海洋分枝桿菌與結核分枝桿菌第七型分泌系統誘發NLRP3發炎小體活化之細菌因子

Abstract

人類結核病主要起因為結核分枝桿菌感染，在人類史上相當早即有感染案例，又稱為「白色瘟疫」，由於其感染後潛伏期長和抗藥性等因素，結核病的診斷及治療開發上仍有相當大的研究空間。而海洋分枝桿菌和結核分枝桿菌親緣關係相近且在第七型分泌系統具有高度保留性，因此許多關於結核分枝桿菌和結核病之研究皆以安全性較高和生長速度較快的海洋分枝桿菌作為模式生物。 在我們的研究中，即先以海洋分枝桿菌探討第七型分泌系統中的ESX-1對NLRP3發炎小體活化之影響，選擇實驗室先前建構之五株ESX-1前段基因突變株分別感染人類巨噬細胞後以酵素免疫分析法測量THP-1細胞受感染後第一型介白素β分泌差異，發現受∆espG感染之細胞其第一型介白素β分泌顯著下降，且細胞破裂情形亦有顯著下降的情形；此外在西方墨點法分析之結果中，亦發現受∆espG感染之細胞上清液可看到第一型胱天冬酶活化態表現下降；而將細胞預處理NLRP3抑制劑再以海洋分枝桿菌感染，從細胞上清液中第一型介白素β的分泌差異可間接證明海洋分枝桿菌誘發之第一型介白素β分泌為NLRP3發炎小體活化所造成，並可推測espG基因和NLRP3發炎小體活化途徑相關。 隨後我們建構結核分枝桿菌espG1缺失突變株及espG1回補菌株組，以相同實驗條件感染THP-1細胞，觀察細胞之第一型介白素β和第一型胱天冬酶蛋白表現差異。雖然espG1缺失確實影響兩者活化態之分泌量下降，回補株卻無法補回基因完整功能。 鑑於先前有文獻指出結核分枝桿菌之ESAT-6蛋白可影響巨噬細胞NLRP3發炎小體活化，於是我們以西方墨點法分析海洋分枝桿菌espG基因和結核分枝桿菌espG1基因所造成之第一型介白素β分泌差異是否和ESAT-6蛋白表現相關。根據實驗結果發現，∆espG和野生株相比，ESX-1主要致病蛋白ESAT-6和CFP-10在細菌上清液中表現皆下降，在細菌裂解液中則表現上升，推測espG可影響ESAT-6和CFP-10分泌；而結核分枝桿菌中亦可發現此趨勢，因此我們推測結核分枝桿菌espG1基因同樣可影響ESAT-6和CFP-10分泌。

Human tuberculosis, also known as 'the white plague', mainly results from Mycobacterium tuberculosis infection which was found in early human history. Due to the long incubation period and drug resistance of M. tuberculosis, the researches of diagnosis and treatment in tuberculosis still remain a considerable development space. Generally, many studies on M. tuberculosis and tuberculosis use Mycobacterium marinum with higher safety and faster growth rate as a model in laboratory works. M. marinum shows high conservation with M. tuberculosis in genetic relationship and the type seven secretion system, which is critical for virulence. In our study, first we used five constructed M. marinum ESX-1 gene deletion mutants: ∆espE, ∆espF, ∆espG, ∆espH and ∆eccA1 to investigate the role of the type seven secretion system ESX-1 in NLRP3 inflammasome activation. After infecting THP-1 differentiated macrophages with these deletion mutants respectively, we found ∆espG induced less IL-1β in cell supernatant through enzyme-linked immunosorbent assay (ELISA). Our data from Western blot also showed ∆espG reduced activated caspase-1 secretion in infected macrophages. Additionally, THP-1 cells pretreated with NLRP3 inhibitor before infected with wildtype M. marinum showed the difference in secretion of IL-1β, while ∆espG-infected cells did not show this pattern. The data could indirectly demonstrate that M. marinum induces IL-1β through activating NLRP3 inflammasomes, and espG may involve in NLRP3 inflammasome activation. Subsequently, we constructed the M. tuberculosis espG1 deletion mutant and the espG1 complemented strains, infected THP-1 cells with M. tuberculosis, and analyzed the protein expression level of IL-1β and caspase-1 in cell lysate and supernatant. Although M. tuberculosis espG1 deletion mutant reduced the secretion of IL-1β significantly, the complemented strain did not repair the function of espG1 completely. In view of the previous studies that ESAT-6 in M. tuberculosis is involved in activation of NLRP3 inflammasomes, we used Western blot to analyze whether the difference in IL-1β secretion is related to the expression of ESAT-6 in M. marinum espG and M. tuberculosis espG1. According to the results, the ESX-1 major virulent proteins, ESAT-6 and CFP-10, showed substantial decrease in both M. marinum ∆espG and M. tuberculosis ∆espG1::pMN437 supernatants respectively, but were increased in bacterial lysates. Thus, we suggested that M. marinum espG gene and M. tuberculosis espG1 gene are involved in secretion of ESAT-6 and CFP-10.