No description available.

While the major events in prokaryotic cell cycle progression are likely to be coordinated with transcriptional and metabolic changes, these processes remain poorly characterized. Unlike many rapidly-growing bacteria, DNA replication and cell division are temporally-resolved in mycobacteria, making these slow-growing organisms a potentially useful system to investigate the prokaryotic cell cycle. To determine if cell-cycle dependent gene regulation occurs in mycobacteria, we characterized the temporal changes in the transcriptome of synchronously replicating populations of Mycobacterium tuberculosis (Mtb). By enriching for genes that display a sinusoidal expression pattern, we discover 485 genes that oscillate with a period consistent with the cell cycle. During cytokinesis, the timing of gene induction could be used to predict the timing of gene function, as mRNA abundance was found to correlate with the order in which proteins were recruited to the developing septum. Similarly, the expression pattern of primary metabolic genes could be used to predict the relative importance of these pathways for different cell cycle processes. Pyrimidine synthetic genes peaked during DNA replication and their depletion caused a filamentation phenotype that phenocopied defects in this process. In contrast, the IMP dehydrogenase guaB2 dedicated to guanosine synthesis displayed the opposite expression pattern and its depletion perturbed septation. Together, these data imply obligate coordination between primary metabolism and cell division, and identify periodically regulated genes that can be related to specific cell biological functions.

After initially being infected with a virus, before an adaptive immune response can be mounted, the innate immune system of a cell recognizes and responds to certain patterns present in pathogenic molecules. I studied the role of two genes—PIR1 and CD200R1—on the innate immune responses in two different mouse models of viral infection, infection with the picornavirus EMCV (encephalomyocarditis virus) and infection with HSV-1 (herpes simplex virus) in a mouse model of herpes simplex encephalitis, respectively. PIR1 is a putative RNA phosphatase that has been shown to play an important role in antiviral small RNA processing in C. elegans. It has also been shown to interact with the RIG-I-like receptor LGP2 in preliminary mammalian experiments. I sought to characterize the effect PIR1 has on the innate immune response to the virus EMCV in mice. By developing a PIR1-null mouse, I have found that the role of PIR1 in the progression of EMCV in mice is limited. However, in vitro studies show that PIR1 might play an important role in regulating foreign RNA recognition during the earliest time points post-infection. CD200R1 is an anti-inflammatory signaling molecule that is expressed on myeloidderived cells, and whose ligand is highly expressed within the central nervous system. I investigated the role of this receptor in an intracranial model of herpes simplex encephalitis. CD200R1KO mice show improved survival following direct intracranial infection with HSV. I found this increased survival can be attributed to decreased levels of viral replication in CD200R1KO compared to wild-type mice. Further investigation has shown that CD200R1 affects the signaling and upregulation of the pattern-recognition receptor TLR-2 (toll-like receptor 2), and thus CD200R1 may impact HSV-1 replication by affecting TLR2 signaling.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes lethal invasive pulmonary disease in immunocompromised hosts and allergic asthma in sensitized individuals. This dissertation explores how eosinophils may protect hosts from acute infection while driving asthma pathogenesis by co-producing IL-23 and IL-17 in both contexts. In an acute model of pulmonary aspergillosis, eosinophils were observed to associate with and kill A. fumigatus spores in vivo. In addition, eosinopenia was correlated with higher mortality rates, decreased recruitment of inflammatory monocytes to the lungs, and decreased expansion of lung macrophages. As IL-17 signaling must occur on a local level to elicit its stereotypical response, such as the up-regulation of antimicrobial peptides and specific chemokines from stromal cells, eosinophils were discovered to be a significant source of pulmonary IL-17 as well as one of its upstream inducers, IL-23. In the context of asthma, this discovery opens a new paradigm whereby eosinophils might be driving asthma pathogenesis.

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRb deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3b sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.

Yersinia pestis, the causative agent of plague, specializes in causing dense bacteremia following intradermal deposition of a small number of bacteria by the bite of an infected flea. This robust invasiveness requires the ability to evade containment by the innate immune system. Of the various mechanisms employed by Y. pestis to subvert the innate immune response and to proliferate rapidly in mammalian tissue, only a few are well-characterized. Here, I present two complementary genetic analyses of Y. pestis adaptations to the mammalian environment. In the first, genome-wide fitness profiling for Y. pestis by Tn-seq demonstrates that the bacterium has adapted to overcome limitation of diverse nutrients during mammalian infection. In the second, a series of combinatorial targeted mutations disentangles apparent functional redundancy among the effectors of the Y. pestis type III secretion system, and we report that YpkA, YopT, and YopJ contribute to virulence in mice. We have also begun to investigate a novel relationship between Y. pestis and mammalian platelets, a highly abundant cell type in plasma. I present evidence that Y. pestis has evolved specific mechanisms to interfere with platelet activation, likely in order to evade immune responses and promote maintenance of bacteremia by undermining platelet thrombotic and innate immune functions. The principles guiding this work – systematic genetic analysis of complex systems, coupled with rational modification of in vitro assays to more closely mimic the in vivo environment – are a generalizable approach for increasing the efficiency of discovering new virulence determinants in bacterial pathogens.

Indiana University-Purdue University Indianapolis (IUPUI) / Anbu is a bacterial protein with significant homology to the sub-units of the 20S proteasome and is predicted to be a novel bacterial proteasome. The goal of this project was to determine if the recombinant Anbu protein from Pseudomonas aeruginosa is a proteasome. Anbu from P. aeruginosa was successfully cloned, expressed and purified. In order to determine the catalytic activity of Anbu, the purified protein was tested with a variety of substrates and conditions. The targets analyzed included fluorescently-labeled substrates, denatured proteins, diubiquitin, and a peptide library in the hopes of obtaining a useful model substrate. Experiments were also conducted to determine what role Anbu has in the cell. Western analysis was performed on the cell lysate of wild type P. aeruginosa and insertional mutants to detect Anbu expression. The level of biofilm formation was compared between the wild type and mutants. Cultures were grown under stress conditions including the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione. Growth rates were monitored in an attempt to detect a phenotypic difference between the wild type and the mutants lacking Anbu, HslV, and the other proteins of interest. While a substrate for Anbu has yet to be found, this protein was found to assemble into a larger structure and P. aeruginosa lacking Anbu was sensitive to the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione.

No description available.

Humanpathogene Sapoviren gehören zur Familie der Caliciviridae und verursachen vor allem bei Klein¬kindern und Senioren Gastroenteritiden. Die Replikationsstrategie von humanpathogenen Sapoviren ist bislang ungeklärt, da weder ein geeignetes Tiermodell noch ein etabliertes Zellkulturmodell zur Verfügung stehen. Aus diesem Grund sollte die Replikation in einem Säugerzellsystem etabliert werden. Die Ergebnisse der Untersuchungen sollen zu einem besseren Verständnis der Replikationsstrategie der humanpathogenen Sapoviren beitragen und können die Grundlage für weitere Unter¬suchungen der Replikationsstrategie der Caliciviren bilden sowie zur Entwicklung geeigneter antiviraler Maßnahmen und Medikamente beitragen. Für die Untersuchung der Replikationsstrategie des humanpathogenen Sapovirus wurde ein Sapovirus-Volle-Länge-Klon aus Patientenmaterial (Stuhlgang-Probe) generiert. Nach der molekularen Charakterisierung konnte der Stamm Hu/SaV/Dresden/pJG-SapI/2004/DE (GenBank-Zugangsnummer AY694184) der Genogruppe I Genotyp 1 der Sapo¬viren zugeordnet werden. Für die Untersuchung der Translation des humanpathogenen Sapovirus in Säugerzellen wurden polyklonale Antikörper in Kaninchen gegen die nichtstrukturellen und strukturellen Sapovirus-Proteine generiert. Im zellfreien System konnte die Sensitivität und Spezifität dieser Antikörper validiert werden. Außerdem wurde die Translation im zellfreien System mit bereits bestehenden Ergebnissen verglichen. Die Prozessierung des ORF1-Polyproteins erfolgte in die nichtstrukturellen Proteine NS1, NS2, NS3NTPase, NS4, NS5VPg, die Fusionsproteine NS1-3, NS2-3, NS4-5, NS4-7, NS5-7 und NS6 7Pro-Pol sowie das strukturelle Protein VP1. Für die Charakterisierung der Replikation des humanpathogenen Sapovirus in Säugerzellen wurden verschiedene Sapovirus-Volle-Länge-cDNA-Klone generiert. Für das Sapovirus-Volle-Länge-RNA-Genom pJG-SapI-T7 konnte eine Translation der Sapovirus-Proteine nach¬gewiesen werden. Die Transfektion von 293T-Zellen erfolgte mit in vitro transkribierter RNA, die ein Cap-Analogon und einen Poly(A)-Schwanz besaß. Durch die dem Sapovirus-Genom vorangestellte Kozak-Sequenz, welche als Ribosomenbindungsstelle dient, konnte auch nach Mutation des aktiven Zentrums des nichtstrukturellen Proteins NS7Pol (RNA-abhängige RNA-Polymerase) eine Translation des Sapovirus-ORF1-Polyproteins nachgewiesen werden. Somit erwies sich dieses Konstrukt als ungeeignet für die Untersuchung der Replikation des humanpathogenen Sapovirus in Säugerzellen. Nach Klonierung des Sapovirus-Volle-Länge-cDNA-Genoms in den pACYC-MCSII-Vektor (pJG-SapI-T7) konnte nach in vitro Transkription ein gekapptes Sapovirus-Volle-Länge-RNA-Genom mit einem Poly(A)-Schwanz generiert werden, welches vermutlich die richtigen 5’- und 3’-Sapovirus-Enden enthält. Nach Transfektion von 293T-Zellen konnten die nichtstrukturellen Fusionsproteine NS2-3, NS4-5, NS4-7 und NS6-7Pro-Pol sowie das strukturelle Protein VP1 im Western Blot nachgewiesen werden. Nach Mutation des aktiven Zentrums des nichtstrukturellen Proteins NS6Pro (Protease) wurde die Prozessierung des ORF1-Polyproteins in Säugerzellen unter¬bunden. Die Replikation der generierten Sapovirus-Volle-Länge-RNA-Genome in Säugerzellen konnte mit Hilfe der quantitativen PCR nicht nachgewiesen werden. Eine Passagierung in verschiedenen Säugerzelllinien war ebenfalls nicht möglich. Weiter wurden verschiedene Sapovirus-Volle-Länge-RNA-Genome direkt aus Patientenmaterial durch RT-PCR generiert und nach in vitro Transkription damit Säugerzellen transfiziert. Bei Sapovirus-Volle-Länge-RNA-Genomen aus drei Patientenproben konnte die Translation und Prozessierung des Sapovirus-ORF1-Polyproteins nachgewiesen werden. Die Replikation konnte mit Hilfe der quantitativen PCR nicht nachgewiesen werden. In einem letzten Schritt wurde aus Patientenmaterial gewonnene RNA direkt für die Transfektion eingesetzt. Hierfür wurden die Patientenproben verwendet, bei denen eine Translation und Prozessierung des Sapovirus-ORF1-Polyproteines nachgewiesen werden konnte. Auch hier konnte keine Replikation mit Hilfe der quantitativen PCR nachgewiesen werden. In der vorliegenden Arbeit konnte erstmals die erfolgreiche Translation und Prozessierung des ORF1-Polyproteins des humanpathogenen Sapovirus (Dresdner Stamm pJG-SapI, GenBank-Zugangsnummer AY694184) in Säugerzellen gezeigt werden. Weitergehende Untersuchungen zur Replikation des humanpathogenen Sapovirus in Säugerzellen könnten mit Hilfe des vorliegenden Dresdner Sapovirus-Stamm pJG-SapI erfolgen, indem weitere rekombinante Systeme etabliert werden. / The human pathogenic sapovirus belongs to the family of the Caliciviridae and is an important agent of gastroenteritis in infants and the elderly. The replication strategy of the human pathogenic sapovirus remains so far unclear, since neither a suitable animal model nor a permissive cell line to cultivate the virus are available. Elucidating the replication strategy of the human pathogenic sapovirus may contribute to a better understanding of its pathogenicity, being also an important pre-requisite for the development of new antiviral strategies against this relevant medical pathogen. In order to investigate the replication strategy of the human pathogenic sapovirus, a cDNA-clone encompassing the entire sapovirus genome was generated from a clinical sample. Based on phylogenetic analysis, the full-length genome of the sapovirus strain Hu/SaV/Dresden/pJG-SapI/2004/DE (GenBank accession number AY694184) was assigned to the Genogruppe I/ Genotype 1. For the investigation of the translation of the human pathogenic sapovirus in mammalian cells, polyclonal antibodies were generated against the nonstructural and structural sapovirus proteins. The sensitivity and specificity of the antibodies were validated using a transcription-translation driven cell free system. Translation of the sapovirus full-length-cDNA clone in the cell free system generated structural and nonstructural sapovirus proteins, in accordance with previously published reports. After translation, the sapovirus ORF1 polyprotein was processed in the nonstructural proteins NS1, NS2, NS3NTPase, NS4, NS5VPg, the fusion proteins NS1-3, NS2-3, NS4-5, NS4-7, NS5-7 and NS6-7Pro-Pol as well as the structural protein VP1. For the characterisation of the replication of the human pathogenic sapovirus in mammalian cells, different sapovirus cDNA-full length clones were generated. Upon transfection in 293-T cells, a translation of the sapovirus proteins was evidenced. However, this translation was not sapovirus-specific, as cDNA clones bearing a mutation in the active site of the sapovirus polymerase NS7Pol were also able to generate viral proteins. In order to further investigate the translation and replication of the sapovirus, the full length cDNA Genome was cloned into the pACYC-MCSII-Vector. Subsequently, a capped sapovirus full length RNA genome with a correct 5’-end and a 3’-end with a poly(A) tail was generated by in vitro transcription. Upon transfection in 293T-cells, the nonstructural fusion proteins NS2-3, NS4-5, NS4-7 and NS6-7Pro-Pol as well as the structural protein VP1 were translated. As a control, mutation of the active site of the nonstructural protein NS6Pro did not lead to processing of the viral enzymes, indicating that the processing of the ORF1-polyprotein in mammalian cells is strictly dependent on the activity of the sapovirus protease NS6Pro. Furthermore, replication of the sapovirus genomic RNA was investigated in mammalian cells. Upon transfection of the sapovirus full-length genomic RNA, replication of the sapovirus full-length RNA genomes was not evidenced in mammalian cells using quantitative real time RT-PCR. In order to exclude a possible flaw in the primary sequence of the viral genome hampering its replication, additional sapovirus full-length genomes were generated by direct amplification of the RNA from stool samples followed by in vitro transcription. Upon transfection in mammalian cells, the translation of sapovirus ORF1-polyprotein was evidenced in three clinical samples. However, replication of the viral genome did not occur. A similar observation was made when the total RNA from the clinical sample was used for transfection of mammalian cells, indicating that the lack of replication of the viral genome may be caused primarily by the cell line used, rather than the viral genome. In conclusion, the present work describes for the first time the successful processing of the ORF1-Polyprotein of the human pathogenic Sapovirus (strain Dresden pJG-SapI, GenBank accession number AY694184) in mammalian cells. This work may be a first step towards understanding the replication strategy of the human pathogenic and non-human pathogenic sapovirus (i.e. the porcine enteric calicivirus), being both important medical pathogens.

Infections caused by antibiotic resistant pathogenic bacteria is an increasing issue inhealthcare, and development of new antimicrobial substances could contribute to combat the continued spread. Plants have historically been used in traditional medicine, have intrinsic defense mechanisms against microbes, and could therefore be a source for new antimicrobial agents. At the Department of Chemistry and Biomedicine at Linnaeus University, Kalmar, a total of 18 extracts made from 9 different combinations of Swedish-growing plants were available.The purpose of the current thesis was to investigate possible antimicrobial effects of the plant extracts in vitro, on a selection of Gram-positive (n=3) and Gram-negative (n=4) bacterial strains. Initial screening of all 18 extracts was performed with agar-based methods including agar well diffusion and direct application on inoculated agar. Detection of concentration-dependent antimicrobial effects was performed with four extracts on Staphylococcus aureus and Enterococcus faecalis. At inhibitory concentrations, viability was estimated as colony forming units/ml (CFU/ml).Screening showed that 11 of 18 extracts affected the growth of at least one of the strains tested. Gram-positive species were affected to a greater extent than Gram-negatives. Estimation of concentration dependency showed inhibitory effects at 50 mg/l in the most potent extracts. Viability estimation revealed an average reduction for both S. aureus and E. faecalis, as compared to the positive control. In conclusion, the study showed possible antimicrobial effects of several extract-bacteria combinations, disclosing potential substances for further investigations.