Immunoreactive Coxiella burnetii Nine Mile proteins separated by 2D electrophoresis and identified by tandem mass spectrometry

Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, USA.
Microbiology (Impact Factor: 2.84). 10/2010; 157(Pt 2):526-42. DOI: 10.1099/mic.0.043513-0
Source: PubMed

ABSTRACT 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.

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Available from: Wendy C Brown, Jul 28, 2015
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    • "Unfortunately, we were unable to express CBU_1718 in our study. Our best diagnostic marker of acute Q fever was CBU_0092 (Appendix S1), which was previously reported as a good marker (Beare et al., 2008; Deringer et al., 2010; Vigil et al., 2010, 2011; Papadioti et al., 2011). Its diagnostic power was strong enough for the general diagnosis of Q fever and might the better specificity for acute Q fever (L = 22). "
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    ABSTRACT: The discriminatory diagnosis of Q fever remains difficult because of the unspecific clinical presentations of the disease. Additionally, the diagnosis is often delayed because serodiagnosis is not sensitive enough in the early stages of the disease when the immune response is not yet efficient. Similarly, the diagnosis of Q fever endocarditis can only be performed in approximately 35%, mainly via serology, which was a criterion postulated by Duke. Owing to the discriminatory diagnosis of Q fever and the high number of tests requested, we focused on expressing several proteins for ELISA studies with Coxiella burnetii-infected sera. Previously, we selected a list of 31 candidates [Sekeyova et al. (2009) Eur J Clin Microbiol Infect Dis 28: 287-295], of which we have successfully cloned and expressed 21. Finally, 15 recombinant proteins were prescreened with the sera of patients with acute Q fever and Q fever endocarditis, respectively. Sera from a control group were also screened. The nine most immunoreactive proteins from the first assay were tested with the sera from a larger group of patients. Our study identified CBU_0092 as the best marker of acute Q fever but failed to isolate a highly specific and sensitive marker of Q fever endocarditis.
    FEMS Immunology & Medical Microbiology 11/2011; 64(1):140-2. DOI:10.1111/j.1574-695X.2011.00912.x · 2.55 Impact Factor
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    • "To date nearly 200 C. burnetii proteins have been documented in the literature, including some from both phase I and II cells [40] [41] [42] [43] [44] [45] [46]. Unfortunately analysis of the two phases has typically been done under different conditions, complicating comparisons. "
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    ABSTRACT: Coxiella burnetii, a category B biological warfare agent, causes multiple outbreaks of the zoonotic disease Q fever world-wide, each year. The virulent phase I and avirulent phase II variants of the Nine Mile RSA 493 and 439 strains of C. burnetii were propagated in embryonated hen eggs and then purified by centrifugation through Renografin gradients. Total protein fractions were isolated from each phase and subjected to analysis by one-dimensional electrophoresis plus tandem mass spectrometry. A total of 235 and 215 non-redundant proteins were unambiguously identified from the phase I and II cells, respectively. Many of these proteins had not been previously reported in proteomic studies of C. burnetii. The newly identified proteins should provide additional insight into the pathogenesis of Q fever. Several of the identified proteins are involved in the biosynthesis and metabolism of components of the extracellular matrix. Forty-four of the proteins have been annotated as having distinct roles in the pathogenesis or survival of C. burnetii within the harsh phagolysosomal environment. We propose that nine enzymes specifically involved with lipopolysaccharide biosynthesis and metabolism, and that are distinctively present in phase I cells, are virulence-associated proteins.
    Journal of proteomics 05/2011; 74(10):1974-84. DOI:10.1016/j.jprot.2011.05.017 · 3.93 Impact Factor
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    ABSTRACT: Q fever is a worldwide zoonosis caused by Coxiella burnetii. The disease most frequently manifests clinically as a self-limited febrile illness, as pneumonia (acute Q fever) or as a chronic illness that presents mainly as infective endocarditis. The extreme infectivity of the bacterium results in large outbreaks, and the recent outbreak in the Netherlands underlines its impact on public health. Recent studies on the bacterium have included genome sequencing, the investigation of host-bacterium interactions, the development of cellular and animal models of infection, and the comprehensive analysis of different clinical isolates by whole genome and proteomic approaches. Current approaches for diagnosing Q fever are based on serological methods and PCR techniques, but the diagnosis of early stage disease lacks specificity and sensitivity. Consequently, different platforms have been created to explore Q fever biomarkers. Several studies using a combination of proteomics and recombinant protein screening approaches have been undertaken for the development of diagnostics and vaccines. In this review, we highlight advances in the field of C. burnetii proteomics, focusing mainly on the contribution of these technologies to the development and improvement of Q fever diagnostics.
    Genome Medicine 07/2011; 3(7):50. DOI:10.1186/gm266 · 4.94 Impact Factor
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