Generation and Screening a Library of Coxiella burnetii Nine Mile Phase II Mutants
Coxiella burnetii is a highly infectious human pathogen that causes a global zoonotic disease called Q fever. Although the organism was first isolated more than 80 years ago, its virulence mechanisms are poorly understood which is mostly due to its intracellular lifestyle. Recent advances such as the development of axenic culture which allows the growth of this bacterium, that was once believed to be an obligate intracellular pathogen, outside of a host-cell, and new genetic manipulation tools has helped scientists make significant progress in studying this microorganism and identification and characterization of its pathogenesis mechanisms. In the work described in this thesis, we generated a library of C. burnetii Nine Mile phase II (NMII) RSA 439 (that is approved for use in a biosafety level two laboratory) transposon insertion mutants and conducted a visual screen to identify genes required for intracellular replication of this bacterium with the help of indirect immunofluorescence assays. Three mutants with severe intracellular growth defects were found although they showed a normal growth in the bacteriological medium. These three mutants had insertions in genes responsible for encoding D-alanyl-D-alanine carboxypeptidase/D-alanyl-D alanine-endopeptidase, ABC transporter ATP-binding protein and LysM peptidoglycan-binding domain-containing protein. To verify our findings, genomic equivalents of NMII mutants were assessed by real-time PCR which showed a significant reduction in their growth. Since quantitative PCR detects the presence of bacterial DNA but does not indicate viability of the bacteria, colony formation assays were conducted and once again verified our findings and showed a decrease in the intracellular growth rate of these mutants.