Tuberculosis is a re-emerging global health problem. Bacille Calmette-Guerin (BCG), the available vaccine against the disease, is only effective short term and is associated with adverse reactions clinically. The development of new effective vaccines will require an understanding of virulence, immunogenic factors and the beneficial immune responses induced in the human host. My thesis investigates phoP and whiB3, two genes associated with virulence and immunogenicity in Mycobacterium tuberculosis. Study of PhoP in a natural phoP mutant, BCG-Prague, and in the clinically safe BCG-Japan, shows that over-expression of PhoP increases the immunogenicity of these vaccine strains. In addition, I found that WhiB3 impacts carbon metabolism in BCG-Birkhaug and BCG-Sweden, although the effect of this on virulence in vivo is still unclear. The characterization of genes involved in virulence and immunogenicity allows us to develop novel approaches for improving the efficacy of BCG, which has important implications for future TB vaccine development.

Tuberculosis is a re-emerging global health problem. Bacille Calmette-Guerin (BCG), the available vaccine against the disease, is only effective short term and is associated with adverse reactions clinically. The development of new effective vaccines will require an understanding of virulence, immunogenic factors and the beneficial immune responses induced in the human host. My thesis investigates phoP and whiB3, two genes associated with virulence and immunogenicity in Mycobacterium tuberculosis. Study of PhoP in a natural phoP mutant, BCG-Prague, and in the clinically safe BCG-Japan, shows that over-expression of PhoP increases the immunogenicity of these vaccine strains. In addition, I found that WhiB3 impacts carbon metabolism in BCG-Birkhaug and BCG-Sweden, although the effect of this on virulence in vivo is still unclear. The characterization of genes involved in virulence and immunogenicity allows us to develop novel approaches for improving the efficacy of BCG, which has important implications for future TB vaccine development.

Creation of a reliable and easy to use serologic test would greatly improve ante mortem diagnosis of Mycobacterium avium subsp. avium and aid in the control of avian mycobacteriosis, particularly in captive birds. In order to determine whether serodiagnostics could be of value in testing ring-neck doves (Streptopelia risoria) for M. a. avium infection, Western blot analysis was used to assess the humoral response of ring-neck doves exposed to M. a. avium, and to evaluate whether an association could be made between humoral response and necropsy findings, histopathology, culture, and PCR testing. Western blot results were examined for reactivity patterns associating the humoral response with infection status, severity and type of lesions (diffuse vs multifocal granulomatous inflammation) and phenotype (white vs non-white). A sensitivity of 88.24% and a specificity of 100% were achieved utilizing Western blot analysis to detect M. a. avium infection in ring-neck doves, offering a negative predictive value of 93% and a positive predictive value of 100%. While Western blot analysis results did not reflect lesion severity, lesion type did partially correspond with the humoral response. The findings of the present study indicate that serologic testing can be used as a valuable ante mortem screening tool for identifying ring-neck doves infected with M. a. avium.

Tuberculosis is the second leading cause of morbidity and mortality worldwide due to an infectious disease. Development of a new tuberculosis (TB) vaccine would be facilitated by a better understanding of the mechanisms of protection induced by the current TB vaccine, Mycobacterium bovis BCG. Recombinant guinea pig (rgp)CCL5 and anti-rgpCCL5 were developed and characterized. The biological activity of rgpCCL5 was determined in a chemotaxis assay using T lymphocytes and pleural exudate cells. The specificity of rabbit anti-rgpCCL5 polyclonal antibody was confirmed by Western blot. RgpCCL5 was used to stimulate alveolar and peritoneal macrophages in vitro. and cytokine/chemokine gene expression was evaluated using real-time PCR. RgpCCL5 stimulated TNFα, IL-1β, CCL2, and CXCL8 mRNA expression and TNFα protein production (as assessed in the L929 cell bioassay) in macrophages. The effect of BCG-vaccination on CCL5 expression and production in leukocytes infected with M. tuberculosis was examined in vitro and in vivo. Polyclonal anti-rgpCCL5 was used to develop an ELISA assay to quantify gpCCL5 protein levels, and real-time PCR was used to detect CCL5 mRNA. Leukocytes isolated from BCG-vaccinated guinea pigs and infected in vitro with virulent M. tuberculosis demonstrated significantly elevated gpCCL5 mRNA and protein compared to cells from naive animals. The response of gpCCL5 to M. tuberculosis in vivo was studied in tuberculous pleural effusions, where peak levels of CCL5 mRNA and protein were reached at day 4 post-induction. Disease severity, cellular differentiation, histology, and cytokine/chemokine mRNA levels in pleural cells and granulomas were analyzed on day 4 in guinea pigs induced with tuberculous pleurisy and treated with either rgpCCL5 or anti-rgpCCL5 by direct intra-pleural injection. In these studies, neutralizing CCL5 resulted in reduced macrophage accumulation, diminished levels of IFNγ, TNFα, and CCL5 mRNA in pleural effusion cells, and reduced spontaneous lymphocyte proliferation. Together these studies suggest an important role for gpCCL5 in activating leukocytes during M. tuberculosis infection.

Mobile inteins and introns are genetic elements capable of self-propagation by “homing” into host genes and occur in entire taxonomy: eubacteria, eukarya, archaea and viruses. The process of “homing” is promoted by an endonuclease encoded by the open reading frame (ORF) embedded within the genetic element. Homing endonucleases are encoded by group I and group II introns, archaeal introns, inteins, and free standing ORFs. They are believed to play a central role in rearrangement of organelle as well as nuclear genomes. Inteins are genetic elements present within protein-coding genes with dual function: protein-splicing and homing endonuclease activities. One hallmark of homing endonucleases is their ability to recognize and cleave extended degenerate asymmetric sequences (14 - 40 bp) in intein- or intron-less alleles. Homing endonucleases are classified into four families based on the presence of LAGLIDADG, GIY-YIG, His-Cys box, or H-N-H conserved motifs. Among these, LAGLIDADG family is the largest, widespread and well-studied class. Structural and biochemical studies have demonstrated that homing endonucleases with one LAGLIDADG motif act as homodimers, whereas enzymes with two such motifs function as monomers during catalysis. In vitro, these enzymes are extremely specific for their recognition sites and they prefer Mg2+ as the metal-ion cofactor. Unlike Escherichia coli, recA of Mycobacterium tuberculosis and Mycobacterium leprae contain in-frame insertion of an intein-coding sequence. In addition to recA,scrutiny of M. tuberculosis genome revealed that intein-coding sequences are present in the ORFs of dnaA and Rv1461 (pps1). M. tuberculosis recA encodes a 85 kDa precursor protein. Amino acid sequence comparison between M. tuberculosis RecA precursor and the prototype E. coli RecA displayed high degree of homology at the amino-terminal (1 -254 amino acid residues) and carboxyl-terminal (694 - 790 amino acid residues)domains of the RecA precursor. The central domain comprising 440 amino acid residues showed significant homology to the members of the LAGLIDADG super-family of intein homing endonucleases. Following the synthesis of precursor protein, RecA intein and active RecA are generated by protein splicing reaction. The protein splicing reaction of RecA intein has been studied extensively; however, its endonuclease activity remained obscure. To identify the biochemical function of M. tuberculosis RecA intein (PI-MtuI), the intervening sequence from recA was cloned, overexpressed in E. coli and purified to homogeneity. The identity of PI-MtuI was ascertained by sequencing 10 amino acid residues at the amino-terminal end and by Western blot analysis using polyclonal antibodies raised against precursor RecA.

La pirazinamida (PZA) es una droga antituberculosa de primera línea, presenta gran actividad in vivo, sin embargo in vitro no es evidente a menos que el pH del medio sea ácido, lo que hace que la susceptibilidad sea difícil de determinar por métodos convencionales. Por lo tanto, el objetivo del presente estudio fue evaluar la prueba molecular Polimorfismo Conformacional de la Hebra simple de ADN (SSCP) en cepas clínicas de Mycobacterium tuberculosis, así como comparar su desempeño con otras pruebas como son el BACTECÔ-460TB, el test de Wayne y el secuenciamiento. Se utilizó la prueba molecular del SSCP para la determinación de la susceptibilidad a PZA trabajando con 157 aislamientos clínicos de M. tuberculosis provenientes del Área de Tuberculosis del Laboratorio de Enfermedades Infecciosas de la Universidad Peruana Cayetano Heredia.

Se caracterizaron los genotipos de cepas de M. tuberculosis resistente, multidrogorresistente (MDR), MDR asociada a resistencia a drogas de segunda línea (MDR plus) y sensible a las drogas que proceden de los distritos de Lima y Callao. Cuarenta y nueve pacientes con TB fueron incluidos en el estudio. Los genotipos de M. tuberculosis fueron establecidos por PCR usando el primer Mtb2 (5’-CGG-CGG-CAA-CGG-CGG-CA-3’) en combinación con primers situados inversamente en los flancos repetitivos de la IS6110. Se revisaron las historias clínicas de los pacientes para la obtención de información epidemiológica. La susceptibilidad a isoniacida, rifampicina, estreptomicina, etambutol, kanamicina, acido p-amin-salicilico, tioacetazona y pirazinamida fueron estudiados. / We characterise the genotypes of Mycobacterium tuberculosis both resistant, multidrug resistant (MDR), multidrug drug resistant plus (MDR plus) and susceptible to drugs strains come from to Lima and Callao. Forty-nine patients with TB were included in the study. The genotypes of the M. tuberculosis isolates were established by PCR using the primer Mtb2 (5’-CGG-CGG-CAA-CGG-CGG-CA-3’) in combination with primers sited at inverted repeats flanking IS6110. Were revised the clinical history of the patients for epidemiological information. The susceptibility to isoniacid, rifampicin, streptomycin, ethambutol, kanamycin, para-amin-salicylic acid, tioacetazon and pyrazinamide was studied.

Reproduction de : Thèse de doctorat : Pharmacie. Santé. Immunologie et Infectiologie : Nantes : 2008. / Bibliogr.

Reproduction de : Thèse d'exercice : Médecine : Nancy 1 : 2003. / Titre provenant de l'écran-titre.

Zugl.: Kaiserslautern, Techn. Univ., Diss., 2009