Pour survivre et se multiplier dans leurs cellules hôtes les bactéries intracellulaires ont élaboré divers mécanismes d'échappement à la réponse immunitaire. L’altération du trafic intracellulaire est une des stratégies utilisée par ces agents pathogènes. Ainsi Coxiella burnetii, bactérie intracellulaire stricte responsable chez l’homme de la fièvre Q, bloque la maturation du phagosome afin de résider et se multiplier dans un compartiment incapable de fusionner avec les lysosomes. Ce défaut de fusion est associé à la virulence bactérienne. Pour analyser le défaut de maturation du phagosome de C. burnetii, j’ai étudié le rôle du lipopolysaccharide (LPS) de C. burnetii dans le trafic intracellulaire de cette bactérie. J’ai montré que le LPS de C. burneti, unique par sa structure, ne permet pas l’activation de la MAPKinase p38α, entrainant un défaut dans le recrutement du complexe HOPS (homotypic fusion and vacuole protein sorting complex) nécessaire à la conversion phagosomale. J’ai en effet montré que le recrutement du complexe HOPS requiert la phosphorylation de la protéine Vps (vacuolar protein sorting) 41. La transfection de macrophages permettant la surexpression d’un activateur de p38 et l’utilisation de mutants phosphomimétiques de Vps41 ont montré une restauration de la conversion phagosomale. Il apparaît ainsi que la MAPK-p38α et son dialogue avec Vps41 jouent un rôle central dans la maturation du phagosome de C. burnetii en phagolysosome. L’utilisation de la structure atypique de son LPS permet ainsi à C. burnetii de se soustraire à la réponse protectrice de l'hôte. / To survive and replicate in their host, microbes have evolved several strategies to hijack the microbicidal properties of the immune cells. C. burnetii, the q fever agent, survive and replicate in macrophages through the alteration of the phago-lysosome biogenesis. To further analyze the nature of the defect phagosome maturation of C. burnetii, I studied the role of lipopolysaccharide (LPS) of C. burnetii in the intracellular trafficking of the bacteria. The LPS is unable to activate the p38α MAPKinase, which explains that the virulent bacteria are not directed to a degradative compartment . The lack of activation of the p38α MAPKinase , which involves a commitment TLR4 antagonist by LPS, has the effect of preventing the recruitment of the HOPS complex ( homotypic fusion and vacuole protein sorting complex) , a complex require for the phagosomal conversion. I have shown that the recruitment of HOPS requires phosphorylation of protein Vps (vacuolar protein sorting) 41. Transfection of macrophages by an activator of p38 and using phosphomimétiques mutants VPS41 showed restoration of phagosome maturation. It thus appears that the p38α MAPK and his dialogue with VPS41 play a central role in phagosome maturation of C. burnetii in the phagolysosome. Use of the unique structure of the LPS allows C. burnetii to evade the protective response of the host.

Les granulomes signent une réponse immune efficace dans de nombreuses maladies infectieuses. C’est le cas de la fièvre Q où ils sont présents dans la forme aiguë de la maladie spontanément résolutive mais où ils sont absents dans sa forme chronique. J’ai mis au point une méthode permettant d’étudier la formation in vitro des granulomes à partir de cellules mononucléées du sang périphérique incubées en présence de billes de Sepharose recouvertes d’extraits de C. burnetii, l’agent de la fièvre Q. Ces granulomes apparaissent en quelques jours avant de disparaître au bout d’une vingtaine de jours. Ils sont composés essentiellement de macrophages et de lymphocytes et, à un moindre degré, de cellules épithélioïdes, de cellules géantes multinucléées et de cellules dendritiques. La méthode d’obtention in vitro des granulomes a permis également d’étudier les premiers stades de leur formation grâce à la mise au point d’une technique en live imaging d’observation en lumière transmise des cellules. J’ai montré que les monocytes, les premières cellules à migrer vers les billes et à entièrement les recouvrir, initient le recrutement des lymphocytes. J’ai également montré que le défaut de formation des granulomes observés chez des patients atteints d’une fièvre Q chronique pourrait être lié à un défaut de migration de leurs monocytes circulants. / Granulomas indicate effective immune response in a large number of infectious diseases. The primo-infection by Coxiella burnetii, the agent of Q fever, is spontaneously resolutive and is characterized by the presence of granulomas, whereas granulomas are absent in the chronic form of the disease. I developed a new method to study in vitro the formation of granulomas using peripheral blood mononuclear cells incubated in the presence of Sepharose beads coated with C. burnetii extracts. Granulomas appeared in few days before disappearing at day 20. They were essentially composed of macrophages and lymphocytes and, in a lesser extent, of epithelioid cells, multinucleated giant cells and dendritic cells. The method to obtain in vitro granulomas allowed the study of the first events of granuloma formation in live imaging microscopy. Monocytes migrated toward Sepharose beads and entirely covered these beads, and initiated the recruitment of lymphocytes. Finally, mononuclear cells from patients with Q fever were unable to generate granulomas due to defective migration of monocytes. We hypothesize that defective migration of monocytes may be responsible for the defective formation of granulomas in Q fever.

Les bactéries pathogènes intracellulaires manipulent les fonctions de la cellule hôte en sécrétant des facteurs de virulence (qu'on appelle effecteurs) dans le cytoplasme de la cellule infectée. Ce processus permet au pathogène de proliférer dans un environnement autrement hostile. L'identification et la caractérisation des effecteurs spécifiques des divers agents pathogènes est donc d'une importance cruciale pour contrer les infections bactériennes. Coxiella burnetii est un agent pathogène à Gram négatif de classe 3, responsable de la Fièvre Q, une zoonose qui entraîne des épidémies majeures, avec un fort impact sur l'économie et la santé. Les réservoirs naturels de Coxiella sont principalement les animaux d’élevage qui peuvent contaminer l’environnement en excrétant la bactérie principalement dans les produits de parturition, le mucus vaginal et les fèces. L’Homme s’infecte ensuite par inhalation de pseudo-spores disséminées dans l’environnement. La nature intracellulaire obligatoire de Coxiella a jusqu'ici sévèrement limité son étude et par conséquent, les facteurs de virulence bactériens impliqués dans le développement et la progression de l'infection restent encore largement inconnus. Coxiella se réplique à l'intérieur des cellules hôtes dans une grande vacuole présentant des caractéristiques autolysosomales. Le développement de la vacuole et la survie de Coxiella dans la cellule hôte sont dépendants de la translocation des effecteurs bactériens par un système de sécrétion de type 4 (SST4) Dot/Icm et de la manipulation de nombreuses voies de trafic et de signalisation de la cellule hôte par ces derniers. Notre équipe a généré et criblé la première banque de mutants par transposition de Coxiella, menant ainsi à l'identification d'un nombre important de potentiels déterminants de virulence et de protéines effectrices. Mon projet de thèse est basé sur la caractérisation de deux effecteurs de Coxiella, CvpF et AnkA, provenant de la banque de mutants générée par l’équipe. Les mutants de ces effecteurs présentent des phénotypes de défaut de réplication intracellulaire et de développement de vacuole. Ici, nous démontrons que l’effecteur CvpF est un substrat du système de sécrétion de type 4 Dot/Icm qui localise aux vacuoles contenant Coxiella (CCV). CvpF est également capable d'interagir avec Rab26, conduisant au recrutement du marqueur autophagosomal LC3B aux CCV. Les mutants de cvpF présentent un défaut de réplication in vitro et in vivo, suggérant que le détournement de l'autophagie par cet effecteur est crucial pour la virulence de Coxiella. Comme pour les mutants de cvpF, les mutants ankA présentent le même défaut de réplication in vitro et la protéine AnkA est un substrat du SST4. L'effecteur AnkA contient des motifs de répétition Ankyrin localisés sur son domaine N-terminal. La bactérie induit une hyperfusion des mitochondries de manière dépendante du SST4 et spécifique de l’effecteur AnkA. Nos résultats montrent que AnkA interagit avec Drp1, une protéine motrice impliquée dans la fission mitochondriale et que cette interaction ainsi que l’hyperfusion des mitochondries seraient dépendant du domaine contenant les répétitions Ankyrine. Le mécanisme par lequel AnkA agit sur Drp1 reste à déterminer. Cependant, les effets observés sur la mitochondrie suggèrent que la manipulation de l’organelle par la bactérie promeut le développement de la vacuole et la réplication intracellulaire du pathogène. En conclusion, notre recherche suggère fortement que de nombreux effecteurs de Coxiella manipulent les voies des cellules hôtes pour assurer le développement intracellulaire efficace de ce pathogène. / Intracellular pathogenic bacteria manipulate host cell functions by secreting virulence factors (known as effectors) into the cytoplasm of the infected cell. This process allows the pathogen to proliferate in an otherwise hostile environment. The identification and characterization of the specific effectors of the various pathogens is therefore of crucial importance to counteract bacterial infections. Coxiella burnetii is a Class 3 gram-negative pathogen that causes Q fever, a zoonosis that causes major epidemics with a high impact on the economy and health. The natural reservoirs of Coxiella are mainly farm animals that can contaminate the environment by excreting the bacteria mainly in parturition products, vaginal mucus and feces. Human is then infected by inhalation of pseudo-spores disseminated in the environment. The obligate intracellular nature of Coxiella has so far severely limited its study, and as result, bacterial virulence factors involved in the development and progression of infection remain largely unknown. Coxiella replicates within host cells in a large vacuole with autolysosomal characteristics. The development of vacuole and survival of Coxiella in the host cell depend on the translocation of bacterial effectors by the type 4 Dot / Icm secretion system (SST4B) and the manipulation of many trafficking and signaling pathways of the host cell. Our team has generated and screened the first library of Coxiella transposon mutants, leading to the identification of a significant number of candidate virulence determinants and effector proteins. My thesis project is based on the characterization of two effectors of Coxiella, CvpF and AnkA, from the mutant library generated by the team. Mutants of these effectors exhibit defect in intracellular replication and vacuole development phenotypes. Here, we demonstrate that the effector CvpF is a substrate of the SST4B that localizes to vacuoles containing Coxiella (CCV). CvpF is also able to interact with Rab26, leading to the recruitment of the LC3B autophagosomal marker to CCV. cvpF mutants exhibit in vitro and in vivo replication deficiencies, suggesting that diversion of autophagy by this effector is crucial for Coxiella virulence. As for cvpF mutants, ankA mutants show the same in vitro defect of replication and the protein AnkA is a substrate of the SST4. AnkA contains Ankyrin repetition patterns located on its N-terminal domain. The bacterium induces an AnkA-dependent hyperfusion of mitochondria. Our results show that AnkA interacts with Drp1, a motor protein involved in mitochondrial fission, and that this interaction as well as mitochondrial hyperfusion is dependent on the domain containing Ankyrin-repeat motifs. The mechanism by which AnkA acts on Drp1 remains to be determined. However, the observed effects on mitochondria suggest that the organelle's manipulation by the bacterium promotes the development of the vacuole and the intracellular replication of the pathogen. To conclude, our research strongly suggests that multiple Coxiella effectors manipulate host cell pathways to ensure the efficient intracellular development of this pathogen.

The aim of this study was to develop improved methods to detect viable Coxiella burnetii in wastes from livestock production. The impetus for this work arose because there is a significant risk of infection for humans attributed to contact with waste products from the livestock production industry. This situation is further compounded by the lack of suitable tools to detect viable C. burnetii in these wastes. In addition, effective disinfection strategies for livestock wastes are also required to reduce the risk of infection with C. burnetii for individuals that come into contact with these waste products. A quantitative real-time PCR system (qPCR) with high sensitivity and specificity was developed to detect the C. burnetii in environmental samples associated with domestic ruminants and native Australian marsupials. Different detection chemistries and procedures were evaluated based on their sensitivity, specificity and reproducibility. Overall it was found that the TaqMan PCR targeting the IS1111a locus provided the most sensitive and reproducible test. The Geneworks PowerSoil(tm) DNA isolation kit provided the best compromise between reproducibility and recovery of DNA from livestock wastes. When combined, the IS1111a TaqMan qPCR and Geneworks PowerSoil DNA Extraction Kit provided a test which was capable of detecting as few as two C. burnetii genome equivalents in 0.2g of soil or faeces. Coxiella burnetii has been shown to display extreme resistance to environmental exposure. Therefore, assessment of the viability of the organism in environmental matrices is more useful for risk assessment programs than detection of DNA alone. A quantitative reverse transcriptase PCR was developed that was able to detect viable C. burnetii cells in soil. The sensitivity of the assay was enhanced by heat-treating the soil samples prior to extraction of RNA. The factor most often associated with transfer of C. burnetii to humans is exposure to livestock or their waste. Therefore, decontamination of waste from livestock production industries is a key factor in preventing outbreaks of Q fever. A system was developed to determine the efficacy of various disinfectant treatments against the environmental pathogen C. burnetii. Treatments evaluated included sodium hypochlorite, ozone, ultraviolet light, peracetic acid (PAA), and Virkon S®. Sodium hypochlorite at a concentration of 0.1 mM reduced the infectivity of C. burnetii by over 92% while treatment with the same sodium hypochlorite concentration in wastewater showed significantly reduced efficacy. Despite this reduced potency, sodium hypochlorite is still useful for control of C. burnetii in the liquid waste of animal production. Commercially available ELISA and CFT assays exist for ruminants but there are no immunological tests available for detecting C. burnetii in marsupials even though Australian marsupials are known to be susceptible to C. burnetii. An indirect ELISA for detecting anti-Coxiella antibodies in kangaroos was developed. Paired serum and faecal samples were taken from 379 ruminants from Western Australia and the serum was tested with a commercially available ELISA and the complement fixation test while the faeces was tested using the qPCR developed during this study. Paired serum and faecal samples were taken from 343 kangaroos from WA and were tested with the antibody-ELISA developed during this study and by qPCR. A very low prevalence of anti-Coxiella antibodies was observed in the ruminants sampled and results from immunological tests correlated poorly with qPCR data. The development of an ELISA for use with kangaroo serum was problematic because of the lack of reference sera from animals known to be infected with C. burnetii. Despite this results from the ELISA developed suggested that the apparent seroprevalence in the WA animals surveyed was approximately 34%. Results from testing kangaroo faeces with the qPCR correlated poorly with the results from the antibody-ELISA. These data suggest that kangaroos may be a significant reservoir of C. burnetii in Western Australia and due to cohabitation of kangaroos and domestic ruminants, may provide a link between the wildlife and domestic cycles of C. burnetii.

Coxiella burnetii is an intracellular pathogen that can cause both acute and chronic disease (Q fever) in humans and infects many animals with varying clinical illness and persistence. A guinea pig aerosol-challenge model of acute Q fever was developed using infection with C. burnetii across a 5-log range of challenge doses. Clinical signs included fever, weight loss, respiratory difficulty, and death, with degree and duration of response corresponding to dose of organism delivered. Histopathologic evaluation revealed coalescing panleukocytic bronchointerstitial pneumonia 7 days after a high-dose challenge, resolving to multifocal lymphohistiocytic interstitial pneumonia by 28 days. Clinical and pathologic changes noted in these guinea pigs were comparable to those seen in human acute Q fever, making this an accurate and valuable animal model. This model was used to compare the relative virulence of eight isolates from four different genotypic groups: I (RSA493, RSA334, and RSA270), IV (Q177 and Q173), V (Q212 and Q217), and VI (5J108-111). Guinea pigs infected with group I acute-diseaseassociated isolates had severe respiratory disease, while no to moderate clinical illness was observed in animals given group IV or V chronic-disease-associated isolates. 5J108- 111 appeared avirulent. These data suggest that C. burnetii isolates have a range of disease potentials and support a distinction in strain virulence between established genotypic groups, though isolates within the same genomic group cause similar pathologic responses. Heterologous protection was confirmed by cross vaccination and challenge with RSA493 and Q217. A marked non-specific suppression of lymphoproliferation was noted at 14 and 28 days post infection with RSA493; similar suppression was seen after infection with Q173 and Q212 but not 5J108-111. Proinflammatory cytokines IFN-γ and TNF-α were produced during early C. burnetii infection, at which time anti-inflammatory cytokines TGF-β and IL-10 were repressed. A vaccine made from phase I C. burnetii was found to be completely protective against lethal infection in the guinea pig model, while vaccination with killed phase II organisms conferred only partial protection, preventing death and reducing but not precluding fever and respiratory illness. Protective vaccination significantly stimulated cell-mediated immunity and elicited increases in IFN-γ, TNF-α, and IL-12p40 mRNA levels.

Thesis (Ph.D.) -- University of Montana, 2008. / Title from author supplied metadata. Description based on contents viewed on June 26, 2009. Includes bibliographical references.

Made available in DSpace on 2015-10-23T12:47:04Z (GMT). No. of bitstreams: 2 ana_silva_ioc_mest_2014.pdf: 1478235 bytes, checksum: 6d52627a1a269a7288d87d75c2559e6c (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-04-14 / Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, / O aumento do consumo de drogas ilícitas na atualidade vem acompanhado não apenas do impacto social e econômico, como também na saúde. As pessoas que injetam drogas apresentam maior susceptibilidade a infecções por diversos agentes, devido ao uso da droga e à subsequente imunossupressão, como também pelo risco de maior exposição a agentes transmitidos por artrópodes e reservatórios vertebrados em ambientes insalubres. São muitos os estudos correlacionando o consumo de drogas à infecção pelo HIV, VHB e VHC, porém raros investigam outros agentes. No presente estudo transversal e retrospectivo, selecionamos aleatoriamente 300 amostras de um estudo multicêntrico da década de 1990 e fizemos a pesquisa sorológica para Bartonella spp., Coxiella burnetii e Hantavirus. O perfil demográfico e social encontrado mostrou-se em concordância com a literatura: homens, jovens, de baixa renda e com histórico de prisão. A maior parte apresentava situações de risco por injetar drogas com dispositivos previamente usados e por injetar em local público. Alguns fatores foram discordantes da literatura internacional e podem ter influenciado os resultados, uma vez que são considerados situações de risco, como escassez de pessoas que injetaram drogas enquanto presas, que residem em instituições, ou sem-teto A evidência de infecção por C. burnetii foi de 9,3%, praticamente o dobro da observada na população geral brasileira, embora, em comparação com dados internacionais, a prevalência tenha sido menor. Não houve diferença estatística significativa em relação às variáveis demográficas entre os grupos sororeativos e soronegativos para febre Q. A presença de anticorpos anti-Bartonella foi de 1%, bem discrepante dos estudos nacionais e internacionais. Pode ser justificado pela não inclusão no teste sorológico de antígeno de B. elizabethae e de B. quintana e pela reduzida frequência de sem-tetos. Não houve correlação do alto consumo de álcool com maior soroprevalência por Bartonella spp., provavelmente pelos motivos acima citados. A soroprevalência para hantavirose de 4% foi semelhante à descrita em estudo brasileiro na população geral. Em relação aos usuários de drogas injetáveis, somente um artigo americano evidenciou uma soroprevalência bem menor, de 0,2%. Isso indica que em meios urbanos brasileiros, hantavírus, provavelmente o Seoul, pode estar circulando. Embora considerando que o estudo foi retrospectivo com base em evidência sorológica e que a possibilidade de reação cruzada não pode ser totalmente descartada, o presente estudo aponta para a importância de se incluir a pesquisa de outros agentes infecciosos além do HIV e dos vírus das hepatites B e C na população injetora de drogas / The increased consumption of illicit drugs today is followed not only by a social and economic impact, as well as in health. People who inject drugs are more susceptible to infections by various agents due to drug use and subsequent immunosuppressi on, as well as the risk of increased exposure to agents transmitted by arthropods and vertebrate reservoirs in unhealthy environments. Many studies link drug use to HIV, HBV and HCV, however only a few investigate other agents. In this re trospective cross - sectional study, we selected randomly 300 samples from a multicenter study from the 1990s and made a serological survey for Bartonella spp., Coxiella burnetii and hantavirus. The demographic and social profile was found in agreement with the literature: me n, young age , low income and with a history of imprisonment . The majority was exposed to risk situations by injecting drugs with previously used devices and by injecting in a public place s . Some factors were dis agreeing with the international literature an d may have influenced the results, since they are considered high - risk situations, such as shortage of people who injected drugs while imprisoned, residing in institutions, or homeless. Evidence of C. burnetii infection was 9.3%, almost double the rate in the general Brazilian population, although in comparison with international data, the prevalence was lower. There was no statistically significant difference in relation to demographic variables among seroreactive and seronegative groups for fever Q. The p resence of antibodies to Bartonella was 1%, significantly discrepant from national/ international studies. Possibly justified by the non inclusion of antigen B. elizabethae and B. quintana in the serological test , and the reduced frequency of homelessness. There was no correlation between high alcohol consumption with higher seroprevalence of Bartonella spp., p robably for the reasons mentioned above. The seroprevalence of hantavirus 4% was similar to that described in a Brazilian study in the general populat ion. Regarding inje c t ing drug users , only an American paper showed a much lower prevalence of 0.2%. This indicates that in Brazilian urban areas, hanta virus can circulate , probably Seoul hantavirus . While considering that the study was retrospective based on serological evidence and the possibility of cross - reactivity cannot be completely ruled out, this study points to the importance of including the research of other infectious agents other than H IV and hepatitis viruses B and C in injectin g drug population

Made available in DSpace on 2016-04-07T13:25:46Z (GMT). No. of bitstreams: 2 maria_guia_ioc_dout_2015.pdf: 33668716 bytes, checksum: 116b84e24b72cbb6e3a9a67cb4657da1 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Febre Q é uma zoonose cosmopolita causada por Coxiella burnetii, uma bactéria intracelular obrigatória gram-negativa da ordem Legionellales. A doença, que ocorre como pequenos surtos ou casos isolados, tem amplo espectro clínico, desde doença febril limitada, pneumonia, hepatite à endocardite e meningoencefalite. Pequenos roedores silvestres são importantes reservatórios, mas a infecção humana está principalmente relacionada a ovelhas, cabras e gado bovino, embora gatos, cães e coelhos estejam implicados em surtos urbanos. Na população humana a transmissão ocorre por aerossóis provenientes de líquido amniótico, placenta e lã, além da urina, fezes, leite e outras secreções animais contendo o agente. No Brasil, a primeira descrição de febre Q ocorreu em 1953. Embora casos esporádicos confirmados por teste sorológico e estudos sorológicos tenham apontado para a presença de C. burnetii no Brasil, somente em 2008 o agente foi identificado por análise molecular em paciente no município de Itaboraí/RJ. Com a consequente caracterização da infecção também nos animais domésticos de propriedade do paciente, durante o período de 2010-2011, o presente estudo foi proposto com o objetivo de: (i) realizar a vigilância de febre Q em pacientes com suspeita de dengue internados no Hospital Municipal Desembargador Leal Júnior, em Itaboraí, durante o período de 24 meses; (ii) verificar a presença de infecção nos familiares dos casos de febre Q; (iii) analisar os animais de propriedade dos casos de febre Q; (iv) investigar a presença de C. burnetii em animais silvestres capturados nas áreas de onde procederam os casos de febre Q e (v) pesquisar C. burnetii nos artrópodes coletados nos animais Neste estudo foram utilizados o teste de imunofluorescência indireta e a análise molecular (PCR) visando os elementos IS1111 transposase no genoma de C. burnetii. Dos 272 pacientes atendidos no período de 2013 a 2014, 26 (10%) apresentaram anticorpos anti-C. burnetii e nove (3,3%) foram PCR positivos. Um dos pacientes apresentou também infecção por dengue. A análise das sequências genômicas obtidas demonstrou a elevada similaridade entre si (99-100%) e com as sequências de C. burnetii depositadas no GenBank. Dos 35 animais domésticos estudados, seis foram sororreativos: 1/13 cães, 3/13 gatos, 2/9 ovelhas. A análise molecular foi positiva em swab anal de um filhote de gato e em amostra de tecido do úbere da ovelha sororreativa com história de aborto. Todos os 59 animais silvestres dos gêneros Didelphis, Philander, Micoureus, Akodon, Oligoryzomys e Nectomys foram negativos na análise molecular. Dos 283 artrópodes analisados, DNA de C. burnetii foi amplificado em oito dos 266 exemplares de Rhipicephalus sanguineus e em um exemplar de oito Amblyomma sculptum e nenhum dos sete Dermacentor nitens e duas pulgas Ctenocephalides canis identificados. Os resultados comprovam a presença de C burnetii em Itaboraí, confirmam a necessidade da inclusão da febre Q no diagnóstico diferencial da dengue e alertam para a necessidade da sensibilização dos profissionais de saúde sobre a ocorrência desta zoonose no Brasil / Q fever is a worldwide zoonosis disease caused by Coxiella burnetii, a gram-negative obligate intracellular bacterium of the legionellales order. The disease, which occurs as individual cases or small outbreaks have broad-spectrum clinical manifestation from limited febrile disease, pneumonia, endocarditis, hepatitis and meningoencephalitis. Small wild rodents are important reservoirs, but human infection is mainly related to sheep, goats and cattle, although cats, dogs and rabbits are involved in urban outbreaks. In human population, transmission occurs by aerosol from amniotic fluid, placenta and wool, as well as urine, feces, milk and other animal secretions, containing the agent. In Brazil, the first Q fever description occurred in 1953. Although sporadic cases confirmed by serological testing and sero-epidemiological studies have pointed to the presence of C. burnetii in Brazil, only in 2008 it was possible identify the agent in a patient's municipality of Itaboraí/RJ by molecular assay. With the consequent characterization of the infection also in domestic animals owned by the patient during the period 2010-2011, this study was proposed with the aim of: (i) conduct surveillance of Q fever in hospitalized patients with suspected dengue in Itaboraí at the Hospital Municipal Desembargador Leal Junior, during the period of 24 months; (ii) to verify the presence of Q fever infection in family cases ; (iii) analyzing, animals the property Q fever cases; (iv) investigate the presence of C. burnetii in wild animals captured in areas from which proceeded cases of Q fever and (v) search C burnetii in arthropods collected from animals. This study used the indirect immunofluorescence assay and molecular analysis (PCR) targeting the IS1111 transposase elements in the genome of C. burnetii. Of the 272 patients assisted from 2013 to 2014, 26 (10%) had anti-C. burnetii antibodies, and nine (3.3%) were PCR positive. One of these patients had also dengue infection. The analysis of the genomic sequences obtained showed high similarity to each other (99-100%) and the sequences of C. burnetii in GenBank. Of the 35 domestic animals studied, six were seroreactive: 1/13 dogs, cats 3/13, 2/9 sheep. Molecular analysis was positive in anal swab of a young kitten and tissue sample from the udder of sororreativa sheep with abortion history. All 59 wild animals Didelphis, Philander, Micoureus, Akodon, Oligoryzomys and Nectomys were negative by molecular analysis. Of the 283 arthropods analyzed, C. burnetii DNA was amplified in eight of 266 Rhipicephalus sanguineus and a specimen of 8 Amblyomma sculptum and none of 7 Dermacentor nitens and 2 fleas Ctenocephalides canis identified. The results show the presence of C. burnetii in Itaboraí, confirm the need for inclusion of Q fever in dengue differential diagnosis and point to the need of awareness among health professionals about the occurrence of this zoonosis in Brazil.

Indiana University-Purdue University Indianapolis (IUPUI) / Coxiella burnetii, the zoonotic agent of human Q fever and chronic endocarditis, is an obligate intracellular bacterial pathogen. The Coxiella intracellular niche, a large, lysosome-like parasitophorous vacuole (PV), is essential for bacterial survival and replication. There is growing evidence that host cell cholesterol trafficking plays a critical role in PV development and maintenance, prompting an examination of the role of cholesterol-binding host protein ORP1L (Oxysterol binding protein-Related Protein 1, Long) during infection. ORP1L is a multi-functional cholesterol-binding protein involved in late endosome/lysosome (LEL) trafficking, formation of membrane contact sites between LEL and the endoplasmic reticulum (ER), and cholesterol transfer from LEL to the ER. ORP1L localizes to the PV at novel membrane contact sites between the ER and the PV membrane. Ectopically expressed ORP1L in Coxiella-infected cells localizes to the PV membrane early during infection, before significant PV expansion and independent of other PV-localized proteins. Further, the N-terminal ORP1L Ankyrin repeats are both necessary and sufficient for PV localization, suggesting that protein-protein interactions, and not protein-lipid interactions, are primarily involved in PV association. Coxiella employs a Type IVB Secretion System (T4BSS) to translocate effector proteins into the host cytoplasm and manipulate various cellular functions. ORP1L is not found on the PV of a Coxiella mutant lacking a functional T4BSS, indicating a secreted bacterial protein is likely responsible for ORP1L recruitment. We identified a Coxiella mutant with a transposon insertion in CBU_0352 that exhibits a 50% decrease in ORP1L recruitment, suggesting that Coxiella CBU_0352 interacts directly or indirectly with ORP1L. Finally, we found that ORP1L depletion using siRNA alters PV dynamics, resulting in smaller yet more fusogenic Coxiella PVs. Together, these data suggest that ORP1L is specifically recruited to the PV, where it plays a novel role in Coxiella PV development and interactions between the PV and the host cell.

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.