Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco, A.C., Guadalajara, Jalisco, Mexico.
Departamento de Patología Experimental, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México City, Mexico.
Vaccine. 2018 Apr 12;36(16):2069-2078. doi: 10.1016/j.vaccine.2018.03.014. Epub 2018 Mar 15.
Pellicles, a type of biofilm, have gathered a renewed interest in the field of tuberculosis as a structure that mimics some characteristics occurring during M. tuberculosis infection, such as antibiotic recalcitrance and chronicity of infection, and as a source of antigens for humoral response in infected guinea pigs. In other bacteria, it has been well documented that the second messenger c-di-GMP modulates the transition from planktonic cells to biofilm formation. In this work, we used the live vaccine Mycobacterium bovis BCG to determine whether deletion of genes involved in c-di-GMP metabolism would affect interaction with macrophages, capacity to induce immune response in a murine cell line and mice, and how the protein profile was modified when grown as surface pellicles. We found that deletion of the BCG1419c (Delta c-di-GMP phosphodiesterase, ΔPDE) gene, or deletion of the BCG1416c (Delta c-di-GMP diguanylate cyclase, ΔDGC) gene, altered production of TNF-α, IL-6, and IL-1β, in murine macrophages, and resulted in attenuation in intra-macrophage replication. Moreover, in addition to the improved immunogenicity of the BCGΔBCG1419c mutant already reported, deletion of the BCG1416c gene leads to increased T CD4 and T CD8 activation. This correlated with protection versus lethality in mice infected with the highly virulent M. tuberculosis 5186 afforded by vaccination with all the tested BCG strains, and controlled the growth of the mildly virulent M. tuberculosis H37Rv in lungs by vaccination with BCGΔBCG1419c during chronic late infection from 4 to 6 months after challenge. Furthermore, when grown as surface pellicles, a condition used to manufacture BCG vaccine, in comparison to BCG wild type, both rBCGs changed expression of antigenic proteins such as DnaK, HbhA, PstS2, 35KDa antigen, GroEL2, as well as AcpM, a protein involved in synthesis of mycolic acids, molecules relevant to modulate inflammatory responses.
生物被膜是生物膜的一种类型，在结核病领域重新引起了人们的兴趣，因为它模拟了结核分枝杆菌感染过程中发生的一些特征，如抗生素耐药性和感染的慢性化，并且是感染豚鼠体液反应的抗原来源。在其他细菌中，已经有充分的文献证明，第二信使 c-di-GMP 调节从浮游细胞向生物膜形成的转变。在这项工作中，我们使用活疫苗卡介苗来确定 c-di-GMP 代谢相关基因的缺失是否会影响与巨噬细胞的相互作用、在小鼠细胞系和小鼠中诱导免疫反应的能力，以及当作为表面被膜生长时蛋白质谱如何被修饰。我们发现，BCG1419c（Δ c-di-GMP 磷酸二酯酶，Δ PDE）基因或 BCG1416c（Δ c-di-GMP 双鸟苷酸环化酶，Δ DGC）基因的缺失改变了 TNF-α、IL-6 和 IL-1β在小鼠巨噬细胞中的产生，并导致巨噬细胞内复制减弱。此外，除了已经报道的 BCGΔBCG1419c 突变体的更好的免疫原性之外，BCG1416c 基因的缺失导致 T CD4 和 T CD8 的激活增加。这与用所有测试的卡介苗菌株接种疫苗对高毒力结核分枝杆菌 5186 感染的小鼠的致死性保护相关，并且在用 BCGΔBCG1419c 接种疫苗控制轻度毒力结核分枝杆菌 H37Rv 在肺部的生长，在挑战后 4 至 6 个月的慢性晚期感染期间。此外，与野生型 BCG 相比，在作为表面被膜生长时（用于制造卡介苗的条件），两种 rBCG 改变了抗原蛋白的表达，如 DnaK、HbhA、PstS2、35KDa 抗原、GroEL2 以及 AcpM，一种参与合成分枝菌酸的蛋白质，这些分子与调节炎症反应有关。
