The obligate human parasite N. gonorrhoeae (the gonococcus) is the causative agent of the sexually transmitted disease gonorrhea. The gonococcal pilus is an essential virulence factor required for infection of human volunteers. The amino acid sequence of the major subunit of the pilus, pilin, can rapidly change of many silent pilin loci to the pilin expression locus by nonreciprocal, homologous recombination. There has been shown in vitro to result form autolysis and transformation of silent sequences into the expressed gene. It has also been proposed that an intracellular recombination system may operate to promote nonreciprocal recombination between the pilin silent and expression loci (gene conversion). We are investigating the specific molecular mechanisms that produce pilin antigenic variation. We have shown that efficient antigenic variation requires a conserved DNA sequence, the Sma/Cla repeat, which is found in every pilin locus and only in pilin loci. This repeat shows high sequence similarity to known site-specific recombination enzyme binding sites, with two sets of binding sequences contained within the repeat. Consistent with this observation, several proteins have been detected that specifically bind the repeat. Mapping the functional sequences of the repeat will determine how many binding sequences are contained in the repeat. Isolating and characterizing the enzymatic activities that act at the site will allow us to understand how the Sma/Cla-specific system is involved in promoting homologous recombination to produce an antigenically variant pilus. In addition, a series of genetic constructs has been generated to provide strains to test if variable pilin sequences transfer more efficiently within a cell (gene conversion) or between cells (autolysis/transformation). Co-culture experiments will also be used to determine if the components of the Sma/Cla-dependent system act within a single cell or between cells. This planned studies will not only refine our models of how gene translocation are produced in the gonococcus, but will also contribute to understanding the mechanisms used to promote high frequency gene translocation in other systems. Moreover, understanding the genetic capabilities of this microbe is essential to understanding the pathogenesis of gonorrhea, since it is the physiological capabilities of this organism that allow it to grow in vivo, evade immune responses, adapt to new environments, and transfer to new hosts. The information gained in these studies will allow a better comprehension of the genetic mechanisms used by this bacterium to promote human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI033493-01A2
Application #
2068521
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1994-05-01
Project End
1998-01-31
Budget Start
1994-05-01
Budget End
1995-01-31
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Northwestern University at Chicago
Department
Microbiology/Immun/Virology
Type
Schools of Dentistry
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Obergfell, Kyle P; Schaub, Ryan E; Priniski, Lauren L et al. (2018) The low-molecular-mass, penicillin-binding proteins DacB and DacC combine to modify peptidoglycan cross-linking and allow stable Type IV pilus expression in Neisseria gonorrhoeae. Mol Microbiol :
Peak, Ian R; Chen, Adrienne; Jen, Freda E-C et al. (2016) Neisseria meningitidis Lacking the Major Porins PorA and PorB Is Viable and Modulates Apoptosis and the Oxidative Burst of Neutrophils. J Proteome Res 15:2356-65
Château, Alice; Seifert, H Steven (2016) Neisseria gonorrhoeae survives within and modulates apoptosis and inflammatory cytokine production of human macrophages. Cell Microbiol 18:546-60
Rotman, Ella; Seifert, H Steven (2015) Neisseria gonorrhoeae MutS affects pilin antigenic variation through mismatch correction and not by pilE guanine quartet binding. J Bacteriol 197:1828-38
Anderson, Mark T; Seifert, H Steven (2013) Phase variation leads to the misidentification of a Neisseria gonorrhoeae virulence gene. PLoS One 8:e72183
Vink, Cornelis; Rudenko, Gloria; Seifert, H Steven (2012) Microbial antigenic variation mediated by homologous DNA recombination. FEMS Microbiol Rev 36:917-48
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
Stohl, Elizabeth A; Gruenig, Marielle C; Cox, Michael M et al. (2011) Purification and characterization of the RecA protein from Neisseria gonorrhoeae. PLoS One 6:e17101
Duffin, Paul M; Seifert, H Steven (2009) ksgA mutations confer resistance to kasugamycin in Neisseria gonorrhoeae. Int J Antimicrob Agents 33:321-7

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