Signature-tagged mutagenesis of Edwardsiella ictaluri identifies virulence-related genes, including a salmonella pathogenicity island 2 class of type III secretion systems

Abstract

Edwardsiella ictaluri is the leading cause of mortality in channel catfish culture, but little is known about its pathogenesis. The use of signature-tagged mutagenesis in a waterborne infection model resulted in the identification of 50 mutants that were unable to infect/survive in catfish. Nineteen had minitransposon insertions in miscellaneous genes in the chromosome, 10 were in genes that matched to hypothetical proteins, and 13 were in genes that had no significant matches in the NCBI databases. Eight insertions were in genes encoding proteins associated with virulence in other pathogens, including three in genes involved in lipopolysaccharide biosynthesis, three in genes involved in type III secretion systems (TTSS), and two in genes involved in urease activity. With the use of a sequence from a lambda clone carrying several TTSS genes, Blastn analysis of the partially completed E. ictaluri genome identified a 26,135-bp pathogenicity island containing 33 genes of a TTSS with similarity to the Salmonella pathogenicity island 2 class of TTSS. The characterization of a TTSS apparatus mutant indicated that it retained its ability to invade catfish cell lines and macrophages but was defective in intracellular replication. The mutant also invaded catfish tissues in numbers equal to those of invading wild-type E. ictaluri bacteria but replicated poorly and was slowly cleared from the tissues, while the wild type increased in number.

FIG. 1.

Overview of the signature-tagged mutagenesis procedures. The two PCR products missing for all three fish (white arrows) represent putative attenuated mutants for further characterization.

FIG. 2.

Agarose gel showing plasmid preparations from wild-type Edwardsiella ictaluri and two mutant strains with transposon insertions in plasmids pEI1 and pEI2. Note the mutant pEI2 plasmid in 166ST carrying the 2,378-bp transposon insertion and the lower relative brightness of the wild-type pEI2 plasmid, suggesting an apparent reduction in copy number. Also note similar results for pEI1 in 217UV. The left lane is a size ladder with numbers of base pairs indicated.

FIG. 3.

Genetic organization of the type III secretion system genes of E. ictaluri compared to homologous regions of other TTSS. Arrows with the same pattern indicate homologous proteins. Note that E. ictaluri gene escC (88) was formerly known as eseA.

FIG. 4.

CCO cells infected with E. ictaluri at 8 h postinfection in a gentamicin survival assay. (A) Cells infected with an E. ictaluri esaU mutant, 65ST, showing typical single bacterium per cell. (B) Cells infected with wild-type E. ictaluri strain 93-146, showing typical large number of bacterial cells per infected CCO cell. Both the mutant and the wild type typically had 1 to 2 bacterial cells per CCO cell immediately following infection.

FIG. 5.

Comparison of the early invasion and persistence of wild-type Edwardsiella ictaluri and an esaU mutant, 65ST, in channel catfish. Head kidney samples were taken from each of five fish at each time point, and the data represent the means of the results, with bars representing the standard errors of the means. ***, significant difference from the corresponding wild type (P > 0.001).