Thesis but not secretion of the T6SS hallmark protein Hcp (Figure 6). Trans-complementation with vasH from N16961, which is more closely related to vasH from RGVC isolates DL2111 and DL2112 (Figure 5), restores Hcp synthesis and secretion in a vasH mutant of V52, but only restores Hcp synthesis (and not secretion) in a vasH mutant of N16961 [19]. Thus, we believe that the inability to restore Hcp secretion in rough strains is not a reflection of the polymorphic nature of VasH. At this 1655472 time, it is unclear whether selective pressures for T6SS regulation exist that drive constitutive T6SS expression in smooth isolates and disable T6SSs in rough V. cholerae strains. V. choleraeLPS’s O-antigen has been shown to induce protective immune responses in humans and experimental animals [31?5]. To counteract the host immune response, V. cholerae may use its T6SS to kill phagocytic immune cells such as macrophages [9]. Because rough isolates lacking O-antigen are frequently isolated from convalescent cholera patients [36], repression of O-antigen biosynthesis may represent an immune evasion mechanism for V. cholerae [37]. Such evasion would allow the pathogen to persist in the host, perhaps in a subclinical state as rough V. cholerae have been shown to be avirulent. In this scenario, rough V. cholerae does not require a functional T6SS, but tolerates mutations that disable its expression. Rough isolates have been shown to revert to a smooth, virulent state [37] but it remains to be determined whether newly reverted smooth bacteria restore expression of their disabled T6SSs. We did not observe restoration of the T6SS in rough isolates through uptake and homologous recombination of chromosomal DNA from a T6SS+ donor, because rough isolates remained T6SS-negative in the presence of smooth T6SS+ V. cholerae strain V52 (data not shown). El Tor strains possess a tightly controlled T6SS [17] and thus differ from the smooth RGVCs that express the T6SS constitutively. As pandemic strains are believed to originate from environmental strains, we speculate that constitutive T6SS expression is prevalent in V. cholerae exposed to microbial competitors and predators until virulence factors such as cholera toxin and toxin-coregulated pilus genes are acquired. However, how pandemic V. cholerae regulate expression of T6SS during their complex life cycle remains to be determined. It is becoming increasingly clear from our investigation and other reports [6,28?0,38] that T6SS-expressing V. cholerae deploy bactericidal effector proteins. Therefore, T6SS expression is likely tied to a protective mechanism, a form of T6SS-immunity that prevents the effector proteins from harming bacteria within a clonal population. We postulate that V52, DL4211, and DL4215 employ unique sets of toxin/antitoxin gene products and therefore form distinct compatibility groups. Members of a T6SS compatibility group could coexist because they encode antitoxins that match the cognate toxins. Conversely, members of different T6SS compatibility buy UKI 1 groups kill each other since the antitoxins of one compatibility group do not protect against the toxins of the other group. Hence, T6SS-mediated selective interstrain killing allows V. cholerae to distinguish self from nonself. This form of kin selection may permit the evolution of distinct lineages, including those that give rise to get ZK 36374 toxigenic strains. The observations presented in this study indicate that the T6SS contributes to V. cholerae’s pathogenesis.Thesis but not secretion of the T6SS hallmark protein Hcp (Figure 6). Trans-complementation with vasH from N16961, which is more closely related to vasH from RGVC isolates DL2111 and DL2112 (Figure 5), restores Hcp synthesis and secretion in a vasH mutant of V52, but only restores Hcp synthesis (and not secretion) in a vasH mutant of N16961 [19]. Thus, we believe that the inability to restore Hcp secretion in rough strains is not a reflection of the polymorphic nature of VasH. At this 1655472 time, it is unclear whether selective pressures for T6SS regulation exist that drive constitutive T6SS expression in smooth isolates and disable T6SSs in rough V. cholerae strains. V. choleraeLPS’s O-antigen has been shown to induce protective immune responses in humans and experimental animals [31?5]. To counteract the host immune response, V. cholerae may use its T6SS to kill phagocytic immune cells such as macrophages [9]. Because rough isolates lacking O-antigen are frequently isolated from convalescent cholera patients [36], repression of O-antigen biosynthesis may represent an immune evasion mechanism for V. cholerae [37]. Such evasion would allow the pathogen to persist in the host, perhaps in a subclinical state as rough V. cholerae have been shown to be avirulent. In this scenario, rough V. cholerae does not require a functional T6SS, but tolerates mutations that disable its expression. Rough isolates have been shown to revert to a smooth, virulent state [37] but it remains to be determined whether newly reverted smooth bacteria restore expression of their disabled T6SSs. We did not observe restoration of the T6SS in rough isolates through uptake and homologous recombination of chromosomal DNA from a T6SS+ donor, because rough isolates remained T6SS-negative in the presence of smooth T6SS+ V. cholerae strain V52 (data not shown). El Tor strains possess a tightly controlled T6SS [17] and thus differ from the smooth RGVCs that express the T6SS constitutively. As pandemic strains are believed to originate from environmental strains, we speculate that constitutive T6SS expression is prevalent in V. cholerae exposed to microbial competitors and predators until virulence factors such as cholera toxin and toxin-coregulated pilus genes are acquired. However, how pandemic V. cholerae regulate expression of T6SS during their complex life cycle remains to be determined. It is becoming increasingly clear from our investigation and other reports [6,28?0,38] that T6SS-expressing V. cholerae deploy bactericidal effector proteins. Therefore, T6SS expression is likely tied to a protective mechanism, a form of T6SS-immunity that prevents the effector proteins from harming bacteria within a clonal population. We postulate that V52, DL4211, and DL4215 employ unique sets of toxin/antitoxin gene products and therefore form distinct compatibility groups. Members of a T6SS compatibility group could coexist because they encode antitoxins that match the cognate toxins. Conversely, members of different T6SS compatibility groups kill each other since the antitoxins of one compatibility group do not protect against the toxins of the other group. Hence, T6SS-mediated selective interstrain killing allows V. cholerae to distinguish self from nonself. This form of kin selection may permit the evolution of distinct lineages, including those that give rise to toxigenic strains. The observations presented in this study indicate that the T6SS contributes to V. cholerae’s pathogenesis.
