The Gram-negative bacterium Neisseria gonorrhoeae is the only causative agent of the sexually transmitted disease gonorrhea. The gonococcal (Gc) pilus is an important virulence factor of this human pathogen. Since Gc do not possess a Type II or Type III secretion apparatus, this Type IV pilus has an important role in host-pathogen interactions. The Gc pilus is involved in many aspects of gonococcal pathogenesis. First, the pilus functions in the initiating events of colonization by enhancing the ability of the bacterium to adhere to and interact with cells of the human host. Second, the pilus is involved in allowing the efficient transport of DNA into the bacterial cell for genetic transformation. Third, it is involved in twitching motility, a specialized form of locomotion expressed by all Type IV piliated bacteria. Much has been learned about the components that make up the Gc pilus assembly apparatus in the past several years, but how the pilus and its assembly apparatus act to promote adherence, DNA transformation, and twitching motility remains largely undefined. We propose a series of experiments to further our understanding of how the Gc pilus provides these functions for pathogenesis.
In Aim 1 will utilize saturating mutagenesis to isolate loss-of-function and gain-of-function mutations in the secretin gene, PilQ. Loss-of-function mutations will define domains of PilQ required for expression, stability, and multimerization. Gain-of-function mutations will be screened for mutations that effect DNA transformation, antibiotic sensitivity, and twitching motility. Mutants will also be tested for changes in epithelial cell adherence.
In Aim 2 will undertake a biochemical, yeast two-hybrid and biophysical analysis of three related predicted NTPases involved in orchestrating pilus function. These studies will provide new findings and insights into the structure and functions of the gonococcal pilus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI055977-03
Application #
7009073
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Hiltke, Thomas J
Project Start
2004-02-01
Project End
2009-01-31
Budget Start
2006-02-01
Budget End
2007-01-31
Support Year
3
Fiscal Year
2006
Total Cost
$290,021
Indirect Cost
Name
Northwestern University at Chicago
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
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Ch√Ęteau, Alice; Seifert, H Steven (2016) Neisseria gonorrhoeae survives within and modulates apoptosis and inflammatory cytokine production of human macrophages. Cell Microbiol 18:546-60
Cahoon, Laty A; Manthei, Kelly A; Rotman, Ella et al. (2013) Neisseria gonorrhoeae RecQ helicase HRDC domains are essential for efficient binding and unwinding of the pilE guanine quartet structure required for pilin antigenic variation. J Bacteriol 195:2255-61
Cahoon, Laty A; Seifert, H Steven (2013) Transcription of a cis-acting, noncoding, small RNA is required for pilin antigenic variation in Neisseria gonorrhoeae. PLoS Pathog 9:e1003074
Duffin, Paul M; Seifert, H Steven (2012) Genetic transformation of Neisseria gonorrhoeae shows a strand preference. FEMS Microbiol Lett 334:44-8
Schook, Paul O P; Stohl, Elizabeth A; Criss, Alison K et al. (2011) The DNA-binding activity of the Neisseria gonorrhoeae LexA orthologue NG1427 is modulated by oxidation. Mol Microbiol 79:846-60
Chen, Adrienne; Seifert, H Steven (2011) Neisseria gonorrhoeae-mediated inhibition of apoptotic signalling in polymorphonuclear leukocytes. Infect Immun 79:4447-58
Duffin, Paul M; Seifert, H Steven (2010) DNA uptake sequence-mediated enhancement of transformation in Neisseria gonorrhoeae is strain dependent. J Bacteriol 192:4436-44
Helm, R Allen; Seifert, H Steven (2010) Frequency and rate of pilin antigenic variation of Neisseria meningitidis. J Bacteriol 192:3822-3
LeCuyer, Brian E; Criss, Alison K; Seifert, H Steven (2010) Genetic characterization of the nucleotide excision repair system of Neisseria gonorrhoeae. J Bacteriol 192:665-73

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