Neisseria gonorrhoeae causes the sexually-transmitted disease gonorrhea as well as pelvic inflammatory disease, sepsis, meningitis, and arthritis. Gonococci generate an incredible degree of genetic diversity and antigenic diversity allowing them to avoid the human immune response. A significant amount of the genetic diversity is created through natural transformation. Natural transformation is a way of life for gonococci. They are constitutively competent for transformation, and they preferentially take up DNA from other gonococci or other Neisseria, i.e., they take up DNA that is most likely to be useful for recombination with the genome. We discovered a type IV secretion system (T4SS) encoded in a genomic island in the gonococcal chromosome. The T4SS secretes chromosomal DNA from the bacteria into their surroundings. The secreted DNA is active in the transformation of other gonococci in the vicinity. We demonstrated that the secreted DNA is single- stranded, blocked at the 5' end, and that for it to be secreted it must first be processed by a relaxase that acts at a single site in the chromosome, the origin of transfer. The T4SS has effects on host cell interactions. It also affects adherence of the bacteria to each other and to cells and surfaces. This proposal is focused on how expression of the T4SS is regulated. As is the case for many other T4SSs, the gonococcal type IV secretion genes are repressed under normal culture conditions. Many T4SS proteins are not detectable by western blot and immune-gold electron microscopy identifies a single spot on a subpopulation of cells. These results suggest that each cell might have one T4SS apparatus or that only a few cells in the culture produce the secretion system. To further study the mechanism of secretion, to identify additional secretion substrates, and to elucidate the effects of the type IV secretion during infection, the mechanism controlling type IV secretion will be determined.
The specific aims of this proposal are: 1) to identify regulators and environmental conditions that affect T4SS expression, 2) to determine the mechanisms and effects of the traH regulatory region on T4SS transcription, translation, and protein localization, and 3) to use model substrates and whole genome sequencing to examine the mechanism and consequences of DNA secretion.

Public Health Relevance

This project will characterize the mechanism for controlling production or function of a secretion apparatus made by the bacterium Neisseria gonorrhoeae. These studies will lead to a better understanding of how genes are transferred between bacteria and how N. gonorrhoeae responds to different conditions during infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI047958-12
Application #
9180664
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Hiltke, Thomas J
Project Start
2000-07-01
Project End
2019-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
12
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Andrade, Warrison A; Agarwal, Sarika; Mo, Shunyan et al. (2016) Type I Interferon Induction by Neisseria gonorrhoeae: Dual Requirement of Cyclic GMP-AMP Synthase and Toll-like Receptor 4. Cell Rep 15:2438-48
Harrison, Odile B; Clemence, Marianne; Dillard, Joseph P et al. (2016) Genomic analyses of Neisseria gonorrhoeae reveal an association of the gonococcal genetic island with antimicrobial resistance. J Infect 73:578-587
Ramsey, Meghan E; Bender, Tobias; Klimowicz, Amy K et al. (2015) Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion. Mol Microbiol 97:1168-85
Pachulec, Emilia; Siewering, Katja; Bender, Tobias et al. (2014) Functional analysis of the Gonococcal Genetic Island of Neisseria gonorrhoeae. PLoS One 9:e109613
Ramsey, Meghan E; Hackett, Kathleen T; Bender, Tobias et al. (2014) TraK and TraB are conserved outer membrane proteins of the Neisseria gonorrhoeae Type IV secretion system and are expressed at low levels in wild-type cells. J Bacteriol 196:2954-68
Kohler, Petra L; Chan, Yolande A; Hackett, Kathleen T et al. (2013) Mating pair formation homologue TraG is a variable membrane protein essential for contact-independent type IV secretion of chromosomal DNA by Neisseria gonorrhoeae. J Bacteriol 195:1666-79
Stohl, Elizabeth A; Chan, Yolande A; Hackett, Kathleen T et al. (2012) Neisseria gonorrhoeae virulence factor NG1686 is a bifunctional M23B family metallopeptidase that influences resistance to hydrogen peroxide and colony morphology. J Biol Chem 287:11222-33
Woodhams, Katelynn L; Benet, Zachary L; Blonsky, Sarah E et al. (2012) Prevalence and detailed mapping of the gonococcal genetic island in Neisseria meningitidis. J Bacteriol 194:2275-85
Jain, Samta; Zweig, Maria; Peeters, Eveline et al. (2012) Characterization of the single stranded DNA binding protein SsbB encoded in the Gonoccocal Genetic Island. PLoS One 7:e35285
Ramsey, Meghan E; Hackett, Kathleen T; Kotha, Chaitra et al. (2012) New complementation constructs for inducible and constitutive gene expression in Neisseria gonorrhoeae and Neisseria meningitidis. Appl Environ Microbiol 78:3068-78

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