The Q fever pathogen Coxiella burnetii inhibits apoptosis (programmed cell death) in host cells in order to replicate in a stable intracellular compartment. Effector proteins secreted by Coxiella into host cell have been shown to inhibit apoptosis, although their mechanisms of action are not fully understood. Additionally, no information is currently available about the roles host microRNAs (miRNAs) potentially play in regulating apoptosis in Coxiella-infected cells. miRNAs are ~22 nt RNAs that regulate several biological processes in human cells, including apoptosis, by binding to target mRNAs and either blocking translation or causing target degradation. Our long-term goal is to understand how non-coding RNAs regulate Coxiella-host interactions. Towards attaining this goal, the objective of this application is to identify miRNAs that have a role in inhibiting apoptosis during Coxiella infection. Based on preliminary data that revealed potential functions for apoptosis-related miRNAs in Coxiella-infected macrophages, our central hypothesis is that miRNAs have a role in delaying apoptosis during Coxiella infection. The objective of this project will be accomplished by three specific aims: (1) Identify miRNAs that are induced or repressed by Coxiella infection. Using RNA-seq and qRT-PCR we will identify all miRNAs that are differentially expressed in Coxiella- infected THP-1 (human monocyte/macrophage) cells. (2) Determine miRNAs that inhibit apoptosis during Coxiella infection. For the miRNAs identified in our preliminary studies and in Aim 1, we will assay their ability to inhibit apoptosis. (3) Define the role of miR-148a during Coxiella infection. To begin to understand at a molecular level how miRNAs facilitate inhibition of apoptosis during Coxiella infection, we will interrogate the function of miR-148a. This miRNA was chosen because it was significantly down-regulated in Coxiella-infected cells in our preliminary analysis, and its target gene (Bcl-2) has critical roles in both C. burnetii development and apoptosis inhibition. The research proposed here is innovative, because it will depart from the status quo to investigate a whole new realm of host-pathogen dynamics: the RNA-level interaction between Coxiella and human cells. This work is significant because this will be the first study to uncover the roles of miRNAs during Coxiella infection, and based on our results, novel pharmaceutical agents that target miRNAs could potentially be developed to control chronic Coxiella infections, which are difficult to treat with currently available antibiotics. Our approach and results could also be broadly applied to studying the functions of miRNAs during infections caused by other intracellular pathogens.
Coxiella burnetii is a zoonotic intracellular bacterium that causes Q fever and chronic endocarditis; however, its pathogenic mechanisms are not clearly understood. The proposed work will investigate the role of host microRNAs in inhibiting apoptosis during Coxiella infection, thereby promoting intracellular growth of the pathogen. Analyses of the RNA-level interaction between host and pathogen will reveal novel data that will vastly improve our knowledge about the unique biology of this intracellular extremophile.
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