Francisco J. Roig

Domain organization and evolution of multifunctional autoprocessing repeats-in-toxin (MARTX) toxin in Vibrio vulnificus.

ABSTRACT The objective of this study was to analyze multifunctional autoprocessing repeats-in-toxin (MARTX) toxin domain organization within the aquatic species Vibrio vulnificus as well as to study the evolution of the rtxA1 gene. The species is subdivided into three biotypes that differ in host range and geographical distribution. We have found three different types (I, II, and III) of V. vulnificus MARTX (MARTXVv) toxins with common domains (an autocatalytic cysteine protease domain [CPD], an α/β-hydrolase domain, and a domain resembling that of the LifA protein of Escherichia coli O127:H6 E2348/69 [Efa/LifA]) and specific domains (a Rho-GTPase inactivation domain [RID], a domain of unknown function [DUF],a domain resembling that of the rtxA protein of Photorhabdus asymbiotica [rtxA PA], and an actin cross-linking domain [ACD]). Biotype 1 isolates harbor MARTXVv toxin types I and II, biotype 2 isolates carry MARTXVv toxin type III, and biotype 3 isolates have MARTXVv toxin type II. The analyzed biotype 2 isolates harbor two identical copies of rtxA1, one chromosomal and the other plasmidic. The evolutionary history of the gene demonstrates that MARTXVv toxins are mosaics, comprising pieces with different evolutionary histories, some of which have been acquired by intra- or interspecific horizontal gene transfer. Finally, we have found evidence that the evolutionary history of the rtxA1 gene for biotype 2 differs totally from the gene history of biotypes 1 and 3.

pilF Polymorphism-Based PCR To Distinguish Vibrio vulnificus Strains Potentially Dangerous to Public Health

ABSTRACT Vibrio vulnificus is a heterogeneous species that comprises strains virulent and avirulent for humans and fish, and it is grouped into three biotypes. In this report, we describe a PCR-based methodology that allows both the species identification and discrimination of those isolates that could be considered dangerous to public health. Discrimination is based on the amplification of a variable region located within the gene pilF, which seems to be associated with potential human pathogenicity, regardless of the biotype of the strain.

Vibrio vulnificus produces quorum sensing signals of the AHL-class

Vibrio vulnificus is an aquatic pathogenic bacterium that can cause vibriosis in humans and fish. The species is subdivided into three biotypes with the fish-virulent strains belonging to biotype 2. The quorum sensing (QS) phenomenon mediated by furanosyl borate diester or autoinducer 2 (AI-2) has been described in human strains of biotype 1, and here we show that the luxS gene which encodes AI-2 is present in all strains of V. vulnificus regardless of origin, biotype or serovar. In this study, we also demonstrate that V. vulnificus produces QS signals of the acylated homoserine lactone (AHL) class (AI-1). AHLs were detected in strains of biotype 1 and 2 from water, fish and human wound infections but not in strains isolated from human septicaemic cases. The AHL compound was identified as N-butanoyl-homoserine-lactone (C(4)-HL) by both reporter strains and by HPLC-high-resolution MS. C(4)-HL was detected when AHL-positive strains were grown in low-nutrient medium [modified sea water yeast extract (MSWYE)] but not in rich media (tryptic soy broth or brain-heart infusion) and its production was enhanced when blood factors were added to MSWYE. C(4)-HL was detected in vivo, in eels infected with AHL-positive biotype 2 strains. No known AHL-related gene was detected by PCR or Southern blot suggesting that AHL-related genes in V. vulnificus are different from those found in other Gram-negative bacteria.

Genome of wild olive and the evolution of oil biosynthesis

Significance We sequenced the genome and transcriptomes of the wild olive (oleaster). More than 50,000 genes were predicted, and evidence was found for two relatively recent whole-genome duplication events, dated at approximately 28 and 59 Mya. Whole-genome sequencing, as well as gene expression studies, provide further insights into the evolution of oil biosynthesis, and will aid future studies aimed at further increasing the production of olive oil, which is a key ingredient of the healthy Mediterranean diet and has been granted a qualified health claim by the US Food and Drug Administration. Here we present the genome sequence and annotation of the wild olive tree (Olea europaea var. sylvestris), called oleaster, which is considered an ancestor of cultivated olive trees. More than 50,000 protein-coding genes were predicted, a majority of which could be anchored to 23 pseudochromosomes obtained through a newly constructed genetic map. The oleaster genome contains signatures of two Oleaceae lineage-specific paleopolyploidy events, dated at ∼28 and ∼59 Mya. These events contributed to the expansion and neofunctionalization of genes and gene families that play important roles in oil biosynthesis. The functional divergence of oil biosynthesis pathway genes, such as FAD2, SACPD, EAR, and ACPTE, following duplication, has been responsible for the differential accumulation of oleic and linoleic acids produced in olive compared with sesame, a closely related oil crop. Duplicated oleaster FAD2 genes are regulated by an siRNA derived from a transposable element-rich region, leading to suppressed levels of FAD2 gene expression. Additionally, neofunctionalization of members of the SACPD gene family has led to increased expression of SACPD2, 3, 5, and 7, consequently resulting in an increased desaturation of steric acid. Taken together, decreased FAD2 expression and increased SACPD expression likely explain the accumulation of exceptionally high levels of oleic acid in olive. The oleaster genome thus provides important insights into the evolution of oil biosynthesis and will be a valuable resource for oil crop genomics.

Plasmid diversity in Vibrio vulnificus biotypes

Vibrio vulnificus is a heterogeneous bacterial species that can be virulent for humans and fish. Virulence in fish seems to rely on a recently described plasmid that can be transmitted between strains, aided by a conjugative plasmid. The main objective of this work was to analyse the plasmid content of a wide collection of strains from the three biotypes of the species, as well as to identify putative conjugative and virulence plasmids by means of Southern hybridization with specific probes and sequence analysis of selected gene markers. We found 28 different plasmid profiles in a total of 112 strains, which were relatively biotype- or serovar-specific. Biotype 1 lacked high-molecular-mass plasmids, with the exception of a putative conjugative plasmid of 48 kb that was present in 42.8% of clinical and environmental strains isolated worldwide. All biotype 2 strains possessed the virulence plasmid, whose molecular mass ranged between 68 and 70 kb, and 89.65% of these strains also had a putative conjugative plasmid with a molecular size of 52-56 kb. Finally, a 48 kb putative conjugative plasmid was present in all biotype 3 strains. Data from partial sequencing of traD, traI and the whole vep07 (a recently described plasmid-borne virulence gene) from a selection of strains suggest that the plasmids of 48-56 kb probably belong to the same family of F-plasmids as pYJ016 and that the gene vep07 is absolutely essential for fish virulence. Additional cryptic plasmids of low molecular mass were present in the three biotypes. In conclusion, plasmids are widespread among V. vulnificus species and could contribute substantially to genetic plasticity of the species.

Novel host‐specific iron acquisition system in the zoonotic pathogen Vibrio vulnificus

Vibrio vulnificus is a marine bacterium associated with human and fish (mainly farmed eels) diseases globally known as vibriosis. The ability to infect and overcome eel innate immunity relies on a virulence plasmid (pVvbt2) specific for biotype 2 (Bt2) strains. In the present study, we demonstrated that pVvbt2 encodes a host-specific iron acquisition system that depends on an outer membrane receptor for eel transferrin called Vep20. The inactivation of vep20 did not affect either bacterial growth in human plasma or virulence for mice, while bacterial growth in eel blood/plasma was abolished and virulence for eels was significantly impaired. Furthermore, vep20 is an iron-regulated gene overexpressed in eel blood during artificially induced vibriosis both in vitro and in vivo. Interestingly, homologues to vep20 were identified in the transferable plasmids of two fish pathogen species of broad-host range, Vibrio harveyi (pVh1) and Photobacterium damselae subsp. damselae (pPHDD1). These data suggest that Vep20 belongs to a new family of plasmid-encoded fish-specific transferrin receptors, and the acquisition of these plasmids through horizontal gene transfer is likely positively selected in the fish-farming environment. Moreover, we propose Ftbp (fish transferrin binding proteins) as a formal name for this family of proteins.

Spontaneous Quinolone Resistance in the Zoonotic Serovar of Vibrio vulnificus

ABSTRACT This work demonstrates that Vibrio vulnificus biotype 2, serovar E, an eel pathogen able to infect humans, can become resistant to quinolone by specific mutations in gyrA (substitution of isoleucine for serine at position 83) and to some fluoroquinolones by additional mutations in parC (substitution of lysine for serine at position 85). Thus, to avoid the selection of resistant strains that are potentially pathogenic for humans, antibiotics other than quinolones must be used to treat vibriosis on farms.

Host-Nonspecific Iron Acquisition Systems and Virulence in the Zoonotic Serovar of Vibrio vulnificus

ABSTRACT The zoonotic serovar of Vibrio vulnificus (known as biotype 2 serovar E) is the etiological agent of human and fish vibriosis. The aim of the present work was to discover the role of the vulnibactin- and hemin-dependent iron acquisition systems in the pathogenicity of this zoonotic serovar under the hypothesis that both are host-nonspecific virulence factors. To this end, we selected three genes for three outer membrane receptors (vuuA, a receptor for ferric vulnibactin, and hupA and hutR, two hemin receptors), obtained single and multiple mutants as well as complemented strains, and tested them in a series of in vitro and in vivo assays, using eels and mice as animal models. The overall results confirm that hupA and vuuA, but not hutR, are host-nonspecific virulence genes and suggest that a third undescribed host-specific plasmid-encoded system could also be used by the zoonotic serovar in fish. hupA and vuuA were expressed in the internal organs of the animals in the first 24 h of infection, suggesting that they may be needed to achieve the population size required to trigger fatal septicemia. vuuA and hupA were sequenced in strains representative of the genetic diversity of this species, and their phylogenies were reconstructed by multilocus sequence analysis of selected housekeeping and virulence genes as a reference. Given the overall results, we suggest that both genes might form part of the core genes essential not only for disease development but also for the survival of this species in its natural reservoir, the aquatic environment.

An Enriched European Eel Transcriptome Sheds Light upon Host-Pathogen Interactions with Vibrio vulnificus

Infectious diseases are one of the principal bottlenecks for the European eel recovery. The aim of this study was to develop a new molecular tool to be used in host-pathogen interaction experiments in the eel. To this end, we first stimulated adult eels with different pathogen-associated molecular patterns (PAMPs), extracted RNA from the immune-related tissues and sequenced the transcriptome. We obtained more than 2x106 reads that were assembled and annotated into 45,067 new descriptions with a notable representation of novel transcripts related with pathogen recognition, signal transduction and the immune response. Then, we designed a DNA-microarray that was used to analyze the early immune response against Vibrio vulnificus, a septicemic pathogen that uses the gills as the portal of entry into the blood, as well as the role of the main toxin of this species (RtxA13) on this early interaction. The gill transcriptomic profiles obtained after bath infecting eels with the wild type strain or with a mutant deficient in rtxA13 were analyzed and compared. Results demonstrate that eels react rapidly and locally against the pathogen and that this immune-response is rtxA13-dependent as transcripts related with cell destruction were highly up-regulated only in the gills from eels infected with the wild-type strain. Furthermore, significant differences in the immune response against the wild type and the mutant strain also suggest that host survival after V. vulnificus infection could depend on an efficient local phagocytic activity. Finally, we also found evidence of the presence of an interbranchial lymphoid tissue in European eel gills although further experiments will be necessary to identify such tissue.

Phylogeny of Vibrio vulnificus from the analysis of the core-genome: Implications for intra-species taxonomy

Vibrio vulnificus (Vv) is a multi-host pathogenic species currently subdivided into three biotypes (Bts). The three Bts are human-pathogens, but only Bt2 is also a fish-pathogen, an ability that is conferred by a transferable virulence-plasmid (pVvbt2). Here we present a phylogenomic analysis from the core genome of 80 Vv strains belonging to the three Bts recovered from a wide range of geographical and ecological sources. We have identified five well-supported phylogenetic groups or lineages (L). L1 comprises a mixture of clinical and environmental Bt1 strains, most of them involved in human clinical cases related to raw seafood ingestion. L2 is formed by a mixture of Bt1 and Bt2 strains from various sources, including diseased fish, and is related to the aquaculture industry. L3 is also linked to the aquaculture industry and includes Bt3 strains exclusively, mostly related to wound infections or secondary septicemia after farmed-fish handling. Lastly, L4 and L5 include a few strains of Bt1 associated with specific geographical areas. The phylogenetic trees for ChrI and II are not congruent to one another, which suggests that inter- and/or intra-chromosomal rearrangements have been produced along Vv evolution. Further, the phylogenetic trees for each chromosome and the virulence plasmid were also not congruent, which also suggests that pVvbt2 has been acquired independently by different clones, probably in fish farms. From all these clones, the one with zoonotic capabilities (Bt2-Serovar E) has successfully spread worldwide. Based on these results, we propose a new updated classification of the species based on phylogenetic lineages rather than on Bts, as well as the inclusion of all Bt2 strains in a pathovar with the particular ability to cause fish vibriosis, for which we suggest the name “piscis.”