C. burnetii is an obligate intracellular bacterium and the causative agent of the zoonosis human Q (query) fever. Acute Q fever normally manifests as a debilitating influenza-like illness. Rare but serious chronic infections can occur that usually present as endocarditis. Adding to the insidious nature of the pathogen is an infective dose approaching one organism and spore-like extracellular stability. These attributes have earned C. burnetii designation as a CDC category B biothreat. Environmental resistance also correlates with resistance to the degradative conditions of the pathogen's intracellular niche: the phagolysosome. Genetically distinct isolates of C. burnetii display different phenotypes with respect to in vitro infectivity/cytopathology and pathogenicity for laboratory animals. Moreover, correlations between C. burnetii genomic groups and human disease presentation (acute versus chronic) have been described, suggesting isolates have distinct virulence characteristics. To provide a more complete understanding of C. burnetii genetic diversity, evolution, and pathogenic potential, we deciphered the whole genome sequences of the K (Q154) and G (Q212) human chronic endocarditis isolates and the naturally attenuated Dugway (5J108-111) rodent isolate. Cross-genome comparisons that included the previously-sequenced Nine Mile (NM) reference isolate (RSA493) revealed both novel gene content and disparate collections of pseudogenes that may contribute to isolate virulence and other phenotypes. While C. burnetii genomes are highly syntenous, recombination between abundant insertion sequence (IS) elements has resulted in genome plasticity manifested as chromosomal rearrangement of syntenic blocks and DNA insertions/deletions. The numerous IS elements, genomic rearrangements, and pseudogenes of C. burnetii isolates is consistent with genome structures of other bacterial pathogens that have recently emerged from non-pathogens with expanded niches. The observation that the severely attenuated Dugway isolate has the largest genome with the fewest pseudogenes and IS elements suggests this isolate lineage is at an earlier stage of pathoadaptation than the NM, K, and G lineages. The lack of methods to genetically manipulate C. burnetii significantly impedes study of the organism. We have successfully transformed C. burnetii to chloramphenicol resistance and mCherry red fluorescent protein expression using the Himar1 transposon (Tn) system. Both chloramphenicol acetyltransferase (CAT) and mCherry were expressed as a single transcriptional unit under control of the C. burnetii Hsp20 promoter p1169. Rescue cloning of the ColE1 origin of replication and DNA sequencing revealed Tn insertion sites scattered throughout the C. burnetii genome. A clone from the transformant mixture was isolated using our micromanipulation cloning method and shown to harbor a Tn insertion within the essential cell division gene ftsZ. Characterization of the FtsZ::Tn mutant revealed a generation time during exponential phase of 19.8 h, almost twice as long as wild type C. burnetii (11.7 h). This is the first description of C. burnetii harboring a defined gene mutation generated by genetic transformation. Importantly, this study shows that the Himar1 transposon system is a robust technique for creating genetic mutations in C. burnetii. While expression of CAT was sufficient to prevent outgrowth of non-transformed bacteria, expression of mCherry was moderate and suboptimal for visualization of transformed organisms by fluorescence microscopy. Therefore, we examined the use of the outer membrane porin P1 (CBU0311) promoter p311 to drive mCherry expression. Moreover, kanamycin resistance as an alternative method of positive selection was tested in addition to host cell-free (axenic) growth of electroporated organisms in acidified citrate cysteine medium (ACCM) as an initial step in expansion of transformants. Expression of mCherry was substantially higher when driven from p311 verses p1169. Indeed, individual organisms were easily visible by fluorescence microscopy. As with infection of Vero cells, C. burnetii transformants initially expanded in ACCM were positive for CAT DNA and resistant to chloramphenicol. However, transformants were detected in 1-2 weeks in ACCM as compared to 4-5 weeks in Vero cells. Kanamycin, which is also not used in the clinical treatment of Q fever, was also tested as an alternative selective marker by transforming C. burnetii with a Himar1 transposon containing the kanamycin resistance gene under control of p1169. Outgrowth of transformants in ACCM containing 250 ug/ml kanamycin was observed with no outgrowth of non-transformed control organisms. Not only does the Himar1 transposon allow random mutagenesis and stable integration of transgenes in C. burnetii, it also provides a tool to test and optimize different aspects of the organisms evolving genetic transformation systems. Moreover, axenic growth of electroporated C. burnetii in ACCM substantially decreases the time of initial outgrowth of transformants in addition to allowing selection of transformants that would otherwise be lethal for growth in host cells. Lipopolysaccharide is the only defined virulence factor of C. burnetii. Virulent phase I organisms, producing full-length LPS, convert to avirulent phase II organisms,synthesizing severely truncated LPS, upon repeated in vitro passages. The genetic lesion(s) accounting for the deep rough phenotype of phase II isolates is unknown. To this end, we generated phase II clones of the high passage Australian and California strains, using our micromanipulation cloning procedure, and hybridized their genomic DNAs to a high-density microarray that contains probe sets encompassing all full-length open reading frames of the Nine Mile phase I strain. These arrays are specifically designed to detect indels (insertions/deletions). A common indel was found within a gene involved in heptose biosynthesis that we believe accounts for phase conversion. A sensitive and specific serodiagnostic test is needed for Q fever that utilizes recombinant C. burnetii protein(s) as antigen. To pursue this goal, we developed a C. burnetii protein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of human C. burnetii infection or vaccination. Transcriptionally active PCR products corresponding to 1988 C. burnetii open reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using an E. coli-based cell-free in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50 C. burnetii proteins including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated and immunoreactivity confirmed by ELISA. This sensitive and high throughput method for identifying immunoreactive C. burnetii proteins will aid development of Q fever serodiagnostic tests based on recombinant antigen. Moreover, testing of microarray-identified antigens for T-cell antigenicity may identify proteins with efficacy as subunit vaccines against Q fever.
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