Publication: Detección, caracterización y análisis funcional de la complejidad clonal en la tuberculosis humana y bovina
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2016-07-04
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Universidad Complutense de Madrid
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Abstract
Las herramientas de genotipado en tuberculosis fueron desarrolladas inicialmente para la realización de estudios epidemiológicos. Sin embargo han permitido asimismo desvelar la complejidad clonal existente en las infecciones causadas por Mycobacterium tuberculosis (MTB) y Mycobacterium bovis (M. bovis), poniendo así en cuestión la asunción de que cada episodio de tuberculosis (TB) estuviera causado por una única cepa. De este modo se comenzaron a describir situaciones de coinfección por más de una cepa (infección mixta) o bien de presencia simultánea de variantes clonales (infección policlonal), pudiendo estas, además, ofrecer una distribución heterogénea de las mismas en los diferentes tejidos infectados (infección compartimentalizada). Sin embargo, son pocos los estudios existentes entorno a estos fenómenos, y los que se han realizado atienden a la mera descripción de casos anecdóticos o al estudio de estos eventos en poblaciones en donde la incidencia de TB es alta. Así, el primer objetivo de esta tesis se centró en dimensionar la complejidad clonal existente en las infecciones por MTB en una población no seleccionada, en un entorno de moderada incidencia, donde las expectativas de encontrar la citada complejidad eran escasas. Mediante el análisis por RFLP-IS6110 y MIRU-VNTR se detectaron infecciones complejas en 11 pacientes con TB pulmonar (1,6%) y en 10 pacientes con TB pulmonar y extrapulmonar (14,1%). De estos 21 casos, 9 correspondieron a infecciones mixtas y 12 a infecciones policlonales. Por último, en 9 casos (5 pacientes con infección mixta y 4 con infección policlonal) se documentó la compartimentalización de la infección. Tras la descripción sistemática de los casos de TB con infecciones complejas, nos centramos en el estudio de una de sus modalidades, la infección policlonal. En concreto decidimos abordar la evaluación del posible significado funcional que pudiera conllevar la adquisición de las sutiles reorganizaciones genéticas identificadas entre variantes clonales, que surgen como resultado de eventos de microevolución...
Genotyping tools in tuberculosis were initially developed for epidemiological studies. However, they have also revealed the existence of clonally complex infections caused by Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (M. bovis), thus questioning the assumption that each episode of tuberculosis (TB) is caused by a single strain. Coinfection by more than one strain (mixed infection), simultaneous presence of clonal variants (polyclonal infection) and even heterogeneous distribution of strains or clonal variants in different infected tissues (compartmentalized infection) have been described. However, there are few studies focused on these phenomena, and those correspond to the description of anecdotal cases or the analysis of these events in populations with high TB incidence. Thus, the first objective of this thesis focused on defining the magnitude in which clonally complex infections by MTB occur in an unselected population, in a context with moderate incidence, where the expectations of detecting this kind of infections are limited. By applying IS6110-RFLP and MIRU-VNTR analysis, clonally complex infections were detected in 11 (1.6%) and 10 (14.1%) patients with exclusively pulmonary TB and both pulmonary and extrapulmonary TB respectively. Nine out of these 21 cases were mixed infections and the remaining 12 corresponded to polyclonal infections. Finally, in 9 cases (5 patients with mixed infection and 4 patients with polyclonal infection) compartmentalization of the infection was documented. Once the systematic description of clonally complex infections was done, we focused on the study of polyclonal infections. Specifically, to evaluate the potential functional meaning associated to the acquisition of subtle genetic rearrangements by microevolution phenomena. First, we evaluated the potential in silico meaning of the variations in the number of IS6110 copies and in the number of repetitions in MIRU-VNTR loci. The analysis detected that some of these variations are located intragenically, which means either a potential impact on the structure and function of the encoded proteins, or even a loss of function when the modification truncates the reading frame of the gene. In addition, variations were also located in intergenic MIRU-VNTR loci, which could mean a potential effect on the expression of adjacent genes...
Genotyping tools in tuberculosis were initially developed for epidemiological studies. However, they have also revealed the existence of clonally complex infections caused by Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (M. bovis), thus questioning the assumption that each episode of tuberculosis (TB) is caused by a single strain. Coinfection by more than one strain (mixed infection), simultaneous presence of clonal variants (polyclonal infection) and even heterogeneous distribution of strains or clonal variants in different infected tissues (compartmentalized infection) have been described. However, there are few studies focused on these phenomena, and those correspond to the description of anecdotal cases or the analysis of these events in populations with high TB incidence. Thus, the first objective of this thesis focused on defining the magnitude in which clonally complex infections by MTB occur in an unselected population, in a context with moderate incidence, where the expectations of detecting this kind of infections are limited. By applying IS6110-RFLP and MIRU-VNTR analysis, clonally complex infections were detected in 11 (1.6%) and 10 (14.1%) patients with exclusively pulmonary TB and both pulmonary and extrapulmonary TB respectively. Nine out of these 21 cases were mixed infections and the remaining 12 corresponded to polyclonal infections. Finally, in 9 cases (5 patients with mixed infection and 4 patients with polyclonal infection) compartmentalization of the infection was documented. Once the systematic description of clonally complex infections was done, we focused on the study of polyclonal infections. Specifically, to evaluate the potential functional meaning associated to the acquisition of subtle genetic rearrangements by microevolution phenomena. First, we evaluated the potential in silico meaning of the variations in the number of IS6110 copies and in the number of repetitions in MIRU-VNTR loci. The analysis detected that some of these variations are located intragenically, which means either a potential impact on the structure and function of the encoded proteins, or even a loss of function when the modification truncates the reading frame of the gene. In addition, variations were also located in intergenic MIRU-VNTR loci, which could mean a potential effect on the expression of adjacent genes...
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Tesis inédita de la Universidad Complutense de Madrid, Facultad de Veterinaria, Sección Departamental de Bioquímica y Biología Molecular I, leída el 17/12/2015