The type VI secretion system of Xanthomonas phaseoli pv. manihotis is involved in virulence and in vitro motility.

BACKGROUND: The type VI protein secretion system (T6SS) is important in diverse cellular processes in Gram-negative bacteria, including interactions with other bacteria and with eukaryotic hosts. In this study we analyze the evolution of the T6SS in the genus Xanthomonas and evaluate its importance of the T6SS for virulence and in vitro motility in Xanthomonas phaseoli pv. manihotis (Xpm), the causal agent of bacterial blight in cassava (Manihot esculenta). We delineate the organization of the T6SS gene clusters in Xanthomonas and then characterize proteins of this secretion system in Xpm strain CIO151. RESULTS: We describe the presence of three different clusters in the genus Xanthomonas that vary in their organization and degree of synteny between species. Using a gene knockout strategy, we also found that vgrG and hcp are required for maximal aggressiveness of Xpm on cassava plants while clpV is important for both motility and maximal aggressiveness. CONCLUSION: We characterized the T6SS in 15 different strains in Xanthomonas and our phylogenetic analyses suggest that the T6SS might have been acquired by a very ancient event of horizontal gene transfer and maintained through evolution, hinting at their importance for the adaptation of Xanthomonas to their hosts. Finally, we demonstrated that the T6SS of Xpm is functional, and significantly contributes to virulence and motility. This is the first experimental study that demonstrates the role of the T6SS in the Xpm-cassava interaction and the T6SS organization in the genus Xanthomonas.

The Ferric Uptake Regulator Represses Type VI Secretion System Function by Binding Directly to the clpV Promoter in Salmonella enterica Serovar Typhimurium.

Type VI secretion systems (T6SSs) are highly conserved and complex protein secretion systems that deliver effector proteins into eukaryotic hosts or other bacteria. T6SSs are regulated precisely by a variety of regulatory systems, which enables bacteria to adapt to varied environments. A T6SS within Salmonella pathogenicity island 6 (SPI-6) is activated during infection, and it contributes to the pathogenesis, as well as interbacterial competition, of Salmonella enterica serovar Typhimurium (S. Typhimurium). However, the regulation of the SPI-6 T6SS in S. Typhimurium is not well understood. In this study, we found that the SPI-6 T6SS core gene clpV was significantly upregulated in response to the iron-depleted condition and during infection. The global ferric uptake regulator (Fur) was shown to repress the clpV expression in the iron-replete medium. Moreover, electrophoretic mobility shift and DNase I footprinting assays revealed that Fur binds directly to the clpV promoter region at multiple sites spanning the transcriptional start site. We also observed that the relieving of Fur-mediated repression on clpV contributed to the interbacterial competition activity and pathogenicity of S. Typhimurium. These findings provide insights into the direct regulation of Fur in the expression and functional activity of SPI-6 T6SS in S. Typhimurium and thus help to elucidate the mechanisms of bacterial adaptability and virulence.

Environmental and cellular factors affecting the localization of T6SS proteins in Burkholderia thailandensis.

The type VI secretion system (T6SS) injects effector proteins into neighboring bacteria and host cells. Effector translocation is driven by contraction of a tubular sheath in the cytoplasm that expels an inner needle across the cell envelope. The AAA + ATPase ClpV disassembles and recycles the contracted sheath. While ClpV-1-GFP of the Burkholderia T6SS-1, which targets prokaryotic cells, assembles into randomly localized foci, ClpV-5-GFP of the virulence-associated T6SS-5 displays a polar distribution. The mechanisms underlying the localization of T6SSs to a particular site in the bacterial cell are currently unknown. We recently showed that ClpV-5-GFP retains its polar localization in the absence of all T6SS-5 components during infection of host cells. Herein, we set out to identify factors involved in the distribution of ClpV-5 and ClpV-1 in Burkholderia thailandensis. We show that focal assembly and polar localization of ClpV-5-GFP is not dependent on the intracellular host cell environment, known to contain the signal to induce T6SS-5 gene expression. In contrast to ClpV-5-GFP, localization of ClpV-1-GFP was dependent on the cognate T6SS. Foci formation of both ClpV5-GFP and ClpV-1-GFP was decreased by D cycloserine-mediated inhibition of peptidoglycan synthesis while treatment of B. thailandensis with A22 blocking the cytoskeletal protein MreB did not affect assembly of ClpV-5 and ClpV-1 into single discrete foci. Furthermore, we found that surface contact promotes but is not essential for localization of ClpV-5-GFP to the pole whereas expression of clpV-1-gfp appears to be induced by surface contact. In summary, the study provides novel insights into the localization of ClpV ATPases of T6SSs targeting prokaryotic and eukaryotic cells.

Mechanistic insight into the roles of Pseudomonas plecoglossicida clpV gene in host-pathogen interactions with Larimichthys crocea by dual RNA-seq.

Large yellow croaker (Larimichthys crocea) is an economical important farmed fish in China. "Visceral White Spot Disease" caused by Pseudomonas plecoglossicida is a disease with a high mortality rate in cage-cultured L. crocea in recent years and resulted in heavy economy lossess. The dual RNA-seq results of previous study showed that the expression of clpV gene in P. plecoglossicida was significantly up-regulated during infection. RNAi significantly reduced the expression of clpV in P. plecoglossicida with maximum silencing efficiency of 96.1%. Compared with the wild type strain, infection of clpV-RNAi strain resulted in a delayed onset time and a 25% reduction in mortality of L. crocea, as well as lessening the symptoms of the spleen. The results of dual RNA-seq of L. crocea infected by clpV-RNAi strain of P. plecoglossicida changed considerably, compared with the counterpart infected with the wild strain. The KEGG enrichment analysis showed that Cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway and MAPK signaling pathway of L. crocea were most affected by the silence of clpV in P. plecoglossicida. RNAi of clpV resulted in the downregulation of genes in flagella assembly pathway and a weaker immune response of host.

clpV is a key virulence gene during in vivo Pseudomonas plecoglossicida infection.

Interaction between bacterial pathogen and aquatic animal host is exceedingly complex, which involves large dynamic changes in gene expression during different stages of the disease. However, research on identifying key virulence genes based on the dynamics of gene expression changes of a one-sided bacterial pathogen in tissue has not been reported so far across different stages of infectious disease. The clpV for the T6SS of Pseudomonas plecoglossicida was identified for a candidate for key virulence gene based on dynamic changes of gene expression. For the Epinephelus coioides infected using clpV-RNAi strain, no deaths were observed up to 20 dpi. The spleens, kidneys and livers of all the E. coioides that received clpV-RNAi strain failed to develop visible nodules at 5-8 dpi, with the swelling gradually disappearing. The burdens of clpV-RNAi strain in the spleen and blood were greatly reduced at most of the time points after injection, and the burdens of clpV-RNAi strain in the head kidneys and trunk kidneys also had a sharp reduction from 72 to 120 hpi. This paper provides a new insight into the discovery of key virulence genes of pathogens in infected tissue systems.

Coevolution of the ATPase ClpV, the sheath proteins TssB and TssC, and the accessory protein TagJ/HsiE1 distinguishes type VI secretion classes.

The type VI secretion system (T6SS) is a bacterial nanomachine for the transport of effector molecules into prokaryotic and eukaryotic cells. It involves the assembly of a tubular structure composed of TssB and TssC that is similar to the tail sheath of bacteriophages. The sheath contracts to provide the energy needed for effector delivery. The AAA(+) ATPase ClpV disassembles the contracted sheath, which resets the systems for reassembly of an extended sheath that is ready to fire again. This mechanism is crucial for T6SS function. In Vibrio cholerae, ClpV binds the N terminus of TssC within a hydrophobic groove. In this study, we resolved the crystal structure of the N-terminal domain of Pseudomonas aeruginosa ClpV1 and observed structural alterations in the hydrophobic groove. The modification in the ClpV1 groove is matched by a change in the N terminus of TssC, suggesting the existence of distinct T6SS classes. An accessory T6SS component, TagJ/HsiE, exists predominantly in one of the classes. Using bacterial two-hybrid approaches, we showed that the P. aeruginosa homolog HsiE1 interacts strongly with ClpV1. We then resolved the crystal structure of HsiE1 in complex with the N terminus of HsiB1, a TssB homolog and component of the contractile sheath. Phylogenetic analysis confirmed that these differences distinguish T6SS classes that resulted from a functional co-evolution between TssB, TssC, TagJ/HsiE, and ClpV. The interaction of TagJ/HsiE with the sheath as well as with ClpV suggests an alternative mode of disassembly in which HsiE recruits the ATPase to the sheath.

Colonization Mediated by T6SS-ClpV Disrupts Host Gut Microbiota and Enhances Virulence of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common foodborne enteric pathogen that infects humans or mammals and colonizes the intestinal tract primarily by invading the host following ingestion. Meanwhile, ClpV is a core secreted protein of the bacterial type VI secretion system (T6SS). Because elucidating ClpV's role in the pathogenesis of T6SS is pivotal for revealing the virulence mechanism of Salmonella, in our study, clpV gene deletion mutants were constructed using a λ-red-based recombination system, and the effect of clpV mutation on SL1344's pathogenicity was examined in terms of stress resistance, motility, cytokine secretion, gut microbiota, and a BALB/c mouse model. Among the results, ClpV affected SL1344's motility and was also involved in cell invasion, adhesion, and intracellular survival in the MDBK cell model but did not affect invasion or intracellular survival in the RAW264.7 cell model. Moreover, clpV gene deletion significantly reduced the transcription levels of GBP2b, IFNB1, IL-6, NLRP3, NOS2, and TNF-α proinflammatory factor levels but significantly increased transcription levels of IL-4 and IL-10 anti-inflammatory factors. Last, ClpV appeared to closely relate to the pathogenicity of S. Typhimurium in vivo, which can change the gut environment and cause dysbiosis of gut microbiota. Our findings elucidate the functions of ClpV in S. Typhimurium and illustrating interactions between T6SS and gut microbiota help to clarify the mechanisms of the pathogenesis of foodborne diseases.

ClpV1 in avian pathogenic Escherichia coli is a crucial virulence factor contributing to meningitis in a mouse model in vivo.

Meningitis associated with avian pathogenic Escherichia coli (APEC) is an infectious disease of poultry that has gained significant attention because of its potential to infect humans. APEC can utilize two type Ⅵ secretion systems (T6SSs) to efficiently transport toxin effectors into hosts. ClpV1 is one of the core components of the T6SS1. To our knowledge, it has not been clarified how the clpV1 gene contributes to the pathogenicity of meningitis-associated APEC. To investigate the function of the clpV1 gene in the process of Escherichia coli meningitis, a mutant TW-XMΔclpV1 strain was constructed and characterized. In this study, the clpV1 deleted strain displayed a significant decrease in both motility and biofilm formation as well as a reduction in the expression of virulence genes fliC, luxS and ibeA. In vivo studies using mouse and duck models found that the clpV1 deleted groups showed decreased proliferation, fewer lesions and lower expression of inflammatory cytokines in the brain suggesting that clpV1 is involved in the pathogenicity of TWXM. Besides, the decreased quantity of Evans Blue (EB) and the down-regulation of tight junctions (TJs) proteins in the mouse clpV1 deleted group demonstrating a more intact blood-brain barrier (BBB). In conclusion, these results suggest that the clpV1 gene is associated with motility and biofilm formation of TWXM strain and contributes to meningitis by damaging the BBB and brain tissues.

Two Type VI Secretion Systems of Enterobacter cloacae Are Required for Bacterial Competition, Cell Adherence, and Intestinal Colonization.

Enterobacter cloacae has emerged as an opportunistic pathogen in healthcare-associated infections. Analysis of the genomic sequences of several E. cloacae strains revealed the presence of genes that code for expression of at least one type VI secretion system (T6SS). Here, we report that E. cloacae strain ATCC 13047 codes for two functional T6SS named T6SS-1 and T6SS-2. T6SS-1 and T6SS-2 were preferentially expressed in tryptic soy broth and tissue culture medium (DMEM), respectively. Mutants in T6SS-1-associated genes clpV1 and hcp1 significantly affected their ability of inter- and intra-bacterial killing indicating that T6SS-1 is required for bacterial competition. In addition, the Hcp effector protein was detected in supernatants of E. cloacae cultures and a functional T6SS-1 was required for the secretion of this protein. A clpV2 mutant was impaired in both biofilm formation and adherence to epithelial cells, supporting the notion that these phenotypes are T6SS-2 dependent. In vivo data strongly suggest that both T6SSs are required for intestinal colonization because single and double mutants in clpV1 and clpV2 genes were defective in gut colonization in mice. We conclude that the two T6SSs are involved in the pathogenesis scheme of E. cloacae with specialized functions in the interaction with other bacteria and with host cells.

ClpV3 of the H3-Type VI Secretion System (H3-T6SS) Affects Multiple Virulence Factors in Pseudomonas aeruginosa.

The type VI secretion system (T6SS) is a toxic effector delivery apparatus widely distributed in Gram-negative bacteria. The opportunistic pathogen Pseudomonas aeruginosa encodes three T6SSs, namely H1-, H2-, and H3-T6SS. Each T6SS possesses its own effectors and their roles are not yet fully understood. Here, we report that an H3-T6SS deletion mutant PAO1(ΔclpV3) significantly affected the virulence-related phenotypes including pyocyanin production, biofilm formation, proteolytic activity, and motilities. Most interestingly, the expression of T3SS genes was markedly affected, indicating a link between H3-T6SS and T3SS. RNA-Sequencing was performed to globally identify the genes differentially expressed when H3-T6SS was inactivated and the results obtained correlated well with the observed phenotypes. Interestingly, the expressions of T2SS, T3SS, H2-T6SS, and H3-T6SS were all significantly decreased, while H1-T6SS was increased in the PAO1(ΔclpV3) strain. We also observed that the intracellular concentration of secondary messenger cAMP was reduced in PAO1(ΔclpV3), and the c-di-GMP level was also decreased as indicated by the decreased cdrA reporter activity. Finally, by using a Galleria mellonella infection model, we show that H3-T6SS plays a key role in the pathogenicity of P. aeruginosa in vivo. Overall, our study highlights the unique connection of H3-T6SS in P. aeruginosa with T3SS, pyocyanin production, biofilm formation and in vivo pathogenicity.

Type VI secretion system of Pseudomonas aeruginosa is associated with biofilm formation but not environmental adaptation.

Pseudomonas aeruginosa encodes three type VI secretion systems (T6SSs), namely H1-, H2-, and H3-T6SS. P. aeruginosa hemolysin-coregulated protein (Hcp) is the effector protein and the hallmark of T6SS. Although T6SS is ubiquitous and affects ecology and human health, its general mechanism and physiological role are still not fully understood. Therefore, in this study, we investigated the impact of the P. aeruginosa T6SS on biofilm formation and environmental adaptation. To this end, we collected P. aeruginosa clinical isolates, divided them into strong biofilm formation (SBF) and nonbiofilm formation (NBF) groups based on their biofilm-forming ability, and compared their associated clinical characteristics. The duration of hospitalization was longer in patients infected with SBF than those infected with NBF strains. The expression levels of T6SS-related genes (hcp1 and hcp3) and a quorum-sensing gene (lasR) were higher in the SBF group as compared to those in the NBF group. In addition, the expression level of lasR was negatively associated with that of hcp1, but was positively associated with those of hcp2 and hcp3. Moreover, we evaluated the expression of T6SS- and biofilm-associated genes in planktonic and biofilm cells of the P. aeruginosa strain PAO1, and constructed strain PAO1△clpV1 to study the adaptation characteristics of H1-T6SS. The expression levels of hcp1, hcp2, hcp3, lasR, and other biofilm-associated genes were significantly higher in PAO1 biofilm cells as compared to those of planktonic cells. However, except for swarming ability as a vital feature for biofilm formation, there were no significant differences in the biofilm-forming ability and expression of biofilm-associated genes, adherence ability, growth characteristics, resistance to acid and osmotic pressure, surface structure, and morphology between the PAO1△clpV1 and PAO1 wild-type strains. Collectively, our results suggest that T6SS might play a role in biofilm formation and that H1-T6SS does not contribute to environmental adaptation in P. aeruginosa.

Polar localization of the ATPase ClpV-5 occurs independent of type VI secretion system apparatus proteins in Burkholderia thailandensis.

OBJECTIVE: ClpV, the ATPase of the type VI secretion system (T6SS) recycles cytoplasmic T6SS proteins following effector translocation. Fluorescent protein fusions to ClpV showed that it localizes to discrete and dynamic foci. ClpV-1-sfGFP of the bacterial cell targeting T6SS-1 of Burkholderia thailandensis exhibits a virtually random localization, whereas ClpV-5-sfGFP of the T6SS-5 targeting host cells is located at one or both poles. The mechanisms underlying the differential localization pattern are not known. Previous analysis of T6SSs, which target bacterial cells revealed that ClpV foci formation is dependent on components of the T6SS. Here, we investigated if the T6SS-5 apparatus confers polar localization of ClpV-5. RESULTS: ClpV-5-sfGFP foci formation and localization was examined in a B. thailandensis mutant harboring a deletion of the entire T6SS-5 gene cluster. We found that ClpV-5-sfGFP localization to discrete foci was not abolished in the absence of the T6SS-5 apparatus. Furthermore, the number of ClpV-5-sfGFP foci displaying a polar localization was not significantly different from that of ClpV-5-sfGFP expressed in the wild type genetic background. These findings suggest the presence of a T6SS-independent localization mechanism for ClpV-5 of the T6SS-5 targeting host cells.