RESUMEN
AIMS: The importance of bacterioferritin in the virulence and pathogenicity of the genus Mycobacterium is still unclear. The aim of this study was to analyse if the expression of a recombinant bacterioferritin from M. tuberculosis (Mtb) by Mycma could improve the capacity of this bacillus to resist the host defence mechanisms. METHODS AND RESULTS: Recombinant Mycma, expressing bacterioferritin (Rv1876) from Mtb, was developed by transformation with pMIP12_Rv1876. To determine bacterioferritin influence on Mycma physiology and virulence, the mycobacteria growth was analysed in vitro and in vivo. It was observed that the expression of bacterioferritin improved the growth rate of recombinant Mycma_BfrA under iron excess and oxidative stress, as compared to the wild type. Furthermore, in the murine model of infection, it was observed that Mycma_BfrA-infected mice had higher bacillary load and a more pronounced lesion in the lungs when compared with the wild type. CONCLUSION: This study showed that bacterioferritin confers additional resistance to stress conditions, resulting in increased pathogenicity of Mycma during mice infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new insights about the importance of bacterioferritin in the virulence and pathogenicity of the Mycobacterium genus.
Asunto(s)
Proteínas Bacterianas/metabolismo , Grupo Citocromo b/metabolismo , Ferritinas/metabolismo , Mycobacterium abscessus/fisiología , Mycobacterium abscessus/patogenicidad , Animales , Carga Bacteriana , Proteínas Bacterianas/genética , Grupo Citocromo b/genética , Ferritinas/genética , Ratones , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/patología , Mycobacterium abscessus/genética , Mycobacterium abscessus/crecimiento & desarrollo , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estrés Fisiológico , VirulenciaRESUMEN
This manuscript reports, at the first time, the photoinactivation evaluation of tetra-cationic and anionic porphyrins as photosensitizers (PS) for the photodynamic inactivation (PDI) of rapidly growing mycobacteria strains. Two different charged porphyrin groups were obtained commercially. PDI experiments in the strains Mycobacterium massiliense e Mycobacterium fortuitum conducted with adequate concentration (without aggregation) of photosensitizer under white light at a fluence rate of 50â¯mW/cm2 over 90â¯min showed that the most effective PS caused a 100 times reduction in the concentration of viable mycobacteria. The present results show that porphyrin with positively charge are more efficient PS than anionic porphyrin (negatively charged) against M. massiliense e M. fortuitum. It is also clear that the effectiveness of the molecule as PS for PDI studies with mycobacteria is strongly related with the porphyrin peripheral charge, and consequently their solubility in physiological media. Cationic PSs might be promising anti-mycobacteria PDI agents with potential applications in medical clinical cases and bioremediation.
Asunto(s)
Mycobacterium/efectos de los fármacos , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Aniones , Cationes , Recuento de Colonia Microbiana , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Humanos , Luz , Pruebas de Sensibilidad Microbiana/métodos , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/efectos de la radiación , Mycobacterium/fisiología , Mycobacterium/efectos de la radiación , Mycobacterium abscessus/efectos de los fármacos , Mycobacterium abscessus/fisiología , Mycobacterium abscessus/efectos de la radiación , Mycobacterium fortuitum/efectos de los fármacos , Mycobacterium fortuitum/fisiología , Mycobacterium fortuitum/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Outbreaks of infections caused by rapidly growing mycobacteria have been reported worldwide generally associated with medical procedures. Mycobacterium abscessus subsp. massiliense CRM0019 was obtained during an epidemic of postsurgical infections and was characterized by increased persistence in vivo. To better understand the successful survival strategies of this microorganism, we evaluated its infectivity and proliferation in macrophages (RAW and BMDM) and alveolar epithelial cells (A549). For that, we assessed the following parameters, for both M. abscessus CRM0019 as well as the reference strain M. abscessus ATCC 19977: internalization, intracellular survival for up 3 days, competence to subvert lysosome fusion and the intracellular survival after cell reinfection. RESULTS: CRM0019 and ATCC 19977 strains showed the same internalization rate (approximately 30% after 6 h infection), in both A549 and RAW cells. However, colony forming units data showed that CRM0019 survived better in A549 cells than the ATCC 19977 strain. Phagosomal characteristics of CRM0019 showed the bacteria inside tight phagosomes in A549 cells, contrasting to the loosely phagosomal membrane in macrophages. This observation holds for the ATCC 19977 strain in both cell types. The competence to subvert lysosome fusion was assessed by acidification and acquisition of lysosomal protein. For M. abscessus strains the phagosomes were acidified in all cell lines; nevertheless, the acquisition of lysosomal protein was reduced by CRM0019 compared to the ATCC 19977 strain, in A549 cells. Conversely, in macrophages, both M. abscessus strains were located in mature phagosomes, however without bacterial death. Once recovered from macrophages M. abscessus could establish a new intracellular infection. Nevertheless, only CRM0019 showed a higher growth rate in A549, increasing nearly 10-fold after 48 and 72 h. CONCLUSION: M. abscessus CRM0019 creates a protective and replicative niche in alveolar epithelial cells mainly by avoiding phagosome maturation. Once recovered from infected macrophages, CRM0019 remains infective and displays greater intracellular growth in A549 cells compared to the ATCC 19977 strain. This evasion strategy in alveolar epithelial cells may contribute to the long survival of the CRM0019 strain in the host and thus to the inefficacy of in vivo treatment.