Esx secretion pathway-dependent growth inhibition of diverse Firmicute species. The structure of one such toxin revealed a previously unobserved protein fold that we demonstrate directs the degradation of a uniquely bacterial molecule required for cell wall biosynthesis, lipid II. Consistent with our functional data linking LXG toxins to interbacterial interactions in S. intermedius, we show that LXG genes are prevalent in the human gut microbiome, a polymicrobial community dominated by Firmicutes. We speculate that interbacterial antagonism mediated by LXG toxins plays a critical role in shaping Firmicute-rich bacterial communities. Most bacteria live in densely colonized environments, such as the human gut, in which they must constantly compete with other microbes for space and nutrients. As a result, bacteria have evolved a wide array of strategies to directly fight their neighbors. For example, some bacteria release antimicrobial compounds into their surroundings, while others ‘inject’ protein toxins directly into adjacent cells. Bacteria can be classified into two groups known as Gram-positive and Gram-negative. Previous studies found that Gram-negative bacteria inject toxins into neighboring cells, but no comparable toxins in Gram-positive bacteria had been identified. Before a bacterium can inject molecules into an adjacent cell, it needs to move the toxins from its interior to the cell surface. It had been suggested that a transport system in Gram-positive bacteria called the Esx pathway may export toxins known as LXG proteins. However, it was not clear whether these proteins help Gram-positive bacteria to compete against other bacteria. Whitney et al. studied the LXG proteins in Gram-positive bacteria known as Firmicutes. The experiments reveal that Firmicutes found in the human gut possess LXG genes. A Firmicute known as Streptococcus intermedius produces three LXG proteins that are all toxic to bacteria. To avoid being harmed by its own LXG proteins, S. intermedius also produces matching antidote proteins. Further experiments show that LXG proteins are exported out of S. intermedius cells and into adjacent competitor bacteria by the Esx pathway. Examining one of these LXG proteins in more detail showed that it can degrade a molecule that bacteria need to make their cell wall. Together, these findings suggest that LXG proteins may influence the species living in many important microbial communities, including the human gut. Changes in the communities of gut microbes have been linked with many diseases. Therefore, understanding more about how the LXG proteins work may help us to develop ways to manipulate these communities to improve human health.

Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.

Bacteroidales use type VI secretion systems (T6SS) to competitively colonize and persist in the colon. We identify a horizontally transferred T6SS with Ntox15 family nuclease effector (Tde1) that mediates interbacterial antagonism among Bacteroidales, including several derived from a single human donor. Expression of cognate (Tdi1) or orphan immunity proteins in acquired interbacterial defense systems protects against Tde1-dependent attack. We find that immunity protein interaction induces a large effector conformational change in Tde nucleases, disrupting the active site and altering the DNA-binding site. Crystallographic snapshots of isolated Tde1, the Tde1/Tdi1 complex, and homologs from Phocaeicola vulgatus (Tde2/Tdi2) illustrate a conserved mechanism of immunity inserting into the central core of Tde, splitting the nuclease fold into two subdomains. The Tde/Tdi interface and immunity mechanism are distinct from all other polymorphic toxin–immunity interactions of known structure. Bacteroidales abundance has been linked to inflammatory bowel disease activity in prior studies, and we demonstrate that Tde and T6SS structural genes are each enriched in fecal metagenomes from ulcerative colitis subjects. Genetically mobile Tde1-encoding T6SS in Bacteroidales mediate competitive growth and may be involved in inflammatory bowel disease. Broad immunity is conferred by Tdi1 homologs through a fold-disrupting mechanism unique among polymorphic effector–immunity pairs of known structure. Bacteroidales are related to inflammatory bowel disease severity and progression. We identify type VI secretion system (T6SS) nuclease effectors (Tde) which are enriched in ulcerative colitis and horizontally transferred on mobile genetic elements. Tde-encoding T6SSs mediate interbacterial competition. Orphan and cognate immunity proteins (Tdi) prevent intoxication by multiple Tde through a new mechanism among polymorphic toxin systems. Tdi inserts into the effector central core, splitting Ntox15 into two subdomains and disrupting the active site. This mechanism may allow for evolutionary diversification of the Tde/Tdi interface as observed in colicin nuclease–immunity interactions, promoting broad neutralization of Tde by orphan Tdi. Tde-dependent T6SS interbacterial antagonism may contribute to Bacteroidales diversity in the context of ulcerative colitis.

Secreted proteins are crucial to the arsenal of bacterial pathogens. Although optimal activity of these proteins is likely to require precise regulation of release, the signalling events that trigger secretion are poorly understood. Here, we identify a threonine phosphorylation event that post-translationally regulates the Hcp secretion island-I-encoded type VI secretion system of Pseudomonas aeruginosa (H-T6SS). We show that a serine–threonine kinase, PpkA, is required for assembly of the H-T6SS and for secretion of Hcp1. PpkA activity is antagonized by PppA, a Ser–Thr phosphatase. These proteins exhibit reciprocal effects on the H-T6SS by acting on an FHA domain-containing protein, termed Fha1. Colocalization experiments with the T6S AAA+ family protein, ClpV1, indicate that Fha1 is a core scaffolding protein of the H-T6SS. Mutations affecting this H-T6S regulatory pathway provide a molecular explanation for the variation in Hcp1 secretion among clinical P. aeruginosa isolates. This mechanism of triggering secretion may be general, as many T6SSs contain orthologues of these proteins. Post-translational regulation of protein secretion by Thr phosphorylation is unprecedented in bacteria, and is likely to reflect the requirement for T6S to respond rapidly and reversibly to its environment.

The type VI secretion system (T6SS) primarily functions to mediate antagonistic interactions between contacting bacterial cells, but also mediates interactions with eukaryotic hosts. This molecular machine secretes antibacterial effector proteins by undergoing cycles of extension and contraction; however, how effectors are loaded into the T6SS and subsequently delivered to target bacteria remains poorly understood. Here, using electron cryomicroscopy, we analysed the structures of the Pseudomonas aeruginosa effector Tse6 loaded onto the T6SS spike protein VgrG1 in solution and embedded in lipid nanodiscs. In the absence of membranes, Tse6 stability requires the chaperone EagT6, two dimers of which interact with the hydrophobic transmembrane domains of Tse6. EagT6 is not directly involved in Tse6 delivery but is crucial for its loading onto VgrG1. VgrG1-loaded Tse6 spontaneously enters membranes and its toxin domain translocates across a lipid bilayer, indicating that effector delivery by the T6SS does not require puncturing of the target cell inner membrane by VgrG1. Eag chaperone family members from diverse Proteobacteria are often encoded adjacent to putative toxins with predicted transmembrane domains and we therefore anticipate that our findings will be generalizable to numerous T6SS-exported membrane-associated effectors. A combination of cryo-EM structures and experiments using liposomes and lipid nanodiscs elucidates how the Tse6 effector is loaded into the type VI secretion system and travels across membranes to intoxicate target cells.