Programmed Secretion Arrest and Receptor-Triggered Toxin Export during Antibacterial Contact-Dependent Growth Inhibition.

Contact-dependent growth inhibition (CDI) entails receptor-mediated delivery of CdiA-derived toxins into Gram-negative target bacteria. Using electron cryotomography, we show that each CdiA effector protein forms a filament extending ∼33 nm from the cell surface. Remarkably, the extracellular filament represents only the N-terminal half of the effector. A programmed secretion arrest sequesters the C-terminal half of CdiA, including the toxin domain, in the periplasm prior to target-cell recognition. Upon binding receptor, CdiA secretion resumes, and the periplasmic FHA-2 domain is transferred to the target-cell outer membrane. The C-terminal toxin region of CdiA then penetrates into the target-cell periplasm, where it is cleaved for subsequent translocation into the cytoplasm. Our findings suggest that the FHA-2 domain assembles into a transmembrane conduit for toxin transport into the periplasm of target bacteria. We propose that receptor-triggered secretion ensures that FHA-2 export is closely coordinated with integration into the target-cell outer membrane. VIDEO ABSTRACT.

An ADP-ribosyltransferase toxin kills bacterial cells by modifying structured non-coding RNAs.

ADP-ribosyltransferases (ARTs) were among the first identified bacterial virulence factors. Canonical ART toxins are delivered into host cells where they modify essential proteins, thereby inactivating cellular processes and promoting pathogenesis. Our understanding of ARTs has since expanded beyond protein-targeting toxins to include antibiotic inactivation and DNA damage repair. Here, we report the discovery of RhsP2 as an ART toxin delivered between competing bacteria by a type VI secretion system of Pseudomonas aeruginosa. A structure of RhsP2 reveals that it resembles protein-targeting ARTs such as diphtheria toxin. Remarkably, however, RhsP2 ADP-ribosylates 2'-hydroxyl groups of double-stranded RNA, and thus, its activity is highly promiscuous with identified cellular targets including the tRNA pool and the RNA-processing ribozyme, ribonuclease P. Consequently, cell death arises from the inhibition of translation and disruption of tRNA processing. Overall, our data demonstrate a previously undescribed mechanism of bacterial antagonism and uncover an unprecedented activity catalyzed by ART enzymes.

Xanthomonas immunity proteins protect against the cis-toxic effects of their cognate T4SS effectors.

Many bacteria kill rival species by translocating toxic effectors into target cells. Effectors are often encoded along with cognate immunity proteins that could (i) protect against "friendly-fire" (trans-intoxication) from neighboring sister cells and/or (ii) protect against internal cis-intoxication (suicide). Here, we distinguish between these two mechanisms in the case of the bactericidal Xanthomonas citri Type IV Secretion System (X-T4SS). We use a set of X. citri mutants lacking multiple effector/immunity protein (X-Tfe/X-Tfi) pairs to show that X-Tfis are not absolutely required to protect against trans-intoxication by wild-type cells. Our investigation then focused on the in vivo function of the lysozyme-like effector X-TfeXAC2609 and its cognate immunity protein X-TfiXAC2610. In the absence of X-TfiXAC2610, we observe X-TfeXAC2609-dependent and X-T4SS-independent accumulation of damage in the X. citri cell envelope, cell death, and inhibition of biofilm formation. While immunity proteins in other systems have been shown to protect against attacks by sister cells (trans-intoxication), this is an example of an antibacterial secretion system in which the immunity proteins are dedicated to protecting cells against cis-intoxication.

Structural basis for effector recognition by an antibacterial type IV secretion system.

Many soil-, water-, and plant-associated bacterial species from the orders Xanthomonadales, Burkholderales, and Neisseriales carry a type IV secretion system (T4SS) specialized in translocating effector proteins into other gram-negative species, leading to target cell death. These effectors, known as X-Tfes, carry a carboxyl-terminal domain of ∼120 residues, termed XVIPCD, characterized by several conserved motifs and a glutamine-rich tail. Previous studies showed that the XVIPCD is required for interaction with the T4SS coupling protein VirD4 and for T4SS-dependent translocation. However, the structural basis of the XVIPCD-VirD4 interaction is unknown. Here, we show that the XVIPCD interacts with the central all-alpha domain of VirD4 (VirD4AAD). We used solution NMR spectroscopy to solve the structure of the XVIPCD of X-TfeXAC2609 from Xanthomonas citri and to map its interaction surface with VirD4AAD Isothermal titration calorimetry and in vivo Xanthomonas citri versus Escherichia coli competition assays using wild-type and mutant X-TfeXAC2609 and X-TfeXAC3634 indicate that XVIPCDs can be divided into two regions with distinct functions: the well-folded N-terminal region contains specific conserved motifs that are responsible for interactions with VirD4AAD, while both N- and carboxyl-terminal regions are required for effective X-Tfe translocation into the target cell. The conformational stability of the N-terminal region is reduced at and below pH 7.0, a property that may facilitate X-Tfe unfolding and translocation through the more acidic environment of the periplasm.

Structural basis for loading and inhibition of a bacterial T6SS phospholipase effector by the VgrG spike.

The bacterial type VI secretion system (T6SS) is a macromolecular machine that injects effectors into prokaryotic and eukaryotic cells. The mode of action of the T6SS is similar to contractile phages: the contraction of a sheath structure pushes a tube topped by a spike into target cells. Effectors are loaded onto the spike or confined into the tube. In enteroaggregative Escherichia coli, the Tle1 phospholipase binds the C-terminal extension of the VgrG trimeric spike. Here, we purify the VgrG-Tle1 complex and show that a VgrG trimer binds three Tle1 monomers and inhibits their activity. Using covalent cross-linking coupled to high-resolution mass spectrometry, we provide information on the sites of contact and further identify the requirement for a Tle1 N-terminal secretion sequence in complex formation. Finally, we report the 2.6-Å-resolution cryo-electron microscopy tri-dimensional structure of the (VgrG)3 -(Tle1)3 complex revealing how the effector binds its cargo, and how VgrG inhibits Tle1 phospholipase activity. The inhibition of Tle1 phospholipase activity once bound to VgrG suggests that Tle1 dissociation from VgrG is required upon delivery.

The dynamics of colicin E9 release from Escherichia coli in native conditions.

Colicin (Col) plasmid contains colicin encoding genes arranged in an operon controlled by an SOS inducible promoter. Therefore, any external stresses to the host cell can induce the expression of the downstream genes in the Col operon, including a lysis gene. The lysis protein is involved in the extracellular release of colicin through lysis of the producer cells, which causes a decline in culture turbidity. However, it is not yet known that E. coli cells with the native pColE9-J plasmid hold the same level of cell death at the population level following a set of induced conditions. In this study, using a mitomycin C sensitivity assay along with a live dead staining method of detection, we showed that the native pColE9-J plasmid, which unusually carries an extended Col operon (ColE9) containing two lysis genes, did not confer a rapid decline in the culture turbidity following induction with mitomycin C. Interestingly a subset of the cells suffered perturbation of their outer membrane, which was not observed from single lysis mutant (∆celE or ∆celI) cells. This observed heterogeneity in the colicin E9 release leading to differential outer membrane perforation may bring a competitive advantage to these cells in a mixed population.

No fitness cost entailed by type VI secretion system synthesis, assembly, contraction, or disassembly in enteroaggregative Escherichia coli.

IMPORTANCE: Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Due to its size and mechanism of action, the T6SS was intuitively thought to be energetically costly. Here, using a combination of mutants and growth measurements in liquid medium, on plates, and in competition experiments, we show that the T6SS does not entail a growth cost to enteroaggregative Escherichia coli.

Quorum-sensing, intra- and inter-species competition in the staphylococci.

In Gram-positive bacteria such as Staphylococcus aureus and the coagulase-negative staphylococci (CoNS), the accessory gene regulator (agr) is a highly conserved but polymorphic quorum-sensing system involved in colonization, virulence and biofilm development. Signalling via agr depends on the interaction of an autoinducing peptide (AIP) with AgrC, a transmembrane sensor kinase that, once phosphorylated activates the response regulator AgrA. This in turn autoinduces AIP biosynthesis and drives target gene expression directly via AgrA or via the post-transcriptional regulator, RNAIII. In this review we describe the molecular mechanisms underlying the agr-mediated generation of, and response to, AIPs and the molecular basis of AIP-dependent activation and inhibition of AgrC. How the environment impacts on agr functionality is considered and the consequences of agr dysfunction for infection explored. We also discuss the concept of AIP-driven competitive interference between S. aureus and the CoNS and its anti-infective potential.

Interbacterial competition mediated by the type VIIb secretion system.

Successful occupancy of a given niche requires the colonising bacteria to interact extensively with the biotic and abiotic environment, including other resident microbes. Bacteria have evolved a range of protein secretion machines for this purpose with eleven such systems identified to date. The type VIIb secretion system (T7SSb) is utilised by Bacillota to secrete a range of protein substrates, including antibacterial toxins targeting closely related strains, and the system as a whole has been implicated in a range of activities such as iron acquisition, intercellular signalling, host colonisation and virulence. This review covers the components and secretion mechanism of the T7SSb, the substrates of these systems and their roles in Gram-positive bacteria, with a focus on interbacterial competition.

Engineering a customizable antibacterial T6SS-based platform in Vibrio natriegens.

Bacterial pathogens are a major risk to human, animal, and plant health. To counteract the spread of antibiotic resistance, alternative antibacterial strategies are urgently needed. Here, we construct a proof-of-concept customizable, modular, and inducible antibacterial toxin delivery platform. By engineering a type VI secretion system (T6SS) that is controlled by an externally induced on/off switch, we transform the safe bacterium, Vibrio natriegens, into an effective antibacterial weapon. Furthermore, we demonstrate that the delivered effector repertoire, and thus the toxicity range of this platform, can be easily manipulated and tested. We believe that this platform can serve as a foundation for novel antibacterial bio-treatments, as well as a unique tool to study antibacterial toxins.

Type VI Secretion Systems: Environmental and Intra-host Competition of Vibrio cholerae.

The Vibrio Type VI Secretion System (T6SS) is a harpoon-like nanomachine that serves as a defense system and is encoded by approximately 25% of all gram-negative bacteria. In this chapter, we describe the structure of the T6SS in different Vibrio species and outline how the use of different T6SS effector and immunity proteins control kin selection. We summarize the genetic loci that encode the structural elements that make up the Vibrio T6SSs and how these gene clusters are regulated. Finally, we provide insights into T6SS-based competitive dynamics, the role of T6SS genetic exchange in those competitive dynamics, and roles for the Vibrio T6SS in virulence.

A membrane-depolarizing toxin substrate of the Staphylococcus aureus type VII secretion system mediates intraspecies competition.

The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date, only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane-depolarizing activity is alleviated by coproduction of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologs at the tspA locus, suggestive of additional roles in intraspecies competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologs.

The ß-encapsulation cage of rearrangement hotspot (Rhs) effectors is required for type VI secretion.

Bacteria deploy rearrangement hotspot (Rhs) proteins as toxic effectors against both prokaryotic and eukaryotic target cells. Rhs proteins are characterized by YD-peptide repeats, which fold into a large ß-cage structure that encapsulates the C-terminal toxin domain. Here, we show that Rhs effectors are essential for type VI secretion system (T6SS) activity in Enterobacter cloacae (ECL). ECL rhs- mutants do not kill Escherichia coli target bacteria and are defective for T6SS-dependent export of hemolysin-coregulated protein (Hcp). The RhsA and RhsB effectors of ECL both contain Pro-Ala-Ala-Arg (PAAR) repeat domains, which bind the ß-spike of trimeric valine-glycine repeat protein G (VgrG) and are important for T6SS activity in other bacteria. Truncated RhsA that retains the PAAR domain is capable of forming higher-order, thermostable complexes with VgrG, yet these assemblies fail to restore secretion activity to ∆rhsA ∆rhsB mutants. Full T6SS-1 activity requires Rhs that contains N-terminal transmembrane helices, the PAAR domain, and an intact ß-cage. Although ∆rhsA ∆rhsB mutants do not kill target bacteria, time-lapse microscopy reveals that they assemble and fire T6SS contractile sheaths at ∼6% of the frequency of rhs+ cells. Therefore, Rhs proteins are not strictly required for T6SS assembly, although they greatly increase secretion efficiency. We propose that PAAR and the ß-cage provide distinct structures that promote secretion. PAAR is clearly sufficient to stabilize trimeric VgrG, but efficient assembly of T6SS-1 also depends on an intact ß-cage. Together, these domains enforce a quality control checkpoint to ensure that VgrG is loaded with toxic cargo before assembling the secretion apparatus.

Oral Commensal Streptococci: Gatekeepers of the Oral Cavity.

Oral commensal streptococci are primary colonizers of the oral cavity. These streptococci produce many adhesins, metabolites, and antimicrobials that modulate microbial succession and diversity within the oral cavity. Often, oral commensal streptococci antagonize cariogenic and periodontal pathogens such as Streptococcus mutans and Porphyromonas gingivalis, respectively. Mechanisms of antagonism are varied and range from the generation of hydrogen peroxide, competitive metabolite scavenging, the generation of reactive nitrogen intermediates, and bacteriocin production. Furthermore, several oral commensal streptococci have been shown to alter the host immune response at steady state and in response to oral pathogens. Collectively, these features highlight the remarkable ability of oral commensal streptococci to regulate the structure and function of the oral microbiome. In this review, we discuss mechanisms used by oral commensal streptococci to interact with diverse oral pathogens, both physically and through the production of antimicrobials. Finally, we conclude by exploring the critical roles of oral commensal streptococci in modulating the host immune response and maintaining health and homeostasis.

Burkholderia multivorans requires species-specific GltJK for entry of a contact-dependent growth inhibition system protein.

Interbacterial antagonism and communication are driving forces behind microbial community development. In many Gram-negative bacteria, contact-dependent growth inhibition (CDI) systems contribute to these microbial interactions. CDI systems deliver the toxic C-terminus of a large surface exposed protein to the cytoplasm of neighboring bacteria upon cell-contact. Termed the BcpA-CT, import of this toxic effector domain is mediated by specific, yet largely unknown receptors on the recipient cell outer and inner membranes. In this study, we demonstrated that cytoplasmic membrane proteins GltJK, components of a predicted ABC-type transporter, are required for entry of CDI system protein BcpA-2 into Burkholderia multivorans recipient cells. Consistent with current CDI models, gltJK were also required for recipient cell susceptibility to a distinct BcpA-CT that shared sequences within the predicted "translocation domain" of BcpA-2. Strikingly, this translocation domain showed low sequence identity to the analogous region of an Escherichia coli GltJK-utilizing CDI system protein. Our results demonstrated that recipient bacteria expressing E. coli gltJK were resistant to BcpA-2-mediated interbacterial antagonism, suggesting that BcpA-2 specifically recognizes Burkholderia GltJK. Using a series of chimeric proteins, the specificity determinant was mapped to Burkholderia-specific sequences at the GltK C-terminus, providing insight into BcpA transport across the recipient cell cytoplasmic membrane.

Activity, delivery, and diversity of Type VI secretion effectors.

The bacterial type VI secretion system (T6SS) system is a contractile secretion apparatus that delivers proteins to neighboring bacterial or eukaryotic cells. Antibacterial effectors are mostly toxins that inhibit the growth of other species and help to dominate the niche. A broad variety of these toxins cause cell lysis of the prey cell by disrupting the cell envelope. Other effectors are delivered into the cytoplasm where they affect DNA integrity, cell division or exhaust energy resources. The modular nature of T6SS machinery allows different means of recruitment of toxic effectors to secreted inner tube and spike components that act as carriers. Toxic effectors can be translationally fused to the secreted components or interact with them through specialized structural domains. These interactions can also be assisted by dedicated chaperone proteins. Moreover, conserved sequence motifs in effector-associated domains are subject to genetic rearrangements and therefore engage in the diversification of the arsenal of toxic effectors. This review discusses the diversity of T6SS secreted toxins and presents current knowledge about their loading on the T6SS machinery.

Bacterial protein secretion systems: Game of types.

Protein trafficking across the bacterial envelope is a process that contributes to the organisation and integrity of the cell. It is the foundation for establishing contact and exchange between the environment and the cytosol. It helps cells to communicate with one another, whether they establish symbiotic or competitive behaviours. It is instrumental for pathogenesis and for bacteria to subvert the host immune response. Understanding the formation of envelope conduits and the manifold strategies employed for moving macromolecules across these channels is a fascinating playground. The diversity of the nanomachines involved in this process logically resulted in an attempt to classify them, which is where the protein secretion system types emerged. As our knowledge grew, so did the number of types, and their rightful nomenclature started to be questioned. While this may seem a semantic or philosophical issue, it also reflects scientific rigour when it comes to assimilating findings into textbooks and science history. Here I give an overview on bacterial protein secretion systems, their history, their nomenclature and why it can be misleading for newcomers in the field. Note that I do not try to suggest a new nomenclature. Instead, I explore the reasons why naming could have escaped our control and I try to reiterate basic concepts that underlie protein trafficking cross membranes.

In Vitro Evolution of Listeria monocytogenes Reveals Selective Pressure for Loss of SigB and AgrA Function at Different Incubation Temperatures.

The alternative sigma factor B (σB) contributes to the stress tolerance of the foodborne pathogen Listeria monocytogenes by upregulating the general stress response. We previously showed that σB loss-of-function mutations arise frequently in strains of L. monocytogenes and suggested that mild stresses might favor the selection of such mutations. In this study, we performed in vitro evolution experiments (IVEE) where L. monocytogenes was allowed to evolve over 30 days at elevated (42°C) or lower (30°C) incubation temperatures. Isolates purified throughout the IVEE revealed the emergence of sigB operon mutations at 42°C. However, at 30°C, independent alleles in the agr locus arose, resulting in the inactivation of Agr quorum sensing. Colonies of both sigB mutants and agr mutants exhibited a greyer coloration on 7-days-old agar plates than those of the parental strain. Scanning electron microscopy revealed a more complex colony architecture in the wild type than in the mutant strains. sigB mutant strains outcompeted the parental strain at 42°C but not at 30°C, while agr mutant strains showed a small increase in competitive fitness at 30°C. Analysis of 40,080 L. monocytogenes publicly available genome sequences revealed a high occurrence rate of premature stop codons in both the sigB and agrCA loci. An analysis of a local L. monocytogenes strain collection revealed 5 out of 168 strains carrying agrCA alleles. Our results suggest that the loss of σB or Agr confer an increased competitive fitness in some specific conditions and this likely contributes to the emergence of these alleles in strains of L. monocytogenes. IMPORTANCE To withstand environmental aggressions, L. monocytogenes upregulates a large regulon through the action of the alternative sigma factor B (σB). However, σB becomes detrimental for L. monocytogenes growth under mild stresses, which confer a competitive advantage to σB loss-of-function alleles. Temperatures of 42°C, a mild stress, are often employed in mutagenesis protocols of L. monocytogenes and promote the emergence of σB loss-of-function alleles in the sigB operon. In contrast, lower temperatures of 30°C promote the emergence of Agr loss-of-function alleles, a cell-cell communication mechanism in L. monocytogenes. Our findings demonstrate that loss-of-function alleles emerge spontaneously in laboratory-grown strains. These alleles rise in the population as a consequence of the trade-off between growth and survival imposed by the activation of σB in L. monocytogenes. Additionally, our results demonstrate the importance of identifying unwanted hitchhiker mutations in newly constructed mutant strains.

The type VI secretion system in Acinetobacter baumannii clinical isolates and its roles in antimicrobial resistance acquisition.

Acinetobacter baumannii is a successful pathogen that can acquire various antibiotic resistance in a short time. However, little is known about how it can evolve from an antibiotic sensitive to a resistant phenotype. In this study, we investigated the roles of the type VI secretion system (T6SS) in the acquisition of antibiotic resistance of A. baumannii. T6SS gene cluster was found to be present in 51 of 77 A. baumannii clinical isolates, of which, it was found in 62% (8/13) of the multiple drug resistant (MDR) isolates, 90% (36/40) of the extensively drug-resistant (XDR) isolates and 26% (6/23) of the antibiotic sensitive isolates. There is a close relationship between the antimicrobial resistance and the presence of T6SS. Besides, T6SS + isolates showed lower biofilm formation activity and higher survival ability in the presence of normal human serum than T6SS- isolates. A. baumannii A152 with complete T6SS can outcompete E.coli effectively and can acquire the antibiotic resistance plasmids released by E.coli. In contrast, the T6SS core gene mutant A152Δhcp showed significantly decreased ability to acquire antimicrobial resistance plasmids from the prey bacteria. These results suggest that T6SS mediated bacterial competition plays important roles in the antimicrobial resistance of A. baumannii, which points out a new direction for us to study the antimicrobial resistance of A. baumannii.

Competitive growth kinetics of Campylobacter jejuni, Escherichia coli O157:H7 and Listeria monocytogenes with enteric microflora in a small-intestine model.

AIMS: The biological events occurring during human digestion help to understand the mechanisms underlying the dose-response relationships of enteric bacterial pathogens. To better understand these events, we investigated the growth and reduction behaviour of bacterial pathogens in an in vitro model simulating the environment of the small intestine. METHODS AND RESULTS: The foodborne pathogens Campylobacter jejuni, Listeria monocytogenes and Escherichia coli O157:H7 were cultured with multiple competing enteric bacteria. Differences in the pathogen's growth kinetics due to the relative amount of competing enteric bacteria were investigated. These growth differences were described using a mathematical model based on Bayesian inference. When pathogenic and enteric bacteria were inoculated at 1 log CFU per ml and 9 log CFU per ml, respectively, L. monocytogenes was inactivated over time, while C. jejuni and E. coli O157:H7 survived without multiplying. However, as pathogen inocula were increased, its inhibition by enteric bacteria also decreased. CONCLUSIONS: Although the growth of pathogenic species was inhibited by enteric bacteria, the pathogens still survived. SIGNIFICANCE AND IMPACT OF THE STUDY: Competition experiments in a small-intestine model have enhanced understanding of the infection risk in the intestine and provide insights for evaluating dose-response relationships.