| Edwardsiella tarda, a rod-shaped Gram-negative bacterium, is responsible for the systemic hemorrhagic septicemia in a variety of freshwater and marine fish species, and even human beings. During the infection to fish, E. tarda colonizes in various organs in both extracellular and intracellular locations. Fishes infected by E. tarda display extensive skin lesions and swelling, necrosis in internal organs, finally developing into mass-mortality outbreaks of edwardsiellosis. The recently completed genome sequencing of E. tarda unravels the genetic basis for habitat adaptation, invasive nature, and virulence determinants of the bacterium. In this study, the pathogenic features of E. tarda EIB202 were analyzed extensively within both animal and cell models and 3 attractive targets for live attenuated vaccine candidate were preliminarily identified to be associated with bacterial two-component system (TCS) and secretion system (TTSS and T6SS). Furthermore, the pathogenic regulations of TCS EsrA-EsrB on T6SS and hemolysin as well as the regulation of TCS QseB-QseC on the adaptation and intracellular survival capabilities mediated by bacterial surface structure were dissected.E. tarda EIB202 isolated from turbot (Scophthamus maximus (L.)) was identified to also be infective to zebra fish (Danio rerio) and Japanese flounder (Paralichthys olivaceus). Moreover, EIB202 was revealed to be capable of invading into EPC cells, resisting the phagocytosis and surviving in murine macrophage J774a. Since TTSS and T6SS as well as TCS were revealed to result in bacterial invasion and intracellular survival, regulation network was believed to contribute the pathogenesis of E. tarda.Based on the genome sequence information and primary knowledge of E. tarda pathogenesis,10 genes associated to virulence, metabolism, regulation, secretion system, stress response and quorum sensing were deleted one by one with homology recombinant technology, and mutantsΔaroC,ΔesrB,ΔtorR,ΔkatB, WED,ΔmgtB,ΔethA,ΔsodC,ΔclpB andΔedwR were constructed. Under the challenge of virulent E. tarda, the relative percentage survival rates ofΔaroC,ΔesrB and WED strains in zebra fish reached 68%,80% and 81%, respectively, indicating that these candidates had great value in serving as an attenuated live vaccine.T6SS has been implicated in numerous apparently disparate pathogenic processes. In this study, sequencing and genetic alignments showed that evpP genes from different E. tarda isolates were highly similar and an evpP homolog was also found in Aeromonas hydrophila 0865 isolated from a diseased eel, suggesting the possible lateral gene transfer of evpP. With reporter strains carrying gfp gene fused to the evpP promoter region, flow cytometric analysis revealed that transcription of evpP was regulated by either the two-component system EsrA-EsrB or the iron concentration in medium. Investigations of infection into Japanese flounder and invasion into EPC cells showed that the evpP deletion mutant was unable to proliferate in vivo and to internalize in EPC cell model in vitro, demonstrating that EvpP in T6SS, regulated by EsrB and Fur, plays critical roles for invasion mechanism of E. tarda.Bacteria possess signal transduction systems for adaptation to external stimuli. In this study, a markedly attenuated AesrB mutant was investigated to exhibit enhanced cell invasion capability, as well as the increased cytotoxicity of its ECPs. Compared with the parental strain, the AesrB mutant unexpectedly displayed the significantly increased hemolytic activity, and the restoration of hemolysin production was observed in the complemented strain esrB+. A hemolysis-associated 147 kDa protein, EthA, was found to be up-regulated in the ECPs of AesrB. These results indicated that the increased production of EthA was responsible for the enhanced cell-invasion related capabilities in AesrB. Furthermore, the expression of EthA in AesrB exhibited a temperature-induced manner, and a nucleoid protein Hha was identified to mediate ethA expression by direct binding to its promoter.The bacterial inter-kingdom communication with the mammalian hosts mediated by AI-3/Epi/NE has recently been described. Here, the qseB and qseC genes of E. tarda inter-kingdom system were found to be co-transcribed and the evolution of QseC strongly correlated to different host niches. Compared with the wild-type and complemented strains, AqseB and AqseC mutants exhibited significant impaired flagellar motilities and Epi was able to stimulate the flagellar motility in E. tarda via QseBC. Hemagglutination caused by fimbriae was induced in AqseB but repressed in AqseC. Moreover, disruption of qseB or qseC down-regulated the intracellular expressions of TTSS elements and impaired their intracellular survival capabilities as well as in vivo competitive abilities. Furthermore, in vitro tests indicated that expression of EseB was induced by Epi via QseBC. Our results revealed that the QseBC system modified the virulence related surface structures and hormone might stimulate the virulence of the pathogen in fish. |
PDF Full Text Request