Bacteria possess an array of defenses against foreign invaders, including a broadly distributed bacteriophage defense system termed CBASS (cyclic oligonucleotide-based anti-phage signaling system). ...In CBASS systems, a cGAS/DncV-like nucleotidyltransferase synthesizes cyclic di- or tri-nucleotide second messengers in response to infection, and these molecules activate diverse effectors to mediate bacteriophage immunity via abortive infection. Here, we show that the CBASS effector NucC is related to restriction enzymes but uniquely assembles into a homotrimer. Binding of NucC trimers to a cyclic tri-adenylate second messenger promotes assembly of a NucC homohexamer competent for non-specific double-strand DNA cleavage. In infected cells, NucC activation leads to complete destruction of the bacterial chromosome, causing cell death prior to completion of phage replication. In addition to CBASS systems, we identify NucC homologs in over 30 type III CRISPR/Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems’ effector complexes.
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•The CBASS-associated nuclease NucC kills phage-infected cells by genome destruction•Binding of a cyclic triadenylate second messenger causes NucC homohexamer assembly•NucC hexamer assembly juxtaposes pairs of active sites to activate DNA cleavage•NucC is found in both CBASS systems and type III CRISPR/Cas systems
Here, Lau et al. show that the cyclic oligonucleotide-based anti-phage signaling system (CBASS)-associated NucC nuclease causes death of phage-infected cells through genome destruction. They show how NucC is allosterically activated by a cyclic triadenylate second messenger synthesized by CBASS upon infection. NucC has also been integrated into type III CRISPR/Cas systems as an accessory nuclease.
Genes necessary for the survival or reproduction of a cell are an attractive class of antibiotic targets. Studying essential genes by classical genetics, however, is inherently problematic because it ...is impossible to knock them out. Here, we screened a set of predicted essential genes for growth inhibition using CRISPR-interference (CRISPRi) knockdown in the human pathogen Vibrio cholerae. We demonstrate that CRISPRi knockdown of 37 predicted essential genes inhibits V. cholerae viability, thus validating the products of these genes as potential drug target candidates. V. cholerae was particularly vulnerable to lethal inhibition of the system for lipoprotein transport (Lol), a central hub for directing lipoproteins from the inner to the outer membrane (OM), with many of these lipoproteins coordinating their own essential processes. Lol depletion makes cells prone to plasmolysis and elaborate membrane reorganization, during which the periplasm extrudes into a mega outer membrane vesicle or “MOMV” encased by OM which dynamically emerges specifically at plasmolysis sites. Ourwork identifies the Lol system as an ideal drug target, whose inhibition could deplete gram-negative bacteria of numerous proteins that reside in the periplasm.
An important question regarding the biologic implications of antibiotic-resistant microbes is how resistance impacts the organism’s overall fitness and virulence. Currently it is generally thought ...that antibiotic resistance carries a fitness cost and reduces virulence. For the human pathogen Pseudomonas aeruginosa , treatment with carbapenem antibiotics is a mainstay of therapy that can lead to the emergence of resistance, often through the loss of the carbapenem entry channel OprD. Transposon insertion-site sequencing was used to analyze the fitness of 300,000 mutants of P. aeruginosa strain PA14 in a mouse model for gut colonization and systemic dissemination after induction of neutropenia. Transposon insertions in the oprD gene led not only to carbapenem resistance but also to a dramatic increase in mucosal colonization and dissemination to the spleen. These findings were confirmed in vivo with different oprD mutants of PA14 as well as with related pairs of carbapenem-susceptible and -resistant clinical isolates. Compared with OprD ⁺ strains, those lacking OprD were more resistant to killing by acidic pH or normal human serum and had increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that an oprD mutant showed dramatic changes in the transcription of genes that may contribute to the various phenotypic changes observed. The association between carbapenem resistance and enhanced survival of P. aeruginosa in infected murine hosts suggests that either drug resistance or host colonization can cause the emergence of more pathogenic, drug-resistant P. aeruginosa clones in a single genetic event.
cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we ...discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of >2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2′–5′- and 3′–5′-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.
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•Cap4 proteins are a major family of nucleotide second messenger receptors in bacteria•Cap4 receptors degrade DNA and mediate phage resistance by CBASS operons•The Cap4 ligand-binding SAVED domain evolved from CRISPR CARF proteins•As in cGAS-STING signaling, bacteria use 2′–5′-linked signals for antiviral immunity
A family of bacterial immune effectors responds to 3′–5′- and 2′–5′-linked nucleotide signals in CBASS antiviral immunity.
YbeY, a highly conserved protein, is an RNase in E. coli and plays key roles in both processing of the critical 3' end of 16 S rRNA and in 70 S ribosome quality control under stress. These central ...roles account for YbeY's inclusion in the postulated minimal bacterial genome. However, YbeY is not essential in E. coli although loss of ybeY severely sensitizes it to multiple physiological stresses. Here, we show that YbeY is an essential endoribonuclease in Vibrio cholerae and is crucial for virulence, stress regulation, RNA processing and ribosome quality control, and is part of a core set of RNases essential in most representative pathogens. To understand its function, we analyzed the rRNA and ribosome profiles of a V. cholerae strain partially depleted for YbeY and other RNase mutants associated with 16 S rRNA processing; our results demonstrate that YbeY is also crucial for 16 S rRNA 3' end maturation in V. cholerae and that its depletion impedes subunit assembly into 70 S ribosomes. YbeY's importance to V. cholerae pathogenesis was demonstrated by the complete loss of mice colonization and biofilm formation, reduced cholera toxin production, and altered expression levels of virulence-associated small RNAs of a V. cholerae strain partially depleted for YbeY. Notably, the ybeY genes of several distantly related pathogens can fully complement an E. coli ΔybeY strain under various stress conditions, demonstrating the high conservation of YbeY's activity in stress regulation. Taken together, this work provides the first comprehensive exploration of YbeY's physiological role in a human pathogen, showing its conserved function across species in essential cellular processes.
Type III secretion systems are toxin delivery systems that are present in a large number of pathogens. A hallmark of all type III secretion systems studied to date is that expression of one or more ...of their components is induced upon cell contact. It has been proposed that this induction is controlled by a negative regulator that is itself secreted by means of the type III secretion machinery. Although candidate proteins for this negative regulator have been proposed in a number of systems, for the most part, a direct demonstration of their role in regulation is lacking. Here, we report the discovery of ExsE, a negative regulator of type III secretion gene expression in Pseudomonas aeruginosa. Deletion of exsE deregulates expression of the type III secretion genes. We provide evidence that ExsE is itself secreted by means of the type III secretion machinery and physically interacts with ExsC, a positive regulator of the type III secretion regulon. Taken together, these data demonstrate that ExsE is the secreted negative regulator that couples triggering of the type III secretion machinery to induction of the type III secretion genes.
The production of virulence factors including cholera toxin and the toxin-coregulated pilus in the human pathogen Vibrio cholerae is strongly influenced by environmental conditions. The ...well-characterized ToxR signal transduction cascade is responsible for sensing and integrating the environmental information and controlling the virulence regulon. We show here that, in addition to the known components of the ToxR signaling circuit, quorum-sensing regulators are involved in regulation of V. cholerae virulence. We focused on the regulators LuxO and HapR because homologues of these two proteins control quorum sensing in the closely related luminous marine bacterium Vibrio harveyi. Using an infant mouse model, we found that a luxO mutant is severely defective in colonization of the small intestine. Gene arrays were used to profile transcription in the V. cholerae wild type and the luxO mutant. These studies revealed that the ToxR regulon is repressed in the luxO mutant, and that this effect is mediated by another negative regulator, HapR. We show that LuxO represses hapR expression early in log-phase growth, and constitutive expression of hapR blocks ToxR-regulon expression. Additionally, LuxO and HapR regulate a variety of other cellular processes including motility, protease production, and biofilm formation. Together these data suggest a role for quorum sensing in modulating expression of blocks of virulence genes in a reciprocal fashion in vivo.
The type 6 secretion system (T6SS) is used by many Gram-negative bacterial species to deliver toxic effector proteins into nearby bacteria prey cells to kill or inhibit their growth. VgrG proteins ...are core conserved secretion substrates of the T6SS and one subset of T6SS effectors consists of VgrG proteins with C-terminal extension domains carrying various enzymatic activities. In Vibrio cholerae, VgrG3 has a hydrolase extension domain and degrades peptidoglycan in the periplasm of target bacteria. In this study, we replaced this domain with a nuclease domain from Salmonella enterica subsp. arizonae. This modified V. cholerae strain was able to kill its parent using its T6SS. This result also demonstrated that V. cholerae T6SS is capable of delivering effectors that could attack substrates found either in the periplasm or cytosol of target bacteria. Additionally, we found that effectors VgrG3 and TseL, despite lacking a classical Sec or TAT secretion signal, were able to reach the periplasm when expressed in the bacterial cytosol. This effector trafficking likely represents an evolutionary strategy for T6SS effectors to reach their intended substrates regardless of which subcellular compartment in the target cell they happen to be delivered to by the T6SS apparatus.
The type 6 secretion system (T6SS) is a dynamic organelle encoded by many gram-negative bacteria that can be used to kill competing bacterial prey species in densely occupied niches. Some predatory ...species, such as
, use their T6SS in an untargeted fashion while in contrast,
assembles and fires its T6SS apparatus only after detecting initial attacks by other bacterial prey cells; this targeted attack strategy has been termed the T6SS tit-for-tat response. Molecules that interact with the
outer membrane such as polymyxin B can also trigger assembly of T6SS organelles via a signal transduction pathway that involves protein phosphorylation. Recent work suggests that a phospholipase T6SS effector (TseL) of
can induce T6SS dynamic activity in
when delivered to or expressed in the periplasmic space of this organism. Here, we report that inhibiting expression of essential genes involved in outer membrane biogenesis can also trigger T6SS activation in
Specifically, we developed a CRISPR interference (CRISPRi) system to knock down expression of
,
, and
and found that these knockdowns activated T6SS activity. This increase in T6SS activity was dependent on the same signal transduction pathway that was previously shown to be required for the tit-for-tat response. We conclude that outer membrane perturbation can be sensed by
to activate the T6SS even when the disruption is generated by aberrant cell envelope biogenesis.
Defense systems that recognize viruses provide important insights into both prokaryotic and eukaryotic innate immunity mechanisms. Such systems that restrict foreign DNA or trigger cell death have ...recently been recognized, but the molecular signals that activate many of these remain largely unknown. Here, we characterize one such system in pandemic Vibrio cholerae responsible for triggering cell density-dependent death (CDD) of cells in response to the presence of certain genetic elements. We show that the key component is the Lamassu DdmABC anti-phage/plasmid defense system. We demonstrate that signals that trigger CDD were palindromic DNA sequences in phages and plasmids that are predicted to form stem-loop hairpins from single-stranded DNA. Our results suggest that agents that damage DNA also trigger DdmABC activation and inhibit cell growth. Thus, any infectious process that results in damaged DNA, particularly during DNA replication, can in theory trigger DNA restriction and death through the DdmABC abortive infection system.
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•DdmABC targeting and degradation of plasmids are dependent on certain cis-acting sequences•DNA sequences that induce DdmABC-based plasmid instability form hairpins•Palindromic sequences include those from related phages that are also restricted by DdmABC•DdmABC is blocked by a PriA allele
Robins et al. report on the identification of cis-acting palindromic DNA sequences in plasmids and phages targeted by the V. cholerae Lamassu DdmABC defense system. A single mutation in the PriA DNA repair-replication protein blocks DdmABC activity. Moreover, DdmABC expression increases UV sensitivity, suggesting DNA repair is one signal for activation.
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