Transcriptomic analysis of host-pathogen relationship in Vibrio vulnificus

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Vibrio vulnificus is a worldwide distributed pathogen traditionally isolated from temperate aquatic ecosystems, whose geographical distribution is currently spreading due to global warming. The species is genetically variable and only the strains that belong to the zoonotic clonal-complex (serovar E within pathovar piscis [formerly biotype 2]) are able to cause disease, with multiple clinical manifestations collectively known as vibriosis, in humans and fishes. The most severe form of vibriosis is hemorrhagic septicemia that, in case of eels it affects healthy animals, while in case of humans mainly affects susceptible patients (those with high levels of free iron in blood [iron-overloaded humans] due to different underlying pathologies). In both cases, septicemia is a very rapid disease that leads to death by sepsis in less than 48 h. The main aim of this thesis is to gain insights into the pathogen’s virulence mechanisms that allow it to cause septicemia in hosts as evolutionary distant as human and fish. To this end, we have selected a representative strain of the zoonotic clonal-complex and applied a combination of transcriptomic and single gene approaches using in vitro and ex vivo models of septicemia to understand how iron and temperature (either outside [water] or inside its host) influence V. vulnificus virulence. First, we have analyzed how Fur (Ferric uptake regulator) mediated the global bacterial response to iron (both iron excess and iron starvation conditions). As a result, we have described the iron stimulon and Fur regulon of V. vulnificus and demonstrated that iron, not always throw Fur, controls the entire life cycle of this pathogen, from its survival in the marine environment (including motility and chemotaxis) to its survival in the blood of their hosts (including host-specific mechanisms of resistance to innate immunity). Second, we have described a host-adapted virulent phenotype that V. vulnificus expresses in the blood of its main susceptible hosts (iron-overloaded humans and healthy eels) by changing metabolism preferences from aerobic to anaerobic (based on amino or glycogen compounds utilization in eels or human blood, respectively) and combining a hostspecific protective envelope (capsule enriched for humans and O-antigen enriched for eels) with the common expression of two toxins (VvhA and RtxA1). Moreover, the zoonotic strains have bypassed the iron requirement of the species for fish infection due to the acquisition of two iron-regulated outer membrane proteins (Ftbp and Fpcrp) involved in resistance to fish innate immunity: Ftbp conferring ability to specifically bind to fish transferrin in order to acquire iron from it; and Fpcrp conferring resistance to fish complement and phagocytosis. Finally, temperature contributes to the host-adapted virulent phenotype, since an increase in temperature (from environmental [around 20ºC] to infective [28ºC for fish and 37ºC for humans] temperatures) entails bacterium fitness which leads to an optimal physiological state that adapts the pathogen for the subsequent host invasion and survival in blood. This, in combination with exogenous iron sources, increases the expression of virulence factors in a host-specific manner. Given that the outcome of an infection depends not only on the virulent mechanisms expressed by the pathogen but also on the host ability to modulate a suitable immune response to eliminate the pathogen, we have also performed ex vivo and in vivo experiments in order to gain insights into the eel immune response against V. vulnificus as well as to understand the host-pathogen relationship. Analyzing the immune transcriptome expressed in eel blood during vibriosis we have proved that eel RBC (red blood cells), as nucleated cells, are transcriptionally active and involved in the immune response against bacterial pathogens not only as antigen recognition, processing and presentation cells but also with ability to produce effectors (i.e., TNF, cytokines, lectins and prostaglandins). Finally, we have also analyzed the immune transcriptome in eel blood during vibriosis caused by a V. vulnificus mutant strain lacking RtxA1 (a strain that cause septicemia but do not kill the animals) and found strong evidence for an early intracellular and atypical immune response induced by RtxA1. This atypical response is mainly carried out by RBC and is based on a rapid cytokine/retrotransposon storm, mediated by a systemic RNAi and an anti-silencing protein, which by unknown mechanisms could lead to the inactivation of miRNA-142a resulting in an exacerbated immune response which eventually causes eel death. In conclusion, V. vulnificus has the ability to express an iron-dependent and host-adapted virulent phenotype based on of a generalist but host-dependent protective envelope plus the common overexpression of RtxA1 and VvhA. In the case of humans, the envelope is enriched in the capsule, while in eels it contains Fpcrp and Ftbp that together confrom a “survival in fish blood kit” which confers specific ability to resist fish innate immunity. Moreover, host-pathogen interaction studies in V. vulnificus and its main host, the eel, have opened the door for new hypothesis and future projects, such as the study of new putative bacterial virulence factors (i.e., T6SS) and the development of new immunotherapeutic tools (i.e., miR-142a) to prevent V. vulnificus outbreaks in fish farms, the major reservoir of this pathogen.
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