Yersinia ruckeri genes that attenuate survival in rainbow trout (Oncorhynchus mykiss) are identified using signature-tagged mutants

Abstract

To identify genes that enable the enteric redmouth disease bacterium, Yersinia ruckeri, to persist in salmonid fish, 1056 signature-tagged mini-Tn5Km2 transposon mutants of a serotype 1 strain of Y. ruckeri, RS1154, were screened in rainbow trout by immersion infection. Two rounds of screening in fish identified 25 mutants that were not re-isolated from the kidney, 7 days post-infection. Six mutants were tested a third time in fish, in 1:1 competitive challenges with the parent strain; 4 failed to establish in kidney and 2 were present at low levels compared to the parent. Sequence analyses from the single transposon insertion sites in each of the 25 mutants identified genes with sequence homologies to genes for ZnuA, a periplasmic zinc-binding protein of ZnuABC transporter; the UvrY response regulator of BarA-UvrY two-component system; a PtrA protease of the insulin-degrading enzyme family; the RcpA protein of type IV bundle-forming pili; the ParA ATPase of a ParAB DNA-partitioning system; a Wzy polymerase; a polysaccharide deacetylase; a transporter belonging to the major facilitator superfamily and 7 hypothetical proteins of unknown function. The products of 5 of these mutated genes have predicted functions associated with cell surfaces or membranes, which could be important for survival of Y. ruckeri in rainbow trout, while other putative gene products could contribute to infection and invasion processes.

Introduction

Yersinia ruckeri infects rainbow trout and other salmonid fish, producing an acute bacterial hemorrhagic septicemia, but the bacterium may also persist in fish as an asymptomatic infection, until conditions of stress trigger disease outbreaks (Busch and Lingg, 1975, Tobback et al., 2007). Several virulence factors have been identified for Y. ruckeri, including a secreted protease, a hemolysin, and a siderophore (reviewed by Fernandez et al., 2007) and a type IV secretion system (Méndez et al., 2009).

We are interested in how the balance between the host and bacterium changes under stress, and so our objective was to identify genes needed for survival and persistence of Y. ruckeri in rainbow trout. Signature-tagged mutagenesis (STM) is an approach that allows identification of mutants that can survive in vitro but not in the host fish. Y. ruckeri mutants made by insertion of mini-Tn5Km2 transposons (Lehoux et al., 1999) have unique short DNA tags which allow each individual mutant in a mixture to be identified by a tag-specific PCR. This allows a competitive screening of multiple mutants in the same fish, using an immersion challenge to model the natural mode of infection. Using this approach, we identified several survival-defective mutants of Y. ruckeri, with mutations in genes with predicted functions in attachment and entry, colonization and spread, and defense against immune system components.