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Background: Type VI secretion system (T6SS) is widely present in Gram-negative bacteria and directly mediates antagonistic prokaryote interactions. PAAR (proline-alanine-alanine-arginine repeats) proteins have been proven essential for T6SS-mediated secretion and target cell killing. Although PAAR proteins are commonly found in A. baumannii, their biological functions are not fully disclosed yet. In this study, we investigated the functions of a PAAR protein termed TagP (T6SS-associated-gene PAAR), encoded by the gene ACX60_RS09070 outside the core T6SS locus of A. baumannii strain ATCC 17978. Methods: In this study, tagP null and complement A. baumannii ATCC 17978 strains were constructed. The influence of TagP on T6SS function was investigated through Hcp detection and bacterial competition assay; the influence on environmental fitness was studied through in vitro growth, biofilm formation assay, surface motility assay, survivability in various simulated environmental conditions; the influence on pathogenicity was explored through cell adhesion and invasion assays, intramacrophage survival assay, serum survival assay, and G. melonella Killing assays. Quantitative transcriptomic and proteomic analyses were utilized to observe the global impact of TagP on bacterial status. Results: Compared with the wildtype strain, the tagP null mutant was impaired in several tested phenotypes such as surface motility, biofilm formation, tolerance to adverse environments, adherence to eukaryotic cells, endurance to serum complement killing, and virulence to Galleria melonella. Notably, although RNASeq and proteomics analysis revealed that many genes were significantly downregulated in the tagP null mutant compared to the wildtype strain, there is no significant difference in their antagonistic abilities. We also found that Histonelike nucleoid structuring protein (H-NS) was significantly upregulated in the tagP null mutant at both mRNA and protein levels. Conclusions: This study enriches our understanding of the biofunction of PAAR proteins in A. baumannii. The results indicates that TagP involved in a unique modulation of fitness and virulence control in A. baumannii, it is more than a classic PAAR protein involved in T6SS, while how TagP play roles in the fitness and virulence of A. baumannii needs further investigation to clarify. [ABSTRACT FROM AUTHOR]

BACKGROUND: Canonical biocontrol bacteria were considered to inhibit pathogenic bacteria mainly by secreting antibiotic metabolites or enzymes. Recent studies revealed that some biocontrol bacteria can inhibit pathogenic bacteria through contact‐dependent killing (CDK) mediated by contact‐dependent secretion systems. The CDK was independent of antibiotic metabolites and often ignored in normal biocontrol activity assay. RESULTS: In this study, we aimed to use a pathogen enrichment strategy to isolate non‐canonical bacteria with CDK ability. Rhizosphere soil samples from Chinese cabbage showing soft rot symptom were collected and Pectobacterium carotovorum subsp. carotovorum (Pcc), the pathogen of cabbage soft rot, were added into these samples to enrich bacteria which attached on Pcc cells. By co‐culture with Pcc, four bacteria strains (named as PcE1, PcE8, PcE12 and PcE13) showing antibacterial activity were isolated from Chinese cabbage rhizosphere. These four bacteria strains showed CDK abilities to different pathogenic bacteria of horticultural plants. Among them, PcE1 was identified as Chryseobacterium cucumeris. Genome sequencing showed that PcE1 genome encoded a type VI secretion system (T6SS) gene cluster. By heterologous expression, four predicted T6SS effectors of PcE1 showed antibacterial activity to Escherichia coli. CONCLUSION: Overall, this study isolated four bacteria strains with CDK activity to various horticultural plant pathogens, and revealed possible involvement of T6SS of Chryseobacterium cucumeris in antibacterial activity. These results provide valuable insight for potential application of CDK activity in biocontrol bacteria. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Abstract Background Acinetobacter baumannii (A. baumannii) is associated with both hospital-acquired infections (HAP) and community-acquired pneumonia (CAP). In this study, we present a novel CAP-associated A. baumannii (CAP-AB) strain causing severe pneumonia in an afore healthy male patient without underlying conditions. Subsequently, we investigated the pathogenicity and immunogenicity of this CAP-AB strain using a mice pneumonia model. Results A 58-year-old male patient with no underlying conditions experienced worsening symptoms of a productive cough, sputum, and fever that developed acutely, in just 24 h. The diagnosis was severe community-acquired pneumonia (CAP) and type-1 respiratory failure. An A. baumannii strain was isolated from his sputum and blood cultures. To gain a deeper understanding of the rapid progression of its pathology, we utilized the CAP-associated A. baumannii strain YC128, a previously obtained hospital-acquired pneumonia A. baumannii (HAP-AB) strain YC156, and a highly virulent A. baumannii control strain LAC-4 to construct a mouse pneumonia model, and subsequently compared the mortality rate of the three groups. Following inoculation with 107 CFU of A. baumannii, the mortality rate for the YC128, LAC-4, and YC156 groups was 60% (6/10), 30% (3/10), and 0%, respectively. The bacterial burden within the pulmonary, liver, and spleen tissues of mice in the YC128 group was significantly higher than that of the YC156 group, and slightly higher than that of the LAC-4 group. Pathological analysis of lung tissue using HE-staining revealed that the inflammatory pathological changes in mice from the YC128 group were significantly more severe than those in the YC156 group. Additionally, CT scan images displayed more pronounced inflammation in the lungs of mice from the YC128 group compared to the YC156 group. Local levels of cytokines/chemokines such as IL-1β, IL-6, TNF-α, and CXCL1 were assessed via RT-qPCR in lung tissues. In comparison with the YC156 strain, the highly virulent YC128 strain induced the expression of proinflammatory cytokines more rapidly and severely. Furthermore, we examined the in vitro anti-phagocytosis ability of YC128 and YC156 strains against mice peritoneal macrophages, revealing that the highly virulent YC128 isolate displayed greater resistance to macrophage uptake in contrast to YC156. Results from Whole Genome Sequencing (WGS) indicated that YC128 harbored a complete type VI secretion system (T6SS) gene cluster, while YC156 lacked the majority of genes within the T6SS gene cluster. The other virulence-related genes exhibited minimal differences between YC128 and YC156. Drawing from previous studies, we postulated that the T6SS is linked to the hypervirulence and robust anti-phagocytic ability of YC128. Conclusions This article reports on the isolation of a novel hypervirulent CAP-AB strain, YC128, from a severe CAP patient. The results demonstrate that this CAP-AB strain, YC128, is capable of inducing fatal pneumonia and extrapulmonary dissemination in a mouse pneumonia model. Moreover, this highly virulent CAP-AB strain exhibits significantly stronger anti-phagocytic abilities compared to the HAP-AB YC156 strain. Genome sequencing comparisons reveal that the heightened hypervirulence and enhanced anti-phagocytosis abilities observed in YC128 may be attributed to the presence of the T6SS.

【Objective】The objective of this study is to investigate the novel effector proteins and their functions in the type VI secretion system（T6SS）.【Method】The secretion mode and function of PA0423 were explored by gene knockout, immunoblotting, coimmunoprecipitation, intraspecific and interspecific competition, Escherichia coli toxicity assays and crystal violet biofilm assays.【Result】 Western blotting and co-immunoprecipitation experiments showed that PA0423 interacted with PA0262, and the secretion of PA0423 depended on H2-T6SS. PA0423 is a H2-T6SS-dependent antimicrobial effector protein with interspecific competitive advantages. PA0423 is toxic to Escherichia coli and PA0422 is its immune protein. PA0423 affected the formation of bacterial biofilms.【Conclusion】PA0423 is a H2-T6SS)dependent antimicrobial effector protein, which has bactericidal function and affects the formation of bacterial biofilms. [ABSTRACT FROM AUTHOR]

Background: Acinetobacter baumannii (A. baumannii) is associated with both hospital-acquired infections (HAP) and community-acquired pneumonia (CAP). In this study, we present a novel CAP-associated A. baumannii (CAP-AB) strain causing severe pneumonia in an afore healthy male patient without underlying conditions. Subsequently, we investigated the pathogenicity and immunogenicity of this CAP-AB strain using a mice pneumonia model. Results: A 58-year-old male patient with no underlying conditions experienced worsening symptoms of a productive cough, sputum, and fever that developed acutely, in just 24 h. The diagnosis was severe community-acquired pneumonia (CAP) and type-1 respiratory failure. An A. baumannii strain was isolated from his sputum and blood cultures. To gain a deeper understanding of the rapid progression of its pathology, we utilized the CAP-associated A. baumannii strain YC128, a previously obtained hospital-acquired pneumonia A. baumannii (HAP-AB) strain YC156, and a highly virulent A. baumannii control strain LAC-4 to construct a mouse pneumonia model, and subsequently compared the mortality rate of the three groups. Following inoculation with 107 CFU of A. baumannii, the mortality rate for the YC128, LAC-4, and YC156 groups was 60% (6/10), 30% (3/10), and 0%, respectively. The bacterial burden within the pulmonary, liver, and spleen tissues of mice in the YC128 group was significantly higher than that of the YC156 group, and slightly higher than that of the LAC-4 group. Pathological analysis of lung tissue using HE-staining revealed that the inflammatory pathological changes in mice from the YC128 group were significantly more severe than those in the YC156 group. Additionally, CT scan images displayed more pronounced inflammation in the lungs of mice from the YC128 group compared to the YC156 group. Local levels of cytokines/chemokines such as IL-1β, IL-6, TNF-α, and CXCL1 were assessed via RT-qPCR in lung tissues. In comparison with the YC156 strain, the highly virulent YC128 strain induced the expression of proinflammatory cytokines more rapidly and severely. Furthermore, we examined the in vitro anti-phagocytosis ability of YC128 and YC156 strains against mice peritoneal macrophages, revealing that the highly virulent YC128 isolate displayed greater resistance to macrophage uptake in contrast to YC156. Results from Whole Genome Sequencing (WGS) indicated that YC128 harbored a complete type VI secretion system (T6SS) gene cluster, while YC156 lacked the majority of genes within the T6SS gene cluster. The other virulence-related genes exhibited minimal differences between YC128 and YC156. Drawing from previous studies, we postulated that the T6SS is linked to the hypervirulence and robust anti-phagocytic ability of YC128. Conclusions: This article reports on the isolation of a novel hypervirulent CAP-AB strain, YC128, from a severe CAP patient. The results demonstrate that this CAP-AB strain, YC128, is capable of inducing fatal pneumonia and extrapulmonary dissemination in a mouse pneumonia model. Moreover, this highly virulent CAP-AB strain exhibits significantly stronger anti-phagocytic abilities compared to the HAP-AB YC156 strain. Genome sequencing comparisons reveal that the heightened hypervirulence and enhanced anti-phagocytosis abilities observed in YC128 may be attributed to the presence of the T6SS. [ABSTRACT FROM AUTHOR]

Abstract The type VI secretion system (T6SS) is a powerful bacterial molecular weapon that can inject effector proteins into prokaryotic or eukaryotic cells, thereby participating in the competition between bacteria and improving bacterial environmental adaptability. Although most current studies of the T6SS have focused on animal bacteria, this system is also significant for the adaptation of plant-associated bacteria. This paper briefly introduces the structure and biological functions of the T6SS. We summarize the role of plant-associated bacterial T6SS in adaptability to host plants and the external environment, including resistance to biotic stresses such as host defenses and competition from other bacteria. We review the role of the T6SS in response to abiotic factors such as acid stress, oxidation stress, and osmotic stress. This review provides an important reference for exploring the functions of the T6SS in plant-associated bacteria. In addition, characterizing these anti-stress functions of the T6SS may provide new pathways toward eliminating plant pathogens and controlling agricultural losses.

Abstract 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.

Francisella tularensis, a Tier 1 select agent of bioterrorism, contains a type VI secretion system (T6SS) encoded within the Francisella pathogenicity island (FPI), which is critical for its pathogenesis. Among the 18 proteins encoded by FPI is IglD, which is essential to Francisella's intracellular growth and virulence, but neither its location within T6SS nor its functional role has been established. Here, we present the cryoEM structure of IglD from Francisella novicida and show that the Francisella IglD forms a homotrimer that is structurally homologous to the T6SS baseplate protein TssK in Escherichia coli. Each IglD monomer consists of an N-terminal β-sandwich domain, a 4-helix bundle domain, and a flexible C-terminal domain. While the overall folds of IglD and TssK are similar, the two structures differ in three aspects: the relative orientation between their β-sandwich and the 4-helix bundle domains; two insertion loops present in TssK's β-sandwich domain; and, consequently, a lack of subunit-subunit interaction between insertion loops in the IglD trimer. Phylogenetic analysis indicates that IglD is genetically remote from the TssK orthologs in other T6SSs. While the other components of the Francisella baseplate are unknown, we conducted pulldown assays showing IglJ interacts with IglD and IglH, pointing to a model wherein IglD, IglH, and IglJ form the baseplate of the Francisella T6SS. Alanine substitution mutagenesis further established that IglD's hydrophobic pocket in the N-terminal β-sandwich domain interacts with two loops of IglJ, reminiscent of the TssK-TssG interaction. These results form a framework for understanding the hitherto unexplored Francisella T6SS baseplate. IMPORTANCE Francisella tularensis is a facultatively intracellular Gram-negative bacterium that causes the serious and potentially fatal zoonotic illness, tularemia. Because of its extraordinarily high infectivity and mortality to humans, especially when inhaled, F. tularensis is considered a potential bioterrorism agent and is classified as a Tier 1 select agent. The type VI secretion system (T6SS) encoded within the Francisella pathogenicity island (FPI) is critical to its pathogenesis, but its baseplate components are largely unknown. Here, we report the cryoEM structure of IglD from Francisella novicida and demonstrate its role as a component of the baseplate complex of the Francisella T6SS. We further show that IglD interacts with IglJ and IglH, and propose a model in which these proteins interact to form the Francisella T6SS baseplate. Elucidation of the structure and composition of the Francisella baseplate should facilitate the design of strategies to prevent and treat infections caused by F. tularensis.

The Type VI Secretion System (T6SS) is a widespread and highly effective mechanism of microbial warfare; it confers the ability to efficiently kill susceptible cells within close proximity. Due to its large physical size, complexity, and ballistic basis for intoxication, it has widely been assumed to incur significant growth costs in the absence of improved competitive outcomes. In this study, we precisely examine the fitness costs of constitutive T6SS firing in the bacterium Vibrio cholerae. We find that, contrary to expectations, constitutive expression of the T6SS has a negligible impact on growth, reducing growth fitness by 0.025 ± 0.5% (95% CI) relative to a T6SS− control. Mathematical modeling of microbial populations demonstrates that, due to clonal interference, constitutive expression of the T6SS will often be neutral, with little impact on evolutionary outcomes. Our findings underscore the importance of precisely measuring the fitness costs of microbial social behaviors and help explain the prevalence of the T6SS across Gram‐negative bacteria. [ABSTRACT FROM AUTHOR]

Bacterial-fungal interactions are pervasive in the rhizosphere. While an increasing number of endohyphal bacteria have been identified, little is known about their ecology and impact on the associated fungal hosts and the surrounding environment. In this study, we characterized the genome of an Enterobacter sp. Crenshaw (En-Cren), which was isolated from the generalist fungal pathogen Rhizoctonia solani, and examined the genetic potential of the bacterium with regard to the phenotypic traits associated with the fungus. Overall, the En-Cren genome size was typical for members of the genus and was capable of free-living growth. The genome was 4.6 MB in size, and no plasmids were detected. Several prophage regions and genomic islands were identified that harbor unique genes in comparison with phylogenetically closely related Enterobacter spp. Type VI secretion system and cyanate assimilation genes were identified from the bacterium, while some common heavy metal resistance genes were absent. En-Cren contains the key genes for indole-3-acetic acid (IAA) and phenylacetic acid (PAA) biosynthesis, and produces IAA and PAA in vitro, which may impact the ecology or pathogenicity of the fungal pathogen in vivo. En-Cren was observed to move along hyphae of R. solani and on other basidiomycetes and ascomycetes in culture. The bacterial flagellum is essential for hyphal movement, while other pathways and genes may also be involved. IMPORTANCE The genome characterization and comparative genomics analysis of Enterobacter sp. Crenshaw provided the foundation and resources for a better understanding of the ecology and evolution of this endohyphal bacteria in the rhizosphere. The ability to produce indole-3-acetic acid and phenylacetic acid may provide new angles to study the impact of phytohormones during the plant-pathogen interactions. The hitchhiking behavior of the bacterium on a diverse group of fungi, while inhibiting the growth of some others, revealed new areas of bacterial-fungal signaling and interaction, which have yet to be explored. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is a powerful bacterial molecular weapon that can inject effector proteins into prokaryotic or eukaryotic cells, thereby participating in the competition between bacteria and improving bacterial environmental adaptability. Although most current studies of the T6SS have focused on animal bacteria, this system is also significant for the adaptation of plant-associated bacteria. This paper briefly introduces the structure and biological functions of the T6SS. We summarize the role of plant-associated bacterial T6SS in adaptability to host plants and the external environment, including resistance to biotic stresses such as host defenses and competition from other bacteria. We review the role of the T6SS in response to abiotic factors such as acid stress, oxidation stress, and osmotic stress. This review provides an important reference for exploring the functions of the T6SS in plant-associated bacteria. In addition, characterizing these anti-stress functions of the T6SS may provide new pathways toward eliminating plant pathogens and controlling agricultural losses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Stenotrophomonas rhizophila CFBP13503 is a seedborne commensal bacterial strain, which is efficiently transmitted to seedlings and can outcompete the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc8004). The type VI secretion system (T6SS), an interference contact‐dependent mechanism, is a critical component of interbacterial competition. The involvement of the T6SS of S. rhizophila CFBP13503 in the inhibition of Xcc8004 growth and seed‐to‐seedling transmission was assessed. The T6SS cluster of S. rhizophila CFBP13503 and nine putative effectors were identified. Deletion of two T6SS structural genes, hcp and tssB, abolished the competitive advantage of S. rhizophila against Xcc8004 in vitro. The population sizes of these two bacterial species were monitored in seedlings after inoculation of radish seeds with mixtures of Xcc8004 and either S. rhizophila wild‐type (wt) strain or isogenic hcp mutant. A significant decrease in the population size of Xcc8004 was observed during confrontation with the S. rhizophila wt in comparison with T6SS‐deletion mutants in germinated seeds and seedlings. We found that the T6SS distribution among 835 genomes of the Stenotrophomonas genus is scarce. In contrast, in all available S. rhizophila genomes, T6SS clusters are widespread and mainly belong to the T6SS group i4. In conclusion, the T6SS of S. rhizophila CFBP13503 is involved in the antibiosis against Xcc8004 and reduces seedling transmission of Xcc8004 in radish. The distribution of this T6SS cluster in the S. rhizophila complex could make it possible to exploit these strains as biocontrol agents against X. campestris pv. campestris. [ABSTRACT FROM AUTHOR]

The Type VI Secretion System (T6SS) is used as weapon by a variety of Gram-negative bacteria in polymicrobial niche competition. Its characterization and study gained more interest in recent years. The system functions as a molecular nano-weapon: it is used in inter-kingdom competition by various bacteria to deliver toxic effectors in target cells. In this context, Photorhabdus luminescens subsp. luminescens strain DJC is a microorganism able to colonize different polymicrobial environments, like nematode guts, plant roots and larvae hemolymph. However, the mechanisms used by this microorganism to compete against other bacteria in the same environment have not been clearly described yet. We hypothesis that the T6SS of Photorhabdus luminescens subsp. luminescens strain DJC can play a role in same-niche environments. In this study we focused our attention on the characterization of the T6SS clusters in P. luminescens and its role in this bacteria lifestyle thought bioinformatic, proteomics analyses and inter-bacterial killing assays. Using bioinformatics analysis, we identified four T6SS gene clusters (T6SS-1, T6SS-2, T6SS-3 and T6SS-4) and multiple orphan T6SS related genes in the genome of P. luminescens. Furthermore, we highlighted 11 T6SS effector-immunity pairs, including three undescribed membrane disrupting effectors, each with putatively different antibacterial activities. By comparing the proteomes of P. luminescens wild type cells and the respective isogenic T6SS-deficient strains, we could point out a putative link between T6SS and other P. luminescens related defense mechanisms such as PVCs, T3SS and pyocins. Furthermore, T6SS-deficiency led to a change in phenotypic traits such as motility and secondary metabolism. Our findings shed light on the T6SS of P. luminescens DJC, suggesting a role of the system in the complex life cycle with a putative cross-link with various defense mechanisms and secondary metabolism. This study could help to gain more knowledge on bacterial T6SSs and better understand the P. luminescens ability to live in polymicrobial environments.

ABSTRACT: The type VI secretion system (T6SS) of avian pathogenic Escherichia coli (APEC) can affect the functions of eukaryotic cells by secreting or injecting effectors. Hemolysin co-regulatory protein (Hcp), one of the markers of the T6SS, is both a structural protein and an effector protein of the T6SS. According to previous studies, mitochondria in eukaryotic cells are targeted by pathogenic bacteria. However, little is known about the regulation of mitochondria in eukaryotic host cells by the T6SS effector protein Hcp of APEC. In our study, DF-1 cells co-incubated with Hcp2a protein for 6 h showed decreased mitochondrial membrane potential, increased Ca2+ concentration, and increased cellular reactive oxygen species (ROS) levels. We therefore conclude that Hcp2a protein causes dysfunction to mitochondria in DF-1 cells. To explain the mechanism that causes mitochondrial dysfunction, we reanalyzed the Hcp2a interaction protein dataset in DF-1 cells, and the Leucine zipper EF-hand-containing transmembrane protein 1 (LETM1), which is associated with mitochondria, was screened. The protein and molecular docking results showed that Hcp2a protein and LETM1 protein have better binding. Finally, subcellular localization results showed that Hcp2a was localized to mitochondria. In summary, Hcp2a effector proteins caused dysfunction to DF-1 cellular mitochondria, and we hypothesize that the interaction of Hcp2a protein with LETM1 protein induces mitochondrial dysfunction and promotes mitochondrial localization of Hcp2a in DF-1 cells.

TssJ-3 is an outer-membrane lipoprotein and is one of the key components of the type VI secretion system in Burkholderia pseudomallei. TssJ translocates effector proteins to target cells to induce innate immune response in the host. However, the tssJ gene has not been identified in B. pseudomallei and its function in this bacterium has not yet been characterized. tssJ-3 knockout and tssJ-3- complemented B. pseudomallei strains were constructed to determine the effects of tssJ-3 on bacterial growth, biofilm formation, flagellum synthesis, motility, host cell infection, and gene expression in B. pseudomallei. We found that the ΔtssJ-3 mutant strain of B. pseudomallei showed significantly suppressed biofilm formation, flagellum synthesis, bacterial growth, motility, and bacterial invasion into host cells (A549 cells). Furthermore, the ΔtssJ-3 mutation downregulated multiple key genes, including biofilm and flagellum-related genes in B. pseudomallei and induced interleukin-8 gene expression in host cells. These results suggest that tssJ-3 , an important gene controlling TssJ-3 protein expression, has regulatory effects on biofilm formation and flagellum synthesis in B. pseudomallei. In addition, B. pseudomallei- derived tssJ-3 contributes to cell infiltration and intracellular replication. This study provides a molecular basis of tssJ-3 for developing therapeutic strategies against B. pseudomallei infections. • We report functional studies of tssJ-3 gene from Burkholderia pseudomallei. • The tssJ-3 gene may influence bacterial quorum sensing and is crucial for biofilm formation. • The tssJ-3 gene has regulatory effects on biofilm formation and flagellum synthesis in Burkholderia pseudomallei. • Burkholderia pseudomallei -derived tssJ-3 contributes to cell infiltration and intracellular replication. • The gene expression of the biofilm-related genes, flagellar genes, and virulence genes were affected in the tssJ-3. [ABSTRACT FROM AUTHOR]

Central nervous system (CNS) melioidosis caused by Burkholderia pseudomallei is being increasingly reported. Because of the high mortality associated with CNS melioidosis, understanding the underlying mechanism of B. pseudomallei pathogenesis in the CNS needs to be intensively investigated to develop better therapeutic strategies against this deadly disease. The type VI secretion system (T6SS) is a multiprotein machine that uses a spring-like mechanism to inject effectors into target cells to benefit the infection process. In this study, the role of the T6SS accessory protein TagAB-5 in B. pseudomallei pathogenicity was examined using the human microglial cell line HCM3, a unique resident immune cell of the CNS acting as a primary mediator of inflammation. We constructed B. pseudomallei tagAB-5 mutant and complementary strains by the markerless allele replacement method. The effects of tagAB-5 deletion on the pathogenicity of B. pseudomallei were studied by bacterial infection assays of HCM3 cells. Compared with the wild type, the tagAB-5 mutant exhibited defective pathogenic abilities in intracellular replication, multinucleated giant cell formation, and induction of cell damage. Additionally, infection by the tagAB-5 mutant elicited a decreased production of interleukin 8 (IL-8) in HCM3, suggesting that efficient pathogenicity of B. pseudomallei is required for IL-8 production in microglia. However, no significant differences in virulence in the Galleria mellonella model were observed between the tagAB-5 mutant and the wild type. Taken together, this study indicated that microglia might be an important intracellular niche for B. pseudomallei, particularly in CNS infection, and TagAB-5 confers B. pseudomallei pathogenicity in these cells. [ABSTRACT FROM AUTHOR]

Bacteria from the genus Pseudomonas have been extensively studied for their capacity to act as biological control agents of disease and pests and for their ability to enhance and promote crop production in agricultural systems. While initial research primarily focused on the human pathogenic bacteria Pseudomonas aeruginosa, recent studies indicate the significance of type VI secretion (T6SS) in other Pseudomonas strains for biocontrol purposes. This system possibly plays a pivotal role in restricting the biological activity of target microorganisms and may also contribute to the bolstering of the survival capabilities of the bacteria within their applied environment. The type VI secretion system is a phage-like structure used to translocate effectors into both prokaryotic and eukaryotic target cells. T6SSs are involved in a myriad of interactions, some of which have direct implications in the success of Pseudomonas as biocontrol agents. The prevalence of T6SSs in the genomes of Pseudomonas species is notably greater than the estimated 25% occurrence rate found in Gram-negative bacteria. This observation implies that T6SS likely plays a pivotal role in the survival and fitness of Pseudomonas. This review provides a brief overview of T6SS, its role in Pseudomonas with biocontrol applications, and future avenues of research within this subject matter. [ABSTRACT FROM AUTHOR]

Background Klebsiella pneumoniae liver abscess (KPLA) with extrahepatic migratory infections is defined as invasive KPLA (IKPLA). The type VI secretion system (T6SS) is involved in the pathogenesis of KPLA. We hypothesized that T6SS plays a role in IKPLA. Methods 16S ribosomal RNA gene sequencing was performed on abscess samples. Polymerase chain reaction (PCR) and reverse-transcription PCR (RT-PCR) was used to validate the expression difference of T6SS hallmark genes. In vitro and in vivo experiments were performed to identify the pathogenic feature of T6SS. Results PICRUSt2 predicted that the T6SS-related genes were notably enriched in the IKPLA group. PCR detection of T6SS hallmark genes (hcp , vgrG , and icmF) showed that 197 (81.1%) were T6SS-positive strains. The T6SS-positive strain detection rate in the IKPLA group was higher than in the KPLA group (97.1% vs 78.4%; P <.05). RT-PCR showed that the hcp expression level was markedly increased in IKPLA isolates (P <.05). The T6SS-positive isolates showed higher survival against serum and neutrophil killing (all P <.05). The T6SS-positive K pneumoniae– infected mice had a shorter survival time, higher mortality, and an increased interleukin 6 expression in the liver and lungs (all P <.05). Conclusions T6SS is an essential virulence factor for K pneumoniae and contributes to IKPLA. [ABSTRACT FROM AUTHOR]

Background and Objectives: The type VI secretion system (T6SS) was identified as a novel virulence factor in many Gram-negative bacteria. This study aimed to investigate the frequency of the T6SS genes in Klebsiella pneumoniae-causing different nosocomial infections, and to study the association between T6SS, antibiotic resistance, and biofilm formation in the isolated bacteria. Materials and Methods: A total of fifty-six non-repetitive K. pneumoniae isolates were collected from different inpatients admitted at Sohag University Hospital from September 2022 to March 2023. Samples were cultured, colonies were identified, and antimicrobial sensitivity was done by VITEK® 2 Compact. Biofilm formation was checked using Congo red agar method. T6SS genes, and capsular serotypes were detected by PCR. Results: Fifty-six K. pneumoniae isolates were obtained in culture. 38 isolates (67.86%) produced biofilm and 44 (78.57%) were positive for T6SS in PCR. There was a significant association between the presence of T6SS and resistance to the following antibiotics: meropenem, ciprofloxacin, and levofloxacin. All biofilm-forming bacteria had T6SS, with significant differences towards T6SS -positive bacteria. There was no significant association between T6SS, and the presence of certain capsular types. Conclusion: The T6SS-positive K. pneumoniae has greater antibiotic resistance, and biofilm-forming ability which is considered a potential pathogenicity of this emerging gene cluster. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is used by bacteria for virulence, resistance to grazing, and competition with other bacteria. We previously demonstrated that the role of the T6SS in interbacterial competition and in resistance to grazing is enhanced in Vibrio cholerae in the presence of subinhibitory concentrations of polymyxin B. Here, we performed a global quantitative proteomic analysis and a targeted transcriptomic analysis of the T6SS-known regulators in V. cholerae grown with and without polymyxin B. The proteome of V. cholerae is greatly modified by polymyxin B with more than 39% of the identified cellular proteins displaying a difference in their abundance, including T6SS-related proteins. We identified a regulator whose abundance and expression are increased in the presence of polymyxin B, vxrB, the response regulator of the two-component system VxrAB (VCA0565-66). In vxrAB, vxrA and vxrB deficient mutants, the expression of both hcp copies (VC1415 and VCA0017), although globally reduced, was not modified by polymyxin B. These hcp genes encode an identical protein Hcp, which is the major component of the T6SS syringe. Thus, the upregulation of the T6SS in the presence of polymyxin B appears to be, at least in part, due to the two-component system VxrAB. [ABSTRACT FROM AUTHOR]

ABSTRACTThe 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.IMPORTANCEGram-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.

Abstract The Type VI Secretion System (T6SS) is a widespread and highly effective mechanism of microbial warfare; it confers the ability to efficiently kill susceptible cells within close proximity. Due to its large physical size, complexity, and ballistic basis for intoxication, it has widely been assumed to incur significant growth costs in the absence of improved competitive outcomes. In this study, we precisely examine the fitness costs of constitutive T6SS firing in the bacterium Vibrio cholerae. We find that, contrary to expectations, constitutive expression of the T6SS has a negligible impact on growth, reducing growth fitness by 0.025 ± 0.5% (95% CI) relative to a T6SS− control. Mathematical modeling of microbial populations demonstrates that, due to clonal interference, constitutive expression of the T6SS will often be neutral, with little impact on evolutionary outcomes. Our findings underscore the importance of precisely measuring the fitness costs of microbial social behaviors and help explain the prevalence of the T6SS across Gram‐negative bacteria.

A surge in the number of anthropogenic pollutants has been caused by increasing industrial activities. Nanoplastics are spotlighted as a new aquatic pollutant that are a threat to microbes and larger organisms. Our previous study showed that the subinhibitory concentrations of aquatic pollutants such as phenol and formalin act as signaling molecules and modulate global gene expression and metabolism. In this study, we aimed to investigate the impact of a new type of anthropogenic contaminant, polystyrene (PS) nanoplastics, on the expression of key virulence factors in zoonotic pathogen Edwardsiella piscicida and the assessment of potential changes in the susceptibility of zebrafish as a model host. The TEM data indicated a noticeable change in the cell membrane indicating that PS particles were possibly entering the bacterial cells. Transcriptome analyses performed to identify the differentially expressed genes upon PS exposure revealed that the genes involved in major virulence factor type VI secretion system (T6SS) were down-regulated. However, the expression of T6SS-related genes was recovered from the PS adapted E. piscicida when nanoplastics are free. This demonstrated the hypervirulence of pathogen in infection assays with both cell lines and in vivo zebrafish model. Therefore, this study provides experimental evidence elucidating the direct regulatory impact of nanoplastics influx into aquatic ecosystems on fish pathogenic bacteria, notably influencing the expression of virulence factors.

ABSTRACTThe formation of reactive oxygen species is harmful and can destroy intracellular macromolecules such as lipids, proteins, and DNA, even leading to bacterial death. To cope with this situation, microbes have evolved a variety of sophisticated mechanisms, including antioxidant enzymes, siderophores, and the type VI secretion system (T6SS). However, the mechanism of oxidative stress resistance in Cupriavidus pinatubonensis is unclear. In this study, we identified Reut_A2805 as an OxyR ortholog in C. pinatubonensis, which positively regulated the expression of T6SS1 by directly binding to its operon promoter region. The study revealed that OxyR-regulated T6SS1 combats oxidative stress by importing iron into bacterial cells. Moreover, the T6SS1-mediated outer membrane vesicles-dependent iron acquisition pathway played a crucial role in the oxidative stress resistance process. Finally, our study demonstrated that the T6SS1 and siderophore systems in C. pinatubonensis exhibit different responses in combating oxidative stress under low-iron conditions, providing a comprehensive understanding of how bacterial iron acquisition systems function in diverse conditions.IMPORTANCEThe ability to eliminate reactive oxygen species is crucial for bacterial survival. Continuous formation of hydroperoxides can damage metalloenzymes, disrupt DNA integrity, and even result in cell death. While various mechanisms have been identified in other bacterial species to combat oxidative stress, the specific mechanism of oxidative stress resistance in C. pinatubonensis remains unclear. The importance of this study is that we elucidate the mechanism that OxyR-regulated T6SS1 combats oxidative stress by importing iron with the help of bacterial outer membrane vesicle. Moreover, the study highlights the contrasting responses of T6SS1- and siderophore-mediated iron acquisition systems to oxidative stress. This study provides a comprehensive understanding of bacterial iron acquisition and its role in oxidative stress resistance in C. pinatubonensis under low-iron conditions.

ABSTRACTAntagonistic 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. Many pathogens deploy the T6SS to escape immune cells, eliminate commensal bacteria, and facilitate infection. Stenotrophomonas maltophilia complex is a Gram-negative opportunistic pathogen that primarily affects immunocompromised people 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 complex clinical and environmental strains possess T6SS genes. We demonstrate that the T6SS of an S. maltophilia complex patient isolate is active and can eliminate other bacteria. We provide evidence that the T6SS contributes to the competitive fitness of S. maltophilia complex against a co-infecting Pseudomonas aeruginosa isolate, and the T6SS alters the organization of S. maltophilia and P. aeruginosa co-cultures. This study expands our knowledge of the mechanisms employed by S. maltophilia complex to secrete antibacterial proteins and compete against other bacteria.IMPORTANCEInfections 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.

ABSTRACT Bacteria are constantly competing to colonize crowded ecological niches, such as the human gut. The type VI secretion system (T6SS) is a critical bacterial weapon in this warfare. It resembles a crossbow with a poisoned arrow allowing bacteria to inject toxic effectors directly into target cells. This machinery is formed by an envelope-spanning complex which recruits the baseplate, an assembly platform allowing the polymerization of a contractile structure. The tail consists of a tube surrounded by a sheath and topped by the needle complex composed of the VgrG and PAAR proteins. In the enteric pathogen enteroaggregative Escherichia coli (EAEC), the Tle1 phospholipase toxin ensures the antibacterial activity of the T6SS-1. For its transport, Tle1 interacts directly with the trimeric spike protein VgrG. However, the importance and the function of the tip protein PAAR in the T6SS remain unclear. Here, we characterized the PAAR protein of EAEC using biochemical, fluorescence microscopy, and antibacterial competition approaches. Using pull-down assays and cryo-electron microscopy analysis of the (VgrG)3-(Tle1)3-PAAR complex, we show that PAAR tops and closes the β-prism structure of the VgrG spike. The PAAR protein structure is further tightened by the zinc atom coordinated via conserved residues essential for its function. We provide evidence that PAAR is necessary for T6SS-1-mediated killing due to its requirement for proper T6SS baseplate assembly and further sheath polymerization. Our results suggest that the PAAR protein is an essential component of T6SS. IMPORTANCE The type VI secretion system (T6SS) is a bacterial contractile injection system involved in bacterial competition by the delivery of antibacterial toxins. The T6SS consists of an envelope-spanning complex that recruits the baseplate, allowing the polymerization of a contractile tail structure. The tail is a tube wrapped by a sheath and topped by the tip of the system, the VgrG spike/PAAR complex. Effectors loaded onto the puncturing tip or into the tube are propelled in the target cells upon sheath contraction. The PAAR protein tips and sharpens the VgrG spike. However, the importance and the function of this protein remain unclear. Here, we provide evidence for association of PAAR at the tip of the VgrG spike. We also found that the PAAR protein is a T6SS critical component required for baseplate and sheath assembly.

ABSTRACTAll strains of the marine bacterium Vibrio parahaemolyticus harbor a type VI secretion system (T6SS) named T6SS2, suggesting that this system plays an important role in the life cycle of this emerging pathogen. Although T6SS2 was recently shown to play a role in interbacterial competition, its effector repertoire remains unknown. Here, we employed proteomics to investigate the T6SS2 secretome of two V. parahaemolyticus strains, and we identified several antibacterial effectors encoded outside of the main T6SS2 gene cluster. We revealed two T6SS2-secreted proteins that are conserved in this species, indicating that they belong to the core secretome of T6SS2; other identified effectors are found only in subsets of strains, suggesting that they comprise an accessory effector arsenal of T6SS2. Remarkably, a conserved Rhs repeat-containing effector serves as a quality control checkpoint and is required for T6SS2 activity. Our results reveal effector repertoires of a conserved T6SS, including effectors that have no known activity and that have not been previously associated with T6SSs.

Background and Objectives: The type VI secretion system (T6SS) was identified as a novel virulence factor in many Gram-negative bacteria. This study aimed to investigate the frequency of the T6SS genes in Klebsiella pneumoniae-causing different nosocomial infections, and to study the association between T6SS, antibiotic resistance, and biofilm formation in the isolated bacteria. Materials and Methods: A total of fifty-six non-repetitive K. pneumoniae isolates were collected from different inpatients admitted at Sohag University Hospital from September 2022 to March 2023. Samples were cultured, colonies were identified, and antimicrobial sensitivity was done by VITEK® 2 Compact. Biofilm formation was checked using Congo red agar method. T6SS genes, and capsular serotypes were detected by PCR. Results: Fifty-six K. pneumoniae isolates were obtained in culture. 38 isolates (67.86%) produced biofilm and 44 (78.57%) were positive for T6SS in PCR. There was a significant association between the presence of T6SS and resistance to the following antibiotics: meropenem, ciprofloxacin, and levofloxacin. All biofilm-forming bacteria had T6SS, with significant differences towards T6SS -positive bacteria. There was no significant association between T6SS, and the presence of certain capsular types. Conclusion: The T6SS-positive K. pneumoniae has greater antibiotic resistance, and biofilm-forming ability which is considered a potential pathogenicity of this emerging gene cluster.

ABSTRACT Direct interspecies electron transfer (DIET) is important in anaerobic communities of environmental and practical significance. Other than the need for close physical contact for electrical connections, the interactions of DIET partners are poorly understood. Type VI secretion systems (T6SSs) typically kill competitive microbes. Surprisingly, Geobacter metallireducens highly expressed T6SS genes when DIET-based co-cultures were initiated with Geobacter sulfurreducens. T6SS gene expression was lower when the electron shuttle anthraquinone-2,6-disulfonate was added to alleviate the need for interspecies contact. Disruption of hcp, the G. metallireducens gene for the main T6SS needle-tube protein subunit, and the most highly upregulated gene in DIET-grown cells eliminated the long lag periods required for the initiation of DIET. The mutation did not aid DIET in the presence of granular-activated carbon (GAC), consistent with the fact that DIET partners do not make physical contact when electrically connected through conductive materials. The hcp-deficient mutant also established DIET quicker with Methanosarcina barkeri. However, the mutant also reduced Fe(III) oxide faster than the wild-type strain, a phenotype not expected from the loss of the T6SS. Quantitative PCR revealed greater gene transcript abundance for key components of extracellular electron transfer in the hcp-deficient mutant versus the wild-type strain, potentially accounting for the faster Fe(III) oxide reduction and impact on DIET. The results highlight that interspecies interactions beyond electrical connections may influence DIET effectiveness. The unexpected increase in the expression of genes for extracellular electron transport components when hcp was deleted emphasizes the complexities in evaluating the electromicrobiology of highly adaptable Geobacter species. IMPORTANCE Direct interspecies electron transfer is an alternative to the much more intensively studied process of interspecies H2 transfer as a mechanism for microbes to share electrons during the cooperative metabolism of energy sources. DIET is an important process in anaerobic soils and sediments generating methane, a significant greenhouse gas. Facilitating DIET can accelerate and stabilize the conversion of organic wastes to methane biofuel in anaerobic digesters. Therefore, a better understanding of the factors controlling how fast DIET partnerships are established is expected to lead to new strategies for promoting this bioenergy process. The finding that when co-cultured with G. sulfurreducens, G. metallireducens initially expressed a type VI secretion system, a behavior not conducive to interspecies cooperation, illustrates the complexity of establishing syntrophic relationships.

Soil salinity is one of the main problems that affects global crop yield. Researchers have attempted to alleviate the effects of salt stress on plant growth using a variety of approaches, including genetic modification of salt-tolerant plants, screening the higher salt-tolerant genotypes, and the inoculation of beneficial plant microbiome, such as plant growth-promoting bacteria (PGPB). PGPB mainly exists in the rhizosphere soil, plant tissues and on the surfaces of leaves or stems, and can promote plant growth and increase plant tolerance to abiotic stress. Many halophytes recruit salt-resistant microorganisms, and therefore endophytic bacteria isolated from halophytes can help enhance plant stress responses. Beneficial plant-microbe interactions are widespread in nature, and microbial communities provide an opportunity to understand these beneficial interactions. In this study, we provide a brief overview of the current state of plant microbiomes and give particular emphasis on its influence factors and discuss various mechanisms used by PGPB in alleviating salt stress for plants. Then, we also describe the relationship between bacterial Type VI secretion system and plant growth promotion. [ABSTRACT FROM AUTHOR]

Acidovorax citrulli, the causative agent of bacterial fruit blotch, can be divided into two main groups based on factors such as pathogenicity and host species preference. PilA is an important structural and functional component of type IV pili (T4P). Previous studies have found significant differences in pilA DNA sequences between group I and group II strains of A. citrulli. In this study, we characterized pilA in the group I strain pslb65 and the group II strain Aac5. pilA mutants, complementation strains, and cross-complementation strains were generated, and their biological phenotypes were analyzed to identify functional differences between pilA in the two groups. pilA deletion mutants (pslb65-ΔpilA and Aac5-ΔpilA) showed significantly reduced pathogenicity compared with the wild-type (WT) strains; pslb65-ΔpilA also completely lost twitching motility, whereas Aac5-ΔpilA only partially lost motility. In King's B medium, there were no significant differences in biofilm formation between pslb65-ΔpilA and WT pslb65, but Aac5-ΔpilA showed significantly reduced biofilm formation compared to WT Aac5. In M9 minimal medium, both mutants showed significantly lower biofilm formation compared to the corresponding WT strains, although biofilm formation was recovered in the complementation strains. The biofilm formation capacity was somewhat recovered in the cross-complementation strains but remained significantly lower than in the WT strains. The interspecies competitive abilities of pslb65-ΔpilA and Aac5-ΔpilA were significantly lower than in the WT strains; Aac5-ΔpilA was more strongly competitive than pslb65-ΔpilA, and the complementation strains recovered competitiveness to WT levels. Furthermore, the cross-complementation strains showed stronger competitive abilities than the corresponding WT strains. The relative expression levels of genes related to T4P and the type VI secretion system were then assessed in the pilA mutants via quantitative PCR. The results showed significant differences in the relative expression levels of multiple genes in pslb65-ΔpilA and Aac5-ΔpilA compared to the corresponding WT stains. This indicated the presence of specific differences in pilA function between the two A. citrulli groups, but the regulatory mechanisms involved require further study. [ABSTRACT FROM AUTHOR]

Abstract The type VI secretion system (T6SS) is a secretion apparatus widely found in pathogenic Gram-negative bacteria and is important for competition among various bacteria and host cell pathogenesis. Hcp is a core component of functional T6SS and transports toxic effectors into target cells by assembling to form tube-like structures. Studies have shown that Hcp simultaneously acts as an effector to influence cellular physiological activities; however, the mechanism of its activity in host cells remains unclear. To investigate the target of effector protein Hcp2a in a chicken fibroblast cell line, we first detected the subcellular localization of Hcp2a in DF-1 cells by indirect immunofluorescence assay. The results showed that Hcp2a protein was localized in the endoplasmic reticulum of DF-1 cells. We also used a streptavidin–biotin affinity pull-down assay combined with LC–MS/MS to screen DF-1 cell lysates for proteins that interact with Hcp2a and analyze the cellular functional pathways affected by them. The results showed that Hcp2a interacted with 52 DF-1 cellular proteins that are involved in multiple intracellular pathways. To further explore the mechanism of Hcp2a protein targeting the endoplasmic reticulum of DF-1 cells, we screened three endoplasmic reticulum-associated proteins (RSL1D1, RPS3A, and RPL23) from 52 prey proteins of Hcp2a for protein–protein molecular docking analysis. The docking analysis showed that the effector protein Hcp2a and the RPL23 protein had good complementarity. Overall, we propose that Hcp2a has strong binding activity to the RPL23 protein in DF-1 cells and this may help Hcp2a anchor to the endoplasmic reticulum in DF-1 cells.

Objective. The comparative analysis of the structure of the regulatory gene vasH of the type VI secretion system and its expression in toxigenic and non-toxigenic V. cholerae O1, biovar El Tor strains. Materials and methods. We used 35 strains isolated from patients and from the environmental samples in the territory of Russia and Ukraine between 1970 and 2017. Analysis of the structure of the vasH gene and the amino acid sequence of the protein was carried out using Ugene 1.32, Mega X, and Bioedit v. 7.0.9.0. The relative level of vasH expression was studied by 2Ct. Results. The The structure of the vasH gene and the amino acid sequence of VasH protein in toxigenic typical strains and genovariants of V. cholerae O1, El Tor biovar (genotype ctxA+tcpA+) have been shown to be identical to the reference V. cholerae n16961 O1, El Tor biovar strain. The vasH sequence is variable in isolates lacking ctxA and tcpA genes (ctxAtcpA), and does not differ from the reference in ctxAtcpA+ (with the exception of one strain). The studied toxigenic typical strains and the genovariants have a similar relative level of expression of the vasH gene. In isolates that do not contain the ctxA and tcpA genes, the expression of this gene is comparable to toxigenic strains, and is 3.1 times higher in ctxAtcpA+ strains than that of ctxAtcpA and 2.142.6 times higher than that of toxigenic ones. Conclusion. The analysis of toxigenic and non-toxigenic V. cholerae O1, biovar El Tor strains isolated in Russia and Ukraine in different periods of the current cholera pandemic confirmed the data of foreign researchers on vasH gene being intact in toxigenic isolates and variable in isolates lacking ctxA and tcpA genes. Meanwhile, the structure of vasH gene has been shown to be identical to that of toxigenic ones in 99% of the studied ctxAtcpA+ strains. The expression of the vasH gene has been detected in all studied strains, being the highest in ctxATtcpA+ strains. Only two non-toxigenic strains presumably synthesizing the functionally inactive VasH protein have been identified.

Abstract The type VI secretion system (T6SS) is a widespread protein secretion apparatus deployed by many Gram-negative bacterial species to interact with competitor bacteria, host organisms, and the environment. Yersinia pseudotuberculosis T6SS4 was recently reported to be involved in manganese acquisition; however, the underlying regulatory mechanism still remains unclear. In this study, we discovered that T6SS4 is regulated by ferric uptake regulator (Fur) in response to manganese ions (Mn2+), and this negative regulation of Fur was proceeded by specifically recognizing the promoter region of T6SS4 in Y. pseudotuberculosis. Furthermore, T6SS4 is induced by low Mn2+ and oxidative stress conditions via Fur, acting as a Mn2+-responsive transcriptional regulator to maintain intracellular manganese homeostasis, which plays important role in the transport of Mn2+ for survival under oxidative stress. Our results provide evidence that T6SS4 can enhance the oxidative stress resistance and virulence for Y. pseudotuberculosis. This study provides new insights into the regulation of T6SS4 via the Mn2+-dependent transcriptional regulator Fur, and expands our knowledge of the regulatory mechanisms and functions of T6SS from Y. pseudotuberculosis.

ABSTRACT Bacteroidales use type VI secretion systems (T6SS) to competitively colonize and persist in the colon. We identify a horizontally transferred T6SS with Ntox15 family nuclease effector (Tde1) that mediates interbacterial antagonism among Bacteroidales, including several derived from a single human donor. Expression of cognate (Tdi1) or orphan immunity proteins in acquired interbacterial defense systems protects against Tde1-dependent attack. We find that immunity protein interaction induces a large effector conformational change in Tde nucleases, disrupting the active site and altering the DNA-binding site. Crystallographic snapshots of isolated Tde1, the Tde1/Tdi1 complex, and homologs from Phocaeicola vulgatus (Tde2/Tdi2) illustrate a conserved mechanism of immunity inserting into the central core of Tde, splitting the nuclease fold into two subdomains. The Tde/Tdi interface and immunity mechanism are distinct from all other polymorphic toxin–immunity interactions of known structure. Bacteroidales abundance has been linked to inflammatory bowel disease activity in prior studies, and we demonstrate that Tde and T6SS structural genes are each enriched in fecal metagenomes from ulcerative colitis subjects. Genetically mobile Tde1-encoding T6SS in Bacteroidales mediate competitive growth and may be involved in inflammatory bowel disease. Broad immunity is conferred by Tdi1 homologs through a fold-disrupting mechanism unique among polymorphic effector–immunity pairs of known structure. IMPORTANCE Bacteroidales are related to inflammatory bowel disease severity and progression. We identify type VI secretion system (T6SS) nuclease effectors (Tde) which are enriched in ulcerative colitis and horizontally transferred on mobile genetic elements. Tde-encoding T6SSs mediate interbacterial competition. Orphan and cognate immunity proteins (Tdi) prevent intoxication by multiple Tde through a new mechanism among polymorphic toxin systems. Tdi inserts into the effector central core, splitting Ntox15 into two subdomains and disrupting the active site. This mechanism may allow for evolutionary diversification of the Tde/Tdi interface as observed in colicin nuclease–immunity interactions, promoting broad neutralization of Tde by orphan Tdi. Tde-dependent T6SS interbacterial antagonism may contribute to Bacteroidales diversity in the context of ulcerative colitis.

Type VI secretion systems (T6SSs) can deliver diverse toxic effectors into eukaryotic and bacterial cells. Although much is known about the regulation and assembly of T6SS, the translocation mechanism of effectors into the periplasm and/or cytoplasm of target cells remains elusive. Here, we use the Agrobacterium tumefaciens DNase effector Tde1 to unravel the mechanism of translocation from attacker to prey. We demonstrate that Tde1 binds to its adaptor Tap1 through the N‐terminus, which harbors continuous copies of GxxxG motifs resembling the glycine zipper structure found in proteins involved in the membrane channel formation. Amino acid substitutions on G39xxxG43 motif do not affect Tde1–Tap1 interaction and secretion but abolish its membrane permeability and translocation of its fluorescent fusion protein into prey cells. The data suggest that G39xxxG43 governs the delivery of Tde1 into target cells by permeabilizing the cytoplasmic membrane. Considering the widespread presence of GxxxG motifs in bacterial effectors and pore‐forming toxins, we propose that glycine zipper‐mediated permeabilization is a conserved mechanism used by bacterial effectors for translocation across target cell membranes. Synopsis: This study describes how the type VI secretion DNase effector Tde1 is translocated from the bacterial attacker into the competitor cell, demonstrating a new and maybe a conserved role of glycine zipper motif(s) in effector delivery. The N‐terminus of the Tde1 effector is necessary and sufficient for its loading onto the secretory machine and for secretion.A glycine zipper motif mediates cytoplasmic membrane permeabilization for target cell delivery.Glycine zipper‐mediated translocation may be a conserved membrane translocation mechanism for bacterial effectors. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus(VP) is a common gram-negative bacterium, mainly distributed in marine, estuary and estuarine sediments, which can cause acute gastroenteritis, wound infection and sepsis in humans, and endanger aquatic product aquaculture. Food poisoning incidents caused by this bacterium occur frequently all over the world, and this bacterium is considered to be one of the main pathogens of microbial food poisoning. The virulence factors that affect the pathogenicity of VP include hemolysin, adhesion factor, secretion system, etc., especially in the secretion systems, Type Ⅲ Secretion Systems (T3SS) and Type Ⅵ Secretion Systems (T6SS). It is closely related to the pathogenicity of VP. T3SS is a multi-subunit needle-like structure that injects secreted proteins directly into the recipient cytoplasm through the donor cytoplasm; T6SS is a contact-dependent protein secretion apparatus that injects toxins directly into target cells. By sorting out the research progress of T3SS and T6SS, starting from the structure of these two secretion systems, and analyzing and comparing their functions and regulatory mechanisms, it was pointed out that the two are involved in effector proteins and regulatory mechanisms. The difference in the production of toxins, the relationship between the two and the colonization of VP were found out, in order to further understand the pathogenic mechanism of VP through the connection between the two, and provide scientific guidance for the prevention and treatment of the bacteria. [ABSTRACT FROM AUTHOR]

Mesenteric adipose tissue (MAT) in Crohn's disease (CD) is associated with transmural inflammation. Extended mesenteric excision can reduce surgical recurrence and improve long‐term outcomes, indicating that MAT plays an important role in the pathogenesis of CD. Bacterial translocation has been reported to occur in the MAT of patients with CD (CD‐MAT), but the mechanisms by which translocated bacteria lead to intestinal colitis remain unclear. Here it is shown that members of Enterobacteriaceae are highly enriched in CD‐MAT compared with non‐CD controls. Viable Klebsiella variicola in Enterobacteriaceae is isolated exclusively in CD‐MAT and can induce a pro‐inflammatory response in vitro and exacerbates colitis both in dextran sulfate sodium (DSS)‐induced colitis mice model and IL‐10−/− spontaneous colitis mice model. Mechanistically, active type VI secretion system (T6SS) is identified in the genome of K. variicola, which can impair the intestinal barrier by inhibiting the zonula occludens (ZO‐1) expression. Dysfunction of T6SS by CRISPR interference system alleviates the inhibitory effect of K. variicola on ZO‐1 expression and attenuated colitis in mice. Overall, these findings demonstrate that a novel colitis‐promoting bacteria exist in the mesenteric adipose tissue of CD, opening a new therapeutic avenue for colitis management. [ABSTRACT FROM AUTHOR]

All bacteria must compete for growth niches and other limited environmental resources. These existential battles are waged at several levels, but one common strategy entails the transfer of growth-inhibitory protein toxins between competing cells. These antibacterial effectors are invariably encoded with immunity proteins that protect cells from intoxication by neighboring siblings. Several effector classes have been described, each designed to breach the cell envelope of target bacteria. Although effector architectures and export pathways tend to be clade specific, phylogenetically distant species often deploy closely related toxin domains. Thus, diverse competition systems are linked through a common reservoir of toxin-immunity pairs that is shared via horizontal gene transfer. These toxin-immunity protein pairs are extraordinarily diverse in sequence, and this polymorphism underpins an important mechanism of self/nonself discrimination in bacteria. This review focuses on the structures, functions, and delivery mechanisms of polymorphic toxin effectors that mediate bacterial competition.

Contact-dependent interbacterial competition is a common strategy used by bacteria to fight for their ecological niches. Interbacterial competition is monitored by a competition assay involving co-culturing the attacker and the recipient bacterial cells on agar, followed by recovery of the surviving recipient cells. Conventional interbacterial competition assays rely on serial dilution, plate spreading, and colony counting experiments for the readout. The high demand for time and labor in a competition assay limits its use for large-scale screening. However, a high-throughput interbacterial competition screening method is required to screen genetic factors involved in an interbacterial competition. Here, using Agrobacterium tumefaciens as an attacker and Escherichia coli as a recipient, we developed a robust, fast, efficient, and high-throughput type VI secretion system-dependent interbacterial competition screening platform. This system allows for 96 simultaneous competition assays without the need for serial dilution and plate spreading. Data analysis of this system relies on only direct and straightforward colony counting. This platform may be easily adapted to identify novel factors involved in any contact-dependent interbacterial competition systems.

The type VI secretion system (T6SS) is an effector delivery system used by Gram-negative bacteria to kill other bacteria or eukaryotic hosts to gain fitness. The plant pathogen Agrobacterium tumefaciens utilizes its T6SS to kill other bacteria, such as Escherichia coli. We observed that the A. tumefaciens T6SS-dependent killing outcome differs when using different T6SS-lacking, K-12 E. coli strains as a recipient cell. Thus, we hypothesized that the A. tumefaciens T6SS killing outcome not only relies on the T6SS activity of the attacker cells but also depends on the recipient cells. Here, we developed a high-throughput interbacterial competition platform to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. Among the 3,909 strains in the E. coli Keio library screened, 16 mutants with less susceptibility to A. tumefaciens C58 T6SS-dependent killing were identified, and four of them were validated by complementation test. Among the four, the clpP encoding ClpP protease, which is universal and highly conserved in both prokaryotes and eukaryotic organelles, was selected for further characterizations. We demonstrated that ClpP is responsible for enhancing susceptibility to the T6SS killing. Because ClpP protease depends on other adapter proteins such as ClpA and ClpX for substrate recognition, further mutant studies followed by complementation tests were carried out to reveal that ClpP-associated AAA+ ATPase ClpA, but not ClpX, is involved in enhancing susceptibility to A. tumefaciens T6SS killing. Moreover, functional and biochemical studies of various ClpP amino acid substitution variants provided evidence that ClpA-ClpP interaction is critical in enhancing susceptibility to the T6SS killing. This study highlights the importance of recipient factors in determining the outcome of the T6SS killing and shows the universal ClpP protease as a novel recipient factor hijacked by the T6SS of A. tumefaciens.

Abstract Background Bacteria deliver effector proteins into the host cell via a secretory system that can directly act on the target to cause disease. As an important pipeline structural protein of the type VI secretion system (T6SS) complex, Hcp acts together with other virulence factors in the target cell. There is growing evidence that T6SS plays a key role in the pathogenic mechanism of APEC. However, the regulatory function played by the effector protein Hcp during its interaction with host cells is not clear. Here, tandem mass tag (TMT) analysis was used to quantify the proteins affected by increased expression of Hcp2a in DF-1 cells. Results The host response was significantly different between the overexpression and null groups at the protein level. A total of 195 differentially expressed proteins (DEPs) were detected in the overexpression group (upregulated, n = 144, downregulated, n = 51). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the biological functions and pathways of differentially expressed proteins. The results showed that these DEPs were mainly enriched in RNA degradation, spliceosome, and mRNA surveillance pathways. Conclusions This study suggests that Hcp2a, the effector protein of APEC, plays an important role in regulating mRNA splicing and protein quality control in DF-1 cells. These findings provide useful clues to elucidate the pathogenic mechanism of effector protein Hcp2a on host target cells.

The bacterial Type VI secretion system (T6SS) is a dynamic macromolecular structure that promotes inter‐ and intra‐species competition through the delivery of toxic effector proteins into neighbouring cells. The T6SS contains 14 well‐characterised core proteins necessary for effector delivery (TssA‐M, PAAR). In this study, we have identified a novel accessory component required for optimal T6SS activity in the opportunistic pathogen Serratia marcescens, which we name TagV. Deletion of tagV, which encodes an outer membrane lipoprotein, caused a reduction in the T6SS‐dependent antibacterial activity of S. marcescens Db10. Mutants of S. marcescens lacking the core component TssJ, a distinct outer membrane lipoprotein previously considered essential for T6SS firing, retained a modest T6SS activity that could be abolished through deletion of tagV. TagV did not interact with the T6SS membrane complex proteins TssL or TssM, but is proposed to bind to peptidoglycan, indicating that the mechanism by which TagV promotes T6SS firing differs from that of TssJ. Homologues of tagV were identified in several other bacterial genera, suggesting that the accessory function of TagV is not restricted to S. marcescens. Together, our findings support the existence of a second, TssJ‐independent mechanism for T6SS firing that is dependent upon the activity of TagV proteins. [ABSTRACT FROM AUTHOR]

Type VI Secretion System (T6SS) plays significant roles in microbial activities via injecting effectors into adjacent cells or environments. T6SS increasingly gained attention due to its important influence on pathogenesis, microbial competition, etc. T6SS-associated research is explosively expanding on numerous grounds that call for an efficient resource. The SecReT6 version 3 provides comprehensive information on T6SS and the interactions between T6SS and T6SS-related proteins such as T6SS regulators and T6SS effectors. To assist T6SS researches like microbial competition and regulatory mechanisms, SecReT6 v3 developed online tools for detection and analysis of T6SS and T6SS-related proteins and estimation of T6SS-dependent killing risk. We have identified a novel T6SS regulator and T6SS-dependent killing capacity in Acinetobacter baumannii clinical isolates with the aid of SecReT6 v3. 17,212 T6SSs and plentiful T6SS-related proteins in 26,573 bacterial complete genomes were also detected, analyzed and incorporated into the database. The database is freely available at https://bioinfo-mml.sjtu.edu.cn/SecReT6/. [ABSTRACT FROM AUTHOR]

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Type VI secretion systems (T6SSs) are cell envelope‐spanning protein complexes that Gram‐negative bacteria use to inject a diverse arsenal of antibacterial toxins into competitor cells. Recently, Wang et al. reported that the H2‐T6SS of Pseudomonas aeruginosa delivers the peptidoglycan recycling amidase, AmpDh3, into the periplasm of recipient cells where it is proposed to act as a peptidoglycan degrading toxin. They further reported that PA0808, the open reading frame downstream of AmpDh3, encodes an immunity protein that localizes to the periplasm where it binds to and inactivates intercellularly delivered AmpDh3, thus protecting against its toxic activity. Given that AmpDh3 has an established role in cell wall homeostasis and that no precedent exists for cytosolic enzymes moonlighting as T6SS effectors, we attempted to replicate these findings. We found that cells lacking PA0808 are not susceptible to bacterial killing by AmpDh3 and that PA0808 and AmpDh3 do not physically interact in vitro or in vivo. Additionally, we found no evidence that AmpDh3 is exported from cells, including by strains with a constitutively active H2‐T6SS. Finally, subcellular fractionation experiments and a 1.97 Å crystal structure reveal that PA0808 does not contain a canonical signal peptide or localize to the correct cellular compartment to confer protection against a cell wall targeting toxin. Taken together, these results cast doubt on the assertion that AmpDh3‐PA0808 constitutes an H2‐T6SS effector–immunity pair. [ABSTRACT FROM AUTHOR]