单核增生李斯特氏菌通过调控线粒体Ca2+摄取蛋白2 (MICU2)介导的致病机制

Mitochondrial calcium uptake 2 (MICU2)-mediated pathogenic mechanism of Listeria monocytogenes

摘要:

目的 探究单核增生李斯特氏菌(Listeria monocytogenes)通过毒力因子溶血素O (listeriolysin O, LLO)调控线粒体Ca²⁺摄取蛋白2 ( MICU2)表达及其互作关系，进而影响线粒体钙稳态和细菌胞内增殖的分子机制。方法 采用Western blotting技术检测单核增生李斯特氏菌EGD-e和Δhly感染HeLa细胞后MICU2的表达变化。运用基因沉默技术和真核过表达技术探究MICU2蛋白水平变化对单核增生李斯特氏菌胞内增殖能力的影响。利用AlphaFold3预测LLO与MICU2的互作位点，并通过免疫共沉淀(co-immunoprecipitation, Co-IP)验证二者的相互作用。结合线粒体钙荧光探针(Rhod-2 AM)分析MICU2对钙稳态的调控作用。结果 EGD-e感染4 h和6 h后MICU2表达显著上调(P<0.001)，而Δhly感染后MICU2的表达量无显著变化(P>0.05)。沉默MICU2可增强细菌增殖能力(P<0.01)并升高线粒体钙水平(P<0.05)；过表达MICU2则减弱细菌增殖能力(P<0.01)并降低线粒体钙水平(P<0.05)。AlphaFold3预测显示，LLO第462位丙氨酸(Ala)与MICU2第119位谷氨酸(Glu)通过氢键结合，Co-IP证实二者存在相互作用。结论 本研究表明，单核增生李斯特氏菌通过LLO上调MICU2表达，MICU2通过减少线粒体钙抑制细菌胞内增殖，LLO与MICU2的互作是此过程的关键分子基础。本研究为深入探究单核增生李斯特氏菌的致病机理提供了参考。

Abstract:

Objective To investigate the molecular mechanism by which Listeria monocytogenes regulates the expression of mitochondrial calcium uptake 2 (MICU2) through the virulence factor listeriolysin O (LLO) and their interaction, thereby affecting mitochondrial calcium homeostasis and bacterial intracellular proliferation.Methods Western blotting was employed to analyze MICU2 expression in HeLa cells infected with L. monocytogenes EGD-e or Δhly. Gene silencing and eukaryotic overexpression approaches were used to examine how MICU2 regulated the intracellular proliferation of L. monocytogenes. AlphaFold3 was used to predict the interaction sites between LLO and MICU2, and co-immunoprecipitation (Co-IP) was performed to verify their interaction. Mitochondrial calcium fluorescence probe (Rhod-2 AM) was used to analyze the regulatory role of MICU2 in calcium homeostasis.Results EGD-e infection upregulated MICU2 expression at 4 h and 6 h post-infection (P<0.001), whereas Δhly showed no effect (P>0.05). The silencing of MICU2 enhanced bacterial proliferation (P<0.01) and elevated mitochondrial calcium levels (P<0.05), whereas overexpression of MICU2 reduced bacterial proliferation (P<0.01) and decreased mitochondrial calcium levels (P<0.05). AlphaFold3 predicted that alanine (Ala) at position 462 of LLO interacted with glutamate (Glu) at position 119 of MICU2 via a hydrogen bond, and Co-IP confirmed their interaction.Conclusion L. monocytogenes upregulates MICU2 expression via LLO, and MICU2 inhibits bacterial intracellular proliferation by reducing mitochondrial calcium levels. The interaction between LLO and MICU2 is a key molecular basis for this process. These findings provide insights into the pathogenic mechanism of L. monocytogenes.