N. gonorrhoeae (GC) infects the mucosal epithelium of the human urogenital tract. It adheres to and invades epithelial cells in a multistep manner. Several bacterial ligands and their cognate epithelial cell receptors have been identified, and the initial events in adhesion and invasion are beginning to be understood. GC next traverses the epithelial cell and exits into the subepithelial matrix. This is a slow process requiring 36 - 48 hours, and the molecular mechanisms underlying it are unknown. We are interested in GC transcellular trafficking, or transcytosis, and have taken a genetic approach to study the process. We first adapted the polarized T84 epithelial cell system as a model epithelial barrier to study GC transcytosis. We then used this system to screen a random bank of mTn-generated GC mutants for fast-trafficking mutants. Four mutants with mTn insertions in three genetic loci were identified in this initial screen. Backcrosses of these mutations show that the fast- trafficking phenotype segregated with the mTn insertion. These mutants do not adhere to or invade cells more quickly, nor do they affect the integrity of the epithelial barrier. These mutants are therefore aberrant in the transcellular trafficking process, not in the initial steps of colonization. Preliminary studies indicate that the loci are likely to play a regulatory role in transcytosis. One mutant is deregulated in its growth within two types of human epithelial cells; its extracellular growth in liquid medium is normal. In this grant application, we propose to further characterize these three loci in order to elucidate their role in GC transcellular trafficking. We also propose to screen the rest of the GC mutant bank for additional fast-trafficking mutants and to characterize their mutated genes. Such studies should shed light on the genetic regulation of the transcytosis process, and hopefully guide the design of novel pharmacologic agents against intracellular GC.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI047260-05
Application #
6751266
Study Section
Special Emphasis Panel (ZRG1-BM-1 (02))
Program Officer
Deal, Carolyn D
Project Start
2000-06-01
Project End
2006-05-31
Budget Start
2004-06-01
Budget End
2006-05-31
Support Year
5
Fiscal Year
2004
Total Cost
$339,750
Indirect Cost
Name
Oregon Health and Science University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
Mattison, Kirsten; Wilbur, J Scott; So, Magdalene et al. (2006) Structure of FitAB from Neisseria gonorrhoeae bound to DNA reveals a tetramer of toxin-antitoxin heterodimers containing pin domains and ribbon-helix-helix motifs. J Biol Chem 281:37942-51
Wilbur, J Scott; Chivers, Peter T; Mattison, Kirsten et al. (2005) Neisseria gonorrhoeae FitA interacts with FitB to bind DNA through its ribbon-helix-helix motif. Biochemistry 44:12515-24
Brettin, Thomas; Altherr, Michael R; Du, Ying et al. (2005) Expression capable library for studies of Neisseria gonorrhoeae, version 1.0. BMC Microbiol 5:50