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1.  The Unusual 23S rRNA Gene of Coxiella burnetii: Two Self-Splicing Group I Introns Flank a 34-Base-Pair Exon, and One Element Lacks the Canonical ΩG▿  
Journal of Bacteriology  2007;189(18):6572-6579.
We describe the presence and characteristics of two self-splicing group I introns in the sole 23S rRNA gene of Coxiella burnetii. The two group I introns, Cbu.L1917 and Cbu.L1951, are inserted at sites 1917 and 1951 (Escherichia coli numbering), respectively, in the 23S rRNA gene of C. burnetii. Both introns were found to be self-splicing in vivo and in vitro even though the terminal nucleotide of Cbu.L1917 is adenine and not the canonical conserved guanine, termed ΩG, found in Cbu.L1951 and all other group I introns described to date. Predicted secondary structures for both introns were constructed and revealed that Cbu.L1917 and Cbu.L1951 were group IB2 and group IA3 introns, respectively. We analyzed strains belonging to eight genomic groups of C. burnetii to determine sequence variation and the presence or absence of the elements and found both introns to be highly conserved (≥99%) among them. Although phylogenetic analysis did not identify the specific identities of donors, it indicates that the introns were likely acquired independently; Cbu.L1917 was acquired from other bacteria like Thermotoga subterranea and Cbu.L1951 from lower eukaryotes like Acanthamoeba castellanii. We also confirmed the fragmented nature of mature 23S rRNA in C. burnetii due to the presence of an intervening sequence. The presence of three selfish elements in C. burnetii's 23S rRNA gene is very unusual for an obligate intracellular bacterium and suggests a recent shift to its current lifestyle from a previous niche with greater opportunities for lateral gene transfer.
doi:10.1128/JB.00812-07
PMCID: PMC2045182  PMID: 17644584
2.  Toxic Introns and Parasitic Intein in Coxiella burnetii: Legacies of a Promiscuous Past▿  
Journal of Bacteriology  2008;190(17):5934-5943.
The genome of the obligate intracellular pathogen Coxiella burnetii contains a large number of selfish genetic elements, including two group I introns (Cbu.L1917 and Cbu.L1951) and an intervening sequence that interrupts the 23S rRNA gene, an intein (Cbu.DnaB) within dnaB and 29 insertion sequences. Here, we describe the ability of the intron-encoded RNAs (ribozymes) to retard bacterial growth rate (toxicity) and examine the functionality and phylogenetic history of Cbu.DnaB. When expressed in Escherichia coli, both introns repressed growth, with Cbu.L1917 being more inhibitory. Both ribozymes were found to associate with ribosomes of Coxiella and E. coli. In addition, ribozymes significantly reduced in vitro luciferase translation, again with Cbu.L1917 being more inhibitory. We analyzed the relative quantities of ribozymes and genomes throughout a 14-day growth cycle of C. burnetii and found that they were inversely correlated, suggesting that the ribozymes have a negative effect on Coxiella's growth. We determined possible sites for ribozyme associations with 23S rRNA that could explain the observed toxicities. Further research is needed to determine whether the introns are being positively selected because they promote bacterial persistence or whether they were fixed in the population due to genetic drift. The intein, Cbu.DnaB, is able to self-splice, leaving the host protein intact and presumably functional. Similar inteins have been found in two extremophilic bacteria (Alkalilimnicola ehrlichei and Halorhodospira halophila) that are distantly related to Coxiella, making it difficult to determine whether the intein was acquired by horizontal gene transfer or was vertically inherited from a common ancestor.
doi:10.1128/JB.00602-08
PMCID: PMC2519523  PMID: 18606739
3.  Ribozyme Stability, Exon Skipping, and a Potential Role for RNA Helicase in Group I Intron Splicing by Coxiella burnetii▿ 
Journal of Bacteriology  2011;193(19):5292-5299.
The 23S rRNA gene of Coxiella burnetii, the agent of Q fever in humans, contains an unusually high number of conserved, selfish genetic elements, including two group I introns, termed Cbu.L1917 (L1917) and Cbu.L1951 (L1951). To better understand the role that introns play in Coxiella's biology, we determined the intrinsic stability time periods (in vitro half-lives) of the encoded ribozymes to be ∼15 days for L1917 and ∼5 days for L1951, possibly due to differences in their sizes (551 and 1,559 bases, respectively), relative degrees of compactness of the respective RNA structures, and amounts of single-stranded RNA. In vivo half-lives for both introns were also determined to be ∼11 min by the use of RNase protection assays and an Escherichia coli model. Intron RNAs were quantified in synchronous cultures of C. burnetii and found to closely parallel those of 16S rRNA; i.e., ribozyme levels significantly increased between days 0 and 3 and then remained stable until 8 days postinfection. Both 16S rRNA and ribozyme levels fell during the stationary and death phases (days 8 to 14). The marked stability of the Coxiella intron RNAs is presumably conferred by their association with ribosomes, a stoichiometric relationship that was determined to be one ribozyme, of either type, per 500 ribosomes. Inaccuracies in splicing (exon 2 skipping) were found to increase during the first 5 days in culture, with a rate of approximately one improperly spliced 23S rRNA per 1.3 million copies. The in vitro efficiency of L1917 intron splicing was significantly enhanced in the presence of a recombinant Coxiella RNA DEAD-box helicase (CBU_0670) relative to that of controls, suggesting that this enzyme may serve as an intron RNA splice facilitator in vivo.
doi:10.1128/JB.05472-11
PMCID: PMC3187410  PMID: 21803999
4.  A Unique Group I Intron in Coxiella burnetii Is a Natural Splice Mutant▿  
Journal of Bacteriology  2009;191(12):4044-4046.
Cbu.L1917, a group I intron present in the 23S rRNA gene of Coxiella burnetii, possesses a unique 3′-terminal adenine in place of a conserved guanine. Here, we show that, unlike all other group I introns, Cbu.L1917 utilizes a different cofactor for each splicing step and has a decreased self-splicing rate in vitro.
doi:10.1128/JB.00359-09
PMCID: PMC2698393  PMID: 19376857
5.  Pentamidine Inhibition of Group I Intron Splicing in Candida albicans Correlates with Growth Inhibition 
We previously demonstrated that pentamidine, which has been clinically used against Pneumocystis carinii, inhibits in vitro a group I intron ribozyme from that organism. Another fungal pathogen, Candida albicans, also harbors a group I intron ribozyme (Ca.LSU) in the essential rRNA genes in almost half of the clinical isolates analyzed. To determine whether pentamidine inhibits Ca.LSU in vitro and in cells, phylogenetically closely related intron-containing (4-1) and intronless (62-1) strains were studied. Splicing in vitro of the Ca.LSU group I intron ribozyme was completely inhibited by pentamidine at 200 μM. On rich glucose medium, the intron-containing strain was more sensitive to growth inhibition by pentamidine than was the intronless strain, as measured by disk or broth microdilution assays. On rich glycerol medium, they were equally susceptible to pentamidine. At pentamidine levels selectively inhibiting the intron-containing strain (1 μM) in glucose liquid cultures, inhibition of splicing and rRNA maturation was detected by quantitative reverse transcription-PCR within 1 min with a 10- to 15-fold accumulation of precursor rRNA. No comparable effect was seen in the intronless strain. These results correlate the cellular splicing inhibition of Ca.LSU with the growth inhibition of strain 4-1 harboring Ca.LSU. Broth microdilution assays of 13 Candida strains showed that intron-containing strains were generally more susceptible to pentamidine than the intronless strains. Our data suggest that ribozymes found in pathogenic microorganisms but absent in mammals may be targets for antimicrobial therapy.
PMCID: PMC89798  PMID: 10722497
6.  Coxiella burnetii Alters Cyclic AMP-Dependent Protein Kinase Signaling during Growth in Macrophages 
Infection and Immunity  2012;80(6):1980-1986.
Coxiella burnetii is the bacterial agent of human Q fever, an acute, flu-like illness that can present as chronic endocarditis in immunocompromised individuals. Following aerosol-mediated transmission, C. burnetii replicates in alveolar macrophages in a unique phagolysosome-like parasitophorous vacuole (PV) required for survival. The mechanisms of C. burnetii intracellular survival are poorly defined and a recent Q fever outbreak in the Netherlands emphasizes the need for better understanding this unique host-pathogen interaction. We recently demonstrated that inhibition of host cyclic AMP-dependent protein kinase (PKA) activity negatively impacts PV formation. In the current study, we confirmed PKA involvement in PV biogenesis and probed the role of PKA signaling during C. burnetii infection of macrophages. Using PKA-specific inhibitors, we found the kinase was needed for biogenesis of prototypical PV and C. burnetii replication. PKA and downstream targets were differentially phosphorylated throughout infection, suggesting prolonged regulation of the pathway. Importantly, the pathogen actively triggered PKA activation, which was also required for PV formation by virulent C. burnetii isolates during infection of primary human alveolar macrophages. A subset of PKA-specific substrates were differentially phosphorylated during C. burnetii infection, suggesting the pathogen uses PKA signaling to control distinct host cell responses. Collectively, the current results suggest a versatile role for PKA in C. burnetii infection and indicate virulent organisms usurp host kinase cascades for efficient intracellular growth.
doi:10.1128/IAI.00101-12
PMCID: PMC3370589  PMID: 22473604
7.  Pentamidine inhibits catalytic activity of group I intron Ca.LSU by altering RNA folding 
Nucleic Acids Research  2002;30(13):2961-2971.
The antimicrobial agent pentamidine inhibits the self-splicing of the group I intron Ca.LSU from the transcripts of the 26S rRNA gene of Candida albicans, but the mechanism of pentamidine inhibition is not clear. We show that preincubation of the ribozyme with pentamidine enhances the inhibitory effect of the drug and alters the folding of the ribozyme in a pattern varying with drug concentration. Pentamidine at 25 µM prevents formation of the catalytically active F band conformation of the precursor RNA and alters the ribonuclease T1 cleavage pattern of Ca.LSU RNA. The effects on cleavage suggest that pentamidine mainly binds to specific sites in or near asymmetric loops of helices P2 and P2.1 on the ribozyme, as well as to the tetraloop of P9.2 and the loosely paired helix P9, resulting in an altered structure of helix P7, which contains the active site. Positively charged molecules antagonize pentamidine inhibition of catalysis and relieve the drug effect on ribozyme folding, suggesting that pentamidine binds to a magnesium binding site(s) of the ribozyme to exert its inhibitory effect.
PMCID: PMC117049  PMID: 12087182
8.  A unique Coxiella burnetii lipoprotein involved in metal binding (LimB) 
Microbiology  2011;157(Pt 4):966-976.
Coxiella burnetii is the bacterial agent of Q fever in humans. Here, we describe a unique, ∼7.2 kDa, surface-exposed lipoprotein involved in metal binding which we have termed LimB. LimB was initially identified as a potential metal-binding protein on far-Western (FW) blots containing whole-cell lysate proteins when probed with nickel-coated horseradish peroxidase (Ni-HRP) and developed with a chemiluminescent HRP substrate. The corresponding identity of LimB as CBU1224a was established by matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry. blast analyses with CBU1224a showed no significant similarity to sequences outside strains of C. burnetii. Additional in silico analyses revealed a putative 20 residue signal sequence with the carboxyl end demarcated by a potential lipobox (LSGC) whose Cys residue is predicted to serve as the N-terminal, lipidated Cys of mature LimB. The second residue of mature LimB is predicted to be Ala, an uncharged envelope localization residue. These features suggest that CBU1224a is synthesized as a prolipoprotein which is subsequently lipidated, secreted and anchored in the outer membrane. Mature LimB is predicted to contain 45 aa, of which there are 10 His and 5 Cys; both amino acids are frequently involved in binding transition metal cations. Recombinant LimB (rLimB) was generated and its Ni-HRP-binding activity demonstrated on FW blots. Ni-HRP binding by rLimB was inhibited by >95 % on FW blots done in the presence of EDTA, imidazole, Ni2+ or Zn2+, and roughly halved in the presence of Co2+ or Fe3+. The limB gene was maximally expressed at 3–7 days post-infection in Coxiella-infected Vero cells, coinciding with exponential phase growth. Two isoforms of LimB were detected on FW and Western blots, including a smaller (∼7.2 kDa) species that was the predominant form in small cell variants and a larger isoform (∼8.7 kDa) in large cell variants. LimB is Sarkosyl-insoluble, like many omps. The predicted surface location of LimB was verified by immunoelectron and immunofluorescence microscopy using anti-rLimB antibodies. Overall, the results suggest that LimB is a unique Coxiella lipoprotein that serves as a surface receptor for divalent metal cations and may play a role in acquiring at least one of these metals during intracellular growth.
doi:10.1099/mic.0.046649-0
PMCID: PMC3139440  PMID: 21212117
9.  Chronic Q Fever in the United States 
Journal of Clinical Microbiology  2006;44(6):2283-2287.
Infections due to Coxiella burnetii, the causative agent of Q fever, are uncommon in the United States. Cases of chronic Q fever are extremely rare and most often manifest as culture-negative endocarditis in patients with underlying valvular heart disease. We describe a 31-year-old farmer from West Virginia with a history of congenital heart disease and recurrent fevers for 14 months who was diagnosed with Q fever endocarditis based on an extremely high antibody titer against Coxiella burnetii phase I antigen. Despite treatment with doxycycline, he continued to have markedly elevated Coxiella burnetii phase I antibody titers for 10 years after the initial diagnosis. To our knowledge, this case represents the longest follow-up period for a patient with chronic Q fever in the United States. We review all cases of chronic Q fever reported in the United States and discuss important issues pertaining to epidemiology, diagnosis, and management of this disease.
doi:10.1128/JCM.02365-05
PMCID: PMC1489455  PMID: 16757641
10.  Q fever — a review 
The Canadian Veterinary Journal  1990;31(8):555-563.
Q or “query” fever is a zoonosis caused by the organism Coxiella burnetii. Cattle, sheep and goats are the most common reservoirs of this organism. The placenta of infected animals contains high numbers (up to 109/g) of C. burnetii. Aerosols occur at the time of parturition and man becomes infected following inhalation of the microorganism. The spectrum of illness in man is wide and consists of acute and chronic forms. Acute Q fever is most often a self-limited flu-like illness but may include pneumonia, hepatitis, or meningoencephalitis. Chronic Q fever almost always means endocarditis and rarely osteomyelitis. Chronic Q fever is not known to occur in animals other than man. An increased abortion and stillbirth rate are seen in infected domestic ungulates.
Four provinces (Nova Scotia, New Brunswick, Ontario and Alberta) reported cases of Q fever in 1989.
A vaccine for Q fever has recently been licensed in Australia.
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PMCID: PMC1480833  PMID: 17423643
11.  Interleukin-10 Stimulates Coxiella burnetii Replication in Human Monocytes through Tumor Necrosis Factor Down-Modulation: Role in Microbicidal Defect of Q Fever 
Infection and Immunity  2001;69(4):2345-2352.
Coxiella burnetii, an obligate intracellular bacterium, is the agent of Q fever. The chronic form of the disease is associated with the overproduction of interleukin-10 and deficient C. burnetii killing by monocytes. We hypothesized that the replication of C. burnetii inside monocytes requires a macrophage-deactivating cytokine such as interleukin-10. In the absence of interleukin-10, C. burnetii survived but did not replicate in monocytes. C. burnetii replication (measured 15 days) was induced in interleukin-10-treated monocytes. This effect of interleukin-10 is specific since transforming growth factor β1 had no effect on bacterial replication. C. burnetii replication involves the down-modulation of tumor necrosis factor (TNF) release. First, interleukin-10 suppressed C. burnetii-stimulated production of TNF. Second, the addition of recombinant TNF to interleukin-10-treated monocytes inhibited bacterial replication. Third, the incubation of infected monocytes with neutralizing anti-TNF antibodies favored C. burnetii replication. On the other hand, deficient C. burnetii killing by monocytes from patients with chronic Q fever involves interleukin-10. Indeed, C. burnetii replication was observed in monocytes from patients with Q fever endocarditis, but not in those from patients with acute Q fever. Bacterial replication was inhibited by neutralizing anti-interleukin-10 antibodies. As monocytes from patients with endocarditis overproduced interleukin-10, the defective bacterial killing is likely related to endogenous interleukin-10. These results suggest that interleukin-10 enables monocytes to support C. burnetii replication and to favor the development of chronic Q fever.
doi:10.1128/IAI.69.4.2345-2352.2001
PMCID: PMC98164  PMID: 11254592
12.  Effector Protein Translocation by the Coxiella burnetii Dot/Icm Type IV Secretion System Requires Endocytic Maturation of the Pathogen-Occupied Vacuole 
PLoS ONE  2013;8(1):e54566.
The human pathogen Coxiella burnetii encodes a type IV secretion system called Dot/Icm that is essential for intracellular replication. The Dot/Icm system delivers bacterial effector proteins into the host cytosol during infection. The effector proteins delivered by C. burnetii are predicted to have important functions during infection, but when these proteins are needed during infection has not been clearly defined. Here, we use a reporter system consisting of fusion proteins that have a β-lactamase enzyme (BlaM) fused to C. burnetii effector proteins to study protein translocation by the Dot/Icm system. Translocation of BlaM fused to the effector proteins CBU0077, CBU1823 and CBU1524 was not detected until 8-hours after infection of HeLa cells, which are permissive for C. burnetii replication. Translocation of these effector fusion proteins by the Dot/Icm system required acidification of the Coxiella-containing vacuole. Silencing of the host genes encoding the membrane transport regulators Rab5 or Rab7 interfered with effector translocation, which indicates that effectors are not translocated until bacteria traffic to a late endocytic compartment in the host cell. Similar requirements for effector translocation were discerned in bone marrow macrophages derived from C57BL/6 mice, which are primary cells that restrict the intracellular replication of C. burnetii. In addition to requiring endocytic maturation of the vacuole for Dot/Icm-mediated translocation of effectors, bacterial transcription was required for this process. Thus, translocation of effector proteins by the C. burnetii Dot/Icm system occurs after acidification of the CCV and maturation of this specialized organelle to a late endocytic compartment. This indicates that creation of the specialized vacuole in which C. burnetii replicates represents a two-stage process mediated initially by host factors that regulate endocytic maturation and then by bacterial effectors delivered into host cells after bacteria establish residency in a lysosome-derived organelle.
doi:10.1371/journal.pone.0054566
PMCID: PMC3547880  PMID: 23349930
13.  Coxiella burnetii blood cultures from acute and chronic Q-fever patients. 
Journal of Clinical Microbiology  1995;33(12):3129-3132.
Q fever, a worldwide zoonosis caused by Coxiella burnetii, may present as either an acute or a chronic disease. We correlated the results of 844 C. burnetii blood cultures with serological, clinical, and therapeutic data. C. burnetii was isolated from 17% of untreated patients with acute Q fever and from 53% of untreated patients with chronic Q fever. C. burnetii was not isolated from patients who were receiving antibiotics active against C. burnetii. For seven culture-positive patients with acute Q fever, serology was negative when C. burnetii was isolated. One patient with acute Q fever had a positive blood culture 25 days after the discontinuation of specific antibiotic therapy, and another had a positive blood culture after the resolution of symptoms. In one case of chronic Q fever, a positive blood culture resulted from noncompliance with treatment. The culture method described in this report is suitable for all laboratories with cell culture facilities. Our findings suggest that blood samples must be collected prior to the initiation of an antibiotic regimen if C. burnetii is to be successfully isolated.
PMCID: PMC228658  PMID: 8586687
14.  Chronic Q Fever-Related Dual-Pathogen Endocarditis: Case Series of Three Patients▿ 
Journal of Clinical Microbiology  2011;49(4):1692-1694.
Following Coxiella burnetii infection, there is a 1 to 5% risk of chronic Q fever. Endocarditis, mycotic aneurysm, and vascular prosthesis infection are common manifestations. We present three patients with endocarditis by C. burnetii concomitant with another bacterial pathogen. Chronic Q fever should therefore be considered in all endocarditis patients in regions where Q fever is endemic.
doi:10.1128/JCM.02596-10
PMCID: PMC3122851  PMID: 21289146
15.  Coxiella burnetii Survives in Monocytes from Patients with Q Fever Endocarditis: Involvement of Tumor Necrosis Factor 
Infection and Immunity  2000;68(1):160-164.
Endocarditis is the most frequent form of chronic Q fever, an infectious disease caused by Coxiella burnetii. As this obligate intracellular bacterium inhabits monocytes and macrophages, we wondered if pathogenesis of Q fever endocarditis is related to defective intracellular killing of C. burnetii by monocytes. Monocytes from healthy controls eliminated virulent C. burnetii within 3 days. In contrast, monocytes from patients with ongoing Q fever endocarditis were unable to eliminate bacteria even after 6 days. In patients who were cured of endocarditis, the monocyte infection was close to that of control monocytes. This killing deficiency was not the consequence of generalized functional impairment, since patient monocytes eliminated avirulent C. burnetii as did control cells. The addition of supernatants of C. burnetii-stimulated monocytes from patients with ongoing endocarditis to control monocytes enabled them to support C. burnetii survival, suggesting that some soluble factor is responsible for bacterial survival. This factor was related to tumor necrosis factor (TNF): expression of TNF mRNA and TNF release were increased in response to C. burnetii in patients with ongoing endocarditis compared to cured patients and healthy controls. In addition, neutralizing anti-TNF antibodies decreased C. burnetii internalization, an early step of bacterial killing, in monocytes from patients with ongoing endocarditis but did not affect delayed steps of intracellular killing. We suggest that Q fever-associated activation of monocytes allows the survival of C. burnetii by modulating early phases of microbial killing.
PMCID: PMC97115  PMID: 10603382
16.  Coxiella burnetii Isolates Cause Genogroup-Specific Virulence in Mouse and Guinea Pig Models of Acute Q Fever▿ † 
Infection and Immunity  2009;77(12):5640-5650.
Q fever is a zoonotic disease of worldwide significance caused by the obligate intracellular bacterium Coxiella burnetii. Humans with Q fever may experience an acute flu-like illness and pneumonia and/or chronic hepatitis or endocarditis. Various markers demonstrate significant phylogenetic separation between and clustering among isolates from acute and chronic human disease. The clinical and pathological responses to infection with phase I C. burnetii isolates from the following four genomic groups were evaluated in immunocompetent and immunocompromised mice and in guinea pig infection models: group I (Nine Mile, African, and Ohio), group IV (Priscilla and P), group V (G and S), and group VI (Dugway). Isolates from all of the groups produced disease in the SCID mouse model, and genogroup-consistent trends were noted in cytokine production in response to infection in the immunocompetent-mouse model. Guinea pigs developed severe acute disease when aerosol challenged with group I isolates, mild to moderate acute disease in response to group V isolates, and no acute disease when infected with group IV and VI isolates. C. burnetii isolates have a range of disease potentials; isolates within the same genomic group cause similar pathological responses, and there is a clear distinction in strain virulence between these genomic groups.
doi:10.1128/IAI.00851-09
PMCID: PMC2786457  PMID: 19786560
17.  Prevalence of Chronic Q Fever in Patients with a History of Cardiac Valve Surgery in an Area Where Coxiella burnetii Is Epidemic 
Chronic Q fever develops in 1 to 5% of patients infected with Coxiella burnetii. The risk for chronic Q fever endocarditis has been estimated to be ∼39% in case of preexisting valvulopathy and is potentially even higher for valvular prostheses. Since 2007, The Netherlands has faced the largest Q fever outbreak ever reported, allowing a more precise risk estimate of chronic Q fever in high-risk groups. Patients with a history of cardiac valve surgery were selected for microbiological screening through a cardiology outpatient clinic in the area where Q fever is epidemic. Blood samples were analyzed for phase I and II IgG against C. burnetii, and if titers were above a defined cutoff level, C. burnetii PCR was performed. Chronic Q fever was considered proven if C. burnetii PCR was positive and probable if the phase I IgG titer was ≥1:1,024. Among 568 patients, the seroprevalence of C. burnetii antibodies (IgG titer greater than or equal to 1:32) was 20.4% (n = 116). Proven or probable chronic Q fever was identified among 7.8% of seropositive patients (n = 9). Valve characteristics did not influence the risk for chronic Q fever. Patients with chronic Q fever were significantly older than patients with past Q fever. In conclusion, screening of high-risk groups is a proper instrument for early detection of chronic Q fever cases. The estimated prevalence of chronic Q fever is 7.8% among seropositive patients with a history of cardiac valve surgery, which is substantially higher than that in nonselected populations but lower than that previously reported. Older age seems to increase vulnerability to chronic Q fever in this population.
doi:10.1128/CVI.00185-12
PMCID: PMC3416079  PMID: 22695158
18.  Immunoreactive Coxiella burnetii Nine Mile proteins separated by 2D electrophoresis and identified by tandem mass spectrometry 
Microbiology  2011;157(Pt 2):526-542.
Coxiella burnetii is a Gram-negative obligate intracellular pathogen and the causative agent of Q fever in humans. Q fever causes acute flu-like symptoms and may develop into a chronic disease leading to endocarditis. Its potential as a bioweapon has led to its classification as a category B select agent. An effective inactivated whole-cell vaccine (WCV) currently exists but causes severe granulomatous/necrotizing reactions in individuals with prior exposure, and is not licensed for use in most countries. Current efforts to reduce or eliminate the deleterious reactions associated with WCVs have focused on identifying potential subunit vaccine candidates. Both humoral and T cell-mediated responses are required for protection in animal models. In this study, nine novel immunogenic C. burnetii proteins were identified in extracted whole-cell lysates using 2D electrophoresis, immunoblotting with immune guinea pig sera, and tandem MS. The immunogenic C. burnetii proteins elicited antigen-specific IgG in guinea pigs vaccinated with whole-cell killed Nine Mile phase I vaccine, suggesting a T cell-dependent response. Eleven additional proteins previously shown to react with immune human sera were also antigenic in guinea pigs, showing the relevance of the guinea pig immunization model for antigen discovery. The antigens described here warrant further investigation to validate their potential use as subunit vaccine candidates.
doi:10.1099/mic.0.043513-0
PMCID: PMC3090129  PMID: 21030434
19.  Q fever endocarditis: A case report and review of the literature 
The case of a 31-year-old man from Alberta diagnosed with Q fever endocarditis is presented. To the authors’ knowledge, this is the first case of Q fever endocarditis diagnosed in the province of Alberta. The patient had undergone open valvulotomy for congenital aortic stenosis as an infant. He presented with congestive heart failure secondary to severe aortic regurgitation and underwent mechanical aortic valve replacement. Early failure of the mechanical prosthesis and numerous laboratory abnormalities prompted an investigation for endocarditis, which was initially negative. Markedly positive serology eventually established the diagnosis of chronic Q fever. The patient subsequently underwent a second aortic valve replacement following initiation of appropriate antimicrobials directed against Coxiella burnetii. The present report reviews the clinical presentation and diagnosis of Q fever endocarditis. It highlights the insidious and nonspecific nature of the presenting symptoms, and emphasizes the use of serology for diagnosis. Increased awareness and earlier diagnosis can significantly decrease the morbidity and mortality associated with this disease.
PMCID: PMC2560519  PMID: 16835673
Coxiella burnetii; Endocarditis; Q Fever
20.  SCID Mouse Model for Lethal Q Fever  
Infection and Immunity  2003;71(8):4717-4723.
Q fever, a worldwide zoonosis caused by Coxiella burnetii, has many manifestations in humans. Endocarditis is the most serious complication of Q fever. Animal models are limited to acute pulmonary or hepatic disease and reproductive disorders. An appropriate experimental animal model for Q fever endocarditis does not yet exist. In this study, severe combined immunodeficient (SCID) mice infected with C. burnetii showed persistent clinical symptoms and died, whereas immunocompetent mice similarly infected became asymptomatic and survived. The SCID mice examined in this study had severe chronic lesions in their primary organs: the heart, lung, spleen, liver, and kidney. The heart lesions of the SCID mice were similar to those in humans with chronic Q fever endocarditis: they had focal calcification and expanded macrophages containing C. burnetii. The 50% lethal dose of C. burnetii in SCID mice was at least 108 times less than that in immunocompetent mice. The SCID mouse is highly susceptible to C. burnetii, and the immunodeficiency of the host enhances the severity of Q fever. This animal model could provide a new tool for the study of chronic Q fever and Q fever in immunodeficient hosts.
doi:10.1128/IAI.71.8.4717-4723.2003
PMCID: PMC166031  PMID: 12874353
21.  A DNA-Binding Peroxiredoxin of Coxiella burnetii Is Involved in Countering Oxidative Stress during Exponential-Phase Growth▿  
Journal of Bacteriology  2010;192(8):2077-2084.
Coxiella burnetii is a Gram-negative, obligate intracellular bacterial pathogen that resides within the harsh, acidic confines of a lysosome-like compartment of the host cell that is termed a parasitophorous vacuole. In this study, we characterized a thiol-specific peroxidase of C. burnetii that belongs to the atypical 2-cysteine subfamily of peroxiredoxins, commonly referred to as bacterioferritin comigratory proteins (BCPs). Coxiella BCP was initially identified as a potential DNA-binding protein by two-dimensional Southwestern (SW) blots of the pathogen's proteome, probed with biotinylated C. burnetii genomic DNA. Confirmation of the identity of the DNA-binding protein as BCP (CBU_0963) was established by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). Recombinant Coxiella BCP (rBCP) was generated, and its DNA binding was demonstrated by two independent methods, including SW blotting and electrophoretic mobility shift assays (EMSAs). rBCP also demonstrated peroxidase activity in vitro that required thioredoxin-thioredoxin reductase (Trx-TrxR). Both the DNA-binding and peroxidase activities of rBCP were lost upon heat denaturation (100°C, 10 min). Functional expression of Coxiella bcp was demonstrated by trans-complementation of an Escherichia coli bcp mutant, as evidenced by the strain's ability to grow in an oxidative-stress growth medium containing tert-butyl hydroperoxide to levels that were indistinguishable from, or significantly greater than, those observed with its wild-type parental strain and significantly greater than bcp mutant levels (P < 0.05). rBCP was also found to protect supercoiled plasmid DNA from oxidative damage (i.e., nicking) in vitro. Maximal expression of the bcp gene coincided with the pathogen's early (day 2 to 3) exponential-growth phase in an experiment involving synchronized infection of an epithelial (Vero) host cell line. Taken as a whole, the results show that Coxiella BCP binds DNA and likely serves to detoxify endogenous hydroperoxide byproducts of Coxiella's metabolism during intracellular replication.
doi:10.1128/JB.01324-09
PMCID: PMC2849434  PMID: 20173000
22.  Coxiella burnetii Expresses a Functional Δ24 Sterol Reductase▿  
Journal of Bacteriology  2010;192(23):6154-6159.
Coxiella burnetii, the etiological agent of human Q fever, occupies a unique niche inside the host cell, where it replicates in a modified acidic phagolysosome or parasitophorous vacuole (PV). The PV membrane is cholesterol-rich, and inhibition of host cholesterol metabolism negatively impacts PV biogenesis and pathogen replication. The precise source(s) of PV membrane cholesterol is unknown, as is whether the bacterium actively diverts and/or modifies host cell cholesterol or sterol precursors. C. burnetii lacks enzymes for de novo cholesterol biosynthesis; however, the organism encodes a eukaryote-like Δ24 sterol reductase homolog, CBU1206. Absent in other prokaryotes, this enzyme is predicted to reduce sterol double bonds at carbon 24 in the final step of cholesterol or ergosterol biosynthesis. In the present study, we examined the functional activity of CBU1206. Amino acid alignments revealed the greatest sequence identity (51.7%) with a Δ24 sterol reductase from the soil amoeba Naegleria gruberi. CBU1206 activity was examined by expressing the protein in a Saccharomyces cerevisiae erg4 mutant under the control of a galactose-inducible promoter. Erg4 is a yeast Δ24 sterol reductase responsible for the final reduction step in ergosterol synthesis. Like Erg4-green fluorescent protein (GFP), a CBU1206-GFP fusion protein localized to the yeast endoplasmic reticulum. Heterologous expression of CBU1206 rescued S. cerevisiae erg4 sensitivity to growth in the presence of brefeldin A and cycloheximide and resulted in new synthesis of ergosterol. These data indicate CBU1206 is an active sterol reductase and suggest the enzyme may act on host sterols during C. burnetii intracellular growth.
doi:10.1128/JB.00818-10
PMCID: PMC2981196  PMID: 20870767
23.  Q fever tricuspid valve endocarditis 
Archives of Disease in Childhood  1997;77(5):448-449.



Q fever is a zoonosis caused by Coxiella burnetii. The most frequent clinical expression of the chronic form is a bacterial culture negative aortic or mitral endocarditis. A case of tricuspid valve endocarditis due to C burnetii is described, with a favourable outcome after treatment with doxycycline and hydroxychloroquine.


PMCID: PMC1717388  PMID: 9487972
24.  High throughput detection of Coxiella burnetii by real-time PCR with internal control system and automated DNA preparation 
BMC Microbiology  2008;8:77.
Background
Coxiella burnetii is the causative agent of Q-fever, a widespread zoonosis. Due to its high environmental stability and infectivity it is regarded as a category B biological weapon agent. In domestic animals infection remains either asymptomatic or presents as infertility or abortion. Clinical presentation in humans can range from mild flu-like illness to acute pneumonia and hepatitis. Endocarditis represents the most common form of chronic Q-fever. In humans serology is the gold standard for diagnosis but is inadequate for early case detection. In order to serve as a diagnostic tool in an eventual biological weapon attack or in local epidemics we developed a real-time 5'nuclease based PCR assay with an internal control system. To facilitate high-throughput an automated extraction procedure was evaluated.
Results
To determine the minimum number of copies that are detectable at 95% chance probit analysis was used. Limit of detection in blood was 2,881 copies/ml [95%CI, 2,188–4,745 copies/ml] with a manual extraction procedure and 4,235 copies/ml [95%CI, 3,143–7,428 copies/ml] with a fully automated extraction procedure, respectively. To demonstrate clinical application a total of 72 specimens of animal origin were compared with respect to manual and automated extraction. A strong correlation between both methods was observed rendering both methods suitable. Testing of 247 follow up specimens of animal origin from a local Q-fever epidemic rendered real-time PCR more sensitive than conventional PCR.
Conclusion
A sensitive and thoroughly evaluated real-time PCR was established. Its high-throughput mode may show a useful approach to rapidly screen samples in local outbreaks for other organisms relevant for humans or animals. Compared to a conventional PCR assay sensitivity of real-time PCR was higher after testing samples from a local Q-fever outbreak.
doi:10.1186/1471-2180-8-77
PMCID: PMC2397412  PMID: 18489780
25.  Isolation of Coxiella burnetii from heart valves of patients treated for Q fever endocarditis. 
Journal of Clinical Microbiology  1995;33(2):428-431.
Coxiella burnetii was isolated from the valve material of two patients who underwent valvectomy because of progressive congestive heart failure due to endocarditis. In each case antibiotic therapy was administered for several months prior to valvectomy. Classical histopathological examination of the valves did not reveal an etiology. However, coxiella-like organisms were demonstrated in valvular material with Köster, Stamp, and Giemsa stains, and the organisms were grown in cell culture. Antibody titers were consistent with the diagnosis of chronic C. burnetii infection. This report illustrates the advantage of simple and fast staining techniques and cell culture for the demonstration and isolation of C. burnetii in the heart valve tissue of patients with Q fever endocarditis.
PMCID: PMC227961  PMID: 7714203

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