Title

Exploring Symbiotic Functions of the Leech Gut Microbiome

Date of Completion

January 2010

Keywords

Biology, Microbiology|Biology, Animal Physiology

Degree

Ph.D.

Abstract

Animal digestive tracts house an array of microorganisms that play a beneficial role in aiding digestion. Studying digestive-tract microbiota poses many challenges including: the vast number of microbes comprising the microbiome, the presence of unculturable microbes, and the lack of genetic tools for many culturable microbes. The availability of a versatile invertebrate model can provide important new insights into digestive-tract symbiosis. The medicinal leech, Hirudo verbana, contains a simple microbial community, dominated by two species, a Rikenella-like bacterium and Aeromonas veronii. The simplicity of this system allows us to study microbe-microbe and microbe-host interactions in a simple, naturally-occurring system. ^ The main focus of this work was to reveal the function of the simple microbial community within the leech gut. One way the function was studied was by identifying genes required for the digestive-tract colonization of the leech gut. Two colonization factors were investigated, mechanisms for iron acquisition and the Type II secretion system (T2SS) in A.veronii Investigating iron acquisition revealed two iron utilization systems in A. veronii, one siderophore-dependent and one siderophore-independent. The siderophore- and heme-utilization mechanisms were studied by generating mutants and double mutants by homologous recombination, demonstrating that heme utilization is an essential colonization factor for A. veronii The second colonization factor was evaluated with a mutant that carried a miniTn5 insertion in exeM, which is involved in forming the inner membrane platform in the T2SS. The mutant was isolated by screening mutants for the loss of β-hemolysis on blood agar plates. Colonization assays suggested that both the T2SS and hemolysin export by the T2SS are critical for initial establishment of A. veronii in the leech gut. ^ A more direct way of examining function was performed by removing symbionts with an antibiotic treatment of juvenile leeches This revealed an unexpected phenotype; the presence of A. vermin is required for the leech to display its characteristic pigmentation. The lack of pigmentation was prevented by adding an antibiotic resistant strain; ensuring that the loss was not due to an indirect effect of the antibiotics.^ Finally, a metagenomic analysis of the leech crop microbiome was done using next generation sequencing. This analysis provided insight into gene content and thus possible metabolic functions that the symbionts contribute to the host.^ These studies have opened up new doors for the leech gut symbiosis, giving answers to many fundamental questions about both beneficial and pathogenic associations. ^