Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xi, 157 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Coxiella burnetii, the etiologic agent of acute Q fever and chronic endocarditis, has a unique biphasic life cycle, which includes a metabolically active intracellular form that occupies a large lysosome-derived acidic vacuole. C. burnetii is the only bacterium known to thrive within such a hostile intracellular niche, and this ability is fundamental to its pathogenicity; however, very little is known about genes that facilitate Coxiella's intracellular growth. This lack of knowledge of Coxiella's basic biology and molecular pathogenesis is a critical barrier to developing more effective therapies. In this study, we aimed to understand both bacterial and host factors that have important roles during C. burnetii infections. Using an evolutionary genomics approach, we identified metabolic pathways that are critical to C. burnetii's ability to grow intracellularly. Among those found, the most promising are fatty acid, biotin, and heme biosyntheses pathways. Coxiella has horizontally acquired extra copies of genes that enhance these processes; when these genes were disrupted, Coxiella's growth was significantly inhibited. Also, by analyzing the host transcriptome, we identified human genes, including microRNA (miRNA) genes that are important during C. burnetii infections. Coxiella induces the expression of multiple anti-apoptotic miRNAs, which likely have a role in inhibiting apoptosis in order to sustain the intracellular replication of the pathogen. The biosynthetic pathways and miRNAs identified in this study are ideal targets for developing more effective therapeutic strategies against Q fever and its chronic and often fatal complications.

Indiana University-Purdue University Indianapolis (IUPUI) / Q fever is a heavily underdiagnosed and underreported infection caused by the obligate intracellular pathogen Coxiella burnetii. Following entry into the host cell, Coxiella replicates in the acidic phagolysosome-like parasitophorous vacuole termed the Coxiella Containing Vacuole (CCV). The CCV is a large and highly fusogenic compartment that actively fuses with the host endocytic pathway during maturation of the phagolysosome. Evidence suggests that the development of the CCV is sensitive to increasing cholesterol levels and leads to CCV acidification and bacterial death. Therefore, we hypothesize that CCV cholesterol concentration is carefully modulated through the Coxiella encoded sterol reductases (CBU1206 and CBU1158). A ∆CBU1206 mutant of Coxiella is hypersensitive to cholesterol and displays growth defects in intracellular replication and CCV development. Following fusion with the host endocytic pathway, the Coxiella NMII Phase II (WT) CCVs readily acquire host proteins such as LAMP1, CD63, Rab7, ORP1L, RILP, and LC3. These heterotypic events with the host endosomal cascade are presumed to provide selected subsets of endocytosed cargo and membrane. Therefore, I investigated whether ΔCBU1206 CCV heterotypic fusion events are defective due to altered lipid content on the CCV membrane. I observed increased accumulation of sterols on the ΔCBU1206 CCV membrane. Similar to WT, the mutant readily fuses host lysosomes and readily acquires the host glycoprotein LAMP1 but displays reduced localization of CD63 (LAMP3). Additionally, reduced localization of the late endosomal markers Rab7, ORP1L, and RILP was observed suggesting that late endosome fusion maybe defective in ΔCBU1206. Further, reduced localization of LC3 was also observed suggesting that the mutant may also be defective in fusing with autophagosomes. Finally, the mutant possesses a functional Type 4 Secretion System that secretes a moderate amount of effector proteins relative to WT. Considering the vast array of functions accomplished by the effectors secreted, the moderate effector secretion by the mutant could influence the endocytic pathway fusion processes as well as CCV development. Collectively, this body of work suggests that the lack of sterol reductase CBU1206 in Coxiella results in defective heterotypic fusion events of the CCV membrane that could alter pathogenesis and CCV expansion.

Acute Q fever is the disease manifestation of Coxiella burnetii infection. This obligate intracellular bacterium is phagocytosed by innate immune cells, where it replicates within the usually bactericidal environment of the phagolysosome. As the immune response is activated, the resultant pro-inflammatory cytokines aid in pathogen clearance but also trigger an acute sickness response in the host. This thesis describes the natural history of acute Q fever in a prospective cohort ??? the Dubbo Infection Outcomes Study (DIOS). In these subjects, the acute febrile illness was characterised by severe headache, drenching sweats and fatigue. In approximately 10% of subjects, symptomatic illness marked by fatigue remained present for months, or occasionally years, after the acute illness. Subjects with more severe acute illness were more likely to develop this post Q fever fatigue syndrome (QFS). The aim of this thesis was to determine whether ongoing infection or aberrant immune activation drive the prolonged symptoms of QFS. Sensitive real time PCR detection of Coxiella DNA revealed a significant minority of subjects had very low copy numbers in circulating monocytes, with an increased prevalence in those with QFS. However, the detection was not consistently found within individual subjects and the copy number was at the threshold of reliable detection. C. burnetii was shown here to stimulate cytokine production in monocytic cells via interaction with Toll-like receptor (TLR)-2 and not TLR-4. Functional polymorphisms in these TLRs were identified in subjects with Q fever, but were not associated with Q fever susceptibility, severity or duration. Phase I-specific responses are believed to be critical in the generation of protective immunity to C. burnetii, yet the phase II-specific responses of innate and adaptive immune components were consistently of higher magnitude. Whole C. burnetii organisms induced antigen-non-specific T cell activation, presumably via the indirect activation of monocytes by C. burnetii LPS. No significant differences were found in the magnitude or kinetics of the host response to infection, or in the carriage of genetic polymorphisms, when comparing subjects who developed QFS with subjects who had promptly resolving illness. It remains unclear what factors mediate the progression of acute Q fever to QFS.

Universiẗat, Diss., 2007--Giessen.

Zugl.: Giessen, Univ., Diss., 2007

Universiẗat, Diss., 2007--Giessen.

No description available.

Orientador: Jane Megid / Resumo: Coxiella burnetii é uma bactéria intracelular obrigatória responsável por causar a febre Q em humanos, mas também é conhecida como coxielose em animais. No Brasil, pouco é sabido a respeito da epidemiologia da enfermidade. O presente estudo objetiva estabelecer a prevalência do patógeno em bovinos abatidos no estado de São Paulo, além da detecção molecular em soro e fetos de bovinos. A sorologia de 1515 amostras de bovinos coletadas em frigoríficos foi realizada por reação de imunofluorescência indireta (RIFI) para anticorpos anti-C. burnetii. Reação em cadeia da polimerase em tempo real (qPCR) visando detectar o gene IS1111, foi utilizada para o diagnóstico molecular de C. burnetii em amostras de soro e fetos de bovinos. A prevalência aparente para anticorpos anti-C. burnetii em bovinos foi de 23,8% [IC95%=21,7% – 25,9%] (n=360), e a prevalência real de 20,0% [IC95%=17,8 – 22,3%]. A qPCR foi realizada nas 360 amostras soropositivas e apresentou resultado positivo em 92 amostras (25,6% [IC95%=20,1% – 28,8%]), demonstrando a presença do micro-organismo na corrente sanguínea dos bovinos amostrados. Entre as 28 amostras de fetos bovinos analisados, 10,7% (n=3) foram positivas na qPCR, confirmado por sequenciamento. A análise por qPCR quantitativo das amostras de abortamentos indicaram concentração entre 500 – 104 células por grama de tecido. Análise de multilocus de repetições em tandem de número variável (MLVA) e Multispacer Sequence Typing (MST) das amostras de fetos e do cont... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Coxiella burnetii is an obligate intracellular bacterium responsible for causing Q fever in humans but is also known as coxiellosis in animals. In Brazil, little is known about the epidemiology of the disease. The present study aims to establish the prevalence of the pathogen in cattle slaughtered in the state of São Paulo, in addition to molecular detection in bovine serum and fetuses. The serology of 1515 bovine samples collected in slaughterhouses was performed by indirect immunofluorescence reaction (IFA) for anti-C. burnetii antibodies. Real-time polymerase chain reaction (qPCR) to detect the IS1111 gene was used for the molecular diagnosis of C. burnetii in bovine serum and fetal samples. The apparent prevalence of anti-C. burnetii antibodies in cattle was 23.8% [95% CI = 21.7% - 25.9%] (n = 360) and the true prevalence was 20.0% [95% = 17.8 - 22.3%]. The qPCR was performed in the 360 seropositive samples and presented a positive result in 92 samples (25.6% [95% CI = 20.1% - 28.8%]), demonstrating the presence of the microorganism in the blood stream of the sampled cattle. Among the 28 samples of bovine fetuses analyzed, 10.7% (n = 3) were positive in qPCR, confirmed by sequencing. Quantitative qPCR analysis of the abortion samples indicated concentration between 500-104 cells per gram of tissue. Multiple-Locus Variable number tandem repeat Analysis (MLVA) and Multispacer Sequence Typing (MST) from fetal samples and positive control revealed new strains of C. burnetii c... (Complete abstract click electronic access below) / Doutor

Ulm, Univ., Diss., 2008.