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Genetic diversity of the Q fever agent Coxiella burnetii Paul Beare

Genetic diversity of the Q fever agent Coxiella burnetii Paul Beare. Coxiella Pathogenesis Section Rocky Mountain Laboratories LICP/NIAID/NIH. Coxiella burnetii (1937- ) Obligate intracellular Gram-negative bacterium Causative agent of query (Q) fever

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Genetic diversity of the Q fever agent Coxiella burnetii Paul Beare

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  1. Genetic diversity of the Q fever agent Coxiella burnetii Paul Beare Coxiella Pathogenesis Section Rocky Mountain Laboratories LICP/NIAID/NIH

  2. Coxiella burnetii (1937- ) • Obligate intracellular Gram-negative bacterium • Causative agent of query (Q) fever • Can manifest as acute or chronic disease • Replicates in a lysosome-like vacoule • Exists as two different developmental forms • small cell and large cell variant • No systems for genetic manipulation • Category B bioterrorism agent Coleman et al, J Bact 2004

  3. Ubiquitous 4-legged “bioterrorists”

  4. Ubiquitous 4-legged “bioterrorists”

  5. Ubiquitous 4-legged “bioterrorists”

  6. Ubiquitous 4-legged “bioterrorists”

  7. Coxiella - Genome characteristics • Sequence of the Nine Mile phase I RSA493 reference isolate has been determined (Seshadri et al, PNAS, 2003) • Genome consists of a 1,995,281 bp circular chromosome and 37,393 bp plasmid • ~2,134 coding sequences (~34% hypothetical) • 29 insertion sequence elements • 83 pseudogenes • Restriction fragment length polymorphism (RFLP) and multispacer sequence typing analyses reveal a range of genetic diversity between different isolates

  8. Coxiella – Isolate differentiation • RFLP analysis • Restriction enzyme fragmentation of genomic DNA followed by an analysis of the associated fragments • Multispacer sequence typing • Based on the sequencing of multiple intergenic regions

  9. Isolates used in this study Genomic group I I I I I I I I I I I I I II III IV IV IV V V V VI UN UN UN UN Plasmid type QpH1 QpH1 QpH1 QpH1 QpH1 QpH1 QpH1 QpH1 QpH1 QpH1 QpH1 QpH1 QpH1 QpH1 QpH1 QpRS QpRS QpRS IP IP IP QpDG QpDV PTU PTU PTU Isolate Nine Mile RSA493 Nine Mile RSA439 Nine Mile RSA514 (Crazy) Australia QD RSA425 D RSA345 Turkey RSA333 African RSA 334 Giroud RSA431 El Tayeb RSA 342 Panama RSA335 California 33 RSA329 California 16 RSA350 Ohio 314 RSA370 Henzerling RSA331 Idaho Goat Q195 MSU Goat Q177 K Q154 P Q173 G Q212 S Q217 Ko Q229 Dugway 7E9-12 Q321 Dugway 5G61-63 BDT1 Le Bruges Original source, yr Montana, tick, 1935 Montana, tick, 1935 Montana, tick, 1935 Australia, human blood, ~1939 USA, human blood, 1938 Turkey, human blood, 1948 Central Africa, human blood, 1949 Central Africa, human blood, 1949 Egypt, tick, 1967 Panama, chiggers, 1961 California, cow’s milk, 1947 California, cow’s milk, 1947 Ohio, cow’s milk, 1956 Italy, human blood, 1945 Idaho, goat, 1981 Montana, goat cotyledon, 1980 Oregon, human heart valve, 1976 California, human heart valve, 1979 Nova Scotia, human heart valve, 1981 Montana, human livery biopsy, 1981 Nova Scotia, human heart valve, 1982 Utah, rodent, 1958 Russia, cow’s milk Utah, tick, 1958 USA, brown dog tick, 1951 France, unknown source Disease - - - Acute Acute Acute Acute Acute - - Persistant Persistant Persistent Acute Abortion Abortion Endocarditis Endocarditis Endocarditis Hepatitis Endocarditis - Persistant - - -

  10. Study aims • Examine the genetic diversity of Coxiella isolates • Phylogenetically group ungrouped isolates • Examine the potential link between pathotype and genome profile • Estimate the core Coxiella genome

  11. The Coxiella microarray • Multipathogen Affymetrix expression array (RMLchip) • Covers 1,988 chromosomal and 36 plasmid ORFs of the Nine Mile phase I RSA493 reference isolate • ~249,690 Coxiella-specific 25-mer probe pairs • Each Coxiella ORF is represented by 16 probe pairs • Maximum of 400 bp coverage for each ORF • Total of ~30% coverage of the Coxiella genome

  12. Comparative genome hybridization - CGH gDNA Mo Bio microbial gDNA isolation kit Fragmented DNA DNaseI treatment Terminal biotinylation RMLchip Expression Array Fragmented, Biotin-labeled DNA Target Hybridization Washing and Staining Scanning and Data Analysis Image adapted from www.affymetrix.com

  13. Key: Present ORF Absent ORF Partial ORF ORF containing a PM or small insertion CGH of 24 Coxiella isolates – chromosome polymorphisms Group IV Group VI Group III Group V Group II Group I Du 5G61-63 Le Bruges Q321 BDT • 24 Coxiella isolates were compared to the Nine Mile phase I (RSA493) reference isolate. • All isolates within a previously defined RFLP genomic group were identical • A total of 139 polymorphisms were identified • PCR validation was carried out on all polymorphisms Beare et al, J Bact, 2006

  14. Group I Group V Group IV Transposase-mediated genome plasticity • Transposase-mediated genome rearrangements result in loss of gene content

  15. Key: Present ORF Absent ORF Partial ORF ORF containing a PM or small insertion CGH of 24 Coxiella isolates – chromosome polymorphisms Group IV Group VI Group III Group V Group II Group I Du 5G61-63 Le Bruges Q321 BDT • 24 Coxiella isolates were compared to the Nine Mile phase I (RSA493) reference isolate. • All isolates within a previously defined RFLP genomic group were identical • A total of 139 polymorphisms were identified • PCR validation was carried out on all polymorphisms Beare et al, J Bact, 2006

  16. Key: Present ORF Absent ORF Partial ORF ORF containing a PM or small insertion CGH of 24 Coxiella isolates – plasmid polymorphisms Du 5G61-63 Group IV Group VI Le Bruges Group III Group V Group II Group I Q321 BDT QpDG QpRS QpDV QpH1 QpH1 QpH1 QpH1 IP • The BDT and Dugway 5G61-63 have the same profile as genomic group I and contain the QpH1 plasmid • Q321 and Le Bruges both have the same unique profile, suggesting that Le Bruges has the QpDV plasmid Beare et al, J Bact, 2006

  17. Phylogenetic analysis of Coxiella isolates • The BDT and Du 5G61- 63 isolates reside in the genomic group I • Q321 and Le Bruges represent two new genomic groups, VII and VIII respectively Beare et al, J Bact, 2006

  18. Functions of polymorphic genes • 67% of polymorphic ORFs encode hypothetical proteins • 12% of polymorphic ORFs reside in pseudogenes • The remaining ORFs have a range of functions; • Ankyrin repeat proteins • Transporters • Transcriptional regulators • Proteins that are part of large gene families

  19. CGH analysis of small DNA samples • Single Coxiella containing vacuoles were isolated by micropipette • DNA contained in the vacuole suspension was amplified using a phi29 polymerase-based whole genome amplification (WGA) kit VERO cell monolayer infected with Coxiella

  20. Reaction Components Phi29 DNA Polymerase Random Hexamer Primers Input DNA Primers bind to template Polymerization begins Polymerization continues Strand displacement Polymerization from new strands New primers bind to newly formed DNA Whole Genome Amplification

  21. CGH analysis of small DNA samples • Single Coxiella containing vacuoles were isolated by micropipette • DNA contained in the vacuole suspension was amplified using a phi29 polymerase-based whole genome amplification (WGA) kit • Following DNA precipitation, 10 mg of DNA was hybridized to the Coxiella microarray • Results obtained using WGA DNA were identical to those of non- amplified genomic DNA VERO cell monolayer infected with Coxiella

  22. Coxiella lipopolysaccharide (LPS) phase variation • LPS is the only defined Coxiella virulence factor • Virulent phase I have full length LPS • Avirulent phase II have severely truncated LPS • In vitro serial passing of virulent phase I results in avirulent phase II

  23. NMC NMII NMI 66.2 45.0 31.0 21.5 14.4 LPS phase variation in Coxiella • Nine Mile phase I (NMI) has a full length LPS and is virulent • Two cloned variants of NMI have been isolated • Nine Mile Crazy (NMC) has an intermediate LPS length and intermediate virulence • Nine Mile phase II (NMII) has a severely truncated LPS and is avirulent

  24. NMI NMII NMC NMC NMII NMI 66.2 45.0 31.0 Region containing genes involved in LPS O-antigen biosynthesis 21.5 14.4 CGH analysis of Coxiella LPS variants • CGH analysis of the NMII and NMC variants indicated the presence of only a single large genome deletion, which has been previously described (Hoover et al, Infect. Immun., 2002) • CGH could not explain the why NMII, with a smaller deletion, produces a more truncated LPS

  25. Nimblegen DNA tiling microarrays • A 27 bp overlapping oligonucleotide tiling microarray was designed based on the NMI genome sequence • NMII gDNA (Test DNA) was compared to NMI gDNA (Reference DNA)

  26. Analysis of LPS variation - Tiling microarray of NMII NMI NLGNLFFLGDLKIGLWAIPITVIVVLANINAMNMIDGQDGLAGGVALGQALLLLLVSVKL 177 NLGNLFFLGDLKIGLWAIPITVIVVLANINAMNMIDGQDGLAGGVALGQA-LLLLVSVKL 176 NMII • NMII has 14 polymorphisms relative to NMI • One polymorphism resides in an ORF (CBU0533) predicted to be involved in LPS biosynthesis (wecA) • This polymorphism is a 3 bp deletion that results in an inframe deletion of L168

  27. The WecA protein of Escherichia coli • A UDP-N-acetylglucosamine (GlcNAc):undecaprenyl-phosphate (Und-P) GlcNAc-1-phosphate transferase • Participates in the biosynthesis of O antigen LPS in many enteric bacteria • Transfers GlcNAc 1-phosphate from UDP-GlcNAc onto Und-P to form Und-P-P-GlcNAc • Product of this reaction serves as an acceptor for the addition of subsequent sugars to complete the biosynthesis of O antigen • Deletion of wecA in E. coli results in a truncated LPS

  28. Analysis of LPS variation - Tiling microarray of NMII NMI NLGNLFFLGDLKIGLWAIPITVIVVLANINAMNMIDGQDGLAGGVALGQALLLLLVSVKL 177 NLGNLFFLGDLKIGLWAIPITVIVVLANINAMNMIDGQDGLAGGVALGQA-LLLLVSVKL 176 SLGYIFGSWEMVLGPFGYFLTLFAVWAAINAFNMVDGIDGLLGGLSCVSFAAIGMILWFD 180 .** :* :: :* :. :*::.* * ***:**:** *** **:: . : :: NMII E. coli • L168 is predicted to be in the transmembrane region (colored green) immediately downstream of the WecA active site • Preliminary evidence in an E. coliwecA mutant indicates that the NMII WecA is not properly inserted into the inner membrane

  29. Conclusions • Genetic variation was observed within Coxiella isolates • Coxiella isolates of unknown genetic backgrounds can be typed and the extent of their genetic variation relative to NMI analyzed • Transposases play an important role in Coxiella genetic variation • Potential pathotype-specific genes have been identified • Using WGA DNA, very small samples (~1 ng of total DNA) can be analyzed by CGH • A mutation potentially involved in Coxiella LPS phase variation was identified • The CBU0533 mutation is also found in a number of isolates producing a severely truncated LPS

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