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Hantaviruses (family Bunyaviridae) are enveloped viruses with a segmented RNA genome of negative polarity. They can cause two different diseases in humans, the hemorrhagic fever with renal syndrome in Europe and Asia and the hantavirus cardiopulmonary syndrome in America. The transmission to humans is mainly indirect by inhalation of aerosolized virus-contaminated rodent excreta. In contrast to the initial assumption that hantaviruses are mainly carried by rodents, during the last years many novel hantaviruses were detected in shrews, moles and recently in bats. These findings raise important questions about the evolutionary history of hantaviruses, their host association and adaptation, the role and frequency of spillover infections and host switch events. This study aims to prove the presence, geographical distribution and host association of the rodent-borne Tula virus (TULV) and the shrew-associated Seewis virus (SWSV) in Central Europe. For this purpose, novel laboratory techniques for molecular and serological hantavirus detection were developed. Initially, a broad-spectrum molecular assay to identify small mammal species from Central Europe was developed. This novel assay is based on PCR amplification using degenerated primers targeting the cytochrome b (cyt b) gene, nucleotide sequence analysis of the amplified cyt b gene portion and followed by pairwise sequence comparison to published sequences using the BLAST function of GenBank. Different small mammal species prevalent in Central Europe could be determined by this new approach, including not only representatives of various Rodentia and Soricomorpha, but also representatives of the orders Erinaceomorpha, Lagomorpha, Carnivora and Chiroptera. For characterization of insectivore-borne hantavirus Thottapalayam virus (TPMV), specific monoclonal antibodies were generated that detect native virus in infected mammalian cells. For the detection of TPMV-specific antibodies, Asian house shrew Suncus murinus immunoglobulin G (IgG)-specific antibodies were produced in laboratory mice and rabbit. Using this anti-shrew IgG and recombinant TPMV nucleocapsid (N) protein, an indirect enzyme-linked immunosorbent assay (ELISA) was developed allowing the detection of TPMV N protein-specific antibodies in immunized and experimentally TPMV infected shrews. A Pan-Hantavirus SYBR-Green RT-qPCR was developed for the search to novel hantaviruses. By this novel RT-qPCR and other conventional RT-PCR approaches, TULV infections were identified for the first time in the Eurasian water vole Arvicola amphibius from different regions in Germany and Switzerland. The phylogenetic analyses of the different partial TULV small (S)-, medium (M)- and large (L)-genome segment sequences from A. amphibius, with those of Microtus arvalis- and M. agrestis-derived TULV lineages, revealed a geographical, but host-independent clustering and may suggest multiple TULV spillover or a potential host switch from M. arvalis or M. agrestis to A. amphibius. In a further comprehensive study, different shrew species (Sorex araneus, S. minutus, S. coronatus, and S. alpinus) were collected in Germany, Czech Republic, and Slovakia and screened by another L-segment-targeting Pan-Hantavirus RT-PCR approach. This screening revealed hantavirus L-segment sequences in a large number of S. araneus and a few S. minutus indicating a broad geographical distribution of this hantavirus. For detailed analyses, S-segment sequences were obtained, from S. araneus and S. minutus. The sequences demonstrated their similarity to SWSV sequences from Hungary, Finland, Austria and Germany. A detailed phylogenetic analysis showed low intra-cluster sequence variability, but high inter-cluster divergence suggesting a long-term SWSV evolution in local shrew populations. In conclusion, the investigations demonstrated a broad geographical distribution and multiple spillover infections of rodent-borne TULV and shrew-borne SWSV in Europe. The finding of putative spillover transmissions described here and in other studies underline the current problem of the hantavirus reservoir host definition. In contrast to the hypothesis of a long-standing hantavirus–rodent (small mammal) host coevolution, the investigations support a more dynamic evolutionary history of hantavirus diversification including spillover infections and host-switch events. In future in vitro and in vivo infection studies as well as field studies has to define factors determining the host specificity of these hantaviruses.
A molecular approach to characterize the arbuscular mycorrhizal fungus, Glomus sp. AMykor isolate
(2012)
The arbuscular mycorrhizal fungi (AMF) interaction with plants has a major impact on the soil ecosystem. However, so far, only a few studies on AMF genetics have been performed and molecular information on the genetic diversity of AMF is limited. In this study a fundamental genetic characterization of the industrial isolate, Glomus sp. AMykor (AMykor GmbH, Bitterfeld, Germany) has been undertaken to increase the understanding of AMF genetic diversity. Based on phylogenetic analysis of partial rDNA sequences, Glomus sp. AMykor isolate was proposed to belong to the G. irregulare species together with the reference isolate, DAOM197198. To investigate if both isolates differ in their ploidy level, fluorescence in situ hybridization (FISH) was performed and mainly one or two hybridization signals per nucleus were observed in both isolates. It is suggested that they harbour at least two major rDNA sites and possibly two minor sites. The DNA content was estimated by means of flow cytometry (FC) and confirmed by Feulgen densitometry (FD). The calculated average DNA content per nucleus is 153.0 ± 3.6 Mb for the G. irregulare AMykor isolate and 154.8 ± 6.2 Mb for the DAOM197198 isolate. Since there are plenty criticisms coming recently of using rDNA sequence for fungal barcoding there is necessity of development other system for the identification to species level of Glomeromycotan fungi. The focus of this part of the study was the GiFRD gene encoding fumarate reductase enzyme for use as a potential candidate for AMP species determination. Unfortunately, observed sequence variations do not allow the discrimination of Glomeromycotan species. However, further analysis of enzyme encoded by GiFRD showed a possible role of fumarate reductase in AMF redox balance maintaining under oxygen deficient conditions. Using a yeast expression system, it has been demonstrated that the protein encoded by GiFRD has fumarate reductase activity. The functional expression of GiFRD in the S. cerevisiae fumarate reductase deletion mutant restored the ability of growth under anaerobiosis which indicated that Gifrdp is able to functionally complement the S. cerevisiae missing genes. The fact that GiFRD expression was present only in the asymbiotic stage confirmed existence of at least one metabolic pathway involved in anaerobic metabolism and suggested that AMF behave as a facultative anaerobe in asymbiotic stage.
The leading hypothesis of why organisms age is the “Free Radical Theory of Aging”, which states that the accumulation of reactive oxygen species (ROS), such as superoxide (O2•-) and hydrogen peroxide (H2O2), causes protein, lipid and DNA damage and leads to the observed age-related decline of cells and tissues. A major obstacle in analyzing the role of oxidative stress in aging organisms is the inability to precisely localize and quantify the oxidants, to identify proteins and pathways that might be affected, and ultimately, to correlate changes in oxidant levels with the lifespan of the organism. To directly monitor the onset and extent of oxidative stress during the lifespan of Caenorhabditis elegans, we utilized the fluorescent H2O2 sensor protein HyPer, which enabled us to quantify endogenous peroxide levels in different tissues of living animals in real time. We made the surprising observation that wildtype C. elegans is exposed to very high peroxide levels during development. Peroxide levels drop rapidly as the animals mature, and low peroxide levels then prevail throughout the reproductive age, after which an age-accompanying increase of peroxide level is observed. These results were in excellent agreement with findings obtained by using the highly quantitative redox proteomic technique OxICAT, which monitors the oxidation status of redox-sensitive proteins as read-out for onset, localization, and protein targets of oxidative stress. By using OxICAT, we detected increased protein thiol oxidation during the development of C. elegans and in aging animals. Many processes in C. elegans might potentially contribute to the elevated peroxide levels observed during development, including cuticle formation, apoptosis, proliferation, gametogenesis, or ROS signaling. The finding that all investigated C. elegans mutants regardless of their lifespan are exposed to high developmental peroxide levels argues for ROS accumulation to be a universal and necessary event. Yet, recovery from the early oxidative boost might determine the subsequent adult lifespan, as we found that long-lived daf-2 mutants transition faster to reducing conditions than short-lived daf-16 mutants, which retain higher peroxide levels throughout their mature life. These results suggest that changes in the cellular oxidant homeostasis, encountered at a very early stage in life, might determine subsequent redox levels and potentially the lifespan of organisms. Manipulation of developmental oxidant levels using glucose restriction or a short bolus of superoxide caused a disruption in developmental growth, a delay in reproduction, and a shortened lifespan. These results suggest that developmental oxidant levels are fine-tuned and optimized. Future experiments are aimed to investigate the sources of developmental hydrogen peroxide, and to elucidate whether active down-regulation of antioxidant enzymes during the larval period might foster peroxide accumulation. Preliminary results indicate that this might indeed be the case for peroxiredoxin 2, whose expression was significantly lower during development than at later stages in life. Finally, we investigated whether the observed variances in the developmental peroxide levels of individual worms within a synchronized wildtype population might be responsible for the observed significant variances in lifespan, and hence could serve as a predictor for adult lifespan. Preliminary results revealed that neither too low nor too high peroxide levels during development are beneficial for the lifespan of wildtype worms, suggesting that ROS level during development might be optimized for maximized lifespan. Future experiments aim to reveal the processes that are affected by ROS and which might influence the individual’s lifespan early in life.
Streptococcus pneumoniae (pneumococci) are Gram-positive cocci and commensals of the human upper respiratory tract. Pneumococcal pathogenesis requires adherence to host cells and dissemination through cellular barriers and to evade host defense mechanisms. The Pneumococcal surface protein C (PspC) is an important virulence factor which has a crucial role in pneumococcal adhesion to host cells and immune evasion by manipulating the host complement system. To elucidate the pneumococcal adherence and uptake mechanism via factor H glycosaminoglycans (dermatan sulfate and heparin) were employed as competitive inhibitors in infection experiments with epithelial cells or human polymorphonuclear leukocytes (PMNs). Glycosaminoglycans significantly inhibited the FH mediated pneumococcal adherence and subsequent invasion to host epithelial cells. Furthermore, the short consensus repeats of FH which promotes the adhesion of pneumococci to host cells were identified by blocking experiments with domain mapped antibodies for specific regions of FH. Moreover, this study indicates that FH acts as adhesion molecule via cellular receptors recognized as integrin CR3 on human PMNs. Binding of Factor H loaded pneumococci to integrins CR3 was assessed by flow cytometry. Pneumococci coated with Factor H showed a significantly increased association with PMNs. This interaction was blocked by anti-CR3 antibodies and Pra1. This project further aims to study mechanisms of pneumococcal endocytosis by host cells, their intracellular fate, and the pathogen induced host cell signal transduction cascades including the calcium signaling upon pneumococcal infection of host cells via the PspC-hpIgR interaction. To assess now the role of protein tyrosine kinases (PTKs) during pneumococcal infection via PspC, cell culture infections were performed in presence of pharmacological inhibitors of PTKs and MAPKs or by employing genetic interference techniques. Blocking the function of Src or ER1/2 and JNK and genetic-knock down of Src and FAK reduced significantly internalization of pneumococci. These data indicated the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells. The impact of host cells intracellular calcium concentrations on pneumococcal PspC-hpIgR mediated internalization was studied. Intracellular calcium measurement of epithelial cells performed in the presence of pneumococci suggested a calcium influx in host epithelial cells and importantly this calcium influx was PspC- hpIgR specific as pspC-deficient pneumococci were unable to mediate calcium mobilization in host cells. The increase in intracellular calcium [Ca2+]i was dependent on phospholipase C as pretreatment of cells with a phospholipase C-specific inhibitor abolished the increase in [Ca2+]i. Furthermore, role of host intracellular calcium concentrations during pneumococcal internalization was demonstrated by employing specific pharmacological inhibitors and calcium chelators in epithelial cell culture infection assays. The results revealed that elevated host cells calcium concentrations diminished pneumococcal internalization while lower calcium concentration in host epithelial cells promoted pneumococcal uptake. This study further demonstrates that dynamin, clathrin and caveolin play a key role during pneumococcal endocytosis into host cells via PspC-hpIgR. The use of specific pharmacological inhibitors or genetic interference approaches against dynamin, clathrin and caveolin in epithelial cell culture infection assays significantly blocked pneumococcal uptake. Furthermore, confocal microscopy revealed that pneumococci co-localize with clathrin. At later stages of the infection the pathogen is sorted to early, late and recycling endosomes as indicated by co-localization of pneumococci with endosomal markers such as Rab5, Rab4, Rab 7, and Lamp1. In order to get further insights into PspC-hpIgR mediated uptake mechanisms, a chimeric PspC was constructed and expressed heterologously on the surface of Lactococcus lactis. Immunofluorescence staining, immunoblot and flow cytometric analysis of L. lactis confirmed the expression of PspC on the bacterial surface. Moreover the ability of recombinant lactococci expressing PspC to adhere to and to invade pIgR-expressing epithelial cells confirmed the functional activity of PspC when exposed on the lactococcal surface. PspC expressing lactococci confirmed the specificity of PspC-hpIgR mediated endocytosis in host epithelial cells as PspC deficient lactococci were not taken up by these host cells. Confocal microscopic analysis demonstrated that only PspC expressing lactococci were sorted to early, late and recycling endosomes, similar to the intracellular fate of S. pneumoniae.
Streptococcus pneumoniae, more commonly known as the pneumococcus, is a Gram-positive bacterium colonizing the human upper respiratory tract as a commensal. However, these apparently harmless bacteria have also a high virulence potential and are known as the etiologic agent of respiratory and life-threatening invasive diseases. Dissemination of pneumococci from the nasopharynx into the lungs or bloodstream leads to community-acquired pneumonia, septicaemia and meningitis. Pneumococcal diseases are treated with antibiotics and prevented with polysaccharide-based vaccines. However, due to the increase of antibiotic resistance and limitations of the current vaccines, the burden of diseases remains high. Interactions of pneumococci with soluble host proteins or cellular receptors are crucial for adherence, colonization, transmigration of host barriers and immune evasion. The pneumococcal surface-exposed proteins are the main players involved in this host-pathogen interaction. Therefore, combating pneumococcal transmission and infections has emphasized the need for a new generation of immunogenic and highly protective pneumococcal vaccines, based on surface-exposed adhesins virtually expressed by all pneumococcal strains and serotypes. The genomic analysis of S. pneumoniae strains helped to identify pneumococcal virulence factors such as pili, PsrP and PavB, which have been demonstrated to interact with human proteins playing an important role during the pathogenic process of pneumococci, and are currently considered as new potential vaccine candidates against S. pneumoniae. A subclass of pneumococcal strains produces pili that are encoded by the pathogenicity islet pilus islet-1 (rlrA islet) and/or the pilus islet-2. Both types of pili are implicated in bacterial adherence to host cells. A further pathogenicity islet encoded protein is PsrP. The presence of the psrP-secY2A2 islet correlated positively with the ability of pneumococci to cause invasive pneumococcal diseases. Recent studies indicated that PsrP is a protective adhesin interacting with keratin 10 on lung epithelial cells. In this study, the genomic loci of the pneumococcal virulence factors pili, PsrP and PavB were molecularly analyzed and used as molecular markers for molecular epidemiology studies of S. pneumoniae. The genotyping results obtained here showed the impact of the PCV7 immunization of children, started in July 2006, on the distribution of these pneumococcal virulence factors among clinical isolates in Germany. These findings gave more insights into the role of pili, PsrP and PavB in pneumococcal pathogenesis and may strongly support the idea of including these pneumococcal constituents in a broad coverage protein-based vaccine against pneumococcal infections produced by invasive serotypes in the future. The mature PavB protein contains a variable number of repetitive sequences referred to as the Streptococcal Surface Repeats (SSURE). PavB has been demonstrated to interact with fibronectin and plasminogen in a dose-dependent manner and it was identified as a surface-exposed adhesin with immunogenic properties, which contributes to pneumococcal colonization and respiratory airways infections. The complete molecular analysis performed here for PavB, allowed to know more accurately its structure and to estimate the real number of SSURE units in different pneumococcal strains. With these findings, a new primary sequence-based structural model was constructed for the PavB protein and its SSURE domain, and, at least for TIGR4, the complete pavB gene and PavB protein sequences with five SSURE units was reported in the GenBank database of the NCBI website. Due to its immediate neighborhood on the pneumococcal genome with the tcs08 genes, PavB is likely linked with this pneumococcal TCS. Here, a significant reduction of the PavB protein expression was observed in delta-tcs08-mutant strains, which may strongly suggest that the TCS08 does play a role in pneumococcal virulence and metabolisme, as further observed in growth behaviour experiments carried out with the TCS08-deficient mutants, cultured in chemically defined medium. Despite several studies suggest that the molecular mechanism underlying the bacterial signal transduction is very sophisticated, the majority of reports in prokaryotic TCS, including those for S. pneumoniae, are still focused in single cognate pairs. The pneumococcal genome encodes 14 TCSs and an orphan response regulator. It is obvious that TCS pathways are often arranged into complex circuits with extensive cross-regulation at a variety of levels, thereby endowing cells with the ability to perform sophisticated information processing tasks. This study established also the experimental and molecular bases for the construction of a comprehensive genome-wide interaction map of the complex TCS pathways for its application in the gene regulation of pneumococcal virulence factors.