Experimental Methods for Studying Kingella kingae

Abstract

Kingella kingae is a naturally competent organism, allowing the use of natural transformation as an efficient mechanism for introducing exogenous DNA into the organism and facilitating genetic manipulation. While no plasmids for the introduction and extrachromosomal replication of cloned DNA into K. kingae have been identified, the generation of targeted mutations in the chromosome is a relatively straightforward process. Gene deletions, site-directed mutations, gene complements, and random transposon libraries have been generated in K. kingae, all relying on insertion of a selectable antibiotic resistance marker into the chromosome. Beyond genetic manipulation, a variety of techniques for isolation of K. kingae surface factors such as type IV pili, outer membrane proteins, polysaccharide capsule, and a secreted exopolysaccharide have been developed, largely based on methods used for research on other gram-negative bacteria. Methods for circumventing the activity of the potent K. kingae RTX toxin to enable investigation of the interaction of the organism with eukaryotic cells in vitro have been developed. Lastly, a juvenile rat intraperitoneal infection model is the only in vivo model shown to demonstrate virulence differences between different clinical isolates of K. kingae and between wild-type and isogenic mutants lacking putative virulence factors.