Vibrio vulnificusfrom water to host

Resumen

Vibrio vulnificus is an aquatic pathogen autochthonous from temperate, tropical and subtropical ecosystems where it lives either as a sessile cell, forming biofilms on biotic/abiotic surfaces, or as a free-swimming cell. From these locations, the pathogen can occasionally infect humans and fish (mainly farmed-eels) causing a disease named vibriosis. The most severe form of human and fish vibriosis is associated with the pathogen’s ability to spread from the infection site to the bloodstream and multiply, process known as invasion. Before invasion, the pathogen has to colonize the mucosal host surface, process that involves not only bacterial attachment/adhesion but also resistance to mucosal immunity, commensal microbiota (competitors) and bacterial predators (mainly amoeba and phages). Recently, Amaro and cols. obtained evidence that supports that mucin, the main protein in mucus, can activate horizontal gene transference (HGT) in V. vulnificus, which could lead to the emergence of new virulent clones in natural mucosal environments. The objective of this thesis was to study the colonization and invasion processes under the global perspective that allow the “omic” technologies. In the first chapter, we focused our attention on a selected host for the pathogen in the aquatic environment, the eel, and analyzed its microbiome by using metagenomics. We describe for the first time the microbiome of the skin mucus of wild- and farmed-eels and compared it with that of the water. We discovered that mucus concentrates, selectively, bacteria present in water (mainly vibrios if present) and identified the genes involved in a successful colonization process, most of which could be considered virulence genes. Then, we developed a protocol to identify MGE and prophages in the metagenomes and described a series of putative ICEs, pathogenicity islands and prophages some of which contained virulence and antibiotic resistance genes. Finally, we were able to describe multiple lytic phages in skin-mucus, which could be considered as a part of the mucosal immunity. The second chapter of this thesis is focused on the invasion, and, in particular, in the ability of this pathogen to grow in blood. The objective was to discover all the pathogen’s genes involved in growth in human blood by applying the “omic” approach known as transposon insertion sequencing (TIS). TIS is a powerful method that couples high-density transposon mutagenesis with next-generation sequencing to comprehensively assess the fitness of thousands of transposon mutants across a genome. TIS considers that the essentiality of a gene in a condition should be proportional to its presence in the library. Accordingly, genes present in both control (LB-1 or inactivated HS) and tested (fresh HS) libraries are not essential or neutral, and genes present only in the control library are essential for growth in blood and, consequently, for invasion. We identified the genes, obtained the corresponding mutants and complemented strains and performed a series of experiments in vitro and in vivo to corroborate the essentiality of the gene and its function (if possible). We identified a series of genes involved in the CPS formation and others with unknown function as essential for human invasion. Moreover, a series of functions have been proposed as important for growth in human blood