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Identification of rifampin resistant-related genes in Mycobacterium smegmatis. / CUHK electronic theses & dissertations collectionJanuary 2012 (has links)
結核病是由結核桿菌感染而引起的慢性傳染病,它是危害人類健康的主要殺手。根據世界衛生組織的報導,目前在全球範圍內有三分之一的人口感染了結核桿菌,每年約有915 萬人口被確診患有結核病。耐藥結核病尤其是對最有效的一線抗結核藥物異煙阱和利福平產生抗藥的耐多藥結核病的出現,令有效的控制結核病更加棘手。 / 在本研究中,我們首先用利福平誘導得到五株伴有明顯生長緩慢的高水平利褔卒耐藥的恥垢分支桿菌。通過比較基因組學研究發現,在編碼區有四個突變,其中兩個位於中rpoB 基因(N484T and 1488F) ,一個位於MSMEG_0436 (V49M) ,一個位於MSMEG_6872 (V181L)。rpoB 基因突變是該恥垢分支桿菌利福平耐藥的主要原因。而生長緩慢主要源於MSMEG_6872基因的影響。更為有趣的是,我們發現一個與MSMEG_6872具有相同的蛋白模序的結核分支桿菌蛋白質Rv1367 在不間的結核分支桿菌菌株之間存在I193V 多態性。193V 主要存在于北京株或者在耐藥的非北京株上。進一步的研究發現,過量表達MSMEG_6872或者Rv1367c 的恥垢分支桿菌形態上呈現為細長棒狀,而他們的親代則為短棒狀。 / 為獲得耐藥性,以及在高濃度的抗生素環境下生存,細菌必須付出一定的生物學代價。本研究中,恥垢分支桿菌以生長缺陷為代價獲得了對利褔平的耐藥,而這個代價可能是由於MSMEG_6872 基因的突變或者過量表達打破了細胞壁延長和分裂的平衡引起。 / Mycobacterium tuberculosis (MTB), which is the pathogen of tuberculosis (TB), remains a major human public health problem throughout the world. According to the report from the World Health Organization, currently about one third of the world's population was infected by MTB and there is globally 9.15 million recorded cases of TB annually. The occurrence of resistance to drugs used to combat TB, particularly multi-drug resistant TB (MDR-TB), defined as resistance to at least isoniazid and rifampin (RIF), has become a significant public health problem in a number of countries and an obstacle to effective global TB control. / In this project, we firstly obtained high level RIF resistant Mycobacterium smegmatis (MSM) strains with obviously growth retardation by repeated drug selection. Comparative analysis of genomic sequences revealed 4 mutations in coding sequences, including two in rpoB (N484T and I488F), one in MSMEG 0436 (y 49M), and one in MSMEG 6872 (y181L). Characterization of these four mutations showed that the two mutations in rpoB were correlated to RIF resistance. The one in MSMEG_6872 can render obviously growth retardation when MSMEG_6872 is over-expressed. Interestingly, we found an MTB protein, Rv1367c, which has the same motif with MSMEG_6872, had an I193V polymorphism in different MTB strains. The 193V variant was mainly found in Beijing/W or drug resistant non-Beijing/W family strains. The transformants, no matter MSMEG_6872 or Rv 1367 c, all exhibited slim and long rod shape compared to stocky and short rod appearance of the parental strain. / Mycobacterial cells must pay biological cost in order to obtain RIF resistance and survive in the high concentration of RIF. In our case, the growth arrest may be due to the mutation of MSMEG_6872 which disrupts the balance of cell wall elongation and cell division. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Guan, Bing. / "November 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 139-143). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.I / Abstract --- p.II / Abstract in Chinese --- p.IV / List of Abbreviations --- p.V / List of Tables --- p.VI / List of Figures --- p.VII / Contents --- p.IX / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Overview of Tuberculosis --- p.1 / Chapter 1.1.1 --- Pathogens --- p.2 / Chapter 1.1.2 --- Syndromes --- p.2 / Chapter 1.1.3 --- Transmission --- p.3 / Chapter 1.1.4 --- Diagnosis --- p.4 / Chapter 1.1.5 --- Epidemiology --- p.6 / Chapter 1.1.6 --- Mortality --- p.8 / Chapter 1.2 --- The Anti-TB Strategies --- p.8 / Chapter 1.2.1 --- Chemotherapy Treatment for MTB --- p.8 / Chapter 1.2.2 --- Vaccine Development for MTB --- p.9 / Chapter 1.3 --- Genome Sequencing of MTB Isolates --- p.9 / Chapter 1.4 --- Drug Resistance of MTB --- p.13 / Chapter 1.4.1 --- MDR-TB and XDR-TB --- p.15 / Chapter 1.4.2 --- Mechanism of Drug Resistance --- p.18 / Chapter 1.4.2.1 --- Intrinsic Resistance of Mycobacterium Species --- p.20 / Chapter 1.4.2.2 --- Acquired Resistance of Mycobacterium Species --- p.22 / Chapter 1.4.3 --- RIF Resistant MTB --- p.24 / Chapter 1.5 --- Useful tool for MTB Research --- p.26 / Chapter 1.6 --- The Biological Cost of Antibiotic Resistance in MTB --- p.27 / Chapter 1.6.1 --- The meaning of Biological Cost --- p.27 / Chapter 1.6.2 --- Factors Involved in Biological Cost of Mycobacterium Species --- p.29 / Chapter 1.17 --- Objectives of the Project and Experimental Strategies --- p.30 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Selection of RIF Resistant MSM mc²155 Strains --- p.31 / Chapter 2.1.1 --- Bacterial Strains, Media, and Growth Conditions --- p.31 / Chapter 2.1.2 --- Selection of RIF Resistant Strain --- p.31 / Chapter 2.2 --- Minimum-Inhibitory-Concentration (MIC) Assay --- p.34 / Chapter 2.3 --- Detection of Mutations in the rpoB Gene of RIF Resistance Strains --- p.36 / Chapter 2.3.1 --- Primers Design --- p.36 / Chapter 2.3.2 --- PCR and Direct Sequencing --- p.36 / Chapter 2.4 --- Characterization of the RpoB Gene --- p.38 / Chapter 2.4.1 --- Construction of Recombinant Clones --- p.38 / Chapter 2.4.2 --- Preparation of MSM competent cell. --- p.38 / Chapter 2.4.3 --- Electroporation of plasmid into MSM competent cells --- p.39 / Chapter 2.4.4 --- Site-directed Mutagenesis of the RpoB Clone --- p.39 / Chapter 2.5 --- Whole Genome Sequencing of Parental and Drug --- p.43 / Chapter 2.5.1 --- MSM Genomic DNA Extraction --- p.43 / Chapter 2.5.2 --- Genomic Sequencing --- p.44 / Chapter 2.5.3 --- Data Analysis and SNPs Identification --- p.45 / Chapter 2.6 --- Validation of Mutations by PCR and Direct Sequencing --- p.46 / Chapter 2.6.1 --- PCR Primers Design --- p.46 / Chapter 2.6.2 --- PCR and Direct Sequencing --- p.46 / Chapter 2.7 --- Characterization of MSMEG 0436 and MSMEG 6872 --- p.47 / Chapter 2.7.1 --- Construction of the recombinant clones --- p.47 / Chapter 2.8 --- Assay of Ethidium Bromide in Intact Cells --- p.48 / Chapter 2.9 --- Quantitative Real-time PCR to Expression of the Measure the ATP-binding Cassette (ABC) Superfamily Efflux Pumps --- p.49 / Chapter 2.9.1 --- RNA Extraction --- p.49 / Chapter 2.9.2 --- Synthesis of Double-stranded cDNA from Total RNA --- p.49 / Chapter 2.9.3 --- Real-time PCR to Quantify the Efflux Pump Gene Expression Level --- p.49 / Chapter 2.10 --- The construction of the Growth Curve --- p.53 / Chapter 2.11 --- Generation of ΔMSMEG_6872 Mutant Strain --- p.54 / Chapter 2.11.1 --- Preparation of Recombination Strain Stocks --- p.54 / Chapter 2.11.2 --- Preparation of the Electrocompetent Cells of the Recombination Strain --- p.54 / Chapter 2.11.3 --- Preparation of Allelic Exchange Substrate (AES) for Generating Gene Replacement Mutants --- p.55 / Chapter 2.12 --- Validation of Rv1367c (MT1414) in MTB --- p.60 / Chapter 2.12.1 --- Primer Design --- p.60 / Chapter 2.12.2 --- Strain Selection --- p.60 / Chapter 2.12.3 --- PCR and Direct Sequencing --- p.60 / Chapter 2.12.4 --- Alignment the Gene Sequence of Rv1367c of Different MTB Strains --- p.61 / Chapter 2.13 --- Model building of the RpoB protein --- p.62 / Chapter 2.14 --- MSM staining method --- p.63 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- dentification of RIF Resistant Related-genes Using Induced RIF Resistant MSM Model --- p.64 / Chapter 3.1.1 --- Emergence ofRIF Resistant Strains after the Prolonged Drug Exposure --- p.64 / Chapter 3.1.2 --- Induced RIF Resistance Were Stable In the Absence of Selection --- p.66 / Chapter 3.1.3 --- The Growth State of 5 RIF Resistance MSM mc²155 Strain --- p.68 / Chapter 3.1.4 --- Involvement of RpoB in the Mechanisms of the Emergence of RIF Resistance in MSM --- p.71 / Chapter 3.1.4.1 --- Mutations in the RpoB Gene --- p.71 / Chapter 3.1.4.2 --- Identical Mutations of RpoB Gene in Different RIF Resistance Isolates from Different Generation --- p.74 / Chapter 3.1.4.3 --- Characterization of RpoB in MSM strains --- p.76 / Chapter 3.1.4.4 --- Rifampin-Binding Pockets of RpoB Protein Model --- p.80 / Chapter 3.1.5 --- Other Genetic Alternations possibly Involved in RIF Resistance --- p.83 / Chapter 3.1.5.1 --- Whole Genome Sequencing of the Patental and P5 MSM mc²155 Strains --- p.83 / Chapter 3.1.5.2 --- Validation of the 32 Selected Alterations --- p.88 / Chapter 3.1.5.3 --- Characterization of MSMEG_0436 and MSMEG_6872 in RIF Resistance --- p.91 / Chapter 3.1.5.4 --- Characterization of MSMEG_0436 in the Growth Rate of MSM --- p.93 / Chapter 3.1.5.5 --- Characterization of MSMEG_6872 in the Growth Rate of MSM --- p.95 / Chapter 3.1.5.6 --- MSMEG_6872 Knock-out Strain Exhibited Normal Phenotype as its Parent --- p.98 / Chapter 3.1.5.7 --- Identification of Mutations in the Beta-Iactamase Gene of Mycobacterium Tuberculosis (MTB) --- p.101 / Chapter 3.1.5.8 --- Characterization of Rv 1367 c in Mycobacterium Growth Rate --- p.108 / Chapter 3.1.5.9 --- Morphology Changes of the Rv1367c and MSMEG_6872 Transformants --- p.110 / Chapter 3.2 --- Genetic Alterations in Non-coding Sequence --- p.112 / Chapter 3.2.1 --- ATP-binding Cassette (ABC) Superfamily Efflux Pumps Up-regulated in Drug Resistant M Smegmatis Strain --- p.112 / Chapter 3.2.2 --- RIF Resistant M smegmatis mc²155 Strain exhibited Low Level Cross-drug Resistance to INH --- p.115 / Chapter 3.2.3 --- RIF Resistant M smegmatis mc²155 Strain Showed Low level Accumulation of Ethidium Bromide --- p.117 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- The Protocol for the Preparation RIF Resistant Strains --- p.121 / Chapter 4.2 --- RIF Induced Stable Chromosomal Mutations in RIF Resistant MSM Strains --- p.123 / Chapter 4.3 --- MIC Levels of the RIF Resistant Strains --- p.125 / Chapter 4.4 --- Factors May involved in RIF Resistant MSM Strains --- p.128 / Chapter 4.5 --- Cell Shape and Growth Regulation --- p.129 / Chapter 4.6 --- MSMEG _6872 and Twin-Arginine Translocase (TAT) Secretion System --- p.135 / Chapter 4.7 --- Conclusion --- p.137 / Chapter 4.8 --- Future Perspectives --- p.138 / REFERENCES --- p.139
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Inativação térmica (75ºC) de Mycobacterium bovis (isolados de origem bovina) em leite integral experimentalmente inoculado / Thermal inactivation (75ºC) of Mycobacterium bovis (isolated from bovine) in whole milk experimentally contaminatedRibeiro, Leandro 11 December 2009 (has links)
A pasteurização do leite destinado ao consumo é obrigatória no Brasil e o sistema rápido (75ºC/15 a 20 segundos) é o mais empregado no país. O processo visa eliminar. Os parâmetros de tempo e temperatura empregados no mundo foram definidos após estudos sobre a resistência térmica do Mycobacterium tuberculosis e da Coxiella burnetti, reconhecidos como os microrganismos patogênicos, não formadores de esporos e que eventualmente podem estar presentes no leite cru, que apresentam a maior resistência térmica. Entretanto, não há estudos sobre a resistência térmica do M. bovis que circula nos bovinos no Brasil. Este estudo propôs-se a avaliar a resistência térmica (75ºC) de cinco espoligotipos de M. bovis, isolados de bovinos abatidos no estado de São Paulo, em leite integral experimentalmente contaminado. Leite UHT foi contaminado com M. bovis e, então, submetido a tratamento térmico em banho-maria a 75ºC por 20 segundos. Cada espoligotipo foi testado 3 vezes. As amostras foram retiradas do banho nos tempos 0 (o momento em que o leite atingiu 75ºC), 5, 10, 15 e 20, correspondendo ao tempo, em segundos, de tratamento térmico. O leite contaminado também foi analisado, para quantificação da carga inicial. O controle do processo envolveu o acompanhamento da temperatura do leite (um tubo com termômetro) e análise das enzimas fosfatase alcalina e peroxidase ao final do tratamento; para tal, amostras de leite cru foram tratadas juntamente com as amostras-teste. Para quantificação, foi realizada a diluição decimal seriada seguida da semeadura em duplicata em meio Stonebrink-Leslie (37ºC/45dias). Os resultados mostraram que foi na fase de aquecimento que ocorreu a maior taxa de morte de todos os espoligotipos. Houve diferença de resistência entre os espoligotipos ao processo que simulou a pasteurização rápida e o espoligotipo BR024 foi o mais resistente. Conclui-se que houve diferença da eficácia da pasteurização, de acordo com o espoligotipo testado, mas que os resultados precisam ser investigados mais detalhadamente. / The pasteurization of milk for consumption is mandatory in Brazil and fast system (75 ° C/15 to 20 seconds) is the most used around the country. The process aims to eliminate. The parameters of time and temperature were set in the world after studies on the thermal resistance of Mycobacterium tuberculosis and Coxiella burnetii, recognized as the pathogenic microorganisms, not spore-forming and eventually may be present in raw milk, with the strongest resistance heat. However, no studies on the thermal resistance of M. bovis circulating in cattle in Brazil. This study aimed to evaluate the thermal resistance (75º C) of five espoligotipos M. bovis isolated from cattle slaughtered in the state of Sao Paulo in experimentally infected whole milk. UHT milk was contaminated with M. bovis and then subjected to heat treatment in a water bath at 75 º C for 20 seconds. Each espoligotipo was tested 3 times. Samples were taken from the bath at 0 (the time when the milk reached 75 ° C), 5, 10, 15 and 20, corresponding to time, in seconds, the heat treatment. The contaminated milk was also analyzed to quantify the initial charge. The control process involves monitoring the temperature of the milk (a tube with a thermometer) and analysis of the enzymes alkaline phosphates and peroxidase the end of treatment, for such raw milk samples were treated with the test samples. For quantification, we performed a ten-fold dilution serial followed by seeding in duplicate in the middle Stonebrink-Leslie (37 C/45dias). The results showed that it was warming up that had the highest death rate of all espoligotipos. There were differences in resistance between espoligotipos the process that simulated pasteurization and rapid espoligotipo br024 was the toughest. It was concluded that there was no difference of the effectiveness of pasteurization, according to the espoligotipo tested, but the results need to be investigated further.
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Avaliação da virulência de estirpes de Mycobacterium avium presentes na população de suínos no sul do Brasil / Evaluation of Mycobacterium avium strains virulence from porcine population of south BrazilOliveira, Eugenia Márcia de Deus 14 December 2001 (has links)
Tendo sido comprovada a existência da famílias molecularmente distintas de M. avium circulando em suínos de região sul do Brasil, e havendo dúvidas a respeito da importância da transmissão horizontal como mecanismo de manutenção da doença, o presente teve por objetivo estudar a virulência dessas estirpes, informação importente para o aperfeiçoamento dos métodos de controle. As estirpes emergiram do estudo caso-controle, onde as tipagens moleculares por RFLP mostraram a existência de quatro famílias de M. avium (PIG-A, B, C e D). Um estirpe representante de cada família foi inoculada pela via intra-peritoneal em 48 hamsters com uma dose de 30.000 U.F.C. por animal. Após 2, 13, 26 e 40 dias da inoculação, 12 hamsters inoculados de cada família foram anestesiados, sacrificados e os agentes foram quantificados no fígado, baço e pulmão. A presença das estirpes foi verificada no sangue e também foram realizados exames histológicos. As estipers PIG-A, B, C e D desenvolveram lesões granulomatosas no fígado e baço nos quatro tempos experimentais; disseminaram-se pela via linfo-hemática, multiplicando-se em fígado, baço e pulmão. Nos quatro tempos experimentais houve diferença entre as contagens de U.F.C./g entre os órgãos (T1: p<0,001; T2: p<0,001; T3: p<0,001 e T4: p<0,001) e as obtidas do baço foram sempre superiores às do fígado e pulmão. Nos quatro tempos experimentais houve diferença entre as contagens de U.F.C./g entre as estirpes (T1: p<0,001; T2: <0,001; T3: p<0,001 e T4: p<0,001) e foi possível construir a seguinte escala decrescente de virulência: PIG-B > PIG-A > PIG-D > PIG-C. / Given that the existence of molecularly different families of M. avium circulating in swine of the south area of Brazil has been proved, and that some doubts remain regarding the importance of the horizontal transmission as mechanism of maintenance of the disease, this work aimed to study the virulence of those strains, an important information for the improvement of the control methods. The strains emerged from a case-control study, when the RFLP molecular typification showed the existence of four families of M. avium (PIG-A, B, C and D). A strain representative of each family was inoculated by intra-peritoneal route in 48 hamsters with a dose of 30.000 C.F.U./animal. After 2, 13, 26 and 40 days post-infection, 12 inoculated hamsters of each family were anesthetized, euthanized and the bacteria were quantified in the liver, spleen and lung. The presence of the strains was verified in the blood and also histological exams were accomplished. The strains PIG-A, B, C and D developed granulomatous lesions in the liver and spleen in the four experimental times; they were disseminated by the linfo-haematic route, multiplying in liver, spleen and lung. In the four experimental times there was difference among the countings of C.F.U./g among the organs (T1: p<0,001; T2: p<0,001; T3: p<0,001 and T4: p<0,001) and that obtained of the spleen were always superiors to the one of the liver and lung. In the four experimental times there was difference among the countings of C.F.U./g among the ancestries (T1: p<0,001; T2: p<0,001; T3: p<0,001 and T4: p<0,001) and was possible to build the following order of decreasing virulence: PIG-B > PIG-A > PIG-D > PIG-C.
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Expressão de antígenos de Schistosoma mansoni em BCG recombinante. / Expression of Schistosoma mansoni antigens in recombinant BCG.Kanno, Alex Issamu 18 October 2013 (has links)
O BCG é um bacilo atenuado de Mycobacterium bovis atualmente investigado na expressão de antígenos heterólogos. Genes oriundos de vírus, bactérias e parasitas são clonados em vetores de expressão micobacterianos e a partir deles, são geradas cepas de BCG recombinante, rBCG. Duas cepas micobacterianas, BCG e M. smegmatis, demonstraram a expressão do antígeno SmStoLP-2 e animais imunizados com este BCG não demonstra diferença na proteção contra a infecção por cercárias, apesar da resposta imune diferenciada. A otimização do códon de SmTSP-2 e SmVAL-5 permitiu sua expressão em M. smegmatis recombinante, mas não em BCG. M. smegmatis transformado com uma biblioteca de plasmídeos contendo o promotor L5p mutagenizado à montante de egfp demonstra acentuada diferença na fluorescência. 3 destes plasmídeos definidos como \'\'forte\'\' e \'\'fraco\'\', com base em sua capacidade de produzir GFP foram utilizados para expressar em BCG o antígeno Sm29 em maior e menor nível. / BCG is an attenuated bacillus derived from Mycobacterium bovis currently being investigated to express foreign antigens. Genes from viruses, bacteria and parasites are cloned to mycobacterial vectors and with them, recombinant BCG strains are created. Recombinant BCG and M. smegmatis strains where capable to express SmStoLP-2 and mice immunized with these rBCGs do not show better protection against infection with cercariae, although the distinct immune response observed. The use of codon optimization made possible the expression of SmTSP-2 and SmVAL-5 in M. smegmatis but not BCG. M. smegmatis transformed with a library of plasmids containing the L5p upstream egfp gene show a wide range in fluorescence. 3 of these plasmids strong and weak defined as their ability to produce GFP were used to express in BCG the antigen Sm29 at different levels by each promoter.
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Utilização de uma técnica rápida para o isolamento de Mycobacterium bovis a partir de amostras de leite experimentalmente inoculadas / Utilization of the fast technique of isolation of Mycobacterium bovis from milk samples experimentally inoculatedDib, Cristina Corsi 28 November 2005 (has links)
A técnica de camada delgada em placas com meio de Middlebrook 7H11 foi comparada com a técnica padrão de cultura em meio de Stonebrink, a fim de se avaliar a sensibilidade e o tempo de detecção de micobactérias em amostras de leite, experimentalmente inoculadas com Mycobacterium bovis (estirpe AN5), em uma diluição 10-2, e submetidas a duas diferentes técnicas de processamento, utilizando-se da gordura (técnica 1)e do sedimento (técnica 2), descontaminadas pelo método de Petroff modificado (adicionado de Tween 80) e confrontadas com a técnica do leite total submetida ao método de Petroff e semeadas nos dois meios. Os resultados destas técnicas (1 e 2) foram comparados entre si pelo teste não paramétrico de Wilcoxon, e entre os resultados obtidos na técnica de leite total submetida ao método tradicional de Petroff, por meio do teste não paramétrico de Mann-Whitney. Posteriormente, amostras de leite nas diluições 10-3, 10-4 e 10-5 foram então submetidas às mesmas técnicas de processamento e descontaminação para averiguação de sensibilidade. Os resultados obtidos demonstraram que: 1) a técnica de cultivo de micobactérias em amostras de leite no meio de Middlebrook 7H11 se mostrou viável frente à tradicional; 2) a técnica de camada delgada permitiu a visualização precoce das micobactérias quando comparadas ao meio de Stonebrink; 3) as técnica 1 e 2 forneceram maior recuperação de micobactérias e maior proporção de cultivos positivos nos dois empregados; 4) a técnica de camada delgada em meio de Middlebrook 7H11 modificado pode ser usada como uma técnica complementar aos métodos tradicionais de diagnóstico da tuberculose bovina em amostras de leite para fins de vigilância epidemiológica. / The modified thin layer Middlebrook 7H11 cultivation technique was compared to the tradicional culture technique using the Stonebrink in order to evaluate the sensibility and the time for the detection of positive cultures of mycobacteria in milk samples, experimentally inoculated by Mycobacterium bovis (strain AN5), 10-2 dilution, were submitted by two types of procedures named technique 1 (milk fat) and 2 (milk sediment), descontamined by modified Petroff method, added with Tween 80, and compared with total milk samples submitted to tradicional Petroff method, in the same media types. The results of the two tecniques were compared within each other by the non-parametric Wilcoxon test, and within the results of standard milk submitted by the tradicional Petroff method, by the non-parametric Mann-Whitney. Milk samples were diluted to 10-3, 10-4 and 10-5 and submitted by the tecniques 1 and 2 descontaminated by the modified Petroff method, and total milk by tradicional Petroff method to averiguate the sensibility. The results found in this experiment demonstrated that 1) the modified thin layer technique of isolation of mycobacteria in milk samples was practicable against the tradicional one; 2) the time needed for the detection of mycobacteria colonies was slightly less with the thin layer technique than the tradicional culture method; 3) techniques 1 and 2 gave more number of colonies and more proportion of positive culture than the tradicional one in all media used; 4) this technique should be used as a complementary method to the tradicional one in the diagnostic of bovine tuberculosis from milk samples in orther to improve epidemiologic vigilance.
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Genetic and Biochemical Insights into the Mycobacterial PrrAB System as a Regulator of Respiration and Central MetabolismJanuary 2019 (has links)
abstract: Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the 10th leading cause of death, worldwide. The prevalence of drug-resistant clinical isolates and the paucity of newly-approved antituberculosis drugs impedes the successful eradication of Mtb. Bacteria commonly use two-component systems (TCS) to sense their environment and genetically modulate adaptive responses. The prrAB TCS is essential in Mtb, thus representing an auspicious drug target; however, the inability to generate an Mtb ΔprrAB mutant complicates investigating how this TCS contributes to pathogenesis. Mycobacterium smegmatis, a commonly used M. tuberculosis genetic surrogate was used here. This work shows that prrAB is not essential in M. smegmatis. During ammonium stress, the ΔprrAB mutant excessively accumulates triacylglycerol lipids, a phenotype associated with M. tuberculosis dormancy and chronic infection. Additionally, triacylglycerol biosynthetic genes were induced in the ΔprrAB mutant relative to the wild-type and complementation strains during ammonium stress. Next, RNA-seq was used to define the M. smegmatis PrrAB regulon. PrrAB regulates genes participating in respiration, metabolism, redox balance, and oxidative phosphorylation. The M. smegmatis ΔprrAB mutant is compromised for growth under hypoxia, is hypersensitive to cyanide, and fails to induce high-affinity respiratory genes during hypoxia. Furthermore, PrrAB positively regulates the hypoxia-responsive dosR TCS response regulator, potentially explaining the hypoxia-mediated growth defects in the ΔprrAB mutant. Despite inducing genes encoding the F1F0 ATP synthase, the ΔprrAB mutant accumulates significantly less ATP during aerobic, exponential growth compared to the wild-type and complementation strains. Finally, the M. smegmatis ΔprrAB mutant exhibited growth impairment in media containing gluconeogenic carbon sources. M. tuberculosis mutants unable to utilize these substrates fail to establish chronic infection, suggesting that PrrAB may regulate Mtb central carbon metabolism in response to chronic infection. In conclusion, 1) prrAB is not universally essential in mycobacteria; 2) M. smegmatis PrrAB regulates genetic responsiveness to nutrient and oxygen stress; and 3) PrrAB may provide feed-forward control of the DosRS TCS and dormancy phenotypes. The data generated in these studies provide insight into the mycobacterial PrrAB TCS transcriptional regulon, PrrAB essentiality in Mtb, and how PrrAB may mediate stresses encountered by Mtb during the transition to chronic infection. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2019
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Rôle et mécanismes moléculaires d'action des lipides de l'enveloppe de Mycobacterium tuberculosis dans la virulence / Role ans molecular mechanisms of action of Mycobacterium tuberculosis cell envelope lipids in virulenceAugenstreich, Jacques 14 September 2018 (has links)
Mycobacterium tuberculosis est la bactérie responsable de la tuberculose (TB), une infection pulmonaire grave. La TB est un problème majeur de santé publique mondial ; un tiers de la population mondiale est porteur de M. tuberculosis et l'émergence de formes de TB résistantes aux antibiotiques confirme la nécessité de développer de nouvelles approches thérapeutiques. Pour atteindre cet objectif, il est crucial de mieux comprendre les mécanismes infectieux de M. tuberculosis. L'équipe de C. Guilhot s'intéresse aux lipides de l'enveloppe bactérienne impliqués dans la virulence de M. tuberculosis, en particulier lors de l'interaction du bacille avec les macrophages de l'hôte. Un type de lipide en particulier s'est démarqué pour son rôle crucial dans cette interaction : les Dimycocérosates de Phthiocérol (DIM). Malgré leur importance, le mécanisme d'action moléculaire des DIM n'est toujours pas connu. Les objectifs de ma thèse ont été 1) d'étudier le rôle des DIM dans le trafic intracellulaire de M. tuberculosis au sein du macrophage, et 2) de comprendre les mécanismes d'action des DIM dans la virulence. Par une combinaison de méthodes biologiques et biophysiques, nous avons montré que les DIM contribuent à l'induction de la rupture du phagosome et de l'apoptose du macrophage infecté par M. tuberculosis, en collaboration avec un autre facteur majeur de virulence bactérien, ESAT-6. Au niveau moléculaire, nous avons confirmé que les DIM sont transférés dans la membrane du macrophage au contact avec la bactérie, et y induisent une rigidification membranaire locale. Nous avons aussi montré que les DIM dans une membrane sont capables de potentialiser l'activité membranolytique d'ESAT-6 et d'autre(s) facteur(s) encore non identifiés. Les DIM ont un rôle pléiotropique dans l'interaction de M. tuberculosis avec le macrophage. Leur mécanisme d'action impliquerait des modifications des propriétés biophysiques membranaires, modifiant l'activité des effecteurs membranaires bactériens et potentiellement de l'hôte. Ces travaux ouvrent la voie à l'étude du mécanisme d'action d'autres lipides de virulence dont certains pourraient également s'insérer dans la membrane du macrophage. / Mycobacterium tuberculosis is the bacterium responsible for tuberculosis (TB), a severe respiratory disease. TB is a major public health threat; one third of the world's population is latently infected by M. tuberculosis, and the emergence of antibiotic-resistant forms of TB confirms that there is a need to develop new therapeutic approaches to control the spread of TB. However, in order to attain that goal, it is crucial to decipher the infectious mechanisms of M. tuberculosis. Research in the team of C. Guilhot focuses on lipids from the envelope of M. tuberculosis which are involved in virulence, in particular those implicated in the interaction of the bacteria with host macrophages. One of these lipids stands out for its crucial role in this interaction: Phthiocerol Dimycocerosate (DIM). Despite its importance, the molecular mechanism of action of DIM is still unknown. The objectives of my PhD were 1) to study the role of DIM in the intracellular trafficking of M. tuberculosis in macrophages, and 2) to decipher the molecular mechanism of action of DIM. By a combination of biological and biophysical techniques, we showed that DIM contributes to phagosomal rupture and induction of apoptosis in macrophages infected with M. tuberculosis, in collaboration with another major virulence factor: ESAT-6. At the molecular level, we confirmed that DIM is transferred to the membrane of the macrophage on contact with M. tuberculosis and induces a local membrane rigidification around the point of contact with the bacterium. We observed that DIM incorporated in membranes is able to promote the membranolytic activity of ESAT-6, and other yet unidentified bacterial factor(s). DIM has a pleiotropic role in the interaction between M. tuberculosis and macrophages, presumably through alterations of the membrane's biophysical properties that influence the activity of membrane effectors from both the bacteria and the host. Thus, this work paves the way for the study of the mechanisms of action of other virulence lipids, some of which could also be inserted in the macrophage membrane.
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Surface-exposed proteins in the pathogenesis of Mycobacterium avium subsp. hominissuisMcNamara, Michael J. 14 March 2012 (has links)
Mycobacterium avium subsp. hominissuis (MAH) is a pervasive environmental bacterium that can cause opportunistic infections in humans. Among the most robust and hardy members of the Mycobacterium genus, M. avium can persist and thrive in a range of challenging environments, including many which place it in direct contact with humans. Surface-exposed proteins are central to the bacterial processes involved in both environmental persistence and pathogenesis. These proteins also play a critical role in how the immune system of the host recognizes and responds to pathogens. Mycobacteria have evolved a specialized mechanism for protein export, a Type VII Secretion System (T7SS), in order to transport their proteins through their thick and impermeable cell envelope. This system is responsible for the export of several classes of proteins, many of which play an integral role in virulence. A central focus of this dissertation is the characterization of a conserved element of the T7SSs in pathogenic mycobacteria, a PPE family protein, whose deletion attenuates virulence in M. avium. Specifically, we examined the localization of this PPE protein (MAV_2928) within the bacterium, screened potential protein-protein interactions with other conserved elements in the adjacent T7SS loci and analyzed the transcriptional regulation of the gene in response to environmental changes.
Seeking to more thoroughly characterize the surface-exposed proteome of M. avium, particularly in the context of early infection, we then developed a method, based on selective biotinylation and affinity purification, to profile the of surface-exposed proteome of the bacterium. We employed this method to analyze the surface-exposed proteomes of M. avium 109 that had been exposed to macrophages to those of M. avium 109 that had been cultured in media. This comparison detected several proteins whose presence at the bacterial surface appeared to be dependent on particular growth conditions. Lastly, in order to establish a more efficient method to isolate biotinylated surface proteins from complex mixtures, we developed a testing paradigm to identify modifications to the original method that might improve our coverage of identified proteins. Through this process, we developed a more robust methodology that yielded improved coverage and depth. We then utilized this technology to profile the surface-exposed proteome of another clinical isolate of M. avium subsp. hominissuis, M. avium 104. Beyond improving our understanding of the basic biology of M. avium, this new data provides independent evidence that PPE family proteins are indeed exported to the surface of M. avium, where they remain associated with the bacterial cell envelope. In total, this analysis represents the most comprehensive profile of the surface-exposed proteins of M. avium generated to date. / Graduation date: 2012
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Tuberculosis in South American sea lions (Otaria flavescens) - diagnostic options and its epidemiologic importance for other mammals within the zoological gardenJurczynski, Kerstin 19 November 2012 (has links) (PDF)
Tuberculosis is a widely spread zoonotic disease caused by acid-fast bacteria of the Mycobacterium tuberculosis complex in a variety of mammalian species. In pinnipeds, tuberculosis has been reported in different captive and wild sea lions and fur seals.
The causative agent, Mycobacterium pinnipedii, is part of the M. tuberculosis complex and has shown pathogenicity in other mammalian species including human beings.
Since 2000 the Heidelberg zoo has been dealing with tuberculosis in its collection of South American sea lions (Otaria flavescens). After a Malayan tapir (Tapirus indicus) was transferred to a zoological institution in France it transmitted the disease to the other tapirs that succumbed to tuberculosis. Culturing and spoligotyping confirmed the origin, the sea lions at the Heidelberg zoo.
An investigation of the sea lion group housed at Heidelberg in addition to different species of mammals living in adjacent exhibits as well as a sea lion, born in Heidelberg but then living in Hamburg, revealed multiple cases of pinniped tuberculosis.
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Transcriptional Analysis Of The Principal Cell Division Gene ftsZ Of Mycobacterium Tuberculosis And Mycobacterium SmegmatisRoy, Sougata 06 1900 (has links)
The success of Mycobacterium tuberculosis as a pathogen is due to its remarkable
ability to: (i). adapt to and survive inside activated macrophages under nonproliferating condition, (ii). put up drug resistance and (iii). enter into hypoxia-induced dormancy and remain in nonproliferating condition, be resistant to drugs, and get reactivated into
proliferation when favourable conditions arise. Thus, regulation of cell division (arrest and resumption) is an obligatory event that is critical to the pathogen for the establishment of successful infection, latency and reactivation process in human host. Therefore, in order to understand and combat the successful survival strategy of the bacterium inside the host macrophages or in granuloma, a basic knowledge of the
regulation of cell division in tubercle bacillus is essential. Bacterial cytokinetic protein FtsZ (a tubulin homologue) is the key regulatory molecule for cell division and its
intracellular level is critical for initiation of cell division in bacteria. Therefore, in order to understand the regulation cell division by expression and maintenance of ftsZ mRNA and protein, we initiated studies on the transcriptional regulation of ftsZ gene in the slow growing pathogen, M. tuberculosis, and in the fast-growing saprophyte M. smegmatis.
Identification of regions containing ftsZMt promoter activity
In order to identify promoter activity-containing regions of ftsZ gene of M. tuberculosis H37Rv (ftsZMt) in vivo, different regions upstream of ftsZMt namely, the ftsQ-ftsZ intergenic region, the ftsQ open reading frame (ORF), and different regions of ftsQ ORF, were cloned in a gfp reporter plasmid and analyzed for gfp expression in M. smegmatis mc2155 cells. Flow cytometric analysis of exponentially grown M. smegmatis mc2155 cells containing these transcription fusion constructs revealed GFP expression in the cells harbouring ftsQ-ftsZ intergenic region (172 bp), the entire ftsQ ORF (945 bp), and 5’ 467 bp and 3’ 217 bp regions of ftsQ ORF. RT-PCR analyses on RNA from M. smegmatis mc2155 cell transformants carrying the entire ftsQ ORF-ftsQ-ftsZ intergenic region containing construct, as well as on total RNA from M. tuberculosis confirmed that the
regions identified indeed elicit promoter activity. RT-PCR analysis on M. tuberculosis RNA as well as semi-quantitative RT-PCR analyses of gfp transcripts driven by cloned MtftsZ promoter regions in M. smegmatis cells showed that about 70% of the total
promoter activity comes from ftsQ ORF and there is co-transcription of ftsQ-ftsZ genes.
Multiple transcripts code for ftsZMt
Primer extension analysis, using primers annealing at different positions in the
ftsQ-ftsZ chromosomal region, on RNA from M. tuberculosis as well as from M.
smegmatis transformants containing 1.117 kb ftsZMtpromoter region in a promoter probe
vector, identified origin of six different transcripts (T1-T6) for the gene. Among them, five transcripts (T1, T2, T3, T4, and T6) were detected in M. tuberculosis cells at exponential phase of growth. T5 could be detected only in M. smegmatis transformants containing 1.117 kb ftsZMt promoter upstream of mycgfp2+ reporter gene. Transcript T1 and T2 originate in the ftsQ-ftsZ intergenic region, while T3, T4, and T6 start in the ftsQ ORF. Analysis of sequence in the –10 and –35 regions of the corresponding promoters for the individual transcripts identified high GC content of the regions, which is characteristic of promoters of M. tuberculosis. All of the individual promoter sequences were independently cloned in a promoter probe vector and confirmed that they are true
promoters, active in M. smegmatis cells, and that the T1-T6 transcripts were not products
of RNA processing.
Differential expression from the multiple ftsZMt promoters
In order to study the activity and regulation of ftsZMt promoters in M. tuberculosis cells, which is a slow grower and also asymptomatically survives as dormant bacteria for decades in human granuloma, a stably genome-integrated plasmid was required where activity of the promoters can be studied by means of stable and enhanced gfp expression. For that purpose, an L5-mycobacteriophage attP (attachment site)-specific integration
proficient promoter probe vector, which contains a stable gfp gene (mycgfp2+) whose codon has been optimized for mycobacterial expression, was generated. Using the vector, all the six promoter regions (P1-P6) were studied in M. smegmatis and M. tuberculosis cells. Flow cytometric and semi-quantitative RT-PCR analyses showed that promoter P5 is unable to elicit activity in M. tuberculosis cells, unlike in M. smegmatis transformants.
Semi-quantitative RT-PCR analyses showed that expression of P3 is only 10-20% of the
total promoter activity. Promoters P1, P2, P4 and P6 showed 50-80% activity of the total promoter activity and their activity were comparable in M. smegmatis and M. tuberculosis.
The presence of multiple promoters reflects the requirement to maintain high
basal level of, or to differentially regulate a critical level of, FtsZ expression during different pathogenic stages of tubercle bacilli. In order to investigate the role of multiple
promoters, we verified the levels of expression of the five transcripts from the five ftsZ promoters in M. tuberculosis cells under conditions of growth inside mouse macrophage cell line and also under various stress conditions mimicking those that exist in the granuloma environment, like conditions of nonreplicating persistence, gradual nutrient depletion stress, oxidative stress, surface tension stress, acidic stress, heat shock, DNA damaging conditions and osmotic stress. For this purpose, individual promoter regions
were cloned into a stably inheritable gfp reporter plasmid vector, and into an L5
mycobacteriophage attP (attachment site)-specific integration-proficient variant of the same vector, for the expression of the promoters from the chromosomal locus in M. smegmatis and M. tuberculosis cells. Quantitative primer extension analyses, semiquantitative RT-PCR analyses on RNA from M. tuberculosis cells grown under these
different conditions, and quantitative GFP fluorescence analyses in these cells showed differential activation of the five promoters under different conditions of growth. Under hypoxic and nutrient-depleted stationary phase of growth, two new promoters, Tdor and Ts, in the ftsQ ORF were identified, and these promoters showed maximal activity only under those specific conditions of growth. None of the ftsZ promoters were found to be responsive to stringent response mediated by overexpression of M. tuberculosis RelA.
None of the promoters were also found to be responsive of overexpression of heat-shock
sigma factor SigH in M. tuberculosis, implicating new pathway of regulation of ftsZ promoters.
Multiple promoters driving expression of ftsZ gene of M. smegmatis
Similar studies, which were carried out on the identification, structural and
functional characterization, regulation of the promoters of cell division gene ftsZ in the fast growing saprophyte M. smegmatis cells, showed the presence of four ftsZ promoters, three of which originates from the 249 bp ftsQ-ftsZ intergenic region and one from the ftsQ ORF. RT-PCR analysis showed that both ftsQ and ftsZ are co-transcribed. Cloning and expression analysis of the individual promoters mapped by primer extension in a GFP based reporter plasmid showed that all the four putative regions are true promoters.
Quantitative primer extension on RNA from a synchronously grown culture identified P2
promoter to be responsive to either initiation of cell division or stress, although expression of P1, P3, and P4 did not vary with respect to synchronous division. Quantitative primer extension analysis and semi-quantitative RT-PCR analysis on the RNA from M. smegmatis cells showed that under various stress conditions, P2 activity goes down significantly. Under nutrient depleted stationary phase and hypoxic nonreplicating persistence stage-2, the levels of P2 and P3 activity could hardly be detected, whereas, expression from P1 and P4 goes down only in hypoxia. Level of total ftsZ mRNA remains almost the same under various stress conditions, although upon hypoxia and stationary phase the level goes down almost two fold. Thus, in fast growing
M. smegmatis too, multiple ftsZ promoters are differentially regulated under various stress conditions and a critical level of ftsZ mRNA is maintained.
Taken together, the study of ftsZ promoters of a slow-growing pathogenic
mycobacterium and a fast growing non-pathogenic mycobacterium indicate that
differential expression of the multiple promoters, along with conditional activation of stage specific promoters like Pdor or Ps, is one of the mechanisms through which the bacilli probably maintain required levels of FtsZ protein that are crucial for the cell survival, probably through cytoskeletal maintenance, and cell division.
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