Interdisciplinary studies on Coxiella burnetii: From molecular to cellular, to host, to one health research.

The Q-GAPS (Q fever GermAn interdisciplinary Program for reSearch) consortium was launched in 2017 as a German consortium of more than 20 scientists with exceptional expertise, competence, and substantial knowledge in the field of the Q fever pathogen Coxiella (C.) burnetii. C. burnetii exemplifies as a zoonotic pathogen the challenges of zoonotic disease control and prophylaxis in human, animal, and environmental settings in a One Health approach. An interdisciplinary approach to studying the pathogen is essential to address unresolved questions about the epidemiology, immunology, pathogenesis, surveillance, and control of C. burnetii. In more than five years, Q-GAPS has provided new insights into pathogenicity and interaction with host defense mechanisms. The consortium has also investigated vaccine efficacy and application in animal reservoirs and identified expanded phenotypic and genotypic characteristics of C. burnetii and their epidemiological significance. In addition, conceptual principles for controlling, surveilling, and preventing zoonotic Q fever infections were developed and prepared for specific target groups. All findings have been continuously integrated into a Web-based, interactive, freely accessible knowledge and information platform (www.q-gaps.de), which also contains Q fever guidelines to support public health institutions in controlling and preventing Q fever. In this review, we will summarize our results and show an example of how an interdisciplinary consortium provides knowledge and better tools to control a zoonotic pathogen at the national level.

Contemporary diagnostics for medically relevant fastidious microorganisms belonging to the genera Anaplasma,Bartonella,Coxiella,OrientiaandRickettsia.

Many of the human infectious pathogens-especially the zoonotic or vector-borne bacteria-are fastidious organisms that are difficult to cultivate because of their strong adaption to the infected host culminating in their near-complete physiological dependence on this environment. These bacterial species exhibit reduced multiplication rates once they are removed from their optimal ecological niche. This fact complicates the laboratory diagnosis of the disease and hinders the detection and further characterization of the underlying organisms, e.g. at the level of their resistance to antibiotics due to their slow growth. Here, we describe the current state of microbiological diagnostics for five genera of human pathogens with a fastidious laboratory lifestyle. For Anaplasma spp., Bartonella spp., Coxiella burnetii, Orientia spp. and Rickettsia spp., we will summarize the existing diagnostic protocols, the specific limitations for implementation of novel diagnostic approaches and the need for further optimization or expansion of the diagnostic armamentarium. We will reflect upon the diagnostic opportunities provided by new technologies including mass spectrometry and next-generation nucleic acid sequencing. Finally, we will review the (im)possibilities of rapidly developing new in vitro diagnostic tools for diseases of which the causative agents are fastidiously growing and therefore hard to detect.

Development of High-Throughput Multiplex Serology to Detect Serum Antibodies against Coxiella burnetii.

The causative agent of Q fever, the bacterium Coxiella burnetii (C. burnetii), has gained increasing interest due to outbreak events and reports about it being a potential risk factor for the development of lymphomas. In order to conduct large-scale studies for population monitoring and to investigate possible associations more closely, accurate and cost-effective high-throughput assays are highly desired. To address this need, nine C. burnetii proteins were expressed as recombinant antigens for multiplex serology. This technique enables the quantitative high-throughput detection of antibodies to multiple antigens simultaneously in a single reaction. Based on a reference group of 76 seropositive and 91 seronegative sera, three antigens were able to detect C. burnetii infections. Com1, GroEL, and DnaK achieved specificities of 93%, 69%, and 77% and sensitivities of 64%, 72%, and 47%, respectively. Double positivity to Com1 and GroEL led to a combined specificity of 90% and a sensitivity of 71%. In a subgroup of seropositives with an increased risk for chronic Q fever, the double positivity to these markers reached a specificity of 90% and a sensitivity of 86%. Multiplex serology enables the detection of antibodies against C. burnetii and appears well-suited to investigate associations between C. burnetii infections and the clinical manifestations in large-scale studies.

Concept of an Active Surveillance System for Q Fever in German Small Ruminants-Conflicts Between Best Practices and Feasibility.

Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii. Inhalation of contaminated dust particles or aerosols originating from animals (esp. small ruminants) is the main source of human infection. Hence, an active early warning system for Q fever in German small ruminant livestock was conceptualized to prevent human infections. First, we describe the best practice for establishing this system before evaluating its feasibility, as the combination of both evokes conflicts. Vaginal swabs from all husbandry systems with a focus on reproductive females should pooled and investigated by PCR to detect C. burnetii-shedding animals. Multistage risk-based sampling shall be carried out at the flock level and within-flock level. At the flock level, all flocks that are at risk to transmit the pathogen to the public must be sampled. At the within-flock level, all primi- and multiparous females after lambing must be tested in order to increase the probability of identifying a positive herd. Sampling should be performed during the main lambing period and before migration in residential areas. Furthermore, individual animals should be tested before migration or exhibition to ensure a negative status. If a flock tests positive in at least one individual sample, then flock-specific preventive measures should be implemented. This approach implies huge financial costs (sample testing, action/control measures). Hence, taking the step to develop more feasible and affordable preventive measures, e.g., vaccinating small ruminant flocks, should replace testing wherever justifiable.

Comparison of Different Commercially Available Enzyme-Linked Immunosorbent Assays with Immunofluorescence Test for Detection of Phase II IgG and IgM Antibodies to Coxiella burnetii.

Several commercially available enzyme-linked immunosorbent assays (ELISAs) for the detection of phase II IgG or IgM antibodies against Coxiella burnetii were compared. In addition, an indirect immunofluorescence test was used as a confirmation test. In all, 70 serum samples for IgG and 43 serum samples for IgM were tested. The ELISAs showed large differences in sensitivity and specificity, which led to a partially high ratio of false-negative determinations. The most convincing test was PanBio from Abbott, which unfortunately can only test IgG but not IgM.

Coxiella-like endosymbiont in argasid ticks (Ornithodoros muesebecki) from a Socotra Cormorant colony in Umm Al Quwain, United Arab Emirates.

Coxiella burnetii is a pathogen causing Q fever in domestic animals and humans. Seabirds have been implicated as possible reservoirs of this bacterium in the Arabian Gulf and in the Western Indian Ocean. Recently, Coxiella species closely related to C. burnetii was detected from ticks collected from oil rigs used as roosting areas by Socotra Cormorants (Phalacrocorax nigrogularis) in the western Arabian Gulf. We collected ticks from the largest breeding colony of Socotra Cormorants in the United Arab Emirates on the eastern extreme of the species' breeding range to determine the prevalence of C. burnetii and evaluate its role as a wild reservoir. All ticks were identified as Ornithodoros muesebecki and genomic DNA was extracted from larval and nymph/adult tick pools. Multiplex PCR tests were performed targeting three C. burnetii specific genes. C. burnetii was not detected although a Coxiella-like endosymbiont was identified that was closely related to Coxiella symbionts from Ornithodoros capensis ticks. Because domestic and wild ungulates are the primary source of C. burnetii, we suggest that the presence of free-ranging, native and non-native ungulates in some off-shore islands in the Arabian Gulf could disseminate C. burnetii to seabirds. More comprehensive studies on seabird colonies are needed to better understand the diversity and prevalence of Coxiella symbionts and to establish if C. burnetii is endemic on some of these islands.

Serological survey of Bartonella spp., Borrelia burgdorferi, Brucella spp., Coxiella burnetii, Francisella tularensis, Leptospira spp., Echinococcus, Hanta-, TBE- and XMR-virus infection in employees of two forestry enterprises in North Rhine-Westphalia, Germany, 2011-2013.

We initiated a survey to collect basic data on the frequency and regional distribution of various zoonoses in 722 employees of forestry enterprises in the German state of North Rhine-Westphalia (NRW) from 2011 to 2013. Exposures associated with seropositivity were identified to give insight into the possible risk factors for infection with each pathogen. 41.2% of participants were found to be seropositive for anti-Bartonella IgG, 30.6% for anti-Borrelia burgdorferi IgG, 14.2% for anti-Leptospira IgG, 6.5% for anti-Coxiella burnetii IgG, 6.0% for anti-Hantavirus IgG, 4.0% for anti-Francisella tularensis IgG, 3.4% for anti-TBE-virus IgG, 1.7% for anti-Echinococcus IgG, 0.0% for anti-Brucella IgG and anti-XMRV IgG. Participants seropositive for B. burgdorferi were 3.96 times more likely to be professional forestry workers (univariable analysis: OR 3.96; 95% CI 2.60-6.04; p<0.001); and participants seropositive for Hantavirus 3.72 times more likely (univariable analysis: OR 3.72; 95% CI 1.44-9.57; p=0.007). This study found a surprisingly high percentage of participants seropositive for anti-B. henselae IgG and for anti-F. tularensis IgG. The relatively high seroprevalence for anti-Leptospira IgG seen in this study could be related to living conditions rather than to exposure at work. No specific risk for exposure to C. burnetii and Echinococcus was identified, indicating that neither forestry workers nor office workers represent a risk population and that NRW is not a typical endemic area. Forestry workers appear to have higher risk for contact with B. burgdorferi-infected ticks and a regionally diverse risk for acquiring Hantavirus-infection. The regional epidemiology of zoonoses is without question of great importance for public health. Knowledge of the regional risk factors facilitates the development of efficient prevention strategies and the implementation of such prevention measures in a sustainable manner.

[Q fever]. / Q-Fieber.

The article summarizes some important recently identified findings about the Coxiella burnetii disease, Q fever. Beside new diagnostic parameters for follow-up issues, the importance of a timely identification of chronic Q fever and the peculiarities of the post Q fever fatigue syndrome are depicted.

[Epidemiological enquiries in two Q fever outbreaks in a community of Baden-Württemberg during 2008 and 2009]. / Epidemiologische Untersuchungen zu zwei Q-Fieber-Ausbrüchen in einer Gemeinde Baden-Württembergs in den Jahren 2008 und 2009.

In 2008 and 2009, two consecutive outbreaks of Q fever in humans were recorded in the district of Freudenstadt, northern Black Forrest, Baden-Württemberg, Germany. In 2008, a total of 41 persons from a single local community fell ill and were found infected with Coxiella burnetii. Although comprehensive diagnostic and epidemiological outbreak investigations were conducted and control measures taken which included vaccination of ruminants at risk in three parts of the affected community, re-occurrence of the disease in 2009 with further 29 confirmed human Q fever cases could not be prevented. While the origin of infection of the first outbreak was probably a flock of 550 sheep moved in the surrounding of the affected villages, the source of infection for the consecutive outbreak in 2009 could not be identified. It seems possible that meadows contaminated with infectious placenta or birth fluids represented the sources of infection.

BH3-only protein Puma contributes to death of antigen-specific T cells during shutdown of an immune response to acute viral infection.

During acute T cell immune responses to viral infection, antigen-specific T cells first proliferate and differentiate into effector cells, but after pathogen clearance most are deleted by apoptosis. The developmentally programmed death of antigen-specific T cells during shutdown of a T cell response is mediated by the Bcl-2-regulated apoptotic pathway and partly depends on the proapoptotic BH3-only protein Bim. However, loss of Bim enhanced survival of antigen-activated T cells to a lesser extent than Bcl-2 overexpression, indicating that other proapoptotic factors must contribute to T cell killing. In this study, we investigated the contributions of several BH3-only proteins to the shutdown of an acute T cell immune response in vivo. After infection with human herpes simplex virus (HSV-1), mice lacking Noxa, Bid, or Bad had a normal increase and subsequent decline in the numbers of antigen-specific CD8(+) T cells. In contrast, Puma-deficient mice showed an abnormally prolonged persistence of antigen-specific CD8(+) T cells in the spleen, associated with enhanced in vitro survival of these cells in the absence of cytokines. Puma was dispensable for viral clearance and also did not play a role in proliferation or activation of HSV-1-specific CD8(+) T cells in vivo. Collectively, these findings show that Puma contributes to the death of antigen-specific T cells during shutdown of an immune response.

Proapoptotic BH3-only protein Bim is essential for developmentally programmed death of germinal center-derived memory B cells and antibody-forming cells.

T cell-dependent B-cell immune responses induce germinal centers that are sites for expansion, diversification, and selection of antigen-specific B cells. During the immune response, antigen-specific B cells are removed in a process that favors the retention of cells with improved affinity for antigen, a cell death process inhibited by excess Bcl-2. In this study, we examined the role of the BH3-only protein Bim, an initiator of apoptosis in the Bcl-2-regulated pathway, in the programmed cell death accompanying an immune response. After immunization, Bim-deficient mice showed persistence of both memory B cells lacking affinity-enhancing mutations in their immunoglobulin genes and antibody-forming cells secreting low-affinity antibodies. This was accompanied by enhanced survival of both cell types in culture. We have identified for the first time the physiologic mechanisms for killing low-affinity antibody-expressing B cells in an immune response and have shown this to be dependent on the BH3-only protein Bim.

Genotype-dependent activation or repression of HBV enhancer II by transcription factor COUP-TF1.

AIM: To study the expression of HBV enhancer II by transcription factor COUP-TF1. METHODS: In order to study the regulation of HBV variants in the vicinity of the NRRE we cloned luciferase constructs containing the HBV enhancer II from variants and from HBV genotypes A and D and cotransfected them together with expression vectors for COUP-TF1 into HepG2 cells. RESULTS: Our findings show that enhancer II of HBV genotype A is also repressed by COUP-TF1. In contrast, two different enhancer II constructs of HBV genotype D were activated by COUP-TF1. The activation was independent of the NRRE because a natural variant with a deletion of nt 1763-1770 was still activated by COUP-TF1. CONCLUSION: Regulation of transcription of the HBV genome seems to differ among HBV genomes derived from different genotypes. These differences in transcriptional control among HBV genotypes may be the molecular basis for differences in the clinical course among HBV genotypes.

Characterization of host cell death induced by Chlamydia trachomatis.

Chlamydia are obligate intracellular bacteria that modulate apoptosis of the host cell. Strikingly, chlamydial infection has been reported both to inhibit and to induce apoptosis. Although the ability to inhibit apoptosis has been corroborated by the identification of cellular targets, confirmation of cell death induction has been complicated by a mixture of apoptotic features and atypical cell death during infection, as well as by differences in the experimental techniques used to measure cell death. Here we use a panel of well-established approaches in the study of apoptosis to define the form of cell death induced by Chlamydia trachomatis infection. Infected cells displayed apoptotic features such as nuclear condensation and fragmentation, as well as positive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining. Fragmentation of genomic DNA occurred, but was atypical. Clear evidence against the activation of effector caspases was found. Nuclear changes were measured in fibroblasts lacking one or both of the effectors of mitochondrial apoptosis, Bax and Bak. A slight reduction in nuclear changes was observed in Bax-deficient cells and in Bax/Bak double-deficient cells. Most surprisingly, this reduction was almost complete in Bak-deficient cells. Finally, dying infected cells were efficiently taken up by professional phagocytes, suggesting that Chlamydia-induced host-cell death could play a role in the immune response. In conclusion, chlamydial infection can induce cell death. Although Chlamydia-induced cell death has certain morphological features of apoptosis, it does not result from activation of the apoptotic pathway.

The NF-kappaB regulator Bcl-3 and the BH3-only proteins Bim and Puma control the death of activated T cells.

Apoptosis of activated T cells is critical for the termination of immune responses. Here we show that adjuvant-stimulated dendritic cells secrete cytokines that prime activated T cells for survival and analyze the roles of the NF-kappaB regulator Bcl-3 and the proapoptotic Bcl-2 family members Bim and Puma. Bcl-3 overexpression increased survival, and activated bcl-3-/- T cells died abnormally rapidly. Cytokines from adjuvant-stimulated dendritic cells induced Bcl-3, but survival through cytokine priming was Bcl-3-independent. Apoptosis inhibition by Bcl-3 involved blockade of Bim activation, because Bim was overactivated in Bcl-3-deficient cells, and Bcl-3 failed to increase survival of bim-/- T cells. However, adjuvants increased survival also in Bim-deficient T cells. This Bim-independent death pathway is at least in part regulated by Puma, as shown by analysis of puma-/- and noxa-/- T cells. IL-1, IL-7, and IL-15 primed T cells for survival even in the absence of Bim or Puma. Our data define interrelations and a Bim-independent pathway to activated T cell death.

Direct inhibition of cytochrome c-induced caspase activation in vitro by Toxoplasma gondii reveals novel mechanisms of interference with host cell apoptosis.

The intracellular parasite Toxoplasma gondii is known to inhibit apoptosis of its host cell. The molecular mechanisms of this interference are, however, not yet completely understood. We show here that viable parasites prominently inhibited the activation of caspase 3/7 induced by cytochrome c, dATP and dithiothreitol in cytosolic extracts of human-derived Jurkat leukemic T cells. In contrast, granzyme B-induced caspase activity was only slightly diminished. De novo protein biosynthesis by T. gondii was dispensable for the inhibition of cytochrome c-induced caspase activation. Furthermore, a complete parasite lysate or, more importantly, molecules released by extracellular parasites mediated the interaction with the caspase cascade. The cell-free system applied here is thus a valuable tool to study the interaction of T. gondii and possibly other intracellular pathogens with host cell apoptosis.

Induction of apoptosis by Legionella pneumophila in mammalian cells requires the mitochondrial pathway for caspase activation.

Legionella pneumophila, the agent of human Legionnaire's disease is a Gram-negative, rod-shaped bacterium. During infection, the bacteria invade human cells and replicate intracellularly. L. pneumophila can induce apoptosis in human myeloid and epitheloid cells and this may contribute to the development of pathology and disease. However, the molecular mechanism of apoptosis induction is still uncertain. Here we investigate this process. Legionella efficiently induced apoptosis in myeloid cells, T cells and fibroblasts. Induction of apoptosis involved activation of the initiator caspase-9 and effector caspases. Caspase activity was required for cell death. Analysis of mutant cells showed that the death receptor pathway was not involved in Legionella-induced apoptosis. Surprisingly, caspase activity was found almost exclusively in cells that did not harbor bacteria. Infection with Legionella caused the activation of the pro-apoptotic protein Bax and the release of cytochrome c. Mouse embryonic fibroblasts deficient for Bax and/or Bak were protected from Legionella-induced caspase activation. These results show a clear contribution of the mitochondrial pathway to Legionella-induced apoptosis.

Apoptosis in infectious disease: how bacteria interfere with the apoptotic apparatus.

Cell death by apoptosis is a common response of a human cell to many extrinsic stimuli. A cell's sensitivity to apoptotic triggers is affected by its activation and differentiation status. Bacteria are recognised by cellular receptors and elicit a multitude of signal transduction events that can, among other effects, alter the cell's response towards apoptotic stimuli. Many different bacteria and bacterial products have been recognised as agents that can act in this way and either induce or inhibit cell death. Besides these common and, as we argue, indirect activities, chlamydiae have been described to have a more specific capacity. These specialists of intracellular life can directly attack the host cell's apoptotic pathway. Here, we will attempt to structure the field of bacterial inhibition of apoptosis and discuss recent advancements in our knowledge of how chlamydiae interfere with the host cell's capacity to undergo apoptosis.

Broad degradation of proapoptotic proteins with the conserved Bcl-2 homology domain 3 during infection with Chlamydia trachomatis.

Chlamydiae are obligate intracellular bacteria that can inhibit apoptosis of their host cell. As shown recently, this inhibition is in part explained by the proteolytic degradation of the proapoptotic Bcl-2 family members (BH3-only proteins) Bim, Puma, and Bad upon chlamydial infection. In this study, we further explore this antiapoptotic mechanism. In cells infected with a Chlamydia trachomatis L2 strain, Bim, Puma, and Bad were degraded with similar kinetics, and the degradation of all three was blocked by inhibition of the proteasome. Furthermore, the BH3-only proteins Bmf, Noxa, and tBid were also targeted by chlamydial infection. The constitutively expressed Bmf disappeared during infection. When Noxa was experimentally induced, the levels were also reduced by infection with C. trachomatis. In death-receptor-induced apoptosis, cleaved and activated tBid was degraded, and this destruction was also prevented by inhibition of the proteasome. These results show that chlamydial infection leads to a broad degradation of BH3-only proteins. This loss of proapoptotic factors can explain the almost general protection of infected cells against apoptotic stimuli.

Toll-like receptor 9 signaling can sensitize fibroblasts for apoptosis.

Toll-like receptors (TLR) are activated by microbial components and transmit signals that induce cell activation and differentiation. A number of recent reports further indicate that TLR also have the potential to induce apoptosis upon ligand binding. Here we investigate the apoptosis-inducing capacity of TLR9, the receptor for microbial CpG-DNA. Unlike ligands for TLR2 and TLR4, CpG-DNA failed to induce apoptosis in RAW264.7 mouse macrophages. In human embryonic kidney fibroblasts transfected stably to express TLR9, CpG-DNA weakly induced apoptosis in one clone but not others without an obvious allocation to differences in TLR-signaling events. Analysis of the apoptotic signaling showed that the mitochondrial pathway of apoptosis was triggered by TLR9, as mitochondrial Bax was activated upstream of caspase-cleavage. CpG-DNA-induced apoptosis was reduced by cycloheximide suggesting that de novo protein synthesis was required. Strikingly, stimulation with CpG-DNA resulted in a strongly increased sensitivity of TLR9-expressing fibroblasts to apoptosis induced by staurosporine and UV-irradiation. These results identify a mitochondrial pathway to apoptosis that can be triggered by TLR9 and that may serve to sensitize cells from the innate immune system to apoptosis in the course of an immune response.

Phagocytosis-induced apoptosis in macrophages is mediated by up-regulation and activation of the Bcl-2 homology domain 3-only protein Bim.

Cell death by apoptosis is important in immune cell homeostasis and in the defense against infectious microorganisms. The physiological event of uptake and intracellular destruction of bacteria is a powerful apoptotic stimulus to macrophages and neutrophil granulocytes. In this study, we provide a molecular analysis of phagocytosis-induced apoptosis. Apoptosis was blocked by Bcl-2 in a mouse macrophage cell line and in primary mouse macrophages. Analysis of the upstream mechanisms revealed that apoptosis was triggered by the Bcl-2 homology domain 3-only protein Bim/Bod. Contact with bacteria or bacterial components induced a strong increase in Bim-expression through TLR and MyD88. Inhibition of the MAPK p38 and JNK reduced both up-regulation of Bim and apoptosis. Phosphorylation of Bim was further observed in mouse macrophages, which appeared to be the result of TLR-dependent phosphatase inhibition. Although TLR-induced Bim was, unlike Bim in resting cells, not bound to the microtubuli cytoskeleton, the up-regulation of Bim was not sufficient to cause apoptosis. A second signal was required that was generated in the process of phagocytosis. Phagocytosis-induced apoptosis was strongly reduced in Bim(-/-) macrophages. These data provide the molecular context of a form of apoptosis that may serve to dispose of terminally differentiated phagocytes.