Espécies de Brucella são causadoras da brucelose, uma das doenças zoonóticas mais difundidas em todo o mundo, que é causa aborto em animais domésticos e infecção potencialmente debilitante em humanos. Apesar da identificação de diferentes espécies dentro do gênero, baseada na diferença de hospedeiro e patogenicidade, o gênero tem sido descrito como geneticamente homogêneo. Técnicas moleculares têm sido empregadas com o intuito de diferenciar e tipificar estes microrganismos, sendo que a abordagem mais promissora visa a identificação de polimorfismos entre as espécies e biovares. O objetivo do trabalho foi analisar o perfil genético de diferentes cepas de B. abortus obtidos a partir da técnica de SE-AFLP a fim de determinar a sequência dos fragmentos obtidos e genes que poderiam estar presentes nos fragmentos selecionados e compará-las com bancos de genes e de proteínas de procariotos. Foram selecionados 19 fragmentos, que foram purificados, sequenciados e, as sequências obtidas, submetidas a diversas ferramentas de bioinformática visando sua caracterização e identificação. Nenhum dos fragmentos de nucleotídeos apresentou similaridade entre si e/ou com outra sequência já conhecida de outros microrganismos, inclusive com sequências conhecidas do gênero Brucella. Das 114 proteínas hipotéticas geradas pela tradução destas sequências genômicas 57% (65 sequências) apresentaram baixo grau de similaridade com proteínas descritas e disponíveis em BLASTp (proteínas não-redundantes e SwissProt), variando entre 29 e 43. Do total de proteínas similares 85% foram atribuídas e proteínas funcionais e 14% a proteínas hipotéticas. Essas novas sequências deverão ser utilizadas em outros trabalhos visando verificar sua abrangência e especificidade dentro das espécies e biovares de Brucella. / Brucella species are the cause of brucellosis, one of the most widespread zoonotic diseases around the world, which is cause abortion in domestic animals and potentially debilitating infection in humans. Despite the identification of different species within the genus, based on the difference of host and pathogenicity, the genre has been described as genetically homogeneous. Molecular techniques have been employed in order to differentiate and classify these organisms, and the most promising approach aims to identify polymorphisms between species and biovars. The objective of this study was to analyze the genetic profile of different strains of B. abortus obtained by the technique of SE-AFLP to determine the sequence of the fragments obtained and genes that may be present in the selected fragments and compare them to databases of genes and proteins in prokaryotes. We selected 19 fragments that were purified, sequenced and the sequences obtained, subject to several bioinformatics tools aiming at its characterization and identification. None of the fragments showed nucleotide similarity between themselves and / or other sequence already known from other organisms, including known sequences of the genus Brucella. Of the 114 hypothetical proteins generated by translation of genomic sequences 57% (65 sequences) showed low level of similarity to proteins described and available in blastp (protein non-redundant and SwissProt), ranging between 29 and 43. Of the proteins similar 85% were attributed to functional proteins and 14% to hypothetical proteins. These new sequences should be used in other studies to verify its completeness and specificity within the species and biovars of Brucella.

Espécies de Brucella são causadoras da brucelose, uma das doenças zoonóticas mais difundidas em todo o mundo, que é causa aborto em animais domésticos e infecção potencialmente debilitante em humanos. Apesar da identificação de diferentes espécies dentro do gênero, baseada na diferença de hospedeiro e patogenicidade, o gênero tem sido descrito como geneticamente homogêneo. Técnicas moleculares têm sido empregadas com o intuito de diferenciar e tipificar estes microrganismos, sendo que a abordagem mais promissora visa a identificação de polimorfismos entre as espécies e biovares. O objetivo do trabalho foi analisar o perfil genético de diferentes cepas de B. abortus obtidos a partir da técnica de SE-AFLP a fim de determinar a sequência dos fragmentos obtidos e genes que poderiam estar presentes nos fragmentos selecionados e compará-las com bancos de genes e de proteínas de procariotos. Foram selecionados 19 fragmentos, que foram purificados, sequenciados e, as sequências obtidas, submetidas a diversas ferramentas de bioinformática visando sua caracterização e identificação. Nenhum dos fragmentos de nucleotídeos apresentou similaridade entre si e/ou com outra sequência já conhecida de outros microrganismos, inclusive com sequências conhecidas do gênero Brucella. Das 114 proteínas hipotéticas geradas pela tradução destas sequências genômicas 57% (65 sequências) apresentaram baixo grau de similaridade com proteínas descritas e disponíveis em BLASTp (proteínas não-redundantes e SwissProt), variando entre 29 e 43. Do total de proteínas similares 85% foram atribuídas e proteínas funcionais e 14% a proteínas hipotéticas. Essas novas sequências deverão ser utilizadas em outros trabalhos visando verificar sua abrangência e especificidade dentro das espécies e biovares de Brucella. / Brucella species are the cause of brucellosis, one of the most widespread zoonotic diseases around the world, which is cause abortion in domestic animals and potentially debilitating infection in humans. Despite the identification of different species within the genus, based on the difference of host and pathogenicity, the genre has been described as genetically homogeneous. Molecular techniques have been employed in order to differentiate and classify these organisms, and the most promising approach aims to identify polymorphisms between species and biovars. The objective of this study was to analyze the genetic profile of different strains of B. abortus obtained by the technique of SE-AFLP to determine the sequence of the fragments obtained and genes that may be present in the selected fragments and compare them to databases of genes and proteins in prokaryotes. We selected 19 fragments that were purified, sequenced and the sequences obtained, subject to several bioinformatics tools aiming at its characterization and identification. None of the fragments showed nucleotide similarity between themselves and / or other sequence already known from other organisms, including known sequences of the genus Brucella. Of the 114 hypothetical proteins generated by translation of genomic sequences 57% (65 sequences) showed low level of similarity to proteins described and available in blastp (protein non-redundant and SwissProt), ranging between 29 and 43. Of the proteins similar 85% were attributed to functional proteins and 14% to hypothetical proteins. These new sequences should be used in other studies to verify its completeness and specificity within the species and biovars of Brucella.

O estudo objetivou avaliar a eficácia do programa de vacinação contra brucelose bovina no estado do Rio Grande do Sul tendo como indicador a prevalência e individualizar os fatores de risco para a doença. O Estado foi dividido em sete regiões. Para cada região foram amostradas aleatoriamente um número preestabelecido de propriedades nas quais foi testado um número também preestabelecido de fêmeas com idade igual ou superior a 24 meses, aleatoriamente selecionadas. O protocolo do sorodiagnóstico foi composto de triagem com o teste do antígeno acidificado tamponado, seguido de teste confirmatório dos sororreagentes com o teste 2-Mercaptoetanol. Nas propriedades foi aplicado um questionário epidemiológico sobre possíveis fatores de riscos associados à brucelose bovina. No estado do Rio Grande do Sul, a prevalência de focos foi de 3,54% [2,49 4,88] e a de animais 0,98% [0,57 1,57]. Nas regiões, as prevalências de focos variaram de 0,66% a 9,03% e a de animais de 0,06% a 2,03%. Rebanhos com 15 ou mais vacas, tipologia corte e compartilhamento de pastagens emergiram como fatores de risco para brucelose bovina no estado. A situação epidemiológica da brucelose bovina no Rio Grande do Sul manteve-se estável desde 2004, a despeito de boas coberturas vacinais terem sido registradas a partir de 2009. Assim, o estado deve continuar seu programa de vacinação, dando ênfase para a qualidade do processo e estimulando a utilização da vacina não indutora de anticorpos. Adicionalmente, o estado deve realizar um grande esforço de educação para que os produtores testem os animais de reprodução para brucelose antes de introduzi-los em suas propriedades e evitem o compartilhamento de pastagens entre rebanhos de condição sanitária desconhecida. / This study aimed to evaluate the effectiveness of a bovine brucellosis vaccination program in Rio Grande do Sul, with prevalence as the indicator, and to identify risk factors for the disease. The state was divided into seven regions. For each region, a predetermined number of properties were randomly sampled, in which a pre-established number of randomly selected females aged over 24 months were tested. The serodiagnosis protocol consisted of a screening test using buffered acidified antigen, followed by a confirmatory test using 2-mercaptoethanol. An epidemiological questionnaire was utilized to identify possible risk factors associated with bovine brucellosis. In the state of Rio Grande do Sul, the prevalence of infected herds was found to be 3.54% [2.49-4.88], and the prevalence of infected animals was 0.98% [0.57-1.57]. In assessments of specific regions, the infected herd prevalence ranged from 0.66% to 3.09%, and among the animals, from 0.06% to 2.03%. In herds comprising 15 or more cows, beef type and pasture sharing emerged as risk factors for bovine brucellosis in the state. The epidemiological status of bovine brucellosis in Rio Grande do Sul has remained unchanged since 2004, even though adequate vaccination coverage has been recorded since 2009. Thus, the state should continue its vaccination program, with emphasis on the quality of the process and on encouraging the use of non-antibody inducing vaccines. In addition, the state must make a greater effort to educate producers on the importance of testing for brucellosis in breeding animals before introducing them onto their properties, and on the importance of avoiding shared grazing among herds whose health conditions are unknown.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2018-08-30T18:28:46Z No. of bitstreams: 1 texto completo.pdf: 13316435 bytes, checksum: b0412364dab9dd00515656dc38ddb78d (MD5) / Made available in DSpace on 2018-08-30T18:28:46Z (GMT). No. of bitstreams: 1 texto completo.pdf: 13316435 bytes, checksum: b0412364dab9dd00515656dc38ddb78d (MD5) Previous issue date: 2018-03-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A brucelose bovina, causada pela bactéria Brucella Abortus, é uma doença presente em to- das as regiões do Brasil e provoca elevados prejuízos econômicos. O Programa Nacional de Controle e Erradicação de Brucelose e Tuberculose Animal (PNCEBT) estabeleceu os testes AAT, 2-ME, FC e DBac para realizar o diagnóstico da brucelose bovina. Na ausência de um teste Padrão Ouro, é necessário que o desempenho desses testes diagnósticos seja validado. O presente estudo, teve como objetivo empregar o modelo de classe latente Bayesiano para es- timar as sensibilidades e as especificidades dos testes diagnósticos AAT, 2-ME, FC e DBac, aplicados em amostras de sangue e carcaças de animais suspeitos de brucelose bovina, bem como a prevalência da doença. O conjunto de dados utilizado foi obtido junto ao Laboratório Nacional Agropecuário de Minas Gerais (LANAGRO-MG). Os testes foram avaliados em dois cenários: individualmente e combinados. Os modelos para a avaliação dos testes combinados foram ajustados considerando-se a independência condicional entre os quatro testes e também incorporando-se ao modelo a dependência condicional entre os testes AAT, 2-ME e FC. As aná- lises foram realizadas em R 3.2.5 usando o pacote R2OpenBUGS. Quanto à avaliação dos testes combinados, os resultados mostraram que os testes AAT, 2-ME e FC são condicionalmente in- dependentes. O teste FC foi o mais sensível, o DBac o menos sensível e os testes AAT, FC e DBac foram os mais específicos. Concluiu-se que nenhum dos quatro testes pode ser utilizado sozinho para o diagnóstico da brucelose bovina. Uma baixa sensibilidade foi encontrada para o teste AAT, resultado que diverge dos relatos geralmente encontrados na literatura. Portanto, recomenda-se que contínuos estudos sejam realizados para que a tomada de decisão dos pesqui- sadores não seja comprometida. Adicionalmente, concluiu-se que o modelo de classe latente bayesiano permitiu estimar os parâmetros de interesse satisfatoriamente. / Bovine brucellosis, caused by the bacterium Brucella Abortus, is a disease present in all regions of Brazil and causes high economic losses. The National Program for the Control and Eradi- cation of Brucellosis and Animal Tuberculosis (PNCEBT) established the AAT, 2-ME, FC and DBac tests for the diagnosis of bovine brucellosis. In the absence of a Gold Standard test, the performance of these diagnostic tests must be validated. The aim of the present study was to use the Bayesian latent class model to estimate the sensitivities and specificities of the AAT, 2- ME, FC and DBac diagnostic tests applied to blood samples and carcasses of animals suspected of bovine brucellosis, as well as the prevalence of the disease. The dataset used was obtained from the National Agricultural Laboratory of Minas Gerais (LANAGRO-MG). The tests were evaluated in two scenarios: individually and in combination. The models for the evaluation of the combined tests were adjusted considering the conditional independence between the four tests and also incorporating to the model the conditional dependence between the AAT, 2-ME and FC tests. Analyses were performed in R 3.2.5 using the R2OpenBUGS package. Regarding the evaluation of the combined tests, the results showed that the AAT, 2-ME and FC tests are conditionally independent. The FC test was the most sensitive, the DBac the least sensitive and the AAT, FC and DBac tests were the most specific. It was concluded that none of the four tests can be used alone for the diagnosis of bovine brucellosis. A low sensitivity was found for the AAT test, a result that diverges from the reports generally found in the literature. Therefore, it is recommended that continuous studies be carried out so that the decision-making of the researchers is not compromised. Additionally, it was concluded that the Bayesian latent class model employed allowed to estimate the parameters of interest satisfactorily.

Brucella abortus and Agrobacterium tumefaciens are pathogenic bacteria that infect animals and plants, respectively. These bacteria are genetically similar and are found within the same Class, Alphaproteobacteria, and Order, Rhizobiales, of the domain Eubacteria; however, they survive and replicate in vastly different environmental niches. In Order to adapt to different environments, bacteria utilize several mechanisms of gene regulation to tightly control gene expression. Two of these mechanisms include transcriptional regulators and small regulatory RNAs (sRNAs), which can activate and repress gene expression through various interactions with DNA, mRNA, and proteins. A well-conserved transcriptional regulator among the Rhizobiales is VtlR, a virulence-associated transcriptional LysR regulator. The objectives of this dissertation were three fold: 1) characterize the known regulon of VtlR in B. abortus with regards to gene regulatory function and virulence, 2) determine the regulon of VtlR in A. tumefaciens and define the mechanism by which this regulation occurs, and 3) define the role of an ABC-type transport system indirectly regulated by VtlR in B. abortus that putatively imports the non-proteinogenic amino acid gamma-aminobutyric acid (GABA). VtlR was characterized in B. abortus as a virulence-associated transcriptional regulator that directly activates four genes: the sRNA AbcR2, and the three small hypothetical proteins BAB1_0914, BAB2_0512, and BAB2_0574; and deletion of vtlR led to a significant defect in the ability of B. abortus to cause infection in vitro and in vivo. Since dysregulation of abcR2 alone could not account for the defect in virulence, it was hypothesized that one or all three hypothetical proteins could be responsible for a virulence phenotype observed in ΔvtlR. This turned out to not be the case, as a deletion of the entire VtlR regulon displayed no difference in virulence compared to the parental strain. Further characterization of the small hypothetical proteins is outlined in Chapter 2 and the data revealed bona fide translation of each small protein, and the deletion strain of the VtlR regulon displayed a growth defect when grown in the presence of the sugar fucose. This phenotype was subsequently observed in ΔvtlR as well. This led to the identification of a putative fucose transport and metabolism locus in B. abortus that has yet to be studied. In A. tumefaciens, VtlR is necessary for proper attachment to plant cells and biofilm formation and regulates over 200 genes, significantly more than the four genes VtlR regulates in B. abortus. The mechanism by which this occurs was unknown, and the relationship between VtlR and AbcR1 or AbcR2 was uncharacterized. The data in Chapter 3 outline the VtlR network by showing that VtlR regulation of myriad genes in A. tumefaciens is primarily indirect via the direct regulation of a few sRNAs. This direct interaction was shown experimentally and a VtlR binding box was identified in the A. tumefaciens genome. This project outlines the divergence of a regulatory element between phylogenetically related organisms that occupy different environmental niches. The AbcR sRNAs are conserved throughout the Rhizobiales and regulate numerous ABC-type transport systems within these bacteria. In A. tumefaciens, one of these transport systems specifically transports the amino acds proline and GABA. B. abortus contains homologs of this system, which led to the hypothesis that the brucellae may also transport GABA but for a yet unknown purpose. The data in Chapter 4 revealed that B. abortus also transports GABA in vitro and this transport is under the regulation of AbcR1 and AbcR2. This transport was increased under extreme nutrient limitations and was uninhibited by the presence of other amino acids. Metabolic studies showed GABA is not utilized by B. abortus under aerobic conditions, and transcriptomic data revealed increased expression of several loci in the presence of GABA. Altogether, this study uncovers a putative signaling role for the amino acid GABA that has been understudied in bacterial pathogens that infect animal hosts. Overall, the work presented in this dissertation is focused on further elucidating the biological role of downstream regulatory targets of both VtlR and the sRNAs AbcR1 and AbcR2 in the related organisms Brucella abortus and Agrobacterium tumefaciens. Findings show that while there are similarities between the two systems, there are also many differences that may be attributed to the vastly different lifestyles of each organism. / Doctor of Philosophy / Brucella abortus and Agrobacterium tumefaciens are two highly related bacterial pathogens that infect mammals and plants, respectively. Although genetically related, both organisms survive and replicate in vastly different environmental niches with one living in the soil (i.e., A. tumefaciens) and the other living within immune cells of the infected host (i.e., B. abortus). In Order to quickly adapt to changing environmental conditions, the bacteria must rapidly control gene expression through multiple regulatory mechanisms. The works presented in this dissertation will focus on further characterizing one of these regulatory systems and comparing the homologous systems shared by B. abortus and A. tumefaciens. This includes uncovering a putative sugar transport and metabolism system, as well as discovering the potential for host-pathogen signaling via the well-studied neurotransmitter GABA.

Neospora caninum is an apicomplexan parasite that causes neuromuscular paralysis in dogs and abortions in cattle. N. caninum is responsible for losses of several million dollars to the dairy and beef industries in several parts of the world. The key players in the host immune response to N. caninum include CD4+ T cells, the Th1 cytokines IL-12, Interferon gamma and IgG2a isotype antibodies. There are currently no chemotherapeutic agents that are effective against adult cattle neosporosis. A commercially available, inactivated vaccine induces the undesirable Th2 type of immunity against N. caninum. Therefore, two approaches towards vaccine development against N. caninum that were designed to induce potent cell mediated immunity have been explored in this dissertation. The first approach consisted of the development of a bivalent recombinant vaccine for both brucellosis and neosporosis, while the second approach involved gamma irradiation of N. caninum tachyzoites for use as an attenuated vaccine against N. caninum. Since N. caninum research has been conducted with several strains of mice and the different strains of mice vary in their susceptibility to infection with N. caninum, there is a need to develop a standard lab animal model for N. caninum. A gerbil and a C57BL/6 mouse model for N. caninum vaccine testing have been developed. It was found that the LD50 of N. caninum tachyzoites in gerbils was 9.3 x105 tachyzoites per gerbil delivered intra-peritoneally, (i.p) while for C57BL/6 mice the LD50 was 1.5 x107 tachyzoites per mouse delivered i.p. Vertical transmission rates in C57BL/6 mice infected with N. caninum tachyzoites during mid-gestation were determined and found to be in the range of 96-100%. Putative protective antigens of N. caninum that included MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in B. abortus strain RB51 to create recombinant vaccine strains. C57BL/6 mice were vaccinated with either the recombinant strains or the irradiated tachyzoites. Antigen specific IgG2a and IgG1 responses and high levels of interferon gamma and IL-10 were induced by vaccination. Mice vaccinated with irradiated tachyzoites, RB51-MIC1 and RB51-GRA6 were completely protected against lethal challenge, while the mice vaccinated with RB51-SRS2, RB51-GRA2 and RB51-MIC3 were partially protected. To determine the efficacy of the vaccines in preventing vertical transmission of N. caninum, mice were vaccinated and bred after administration of a booster dose four weeks after the primary vaccination. Antigen specific IgG1 and IgG2a and significant levels of IFN-ã and IL-10 were detected in vaccinated, pregnant mice. Pregnant mice were challenged with 5 x 106 N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups three weeks after birth and examined for the presence of N. caninum by a semi-nested PCR. Protection against vertical transmission elicited by the RB51-GRA6, RB51-MIC3, irradiated tachyzoite, RB51-GRA2, RB51-MIC1 and RB51-SRS2 vaccinated groups were 43%, 38%, 34%, 34%, 18%, and 7% respectively. Since not all the antigens that were highly protective against acute disease were not very effective in preventing vertical transmission, the role of the selected antigens in preventing acute disease and vertical transmission appear to differ. Only GRA6 was found to be effective in protecting against an acute lethal challenge as well as preventing vertical transmission 43% of the time. In summary, two animal models for the testing of N. caninum vaccines were developed. N. caninum protective antigens were successfully expressed in B. abortus strain RB51. The irradiated tachyzoite and recombinant RB51-Neospora vaccines were highly effective in protecting against acute neosporosis and partially protective against vertical transmission. Therefore, both these approaches show great promise as practical and effective means to achieve the goal of successful prophylaxis against N. caninum induced abortions and reduce the chances of vertical transmission. / Ph. D.

Brucella abortus is a facultative, intracellular zoonotic pathogen, which can cause undulant fever in humans and abortion in cattle. Despite all of the progress in brucellosis research, there are still many unanswered questions regarding the molecular mechanisms involved in the pathogenesis of Brucella infections. To better understand the Brucella antigens involved in virulence and/or immunity, genetic and immunologic characterization of a 16 kDa protein of B. abortus was performed. Using PCR methods, the gene encoding the 16 kDa protein was cloned and sequenced. PCR and Southern blot analysis revealed that the gene is conserved among the 6 nomen species of Brucella. Overexpression of this protein in B. abortus vaccine strain RB51 was achieved using Brucella groE and sodC promoters as well as its own promoter. Protection and clearance studies were performed in mice to determine the role of this protein in Brucella immunity and pathogenesis. Inoculation with either strain RB51 overexpressing the 16 kDa protein or a DNA vaccine encoding the 16 kDa protein gene failed to provide significant protection. No difference was noted between the splenic clearance of B. abortus strain 2308 and its recombinant overexpressing the 16 kDa protein. A mutant of strain 2308 (2308D16) was created by disrupting the 16 kDa protein's gene with a chloramphenicol resistance cassette. Western blot analysis indicated that the O antigen profile of strain 2308D16 differed from that of strain 2308. Mice cleared strain 2308D16 faster than strain 2308 indicating the potential attenuation of the disruption mutant. Purified 16 kDa protein was obtained by overexpressing it in E. coli via the pRSET expression system. Western blotting results initially identified this protein as an immunoglobulin-binding protein. Hemagglutination assay revealed that the 16 kDa protein exhibits lectin-like properties. Preliminary studies using hemagglutination inhibition identified mannose as a possible sugar to which the 16 kDa protein can interact. The lectin-like properties exhibited by the 16 kDa protein appears to influence smooth lipopolysaccharide production, and thereby may be involved in virulence. / Master of Science

Brucella abortus is a facultative intracellular bacterial pathogen, which causes abortion in cattle and undulant fever in human. B. abortus strain RB51 (Strain RB51) is the official vaccine for bovine brucellosis in the USA. B. abortus strain RB51 can be used as a vector for the over-expression of its own (homologous) as well as heterologous protective antigens. The immune system can detect these heterologous antigens and produce a response. Expressing a protein in different bacterial compartments has been shown to affect its accessibility to the immune system and the way the antigen is processed by antigen presenting cells. In order to determine if the immune response is affected by the localization of the antigen, green fluorescent protein (GFP) was expressed at three different locations in B. abortus strain RB51, outer-membrane (OM), periplasmic space (PS) and in the cytoplasmic region (CR) of B. abortus strain RB51. This localization was obtained by transforming strain RB51 with plasmids pBBg18sGFP and pBBgSsGFP, in which the 18 kDa Brucella lipoprotein and the Brucella Cu/Zn SOD protein signal sequences were added to the GFP sequence to cause OM and PS expression respectively. No signal sequences were added to the plasmid pBBgGFP for CR only expression. Expression and localization of GFP in the different compartments in recombinant B. abortus strain RB51 were confirmed by electron microscopy and antibody absorption experiments. Groups of 5 female BALB/c mice each were injected and boosted with three recombinant strains and appropriate controls. Mice were bled and their anti-GFP antibody production was assessed. None of the immunized mice produced specific antibodies against GFP, probably due to the low expression of the heterologous antigen observed in this study by strain RB51 observed in this study. It will be necessary to produce new recombinants which are able to express higher amounts of GFP to answer if localization of heterologous antigen within the recombinant RB51 affects the level of a specific immune response. / Master of Science

Brucella causes undulant fever in humans and uterine and systemic infection leading to abortions in domestic animals and wild life. For the acquisition of iron in mammalian hosts, species of Brucella are known to produce two siderophores, 2, 3-dihydroxy benzoic acid (2, 3-DHBA) and brucebactin. Inability to synthesize of 2, 3-DHBA affects the ability of pathogen to metabolize erythritol, replicate in trophoblast cells and cause abortion in pregnant ruminant host. The entF gene has been implicated in the unresolved pathway allowing brucebactin biosynthesis in Brucella. The research effort presented in this thesis tries to relate the role of entF in iron acquisition and potential relation with erythritol metabolism by wild type B. abortus 2308. An entF deletion mutant (BAN1) of B. abortus 2308, generated using cre-lox methodology was found to be growth inhibited in iron minimal media compared to wild type strain. Growth inhibition was further enhanced with the addition of an iron chelator or 0.1% erythritol. Compared to wild type strain, no growth inhibition of BAN1 mutant was found in murine J774A.1 macrophages, which suggests that Brucella could acquire iron inside mammalian cells. The entF gene complemented mutant strains of BAN1 (BAN2A and BAN2B) were found to be intermediate in their ability to grow in iron minimal media supplemented with 0.0.05% erythritol, when compared to wild type and BAN1 strain. The results from the present thesis demonstrate that entF gene plays an important role in iron acquisition and erythritol metabolism by B. abortus 2308 under iron limiting conditions. / Master of Science

The gram-negative bacterial pathogen Brucella abortus is a zoonotic pathogen causing brucellosis in a variety of animal species including humans. The loss of the O-side chain in the lipopolysaccharide of the outer membrane decreases Brucella virulence. To understand the genetics of O-side chain biosynthesis and its relationship to virulence, studies were initiated to characterize specific O-side chain mutants. B. abortus rough mutant strain RA2 was derived by transposon (Tn5) mutagenesis of smooth B. abortus 2308. The chromosomal region of strain RA2 with the Tn5 and flanking chromosomal region was cloned into the sequencing vector pGEM-3Z to create a suicide plasmid pNW-2. The plasmid pNW-2, or a derivative of it (pNW-3), in which Tn5 was replaced with a Kank gene, were electroporated into wild type smooth B. abortus 2308 in order to assess the phenotypic conversion from smooth to rough. The electroporation parameters such as cell growth stage, pulse field strength and pulse length were optimized. It was determined that using late log phase cells (approximately 70-77 Klett units), 10 ms and 13 KV/cm were the best conditions for achieving transformation by pNW-2 or pNW3. Kanamycin resistant and ampicillin sensitive Brucella were screened for double reciprocal crossovers between the suicide plasmids (pNW-2 and pNW-3) and Brucella chromosomal DNA. The recombinants were checked for their O-side chain by crystal violet uptake and immunoblotting with monoclonal antibody specific for the O-side chain. The locations of Tn5 and the flanking region in the genome of these recombinants were characterized by Southern blot using either a Tn5 probe or a flanking region probe. An analysis of KanR colonies showed that none of the recombinants were rough. The B. abortus DNA in pNW-2 was sequenced and compared with other genes. No Significant homology was found between the Brucella DNA in pNW-2 and gene sequences in the gene bank. Analysis of the recombinants suggests no linkage between the Tn5 element in strain RA2 and the rough phenotype. / Master of Science