Browsing by Subject "Brucella"
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Item Concerning Brucella LPS: genetic analysis and role in host- agent interaction(Texas A&M University, 2006-10-30) Turse, Joshua EdwardB rucella lipopolysaccharide is an important component of virulence in brucellosis. Recent research in macrophage models has shown that Brucella LPS does not behave like classical LPS by stimulating potent inflammatory responses. The central hypothesis of this work is that O-antigen is dynamic signaling molecular and participates in complex interactions with the host to promote productive infection. A corollary to this is that the host environment is dynamic, and Brucella has evolved mechanisms to cope with changing environments. In an effort to understand the contribution of Brucella LPS to virulence and pathogenesis, the function of a metabolic locus important in the synthesis of LPS has been demonstrated and complemented. The spontaneous loss of LPS expression has been characterized. Contribution of LPS to acquisition of the host environment in tissue culture and mouse models has been explored. This work demonstrated that genes outside the O-antigen biosynthesis ( manBA) cluster contribute to LPS biosynthesis. Further high frequency mutation involving manBA is partly responsible for observed dissociation of Brucella strains. Finally, work herein attempts to look at the role of LPS in acquisition of the host environment and shows that LPS is important for recruiting particular cell populations within a host model of brucellosis.Item Evaluation of microencapsulation as an improved vaccination strategy against brucellosis(2009-05-15) Arenas Gamboa, Angela MariaBrucellosis is an important zoonotic disease of nearly worldwide distribution. Despite the availability of live vaccine strains for bovine (S19, RB51) and small ruminants (Rev 1), these vaccines have several drawbacks including residual virulence for animals and humans. Safe and efficacious immunization systems are therefore needed to overcome these disadvantages. Brucella melitensis and Brucella abortus mutants in the luxR gene were generated and investigated for theri potential use as improve vaccine candidates. Immunization with a sustained release vehicle to enhance vaccination efficacy was evaluated utilizing the live mutants in encapsulated alginate microspheres containing a non-immunogenic eggshell precursor protein of the parasite Fasciola hepatica (Vitelline protein B, VpB). BALB/c mice were immunized with either encapsulated or nonencapsulated vaccine candidates to evaluate immunogenicity, safety and protective efficacy. The results suggest that luxR mutants, are attenuated in the mouse and macrophage model and appear good and safe vaccine candidates when the immunogen is given in a microencapsulated format. We were also able to demonstrate the utility of microencapsulation in oral delivery by increasing vaccine performance of current licensed vaccine strains in a natural host, the Red Deer. Together, these results suggest that microencapsulation of live Brucella produces an enhanced delivery vaccine system against brucellosis increasing the efficacy of poorly-performing nonencapsulated vaccine candidates.Item Evaluation of unmarked deletion mutants as improved Brucella vaccine strains in the mouse and goat models(Texas A&M University, 2006-10-30) Kahl, Melissa MarieHistorical data suggests that prolonged survival of Brucella vaccine organisms in the target host enhances immune protection. Recent research has focused upon the development of rough vaccine strains to avoid interference with standard diagnostic tests. Rough organisms are rapidly cleared from the host, however. In an effort to develop improved vaccine strains, we have screened signature tagged mutagenesis banks to identify mutants with varying survival characteristics. We hypothesize that in order for a vaccine to be efficacious, it must survive in the host. In order to test this, we constructed marked and unmarked deletion mutants of B. abortus and B. melitensis in genes previously demonstrated by transposon mutagenesis to attenuate in vivo and in vitro virulence. Survival and efficacy of these novel deletion mutants were then evaluated in the mouse model. The asp24 mutants, which persist for extended periods in vivo, appear superior as a vaccine candidate compared to approved vaccine strains S19 and Rev1 in the mouse model against either homologous or heterologous challenges. Once enhanced protection against infection was demonstrated in the mouse, components of immune function that appeared to be most important were identified to correlate the immune response with the observed protection. We demonstrated that the most persistent mutant, delta-asp24, affords the greatest protection in mice against virulent challenge. In order to evaluate safety of the novel vaccine strains as well as protection against infection and abortion, we tested selected B. melitensis unmarked deletion mutants in a natural host, the goat. The delta-asp24 mutant was shown to be safe in pregnant goats while providing significant protection against infection and abortion.Item Host and pathogen transcriptional profiles of acute Brucella melitensis infection(2009-05-15) Rossetti, Carlos AlbertoThe parallel gene expression profiles of Brucella melitensis and the host have not been elaborated. In this study, I analyze and discuss the transcriptional profiles of B. melitensis invasive-associated genes, the expression profile of intracellular B. melitensis and B. melitensis-infected non-phagocytic cells in the first 12 h post-infection (PI), and the in vivo temporal global transcriptome of both B. melitensis and the infected bovine host in the first 4 h PI. The initial study found that B. melitensis at late-log phase of growth were more invasive in non-phagocytic cells than at early-log or stationary growth phase. Microarray-based studies identified 454 Brucella genes differentially expressed between the most and the least invasive growth phases. Additionally, B. melitensis strains with transposon interrupted in loci BMEII0380 (acrA) and BMEI1538 (hypothetical protein) were found to be deficient in internalization compare with the wild-type strain. A second experiment was designed with the goal of characterizing host and pathogen transcriptome in parallel. For detecting intracellular Brucella gene expression, a combined protocol consisting of a linear amplification of sense-stranded RNA biased to pathogen transcripts to the previously enriched host:pathogen RNA mixed sample, was developed. RNA samples were hybridized on human and Brucella cDNA microarrays, which analysis revealed a common down-regulation transcriptional profile at 4 h PI that was reverse at 12 h PI. The integrity of B. melitensis virB operon and the expression of host MAPK1 were confirmed as critical for early B. melitensis intracellular survival and replication in non-phagocytic cells. Finally, a temporal morphological and molecular characterization of the initial B. melitensis:bovine host interaction using a calf ileal loop model was performed. B. melitensis was isolated from intestinal Peyer?s patches as soon as 15 min and from systemic blood after 30 min postintra luminal inoculation. Microarray results revealed a common transcriptional profile in Brucella, but two different transcriptional profiles were identified in the host in the first 4 h PI. The importance of differentially expressed biological processes, pathways and individual genes in the initial Brucella pathogenesis is discussed.Item Identification and Evaluation of Brucella Recombinant Outer Membrane Proteins as Subunit Vaccinogen Candidates in the Mouse Model of Brucellosis(2013-02-26) Gomez, GabrielDespite being amongst the most common zoonotic diseases in the world, brucellosis is a neglected disease for which an approved vaccine for human use does not exist. Thus far, the traditional approaches to Brucella antigen selection for subunit vaccine development have yielded unacceptable results. In this work, we evaluated the predictive ability of a multistep Brucella antigen selection process with in vitro immunological and invasion assays and in vivo protection experiments. Initial in silico screening for antigens was performed via genomic sequence analysis where 27 Brucella melitensis open reading frames (ORF) coding for outer membrane proteins bearing MHC epitopes, adhesin and conserved properties were identified. Evidence for a role in any aspect of Brucella virulence (i.e., invasion, co-regulation/expression with known Brucella virulence factors, intracellular adaptation) was then used to narrow the list of candidate antigens. To further increase confidence in the candidate ORF putative role in Brucella pathogenesis, differential expression of candidate ORF was evaluated using previously generated global transcriptomics data in in vitro HeLa and in vivo bovine models of acute Brucella infection. Protein expression in the E. coli heterologous system resulted in the successful expression of OmpW, BtuB, Omp22, Hia, and FlgK. With regards to virulence, the two proteins with the highest predicted adhesin scores conferred an invasive phenotype to the non-invasive BL-21 E. coli strain in alveolar epithelial cells. From an immunogenicity standpoint, all proteins elicited IgG production in Brucella-exposed goats, mouse and humans. Antigen-specific recall responses in splenocytes from C57BL/6 mice immunized with a cocktail of the three proteins with highest MHC scores revealed a mixed Th1/Th2 response with a comparatively greater Th1 response. In protection studies, subcutaneous (SQ) immunization with BtuB, Hia and FlgK, individually, promoted bacterial clearance following a robust intraperitoneal challenge dose of Brucella melitensis 16M. In addition, single SQ inoculation of FlgK enhanced protective efficacy of the vaccine strain B. abortus S19. In contrast, immunization of mice with the three protective antigens in a cocktail formulation elicited immune responses but no protection against intraperitoneal challenge with Brucella melitensis 16M in the spleen and liver. In conclusion, our results indicate that our combinatorial in silico, in vitro and in vivo antigen selection and identification modeling approach provides strong evidence for prediction of Brucella protective antigens, and represent a novel strategy with broad application to other major pathogens.Item Structural studies of Mycobacterium tuberculosis KatG, an INH drug activator, and Brucella abortus VirB11, an ATPase of type IV translocation system(2009-05-15) Yu, HongCatalase-peroxidase (KatG) of Mycobacterium tuberculosis is a bifunctional heme enzyme that has been shown to play an important role in the activation of a first line drug, isoniazid (INH), used in the treatment of tuberculosis infection. Mutations in the katG gene have been found to be associated with INH resistance. The most commonly encountered mutation is the Ser315Thr point mutation. In this dissertation, the x-ray crystallographic structures of MtbKatG and the mutant enzyme KatG[S315T] are presented to explore the molecular basis of the INH activation and resistance. The structure is dimeric and contains a heme cofactor in each subunit of the dimer. The most important change in KatG[S315T] is due to the presence of the methyl group of the threonine 315 side chain, which is located at the narrowest part of the substrate channel. The protruding methyl group effectively constricts the accessibility to the heme by closing down the dimensions of the channel, constraining the substrate entrance. VirB11 of Brucella abortus is a hexameric ATPase that belongs to the type IV secretion system. The crystal structure of BaVirB11 was found to contain six molecules per asymmetric unit. The Walker A (P loop), His box, and Glu box from the C-terminal domain are located at the interface of the N- and C-terminal domain. A large conformational change was found in the linker region when compared with that of HP0525 structure, the VirB11 analogous from H. pylori. To elucidate the functional role of each domain, seven functional mutations were generated and used for biochemical studies. The GER motif and the linker region were found to be crucial for ATP hydrolysis activity of BaVirB11. Mutations in the GER motif (R101Q) and the linker region (R120E) of BaVirB11 completely abolish the ATP hydrolysis activity of the enzyme. The binding affinities of the two mutants to the ATP; however, are similar to that of the wild-type enzyme, indicating that mutation in the GER motif or the linker region has no effect on ATP binding.Item Utilization of the persistent nature of Brucella in the development of live vaccines(Texas A&M University, 2006-10-30) Hong, Priscilla ChristineThe roles of genes responsible for the survival and persistence of Brucella in the host and the relationship between these genes and the disease were investigated via signature-tagged transposon mutagenesis. As much as 8% of the Brucella genome is important for survival of this organism in the host. This is an unusually high number and may help to explain the chronic or persistent nature of Brucella infections. Mutants attenuated in the mouse model were divided into two groups. The early mutants failed to establish infection or colonize the host. The late mutants colonized the host but failed to maintain infection. The vaccine potential of two mutants (virB10 and gcvH) that were unable to sustain infection was compared to that of a vaccine strain, S19. Survival of strain S19 in vivo was up to 12 weeks while virB10 and gcvH mutants were cleared from spleen at 8, and 24 weeks post-inoculation, respectively. Mice were vaccinated with individual mutants and then challenged with virulent S2308 at 8, 16, and 24 weeks postvaccination. As a result, protective immunity correlated with persistence of the mutant strain [gcvH>virB10]. These results suggest that survival is one of several factors that may influence protective immunity making it difficult to compare strains. For example, examination of host immune response revealed a similar pattern of host immune function (TH1 over TH2) in all mice except those vaccinated with virB10 mutant. Since gcvH mutant provided the best immunity, experiments were designed to explore its contribution of persistence to protection. In an effort to reduce non-specific activation induced by prolonged survival of gcvH mutant, protection was monitored after different periods of vaccination exposure followed with doxycycline treatment. In these studies, persistence of gcvH mutant enhanced protection against challenge. Overall, defined mutations in genes affecting survival may render mutants as vaccine candidates capable of stimulating protective immunity equal to or better than fortuitously isolated attenuated strains. Future studies should focus on characterization of these and other genes responsible for the persistence of Brucella to improve the safety and efficacy of live vaccines.