A leptospirose é uma zoonose disseminada mundialmente, causada por bactérias do gênero Leptospira. A melhor maneira de contornar o problema é por de medidas preventivas, já que a contenção da proliferação de roedores é inviável e não há vacina eficaz disponível. Estratégias de genômica funcional têm identificado um grande número de proteínas a serem estudadas. Esse fato aliado a dados da literatura, que identificaram proteínas envolvidas na patogenicidade e que são expressas somente em condição de virulência, levou à proposição da utilização da proteômica para canalizar os estudos. A metodologia envolveu obtenção de extratos protéicos de leptospiras retiradas de animais infectados, sua separação por gel bidimensional, e identificação dos spots por espectrometria de massas. O objetivo central foi a identificação de proteínas expressas em bactérias virulentas e ausentes nas não-virulentas. A identificação dessas proteínas pode facilitar a busca de proteínas envolvidas na virulência e infecção da leptospirose. Adicionalmente, é um avanço no esclarecimento da biologia e patogenicidade das leptospiras, bem como para o reconhecimento de candidatos potenciais para a composição de vacinas e/ou métodos diagnósticos mais eficientes. / Leptospirosis, one of the most spread zoonosis worldwide, is caused by bacteria of the genus Leptospira. Preventive measures are the best way to control the disease due to the difficulty to impair the proliferation of rodents and because no efficient vaccine is currently available. Functional genomics strategies have pointed a large number of proteins that could be important immunogens. This fact allied to published data reporting the identification of proteins involved in pathogenesis that are expressed only in virulent strains, led us to propose the use of proteomics as a tool to narrow down these studies. The methodology involved the preparation of protein extracts from tissuederived leptospires, separation by two-dimensional gel and identification of the spots by mass spectrometry. The central objective was to identify proteins expressed only in virulent bacteria. The identification of these proteins could help the search for proteins involved in virulence with a role during infection. Additionally, the data presented here represent a large step to clarify the biology and pathogenicity of leptospires, as well as the identification of potentially important vaccine candidates and/or proteins to compose more efficient diagnostic methods.

Leptospirose é uma zoonose mundial que causa grandes prejuízos econômicos e sociais. Os mecanismos de patogenicidade da leptospira ainda não estão totalmente elucidados. Nesse trabalho foi avaliado o papel de três proteínas hipotéticas de superfície na patogenia da leptospirose: LIC11009, LIC11360 e LIC11975. Os genes foram clonados a partir do DNA da L. interrogans sorovar Copenhageni e as proteínas recombinantes foram purificadas por cromatografia de afinidade. RT-qPCR mostrou a transcrição dos genes em leptospiras. As proteínas recombinantes reagiram com soro de humanos diagnosticados com leptospirose. rLIC11009 induziu somente resposta imune Th1 em camundongos, enquanto que rLIC11360 e rLIC11975 induziram resposta Th1 e Th2. As três proteínas recombinantes se ligaram à laminina e plasminogênio, enquanto rLIC11360 e rLIC11975 também se ligaram à fibronectina plasmática e fibrinogênio. Em adição, rLIC11360 se ligou ainda aos reguladores do sistema complemento fator H e C4BP. Os resultados sugerem que as proteínas estudadas podem auxiliar a leptospira a evadir e invadir o hospedeiro. / Leptospirosis is a worldwide zoonosis that causes great economic and social losses. The pathogenic mechanisms of leptospira are not yet fully elucidated. In this study we evaluated the role of three hypothetical surface proteins in the leptospiral pathogenesis: LIC11009, LIC11360 and LIC11975. Genes were cloned from DNA of L. interrogans serovar Copenhageni and the recombinant proteins were purified by affinity chromatography. RT - qPCR data have shown that the genes are fully transcribed in leptospires. Recombinant proteins reacted with sera from humans diagnosed with leptospirosis. rLIC11009 induced Th1 immune response in mice, whereas rLIC11360 and rLIC11975 promoted both Th1 and Th2. The three recombinant proteins interacted with laminin and plasminogen while, rLIC11360 and rLIC11975, also interacted with plasma fibronectin and fibrinogen. In addition, rLIC11360 interacted with the complement regulators factor H and C4BP. These results suggest that the proteins tested can help leptospires to evade and invade the host.

Japanese encephalitis virus (JEV) is the causative agent of Japanese encephalitis (JE), the leading cause of vaccine-preventable neurological disease. JEV is a flavivirus that is primarily transmitted through the bite of infected mosquitoes, similar to dengue virus (DENV), St. Louis encephalitis virus (SLEV), West Nile virus (WNV), and Zika virus (ZIKV). The two viral characteristics that dictate virulence are (1) neuroinvasiveness, the ability of the virus to invade the central nervous system(CNS), and (2) neurovirulence, the capacity of the virus to kill resident cells in the CNS. The clinically proven live-attenuated JEV vaccine, SA14-14-2, lacks both pathogenic characteristics unlike its virulent parental virus, SA14. Previous work has revealed the viral E gene as the main determinant of these two pathogenic properties, though the molecular mechanisms behind their attenuation remain unclear. The E gene encodes for the viral envelope glycoprotein that is involved in viral entry into susceptible host cells. The E protein of SA14-14-2 differs from SA14 by nine amino acids. To investigate the role of these mutations in JEV virulence, we created a series of SA14E mutants using infectious cDNA technology. Here, we report the independent function of domains I (DI) and II (DII) of the viral E protein in JEV neurotropism. We reveal that an individual mutation in DI, E138K,and synergism between two mutations in DII, E244G and K279M,are independently sufficient for the attenuation of JEV neuroinvasion. Also, we report that multiple E mutations are required for full attenuation of JEV neurovirulence. Overall, our findings show the direct relationship between genetic factors and JEV neuroinvasion. These results provide a solid foundational base for the logical development of other, currently non-existing, live-attenuated neurotropic flavivirus vaccines and antivirals.

OBJECTIVE: The principal objective of this study was to measure various aspects of immunity in children with invasive infections due to Haemophilus influenzae type b and Staphylococcus aureus. These serious infections are a significant cause of childhood morbidity and mortality in all populations and affect healthy as well as compromised children. Evidence suggests that imbalances or deficiencies in certain aspects of immunity such as IgG subclasses, the capacity to make specific subclass antibodies, antibody affinities, complement isotypes, immunoglobulin allotypes or mannose binding protein may place certain children at risk for developing invasive disease. Investigation of these factors in a group of children with infection necessitated that normal ranges be established for children of comparable ages from the same population. A secondary objective of this study has therefore been to establish normal percentiles for the IgG subclasses in age, race and sex matched healthy controls. METHODS: Patients admitted to the Red Cross War Memorial Children's Hospital with septic meningitis due to Haemophilus influenzae type b and osteomyelitis/septic arthritis due to Haemophilus influenzae type b or Staphylococcus aureus formed the study population. Section A of this thesis describes the methods for establishing, validating and standardizing ELISAs for measuring the IgG subclasses (lgGl, IgG2, IgG3 and IgG4) and subclass antibodies specific to Haemophilus influenzae polyribosylribitol phosphate, Staphylococcus aureus teichoic acid and tetanus toxoid. The relative affinity of antibodies in these ELISAs was determined by the incorporation of diethylamine (DEA). In order to determine the immunoglobulin allotypes ELISAs were developed to measure the G1m(f), G2m(n) and Km(3) allotypes. The frequency of these allotypic markers in the different ethnic groups was established. The relationship between immunoglobulin allotypes and IgG subclass values were investigated in both patient and control groups. RESULTS: ELISA assays to measure IgG subclasses; IgG, IgG 1 and IgG4 tetanus toxoid antibodies; IgG, IgG 1 and IgG2 H. influenzae type b polyribosylribitol phosphate capsular polysaccharide antibodies; IgG, IgG1 and IgG2 S. aureus teichoic acid antibodies and G1m(f), G2m(n) and Km(3) allotypes were successfully established. Where possible the assays were standardized with reference sera and specimens were exchanged with international laboratories. Age, race and sex related percentile charts and tables of normal ranges for IgG and IgG subclasses of Black and Coloured children were established. The IgG and IgG 1 values were higher than those previously reported for children in developed countries. Black children with H. influenzae meningitis had significantly lower IgG 1, IgG2 and IgG3 levels compared to the controls and although similar trends were seen for IgG and IgG4 levels they were not statistically significant. Coloured children with H. influenzae meningitis and Coloured and Black children with H. influenzae osteomyelitis/septic arthritis also showed a similar tendency of lower IgG and IgG subclass levels than the controls but these trends were also not significantly different. All patients responded to tetanus toxoid antigen suggesting normal immunocompetence to protein antigens. H. influenzae type b capsular polysaccharide antibodies were low in children with H. influenzae type b meningitis and osteomyelitis/septic arthritis and did not increase during the illness. IgG and IgG 1 teichoic acid antibodies were raised in patients with S. aureus osteomyelitis/septic arthritis although no further rise in these antibodies was seen when measured several weeks after the illness. The antibody affinity ELISAs showed that IgG 1 tetanus toxoid antibody had a greater affinity than IgG4 tetanus toxoid antibody, the IgG 1 and IgG2 H. influenzae capsular polysaccharide antibodies were of similar affinity and the IgG 1 teichoic acid antibody was of higher affinity than the IgG2 antibody. The G1m(f) and G2m(n) positive allotypes were uncommon in Black but common in the Coloured populations whereas Km(3) was common in both groups. There was a significantly decreased frequency of the G2m(n) positive allotype in Coloured patients with H. influenzae type b meningitis and H. influenzae type b osteomyelitis/septic arthritis which was not found in patients with S. aureus osteomyelitis/septic arthritis. In both Coloured and Black children with H. influenzae meningitis there was a significantly decreased frequency of the Km(3) allotype. No differences in C4 isotypes and mannose binding protein levels were evident in the patient and control groups. CONCLUSION: This study has developed simple, specific and reproducible ELISAs to measure IgG subclasses and subclass antibodies specific to tetanus toxoid, H. influenzae polyribosylribitol phosphate and S. aureus teichoic acid. Age, sex and race related normal ranges for IgG subclasses in the local Black and Coloured populations have been established. Black children with H. influenzae type b meningitis had significantly lower IgG 1, IgG2 and IgG3 levels compared to the controls. There was a clear association between a decrease of the G2m(n) allotype and the Km(3) allotype and susceptibility to invasive infections caused by H. influenzae.

No description available.

Influenza viruses cause between 3 and 5 million cases of respiratory infection each year and are responsible for between 250 and 500 thousand deaths. There are principally two avenues for the treatment and prevention of influenza. They are vaccination and antiviral regimens. Prevention of infection is largely accomplished through vaccination. While vaccines remain the preferred method for controlling the spread of influenza, treatment with antiviral drugs is important for treatment of severe infections that are caused by viruses that are different from the vaccination strains. The two major classes of antiviral drugs for influenza treatment are the adamantanes and the neuraminidase inhibitors. While most viruses have become resistant to the adamantanes, the neuraminidase inhibitors remain the primary choice for treatment of infections. Oseltamivir is the most important of the neuraminidase inhibitors. Data from an experiment run at Utah State University displayed a characteristic that is reflected in other published data. Oseltamivir, which has been shown to be effective against influenza virus strains in vitro, is unable to sufficiently protect mice from lethal infections. The focus of the present research was to identify viral differences that might explain for this discrepancy. Four viral strains were chosen that display differing susceptibilities to oseltamivir in mice. The viruses used were Influenza A/Duck/MN/1525/81 (H5N1), A/Victoria/3/75 (H3N2), A/NWS/33 (H1N1) and A/California/04/2009 (Pandemic H1N1). Oseltamivir was unable to protect mice that were infected with the A/Duck/MN/1525/81 and A/Victoria/3/75 viruses. These two viruses, along with the A/NWS/33 and the A/California/04/2009 viruses, were compared in vitro using virus replication kinetics, neuraminidase inhibition assays, and antiviral assays in cell culture. The viruses were studied in vivo by comparing survival, weight loss, lung scores and weights, lung virus titers, complete blood counts, cytokine assays, and histopathology. A second in vivo experiment was run to determine the effects of oseltamivir on survival, weight loss, lung scores and weights, lung virus titers, and histopathology. The two in vivo experiments summarized in this study confirmed previous data since oseltamivir was unable to protect mice infected with the influenza A/Duck and A/Victoria viruses. Overall, the virus infections behaved remarkably similar. The most interesting difference was that the A/Duck/MN/1525/81 and A/Victoria/3/75 viruses were able to induce more severe histopathological damage in mouse lungs earlier in the infection. The ability to cause severe disease more quickly might explain why the A/Duck/MN/1525/81 and A/Victoria/3/75 viruses remain lethal, despite oseltamivir treatment.

Fusarium virguliforme (Aoki, O’Donnell, Homma & Lattanzi), the causal agent of sudden death syndrome (SDS) of soybean (Glycine max [L.] Merrill), is responsible for major soybean yield losses in North America and South America. Despite the importance of SDS, few agronomic practices have been used to manage SDS successfully. Understanding the pathogen and the mechanisms it uses to cause disease is vital to devise effective disease control strategies. However, our knowledge of the pathogenicity mechanisms used by F. virguliforme is limited. The identification of pathogenic genes will shed light on the molecular basis of the interaction between F.virguliforme and soybean, which may ultimately lead to better management of SDS. Therefore, the studies presented in this thesis were aimed at identifying and characterizing candidate pathogenicity genes in F. virguliforme.To fulfill this objective, 40 candidate pathogenicity genes of F. virguliforme were identified based on a combined approach, which included hands-on literature and database mining, functional genomics as well as transcriptome analyses. From these genes, the FvSNF1gene (a sucrose non-fermenting protein kinase 1 ortholog), the Fvstr1 gene (a striatin protein ortholog) were functionally characterized through a gene knock-out strategy. Targeted disruption of the FvSNF1 locus in wild type F. virguliforme strain reduced virulence significantly on soybean and abolished galactose utilization. In addition, the FvΔSNF1 mutant displayed significant reduction in expression of several CWDEs genes and was defective in colonizing the vascular system of the roots. To identify putative target genes regulated by FvSNF1, transcriptome analyses were performed in the FvSNF1 deletion mutant and in the wild-type. Disruption of FvSNF1 affected the level of transcription of 393 genes and a majority of the genes were involved in carbohydrate metabolism, lignin degradation, and cellular signaling pathway. The disruption of Fvstr1, a striatin ortholog in F. virguliforme, resulted in a complete loss of virulence as well as impaired conidiation, conidiophore development and pigmentation in the fungus. The FvΔstr1 mutant also failed to colonize the vascular tissues of roots of inoculated soybean plants. The results suggest that FvSNF1and Fvstr1 have critical roles in pathogenicity. Another part of the study was to investigate the efficacy of ILeVO®, a succinate dehydrogenase inhibitor fungicide from Bayer CropScience, against F. virguliforme. Our results showed that ILeVO® was very active against F. virguliforme in vitro and was very effective in minimizing F. virguliforme infection thus providing yet another tool to combat SDS.

The ultimate goal of this research is to develop an efficient, reproducible and low cost method for analysis of VOCs in complex mixtures such as those in exhaled breath and in headspace of fungi cultures. In Chapter I; analytical methods for volatile biomarkers identification is reviewed In Chapter II, active SPME GCMS was employed to analyze VOCs in the breath of a single healthy male and a single female. The goal was to determine the extent of intra-individual variations in the VOC profiles. In Chapter III, a preliminary study was carried out in a greenhouse to determine the pathogenicity of different isolates of M. phaseolina on soybeans. This will allow, in future studies, the matching of VOC profiles of different isolates of M. phaseolina with their relative pathogenicity. This is a key step towards the development of an early warning system for the detection of pathogenic M. phaseolina fungus contaminations.

No description available.

<p>Bacteria demonstrate an extraordinary capacity to survive and adapt to changing environments. In part, this ability to adapt can be attributed to horizontal gene transfer, a phenomenon which introduces novel genetic information that can be appropriated for use in particular niches. Nowhere is this more relevant than in pathogenic bacteria, whose acquisition of virulence genes have provided an arsenal that permits them to thrive within their selected host. Regulatory evolution is necessary for timely regulation of these acquired virulence genes in the host environment. <em>Salmonella enterica</em> serovar Typhimurium is an intracellular pathogen which possesses numerous horizontally-acquired genomic islands encoding pathogenic determinants that facilitate its host lifestyle. One island, <em>Salmonella</em> Pathogenicity Island (SPI)-2, encodes a type-III secretion system (T3SS) which is regulated by the two-component regulatory system SsrA-SsrB. This system coordinates expression of the SPI-2 T3SS as well as an array of virulence effectors encoded in horizontally-acquired regions throughout the <em>Salmonella</em> genome. The studies presented here investigated the mechanisms in which the transcription factor SsrB functions to integrate virulence processes through regulatory adaptation. This work identified the regulatory logic controlling SsrB and defined the associated SsrB regulon. Furthermore, SsrB was found to induce a regulatory cascade responsible for the expression of bacteriophage genes encoded within SPI-12, an island that also contributes to <em>Salmonella</em> virulence. These findings demonstrate the important contribution of regulatory evolution in pathogen adaptation to the host, and show that horizontally-acquired genes, once integrated into appropriate regulatory networks, can contribute to pathogen fitness in specific niche environments.</p> / Doctor of Philosophy (PhD)