Coxiella burnetii is an extremely infectious, intracellular bacterium that causes Q fever in humans and is classified as a select agent. Q fever typically presents as a debilitating, flu-like illness accompanied by pneumonia or hepatitis, but in a minority of cases a severe, chronic infection occurs with life-threatening endocarditis as the predominant manifestation. Little is known about Coxiella's virulence determinants or how the bacterium subverts the host cell, despite the central role for these factors and activities in the pathogen's survival. Our overall goals are to identify novel virulence determinants of C. burnetii and characterize the host's innate immune response against the bacterium during infection. To that end, we recently developed a novel animal model for Coxiella infection in Caenorhabditis elegans. When C. elegans nematodes feed on C. burnetii, the intestines become persistently colonized. Moreover, infection significantly decreases the worm's lifespan and produces a visible and pronounced pathology (deformed anal region or DAR). Previous reports showing that virulence factors identified in the C. elegans infection model are similarly utilized in higher-order hosts and that signaling pathways of C. elegans' innate immune response (IR) against pathogens are conserved in higher eukaryotes, underscores the value of this model for investigating human pathogens. We hypothesize that the C. elegans model of infection can be utilized in a high-throughput fashion to identify novel virulence factors of Coxiella and to analyze effectors of the worm's innate immune response against this enigmatic bacterium. To address the hypothesis, Aim 1 will screen a library of C. burnetii Himar1 TnA7 mutants for reduced fitness in C. elegans to identify potential virulence factors. Once identified, the corresponding Coxiella genes will be investigated using molecular Koch's postulates to verify their role in potentiating virulence. TNSeq will also be done to analyze the contribution of every mutagenized gene in the Coxiella library to bacterial fitness over the course of infection.
I Aim 2, we will analyze the infection-specific transcriptome of C. elegans by RNASeq to identify innate IR effectors against C. burnetii.
In Aim 3, we will investigate the functionality of these innate IR effectors using a combination of RNAi and mutant strains of C. elegans to investigate host susceptibility to a Coxiella infection, relative to wild-type nematodes. In the end, this stud will significantly move the field forward by: a) identification and analysis of novel Coxiella virulence determinants, b) providing a new animal model of C. burnetii infection that will facilitae screening for virulence factors and other desirable phenotypes, and c) characterizing potentially novel, innate IR effectors that are utilized by a host to counter the pathogen.
Coxiella burnetii is the bacterial agent of Q fever and an HHS select agent. The proposed research will use our Caenorhabditis elegans model of Coxiella infection in a high-throughput fashion to rapidly identify novel virulence determinants of the bacterium and analyze immune effectors of the nematode. Results will provide valuable information regarding virulence factors employed by the pathogen and effectors of the host's innate immune response, and therefore aid in the future design of immunological strategies to counter Q fever.
| Battisti, James M; Watson, Lance A; Naung, Myo T et al. (2017) Analysis of the Caenorhabditis elegans innate immune response to Coxiella burnetii. Innate Immun 23:111-127 |
