Isolation of Neisseria gonorrhoeae mutants that show enhanced trafficking across polarized T84 epithelial monolayers

Initiation of a gonococcal infection involves attachment of Neisseria gonor rhoeae to the plasma membrane of an epithelial cell in the mucosal epitheli um and its internalization, transepithelial trafficking, and exocytosis fro m the basal membrane. Piliation and expression of certain Opa proteins and the immunoglobulin Al protease influence the transcytosis process. We are i nterested in identifying other genetic determinants of N. gonorrhoeae that play a role in transcellular trafficking. Using polarized T84 monolayers as a model epithelial barrier, we have assayed an N, gonorrhoeae FA1090 minit ransposon (mTn) mutant bank for isolates that traverse the monolayer more q uickly than the isogenic wild-type (WT) strain. From an initial screen, we isolated four mutants, defining three genetic loci, that traverse monolayer s significantly more quickly than their WT parent strain. These mutants adh ere to and invade cells normally and do not affect the integrity of the mon olayer barrier. Backcrosses of the mutations into the WT FA1090 strain yiel ded mutants with a similar fast-trafficking phenotype. In two mutants, the mTns had inserted 370 bp apart into the same locus, which we have named fit , for fast intracellular trafficker. Backcrosses of one of these mutants in to the MS11A genetic background also yielded a fast-tracking mutant. The fi t locus contains two overlapping open reading frames, fitA and fitB, whose deduced amino acid sequences have predicted molecular weights of 8.6 and 15 .3, respectively. Neither protein contains a signal sequence. FitA has a po tential helix-turn-helix motif, while the deduced sequence of FitB offers n o clues to its function. fitA or fitB homologues are present in the genomes of Pseudomonas syringae and Rhizobium meliloti, but not Neisseria meningit idis. Replication of the MS11A fitA mutant in A431 and T84 cells is signifi cantly accelerated compared to that of the isogenic WT strain. In contrast, growth of this mutant in Liquid media is normal. Taken together, these res ults strongly suggest that traversal of N. gonorrhoeae across an epithelial barrier is linked to intracellular bacterial growth.