DUAL FLAA1 FLAB1 MUTANT OF SERPULINA-HYODYSENTERIAE EXPRESSING PERIPLASMIC FLAGELLA IS SEVERELY ATTENUATED IN A MURINE MODEL OF SWINE-DYSENTERY

The motility imparted by the periplasmic flagella (PF) of Serpulina hy odysenteriae is thought to play a pivotal role in the enteropathogenic ity of this spirochete. The complex PF are composed of multiple class A and class B poly peptides. Isogenic strains containing specifically disrupted flaA1 or flaB1 alleles remain capable of expressing PF, alth ough such mutants display aberrant motility in vitro. To further exami ne the role that these proteins play in the maintenance of periplasmic flagellar structural integrity, motility, and fitness for intestinal colonization, we constructed a novel strain of S. hyodysenteriae which is deficient in both FlaA1 and FlaB1, To facilitate construction of t his strain, a chloramphenicol gene cassette, with general application as a selectable marker in prokaryotes, was developed, The cloned flaA1 and flaB1 genes were disrupted by replacement of internal fragments w ith chloramphenicol and kanamycin gene cassettes, respectively, The in activated flagellar genes were introduced into S. hyodysenteriae, and allelic exchange at the targeted chromosomal flaA1 and flaB1 loci was verified by PCR analysis, Immunoblots of cell lysates with antiserum r aised against purified FlaA or FlaB confirmed the absence of the corre sponding sheath and core proteins in this dual flagellar mutant, These mutations selectively abolished the expression of the targeted genes without affecting the synthesis of other immunologically related FlaB proteins, The resulting flaA1 flaB1 mutant exhibited altered motility in vitro, Surprisingly, it was capable of assembling periplasmic flage lla that were morphologically normal as evidenced by electron microsco py, The virulence of this strain was assessed in a murine model of swi ne dysentery by determining the incidence of cecal lesions and the per sistence of S. hyodysenteriae in the gut, Mice challenged with the wil d-type strain or a passage control strain showed a dose-related respon se to the challenge organism, The dual flagellar mutant was severely a ttenuated in murine challenge experiments, suggesting that the FlaA1 a nd FlaB1 proteins are dispensable for flagellar assembly but critical for normal flagellar function and colonization of mucosal surfaces of the gastrointestinal tract, This strain represents the first spirochet e engineered to contain specifically defined mutations in more than on e genetic locus.