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Infection and Immunity, February 2000, p. 896-905, Vol. 68, No. 2
Department of Molecular Microbiology and Immunology, Oregon
Health Sciences University, Portland, Oregon
97201-3098,1 and Department of
Microbiology-Immunology, Northwestern University Medical School,
Chicago, Illinois 606112
Received 15 September 1999/Returned for modification 15 October
1999/Accepted 5 November 1999
Initiation of a gonococcal infection involves attachment of
Neisseria gonorrhoeae to the plasma membrane of an
epithelial cell in the mucosal epithelium and its internalization,
transepithelial trafficking, and exocytosis from the basal membrane.
Piliation and expression of certain Opa proteins and the immunoglobulin A1 protease influence the transcytosis process. We are interested 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 minitransposon (mTn) mutant bank for isolates that traverse the monolayer more quickly than the isogenic wild-type (WT) strain. From an initial screen, we isolated four mutants, defining
three genetic loci, that traverse monolayers significantly more quickly
than their WT parent strain. These mutants adhere to and invade cells
normally and do not affect the integrity of the monolayer barrier.
Backcrosses of the mutations into the WT FA1090 strain yielded 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 into the MS11A genetic background also yielded a
fast-trafficking mutant. The fit 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 potential helix-turn-helix motif, while the deduced sequence
of FitB offers no clues to its function. fitA or
fitB homologues are present in the genomes of
Pseudomonas syringae and Rhizobium meliloti,
but not Neisseria meningitidis. Replication of the MS11A
fitA mutant in A431 and T84 cells is significantly
accelerated compared to that of the isogenic WT strain. In contrast,
growth of this mutant in liquid media is normal. Taken together, these
results strongly suggest that traversal of N. gonorrhoeae
across an epithelial barrier is linked to intracellular bacterial growth.
0019-9567/00/$04.00+0
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Isolation of Neisseria gonorrhoeae
Mutants That Show Enhanced Trafficking across Polarized T84
Epithelial Monolayers
*
Corresponding author. Mailing address: Department of
Molecular Microbiology and Immunology, L220, Oregon Health Sciences
University, 3181 SW Sam Jackson Park Rd., Portland, OR 97201-3098. Phone: (503) 494-6840. Fax: (503) 494-6862. E-mail:
hoppers{at}ohsu.edu.
Present address: Business Development Center, Pharmacogenomic
Services, Laboratory Corporation of America, Research Triangle Park, NC 27709.
Infection and Immunity, February 2000, p. 896-905, Vol. 68, No. 2
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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