Genome-Wide SNP-genotyping array to study the evolution of the human pathogen Vibrio vulnificus Biotype 3

Vibrio vulnificus is an aquatic bacterium and an important human pathogen. Strains Of V. vulnificus are classified into three different biotypes. The newly emerged biotype 3 has been found to be clonal and restricted to Israel. In the family Vibrionaceae , horizontal gene transfer is the main mechanism responsible for the emergence of new pathogen groups. To better understand the evolution of the bacterium, and in particular to trace the evolution of biotype 3, we performed genome-wide SNP genotyping of 254 clinical and environmental V. vulnificus isolates with worldwide distribution recovered over a 30-year period, representing all phylogeny groups. A custom single-nucleotide polymorphism (SNP) array implemented on the Illumina GoldenGate platform was developed based on 570 SNPs randomly distributed throughout the genome. In general, the genotyping results divided the V. vulnificus species into three main phylogenetic lineages and an additional subgroup, clade B, consisting of environmental and clinical isolates from Israel. Data analysis suggested that 69% of biotype 3 SNPs are similar to SNPs from clade B, indicating that biotype 3 and clade B have a common ancestor. The rest of the biotype 3 SNPs were scattered along the biotype 3 genome, probably representing multiple chromosomal segments that may have been horizontally inserted into the clade B recipient core genome from other phylogroups or bacterial species sharing the same ecological niche. Results emphasize the continuous evolution of V. vulnificus and support the emergence of new pathogenic groups within this species as a recurrent phenomenon. Our findings contribute to a broader understanding of the evolution of this human pathogen

VIBRIO VULNIFICUS VIRULENCE AND SURVIVAL MECHANISMS REVEALED THROUGH COMPARATIVE MICROBIAL GENOMIC ANALYSIS

A sound genome assembly and robust annotations are essential to the differential analysis of bacterial genomes. Using a case study data set of newly sequenced Vibrio vulnificus genomes, both the biology of these bacteria, and the bioinformatics processes that support identification of the similarities and differences found within the different isolates of V. vulnificus, were examined. The two main themes of this research are 1) identification of the virulence and survival mechanisms of clinical and environmental biotypes of Vibrio vulnificus and 2) quantification of the impact of different analysis choices on the overall biological conclusions of the study. Whole genome sequencing, in conjunction with comparative genomics, are current techniques used to capture the genetic and functional repertoire of organisms. It is important to consider and track analytic provenance in bacterial genomics because the impact of making alternate workflow choices can involve changing the biological interpretation of hundreds of genes, even in relatively simple bacterial genomes. Chapter 1 describes the bioinformatics analyses used to determine the draft genome sequences of three environmental genotype Vibrio vulnificus reference genomes and to identify genotype-specific genomic regions. Chapter 1 also highlights the functional systems including the virulence and survival genes that differentiate between clinical and environmental Vibrio vulnificus genotypes. Chapter 2 explores the direct impact of the parameter and methods selected during the assembly and annotation stage of a genome project. Despite decades of advances in ab initio gene prediction, method and parameter choices still strongly influence the identification of genes, and therefore the biologically significant results in a comparative genomics analysis. Using a benchmarking approach based on simulation studies with a related genome, it is possible to identify an optimal assembly-to-annotation pipeline for the collection of V. vulnificus strains. A software framework for comparing the outcomes of different assembly-to-annotation workflows was constructed in the Taverna workflow management system and used to carry out the bioinformatics experiments described in Chapter 3. Chapter 3 expands on the analysis performed in Chapter 1 by performing an extensive comparative genomics analysis of newly sequenced Vibrio vulnificus genomes, each ones represents the different biological classifications found within this species. The analysis of these genomes reveals genes that are specific to each of the biotypes. Comparative analysis of representative strains from each of the established Vibrio vulnificus biotypes is used to identify differentiating genes, which may relate to the apparent host-specificity of the different biotypes

Iron and virulence in the zoonotic pathogen Vibrio vulnificus

Vibrio vulnificus es un patógeno humano emergente que es autóctono de ecosistemas acuáticos salobres de climas templados, tropicales y subtropicales. La especie se subdivide en 3 biotipos (Bt), de los cuales el Bt2 contiene un grupo de cepas que, además de poder infectar al hombre, pueden causar infecciones en peces. Este grupo es un complejo clonal serológicamente homogéneo que denominamos serovar E o serovar zoonótica (VvBt2SerE). Como patógeno humano, VvBt2SerE causa casos esporádicos de infecciones graves en heridas que pueden derivar en septicemia secundaria en pacientes inmunocomprometidos y como patógeno de peces, brotes o epizootias de una septicemia hemorrágica conocida como vibriosis de aguas cálidas. La presente Tesis se centra en averiguar el papel en la virulencia de VvB2SerE de genes seleccionados en base al conocimiento que existe sobre los otros biotipos de la especie, en especial el Bt1, y su patogenicidad para humanos. En concreto, se han seleccionado los genes rtxA13, hupA, hutR, vuuA, vep20 y fur. Los resultados más relevantes que se han encontrado, se discuten a continuación. rtxA13 codifica una toxina de la familia MARTX (Multifunctional Autoprocessatve Repeat in Toxin) única en la especie (tipo III) que en el Bt1 (tipo I) está relacionada con invasión y resistencia a la fagocitosis. Nuestro trabajo demuestra que la toxina tipo III ejerce la misma función que la tipo I en mamíferos y una función diferente en peces. Encontramos que la mutación del gen no produce efectos en la capacidad de colonización e invasión de VvBt2SerE pero anula su virulencia para anguila, lo que unido a que el patógeno causa la muerte sin alcanzar los tamaños poblacionales en órgano interno propios de otros vibrios, sugiere que la toxina produce la muerte de los animales por choque tóxico. Demostramos que el gen rtxA13 se expresa in vivo y que se activa sólo tras el contacto directo de la bacteria con células eucarióticas y lo relacionamos con citotoxicidad para distintos tipos celulares, incluyendo células de defensa (eritrocitos y neutrófilos) por lo que hipotetizamos que el choque tóxico se produce porque la toxina desencadena una tormenta de citoquinas como consecuencia de la interacción de la bacteria con las células de defensa. Asimismo, pudimos relacionar la toxina con resistencia a predación por amebas y ampliar su papel de factor de virulencia a factor de supervivencia fuera del hospedador, lo que explicaría por qué el gen rtxA13 está presente en todos los clones y complejos clonales del Bt2 y por duplicado, en plásmido y cromosoma. hupA, hutR y vuuA son tres genes cromosómicos que en el Bt1 codifican para tres receptores relacionados con crecimiento en condiciones restrictivas en hierro. vep20 es un gen plasmídico, no estudiado, que presenta homología con receptores para hemina/hemoglobina y transferrina. Este trabajo relaciona hupA con captación de hemina (hutR es un gen secundario) y vuuA con captación de vulnibactina por VvBt2SerE y demuestra que la anulación del sistema de captación de hemina dependiente de HupA o del sistema de captación de vulnibactina por mutación de los receptores reduce la virulencia para peces y mamíferos mientras que la anulación de ambos sistemas atenúa aún más la virulencia para peces y elimina completamente la virulencia de la bacteria para mamíferos. Además, los resultados obtenidos con el mutante en vep20 sugieren que hay un tercer sistema de captación de hierro en la serovariedad zoonótica, esta vez plasmídico, que probablemente depende del reconocimiento de una proteína almacenadora de hierro o, lo más probable, del quelante transferrina, específicamente para peces. La secuenciación de los tres genes en una amplia colección de cepas de la especie y su posterior análisis filogenético demuestra que hupA y vuuA son genes antiguos que pertenecen al core de la especie y que presentan un grado de variación indicativo de presión de selección relacionada con procedencia del aislado (competencia por sideróforos, adaptación a hemoglobina de los peces…) mientras que vep20 es un gen de adquisición reciente y no presenta variación. Finalmente, dada la importancia que el hierro tiene en la virulencia de esta especie para peces y mamíferos, hemos obtenido un mutante en el gen regulador fur, que hemos caracterizado fenotípicamente y valorado usando un microarray diseñado específicamente para VvBt2SerE. Los resultados preliminares confirman que hay cientos de genes regulados por Fur, de forma dependiente o no de hierro, y regulados por hierro, y que Fur, además de un represor, puede actuar como activador y que, en conjunto, controlan funciones tan dispares como: movilidad, quimiotaxis, producción de cápsula y lípido A, resistencia a péptidos microcidas y a formas reactivas del oxígeno, resistencia al suero, al choque térmico, etc. Parte de estas funciones han sido confirmadas diseñando experimentos y comparando las diferencias entre cepa parental y mutante o entre condiciones de crecimiento, con y sin hierro. En conclusión, VvBt2SerE posee un set de genes que le capacita tanto para sobrevivir en el medio ambiente como para infectar hospedadores tan distintos como peces y mamíferos, que contribuyen directamente a la colonización, invasión y destrucción de los tejidos/órganos del hospedador, siendo este proceso y otros tantos regulados por la concentración de hierro y el regulador global Fur.Vibrio vulnificus is an emerging human pathogen that inhabits aquatic ecosystems in temperate, tropical and subtropical climates. The species is subdivided in three biotypes (Bt), of which the Bt2 comprises a group of strains that can infect both human and fish. This group is a clonal complex, serologically homogeneous, denominated serovar E or zoonotic serovar (VvBt2SerE). As human pathogen, VvBt2SerE causes sporadic cases of wound infections that can derive to secondary septicemia in immunocompromised patients, and as fish pathogen causes outbreaks of a primary septicemia known as warm-water vibriosis. The present Thesis is focused on find out the role in the VvBt2SerE virulence of selected genes, chosen in basis of the other biotypes of the species, specially the Bt1, and its pathogenicity for humans. Specifically, the genes rtxA13, hupA, hutR, vuuA, vep20 and fur were selected. The results are discussed below. rtxA13 codifies for a toxin of the MARTX family (Multifunctional Autoprocessatve Repeat in Toxin) exclusive in the species (type III) that in the Bt1 (type I) is involved in invasion and resistance to phagocytosis. Our work demonstrates that type III toxin exerts the same function that type I in mammals but a different one in fish. We found that gene mutation did not produce effects in colonization and invasion of the VvBt2SerE but abolished the virulence for eels, and considering that the pathogen causes the animal death without rising a high number population common in other Vibrio species, the results suggest that the toxin produce the animal death by toxic shock. We demonstrated that the gene rtxA13 is expressed in vivo and only when the bacteria is in direct contact with eukaryotic cells, and that present a high cytotoxic activity towards different cellular types, including cells of the immune system (erythrocytes and neutrophils), so we hypothesized that the toxic shock is produced because the toxin triggers a cytokine storm as consequence of the interaction with immune cells. Moreover, we could relate the toxin with the resistance to amoeba predation and extend its role of virulence factor as a survival factor outside the host, what is according with the fact that the gene rtxA13 is present in all clones and clonal complexes of the Bt2 and by duplicated, in the plasmid and the chromosome. hupA, hutR and vuuA are chromosomic genes that in the Bt1 codify for three receptors related with growth in iron restricted conditions. Vep20 is an uncharacterized plasmidic gene that present homology with hemin/hemoglobin and transferrin receptors. This work relates hupA with the use of hemin (hutR is a secondary heme-receptor) and vuuA with the use of ferric vulnibactin by VvBt2SerE and demonstrate that the inactivation of the HupA- or VuuA-dependent systems reduce the virulence degree for fish and mammals while the inactivation of both decreases even more the virulence degree for fish and abolishes completely the virulence for mammals. Moreover, the results obtained with the vep20 mutant suggest that there is a third iron acquisition system in the zoonotic serovar, in this case plasmidic, that is probably involved in the scavenge of iron from transferrin, specifically for fish. The sequencing of the three genes in a wide group of strains and the phylogenetic analysis demonstrate that hupA and vuuA belong to the core genes of the species and present sequence variability, while vep20 is a recently acquired gene without variation. Finally, given the importance of iron in the virulence of this species for fish and mammals, we obtained a mutant in the fur gene to characterize it phenotypically and use it in a microarray designed specifically for VvBt2SerE. The preliminary results confirm that there are hundreds of genes under control of Fur and iron, and that Fur can also work as an activator in addition to as a repressor, controlling phenotypes such as: motility, chemotaxis, capsule and lipid A synthesis, resistance to microcide peptides, plasma, heat shock, etc. Part of these functions have been confirmed with phenotypic assays by comparing the effects of fur mutation and the presence/absence of iron. In conclusion, VvBt2SerE possesses a set of genes that enables both survive in the environment and infect different hosts such as fish and mammals, contributing directly to the colonization, invasion and destruction of hosts tissues/organs, being this process and many others regulated by the iron concentration and the global regulator Fur

Insight into the Evolution of Vibrio vulnificus biotype 3's Genome

Vibrio vulnificus is an aquatic bacterium and an important human pathogen. Strains of V.vulnificus are biochemically classified into three biotypes. The newly emerged biotype 3 appears to be rather clonal and geographically restricted to Israel, where it caused an outbreak of wound infections and bacteremia. To understand the evolution of the bacterium's genome, we sequenced and analyzed the genome of biotype 3 strain VVyb1(BT3), and then conducted a microbial environmental survey of the hypothesized niche from which it probably evolved. The genome of this environmental isolate revealed higher similarity to the published biotype 1 genomes of clinical strains (90%) than to the environmental strains (87%), supporting the virulence of the biotype 3 group. Moreover, 214 of the total 5361 genes were found to be unique to strain VVyb1(BT3), having no sequence similarity to any of the known genomes of V.vulnificus; 35 of them function in DNA mobility and rearrangement, supporting the role of horizontal gene transfer in genome evolution. Interestingly, 29 of the ‘unique’ genes had homologies among Shewanella species. In a survey conducted in aquaculture ponds in Israel, we successfully co-isolated Shewanella and V.vulnificus from the same niche, further supporting the probable contribution of Shewanella to the genome evolution of biotype 3. Indeed, one gene was found in a S.algae isolate. Surprisingly, molecular analysis revealed that some of the considered unique genes are harbored by nonsequenced biotype 1 strains isolated from the same environment. Finally, analyses of the biotype 3 genome together with the environmental survey suggested that its genome originated from a biotype 1 Israeli strain that acquired a rather small number of genes from other bacterial species in the niche, such as Shewanella. Therefore, aquaculture is likely to play a major role as a man-made ecological niche in bacterial evolution, leading the emergence of new pathogenic groups in V.vulnificus