Tao G. Dong

Generation of reactive oxygen species by lethal attacks from competing microbes

Significance How microbes respond to lethal attacks from competing species is not fully understood. Here, we investigated the response of Escherichia coli to attacks from the type VI secretion system (T6SS), bacteriophage P1vir, and polymyxin B. We report that generation of reactive oxygen species (ROS) is a general outcome of potentially lethal activities mediated by contact-dependent or contact-independent interactions of aggressive competing bacterial species and phage. An ROS response gene, soxS, is highly induced in response to all sources of attacks tested. This discovery will likely prompt other investigations into why evolution has selected expression of this gene as a “first responder” to potentially lethal interspecies competition. Whether antibiotics induce the production of reactive oxygen species (ROS) that contribute to cell death is an important yet controversial topic. Here, we report that lethal attacks from bacterial and viral species also result in ROS production in target cells. Using soxS as an ROS reporter, we found soxS was highly induced in Escherichia coli exposed to various forms of attacks mediated by the type VI secretion system (T6SS), P1vir phage, and polymyxin B. Using a fluorescence ROS probe, we found enhanced ROS levels correlate with induced soxS in E. coli expressing a toxic T6SS antibacterial effector and in E. coli treated with P1vir phage or polymyxin B. We conclude that both contact-dependent and contact-independent interactions with aggressive competing bacterial species and viruses can induce production of ROS in E. coli target cells.

Identification of divergent type VI secretion effectors using a conserved chaperone domain

Significance How different microbial species compete for specific niches is not fully understood. Here, we focus on the type VI secretion system (T6SS), a specialized protein delivery system, which many Gram-negative bacteria use to kill eukaryotic and prokaryotic competitors by translocating toxic protein molecules to target cells. Identification of effectors is required for understanding the pivotal role that the T6SS plays in dictating interbacterial and bacterial–host dynamics. In this study, we describe a new approach to systematically identify T6SS effectors. We also demonstrate that secretion of effectors requires interaction with a set of cognate effector-binding chaperone proteins that we define in this study, providing important insights for understanding the mechanism of T6SS effector delivery. The type VI secretion system (T6SS) is a lethal weapon used by many bacteria to kill eukaryotic predators or prokaryotic competitors. Killing by the T6SS results from repetitive delivery of toxic effectors. Despite their importance in dictating bacterial fitness, systematic prediction of T6SS effectors remains challenging due to high effector diversity and the absence of a conserved signature sequence. Here, we report a class of T6SS effector chaperone (TEC) proteins that are required for effector delivery through binding to VgrG and effector proteins. The TEC proteins share a highly conserved domain (DUF4123) and are genetically encoded upstream of their cognate effector genes. Using the conserved TEC domain sequence, we identified a large family of TEC genes coupled to putative T6SS effectors in Gram-negative bacteria. We validated this approach by verifying a predicted effector TseC in Aeromonas hydrophila. We show that TseC is a T6SS-secreted antibacterial effector and that the downstream gene tsiC encodes the cognate immunity protein. Further, we demonstrate that TseC secretion requires its cognate TEC protein and an associated VgrG protein. Distinct from previous effector-dependent bioinformatic analyses, our approach using the conserved TEC domain will facilitate the discovery and functional characterization of new T6SS effectors in Gram-negative bacteria.

Secretome Analysis of Vibrio cholerae Type VI Secretion System Reveals a New Effector-Immunity Pair

ABSTRACT The type VI secretion system (T6SS) is a dynamic macromolecular organelle that many Gram-negative bacteria use to inhibit or kill other prokaryotic or eukaryotic cells. The toxic effectors of T6SS are delivered to the prey cells in a contact-dependent manner. In Vibrio cholerae, the etiologic agent of cholera, T6SS is active during intestinal infection. Here, we describe the use of comparative proteomics coupled with bioinformatics to identify a new T6SS effector-immunity pair. This analysis was able to identify all previously identified secreted substrates of T6SS except PAAR (proline, alanine, alanine, arginine) motif-containing proteins. Additionally, this approach led to the identification of a new secreted protein encoded by VCA0285 (TseH) that carries a predicted hydrolase domain. We confirmed that TseH is toxic when expressed in the periplasm of Escherichia coli and V. cholerae cells. The toxicity observed in V. cholerae was suppressed by coexpression of the protein encoded by VCA0286 (TsiH), indicating that this protein is the cognate immunity protein of TseH. Furthermore, exogenous addition of purified recombinant TseH to permeabilized E. coli cells caused cell lysis. Bioinformatics analysis of the TseH protein sequence suggest that it is a member of a new family of cell wall-degrading enzymes that include proteins belonging to the YD repeat and Rhs superfamilies and that orthologs of TseH are likely expressed by species belonging to phyla as diverse as Bacteroidetes and Proteobacteria. IMPORTANCE The Gram-negative bacterium Vibrio cholerae causes cholera, a severe and often lethal diarrheal disease. The 2010-2012 epidemic in Haiti and new explosive epidemics in Africa show that cholera remains a significant global public health problem. The type VI secretion system (T6SS) is a dynamic organelle expressed by many Gram-negative bacteria, which use it to inject toxic effector proteins into eukaryotic and bacterial prey cells. In this study, we applied a comparative proteomics approach to the V. cholerae T6SS secretome to identify new substrates of this secretion apparatus. We show that the product of the gene VCA0285 is likely a new peptidoglycan hydrolase that is secreted by T6SS and that its cognate immunity protein is encoded by the gene that is immediately downstream (VCA0286). Bioinformatics analysis shows that VCA0285 carries four conserved motifs that likely define a large family of hydrolases with antibacterial activity. The identification of new antibacterial T6SS effectors provides useful information for the development of novel antibiotics and therapeutic agents. The Gram-negative bacterium Vibrio cholerae causes cholera, a severe and often lethal diarrheal disease. The 2010-2012 epidemic in Haiti and new explosive epidemics in Africa show that cholera remains a significant global public health problem. The type VI secretion system (T6SS) is a dynamic organelle expressed by many Gram-negative bacteria, which use it to inject toxic effector proteins into eukaryotic and bacterial prey cells. In this study, we applied a comparative proteomics approach to the V. cholerae T6SS secretome to identify new substrates of this secretion apparatus. We show that the product of the gene VCA0285 is likely a new peptidoglycan hydrolase that is secreted by T6SS and that its cognate immunity protein is encoded by the gene that is immediately downstream (VCA0286). Bioinformatics analysis shows that VCA0285 carries four conserved motifs that likely define a large family of hydrolases with antibacterial activity. The identification of new antibacterial T6SS effectors provides useful information for the development of novel antibiotics and therapeutic agents.

Manganese scavenging and oxidative stress response mediated by type VI secretion system in Burkholderia thailandensis

Significance As an essential micronutrient, Gram-negative bacteria must concentrate Mn2+ into the cytosol via active transport systems to meet cellular demands. Whereas inner membrane Mn2+ transporters have been characterized, an active transporter for translocation of Mn2+ across the outer membrane has not been described. Here we report a Mn2+-scavenging pathway consisting of a newly identified TonB-dependent outer membrane manganese transporter, MnoT, and a type VI secretion system (T6SS)-secreted Mn2+-binding protein, TseM. Traditionally, T6SS is recognized as a contact-dependent nanomachine to inject effectors into eukaryotic or prokaryotic cells for virulence or for interbacterial competition. The contact-independent functions of T6SS for metal acquisition and bacteria–bacteria competition, reported here, suggest that T6SS may have been retrofitted by some bacteria to gain additional adaptive functions during evolution. Type VI secretion system (T6SS) is a versatile protein export machinery widely distributed in Gram-negative bacteria. Known to translocate protein substrates to eukaryotic and prokaryotic target cells to cause cellular damage, the T6SS has been primarily recognized as a contact-dependent bacterial weapon for microbe–host and microbial interspecies competition. Here we report contact-independent functions of the T6SS for metal acquisition, bacteria competition, and resistance to oxidative stress. We demonstrate that the T6SS-4 in Burkholderia thailandensis is critical for survival under oxidative stress and is regulated by OxyR, a conserved oxidative stress regulator. The T6SS-4 is important for intracellular accumulation of manganese (Mn2+) under oxidative stress. Next, we identified a T6SS-4–dependent Mn2+-binding effector TseM, and its interacting partner MnoT, a Mn2+-specific TonB-dependent outer membrane transporter. Similar to the T6SS-4 genes, expression of mnoT is regulated by OxyR and is induced under oxidative stress and low Mn2+ conditions. Both TseM and MnoT are required for efficient uptake of Mn2+ across the outer membrane under Mn2+-limited and -oxidative stress conditions. The TseM–MnoT-mediated active Mn2+ transport system is also involved in contact-independent bacteria–bacteria competition and bacterial virulence. This finding provides a perspective for understanding the mechanisms of metal ion uptake and the roles of T6SS in bacteria–bacteria competition.

Microbial Herd Protection Mediated by Antagonistic Interaction in Polymicrobial Communities

ABSTRACT In host and natural environments, microbes often exist in complex multispecies communities. The molecular mechanisms through which such communities develop and persist, despite significant antagonistic interactions between species, are not well understood. The type VI secretion system (T6SS) is a lethal weapon commonly employed by Gram-negative bacteria to inhibit neighboring species through the delivery of toxic effectors. It is well established that intraspecies protection is conferred by immunity proteins that neutralize effector toxicities. In contrast, the mechanisms for interspecies protection are not clear. Here we use two T6SS-active antagonistic bacterial species, Aeromonas hydrophila and Vibrio cholerae, to demonstrate that interspecies protection is dependent on effectors. A. hydrophila and V. cholerae do not share conserved immunity genes but could coexist equally in a mixture. However, mutants lacking the T6SS or effectors were effectively eliminated by the competing wild-type strain. Time-lapse microscopic analyses showed that mutually lethal interactions drive the segregation of mixed species into distinct single-species clusters by eliminating interspersed single cells. Cluster formation provides herd protection by abolishing lethal interactions inside each cluster and restricting the interactions to the boundary. Using an agent-based modeling approach, we simulated the antagonistic interactions of two hypothetical species. The resulting simulations recapitulated our experimental observations. These results provide mechanistic insights regarding the general role of microbial weapons in determining the structures of complex multispecies communities. IMPORTANCE Investigating the warfare of microbes allows us to better understand the ecological relationships in complex microbial communities such as the human microbiota. Here we use the T6SS, a deadly bacterial weapon, as a model to demonstrate the importance of lethal interactions in determining community structures and the exchange of genetic materials. This simplified model elucidates a mechanism of microbial herd protection by which competing antagonistic species can coexist in the same niche, despite their diverse mutually destructive activities. Our results also suggest that antagonistic interactions impose strong selection that could promote multicellular organism-like social behaviors and contribute to the transition to multicellularity during evolution.

The Type VI Secretion System Engages a Redox-Regulated Dual-Functional Heme Transporter for Zinc Acquisition

The type VI secretion system was recently reported to be involved in zinc acquisition, but the underlying mechanism remains unclear. Here, we report that Burkholderia thailandensis T6SS4 is involved in zinc acquisition via secretion of a zinc-scavenging protein, TseZ, that interacts with the outer membrane heme transporter HmuR. We find that HmuR is a redox-regulated dual-functional transporter that transports heme iron under normal conditions but zinc upon sensing extracellular oxidative stress, triggered by formation of an intramolecular disulfide bond. Acting as the first line of defense against oxidative stress, HmuR not only guarantees an immediate response to the changing environment but also provides a fine-tuned mechanism that allows a gradual response to perceived stress. The T6SS/HmuR-mediated active zinc transport system is also involved in bacterial virulence and contact-independent bacterial competition. We describe a sophisticated bacterial zinc acquisition mechanism affording insights into the role of metal ion transport systems.

Vibrio cholerae type 6 secretion system effector trafficking in target bacterial cells

Significance This work demonstrates that it is possible to alter the prey target range of the antibacterial activity of the type 6 secretion system (T6SS). Being able to change the T6SS target specificity is the first step toward using the T6SS as an antipathogen or commensal therapeutic, prophylactic, or probiotic. This work also uncovers a cryptic secretion mechanism(s) for delivering protein substrates from the bacterial cytosol to the periplasm. This mechanism is exploited by some T6SS effectors as an alternative pathway for reaching periplasmic targets when they are by chance delivered into the cytosol of target cells. 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.

Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg2+ or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities.

A type VI secretion system effector delivery mechanism dependent on PAAR and a chaperone–co-chaperone complex

The type VI secretion system (T6SS) is used by many Gram-negative bacteria as a molecular weapon to modulate neighbouring bacterial and eukaryotic cells, thereby affecting the dynamics of community structure in multiple species environments. The T6SS injects its inner-needle Hcp tube, the sharpening tip complex consisting of VgrG and PAAR, and toxic effectors into neighbouring cells. Its functions are largely determined by the activities of its delivered effectors. Six mechanisms of effector delivery have been described: two mediated by the inner tube and the others mediated by the VgrG and PAAR tip complex. Here, we report an additional effector delivery mechanism that relies on interaction with a chaperone complex and a PAAR protein as a carrier. The Pseudomonas aeruginosa PAO1 TOX-REase-5 domain-containing effector TseT directly interacts with PAAR4 and the chaperone TecT for delivery, and an immunity protein, TsiT, for protection from its toxicity. TecT forms a complex with its co-chaperone, co-TecT, which is disrupted by the carboxy-terminal tail of PAAR4. In addition, we delineate a complex, multilayered competitive process that dictates effector trafficking. PAAR delivery provides an additional tool for engineering cargo protein translocation.The Pseudomonas aeruginosa effector TseT is loaded into the type VI secretion system (T6SS) by the chaperone TecT. Interactions between the T6SS needle-tip protein PAAR and TecT disrupt binding between TecT and its co-chaperone, co-TecT, and promote binding of TseT to PAAR.

Vibrio parahaemolyticus RhsP represents a widespread group of pro-effectors for type VI secretion systems

Type VI secretion systems (T6SSs) translocate effector proteins, such as Rhs toxins, to eukaryotic cells or prokaryotic competitors. All T6SS Rhs-type effectors characterized thus far contain a PAAR motif or a similar structure. Here, we describe a T6SS-dependent delivery mechanism for a subset of Rhs proteins that lack a PAAR motif. We show that the N-terminal Rhs domain of protein RhsP (or VP1517) from Vibrio parahaemolyticus inhibits the activity of the C-terminal DNase domain. Upon auto-proteolysis, the Rhs fragment remains inside the cells, and the C-terminal region interacts with PAAR2 and is secreted by T6SS2; therefore, RhsP acts as a pro-effector. Furthermore, we show that RhsP contributes to the control of certain “social cheaters” (opaR mutants). Genes encoding proteins with similar Rhs and PAAR-interacting domains, but diverse C-terminal regions, are widely distributed among Vibrio species.It is unclear how Rhs toxins lacking a PAAR motif are secreted by Type VI secretion systems. Here, the authors show for one of these proteins that the mechanism requires removal of an N-terminal fragment by auto-proteolysis, followed by interaction with a PAAR protein and then secretion.