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.

SummaryThe IgA1 protease secreted by the pathogenic Neisseriae cleaves Lamp1, a major integral membrane glycoprotein of lysosomes, and significantly reduces its steady-state levels in an infected cell. IgA1 protease hydrolysis of Lamp1 is inefficient at the low pH of lysosomes, strongly suggesting that the enzyme is unlikely to reduce Lamp1 levels within lysosomes to any appreciable extent. We therefore explored the possibility that the protease may reach Lamp1 through an alternative route. We demonstrate that Neisseria pili induce a transient increase in the levels of cytosolic free Ca 21 in A431 human epithelial cells, as demonstrated previously for ME180 cells. This Ca 21 flux triggers lysosome exocytosis, quickly altering the cellular distribution of Lamp1 and increasing surface Lamp1 levels. Finally, we demonstrate that surface Lamp1 is cleaved by IgA1 protease secreted by adherent bacteria. We conclude that the pilus-induced Ca 21 flux increases the amount of Lamp1 that is cleavable by the IgA1 protease.

We previously demonstrated that the Neisseria IgA1 protease cleaves LAMP1 (lysosome-associated membrane protein 1), a major integral membrane glycoprotein of lysosomes, thereby accelerating its degradation rate in infected A431 human epidermoid carcinoma cells and resulting in the alteration of lysosomes in these cells. In this study, we determined whether the IgA1 protease also affects the trafficking of Neisseria gonorrhoeae across polarized T84 epithelial monolayers. We report that N. gonorrhoeae infection of T84 monolayers, grown on a solid substrate or polarized on semiporous membranes, also results in IgA1 protease-mediated reduction of LAMP1. We demonstrate that iga mutants in two genetic backgrounds exited polarized T84 monolayers in fewer numbers than the corresponding wild-type strains. Finally, we present evidence that these mutants have a statistically significant and reproducible defect in their ability to traverse T84 monolayers. These results add to our previous data by showing that the IgA1 protease alters lysosomal content in polarized as well as unpolarized cells and by demonstrating a role for the protease in the traversal of epithelial barriers by N. gonorrhoeae.Neisseria gonorrhoeae (i.e., the gonococcus [GC]) causes gonorrhea in humans, its only host, and gains entry into the body via the mucosal surfaces. There is no animal model for GC disease. Studies on the molecular requirements of GC interactions with the epithelium rely on human fallopian tube organ cultures (hFTOC), immortalized human epithelial cells grown on solid substrates, and the human challenge model of urethral infection. Such studies reveal that GC attach to and invade nonciliated cells of the mucosal epithelium through the coupling of several bacterial adhesins (type IV pili, PilC, and certain Opa variants) with their cognate host cell receptors (CD46, CD66, and heparan sulfate proteoglycans), which are present in a large number of human

Iron is an essential element for nearly all organisms. In mammals, iron is transported to body tissues by the serum glycoprotein transferrin. Transferrin‐iron is internalized by binding to specific receptors followed by endocytosis. In vitro, Neisseria meningitidis and Neisseria gonorrhoeae can use iron from a variety of iron‐containing compounds, including human transferrin. In vivo, transferrin is an important source of iron for N. gonorrhoeae: a mutant that is unable to bind and use transferrin‐iron is unable to colonize the urethra of men or initiate disease at this site. As pathogenic Neisseria and its human host derive much of their iron from transferrin, we reasoned that a competition may exist between microbe and host epithelial cells for transferrin‐iron at certain stages of infection. We therefore tested the hypothesis that N. meningitidis and N. gonorrhoeae may actively interfere with host transferrin‐iron metabolism. We report that Neisseria‐infected human epithelial cells have reduced levels of transferrin receptor messenger RNA and cycling transferrin receptors. The ability of infected cells to internalize transferrin receptor is also reduced. Finally, the relative distribution of surface and cycling transferrin receptors is altered in an infected cell. We conclude that Neisseria infection alters epithelial cell transferrin‐iron homeostasis at multiple levels.

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