REGULATION OF LEUKOTOXIN IN A ACTINOMYCETEMCOMITANS

Project: Research project

Project Details

Description

Periodontitis, an inflammatory disease of tissues in the subgingival
crevice, can lead to soft tissue damage and dramatic bone loss.
Development of periodontitis is associated with a dramatic shift in the
subgingival microflora; gram negative microorganisms replace gram
positive bacteria as the primary flora. In order to understand why
certain bacteria are successful periodontopathogens, it is important to
determine how their virulence determinants might be regulated by
components of the complex and unique subgingival microenvironment. This
proposal focuses on regulation in the gram negative, coccobacillus
Actinobacillus actinomycetemcomitans (Aa) for several reasons: (1) Aa
is the primary candidate pathogen in several forms of periodontitis, (2)
Aa is the only periodontopathogen to produce a leukotoxin, and (3) the
Aa leukotoxin gene has been cloned, so both molecular genetic and
biochemical approaches can be used to study its regulation. Our hypothesis is that changes in environmental cues will alter the
expression of Aa leukotoxin. Aa strains in which both leukotoxin and
beta-galactosidase are transcribed from the leukotoxin promoter have been
generated. This will allow leukotoxin promoter activity to be assessed
using a simple, quantitative beta-galactosidase assay. These Aa strains
will be grown in media with varying amounts of iron, in media at
different pHs and in cultures which have been stressed (heat shocked).
Beta-galactosidase activity will be assayed to determine those culture
changes that modulate leukotoxin expression. then RNA and protein will
be isolated from the same cultures and analyzed using specific DNA and
antibody probes to determine the level at which leukotoxin regulation
occurs. the regulation of cell envelope proteins will also be examined
from the same cells. The results will show how the virulence factor,
leukotoxin, is regulated by changes in these three environmental signals
and will also identify other regulated proteins. the other regulated
proteins are candidate virulence factors. Using the cloned leukotoxin gene as a starting point, molecular genetic
approaches will then be used to determine the DNA sequences and
regulatory proteins involved in regulating leukotoxin transcription in
Aa. Deletion and point mutant analyses of the leukotoxin promoter will
define important cis elements. Transposon mutagenesis will be used to
identify genes which regulate the transcriptional response of the
leukotoxin gene. The results from these studies will define the cast of
characters used by Aa to regulate a virulence factor, leukotoxin. this
will enable us, in future years, to delineate the molecular mechanisms
by which environmental cues regulate leukotoxin and other virulence
factors of Aa.
StatusFinished
Effective start/end date9/1/938/31/02

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $129,987.00
  • National Institutes of Health: $222,947.00
  • National Institutes of Health: $233,933.00
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Dentistry(all)

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