Investigation of factors influencing virulence in Vibrio anguillarum O1 : from cryo-storage to metals and the search for novel drug targets

Abstract

Long-term preservation of bacterial stock cultures is important for applied and environmental microbiology as it underpins confirmation of earlier results, ensures strains are not lost and catalogues biodiversity for future research or commercial use. A key assumption of such methods is that organism characteristics are unaffected by storage. Evidence from previous work suggests this assumption isn't always true for human pathogens, including Salmonella typhimurium, Shigella spp. and Bacillus anthracus, whilst there is also suggestion that such attenuation with storage occurs in fish pathogens such as Flavobacterium psychrophilum and some Vibrio species, including Vibrio (Listonella) anguillarum. The present work, for the first time, evaluated and characterised long-term (ca. 20 years) changes in the virulence stability of the significant fish pathogen V. anguillarum serotype O1 after storage, as well as identifying underlying mechanisms. Vibrio anguillarum O1 isolates (n = 20) from an in-house culture collection were selected based on having been classified as high virulence (100% mortalities after 24h on in vivo challenge in salmonid fish) immediately before cryopreservation in 1995 (-70oC; 15% glycerol in TSB). Isolates re-cultured after ~20 years showed both partial and total loss, as well as maintenance, of virulence (in vivo challenge in rainbow trout, Oncorhynchus mykiss) after storage, with complete virulence loss being associated with the absence of the pJM1 plasmid on reculture. Maximum in vitro siderophore production in tested isolates of V. anguillarum O1 occurred under conditions of 20% Glucose, 1 mM of FeCl3.6H2O, 200 rpm agitation, 30ºC and 0.03 g/ml Ethylenediaminetetraacetic acid (EDTA; as iron chelator) in minimum medium (MM9). These conditions led plasmid-positive V. anguillarum O1 isolates to produce 77.28 % units of siderophore in the exponential growth phase. Virulence plasmid status and siderophore production level did not affect in vitro antibiotic susceptibility. However, the iron chelator EDTA, enhanced the in vitro antimicrobial effect of Gentamycin sulphate and Kanamycin sulphate against all V. anguillarum O1 isolates. Adding 0.061g EDTA/100 ml TSB gave fractional inhibitory concentrations (FIC) of 0.75 (gentamycin) and 0.8 (kanamycin), indicating an additive or weak synergistic effect of EDTA with the antibiotics. Growth inhibition of V. anguillarum O1 by EDTA alone was also determined, with 0.88 g/l EDTA in TSA inhibiting growth of virulent isolates, whereas only 0.66 g/l was required for inhibition of non-virulent isolates. In MHA medium, equivalent concentrations of EDTA were 0.40 g/l (virulent) and 0.20 g/l (non-virulent). This indicated greater resistance of virulent (plasmid-harbouring) isolates to EDTA action, linked to siderophore sequestration of trace iron. Adding CuCl2 (0.2-0.6 g/l in Tryptone Soya Broth (TSB), Luria broth (LB), Tryptone Soya Agar (TSA) and Mueller Hinton Agar (MHA) media led to the recovery of in vitro growth by both plasmid-positive and plasmid-negative strains under iron-limited conditions in both liquid and solid media. Both isolate types survived up to doses exceeding 1.55 g EDTA /l agar media when 0.2-0.6 g/l CuCl2 was added. However, the addition of 0.77 g/l of CuCl2 inhibited growth in plasmid-negative strains, whereas a greater concentration (0.89 g/l CuCl2) was necessary to inhibit the growth of plasmid-positive strains. Therefore, exposure of V. anguillarum O1 isolates to CuCl2 was shown to support their growth under iron limited conditions, potentially explaining, in part, previously observed increases in disease virulence under copper pollution conditions. Building on the above findings, in silico bioinformatic tools, were used to investigate the possibility for targeting the iron uptake system, and especially siderophore production, as a novel strategy for controlling V. anguillarum O1 infection and overcoming some problems of antibiotic resistance. This was achieved by focussing on the VabC protein (Chorismate synthetase). Using bioinformatic software, the previously unresearched structure of the VabC protein was generated and explored in silico, and the resulting predicted tertiary structure revealed several candidate ligand-binding pockets in the active site of this enzyme. Further application of recent ligand-binding predictive software showed some of these to have high druggability scores, suggesting that this protein would make a good potential target for existing and novel drug compounds. Several ligand classes were also identified which could form the basis of future drug trials. Overall, virulence of V. anguillarum O1 strains was reduced after long-term storage, with complete virulence loss being linked to an inability to produce siderophores from plasmid pJM1. This highlighted the significance of siderophore as a virulence factor in V. anguillarum O1 and focussed attention on possibilities for targeting the siderophore synthesis pathway, as a means of control and treatment of this commercially damaging microorganism.