Analysis of the Coxiella burnetii type IV secretion system region I during infection

Abstract

Scope and Method of Study: Scope - To determine the temporal regulation of Coxiella burnetii type IV secretion system Region I homologs during infection of host cells over a time course of infection. Methods for microbiological and molecular analyses include: eukaryotic and prokaryotic cell culture, digitonin/GeneLock TM cell lysis, Deoxyribonucleic acid (DNA), Ribonucleic acid (RNA), Polymerase Chain Reaction (PCR), reverse transcriptase PCR, quantitative reverse transcriptase PCR, recombinant protein generation, recombinant polyclonal antibody generation, indirect immunofluorescent antibody (IFA), immunoelectron microscopy, fluorescent and light microscopy, bio-analyzer, etc.

Findings and Conclusions: Findings - I found a combination of Digitonin lysis with GeneLock TM performed on ice, significantly enriches the relative quantity and quality of intracellular specific bacterial RNA. I also determined that the Coxiella burnetii Dot/Icm type IV RI contains 3 operons that are expressed and coordinately regulated over a time course of infection as the bacteria shifts forms. Which led to my discovery that Coxiella burnetii Dot/Icm type IV RI protein expression over a time course of infection shows a tight correlation to RI RNA expression over correlating time points during host cell infection. Additionally I discovered that the Coxiella burnetii Dot/Icm type IV secretion system is polarly expressed on the LCV bacterial form. Conclusions - I conclude that the C. burnetii type IV secretion system region I is temporally regulated at both the RNA and protein level within its host over its time course of infection correlating to phase shift from SCV->LCV->SCV bacterial forms and that the C. burnetii type IV system is expressed on the bacterial poles in the LCV form.