In vitro and in vivo expression of virulence genes in Vibrio isolates belonging to the Harveyi clade in relation to their virulence towards gnotobiotic brine shrimp (Artemia franciscana)

Abstract

Vibrios belonging to the Harveyi clade are pathogenic marine bacteria affecting both vertebrates and invertebrates, thereby causing a severe threat to the aquaculture industry. In this study, the expression of haemolysin, metalloprotease, serine protease, the quorum sensing master regulator LuxR and the virulence regulator ToxR in different Harveyi clade isolates was measured with reverse transcriptase real-time PCR with specific primers. There was relatively low variation in the in vitro expression levels of the quorum sensing master regulator luxR (sevenfold), whereas for the other genes, the difference in expression between the isolates showing lowest and highest expression levels was over 25-fold. Furthermore, there was a significant correlation between expression levels of toxR and luxR and between the expression levels of these regulators and the protease genes. The expression levels of luxR, toxR and haemolysin were negatively correlated with the survival of brine shrimp larvae challenged with the isolates. Finally, a non-virulent, a moderately virulent and a strongly virulent isolate were selected to study in vivo expression of the virulence genes during infection of gnotobiotic brine shrimp larvae. The in vivo gene expression study showed a clear difference in virulence gene expression between both virulent isolates and the non-virulent isolate.

Monitoring of Vibrio harveyi quorum sensing activity in real time during infection of brine shrimp larvae

“…Shrimp-associated wild-type Vibrio harveyi showed a peak in quorum sensing activity early during infection-well before the first mortality is observed. This is consistent with our previous work, in which we found that there is a peak in expression of the quorum sensing master regulator gene luxR and different virulence genes early during infection of brine shrimp larvae (Ruwandeepika et al, 2011b). The in vivo quorum sensing activity of mutants defective in the production of one of the three signal molecules is consistent with their virulence, with no detectable in vivo quorum sensing activity in AI-2-and CAI-1-deficient mutants.…”

“…As brine shrimp are particle filter feeders, the challenge procedure allows infection to develop in a natural way, with the vibrios being accumulated in the gastro-intestinal tract and causing damage to the gut epithelium (Gunasekara et al, 2012). Recently, we developed an experimental procedure allowing us to measure virulence gene expression of vibrios in vivo during infection of brine shrimp larvae and found that the Vibrio harveyi quorum sensing master regulator gene luxR showed a peak in in vivo expression levels in virulent isolates early during infection, whereas the expression levels remained low in an avirulent isolate (Ruwandeepika et al, 2011b). However, to obtain good quantification, we needed to take 500 larvae per sample for these analyses.…”

RpoS and Indole Signaling Control the Virulence of Vibrio anguillarum towards Gnotobiotic Sea Bass (Dicentrarchus labrax) Larvae

“…Gene expression was determined with qRT-PCR as described previously [31]. V. anguillarum cultures grown in LB 20 medium were collected at 6 h, 12 h and 24 h. Three different V. anguillarum cultures were sampled for each treatment.…”

Single cell analysis of Vibrio harveyi uncovers functional heterogeneity in response to quorum sensing signals

“…vscP ) [30] and exoproteolytic activity (e.g. vhp ) [17,31], in addition to bioluminescence (using the lux system) [32]. …”