Microbiology

CRIS AD-421 Research Reports Submitted for 98-35204-6979 on 01/29/01

ROLE OF TYPE IV PILI IN AEROMONAS SALMONICIDA PATHOGENESIS AND USE AS A VACCINE

Investigators: Strom, M. S.

Termination Date: 11/30/00
Reporting period: 12/01/98 to 11/30/00

Termination Report:
The goal of this research is to clone and characterize the genes that encode factors involved in expression of type IV pili and extracellular protein secretion from the pathogen Aeromonas salmonicida, which causes the disease furunculosis in salmonid fish. Pili are fibers that protrude from bacterial cells that are often involved in the initial attachment or colonization of pathogenic bacteria to host cells. These same bacteria are often capable of secreting a number of toxins and degradative enzymes involved in virulence through a related pathway. A demonstration that these factors are important for the pathogenesis of A. salmonicida could form the basis for an effective vaccine against the pathogen. A specific tapA mutant of A. salmonicida was constructed by allelic exchange and shown not to transcribe tapA mRNA by Northern blot. Highly specific anti TapA antisera was prepared from recombinant TapA protein (purified from a tapA:FLAG fusion construct). The antisera was shown to recognize TapA from wild-type A. salmonicida while no TapA was detected in the tapA mutant. However, the level of TapA expressed under the conditions examined to date (growth on BHI broth at 22 C) shows that much less of the protein is expressed than what is seen in other type IV piliated bacteria. Preliminary experiments have been carried out to assess the phenotype of the A. salmonicida tapA mutant strain. An in vitro bacterial adherence assay was attempted with A. salmonicida using the CHSE-214 (chinook salmon embryo) salmonid cell line. Low but consistent adherence values were obtained for A. salmonicida but there was no difference between the wild-type and mutant strains. However, we are currently exploring the use of other cell lines. Preliminary virulence challenge studies have also been initiated. Rainbow trout (5 g) have been challenged with wild-type A. salmonicida A450N1 and a tapA mutant to determine the difference in infective dose in an immersion challenge. There is a small, but consistent and significant difference in virulence between the wild-type and mutant strains, with the latter requiring 35-50% more colony forming units to achieve the same level of mortality in waterborne challenge of rainbow trout. At this time, challenge experiments are being performed to determine whether fish surviving challenge with wild-type are more resistant to subsequent re-challenge with the wild-type pathogen than those originally challenged with the tapA mutant. We have yet to construct a mutant strain unable to express TapD, the type IV prepilin peptidase. All candidates recovered contain the mutated and wild-type copy of the tapD gene. This has led us to speculate that tapD is essential for viability of A. salmonicida under in vitro growth conditions. This is significant in that while type IV prepilin peptidases are generally involved in both type IV pilus biogenesis and extracellular secretion of a number of exotoxins (including aerolyisin), none been shown to be essential for in vitro growth. We are currently developing vectors and methodologies to test this hypothesis, including inducible overexpression of a dominant-negative allele of TapD.

Impact:
A demonstration that type IV pili are involved in the intial interaction or colonization of the salmonid fish host in infections caused by Aeromonas salmonicida will be an advance in the understanding of the mechanisms used by this pathogen to cause disease. It could also potentially lead to the development of a safe, efficacious, and easy to administer vaccine against furunculosis.

Publications:
(No publications reported.)