Complete genome sequence of Stenotrophomonas maltophilia complex strain STEN00241.

We report here the complete, closed genome sequence of Stenotrophomonas maltophilia complex strain STEN00241, which was isolated from patient sputum. The genome is a single contig with a total length of 4,751,329 nucleotides and a GC content of 66.5%.

The type VI secretion system of the emerging pathogen Stenotrophomonas maltophilia complex has antibacterial properties.

IMPORTANCE: Infections with the opportunistic pathogen Stenotrophomonas maltophilia complex can be fatal for immunocompromised patients. The mechanisms used by the bacterium to compete against other prokaryotes are not well understood. We found that the type VI secretion system (T6SS) allows S. maltophilia complex to eliminate other bacteria and contributes to the competitive fitness against a co-infecting isolate. The presence of T6SS genes in isolates across the globe highlights the importance of this apparatus as a weapon in the antibacterial arsenal of S. maltophilia complex. The T6SS may confer survival advantages to S. maltophilia complex isolates in polymicrobial communities in both environmental settings and during infections.

The Type VI Secretion System of the Emerging Pathogen Stenotrophomonas maltophilia has Antibacterial Properties.

Antagonistic behaviors between bacterial cells can have profound effects on microbial populations and disease outcomes. Polymicrobial interactions may be mediated by contact-dependent proteins with antibacterial properties. The Type VI Secretion System (T6SS) is a macromolecular weapon used by Gram-negative bacteria to translocate proteins into adjacent cells. The T6SS is used by pathogens to escape immune cells, eliminate commensal bacteria, and facilitate infection. Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that causes a wide range of infections in immunocompromised patients and infects the lungs of patients with cystic fibrosis. Infections with the bacterium can be deadly and are challenging to treat because many isolates are multidrug-resistant. We found that globally dispersed S. maltophilia clinical and environmental strains possess T6SS genes. We demonstrate that the T6SS of an S. maltophilia patient isolate is active and can eliminate other bacteria. Furthermore, we provide evidence that the T6SS contributes to the competitive fitness of S. maltophilia against a co-infecting Pseudomonas aeruginosa isolate, and that the T6SS alters the cellular organization of S. maltophilia and P. aeruginosa co-cultures. This study expands our knowledge of the mechanisms employed by S. maltophilia to secrete antibacterial proteins and compete against other bacteria. IMPORTANCE: Infections with the opportunistic pathogen Stenotrophomonas maltophilia can be fatal for immunocompromised patients. The mechanisms used by the bacterium to compete against other prokaryotes are not well understood. We found that the T6SS allows S. maltophilia to eliminate other bacteria and contributes to the competitive fitness against a co-infecting isolate. The presence of T6SS genes in isolates across the globe highlights the importance of this apparatus as a weapon in the antibacterial arsenal of S. maltophilia . The T6SS may confer survival advantages to S. maltophilia isolates in polymicrobial communities in both environmental settings and during infections.

Pseudomonas aeruginosa Antivirulence Strategies: Targeting the Type III Secretion System.

The Pseudomonas aeruginosa type III secretion system (T3SS) is a complex molecular machine that delivers toxic proteins from the bacterial cytoplasm directly into host cells. This apparatus spans the inner and outer membrane and employs a needle-like structure that penetrates through the eucaryotic cell membrane into the host cell cytosol. The expression of the P. aeruginosa T3SS is highly regulated by environmental signals including low calcium and host cell contact. P. aeruginosa strains with mutations in T3SS genes are less pathogenic, suggesting that the T3SS is a virulence mechanism. Given that P. aeruginosa is naturally antibiotic resistant and multidrug resistant isolates are rapidly emerging, new antibiotics to target P. aeruginosa are needed. Furthermore, even if new antibiotics were to be developed, the timeline between when an antibiotic is released and resistance development is relatively short. Therefore, the concept of targeting virulence factors has garnered attention. So-called "antivirulence" approaches do not kill the microbe but instead focus on rendering it harmless and therefore unable to cause damage. Since these therapies target a particular system or pathway, the normal microbiome is unlikely to be affected and there is less concern about the spread to other microbes. Finally, and most importantly, since any antivirulence drug does not kill the microbe, there should be less selective pressure to develop resistance to these inhibitors. The P. aeruginosa T3SS has been well studied due to its importance for pathogenesis in numerous human and animal infections. Thus, many P. aeruginosa T3SS inhibitors have been described as potential antivirulence therapeutics, some of which have progressed to clinical trials.

Antibacterial contact-dependent proteins secreted by Gram-negative cystic fibrosis respiratory pathogens.

Cystic fibrosis (CF) is a genetic disease that affects almost 100 000 people worldwide. CF patients suffer from chronic bacterial airway infections that are often polymicrobial and are the leading cause of mortality. Interactions between pathogens modulate expression of genes responsible for virulence and antibiotic resistance. One of the ways bacteria can interact is through contact-dependent systems, which secrete antibacterial proteins (effectors) that confer advantages to cells that harbor them. Here, we highlight recent work that describes effectors used by Gram-negative CF pathogens to eliminate competitor bacteria. Understanding the mechanisms of secreted effectors may lead to novel insights into the ecology of bacteria that colonize respiratory tracts and could also pave the way for the design of new therapeutics.

A New Contact Killing Toxin Permeabilizes Cells and Belongs to a Broadly Distributed Protein Family.

Vibrio cholerae is an aquatic Gram-negative bacterium that causes severe diarrheal cholera disease when ingested by humans. To eliminate competitor cells in both the external environment and inside hosts, V. cholerae uses the type VI secretion system (T6SS). The T6SS is a macromolecular contact-dependent weapon employed by many Gram-negative bacteria to deliver cytotoxic proteins into adjacent cells. In addition to canonical T6SS gene clusters encoded by all sequenced V. cholerae isolates, strain BGT49 encodes another locus, which we named auxiliary (Aux) cluster 4. The Aux 4 cluster is located on a mobile genetic element and can be used by killer cells to eliminate both V. cholerae and Escherichia coli cells in a T6SS-dependent manner. A putative toxin encoded in the cluster, which we name TpeV (type VI permeabilizing effector Vibrio), shares no homology to known proteins and does not contain motifs or domains indicative of function. Ectopic expression of TpeV in the periplasm of E. coli permeabilizes cells and disrupts the membrane potential. Using confocal microscopy, we confirm that susceptible target cells become permeabilized when competed with killer cells harboring the Aux 4 cluster. We also determine that tpiV, the gene located immediately downstream of tpeV, encodes an immunity protein that neutralizes the toxicity of TpeV. Finally, we show that TpeV homologs are broadly distributed across important human, animal, and plant pathogens and are localized in proximity to other T6SS genes. Our results suggest that TpeV is a toxin that belongs to a large family of T6SS proteins. IMPORTANCE Bacteria live in polymicrobial communities where competition for resources and space is essential for survival. Proteobacteria use the T6SS to eliminate neighboring cells and cause disease. However, the mechanisms by which many T6SS toxins kill or inhibit susceptible target cells are poorly understood. The sequence of the TpeV toxin that we describe here is unlike any previously described protein. We demonstrate that it has antimicrobial activity by permeabilizing cells, eliminating membrane potentials, and causing severe cytotoxicity. TpeV homologs are found near known T6SS genes in human, animal, and plant bacterial pathogens, indicating that the toxin is a representative member of a broadly distributed protein family. We propose that TpeV-like toxins contribute to the fitness of many bacteria. Finally, since antibiotic resistance is a critical global health threat, the discovery of new antimicrobial mechanisms could lead to the development of new treatments against resistant strains.

Glucose confers protection to Escherichia coli against contact killing by Vibrio cholerae.

Evolutionary arms races are broadly prevalent among organisms including bacteria, which have evolved defensive strategies against various attackers. A common microbial aggression mechanism is the type VI secretion system (T6SS), a contact-dependent bacterial weapon used to deliver toxic effector proteins into adjacent target cells. Sibling cells constitutively express immunity proteins that neutralize effectors. However, less is known about factors that protect non-sibling bacteria from T6SS attacks independently of cognate immunity proteins. In this study, we observe that human Escherichia coli commensal strains sensitive to T6SS attacks from Vibrio cholerae are protected when co-cultured with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find that cells lacking the cAMP receptor protein (CRP), which regulates expression of hundreds of genes in response to glucose, survive significantly better against V. cholerae T6SS attacks even in the absence of glucose. Finally, we show that the glucose-mediated T6SS protection varies with different targets and killers. Our findings highlight the first example of an extracellular small molecule modulating a genetically controlled response for protection against T6SS attacks. This discovery may have major implications for microbial interactions during pathogen-host colonization and survival of bacteria in environmental communities.

The Vibrio cholerae type VI secretion system: toxins, regulators and consequences.

The type VI secretion system (T6SS) is a proteinaceous weapon used by many Gram-negative bacteria to deliver toxins into adjacent target cells. Vibrio cholerae, the bacterium responsible for the fatal water-borne cholera disease, uses the T6SS to evade phagocytic eukaryotes, cause intestinal inflammation, and compete against other bacteria with toxins that disrupt lipid membranes, cell walls and actin cytoskeletons. The control of T6SS genes varies among V. cholerae strains and typically includes inputs from external signals and cues, such as quorum sensing and chitin availability. In the following review, we highlight the repertoire of toxic T6SS effectors and the diverse genetic regulation networks among different isolates of V. cholerae. Finally, we discuss the roles played by the T6SS of V. cholerae in both natural environments and hosts.

Analysis of Vibrio cholerae genomes identifies new type VI secretion system gene clusters.

BACKGROUND: Like many bacteria, Vibrio cholerae deploys a harpoon-like type VI secretion system (T6SS) to compete against other microbes in environmental and host settings. The T6SS punctures adjacent cells and delivers toxic effector proteins that are harmless to bacteria carrying cognate immunity factors. Only four effector/immunity pairs encoded on one large and three auxiliary gene clusters have been characterized from largely clonal, patient-derived strains of V. cholerae. RESULTS: We sequence two dozen V. cholerae strain genomes from diverse sources and develop a novel and adaptable bioinformatics tool based on hidden Markov models. We identify two new T6SS auxiliary gene clusters and describe Aux 5 here. Four Aux 5 loci are present in the host strain, each with an atypical effector/immunity gene organization. Structural prediction of the putative effector indicates it is a lipase, which we name TleV1 (type VI lipase effector Vibrio). Ectopic TleV1 expression induces toxicity in Escherichia coli, which is rescued by co-expression of the TliV1a immunity factor. A clinical V. cholerae reference strain expressing the Aux 5 cluster uses TleV1 to lyse its parental strain upon contact via its T6SS but is unable to kill parental cells expressing the TliV1a immunity factor. CONCLUSION: We develop a novel bioinformatics method and identify new T6SS gene clusters in V. cholerae. We also show the TleV1 toxin is delivered in a T6SS manner by V. cholerae and can lyse other bacterial cells. Our web-based tool can be modified to identify additional novel T6SS genomic loci in diverse bacterial species.