Biomarker zur Beurteilung der Virulenz von Coxiella burnetii in verschiedenen Wirten

Coxiella burnetii (C. b.) ist ein obligat intrazelluläres gram-negatives Bakterium und der Erreger der Zoonose Q-Fieber. Wichtigste Infektionsquelle für den Menschen sind Wiederkäuer, wie Schafe, Ziegen und Rinder. In der Arbeit wurde ein In vitro-Zellkultursystem mit bovinen monocyte derived macrophages (MDM) etabliert, um die Interaktion von C. b. mit seinem Wirt zu untersuchen. Nach Infektion mit 13 heterogenen C. b.-Stämmen wurde die bakterielle Replikation und die Wirtszellantwort analysiert, um potentielle Biomarker zur Beurteilung der Virulenz von C. b. abzuleiten. Wirtsspezifische Unterschiede wurden an humanen MDM überprüft. Die Replikation der Bakterien in den MDM verlief stamm- und speziesspezifisch. In humanen MDM vermehrten sich Coxiellen effizienter als in bovinen MDM. Unabhängig von der Spezies korrelierte das bakterielle Wachstum mit den Genotypen der Stämme. Isolate der MLVA-Gruppe IV replizierten in beiden MDM besonders effizient. Dagegen zeigten Stämme der MLVA-Gruppe III ein sehr schwaches Wachstum auf. Die Wirtsreaktion unterschied sich stamm- bzw. speziesspezifisch und verlief unabhängig von der Replikation. Die Expression von pro-inflammatorischen Zytokinen, wie IL-1β, IL-8, IL-12 und TNF-α, dominierte in der frühen Infektionsphase (3 h p. i.) und korrelierte mit den Genotypen der Stämme. Eine sehr starke Wirtszellreaktion wurde vor allem von Isolaten aus der MLVA-Gruppe IV hervorgerufen. Dagegen reagierten MDM auf die Infektion mit Stämmen der MLVA-Gruppe I mit einer sehr schwachen Wirtsantwort. Die Expression von Aktivierungsmarkern (CD40, CD80, CD86, MHCI/II) auf bovinen und humanen MDM wurde durch die C. b.-Infektion kaum verändert. Zusammenfassend konnte eine Korrelation zwischen den Genotypen der Stämme und der Replikationseffizienz sowie der induzierten Wirtsantwort abgeleitet werden. Damit stellt der Genotyp einen potentiellen Biomarker für C. b. dar

The inhibition of the apoptosis pathway by the Coxiella burnetii effector protein CaeA requires the EK repetition motif, but is independent of survivin

Coxiella burnetii is an obligate intracellular bacterium that causes Query (Q) fever, a zoonotic disease. It requires a functional type IV secretion system (T4SS) which translocate bacterial effector proteins into the host cell cytoplasm and thereby facilitates bacterial replication. To date, more than 130 effector proteins have been identified, but their functions remain largely unknown. Recently, we demonstrated that one of these proteins, CaeA (CBU1524) localized to the host cell nucleus and inhibited intrinsic apoptosis of HEK293 or CHO cells. In the present study we addressed the question whether CaeA also affects the extrinsic apoptosis pathway. Ectopic expression of CaeA reduced extrinsic apoptosis and prevented the cleavage of the executioner caspase 7, but did not impair the activation of initiator caspase 9. CaeA expression resulted in an up-regulation of survivin (an inhibitor of activated caspases), which, however, was not causal for the anti-apoptotic effect of CaeA. Comparing the sequence of CaeA from 25 different C. burnetii isolates we identified an EK (glutamic acid/ lysine) repetition motif as a site of high genetic variability. The EK motif of CaeA was essential for the anti-apoptotic activity of CaeA. From these data, we conclude that the C. burnetii effector protein CaeA interferes with the intrinsic and extrinsic apoptosis pathway. The process requires the EK repetition motif of CaeA, but is independent of the upregulated expression of survivin.This work was supported by the Deutsche Forschungsgemeinschaft (SFB796 project B8) to AL and by the ERA-NET PathoGenoMics 3rd call to AL and JPG

Phenotype of Coxiella burnetii Strains of Different Sources and Genotypes in Bovine Mammary Gland Epithelial Cells

Despite the high prevalence of C. burnetii in dairy herds and continuous shedding via milk by chronically infected cows, bovine milk is not recognized as a relevant source of human Q fever. We hypothesized that the bovine mammary gland epithelial cell line PS represents a suitable in vitro model for the identification of C. burnetii-strain-specific virulence properties that may account for this discrepancy. Fifteen C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA) genotypes (I, II, III and IV). The replication efficiencies of all strains were similar, even though strains of the MLVA-genotype II replicated significantly better than genotype I strains, and bovine and ovine isolates replicated better than caprine ones. Bovine milk isolates replicated with similar efficiencies to isolates from other bovine organs. One sheep isolate (Cb30/14, MLVA type I, isolated from fetal membranes) induced a remarkable up-regulation of IL-1β and TNF-α, whereas prototypic strains and bovine milk isolates tended to suppress pro-inflammatory responses. While infection with strain Nine Mile I rendered the cells partially refractory to re-stimulation with E. coli lipopolysaccharide, Cb30/14 exerted a selective suppressive effect which was restricted to IL-6 and TNF-α and spared IL-1β. PS cells support the replication of different strains of C. burnetii and respond in a strain-specific manner, but isolates from bovine milk did not display a common pattern, which distinguishes them from strains identified as a public health concern

Interaction of Coxiella burnetii Strains of Different Sources and Genotypes with Bovine and Human Monocyte-Derived Macrophages

Most human Q fever infections originate from small ruminants. By contrast, highly prevalent shedding of Coxiella (C.) burnetii by bovine milk rarely results in human disease. We hypothesized that primary bovine and human monocyte-derived macrophages (MDM) represent a suitable in vitro model for the identification of strain-specific virulence properties at the cellular level. Twelve different C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA) genotypes. Infection efficiency and replication of C. burnetii were monitored by cell culture re-titration and qPCR. Expression of immunoregulatory factors after MDM infection was measured by qRT-PCR and flow cytometry. Invasion, replication and MDM response differed between C. burnetii strains but not between MDMs of the two hosts. Strains isolated from ruminants were less well internalized than isolates from humans and rodents. Internalization of MLVA group I strains was lower compared to other genogroups. Replication efficacy of C. burnetii in MDM ranged from low (MLVA group III) to high (MLVA group IV). Infected human and bovine MDM responded with a principal up-regulation of pro-inflammatory cytokines such as IL-1β, IL-12, and TNF-α. However, MLVA group IV strains induced a pronounced host response whereas infection with group I strains resulted in a milder response. C. burnetii infection marginally affected polarization of MDM. Only one C. burnetii strain of MLVA group IV caused a substantial up-regulation of activation markers (CD40, CD80) on the surface of bovine and human MDM. The study showed that replication of C. burnetii in MDM and the subsequent host cell response is genotype-specific rather than being determined by the host species pointing to a clear distinction in C. burnetii virulence between the genetic groups

Limitation of TCA Cycle Intermediates Represents an Oxygen-Independent Nutritional Antibacterial Effector Mechanism of Macrophages

Summary: In hypoxic and inflamed tissues, oxygen (O2)-dependent antimicrobial defenses are impaired due to a shortage of O2. To gain insight into the mechanisms that control bacterial infection under hypoxic conditions, we infected macrophages with the obligate intracellular pathogen Coxiella burnetii, the causative agent of Q fever. Our experiments revealed that hypoxia impeded C. burnetii replication in a hypoxia-inducible factor (HIF) 1α-dependent manner. Mechanistically, under hypoxia, HIF1α impaired the activity of STAT3, which in turn reduced the intracellular level of TCA cycle intermediates, including citrate, and impeded C. burnetii replication in macrophages. However, bacterial viability was maintained, allowing the persistence of C. burnetii, which is a prerequisite for the development of chronic Q fever. This knowledge will open future research avenues on the pathogenesis of chronic Q fever. In addition, the regulation of TCA cycle metabolites by HIF1α represents a previously unappreciated mechanism of host defense against intracellular pathogens. : The mechanisms that control bacterial infection under hypoxic conditions are only partially understood. Hayek et al. show that hypoxia-mediated stabilization of HIF1α results in the inhibition of STAT3 activation and the reduction of TCA metabolite levels, including citrate, in macrophages. This prevents C. burnetii replication without reducing bacterial viability. Keywords: Coxiella burnetii, HIF1α, STAT3, citrate, macrophag

MesSBAR—Multicopter and Instrumentation for Air Quality Research

Air quality measurements usually consist of ground-based instrumentation at fixed locations. However, vertical profiles of pollutants are of interest for understanding processes, distribution, dilution and concentration. Therefore, a multicopter system has been developed to investigate the vertical distribution of the concentration of aerosol particles, black carbon, ozone, nitrogen oxides (NOx) and carbon monoxide and the meteorological parameters of temperature and humidity. This article presents the requirements by different users, the setup of the quadrocopter system, the instrumentation and the results of first applications. The vertical distribution of particulate matter next to a highway was strongly related to atmospheric stratification, with different concentrations below and above the temperature inversion present in the morning. After the qualification phase described in this article, two identically equipped multicopters will be used upwind and downwind of line or diffuse sources such as highways or urban areas to quantify the influence of their emissions on the local air quality

Permissiveness of bovine epithelial cells from lung, intestine, placenta and udder for infection with Coxiella burnetii

International audienceRuminants are the main source of human infections with the obligate intracellular bacterium Coxiella (C.) burnetii. Infected animals shed high numbers of C. burnetii by milk, feces, and birth products. In goats, shedding by the latter route coincides with C. burnetii replication in epithelial (trophoblast) cells of the placenta, which led us to hypothesize that epithelial cells are generally implicated in replication and shedding of C. burnetii. We therefore aimed at analyzing the interactions of C. burnetii with epithelial cells of the bovine host (1) at the entry site (lung epithelium) which govern host immune responses and (2) in epithelial cells of gut, udder and placenta decisive for the quantity of pathogen excretion. Epithelial cell lines [PS (udder), FKD-R 971 (small intestine), BCEC (maternal placenta), F3 (fetal placenta), BEL-26 (lung)] were inoculated with C. burnetii strains Nine Mile I (NMI) and NMII at different cultivation conditions. The cell lines exhibited different permissiveness for C. burnetii. While maintaining cell viability, udder cells allowed the highest replication rates with formation of large cell-filling Coxiella containing vacuoles. Intestinal cells showed an enhanced susceptibility to invasion but supported C. burnetii replication only at intermediate levels. Lung and placental cells also internalized the bacteria but in strikingly smaller numbers. In any of the epithelial cells, both Coxiella strains failed to trigger a substantial IL-1β, IL-6 and TNF-α response. Epithelial cells, with mammary epithelial cells in particular, may therefore serve as a niche for C. burnetii replication in vivo without alerting the host’s immune response