[Show abstract][Hide abstract] ABSTRACT: Coxiella burnetii, the etiological agent of Q fever, is a small, Gram-negative, obligate intracellular bacterium. Replication of C. burnetii during infection has been shown to be increased by decreasing oxidative stress using p47(phox -/-) and iNOS(-/-) mice in vivo and by pharmacologic inhibitors in vitro. Building upon this model, we investigated the role polymorphonuclear leukocytes (PMN) play in the control of infection, since NADPH oxidase-mediated release of reactive oxygen intermediates (ROI) is a primary bactericidal mechanism for these cells that is critical for early innate clearance. Earlier studies suggested that C. burnetii actively inhibited release of ROI from PMN through expression of an unidentified acid phosphatase (ACP). Recent genomic annotations identified one open reading frame (CBU0335) which may encode a Sec- and type II-dependent secreted ACP. To test this model, viable C. burnetii propagated in tissue culture host cells or axenic media, C. burnetii extracts, or purified recombinant ACP (rACP) was combined with human PMN induced with 4-phorbol 12-myristate 13-acetate (PMA). The release of ROI was inhibited when PMN were challenged with viable C. burnetii, C. burnetii extracts, or rACP but not when PMN were challenged with electron beam-inactivated C. burnetii. C. burnetii extracts and rACP were also able to inhibit PMA-induced formation of NADPH oxidase complex on PMN membranes, suggesting a molecular mechanism responsible for this inhibition. These data support a model in which C. burnetii eludes the primary ROI killing mechanism of activated PMN by secreting at least one acid phosphatase.
Infection and immunity 11/2010; 79(1):414-20. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Infection and immunity 09/2009; 77(12):5640-50. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Humoral and cellular immune responses to 11 recombinant proteins were evaluated in this study. Although none of the individual antigens provided complete detection of all positive serum samples, one or more antigens reacted with each serum, indicating that combinations of two or more antigens could increase sensitivity. In accordance with previous studies showing that murine and human MHCs recognize different epitopes , we found that antigen-specific CD4+ T-cells were generated differently in HLA-DR4 transgenic mice and wild-type mice; this confirms that the HLA transgenic mouse is a more relevant model for screening human T-cell antigens. Most proteins with strong antibody responses also strongly induced IFN-c ⁄ recall responses in purified CD4+ T-cells of vaccinated mice, which means that immunoreactive antigen screening based on serology testing, such as protein microarray, can aid in the discovery of T-cell antigens. We have developed a sensitive, high-throughput approach for screening immunoreactive C. burnetii antigens for strong humoral and cellular immune responses. This platform will be used for the rational design of effective subunit vaccines and serodiagnostic tools.
[Show abstract][Hide abstract] ABSTRACT: Acute Q fever is a zoonotic disease caused by the obligate intracellular bacterium Coxiella burnetii and can manifest as a flu-like illness, pneumonia, or hepatitis. A need exists in Q fever research for animal models mimicking both the typical route of infection (inhalation) and the clinical illness seen in human cases of Q fever. A guinea pig aerosol challenge model was developed using C. burnetii Nine Mile phase I (RSA 493), administered using a specialized chamber designed to deliver droplet nuclei directly to the alveolar spaces. Guinea pigs were given 10(1) to 10(6) organisms and evaluated for 28 days postinfection. Clinical signs included fever, weight loss, respiratory difficulty, and death, with the degree and duration of response corresponding to the dose of organism delivered. Histopathologic evaluation of the lungs of animals infected with a high dose showed coalescing panleukocytic bronchointerstitial pneumonia at 7 days postinfection that resolved to multifocal lymphohistiocytic interstitial pneumonia by 28 days. Guinea pigs receiving a killed whole-cell vaccine prior to challenge with the highest dose of C. burnetii were protected against lethal infection and did not develop fever. Clinical signs and pathological changes noted for these guinea pigs were comparable to those seen in human acute Q fever, making this an accurate and valuable animal model of human disease.
Infection and Immunity 12/2006; 74(11):6085-91. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper studies the functional and regulatory role of RecA, but further investigations are necessary to clarify the control mechanism of the SOS response gene expression in C. burnetii.
Annals of the New York Academy of Sciences 01/2006; 1063:73-5. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Coxiella burnetii is an obligate intracellular bacterium that causes acute Q fever and occasional chronic infections in humans. To determine the immunodominant antigens during infection with C. burnetii, sera from mice experimentally infected with Nine Mile phase I were tested by immunoblotting. The mouse sera recognized antigens with a variety of molecular weights, including proteins of 14, 22, 28, 34, and 60 kDa as immunodominant antigens. In order to clone potential protective antigens, a genomic DNA library of Nine Mile phase I was constructed in the expression vector Lambda ZAP Express and screened with sera from mice that recovered from C. burnetii infection. A total of 102 immunoreactive clones with various signal intensities were identified from about 8,000 plaques. These clones were purified and expressed in the excised plasmid pBK-CMV. The proteins expressed by these recombinant plasmids were analyzed by SDS-PAGE and immunoblotting. Fifty-four clones expressed immunoreactive proteins of molecular masses ranging from approximately 14 to 60 kDa. Sequence analysis and BLAST search of the recently completed genome sequence identified a variety of novel immunoreactive proteins. These proteins are logical vaccine candidates for testing protective activity against C. burnetii challenge. We established a sublethal challenge model in BALB/c mice with protection from the development of severe splenomegaly as an indicator of vaccinogenic activity. Further characterization of these proteins will provide essential information for developing novel, specific diagnostic reagents and potential subunit vaccine candidates against C. burnetii infection.
Annals of the New York Academy of Sciences 07/2003; 990:510-20. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The agent of acute and chronic Q fever, Coxiella burnetii, occupies a unique niche among intracellular pathogens. The mechanisms the organism employs to cause disease are unclear but involve persistence in a parasitophorous vacuole and the subsequent host response. Studies designed to model molecular mechanisms of pathogenesis have relied upon indirect evidence for testing the role of virulence factors since methods for generation of defined mutations have not been developed. Evidence suggests replication involving a developmental lifecycle is critical for intra- and extracellular survival but this cycle is incompletely defined. It has been proposed that survival in the phagolysosomal-like parasitophorous vacuole requires specific iron uptake systems, secretion of enzymes to detoxify the compartment (catalase and SOD), and down-regulation of an oxidative burst (acid phosphatase). Studies to test these potential virulence mechanisms can be accelerated with the recent development of the complete genome sequence for the prototype acute disease isolate, Nine Mile. Proteins differentially expressed during the developmental cycle can more readily be identified with MALDI-TOF description of proteomic profiles. Genes encoding secreted Cu/Zn SOD, catalase, and acid phosphatase are predicted and can be tested for function and expression. An iron regulon is predicted based upon Fur-regulated open reading frames. The specific role the iron-regulated genes play in iron acquisition can be tested. Confirmation of the iron regulon and others can be tested using microarrays based upon the genomic ORF predictions. These are examples of how we are rapidly changing the experimental approaches used to investigate C. burnetii to improve our understanding of the biology of this unusual and highly adapted organism.
Annals of the New York Academy of Sciences 07/2003; 990:653-63. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Coxiella burnetii is an obligate intracellular bacterium that resides in an acidified phagolysosome and has a remarkable ability to persist in the extracellular environment. C. burnetii has evolved a developmental cycle that includes at least two morphologic forms, designated large cell variants (LCV) and small cell variants (SCV). Based on differential protein expression, distinct ultrastructures, and different metabolic activities, we speculated that LCV and SCV are similar to typical logarithmic- and stationary-phase growth stages. We hypothesized that the alternate sigma factor, RpoS, a global regulator of genes expressed under stationary-phase, starvation, and stress conditions in many bacteria, regulates differential expression in life cycle variants of C. burnetii. To test this hypothesis, we cloned and characterized the major sigma factor, encoded by an rpoD homologue, and the stress response sigma factor, encoded by an rpoS homologue. The rpoS gene was cloned by complementation of an Escherichia coli rpoS null mutant containing an RpoS-dependent lacZ fusion (osmY::lacZ). Expression of C. burnetii rpoS was regulated by growth phase in E. coli (induced upon entry into stationary phase). A glutathione S-transferase-RpoS fusion protein was used to develop polyclonal antiserum against C. burnetii RpoS. Western blot analysis detected abundant RpoS in LCV but not in SCV. These results suggest that LCV and SCV are not comparable to logarithmic and stationary phases of growth and may represent a novel adaptation for survival in both the phagolysosome and the extracellular environment.
Infection and Immunity 09/2001; 69(8):4874-83. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Coxiella burnetii replicates as distinct morphological forms, which may allow potential life cycle variants to survive the harsh environment of the phagolysosome. Monoclonal antibodies (MAbs) were compared by Western blotting for reactivity with large cell variant (LCV) and small cell variant (SCV) antigens to characterize proteins differentially expressed by C. burnetii. MAb NM7.3 reacted with a approximately 32-kDa LCV-upregulated antigen, and MAb NM183 reacted with a approximately 45-kDa LCV-specific antigen. MAb NM7.3 was used to screen a lambdaZapII C. burnetii DNA expression library, and an immunoreactive clone was identified with sequence similarity to the Escherichia coli tsf gene, which encodes elongation factor Ts (EF-Ts). Since a similar screen with MAb NM183 did not identify immunoreactive clones, an alternate strategy was devised to clone the reactive antigen based on observations of cross-reactivity with the 45-kDa elongation factor Tu (EF-Tu) protein from Chlamydia trachomatis. The highly conserved nature of EF-Tu among eubacteria allowed PCR amplification of a tuf gene fragment (encoding approximately 95% of the predicted EF-Tu open reading frame) from C. burnetii using degenerate primers. The product of the cloned tuf gene fragment reacted with MAb NM183 in Western blot analysis, confirming the identity of the 45-kDa LCV-specific antigen. Identification of two proteins differentially expressed by C. burnetii, EF-Tu and EF-Ts, both essential components of the translational machinery of the cell, supports the hypothesis that LCVs are metabolically more active than SCVs.
Infection and Immunity 12/1999; 67(11):6026-33. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The gene for an approximately 27-kDa outer membrane-associated, immunoreactive protein was cloned from the rickettsial pathogen Coxiella burnetii. The gene, designated com1 for Coxiella outer membrane protein 1, was expressed in Escherichia coli, presumably by its own promoter. The complete nucleotide sequence of the gene was determined. The deduced amino acid sequence of 252 residues includes a putative leader sequence. The leader sequence is recognized in and removed by E. coli on the basis of the difference in the molecular mass of the protein produced in an in vitro transcription-translation system (27.6 kDa) and that of the protein immunoprecipitated from an iodinated E. coli clone (25.7 kDa). The Com1 protein expressed in E. coli was proteinase K sensitive in nondisrupted cells and soluble in 1% Sarkosyl, suggesting a loose association with the outer membrane. While the complete predicted sequence of the Com1 protein does not show any overall similarity to those of previously described proteins, a region which includes the only two cysteines in Com1 is homologous to the catalytic site of protein disulfide oxidoreductases.
Infection and Immunity 03/1993; 61(2):470-7. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thirty-two isolates of Coxiella burnetii collected from various hosts ranging from arthropods to man were compared by restriction endonuclease (RE) digestion patterns of chromosomal DNA using SDS-PAGE. SDS-PAGE provided better DNA fragment separation than agarose gel electrophoresis and enabled the differentiation of these isolates into six distinct groups on the basis of DNA restriction fingerprints. Two groups of chronic disease isolates could be distinguished, each having unique RE digestion patterns of chromosomal DNA. Three similar but distinct RE digestion patterns were seen among the group of acute disease isolates. Three additional isolates included in this study exhibited a unique RE digestion pattern and also had a unique plasmid type, designated QpDG. DNA-DNA hybridization on selected isolates quantified the relatedness between several groups and supported the classification of these groups as distinct strains.
Journal of general microbiology 03/1991; 137(2):269-76.
[Show abstract][Hide abstract] ABSTRACT: Isolation of Coxiella Burnetii in the standard laboratory setting is hazardous; therefore most diagnoses are based on retrospective detection of a rising antibody titer to C. burnetti. As a result, this disease is usually undiagnosed or misdiagnosed. Methods for the rapid detection of C. burnetti have now been developed that utilize specific hybridization of labeled DNA probes to nucleic acid in clinical samples. One method detects the presence of C. burnetii 16S ribosomal RNA (rRNA); another uses plasmid sequences. We have developed a probe that detects C. burnetii and one that differentiates between Coxiella strains capable of causing chronic disease and those that cause the acute form. Using these probes, C. burnetii can be identified in blood, urine, and tissue samples. The plasmid-derived probes detect as few as 10(4) organisms and less than 1 ng of Coxiella DNA. A third method differentiates between chronic (endocarditis-causing) strains and those that cause acute Q fever. This method uses the polymerase chain reaction (PCR), in which the target regions of DNA are amplified by iterative cycles of Taq I DNA polymerase chain extension to produce up to a 10(6) amplification of the target sequences. When Southern blotting is used in conjunction with PCR, the test detects as few as 2-9 C. burnetti cells.
Annals of the New York Academy of Sciences 02/1990; 590:445-58. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Patients with acute Q fever develop Coxiella burnetii-specific antibody, and chronic disease patients often develop extremely high levels of C. burnetii-specific antibody. Antibody-reactive LPS has been well characterized, but only a few immunoreactive proteins have been identified. An immunoreactive ca. 62-kDa protein has been cloned, sequenced, and shown to be related to an Escherichia coli heat-shock protein. A ca. 27-kDa immunoreactive outer membrane protein(s) has also been identified. We have begun characterizing C. burnetii immunoreactive proteins by gene-cloning methods. A gene bank of total C. burnetii Nine Mile phase 1 (acute strain) DNA was created using the lambda vector EMBL3. This bank was screened for plaques which reacted with E. coli-pre-absorbed rabbit antisera specific for purified, Nine Mile C. burnetii whole cells. Twenty-three immunoreactive plaques were identified from a screening of 3000 plaques. Twenty-two plaques produced a ca. 60-65-kDa immunoreactive protein. One plaque produced a ca. 27-kDa immunoreactive protein, as well as a ca. 60-65-kDa immunoreactive protein. Phage extracts of this plaque were used to enrich the antisera to produce a ca. 27-kDa-specific antisera. This enriched sera reacted with a ca. 27-kDa protein in all C. burnetii isolates tested, comprising isolates from both chronic and acute strains. The DNA contained in the immunoreactive clone was C. burnetii-specific, as shown by DNA hybridization. We are currently subcloning the ca. 27-kDa protein-coding region for sequencing to determine if this gene encodes the previously identified ca. 27-kDa immunodominant protein. This protein may ultimately have both diagnostic and vaccinogenic potential.
Annals of the New York Academy of Sciences 02/1990; 590:534-40. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Small subunit rRNA sequences have been determined for representative strains of six species of the family Rickettsiaceae: Rickettsia rickettsii, Rickettsia prowazekii, Rickettsia typhi, Coxiella burnetii, Ehrlichia risticii, and Wolbachia persica. The relationships among these sequences and those of other eubacteria show that all members of the family Rickettsiaceae belong to the so-called purple bacterial phylum. The three representatives of the genus Rickettsia form a tight monophyletic cluster within the alpha subdivision of the purple bacteria. E. risticii also belongs to the alpha subdivision and shows a distant yet specific relationship to the genus Rickettsia. However, the family as a whole is not monophyletic, in that C. burnetii and W. persica are members of the gamma subdivision. The former appears to show a specific, but rather distant, relationship to the genus Legionella.
Journal of Bacteriology 09/1989; 171(8):4202-6. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The rickettsial pathogen Coxiella burnetii undergoes a variation in which virulent isolates (phase 1) become avirulent (phase 2) after repeated passage in a non-immunologically competent host. Biochemically, this variation is associated with a lipopolysaccharide modification and possibly other factors. Genetically, the regions of DNA responsible for phase variation have not been identified. We have sought to determine whether the plasmid identified in acute disease isolates, QpH1, which represents approximately 5% of the coding capacity of this organism is involved in phase variation. Plasmids from phase 1 and phase 2 variants (designated QpH1 and QpH2, respectively) were compared by restriction endonuclease digestion and Southern blot hybridization to determine whether sequence changes in the phase 2 plasmid might account for changes in the virulence of phase 2 organisms compared with that of phase 1 cells. Using over 20 different restriction enzymes, no changes in DNA restriction fragment patterns were detected regardless of whether the phase change occurred during egg or tissue culture passage. The plasmid-specific mRNAs produced from metabolically active, purified cells were identical for each phase type. Using QpH1 or QpH2 DNA as a template, the mRNA produced by an E. coli extract was also identical. Finally, the proteins encoded by either plasmid in an in vitro transcription/translation reaction were identical. These data indicate that within the limits of our analysis, the plasmid DNA from C. burnetii phase variants is structurally and functionally the same and is therefore unlikely to be involved in phase variation.
Journal of general microbiology 08/1988; 134(7):1795-1805.
[Show abstract][Hide abstract] ABSTRACT: The obligate intracellular bacterium Coxiella burnetii is the etiological agent of acute Q-fever and chronic endocarditis in humans and of several zoonotic infections. The DNA from a variety of these disease isolates was compared for homology to the plasmid QpH1, found in the Nine Mile strain. Three patterns of homology were found in these isolates, i.e., one pattern identical to that of QpH1, one common to several endocarditis isolates and goat abortion isolates, and one common to the remaining group of endocarditis isolates. Plasmid DNA from the endocarditis-abortion isolate group, designated QpRS, was mapped by restriction enzyme analysis and compared with QpH1. These data show that QpRS was 2 to 3 kilobase pairs larger, contained DNA not found in QpH1, but was not generated from QpH1 by a single insertional event. Isolation of plasmid DNA from the second endocarditis group of isolates was not successful and may indicate that the plasmid has integrated into the chromosome. This analysis provides the first clear evidence that differences exist between C. burnetii isolates which cause various diseases, indicating that different C. burnetii strains may have unique virulence characteristics.
Infection and Immunity 10/1985; 49(3):775-9. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chinese hamster ovary (CHO) cells were exposed to 60-Hz ac electromagnetic fields at strengths from 0.15 V/m to 10.9 V/m, using an exposure system which magnetically induces an electric field in the culture medium without the use of electrodes. Electric-field exposure with this system had no detectable cytotoxic or mutagenic effects. Mutation frequencies were not significantly increased at any of the electric-field strengths or exposure durations. However, cell viability, as measured by plating efficiency (ability of a cell to produce a colony), was significantly reduced. The threshold of detectability for this effect was a 24-h exposure at 0.7 V/m. As a result of these findings, and because of limitations of the exposure system, we designed and tested another exposure system which allowed exposure at higher field strengths. This system employs carbon electrodes directly coupled to the culture medium. To assure that electrode effects did not perturb the experiment, agar bridges were interposed between the electrodes and the cell suspension. The plating efficiency of cells exposured to 3.5 V/m in the agar-bridge graphite-electrode exposure system was not significantly different from those of control cultures. Therefore, the decreased cloning efficiency observed with the magnetically induced electric field may be either artifactual or a function of the system itself (i.e., the greater magnetic component relative to that of the agar-bridge exposure chamber). 22 refs., 9 figs., 2 tabs.