Institute of Molecular Pathogenesis

Jena, Germany

Institute of Molecular Pathogenesis

Jena, Germany

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Jacobsen I.D.,Leibniz Institute for Natural Product Research and Infection Biology | Grosse K.,Leibniz Institute for Natural Product Research and Infection Biology | Slesiona S.,Leibniz Institute for Natural Product Research and Infection Biology | Hube B.,Leibniz Institute for Natural Product Research and Infection Biology | And 2 more authors.
Infection and Immunity | Year: 2010

Infection models are essential tools for studying microbial pathogenesis. Murine models are considered the "gold standard" for studying in vivo infections caused by Aspergillus species, such as A. fumigatus. Recently developed molecular protocols allow rapid construction of high numbers of fungal deletion mutants, and alternative infection models based on cell culture or invertebrates are widely used for screening such mutants to reduce the number of rodents in animal experiments. To bridge the gap between invertebrate models and mice, we have developed an alternative, low-cost, and easy-to-use infection model for Aspergillus species based on embryonated eggs. The outcome of infections in the egg model is dose and age dependent and highly reproducible. We show that the age of the embryos affects the susceptibility to A. fumigatus and that increased resistance coincides with altered chemokine production after infection. The progress of disease in the model can be monitored by using egg survival and histology. Based on pathological analyses, we hypothesize that invasion of embryonic membranes and blood vessels leads to embryonic death. Defined deletion mutant strains previously shown to be fully virulent or partially or strongly attenuated in a mouse model of bronchopulmonary aspergillosis showed comparable degrees of attenuation in the egg model. Addition of nutrients restored the reduced virulence of a mutant lacking a biosynthetic gene, and variations of the infectious route can be used to further analyze the role of distinct genes in our model. Our results suggest that embryonated eggs can be a very useful alternative infection model to study A. fumigatus virulence and pathogenicity. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Jacobsen I.D.,Leibniz Institute for Natural Product Research and Infection Biology | Grosse K.,Leibniz Institute for Natural Product Research and Infection Biology | Berndt A.,Institute of Molecular Pathogenesis | Hube B.,Leibniz Institute for Natural Product Research and Infection Biology | Hube B.,Friedrich - Schiller University of Jena
PLoS ONE | Year: 2011

Alternative models of microbial infections are increasingly used to screen virulence determinants of pathogens. In this study, we investigated the pathogenesis of Candida albicans and C. glabrata infections in chicken embryos infected via the chorio-allantoic membrane (CAM) and analyzed the virulence of deletion mutants. The developing immune system of the host significantly influenced susceptibility: With increasing age, embryos became more resistant and mounted a more balanced immune response, characterized by lower induction of proinflammatory cytokines and increased transcription of regulatory cytokines, suggesting that immunopathology contributes to pathogenesis. While many aspects of the chicken embryo response resembled murine infections, we also observed significant differences: In contrast to systemic infections in mice, IL-10 had a beneficial effect in chicken embryos. IL-22 and IL-17A were only upregulated after the peak mortality in the chicken embryo model occurred; thus, the role of the Th17 response in this model remains unclear. Abscess formation occurs frequently in murine models, whereas the avian response was dominated by granuloma formation. Pathogenicity of the majority of 15 tested C. albicans deletion strains was comparable to the virulence in mouse models and reduced virulence was associated with significantly lower transcription of proinflammatory cytokines. However, fungal burden did not correlate with virulence and for few mutants like bcr1Δ and tec1Δ different outcomes in survival compared to murine infections were observed. C. albicans strains locked in the yeast stage disseminated significantly more often from the CAM into the embryo, supporting the hypothesis that the yeast morphology is responsible for dissemination in systemic infections. These data suggest that the pathogenesis of C. albicans infections in the chicken embryo model resembles systemic murine infections but also differs in some aspects. Despite its limitations, it presents a useful alternative tool to pre-screen C. albicans strains to select strains for subsequent testing in murine models. © 2011 Jacobsen et al.


Berens C.,Institute of Molecular Pathogenesis | Groher F.,TU Darmstadt | Suess B.,TU Darmstadt
Biotechnology Journal | Year: 2015

RNA utilizes many different mechanisms to control gene expression. Among the regulatory elements that respond to external stimuli, riboswitches are a prominent and elegant example. They consist solely of RNA and couple binding of a small molecule ligand to the so-called "aptamer domain" with a conformational change in the downstream "expression platform" which then determines system output. The modular organization of riboswitches and the relative ease with which ligand-binding RNA aptamers can be selected in vitro against almost any molecule have led to the rapid and widespread adoption of engineered riboswitches as artificial genetic control devices in biotechnology and synthetic biology over the past decade. This review highlights proof-of-principle applications to demonstrate the versatility and robustness of engineered riboswitches in regulating gene expression in pro- and eukaryotes. It then focuses on strategies and parameters to identify aptamers that can be integrated into synthetic riboswitches that are functional in vivo, before finishing with a reflection on how to improve the regulatory properties of engineered riboswitches, so that we can not only further expand riboswitch applicability, but also finally fully exploit their potential as control elements in regulating gene expression. Small molecule-binding RNA aptamers can be exploited for the engineering of synthetic riboswitches. In vitro selection combined with in vivo screening helps to identify RNA aptamers with regulatory potential. This review highlights proof-of-principle applications, focuses on strategies and parameters to identify aptamers to be integrated into synthetic riboswitches and discusses how to improve the regulatory properties of engineered riboswitches to effectively employ them for regulating gene expression. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Kalmar I.,Ghent University | Berndt A.,Institute of Molecular Pathogenesis | Yin L.,Ghent University | Chiers K.,Ghent University | And 2 more authors.
Veterinary Immunology and Immunopathology | Year: 2015

Although Chlamydia (C.) psittaci infections are recognized as an important factor causing economic losses and impairing animal welfare in poultry production, the specific mechanisms leading to severe clinical outcomes are poorly understood. In the present study, we comparatively investigated pathology and host immune response, as well as systemic dissemination and expression of essential chlamydial genes in the course of experimental aerogeneous infection with C. psittaci and the closely related C. abortus, respectively, in specific pathogen-free chicks. Clinical signs appeared sooner and were more severe in the C. psittaci-infected group. Compared to C. abortus infection, more intense systemic dissemination of C. psittaci correlated with higher and faster infiltration of immune cells, as well as more macroscopic lesions and epithelial pathology, such as hyperplasia and erosion. In thoracic air sac tissue, mRNA expression of immunologically relevant factors, such as IFN-γ, IL-1β, IL-6, IL-17, IL-22, LITAF and iNOS was significantly stronger up-regulated in C. psittaci- than in C. abortus-infected birds between 3 and 14 days post-infection. Likewise, transcription rates of the chlamydial genes groEL, cpaf and ftsW were consistently higher in C. psittaci during the acute phase. These findings illustrate that the stronger replication of C. psittaci in its natural host also evoked a more intense immune response than in the case of C. abortus infection. © 2015 Elsevier B.V.


Pieper J.,Jena University of Applied Sciences | Methner U.,Institute of Bacterial Infections and Zoonoses | Berndt A.,Institute of Molecular Pathogenesis
Infection and Immunity | Year: 2011

Avian γδ T lymphocytes are frequently found in blood and organs and are assumed to be crucial to the immune defense against Salmonella infections of chicks. To elucidate the so-far-unknown immunological features of subpopulations of avian γδ T cells in the course of infection, day-old chicks were infected orally with Salmonella enterica serovar Typhimurium. Until 11 days after infection, the occurrence as well as transcription of the CD8 antigen and immunologically relevant protein genes of CD8α- and CD8α+high (CD8αα + CD8αβ+) γδ cells were analyzed using flow cytometry and quantitative real-time reverse transcription-PCR (RT-PCR) with blood, spleen, thymus, and cecum samples. After infection, an increased percentage of CD8α+high γδ T lymphocytes was found in blood, in spleen, and, with the highest values and most rapidly, in cecum. Within the CD8α+high subset, a significant rise in the number of CD8αα+ cells was accompanied by enhanced CD8α antigen expression and reduced gene transcription of the CD8β chain. CD8αα+ and CD8αβ+ cells showed elevated transcription for Fas, Fas ligand (FasL), interleukin-2 receptor α (IL-2Rα), and gamma interferon (IFN-γ). While the highest fold changes in mRNA levels were observed in CD8αα+ cells, the mRNA expression rates of CD8αβ+ cells never significantly exceeded those of the CD8αα+ cells. In conclusion, both CD8α+high γδ T-cell subpopulations (CD8αα+ and CD8αβ+) might be a potential source of IFN-γ in Salmonella-infected chicks. However, due to their prominent frequency in blood and organs after infection, the avian CD8αα+ gamma;δ T-cell subset seems to be unique and of importance in the course of Salmonella Typhimurium infection of very young chicks. Copyright © 2011, American Society for Microbiology. All Rights Reserved.


Fritsch I.,Institute of Molecular Pathogenesis | Fritsch I.,Universitatsklinikum Jena | Luyven G.,Chemical and Veterinary Laboratories Agency Rhein Ruhr Wupper | Kohler H.,Institute of Molecular Pathogenesis | And 2 more authors.
Applied and Environmental Microbiology | Year: 2012

Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit-variable-number tandemrepeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n=13), six cattle herds living in the area of this park (n=23), and five cattle herds without any contact with these red deer (n=17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable interpretations of epidemiological studies on paratuberculosis. © 2012, American Society for Microbiology.


Prohl A.,Institute of Molecular Pathogenesis | Ostermann C.,Institute of Molecular Pathogenesis | Lohr M.,Institute of Molecular Pathogenesis | Reinhold P.,Institute of Molecular Pathogenesis
Journal of Visualized Experiments | Year: 2014

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals. The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions. Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host. © JoVE 2006-2014. All Rights Reserved.


Ostermann C.,Institute of Molecular Pathogenesis | Linde S.,Institute of Molecular Pathogenesis | Siegling-Vlitakis C.,Free University of Berlin | Reinhold P.,Institute of Molecular Pathogenesis
Multidisciplinary Respiratory Medicine | Year: 2014

Background: Chlamydia psittaci (Cp) is a respiratory pathogen capable of inducing acute pulmonary zoonotic disease (psittacosis) or persistent infection. To elucidate the pathogenesis of this infection, a translational large animal model was recently introduced by our group. This study aims at quantifying and differentiating pulmonary dysfunction and acid-base imbalances induced by Cp. Methods: Forty-two calves were grouped in (i) animals inoculated with Cp (n = 21) and (ii) controls shaminoculated with uninfected cell culture (n = 21). For pulmonary function testing, impulse oscillometry, capnography, and FRC (functional residual capacity) measurement were applied to spontaneously breathing animals. Variables of acid-base status were assessed in venous blood using both (i) traditional Henderson-Hasselbalch and (ii) strong ion approach. Results: Both obstructive and restrictive pulmonary disorders were induced in calves experimentally inoculated with Cp. Although disorders in respiratory mechanics lasted for 8-11 days, the pattern of spontaneous breathing was mainly altered in the period of acute illness (until 4 days post inoculation, dpi). Expiration was more impaired than inspiration, resulting in elevated FRC. Ventilation was characterised by a reduction in tidal volume (-25%) combined with an increased percentage of dead space volume and a significant reduction of alveolar volume by 10%. Minute ventilation increased significantly (+50%) due to a compensatory doubling of respiratory rate. Hyperventilatory hypocapnia at 2-3 dpi resulted in slightly increased blood pH at 2 dpi. However, the acid-base equilibrium was additionally influenced by metabolic components, i.e. the systemic inflammatory response, all of which were detected with help of the strong ion theory. Decreased concentrations of albumin (2-10 dpi), a negative acute-phase marker, resulted in a decrease in the sum of non-volatile weak acids (Atot), revealing an alkalotic effect. This was counterbalanced by acidic effects of decreased strong ion difference (SID), mediated by the interplay between hypochloraemia (alkalotic effect) and hyponatraemia (acidic effect). Conclusions: This bovine model was found to be suitable for studying pathophysiology of respiratory Cp infection and may help elucidating functional host-pathogen interactions in the mammalian lung. © 2014 Ostermann et al.; licensee BioMed Central Ltd.


Munoz L.E.,Friedrich - Alexander - University, Erlangen - Nuremberg | Herrmann M.,Friedrich - Alexander - University, Erlangen - Nuremberg | Berens C.,Friedrich - Alexander - University, Erlangen - Nuremberg | Berens C.,Institute of Molecular Pathogenesis
Clinical and Experimental Immunology | Year: 2015

Summary: In an organism, cell death occurs at many different sites and in many different forms. It is frequently part of normal development or serves to maintain cell homeostasis. In other cases, cell death not only occurs due to injury, disease or infection, but also as a consequence of various therapeutic interventions. However, in all of these scenarios, the immune system has to react to the dying and dead cells and decide whether to mount an immune response, to remain quiet or to initiate healing and repopulation. This is essential for the organism, testified by many diseases that are associated with malfunctioning in the cell death process, the corpse removal, or the ensuing immune responsiveness. Therefore, dying cells generally have to be considered as instructors of the immune system. How this happens and which signals and pathways contribute to modulate or shape the immune response is still elusive in many conditions. The articles presented in this Special Issue address such open questions. They highlight that the context in which cell death occurs will not only influence the cell death process itself, but also affect the surrounding cellular milieu, how the generation and presence of 'eat me' signals can have an impact on cell clearance, and that the exact nature of the residual 'debris' and how it is processed are fundamental to determining the immunological consequences. Hopefully, these articles initiate new approaches and new experiments to complete our understanding of how cell death and the immune system interact with each other. © 2015 British Society for Immunology.


Elze J.,Institute of Molecular Pathogenesis | Liebler-Tenorio E.,Institute of Molecular Pathogenesis | Ziller M.,Friedrich Loeffler Institute | Kohler H.,Institute of Molecular Pathogenesis
Epidemiology and Infection | Year: 2013

The objective of this study was to identify the most reliable approach for prevalence estimation of Mycobacterium avium ssp. paratuberculosis (MAP) infection in clinically healthy slaughtered cattle. Sampling of macroscopically suspect tissue was compared to systematic sampling. Specimens of ileum, jejunum, mesenteric and caecal lymph nodes were examined for MAP infection using bacterial microscopy, culture, histopathology and immunohistochemistry. MAP was found most frequently in caecal lymph nodes, but sampling more tissues optimized the detection rate. Examination by culture was most efficient while combination with histopathology increased the detection rate slightly. MAP was detected in 49/50 animals with macroscopic lesions representing 1·35% of the slaughtered cattle examined. Of 150 systematically sampled macroscopically non-suspect cows, 28·7% were infected with MAP. This indicates that the majority of MAP-positive cattle are slaughtered without evidence of macroscopic lesions and before clinical signs occur. For reliable prevalence estimation of MAP infection in slaughtered cattle, systematic random sampling is essential. Copyright © Cambridge University Press 2012.

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