Research

The research in my laboratory centers on sexually transmitted pathogen Neisseria gonorrhoeae (GC, gonococcus). There are three directions of focus in my laboratory which are inter-related, and all share the long range goal of more fully understanding the biology of GC with an emphasis on how it causes disease.

Protein Targeting

The first area of research involves the study of protein targeting in this bacterium. While most non-cytoplasmic proteins synthesized by bacteria are targeted out of the cytosol via the well-characterized General Secretory Pathway (GSP), a subset of cytoplasmic membrane proteins have been shown to use a second targeting pathway, the Signal Recognition Particle (SRP) pathway, which was first identified based on its similarity to the eukaryotic SRP, which targets proteins out of the cytosol via the endoplasmic reticulum.

We are currently exploring the interactions between components of the gonococcal SRP, and in particular are focussing on the role of the SRP receptor in the process. We have recently shown that the Neisseria SRP receptor, PilA, binds both RNA (4.5S) and DNA in a sequence specific manner. A consequence of this binding is a stimulation of the GTPase activity of the protein, an activity which is essential for its function in targeting. We propose that in addition to the translation and translocation machinery, the targeting complex might also include the transcription machinery, thus coupling transcription, translation, and translocation, thus compartmentalizing the entire process. Experiments designed to test this hypothesis are in progress.

Gly1

Another project in the laboratory began as a hunt for potential gonococcal cytotoxins. We have identified and characterized a locus, gly1, that confers a hemolytic phenotype on the E. coli strain into which it was transformed. Analysis of this locus revealed that it contains two open reading frames (ORFs), ORF1, which encodes a protein that is either outer membrane associated or secreted by N. gonorrhoeae, and ORF2, which encodes a putative homolog of HemD. Gly1ORF1 has no significant homology to any proteins in the GenBank database, indicating it is a novel protein. Additionally, the gly1 genes are only present in the two pathogenic Neisseria species, suggesting they are involved in virulence. Analysis of gonococcal strains deleted for gly1 in a human fallopian tube organ culture (HFTOC) model (in collaboration with Dr. Morris Cooper at Southern Illinois University) demonstrated that the mutants show dramatically increased toxicity when compared to the wild-type strains. These results implicate the gly1 locus as having a role in the toxicity of N. gonorrhoeae to human fallopian tubes. Experiments are in progress to determine the molecular basis for this observation. Structural analysis of the Gly1ORF1 protein is one approach we are taking to do this in collaboration with Dr. Dennis Arvidson here at MSU.

Regulation of gene expression

The newest project in the laboratory centers on how the gonococcus senses and responds to its environment. The first two things the gonococcus comes in contact with when it enters a new host are the organisms that comprise the normal flora and the mucosal epithelia. It is likely that this initial contact serves as a signal for the bacterium to modulate the expression of genes that are important to the survival of the organism in its host. This provides a selective advantage to an organism that is, at least initially, outnumbered, and must deal with differences in nutrient availability as well as with the (often hostile) response of the host.

As part of this project, we are collaborating with a group of investigators to construct a set of DNA arrays which we intend to make available to all interested investigators. Utilization of the information from the recently annotated genome sequence of N. gonorrhoeae strain FA1090 http://www.stdgen.lanl.gov/ is also a key part of this work. We are using a combination of approaches to dissect the regulatory networks involved in the response of gonococci to envirnmental stimuli, including cell culture, reporter genes, as well as the DNA arrays described above. Since there are few well-characterized virulence regulatory systems identified in the pathogenic Neisseriae, the results of these experiments will provide new and vital information in understanding how these pathogens respond to their environment and how this is related to the disease process.

Publications

Search PubMed at the National Library of Medicine: