Investigating survival mechanisms of Yersinia ruckeri in rainbow trout, Oncorhynchus mykiss
Yersinia ruckeri' causes hemorrhagic septicemia of salmonid fish. We hypothesized that the ability to persist inside infected fish was a key component of virulence strategy of 'Y. ruckeri' and aimed to identify survival-essential genes of 'Y. ruckeri' serotype 1 strain, RS1154. After two rounds of screening of 1056 miniTn5Km2 signature-tagged mutants, 25 mutants that did not survive in kidney at 7 days post-infection in immersion infected rainbow trout were selected for further study. Sequencing of interrupted genes in selected mutants identified genes homologous to ' znuA', which encodes a component of a zinc transporter in ' Escherichia coli', and 'uvrY', which encodes the response regulator of BarA-UvrY two-component system (TCS) in 'E. coli'. The 'uvrY' mutant was hypersensitive to H2O 2-mediated killing and was less invasive to 'Epithelioma papulosum cyprini' fish cells than wild type (WT) bacteria, but was not affected in serum sensitivity or growth under iron-limiting conditions. In a competitive infection with WT, the 'uvrY' mutant had lower infection loads in rainbow trout kidney. When present in a low-copy plasmid, the 'znuACB' locus of 'Y. ruckeri' fully restored growth of a zinc-transport deficient [Delta]' znuACB' mutant of 'E. coli' in Luria-Bertani (LB) medium supplemented with 2.0 mM ethylenediamine tetraacetic acid (EDTA), indicating that 'znuACB' locus of 'Y. ruckeri' is likely involved in zinc transport. Unlike [Delta]'znuACB' mutants of 'E. coli', [Delta]'znuACB' mutant of ' Y. ruckeri' did not show poor growth in zinc-deficient M9 medium and LB medium supplemented with metal chelators, EDTA and tetrakis-(2-pyridylmethyl)-ethylenediamine, suggesting presence of additional zinc transporters in 'Y. ruckeri '. The 'znuA' mutant of 'Y. ruckeri' was out-competed by WT in rainbow trout kidney. Survival of 'Y. ruckeri' in rainbow trout was also reduced with mutations in gene homologs encoding an O-antigen polymerase (' wzy'), peptidoglycan deacetylase ('pdaA'), protease ('ptrA'), bundle-forming pili ('rcpA'), ATPase of DNA segregation ('parA') and transposase of Tn7, bacteriophage tail fiber-like protein and genes of unknown functions. Characterization of survival-essential genes in fish helps identify new virulence genes and contributes to understanding survival strategies and pathogenic mechanisms of ' Y. ruckeri'.