My doctoral graduate research activities involved studies on type A
streptococcal exotoxin produced by group A streptococci. I was able to
purify the type A toxin and to develop the ELISA technique for the
detection of the toxin. This technique was
used to monitor the kinetics of toxin synthesis in strains of
streptococci. In addition, several biochemical and biological studies
were conducted on the toxin.
As a postgraduate, my research has
included the development of a very sensitive
antigenic assay for the detection of Salmonella toxin (ELISA).
This assay was employed as a probe to determine the effect of heating on
the antigenic structure of Salmonella toxin as well as the isoelectric
point and molecular weight of the toxin.
Data were obtained which suggested that mitomycin C (MTC) caused
cell lysis, resulting in the release of intracellular Salmonella toxin
rather than an increase in toxin synthesis. The mechanism of this
phenomenon was substantiated by the appearance
of bacteriophage concomitantly with the detection of Salmonella
toxin subsequent to the addition of MTC to Salmonella broth cultures.
Experiments were conducted which indicated that the gene which codes for
Salmonella toxin synthesis was not associated
with a bacteriophage.
Presently, the long term objective of our laboratory is
to determine the role of Aeromonas hydrophila virulence factors in the
pathogenesis of disease in man. The virulence factors presently reported
in the literature associated with
A. hydrophila have not been defined clearly. A. hydrophila, a
member of the family Vibrionaceae, is considered to be a significant
human pathogen. This microorganism is responsible for a variety of
diseases including acute bacterial diarrhea,
septicemia, meningitis, endocarditis, corneal ulcers,
peritonitis, and wound infections. These diseases have occurred in
immunocompetent and well as immunocompromised individuals. A. hydrophila
produce as well as possess a number of factors which
contribute to the overall virulence of the organism. We have
characterized two enterotoxins biochemically, biologically and
antigenically. We have identified the genes for both enterotoxins and
have cloned them. A complete nucleotide sequence
analysis of the cytolytic enterotoxin gene has been
accomplished. Our preliminary results regarding the mechanism of action
of our cytotonic enterotoxin as been published. Our goals for the future
will be to define the domains on the cytolytic
enterotoxin that are responsible for various biological
activities associated with this toxin molecule as well as to locate the
binding region on the cytolytic enterotoxin which interacts with the
host eukaryotic cell membrane; to study the regulation
of expression of the cytolytic enterotoxin gene; to define the
area in a cloned DNA fragment that codes for a cytotonic enterotoxin
gene and subject it to nucleotide sequence analysis; to overexpress both
the cytolytic and cytotonic enterotoxins
using several expression vector systems i.e., pET, pGEX or
pTRXFUS; and, to initiate studies to establish the precise role of these
enterotoxins in the pathogenesis of Aeromonas-mediated diseases.