Coxiella burnetii is an extremely infectious, obligate intracellular bacterium that causes Q fever, and is classified as a Select B Agent. Q fever is categorized as acute (pneumonitis, malaise) or chronic (hepatitis, endocarditis). Long-term goals are to identify bacterial factors that contribute to C. burnetii's developmental cycle, slow growth rate, and persistence in the environment. three interwoven virulence attributes. Preliminary data show that Coxiella's single 23S rDNA encodes two self-splicing, group I introns that inhibit ribosome function. We hypothesize that group I introns play central roles in C. burnetii's biology by: 1) helping maintain a slow growth rate, thereby fostering chronic infection, 2) promoting dormancy and environmental persistence by inhibiting translation as the bacterium transitions from a physiologically-active cell in the exponential phase (LCV) to a persistent, dormant-cell (SCV) during stationary phase, and 3) being involved in growth and development, since intron RNA (ribozyme) splicing is required to form mature 23S rRNA.
Aim 1 will examine ribosome inhibition by ribozymes. Preliminary data show that both ribozymes associate with ribosomes of Coxiella or E. coli. Data also show that ribozymes retard growth of both bacteria.
Aim 1 will examine ribozyme - ribosome interactions in detail to better understand the mechanisms involved. The first goal will build on preliminary data showing that electroporation of Cbu.L1917 RNA impairs C. burnetii growth. Dose-dependency and the possibility that Cbu.L1917 and Cbu.L1951 elicit differential levels of inhibition will be determined. A second goal will analyze the ribosome subunit targets of each ribozyme. Finally, the role of the internal guide sequence (IGS) in ribosome targeting will be analyzed with IGS mutants and wild-type introns in in vivo and in vitro assays.
Aim 2 will elucidate the role of introns in development. Preliminary data suggest that ribozyme quantities are inversely proportional to Coxiella genomes during exponential phase. We will examine this relationship in long-term cultures to determine whether the opposite occurs as the cell approaches stationary phase. In addition to ribozyme-mediated inhibition of the ribosome, an alternative strategy is to produce fewer, functional 23S rRNAs by decreased splicing efficiency in stationary phase, and this possibility will be examined. Finally, we will determine whether exon skipping occurs;an event that would prevent mature 23S rRNA formation by exclusion of a 34-bp exon flanked by the two introns.
Aim 3 will analyze unusual attributes of each intron. Cbu.L1917 (unique among group I introns in possessing a terminal &A instead of &G) will be assayed for self-splicing efficiency relative to a mutated intron with &G. Cofactors will be analyzed by varying the RNA splicing buffer. Two final goals will follow expression of a potential homing endonuclease (HE) encoded by Cbu.L1951, during development and assay HE for DNA endonuclease (homing) and maturase (splice facilitation) functions in vitro. This study will provide valuable information on the molecular biology of Coxiella and group I introns, and elucidate a possible target for therapeutic intervention of Q fever.
Coxiella burnetii is a zoonotic, bacterial pathogen that causes Q fever in humans and is classified as a select B agent by the CDC. The proposed research will investigate two genetic elements and associated events, hypothesized to contribute to C. burnetii's slow growth rate (fosters formation of chronic infection) and developmental cycle (provides for environmental persistence and enhanced transmission of the agent). In addition to helping address a dearth of information available on C. burnetii's molecular biology, interruption of these genetic elements and events will provide novel targets and strategies for therapeutic intervention of Q fever.
|Warrier, Indu; Walter, Mathias C; Frangoulidis, Dimitrios et al. (2016) The Intervening Sequence of Coxiella burnetii: Characterization and Evolution. Front Cell Infect Microbiol 6:83|
|Hicks, Linda D; Warrier, Indu; Raghavan, Rahul et al. (2011) Ribozyme stability, exon skipping, and a potential role for RNA helicase in group I intron splicing by Coxiella burnetii. J Bacteriol 193:5292-9|
|Minnick, Michael F; Raghavan, Rahul (2011) Genetics of Coxiella burnetii: on the path of specialization. Future Microbiol 6:1297-314|
|Minnick, Michael F; Hicks, Linda D; Battisti, James M et al. (2010) Pentamidine inhibits Coxiella burnetii growth and 23S rRNA intron splicing in vitro. Int J Antimicrob Agents 36:380-2|
|Raghavan, Rahul; Minnick, Michael F (2009) Group I introns and inteins: disparate origins but convergent parasitic strategies. J Bacteriol 191:6193-202|