Date of Award

2017

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Gilchrist, Sandra

Keywords

Legionella pneumophila, Type IV Secretion System, Acanthamoeba castellanii, Legionnaire’s Disease

Area of Concentration

Biology

Abstract

Research dating back over twenty years demonstrated the bacterium, Legionella pneumophila, when co-cultured with natural host amoebae, Acanthamoeba castellanii, was rendered significantly hypervirulent to macrophages in cell culture when compared to L. pneumophila cultured in laboratory media alone. Therefore, it was hypothesized that the most significant virulence factors were not produced in L. pneumophila cultured in artificial media, but instead were upregulated and secreted by a distinct Dot/Icm type IV secretion system following intracellular growth in A. castellanii. Work in the Cambronne laboratory validated this argument with the description of the SidGV effector complex (Drennan et al., submitted), which was specifically activated during intracellular growth in A. castellanii. Using sid GV transcriptional profiling, an amoebae-cued regulatory circuit was defined. Subsequent proteomic analysis revealed several dot/icm loci to be co-regulated in response to the amoebae host in a manner similar to sid GV. To evaluate the contribution to pathogenesis of these uncharacterized effector proteins, wild-type L. pneumophila was subjected to mutagenesis via homologous recombination. An in-frame deletion of the effector encoding lpg1496 was generated using a triparental mating scheme. After validation of gene disruption via PCR, the CR39Δlpg1496 strain was used for infections of A. castellanii to determine the efficacy of infection over a time course and compared to the virulence of the wild-type strain. Acanthamoeba castellanii infection assays were performed and visualized using fluorescence microscopy. The mutant, CR39Δlpg1496, exhibited a unique, hyper-virulent phenotype compared to the positive and negative control strains CR39 (WT) and CR39Δ cpxA (attenuated). These findings suggest that Lpg1496 plays a critical role in the regulation of virulence effectors.

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