Since Neisseria gonorrhoeae is an obligate human pathogen, there is presently no animal model system for gonococcal infection which has severely restricted the ability to identify gonococcal gene products that are important for virulence. We recently extended an in vitro model system, the invasion of the endometrial adenocarcinoma cell line HeclB, described by Shaw and Falkow, and found that preincubation of the gonococcus with fixed HeclB cells resulted in the induction of one or more cellular components that enable the bacterium to invade this cell line. This induction 1) requires HecIB cells and not simply tissue culture medium; 2) is prevented by protein synthesis inhibitors; and 3) appears to be specific for endometrial or cervical cell lines, especially HecIB cells, and is not observed with HEp-2, HeLA, CHO< or Change cell lines. We propose to pursue our studies of he molecular mechanisms of gonococcal invasion of HecIB cells and the induction of the invasion phenotype as follows:1). Characterization of gonococcal components induced by preincubation with HEcIB cells. We will emphasize characterization of the new adhesion that appears to be use pulse labelling techniques followed by two-dimensional electrophoresis of total protein extracts and outer membranes from reincubated gonococci to identify new proteins that are synthesized and will determine if there are changes in LOS. 2) Cloning the gene for the new adhesion. We will attempt to isolate the adhesion gene from a variety of gene banks, including those constructed with lambdagt11, a plasmid vector (lambdaSE6), and an expression vector (pGEX-2T) that will produce protein fusions to glutathione-S transferase. 3.) Construction and characterization of insertion mutations in gonococcal inv genes. We will use in vitro and/or in vivo insertion mutagenesis techniques to construct a gonococcal library of mutants that will be screened for defects in invasin. We will also screen for transpositional mutants that are increased in invasion due to insertional inactivation and/or insertion of a transposon generating constitutive expression of gonococcal genes. 4) Cloning and characterization of gonococcal genes required for invasin of HecIB cells. We will clone the gonococcal genes involved in vision from the mutants. with section in . coli for the antibiotic resistance gene inserted into the inv gene. The clone genes will be sequenced an compared to genes from other invasive bacteria. We will determine whether these genes produce a gene product that is required for invasion or a regulatory gene that is required for induction of multiple proteins. The successful completion of this study should reveal significant information regarding what appears to be a relatively novel bacterial invasin system and should also provide new insight into the pathogenesis of N. gonorrhoeae.
| Spence, Janice M; Tyler, Ryan E; Domaoal, Robert A et al. (2002) L12 enhances gonococcal transcytosis of polarized Hec1B cells via the lutropin receptor. Microb Pathog 32:117-25 |
| Spence, J M; Clark, V L (2000) Role of ribosomal protein L12 in gonococcal invasion of Hec1B cells. Infect Immun 68:5002-10 |
| Spence, J M; Chen, J C; Clark, V L (1997) A proposed role for the lutropin receptor in contact-inducible gonococcal invasion of Hec1B cells. Infect Immun 65:3736-42 |
| Silver, L E; Clark, V L (1995) Construction of a translational lacZ fusion system to study gene regulation in Neisseria gonorrhoeae. Gene 166:101-4 |
