[Regulation mechanism of the quorum sensing regulator AphA on the type â ¥ secretion system VflT6SS2 in Vibrio fluvialis].

Objective: To explore the regulation mechanism of the quorum sensing regulator AphA on the functional activity of type Ⅵ secretion system VflT6SS2 in Vibrio fluvialis. Methods: Western Blot analysis was used to detect the relative expression and secretion of VflT6SS2 signature component hemolysin-coregulated protein (Hcp) in wild type (WT), ΔaphA, and corresponding complementary strains. Quantitative reverse transcription PCR and luminescence activity assay of the promoter-lux fusion system was used to measure the mRNA expression levels and promoter activity of the VflT6SS2 core and accessory gene-cluster representative genes tssB2, hcp (tssD2) and vgrG (tssI2), and the quorum sensing regulator HapR in WT and ΔaphA strains. A point mutation experiment combined with a luminescence activity assay was used to verify the regulatory binding site of AphA in the tssD2b promoter region. Electrophoretic mobility shift assay (EMSA) was used to determine AphA binding to the hapR promoter. Results: The mRNA expression levels of tssB2, hcp(tssD2), vgrG (tssI2), and hapR as well as the protein expression and secretion levels of Hcp in ΔaphA strain, were significantly higher than those in the WT strain. The promoter activities of the VflT6SS2 core cluster, tssD2a, tssI2a, and hapR were higher in ΔaphA strain than in the WT strain, while the promoter activity of tssD2b showed the opposite trend. The promoter sequence analysis of tssD2a and tssD2b found significant differences in the region from -335 bp to -229 bp, and two potential AphA binding sites on tssD2b. The promoter activity of tssD2b decreased significantly after the point mutation of the two potential AphA binding sites. EMSA results showed that AphA binds directly to the promoter region of hapR. Conclusions: AphA indirectly inhibits the regulation of the VflT6SS2 core and accessory gene clusters at the promoter level by directly repressing the expression of hapR. AphA showed opposite regulation patterns for tssD2a and tssD2b, and AphA could positively regulate the expression of tssD2b by directly binding to the tssD2b promoter region (-335 bp to -229 bp).

Type 1 fimbriae-mediated collective protection against type 6 secretion system attacks.

Bacterial competition may rely on secretion systems such as the type 6 secretion system (T6SS), which punctures and releases toxic molecules into neighboring cells. To subsist, bacterial targets must counteract the threats posed by T6SS-positive competitors. In this study, we used a comprehensive genome-wide high-throughput screening approach to investigate the dynamics of interbacterial competition. Our primary goal was to identify deletion mutants within the well-characterized E. coli K-12 single-gene deletion library, the Keio collection, that demonstrated resistance to T6SS-mediated killing by the enteropathogenic bacterium Cronobacter malonaticus. We identified 49 potential mutants conferring resistance to T6SS and focused our interest on a deletion mutant (∆fimE) exhibiting enhanced expression of type 1 fimbriae. We demonstrated that the presence of type 1 fimbriae leads to the formation of microcolonies and thus protects against T6SS-mediated assaults. Collectively, our study demonstrated that adhesive structures such as type 1 fimbriae confer collective protective behavior against T6SS attacks.IMPORTANCEType 6 secretion systems (T6SS) are molecular weapons employed by gram-negative bacteria to eliminate neighboring microbes. T6SS plays a pivotal role as a virulence factor, enabling pathogenic gram-negative bacteria to compete with the established communities to colonize hosts and induce infections. Gaining a deeper understanding of bacterial interactions will allow the development of strategies to control the action of systems such as the T6SS that can manipulate bacterial communities. In this context, we demonstrate that bacteria targeted by T6SS attacks from the enteric pathogen Cronobacter malonaticus, which poses a significant threat to infants, can develop a collective protective mechanism centered on the production of type I fimbriae. These adhesive structures promote the aggregation of bacterial preys and the formation of microcolonies, which protect the cells from T6SS attacks.

Role of Type VI secretion system in pathogenic remodeling of host gut microbiota during Aeromonas veronii infection.

Intestinal microbial disturbance is a direct cause of host disease. The bacterial Type VI secretion system (T6SS) often plays a crucial role in the fitness of pathogenic bacteria by delivering toxic effectors into target cells. However, its impact on the gut microbiota and host pathogenesis is poorly understood. To address this question, we characterized a new T6SS in the pathogenic Aeromonas veronii C4. First, we validated the secretion function of the core machinery of A. veronii C4 T6SS. Second, we found that the pathogenesis and colonization of A. veronii C4 is largely dependent on its T6SS. The effector secretion activity of A. veronii C4 T6SS not only provides an advantage in competition among bacteria in vitro, but also contributes to occupation of an ecological niche in the nutritionally deficient and anaerobic environment of the host intestine. Metagenomic analysis showed that the T6SS directly inhibits or eliminates symbiotic strains from the intestine, resulting in dysregulated gut microbiome homeostasis. In addition, we identified three unknown effectors, Tse1, Tse2, and Tse3, in the T6SS, which contribute to T6SS-mediated bacterial competition and pathogenesis by impairing targeted cell integrity. Our findings highlight that T6SS can remodel the host gut microbiota by intricate interplay between T6SS-mediated bacterial competition and altered host immune responses, which synergistically promote pathogenesis of A. veronii C4. Therefore, this newly characterized T6SS could represent a general interaction mechanism between the host and pathogen, and may offer a potential therapeutic target for controlling bacterial pathogens.

EvfG is a multi-function protein located in the Type VI secretion system for ExPEC.

The Type VI secretion system (T6SS) functions as a protein transport nanoweapon in several stages of bacterial life. Even though bacterial competition is the primary function of T6SS, different bacteria exhibit significant variations. Particularly in Extraintestinal pathogenic Escherichia coli (ExPEC), research into T6SS remains relatively limited. This study identified the uncharacterized gene evfG within the T6SS cluster of ExPEC RS218. Through our experiments, we showed that evfG is involved in T6SS expression in ExPEC RS218. We also found evfG can modulate T6SS activity by competitively binding to c-di-GMP, leading to a reduction in the inhibitory effect. Furthermore, we found that evfG can recruit sodA to alleviate oxidative stress. The research shown evfG controls an array of traits, both directly and indirectly, through transcriptome and additional tests. These traits include cell adhesion, invasion, motility, drug resistance, and pathogenicity of microorganisms. Overall, we contend that evfG serves as a multi-functional regulator for the T6SS and several crucial activities. This forms the basis for the advancement of T6SS function research, as well as new opportunities for vaccine and medication development.

A combination of genomics and transcriptomics provides insights into the distribution and differential mRNA expression of type VI secretion system in clinical Klebsiella pneumoniae.

The type VI secretion system (T6SS) serves as a crucial molecular weapon in interbacterial competition and significantly influences the adaptability of bacteria in their ecological niche. However, the distribution and function of T6SS in clinical Klebsiella pneumoniae, a common opportunistic nosocomial pathogen, have not been fully elucidated. Here, we conducted a genomic analysis of 65 clinical K. pneumoniae isolates obtained from patients with varying infections. Genes encoding a T6SS cluster present in all analyzed strains of K. pneumoniae, and strains with identical sequence type carried structurally and numerically identical T6SS. Our study also highlights the importance of selecting conserved regions within essential T6SS genes for PCR-based identification of T6SS in bacteria. Afterward, we utilized the predominant sequence type 11 (ST11) K. pneumoniae HS11286 to investigate the effect of knocking out T6SS marker genes hcp or vgrG. Transcriptome analysis identified a total of 1,298 co-upregulated and 1,752 co-downregulated differentially expressed genes in both mutants. Pathway analysis showed that only Δhcp mutant exhibited alterations in transport, establishment of localization, localization, and cell processes. The absence of hcp or vgrG gene suppressed the expression of other T6SS-related genes within the locus I cluster. Additionally, interbacterial competition experiments showed that hcp and vgrG are essential for competitive ability of ST11 K. pneumoniae HS11286. This study furthers our understanding of the genomic characteristics of T6SS in clinical K. pneumoniae and suggests the involvement of multiple genes in T6SS of strain HS11286. IMPORTANCE: Gram-negative bacteria use type VI secretion system (T6SS) to deliver effectors that interact with neighboring cells for niche advantage. Klebsiella pneumoniae is an opportunistic nosocomial pathogen that often carries multiple T6SS loci, the function of which has not yet been elucidated. We performed a genomic analysis of 65 clinical K. pneumoniae strains isolated from various sources, confirming that all strains contained T6SS. We then used transcriptomics to further study changes in gene expression and its effect on interbacterial competition following the knockout of key T6SS genes in sequence type 11 (ST11) K. pneumoniae HS11286. Our findings revealed the distribution and genomic characteristics of T6SS in clinical K. pneumoniae. This study also described the overall transcriptional changes in the predominant Chinese ST11 strain HS11286 upon deletion of crucial T6SS genes. Additionally, this work provides a reference for future research on the identification of T6SS in bacteria.

The role of type VI secretion system genes in antibiotic resistance and virulence in Acinetobacter baumannii clinical isolates.

Introduction: The type VI secretion system (T6SS) is a crucial virulence factor in the nosocomial pathogen Acinetobacter baumannii. However, its association with drug resistance is less well known. Notably, the roles that different T6SS components play in the process of antimicrobial resistance, as well as in virulence, have not been systematically revealed. Methods: The importance of three representative T6SS core genes involved in the drug resistance and virulence of A. baumannii, namely, tssB, tssD (hcp), and tssM was elucidated. Results: A higher ratio of the three core genes was detected in drug-resistant strains than in susceptible strains among our 114 A. baumannii clinical isolates. Upon deletion of tssB in AB795639, increased antimicrobial resistance to cefuroxime and ceftriaxone was observed, alongside reduced resistance to gentamicin. The ΔtssD mutant showed decreased resistance to ciprofloxacin, norfloxacin, ofloxacin, tetracycline, and doxycycline, but increased resistance to tobramycin and streptomycin. The tssM-lacking mutant showed an increased sensitivity to ofloxacin, polymyxin B, and furazolidone. In addition, a significant reduction in biofilm formation was observed only with the ΔtssM mutant. Moreover, the ΔtssM strain, followed by the ΔtssD mutant, showed decreased survival in human serum, with attenuated competition with Escherichia coli and impaired lethality in Galleria mellonella. Discussion: The above results suggest that T6SS plays an important role, participating in the antibiotic resistance of A. baumannii, especially in terms of intrinsic resistance. Meanwhile, tssM and tssD contribute to bacterial virulence to a greater degree, with tssM being associated with greater importance.

A PilZ domain protein interacts with the transcriptional regulator HinK to regulate type VI secretion system in Pseudomonas aeruginosa.

Type VI secretion systems (T6SS) are bacterial macromolecular complexes that secrete effectors into target cells or the extracellular environment, leading to the demise of adjacent cells and providing a survival advantage. Although studies have shown that the T6SS in Pseudomonas aeruginosa is regulated by the Quorum Sensing system and second messenger c-di-GMP, the underlying molecular mechanism remains largely unknown. In this study, we discovered that the c-di-GMP-binding adaptor protein PA0012 has a repressive effect on the expression of the T6SS HSI-I genes in P. aeruginosa PAO1. To probe the mechanism by which PA0012 (renamed TssZ, Type Six Secretion System -associated PilZ protein) regulates the expression of HSI-I genes, we conducted yeast two-hybrid screening and identified HinK, a LasR-type transcriptional regulator, as the binding partner of TssZ. The protein-protein interaction between HinK and TssZ was confirmed through co-immunoprecipitation assays. Further analysis suggested that the HinK-TssZ interaction was weakened at high c-di-GMP concentrations, contrary to the current paradigm wherein c-di-GMP enhances the interaction between PilZ proteins and their partners. Electrophoretic mobility shift assays revealed that the non-c-di-GMP-binding mutant TssZR5A/R9A interacts directly with HinK and prevents it from binding to the promoter of the quorum-sensing regulator pqsR. The functional connection between TssZ and HinK is further supported by observations that TssZ and HinK impact the swarming motility, pyocyanin production, and T6SS-mediated bacterial killing activity of P. aeruginosa in a PqsR-dependent manner. Together, these results unveil a novel regulatory mechanism wherein TssZ functions as an inhibitor that interacts with HinK to control gene expression.

Trojan horselike T6SS effector TepC mediates both interference competition and exploitative competition.

The type VI secretion system (T6SS) is a bacterial weapon capable of delivering antibacterial effectors to kill competing cells for interference competition, as well as secreting metal ion scavenging effectors to acquire essential micronutrients for exploitation competition. However, no T6SS effectors that can mediate both interference competition and exploitation competition have been reported. In this study, we identified a unique T6SS-1 effector in Yersinia pseudotuberculosis named TepC, which plays versatile roles in microbial communities. First, secreted TepC acts as a proteinaceous siderophore that binds to iron and mediates exploitative competition. Additionally, we discovered that TepC has DNase activity, which gives it both contact-dependent and contact-independent interference competition abilities. In conditions where iron is limited, the iron-loaded TepC is taken up by target cells expressing the outer membrane receptor TdsR. For kin cells encoding the cognate immunity protein TipC, TepC facilitates iron acquisition, and its toxic effects are neutralized. On the other hand, nonkin cells lacking TipC are enticed to uptake TepC and are killed by its DNase activity. Therefore, we have uncovered a T6SS effector, TepC, that functions like a "Trojan horse" by binding to iron ions to provide a valuable resource to kin cells, whereas punishing cheaters that do not produce public goods. This lure-to-kill mechanism, mediated by a bifunctional T6SS effector, may offer new insights into the molecular mechanisms that maintain stability in microbial communities.

Assembly of a unique membrane complex in type VI secretion systems of Bacteroidota.

The type VI secretion system (T6SS) of Gram-negative bacteria inhibits competitor cells through contact-dependent translocation of toxic effector proteins. In Proteobacteria, the T6SS is anchored to the cell envelope through a megadalton-sized membrane complex (MC). However, the genomes of Bacteroidota with T6SSs appear to lack genes encoding homologs of canonical MC components. Here, we identify five genes in Bacteroides fragilis (tssNQOPR) that are essential for T6SS function and encode a Bacteroidota-specific MC. We purify this complex, reveal its dimensions using electron microscopy, and identify a protein-protein interaction network underlying the assembly of the MC including the stoichiometry of the five TssNQOPR components. Protein TssN mediates the connection between the Bacteroidota MC and the conserved baseplate. Although MC gene content and organization varies across the phylum Bacteroidota, no MC homologs are detected outside of T6SS loci, suggesting ancient co-option and functional convergence with the non-homologous MC of Pseudomonadota.

High genetic diversity and different type VI secretion systems in Enterobacter species revealed by comparative genomics analysis.

The human-pathogenic Enterobacter species are widely distributed in diverse environmental conditions, however, the understanding of the virulence factors and genetic variations within the genus is very limited. In this study, we performed comparative genomics analysis of 49 strains originated from diverse niches and belonged to eight Enterobacter species, in order to further understand the mechanism of adaption to the environment in Enterobacter. The results showed that they had an open pan-genome and high genomic diversity which allowed adaptation to distinctive ecological niches. We found the number of secretion systems was the highest among various virulence factors in these Enterobacter strains. Three types of T6SS gene clusters including T6SS-A, T6SS-B and T6SS-C were detected in most Enterobacter strains. T6SS-A and T6SS-B shared 13 specific core genes, but they had different gene structures, suggesting they probably have different biological functions. Notably, T6SS-C was restricted to E. cancerogenus. We detected a T6SS gene cluster, highly similar to T6SS-C (91.2%), in the remote related Citrobacter rodenitum, suggesting that this unique gene cluster was probably acquired by horizontal gene transfer. The genomes of Enterobacter strains possess high genetic diversity, limited number of conserved core genes, and multiple copies of T6SS gene clusters with differentiated structures, suggesting that the origins of T6SS were not by duplication instead by independent acquisition. These findings provide valuable information for better understanding of the functional features of Enterobacter species and their evolutionary relationships.

Transcriptome profiling of type VI secretion system core gene tssM mutant of Xanthomonas perforans highlights regulators controlling diverse functions ranging from virulence to metabolism.

IMPORTANCE: T6SS has received attention due to its significance in mediating interorganismal competition through contact-dependent release of effector molecules into prokaryotic and eukaryotic cells. Reverse-genetic studies have indicated the role of T6SS in virulence in a variety of plant pathogenic bacteria, including the one studied here, Xanthomonas. However, it is not clear whether such effect on virulence is merely due to a shift in the microbiome-mediated protection or if T6SS is involved in a complex virulence regulatory network. In this study, we conducted in vitro transcriptome profiling in minimal medium to decipher the signaling pathways regulated by tssM-i3* in X. perforans AL65. We show that TssM-i3* regulates the expression of a suite of genes associated with virulence and metabolism either directly or indirectly by altering the transcription of several regulators. These findings further expand our knowledge on the intricate molecular circuits regulated by T6SS in phytopathogenic bacteria.

Type VI secretion system drives bacterial diversity and functions in multispecies biofilms.

Type VI secretion system (T6SS) plays an essential role in interspecies interactions and provides an advantage for a strain with T6SS in multispecies biofilms. However, how T6SS drives the bacterial community structure and functions in multispecies biofilms still needs to be determined. Using gene deletion and Illumina sequencing technique, we estimated bacterial community responses in multispecies biofilms to T6SS by introducing T6SS-containing Pseudomonas putida KT2440. Results showed that the niche structure shifts of multispecies biofilms were remarkably higher in the presence of T6SS than in the absence of T6SS. The presence of T6SS significantly drove the variation in microbial composition, reduced the alpha-diversity of bacterial communities in multispecies biofilms, and separately decreased and increased the relative abundance of Proteobacteria and Bacteroidota. Co-occurrence network analysis with inferred putative bacterial interactions indicated that P. putida KT2440 mainly displayed strong negative associations with the genera of Psychrobacter, Cellvibrio, Stenotrophomonas, and Brevundimonas. Moreover, the function redundancy index of the bacterial community was strikingly higher in the presence of T6SS than in the absence of T6SS, regardless of whether relative abundances of bacterial taxa were inhibited or promoted. Remarkably, the increased metabolic network similarity with T6SS-containing P. putida KT2440 could enhance the antibacterial activity of P. putida KT2440 on other bacterial taxa. Our findings extend knowledge of microbial adaptation strategies to potential bacterial weapons and could contribute to predicting biodiversity loss and change in ecological functions caused by T6SS.

Distribution and diversity of type VI secretion system clusters in Enterobacter bugandensis and Enterobacter cloacae.

Gram-negative bacteria use type VI secretion systems (T6SSs) to antagonize neighbouring cells. Although primarily involved in bacterial competition, the T6SS is also implicated in pathogenesis, biofilm formation and ion scavenging. Enterobacter species belong to the ESKAPE pathogens, and while their antibiotic resistance has been well studied, less is known about their pathogenesis. Here, we investigated the distribution and diversity of T6SS components in isolates of two clinically relevant Enterobacter species, E. cloacae and E. bugandensis. T6SS clusters are grouped into four types (T6SSi-T6SSiv), of which type i can be further divided into six subtypes (i1, i2, i3, i4a, i4b, i5). Analysis of a curated dataset of 31 strains demonstrated that most of them encode T6SS clusters belonging to the T6SSi type. All T6SS-positive strains possessed a conserved i3 cluster, and many harboured one or two additional i2 clusters. These clusters were less conserved, and some strains displayed evidence of deletion. We focused on a pathogenic E. bugandensis clinical isolate for comprehensive in silico effector prediction, with comparative analyses across the 31 isolates. Several new effector candidates were identified, including an evolved VgrG with a metallopeptidase domain and a Tse6-like protein. Additional effectors included an anti-eukaryotic catalase (KatN), M23 peptidase, PAAR and VgrG proteins. Our findings highlight the diversity of Enterobacter T6SSs and reveal new putative effectors that may be important for the interaction of these species with neighbouring cells and their environment.

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.

Characterization of Type VI secretion system in Edwardsiella ictaluri.

Edwardsiella ictaluri is a Gram-negative facultative intracellular fish pathogen causing enteric septicemia of catfish (ESC). While various secretion systems contribute to E. ictaluri virulence, the Type VI secretion system (T6SS) remains poorly understood. In this study, we constructed 13 E. ictaluri T6SS mutants using splicing by overlap extension PCR and characterized them, assessing their uptake and survival in channel catfish (Ictalurus punctatus) peritoneal macrophages, attachment and invasion in channel catfish ovary (CCO) cells, in vitro stress resistance, and virulence and efficacy in channel catfish. Among the mutants, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO exhibited reduced replication inside peritoneal macrophages. EiΔevpM, EiΔevpN, and EiΔevpO showed significantly decreased attachment to CCO cells, while EiΔevpN and EiΔevpO also displayed reduced invasion of CCO cells (p < 0.05). Overall, T6SS mutants demonstrated enhanced resistance to oxidative and nitrosative stress in the nutrient-rich medium compared to the minimal medium. However, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO were susceptible to oxidative stress in both nutrient-rich and minimal medium. In fish challenges, EiΔevpD, EiΔevpE, EiΔevpG, EiΔevpJ, and EiΔevpK exhibited attenuation and provided effective protection against E. ictaluri wild-type (EiWT) infection in catfish fingerlings. However, their attenuation and protective efficacy were lower in catfish fry. These findings shed light on the role of the T6SS in E. ictaluri pathogenesis, highlighting its significance in intracellular survival, host cell attachment and invasion, stress resistance, and virulence. The attenuated T6SS mutants hold promise as potential candidates for protective immunization strategies in catfish fingerlings.

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.

Genome wide analysis revealed conserved domains involved in the effector discrimination of bacterial type VI secretion system.

Type VI secretion systems (T6SSs) deliver effectors into target cells. Besides structural and effector proteins, many other proteins, such as adaptors, co-effectors and accessory proteins, are involved in this process. MIX domains can assist in the delivery of T6SS effectors when encoded as a stand-alone gene or fused at the N-terminal of the effector. However, whether there are other conserved domains exhibiting similar encoding forms to MIX in T6SS remains obscure. Here, we scanned publicly available bacterial genomes and established a database which include 130,825 T6SS vgrG loci from 45,041 bacterial genomes. Based on this database, we revealed six domain families encoded within vgrG loci, which are either fused at the C-terminus of VgrG/N-terminus of T6SS toxin or encoded by an independent gene. Among them, DUF2345 was further validated and shown to be indispensable for the T6SS effector delivery and LysM was confirmed to assist the interaction between VgrG and the corresponding effector. Together, our results implied that these widely distributed domain families with similar genetic configurations may be required for the T6SS effector recruitment process.

An Orphan VrgG Auxiliary Module Related to the Type VI Secretion Systems from Pseudomonas ogarae F113 Mediates Bacterial Killing.

The model rhizobacterium Pseudomonas ogarae F113, a relevant plant growth-promoting bacterium, encodes three different Type VI secretion systems (T6SS) in its genome. In silico analysis of its genome revealed the presence of a genetic auxiliary module containing a gene encoding an orphan VgrG protein (VgrG5a) that is not genetically linked to any T6SS structural cluster, but is associated with genes encoding putative T6SS-related proteins: a possible adaptor Tap protein, followed by a putative effector, Tfe8, and its putative cognate immunity protein, Tfi8. The bioinformatic analysis of the VgrG5a auxiliary module has revealed that this cluster is only present in several subgroups of the P. fluorescens complex of species. An analysis of the mutants affecting the vgrG5a and tfe8 genes has shown that the module is involved in bacterial killing. To test whether Tfe8/Tfi8 constitute an effector-immunity pair, the genes encoding Tfe8 and Tfi8 were cloned and expressed in E. coli, showing that the ectopic expression of tfe8 affected growth. The growth defect was suppressed by tfi8 ectopic expression. These results indicate that Tfe8 is a bacterial killing effector, while Tfi8 is its cognate immunity protein. The Tfe8 protein sequence presents homology to the proteins of the MATE family involved in drug extrusion. The Tfe8 effector is a membrane protein with 10 to 12 transmembrane domains that could destabilize the membranes of target cells by the formation of pores, revealing the importance of these effectors for bacterial interaction. Tfe8 represents a novel type of a T6SS effector present in pseudomonads.