GONOCOCCAL PIA--ROLE IN INVASION OF FALLOPIAN TUBE CELL

Project: Research project

Project Details

Description

Neisseria gonorrhoeae (GC) is able to attach to and invade non-ciliated
epithelial cells of the Fallopian tube organ culture model (FTOC). The
mechanism of this interaction remains to be defined. GC Por or PI, the
focus of this proposal, is an outer membrane porin protein that comes in
two forms, PIA or PIB. It does not vary within a single GC strain. It can
transfer spontaneously from GC to lipid bilayers and human cells. It is
able to bind calmodulin, but the impact of this on pathogenesis is
unknown. Two surface-exposed regions of PIA have amino acid sequences
compatible with a basic amphiphilic a-helical conformation, a motif that
calmodulin recognizes in binding to a large variety of proteins. This
motif also allows peptides and some transmembrane receptors to activate
G proteins. PIA has been cloned and expressed in E. coli. In the FTOC
model, these E. coli fail to attach, which has prevented assessment of
PIA's invasion-triggering properties.

GC opacity-associated proteins (Opa) and lipooligosaccharides (LOS), have
been implicated as attachment and invasion factors in some cell culture
models. Opa and LOS undergo rapid phase and antigenic variation. One
type of LOS defined by the 3F11 monoclonal antibody binds to the
asialoglycoprotein receptor in liver cell lines. It is not known if the
3F11 epitope mediates attachment to or invasion of fallopian tube
epithelium. Opas and the 3F11 LOS have been cloned and expressed in E.
coli. Opa production facilitates attachment of E. coli in the human FTOC
model, but observed bacterial transport through the cells has been much
less than that seen with GC.

Thus, we hypothesize that following intimate Opa- and/or LOS-mediated
attachment, PI insertion into host cells triggers significant GC invasion
via calmodulin- and/or G protein-mediated pathways. By providing for
bacterial attachment and eliminating phase and antigenic variation via
subcloning of Opa and/or LOS into PIA-bearing E. coli, "GC surrogates"
will be constructed to study invasion-triggering by PIA. Variants which
express Opa, 3F11 LOS, and PIA in various combinations will be tested in
the FTOC model for invasiveness as measured by computerized image
analysis in conjunction with digital confocal microscopy. Invasive
subclones of E. coli and GC will be tested in the FTOC model with G
protein and calmodulin activators or inhibitors to determine if these
pathways are connected to invasion through GC PIA. Site-directed
mutagenesis of putative basic amphiphilic alpha-helical PIA regions with
a-helix breaking proline residues should ablate calmodulin-binding
properties and invasiveness. Morphometric comparisons of these mutants
to E. coli expressing wild-type PIA in the FTOC model will confirm the
pathogenic role of these regions.
StatusFinished
Effective start/end date9/30/958/31/01

Funding

  • National Institute of Allergy and Infectious Diseases
  • National Institute of Allergy and Infectious Diseases
  • National Institute of Allergy and Infectious Diseases
  • National Institute of Allergy and Infectious Diseases
  • National Institute of Allergy and Infectious Diseases

Fingerprint

Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.