Effects of Coxiella burnetii on MAPKinases phosphorylation

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is characterized by efficient immune response, whereas chronic Q fever is characterized by dysregulated immune response as demonstrated by the lack of granulomas, the failure of C. burnetii to induce lymphoproliferation, and interferon-γ production. The mitogen-activated protein kinase (MAPK) signaling pathway plays crucial roles in innate immune responses and control of bacterial infections. However, its role in Q fever has not been addressed. First, we investigated the activation of MAPKs p38, c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in murine macrophages stimulated with C. burnetii. Coxiella burnetii NM phase I (virulent) and NM phase II (avirulent) induced the activation of JNK and ERK1/2. Avirulent C. burnetii activate p38, whereas C. burnetii did not induce the phosphorylation of p38. Second, the level of p38 activation was studied in Q fever patients. We found that p38 was activated in monocyte-derived macrophages from healthy donors and patients with acute Q fever in response to a potent agonist such as lipopolysaccharide. Interestingly, p38 was not activated in patients with active chronic Q fever and was activated in patients with cured chronic Q fever. These results suggest that the determination of p38 activation may serve as a tool for measuring Q fever activity.     

Coxiella burnetii stimulates production of RANTES and MCP-1 by mononuclear cells: modulation by adhesion to endothelial cells and its implication in Q fever

Q fever is an infectious disease caused by Coxiella burnetii, which may become chronic when cytokine network and cell-mediated immune responses are altered. Chemokines, such as Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES, CCL5) and Monocyte Chemoattractant Protein-1 (MCP-1, CCL2), are specialized in the trafficing of peripheral blood mononuclear cells (PBMC), and are associated with T cell polarization that is essential for intracellular survival of C. burnetii. The present study investigated whether or not the infection status (no infection and acute or chronic infection with C. burnetii) of donors, affected the production of the two chemokines by PBMC with or without stimulation with virulent and avirulent C. burnetii. Our findings indicate that in vitro exposure to virulent or avirulent C. burnetii stimulated the production of RANTES and MCP-1 in PBMC obtained from healthy adults. The co-cultivation of endothelial cells and human PBMC resulted in an increased production of MCP-1 and the up-regulation of RANTES, which were contact-dependent. Unstimulated PBMC from patients with acute or chronic Q fever overproduced MCP-1. Interestingly, the addition of C. burnetii resulted in an increased production of RANTES and MCP-1 by PBMC obtained from patients with chronic Q fever, and the co-cultivation of PBMC with endothelial cells amplified increased production of chemokines. Circulating levels of RANTES and MCP-1 were also increased in chronic Q fever. We suggest that the overproduction of RANTES and MCP-1 secondary to the contact of PBMC with endothelium may perpetuate exaggerated inflammatory responses leading to inappropriate PBMC trafficking and to the pathogenesis of Q fever.     

Foxp3(+)CD4(+)CD25(+) regulatory T cells are increased in patients with Coxiella burnetii endocarditis

Chronic Q fever, which principally manifests as endocarditis, is characterized by Coxiella burnetii persistence and an impaired cell-mediated immune response. The long-term persistence of pathogens has been associated with the expansion of regulatory T cells (Tregs), the CD4(+) T-cell subset that is characterized by the expression of CD25 and Foxp3. We investigated the presence of Tregs in patients with acute Q fever (n?=?17), known to exhibit an efficient immune response, patients with Q fever endocarditis (n?=?54) and controls (n?=?27) by flow cytometry. The proportion of CD3(+) , CD4(+) and CD8(+) T cells was similar in controls and patients with Q fever. The percentage of CD4(+) T cells that expressed CD25 was similar in controls and patients with Q fever. The population of CD4(+) T cells that expressed both CD25 and Foxp3 was significantly (P?相似文献   

Profiling the humoral immune response of acute and chronic Q fever by protein microarray

Antigen profiling using comprehensive protein microarrays is a powerful tool for characterizing the humoral immune response to infectious pathogens. Coxiella burnetii is a CDC category B bioterrorist infectious agent with worldwide distribution. In order to assess the antibody repertoire of acute and chronic Q fever patients we have constructed a protein microarray containing 93% of the proteome of Coxiella burnetii, the causative agent of Q fever. Here we report the profile of the IgG and IgM seroreactivity in 25 acute Q fever patients in longitudinal samples. We found that both early and late time points of infection have a very consistent repertoire of IgM and IgG response, with a limited number of proteins undergoing increasing or decreasing seroreactivity. We also probed a large collection of acute and chronic Q fever patient samples and identified serological markers that can differentiate between the two disease states. In this comparative analysis we confirmed the identity of numerous IgG biomarkers of acute infection, identified novel IgG biomarkers for acute and chronic infections, and profiled for the first time the IgM antibody repertoire for both acute and chronic Q fever. Using these results we were able to devise a test that can distinguish acute from chronic Q fever. These results also provide a unique perspective on isotype switch and demonstrate the utility of protein microarrays for simultaneously examining the dynamic humoral immune response against thousands of proteins from a large number of patients. The results presented here identify novel seroreactive antigens for the development of recombinant protein-based diagnostics and subunit vaccines, and provide insight into the development of the antibody response.     

Developing a new clinical tool for diagnosing chronic Q fever: the Coxiella ELISPOT

Definitively establishing a clinical diagnosis of chronic Q fever remains challenging, as the diagnostic performance of both conventional serological tests and PCR is limited. Given the importance of an early diagnosis of chronic Q fever, there is a need for a reliable diagnostic test. We developed an enzyme-linked immunospot assay to measure Coxiella burnetii (C.?burnetii)-specific T-cell responses (Coxiella ELISPOT) to both phase I and phase II antigens and tested convalescent Q fever patients (without chronic disease, n?=?9) and patients with an established diagnosis of chronic Q fever (n?=?3). The Coxiella ELISPOT adequately identified convalescent Q fever patients from healthy controls by demonstrating C.?burnetii-specific T-cell interferon-γ production to both phase I and phase II antigens. Compared to convalescent Q fever patients, chronic Q fever patients showed a distinct Coxiella ELISPOT profile characterized by a much higher spot count for both phase I and phase II (18-fold for phase II, 8-fold higher for phase I) and a consistent shift towards more phase I reactivity. The diagnostic potential of the Coxiella ELISPOT is promising and warrants further investigation.     

Serotyping Coxiella burnetii isolates from acute and chronic Q fever patients by using monoclonal antibodies

Abstract Four mouse monoclonal antibodies reacting with Coxiella burnetii lipopolysaccharide antigens were produced and used in serotyping 17 C. burnetii isolates from acute Q fever and Q fever endocarditis patients in France. Two monoclonal antibodies (1B2 and 3B6) were considered specific for the Priscilla strain, a representative of Q fever endocarditis isolates, and did not react with the Nine Mile strain, which is representative of acute Q fever isolates. Monoclonal antibodies Nos. 1B2 and 3B6 reacted with 75% (3/4) acute Q fever isolates and 85% (11/13) of endocarditis isolates from France. It is reasonable to conclude that Priscilla-like strains cause both acute Q fever and Q fever endocarditis. The hypothesis that Priscilla-like strains only are associated with Q fever endocarditis should be reconsidered.     

Effects of X-irradiation on the immune response of guinea pigs to Q fever vaccine

The immune response of guinea pigs to Q fever vaccine following 75 to 250 R (60 to 180 rads) of acute whole-body irradiations was investigated. Complement-fixing (CF) antibody titers and protection against febrile response to challenge with virulent Coxiella burnetii were studied. Exposures ranging from 75 to 250 R, 24 hours prior to inoculation, did not detectably alter the CF antibody response. Similar results were observed with 175 R delivered 48 or 72 hours before immunization. Protection against febrile response to challenge with 10(3) median fever doses of C. burnetii was seen in animals irradiated with 175 R, 24 or 72 hours before immunization. Significant protection was detectable at 14, 21, and 42 days after immunization in both irradiated and nonirradiated animals. Acute irradiation of the degree studied increases the mortality in normal animals infected 15 to 17 days later with virulent C. burnetii. The lethal effect could be prevented by use of Q fever vaccine.     

The immune response in a cat-related outbreak of Q fever as measured by the indirect immunofluorescence test and the enzyme-linked immunosorbent assay

The isotypic immune response of 16 individuals who developed Q fever pneumonia following exposure to an infected parturient cat was studied. The enzyme-linked immunosorbent (ELISA) test was used to detect IgM, IgA, and IgG antibodies to phase I and phase II Coxiella burnetii whole-cell antigens and to the phase I lipopolysaccharide. The indirect immunofluorescent antibody (IFA) test was also used to detect antibodies to phase I and phase II whole cells. None of the 16 subjects developed antibodies to the phase I lipopolysaccharide. The ELISA was more sensitive than the IFA test. IgM antibodies to phase II antigen were detectable by ELISA in 80% of the subjects at the time of onset of symptoms and were still present in 7 of the 8 tested at 32 weeks following the onset of symptoms. In all instances (ELISA: IgG, IgM; IFA: IgG, IgM) phase II antibodies developed earlier and reached higher levels than did phase I antibodies. The absence of antibodies to phase I lipopolysaccharide in acute Q fever combined with our unpublished findings of antibodies to phase I lipopolysaccharide in chronic Q fever suggests that this test may be used to distinguish acute from chronic Q fever.     

The uptake of apoptotic cells drives Coxiella burnetii replication and macrophage polarization: a model for Q fever endocarditis

Patients with valvulopathy have the highest risk to develop infective endocarditis (IE), although the relationship between valvulopathy and IE is not clearly understood. Q fever endocarditis, an IE due to Coxiella burnetii, is accompanied by immune impairment. Patients with valvulopathy exhibited increased levels of circulating apoptotic leukocytes, as determined by the measurement of active caspases and nucleosome determination. The binding of apoptotic cells to monocytes and macrophages, the hosts of C. burnetii, may be responsible for the immune impairment observed in Q fever endocarditis. Apoptotic lymphocytes (AL) increased C. burnetii replication in monocytes and monocyte-derived macrophages in a cell-contact dependent manner, as determined by quantitative PCR and immunofluorescence. AL binding induced a M2 program in monocytes and macrophages stimulated with C. burnetii as determined by a cDNA chip containing 440 arrayed sequences and functional tests, but this program was in part different in monocytes and macrophages. While monocytes that had bound AL released high levels of IL-10 and IL-6, low levels of TNF and increased CD14 expression, macrophages that had bound AL released high levels of TGF-beta1 and expressed mannose receptor. The neutralization of IL-10 and TGF-beta1 prevented the replication of C. burnetii due to the binding of AL, suggesting that they were critically involved in bacterial replication. In contrast, the binding of necrotic cells to monocytes and macrophages led to C. burnetii killing and typical M1 polarization. Finally, interferon-gamma corrected the immune deactivation induced by apoptotic cells: it prevented the replication of C. burnetii and re-directed monocytes and macrophages toward a M1 program, which was deleterious for C. burnetii. We suggest that leukocyte apoptosis associated with valvulopathy may be critical for the pathogenesis of Q fever endocarditis by deactivating immune cells and creating a favorable environment for bacterial persistence.     

Genomotyping of Coxiella burnetii using microarrays reveals a conserved genomotype for hard tick isolates

C. burnetii is a Gram-negative intracellular Y-proteobacteria that causes the zoonotic disease Q fever. Q fever can manifest as an acute or chronic illness. Different typing methods have been previously developed to classify C. burnetii isolates to explore its pathogenicity. Here, we report a comprehensive genomotyping method based on the presence or absence of genes using microarrays. The genomotyping method was then tested in 52 isolates obtained from different geographic areas, different hosts and patients with different clinical manifestations. The analysis revealed the presence of 10 genomotypes organized into 3 groups, with a topology congruent with that obtained through multi-spacer typing. We also found that only 4 genomotypes were specifically associated with acute Q fever, whereas all of the genomotypes could be associated to chronic human infection. Serendipitously, the genomotyping results revealed that all hard tick isolates, including the Nine Mile strain, belong to the same genomotype.     

Differentiation of Coxiella burnetii by Sequence Analysis of the Gene (com1) Encoding a 27-kDa Outer Membrane Protein

The gene (com1) encoding a 27-kDa outer membrane protein in 21 strains of Coxiella burnetii from a variety of clinical and geographical sources was sequenced for strain differentiation. The com1 gene was highly conserved among all the strains tested but there were several differences in nucleotide and deduced amino acid sequences. Based on the com1 gene-specific nucleotides and deduced amino acids, the 21 strains were divided into four groups. Group 1 contained 14 strains originating from ticks, cattle and human cases of acute Q fever. Groups 2 and 3 included 2 and 3 strains, respectively, originating from human cases of chronic Q fever. Group 4 contained 2 strains originating from a human case of acute Q fever and a goat with abortion. The results indicated that the strains originating from ticks, cattle and human cases of acute Q fever differed at the molecular level from those of human chronic Q fever. This study suggests that a sequence analysis of the com1 gene can be used for strain differentiation of C. burnetii.     

Differentiation of Coxiella burnetii isolates by sequence determination and PCR-restriction fragment length polymorphism analysis of isocitrate dehydrogenase gene

The isocitrate dehydrogenase (icd) gene of Coxiella burnetii was cloned and sequenced to differentiate between isolates with various geographic origins and phenotypic properties. Based on the gene sequences all 19 isolates studied could be divided into three groups. Group 1 contained isolates originating from acute cases of Q fever, ticks and cows. Groups 2 and 3 included isolates from chronic Q fever patients and a prototype strain from an aborted goat. Although the icd gene profiles were different among isolates of the latter two groups, there were two base differences common for both groups which could be used as markers to distinguish them from group 1 isolates. Based on one of the markers a simple method using PCR-restriction fragment length polymorphism analysis was developed for rapid differentiation of C. burnetii isolates as well as for direct detection and differentiation of the bacterium in human serum samples. Taken together, the study results suggest that the icd-based differentiation method may be useful in clinical investigation of Coxiella infections.     

Cellular immunity in Q fever: modulation of responsiveness by a suppressor T cell-monocyte circuit

Human infection with the rickettsia Coxiella burnetii presents as an acute flulike primary Q fever, as a subacute granulomatous hepatitis, or, rarely, as chronic endocarditis. We have previously described lymphocyte unresponsiveness to Coxiella antigen in patients with Q fever endocarditis. This unresponsiveness was antigen specific and was mediated in part by adherent suppressor cells. In this report we show that the adherent suppressor cells work via prostaglandin E2 (PGE2)4 production. Addition of the cyclooxygenase inhibitor indomethacin to cultures of PBMC from patients with endocarditis or chronic laboratory exposure resulted in consistent increases in Coxiella-specific lymphocyte proliferation. The degree of increase in proliferation induced by indomethacin correlated strongly with the amount of PGE2 produced in a 4-hr culture stimulated by Coxiella antigen, but it also correlated with the sensitivity to inhibition of mitogenesis by PGE2. The suppressor mechanism was antigen nonspecific, because induction of suppression in vitro by Coxiella antigen also suppressed Candida-induced proliferation when both antigens were present in the same culture. Addition of indomethacin to these antigen cocultures totally reversed the Coxiella-induced suppression, confirming the evidence above that the nonspecific effector mechanism of suppression was prostaglandin (PG)-mediated. Elicitation of suppression, however, was antigen specific and involved a T cell-monocyte suppressor circuit. Supernatants from Coxiella-stimulated immune T cells and from the suppressor subset (OKT8+-enriched) of those T cells, but not unstimulated immune cells, induced augmented PGE2 production by unrelated nonimmune PBMC. We conclude that the lymphocyte unresponsiveness characterizing patients with Q fever endocarditis is modulated in part by an antigen-specific T suppressor cell which secretes a lymphokine to stimulate PGE2 production by adherent cells.     

Coxiella burnetii, the agent of Q fever in Brazil: its hidden role in seronegative arthritis and the importance of molecular diagnosis based on the repetitive element IS1111 associated with the transposase gene

Coxiella burnetii is the agent of Q fever , an emergent worldwide zoonosis of wide clinical spectrum. Although C. burnetii infection is typically associated with acute infection, atypical pneumonia and flu-like symptoms, endocarditis, osteoarticular manifestations and severe disease are possible, especially when the patient has a suppressed immune system; however, these severe complications are typically neglected. This study reports the sequencing of the repetitive element IS1111 of the transposase gene of C. burnetii from blood and bronchoalveolar lavage (BAL) samples from a patient with severe pneumonia following methotrexate therapy, resulting in the molecular diagnosis of Q fever in a patient who had been diagnosed with active seronegative polyarthritis two years earlier. To the best of our knowledge, this represents the first documented case of the isolation of C. burnetii DNA from a BAL sample.     

First case of Q fever endocarditis in Croatia and a short review

We present a 70-year-old man from Dalmatia, Croatia, with a history of prolonged high fever diagnosed as Q fever endocarditis. As far as we know, this is the first case of chronic Q fever in Croatia. The treatment was started as for culture-negative endocarditis, but was without clinical response. After significantly high anti-phase I IgG plus IgA antibodies titers to Coxiella burnetii were shown, the initial treatment with doxycycline was changed and ciprofloxacin was started with good clinical response.     

Coxiella burnetii: international pathogen of mystery

Coxiella burnetii is an intracellular bacterium that causes acute and chronic Q fever. This unique pathogen has been historically challenging to study due to obstacles in genetically manipulating the organism and the inability of small animal models to fully mimic human Q fever. Here, we review the current state of C. burnetii research, highlighting new approaches that allow the mechanistic study of infection in disease relevant settings.     

A proteomic approach to investigate the differential antigenic profile of two Coxiella burnetii strains

Q fever is a widespread zoonosis caused by Coxiella burnetii, an obligate intracellular Gram-negative bacterium. Current diagnostics of Q fever is based on serological testing of patient serum. Biological distinction among C. burnetii strains has been referred at the genetic level as well as in virulence in animal models of Q fever. Disclosure of strain specific antigens might show insight into the biology and pathogenesis of this query pathogen, as well as it can provide the literature with potential serodiagnostic markers. In the present study, we sought to obtain an outer membrane enriched fraction of two C. burnetii reference strains, which originate from different sources, in order to investigate the way in which their antigenic profile is differentiated against a patient serum. We systematically analyzed the sarcosyl-insoluble fraction, enriched in outer membrane proteins, of the two C. burnetii strains using doubled SDS-PAGE combined with MS/MS analysis. In total, twenty-two outer membrane proteins were identified, representing 26% of the overall 86 identified proteins. The sarcosyl-insoluble fraction was then separated on 2DE IEF/SDS-PAGE and probed with serum from an infected patient. Different immuno-reactive proteins between the two C. burnetii strains were identified and included 2 outer membrane proteins, a hypothetical protein (CBU_0937) with unknown function and OmpH (CBU_0612), a previously identified marker for Q fever endocarditis. This approach can be used to reveal strain-specific proteins involved in pathogenesis and new serodiagnostic markers.     

A new plasmid (QpDV) common to Coxiella burnetii isolates associated with acute and chronic Q fever

Abstract Genetic studies of Coxiella burnetii strains suggested the possibility of differentiating new isolates according to their plasmid DNA content. Virulence and/or clinical manifestations ('chronic' and 'acute' Q fever) had been claimed to correlate with this plasmid typing. A new plasmid, named QpDV, was found to be common to C. burnetii isolates obtained from acute and chronic Q fever. According to the results obtained, plasmid usage for detection and differentiation of respective pathovars of C. burnetii and the correlation between gene specificity and pathovar has to be revised. Closer studies suggested a common origin of C. burnetii plasmids, but also showed some differences characteristic for each plasmid, probably reflecting divergent evolution.