Development and evaluation of a diagnostic PCR-assay for detection of specific Paracoccidioides brasiliensis-DNA

Das Ziel der vorliegenden Arbeit war der Aufbau einer PCR-Methode zum Nachweis von DNA des dimorphen humanpathogenen Pilzes Paracoccidioides brasiliensis. Es wurde zuerst erwogen die Zielsequenz in die kleine Untereinheit der ribosomalen DNA (18S rDNA) zu legen, da die Anwesenheit multipler Kopien pro Genom eine hohe Sensitivität der Methode gewährleistet. Die erstmalig durchgeführte Sequenzierung des Gens zeigte jedoch eine ausgesprochene Homologie zu nahe verwandten Pilzarten. Da eine diagnostische PCR eine Unterscheidung der DNA insbesondere zu anderen dimorphen Pilzen leisten sollte, wurde als Alternative das Gen des P. brasiliensis spezifischen Glykoproteins gp43 gewählt. Die nested-PCR amplifizierte DNA ausschließlich von P. brasiliensis Isolaten, so dass sie zur Stammidentifizierung verwendet werden konnte. Aus 21 von 23 Lungenhomogenaten infizierter Mäuse konnte P. brasiliensis spezifische DNA amplifiziert werden, jedoch weder aus Lungenhomogenaten von 20 mit H. capsulatum intravenös infizierten Mäusen noch aus denen von zwei Kontrolltieren. Die Nachweisgrenze von 0,5 fg wurde mit klonierter Plasmid-DNA ermittelt. Diese konnte im Tiermodell mit Nachweis spezifischer DNA aus Lungenhomogenaten mit mindestens 1 x 103 bis maximal 1,3 x 107 Kolonie-bildenden Einheiten pro Gramm Gewebe bestätigt werden. Die Sequenzierung aller nested-PCR Produkte aus Lungenhomogenaten und von sieben Isolaten ergab in allen Fällen eine 100%ige Übereinstimmung mit dem gp43-Gen von P. brasiliensis in der Datenbank GenBank. Die erstmalig ermittelte Nukleotidsequenz der 18S rDNA von fünf P. brasiliensis Isolaten wurde mit den bekannten 18S rDNA-Sequenzen verwandter Pilze der Familien Gymnoascaceae, Arthrodermaceae und Onygenaceae in der Ordnung Onygenales verglichen. Unter Verwendung von drei dafür üblicherweise eingesetzten Methoden (Neighbor Joining, Maximum Parsimony und Maximum Likelihood) sowie unter Anwendung des Kishino-Hasegawa Tests wurde gezeigt, dass P. brasiliensis näher mit B. dermatitidis und E. parva verwandt ist als B. dermatitidis mit H. capsulatum, deren beide teleomorphen Formen zur Gattung Ajellomyces zählen. Übereinstimmend mit den Ergebnissen anderer Autoren wurde eine eindeutige Zuordnung von P. brasiliensis zur Familie der Onygenaceae festgestellt. Es finden sich Hinweise in der phylogenetischen Analyse, dass die Gruppe der Gattungen mit humanpathogenen Spezies, also Paracoccidioides, Emmonsia, Blastomyces und Histoplasma einen getrennten evolutionären Hintergrund haben. Dennoch sind die anderen Gattungen Auxarthron, Coccidioides, Malbranchea, Onygena, Renispora und Uncinocarpus, der Familie Onygenaceae nahe verwandt mit der ersten Gruppe. Nachdem P. brasiliensis auch anhand seiner 18S rDNA Sequenz taxonomisch in der Familie der Onygenaceae eingeordnet werden konnte, wurde eine spezifische und sensitive nested-PCR mit Zielsequenz im Gen des immundominanten gp43 Glykoproteins aufgebaut. Diese soll in der Diagnostik der Parakokzidiodomykose und zur Differenzialdiagnostik anderer endemischer Systemmykosen des Menschen in weiteren Studien evaluiert werden.The goal of this study was to develop a specific and diagnostic PCR assay for detection of the dimorphic human pathogenic fungus P. brasiliensis. A common target used for diagnostic fungal PCR assays has been the 18S rRNA gene (rDNA) because its high frequency in the genome guarantees a high sensitivity. To determine the specificity of the 18s rDNA gene, we, for the first time, performed phylogenetic homology analysis by sequencing the 18S rDNA gene from five isolates of P. brasiliensis, and closely related fungi. Sequence alignment of the entire 18S rDNA of P. brasiliensis and closely related fungi including Blastomyces dermatitidis and Histoplasma capsulatum revealed high homology. Thus indicating that P. brasiliensis, B. dermatitidis, and Emmonsia parva were more closely related than H. capsulatum and B. dermatitidis, whose teleomorphic forms belong to one genus, Ajellomyces. In accordance with the work of other investigators, our data showed that P. brasiliensis must be grouped within the order Onygenales and is closely related to members of the family Onygenaceae. In addition, detailed molecular phylogenetic analysis suggested that the family Onygenaceae might be further divided into two families. The subgroup that includes P. brasiliensis comprises all zoopathogenic species. These groups can help to identify species of the members of the family Onygenaceae pathogenic for humans, but the findings also indicate that the reliance on gene probes within the 18S rDNA sequences has limited specificity. Therefore another target, the gp43 gene, which encodes an outer membrane protein that is highly immunogenic, unique and of diagnostic value in paracoccidioidomycosis, was evaluated by nested-PCR assay. The pg43 gene was amplified by nested PCR from lung homogenates of 21 out of 23 infected mice with concentrations ranging from 1 × 103 to 1.3 × 107 CFU of P. brasiliensis per g of lung. Uninfected and lung samples from 20 H. capsulatum-infected mice, as well as DNA from closely related fungi, gave negative results. A detection limit of 0.5 fg of specific DNA was determined using a standard curve developed from cloned plasmid DNA. Sequencing of all nested PCR products from lung homogenates and from seven P. brasiliensis isolates demonstrated 100% identity to the gp43 sequences of P. brasiliensis present in GenBank. In summary, we have developed a highly sensitive and specific nested PCR assay for the detection of P. brasiliensis in tissue samples using a sequence of the immunogenic gp43 gene. This assay may be useful for diagnosis of paracoccidioidomycosis in human tissue samples

Experimental Evolution of Human Influenza Virus H3 Hemagglutinin in the Mouse Lung Identifies Adaptive Regions in HA1 and HA2▿

The genetic basis for virulence and host switching in influenza A viruses (FLUAV) is largely unknown. Because the hemagglutinin (HA) protein is a determinant of these properties, HA evolution was mapped in an experimental model of mouse lung adaptation. Variants of prototype A/Hong Kong/1/68 (H3N2) (wild-type [wt] HK) human virus were selected in both longitudinal and parallel studies of lung adaptation. Mapping of HA mutations found in 11 independently derived mouse-adapted populations of wt HK identified 27 mutations that clustered within two distinct regions in or near the globular frameworks of the HA1 and HA2 subunits. The adaptive mutations demonstrated multiple instances of convergent evolution involving four amino acid positions (162, 210, and 218 in HA1 and 154 in HA2). By use of reverse genetics, convergent HA mutations were shown to affect cell tropism by enhancing infection and replication in primary mouse tracheal epithelial cells in vitro and mouse lung tissue in vivo. Adaptive HA mutations were multifunctional, affecting both median pH of fusion and receptor specificity. Specific mutations within both adaptive regions were shown to increase virulence in a mouse lung model. The occurrence of mutations in the HA1 and HA2 adaptive regions of natural FLUAV host range and virulent variants of avian and mammalian viruses is discussed. This study has identified adaptive sites and regions within the HA1 and HA2 subunits that may guide future studies of viral adaptation and evolution in nature

In Vitro and Murine Efficacy and Toxicity Studies of Nebulized SCC1, a Methylated Caffeine-Silver(I) Complex, for Treatment of Pulmonary Infections ▿

The expanding clinical challenge of respiratory tract infections due to resistant bacteria necessitates the development of new forms of therapy. The development of a compound composed of silver coupled to a methylated caffeine carrier (silver carbene complex 1 [SCC1]) that demonstrated in vitro efficacy against bacteria, including drug-resistant organisms, isolated from patients with respiratory tract infections was described previously. The findings of current in vitro studies now suggest that bactericidal concentrations of SCC1 are not toxic to airway epithelial cells in primary culture. Thus, it was hypothesized that SCC1 could be administered by the aerosolized route to concentrate delivery to the lung while minimizing systemic toxicity. In vivo, aerosolized SCC1 delivered to mice resulted in mild aversion behavior, but it was otherwise well tolerated and did not cause lung inflammation following administration over a 5-day period. The therapeutic efficacy of SCC1 compared to that of water was shown in a 3-day prophylaxis protocol, in which mice infected with a clinical strain of Pseudomonas aeruginosa had increased survival, decreased amounts of bacteria in the lung, and a lower prevalence of bacteremia. Similarly, by using an airway infection model in which bacteria were impacted in the airways by agarose beads, the administration of SCC1 was significantly superior to water in decreasing the lung bacterial burden and the levels of bacteremia and markers of airway inflammation. These observations indicate that aerosolized SCC1, a novel antimicrobial agent, warrants further study as a potential therapy for bacterial respiratory tract infections

Maintenance of Airway Epithelium in Acutely Rejected Orthotopic Vascularized Mouse Lung Transplants

Lung transplantation remains the only therapeutic option for many patients suffering from end-stage pulmonary disease. Long-term success after lung transplantation is severely limited by the development of bronchiolitis obliterans. The murine heterotopic tracheal transplantation model has been widely used for studies investigating pathogenesis of obliterative airway disease and immunosuppressive strategies to prevent its development. Despite its utility, this model employs proximal airway that lacks airflow and is not vascularized. We have developed a novel model of orthotopic vascularized lung transplantation in the mouse, which leads to severe vascular rejection in allogeneic strain combinations. Here we characterize differences in the fate of airway epithelial cells in nonimmunosuppressed heterotopic tracheal and vascularized lung allograft models over 28 days. Up-regulation of growth factors that are thought to be critical for the development of airway fibrosis and interstitial collagen deposition were similar in both models. However, while loss of airway epithelial cells occurred in the tracheal model, airway epithelium remained intact and fully differentiated in lung allografts, despite profound vascular rejection. Moreover, we demonstrate expression of the anti-apoptotic protein Bcl-2 in airway epithelial cells of acutely rejected lung allografts. These findings suggest that in addition to alloimmune responses, other stimuli may be required for the destruction of airway epithelial cells. Thus, the model of vascularized mouse lung transplantation may provide a new and more physiologic experimental tool to study the interaction between immune and nonimmune mechanisms affecting airway pathology in lung allografts