Expression of virulence genes of vibrios belonging to the Harveyi clade in the brine shrimp Artemia

Vibrios belonging to the Harveyi clade are pathogenic marine bacteria affecting both vertebrates and invertebrates. These bacteria are severe threat to the aquaculture industry with multibillion dollar losses. Many virulence factors have been identified in Harveyi clade vibrios, including phospholipases, hemolysins, proteases, (cysteine protease, metalloprotease, serine protease), lipopolysaccharide, presence of bacteriophages, bacteriocin-like substance (BLIS) and chitinase. The virulence of vibrios is controlled by different regulatory systems, including quorum sensing and the transmembrane transcriptional regulator ToxR. However, detailed knowledge on the expression and regulation of virulence genes, especially under in vivo conditions (i.e. during infection of a host) is still lacking. For the development of specific biocontrol measures to overcome this major aquaculture disease, it is of utmost importance to understand the molecular basics of virulence expression. This study aimed at investigating virulence factors (virulence genes) and their expression in vitro and in vivo within isolates belonging to the Harveyi clade. In the first step of this study, an overview is given of the current knowledge on pathogenesis of bacteria belonging to the Harveyi clade, their impact on the aquaculture sector, the virulence factors that are involved in pathogenicity and their regulation. The first part of the research aimed at checking for the presence of several virulence genes that has been described before in V. harveyi in different isolates belonging to the Harveyi clade. The presence of virulence genes of other vibrios (i.e. atypical virulence genes) in the genomes of the isolates was also studied. It was found that all the virulence genes that are typical for the Harveyi clade vibrios (including the quorum sensing master regulator luxR, the virulence regulator toxRVh, vhp metalloprotease, chiA chitinase, srp serine protease and vhh hemolysin) were present in all the isolates tested and some atypical virulence genes were also found in some of the isolates tested. These results suggest that vibrios belonging to the Harveyi clade might have acquired virulence genes from other vibrios in the aquatic environment through horizontal gene transfer. As all tested Harveyi clade isolates have been positive for all typical virulence genes, their different specific virulence cannot be linked to the presence of (one of) these genes. In a further study, the expression of virulence genes in these bacteria was quantified both in vitro and in vivo and the relation between virulence gene expression and survival of challenged brine shrimp (Artemia franciscana) was determined. A standardized a method was developed for quantifying in vivo gene expression using Artemia as an invertebrate gnotobiotic host model, which facilitates the study of virulence gene expression and regulation under natural conditions in order to gain more relevant data. The results showed that there is variation in the expression level of genes coding for virulence regulators and virulence factors in Vibrio isolates belonging to the Harveyi clade. In vitro expression of the regulators luxR and toxR and the vhh hemolysin gene were correlated with the survival of challenged brine shrimp larvae, indicating that variation in the expression of these genes might be the cause of the variation in the virulence of the isolates. The in vivo virulence gene expression in three selected isolates (one avirulent, one moderately virulent and one strongly virulent) during infection of brine shrimp larvae revealed a peak in expression after 24h in the virulent isolates and a constantly low expression in the avirulent isolate. Therefore, it can be concluded that the expression levels of the virulence genes studied here are strongly linked to virulence of Harveyi clade vibrios towards brine shrimp. Quorum sensing is one of the regulatory mechanisms in V. harveyi and other vibrios. Quorum sensing regulation of virulence genes was investigated by determining the expression levels of virulence genes in wild type V. harveyi and different quorum sensing mutants, both in vitro and in vivo during infection of brine shrimp (Artemia franciscana) larvae. This study revealed that the vhp metalloprotease gene is positively regulated by quorum sensing. The vhh hemolysin gene did not show a regulation by the three-channel QS mechanism (no difference in expression in the mutants which have either maximum or minimum activity of the three-channel system). But there was a lower expression in the AI-2 synthase and receptor mutants when compared to the other strains. This suggests that vhh hemolysin might be specifically regulated by AI-2 quorum sensing via the AI-2 receptor LuxPQ but not via the three-channel signal transduction system. There was no difference between the strains in expression levels of the virulence regulator gene toxR and the srp serine protease gene, which indicated that these genes are independent of quorum sensing. Under in vivo conditions, the luxR and vhp metalloprotease genes also showed to be under quorum sensing regulation and the vhh hemolysin gene followed the same pattern of expression as in in vitro, having lower expression in an AI-2 deficient mutant. toxR and the srp serine protease gene did not show quorum sensing regulation under in vivo conditions. Further, the expression levels of the virulence genes in luminescent and non-luminescent isogenic strains were investigated. The study revealed that the non-luminescent variants produced lower levels of the quorum sensing master regulator luxR and the vhp metalloprotease gene. Challenge tests with gnotobiotic brine shrimp larvae showed that the non-luminescent strains are less virulent than their luminescent counterparts. In a last study, the quorum sensing regulation of the chitin-degrading enzyme chitinase, which might be involved in colonization and infection of host organisms, especially crustaceans (which contain chitin in their shells) was investigated. This study indicated that chitinase A is negatively regulated by quorum sensing