The Molecular Mechanism Of Curcumin Against The Activity Of Listeriolysin O From Listeria Monocytogenes

The gram-positive opportunistic bacterium Listeria monocytogenes(L.monocytogenes) is recognized as a facultative intracytosolic pathogen of zoonosis, which is responsible for the causative of listeriosis. This disease occurs predominantly in immunocompromised individuals, the elderly and pregnant women. Despite the administration of current antibiotic therapy which calls for high-dose antibiotics, the mortality rate of L.monocytogenes infections reaching up to 30%. The continuing outbreak and prevalence of L.monocytogenes infection all over the world and antibiotic resistant strains isolated from human and environment make L.monocytogenes a major concern for public health.As an typical intracellular pathogen, L.monocytogenes expresses several virulence factors that highly associated with cellular invasion and virulence. This pathogen first internalization into targeting cells, including both phagocytic cells and nonphagocytica cells, and subsequent escaping from phagosomes to cell cytosol. The bacteria utilizes actin cytoskeleton in host cell via its surface protin Act A to form F-Actin that provides the bacterial motility, resulting in spreading to neighboring cell. In order to escape from secondary vesicle before degradation in the adjacent cells, the bacteria expresses pore-forming toxin listeriolysin(Listeriolysin O, LLO) and PLCs(PI-PLC and PC-PLC) that contribute to its survival. It is notable that if L.monocytogenes were fail to escape, activated macrophages will kill intracellular bacteria by generating NOS and ROS, leading to the effectively degradation of the bacteria. L.monocytogenes resides in the host cell cytosol will undergo a novel round of proliferation, and in this manner, L.monocytogenes is capable of completing its intracellular life cycle and avoiding exposing to the extracellular environment which ultimately killed by the host immune response. Though its intracellular life cycle, LLO is the critical virulence factor for bacterial intracellular survival. Several studies have demonstrated that L.monocytogenes lacking LLO remain trapped in the phagocytic and displayed defect in bacterial intracellular growth in the host cell. Similarly, compared with wild-type strains, LLO-defective stains were fail to cause mortality and significantly lower bacterial burden in mouse model. LLO plays important role in L.monocytogenes infection diseases, which renders this virulence factor a promising target for the development of treating L.monocytogenes infection.In this work, by developing hemolysis assay, we demonstrated five flavonoid components, including myricetin, morin, baicalein, chrysin and naringenin,could decrease the pore-forming activity of purified LLO by directly neutralizing this virulence. The results indicated that the engagement of those five components in Loop 3 impaired the activity of LLO. Because the drug effect is highly associate with its structure, we further investigated if other nature components have similar effect on LLO. We found Curcumin, a polyphenol, could effective decrease LLO pore-forming activity as well as protect J774 cell from cell death caused by L.monocytogenes.Because Curcmin could effective decrease LLO pore-forming activity, it is reasonable to hypothesize that Curcumin is capable of decreasing the number of intracellur bacteria. The result from the growth curves of L.monocytogenes assay indicated that the inhibition of bacterial intracellular multiplication by Curcumin was concentration-dependent. To investigate the impact of Curcumin on L.monocytogenes intracellular life cycle, J774 cell were co-cultured with Curcumin and L.monocytogenes(MOI of 2.5), then fixed, and stained for fluorescence microscopy-based assay at 0.5 h,3 h, and 5 h. 0.5 hours after infection, no significant difference were observed between wild-type L.monocytogens group and Curcumin-treated group, suggesting that Curcumin did not effect the phagocytosis of L.monocytogenes. The result from 3 hours after infection shown that about 56.71% of wild-type L.monocytogenes escaped in the cytoplasm, and all of the LLO-/- mutants remain in the phagosomal. After treatment with 16 μg/ml, the number of bacterial escape reduced to 23.94%, which indicated that Curcumin could counteract bacteria-induced phagosomal escape, and the protective effective of Curcumin on this pathogen was due to the impact on LLO activity. At 5 hours after infection, bacteria proliferation rapidly in macrophages, and the majority of bacteria strained with F-Actin. Treatment with 16 μg/ml Curcumin significant reduced the number of the bacteria. Further stained intercellular bacteria by PI and SYTO 9, the number of live bacteria in Curcumin-treated group decrease from 96.7% to 37.29%, indicating that treatment with indicated concentrations of Curcumin facilitated macrophages to clear intracellular L.monocytogenes.Furthermore, Curcumin effectively protected mice from L.monocytogenes infection in a mouse infection model. Compared with wild-type strains infection mice, Curcumin-treated group had significant lower bacterial burden in spleen and liver, and when Curcumin was given at 200 mg/kg to mice effective alleviated pathological injury caused by L.monocytogenes. Treatment mice with Curcumin after infection increased the survival time of mice.The result from computational biology revealed that the molecular mechanism of Curcumin against the activity of LLO was due to the engagement of Curcumin to V100 and L503. Curcumin interaction with the cleft between domain 3 and domain 4 of LLO, thereby neutralized LLO activity by interfering with LLO oligomerization, which is different from five flavonoid components. After two amino-acid mutations(V100A and L503A) were created, we developed hemolysis assay, and the result suggesting that Curcumin has significant lower ability to against the hemolytic activity of LLO mutains than wild type LLO. Consist with this findings, the same results were observed on LDH release assay, indicating that V100 A and L503 A were two important amino-acid which Curcumin interfered with LLO. Additionally, membrane-binding assay and LLO oligomerization assay demonstrated that Curcumin has no obvious impact on LLO membrane binding, while this compound could reduce the oligomer formed by LLO. All the results demonstrated that Curcumin could neutralize LLO activity by interfering with LLO oligomerization.Taken together, these results suggest that Curcumin is a potent candidate against L.monocytogenes and this agent may be a valuable alternative or adjuncts to current antibiotic therapies.