Title

Investigating the function of the type III secretion system in beneficial and pathogenic associations

Date of Completion

January 2008

Keywords

Biology, Molecular|Biology, Genetics|Biology, Microbiology

Degree

Ph.D.

Abstract

The digestive tract of animals is usually colonized by a complex community of beneficial microorganisms, making it challenging to reveal the molecular interactions between microbe and host. The focus of this work was centered on the use of a simple model system, the medicinal leech, to study beneficial microbe-host associations allowing an investigation into the molecular interactions behind the symbiosis. ^ Signature-tagged mutagenesis (STM) was used to identify the genes required for the digestive-tract colonization of the medicinal leech by Aeromonas veronii. The screen identified 20 serum-resistant colonization mutants that were grouped into five categories based on the presumed function of the inactivated gene: surface modification, regulatory, nutritional, host interaction, and unknown function. The detailed characterization of one of these mutants, JG736, suggests additional antimicrobial compounds are released by the leech or bacterial symbionts in the crop, further contributing to the specificity of the symbiosis. ^ Another STM mutant, with a Tn insertion in ascU, encoding an inner membrane component of the type III secretion system (T3SS), was characterized in detail. This mutant was severely attenuated in its ability to colonize the leech crop, prevent phagocytosis by leech hemocytes, confer cytoxicity toward mouse macrophage, and confer virulence in mice. The results show how animals utilize the innate immune system to control pathogenic as well as beneficial associations, which revealed another factor, the removal of sensitive bacteria from the crop by leech hemocytes, that contributes to the specificity of the A. veronii-medicinal leech symbiosis. ^ The prevalence of the T3SS, AexT, and AexU was evaluated in the Aeromonas veronii group. All 20 isolates obtained from environmental and clinical sources possessed the T3SS and both effectors, which indicates a much greater prevalence than previous studies have reported. ^ Phylogenies from housekeeping and T3SS associated genes were constructed, however, before phylogenetic comparisons can be made between the phylogenies further work needs to be done in order to determine if consensus trees can be constructed. ^ These results have not only enhanced our understanding of the A. veronii-leech symbiosis but have demonstrated that animals use conserved mechanisms to defend against pathogens and to maintain a mutualistic symbiotic community. ^