Citrate lyase CitE in Mycobacterium tuberculosis contributes to mycobacterial survival under hypoxic conditions

Mycobacterium tuberculosis is the causative agent of tuberculosis and has evolved an ability to survive in hostile host environments. M. tuberculosis is thought to utilize the rTCA cycle to sustain its latent growth during infection, but the enzymatic characteristics and physiological function for the key citrate lyase of the rTCA cycle, MtbCitE, in the important pathogen remain unclear. In this study, we investigated the function of MtbCitE based on its structural properties and sequence comparisons with other bacterial citrate lyase subunits. We showed that several amino acid residues were important for the citrate cleavage activity of MtbCitE. Strikingly, the citrate cleavage activity of MtbCitE was inhibited by ATP, indicating that energy metabolism might couple with the regulation of MtbCitE activity, which differed from other CitEs. More interestingly, deletion of citE from Mycobacterium bovis BCG decreased the mycobacterial survival rate under hypoxic conditions, whereas complementation with citE restored the phenotype to wild-type levels. Consistently, three key rTCA cycle enzymes were positively regulated under hypoxic conditions in mycobacteria. Therefore, we characterized a unique citrate lyase MtbCitE from M. tuberculosis and found that the CitE protein significantly contributed to mycobacterial survival under hypoxic conditions.

结核分枝杆菌（Mycobacterium tuberculosis）是结核病的致病菌，已演化出在恶劣宿主环境中存活的能力。学界普遍认为，结核分枝杆菌可借助反向三羧酸循环（rTCA cycle）维持感染过程中的潜伏生长，但针对该重要病原菌中反向三羧酸循环关键柠檬酸裂解酶亚基MtbCitE的酶学特性与生理功能，目前仍尚不明确。本研究通过解析MtbCitE的结构特性，并与其他细菌的柠檬酸裂解酶亚基开展序列比对，对其功能进行了系统探究。研究结果证实，多个氨基酸残基对MtbCitE的柠檬酸裂解活性具有关键作用。尤为关键的是，MtbCitE的柠檬酸裂解活性可被三磷酸腺苷（ATP）抑制，这提示能量代谢或与MtbCitE的活性调控存在耦合关联，该特性与其他CitE蛋白截然不同。更具研究价值的是，在牛分枝杆菌卡介苗（Mycobacterium bovis BCG）中敲除citE基因后，分枝杆菌在低氧条件下的存活率显著下降；而通过citE基因进行功能互补，可使该表型恢复至野生型水平。与此一致的是，分枝杆菌体内的三种核心反向三羧酸循环酶在低氧条件下均呈现正向调控趋势。综上，本研究解析了结核分枝杆菌中一种独特的柠檬酸裂解酶MtbCitE，并证实该CitE蛋白对分枝杆菌在低氧条件下的存活具有显著的促进作用。