Virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata involves both quorum sensing and a type III secretion system

Abstract

Environmental Vibrio strains represent a major threat in aquaculture, but the understanding of their virulence mechanisms heavily relies on the transposition of knowledge from human-pathogen vibrios. Here, the genetic bases of the virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata were characterized. We demonstrated that luxO, encoding a major regulator of the quorum sensing system, is crucial for the virulence of this strain, and that its deletion leads to a decrease in swimming motility, biofilm formation, and exopolysaccharide production. Furthermore, the biofilm formation by V. harveyi ORM4 was increased by abalone serum, which required LuxO. The absence of LuxO in V. harveyi ORM4 yielded opposite phenotypes compared with other Vibrio species including V. campbellii (still frequently named V. harveyi). In addition, we report a full type III secretion system (T3SS) gene cluster in the V. harveyi ORM4 genome. LuxO was shown to negatively regulate the promoter activity of exsA, encoding the major regulator of the T3SS genes, and the deletion of exsA abolished the virulence of V. harveyi ORM4. These results unveil virulence mechanisms set up by this environmentally important bacterial pathogen and pave the way for a better molecular understanding of the regulation of its pathogenicity.

LuxT Is a Global Regulator of Low-Cell-Density Behaviors, Including Type III Secretion, Siderophore Production, and Aerolysin Production, in Vibrio harveyi

“…Type III secretion systems (T3SSs) are syringe-like molecular machines that ferry toxic effector proteins across bacterial inner and outer membranes and into target eukaryotic cells ( 25 – 27 ). In V. harveyi and other vibrios, type III secretion is crucial for virulence ( 28 – 30 ). The structural proteins comprising the V. harveyi T3SS are encoded in four operons (T3SS.1, T3SS.2, T3SS.3, and T3SS.4) ( Fig.…”

Genetic and physiological insights into the diazotrophic activity of a non-cyanobacterial marine diazotroph

“…Standard procedures were used for all molecular techniques, following the reagent suppliers recommendations. Deletion of nifH was performed by double homologous recombination between a suicide plasmid and the chromosome of V. diazotrophicus, following a previously established protocol (Morot et al, 2021), with some adaptations: V. diazotrophicus was grown overnight at 30°C (it does not grow at 37°C), cointegrates were incubated in LB+L-ara liquid before plating on LB+L-ara, and the temperature used to incubate the filter-deposited LB+DAP plates was 30°C. The suicide plasmid pFD114 (Table S2) was constructed by amplifying around 750 bp of the upstream and downstream regions of the nifH gene of V.…”

“…Complementation of nifH was achieved by amplifying the nifH gene and its dedicated promoter, using primers 210204 and 210205 (Table S3). The P nifH -nifH amplicon was cloned within the pGEM-T vector (Promega), verified by sequencing and cloned within pFD086 (Morot et al, 2021) to replace P lac -gfp, using BamHI and XhoI. The resulting pFD120 plasmid was introduced by biparental mating as described above, using β3914 containing pFD120 as a donor strain.…”

“…Plates were placed in a microplate reader (TECAN Infinite M1000) for 48 hours at 30°C without shaking, and OD 600nm was measured every 30 minutes. After 48 hours, plates were treated following an already established protocol (Morot et al, 2021). Biofilm production was corrected by dividing the OD 550nm measured after crystal violet staining by the maximal OD 600nm measured during the growing time of the corresponding well.…”

“…However, besides the description of its isolation in 1981 and its taxonomical characterization one year latter (Guerinot et al, 1982), no further physiological or genetic characterization of any member of this species has been performed. Because of its presence in various oceanic environments and because many genetic and molecular tools are available for this genus (Delavat et al, 2018;Morot et al, 2021), we think that V. diazotrophicus can constitute an ideal model to assess the ecophysiology of marine NCD and their diazotrophic activity.…”