Study On The Function Of Bacterial Manganese Transport Systems In Bacteria-Host Interactions

Salmonella enterica serovar Typhimurium（S.Typhimurium）and Enterohemorrhagic Escherichia coli（EHEC）are common foodborne enteropathogens.Bacterial manganese transport systems are essential for bacteria to acquire manganese that adapts to different environments.The main manganese transport proteins in S.Typhimurium are Mnt H and Sit ABCD and in EHEC is Mnt H.Manganese plays an important role in bacteria-host interaction.It can regulate the innate immune response of host cells and bacteria can escape the innate immune response by transporting manganese.In host cells,Manganese is released from organelles to the cytoplasm when infected with bacteria,thereby activating the cGAS-STING signaling pathway and innate immune responses.Whether manganese transport systems in bacteria can affect bacterial pathogenicity and host innate immune responses during EHEC and S.Typhimurium infection is not clear.Therefore,revealing the function of bacterial manganese transport systems during EHEC and S.Typhimurium infection will provide new insight into ion-mediated bacteria-host interactions.In this study,EHEC and S.Typhimurium were used to study the function of the manganese transport system in bacterial physiology.We further explored the effects of manganese and bacterial manganese transport systems on host innate immune responses.We revealed a mechanism of bacterial manganese transport systems that affect bacterial pathogenicity and host innate immune responses.The main findings were as follows:（1）To detect the functions of manganese transport systems in S.Typhimurium and EHEC,q RT-PCR was performed to detect the expression of mnt H and sit A in bacteria in different manganese and oxidative stress environments.The results showed that a low manganese environment and oxidative stress activate the expression of mnt H and sit A,while a high manganese environment inhibits these gene expressions.Then we constructed the S.Typhimurium mnt H（sl1344＿2376）and sit A（sl1344＿2841）double gene mutants and the EHEC mnt H（z3658）single gene mutant.We found the deletion of these genes decreased intracellular manganese in bacteria.Through bacterial competition and stress experiments,we found Mnt H and Sit ABCD affect the growth of bacteria and the nutritional competitiveness of bacteria in different extreme environments.（2）To detect how manganese affects host cell resistance to S.Typhimurium and EHEC infection,q RT-PCR was performed to detect the activation of genes that related to the innate immune response in S.Typhimurium or EHEC-infected mouse peritoneal macrophages（PMs）and macrophages（RAW264.7）.The results showed that S.Typhimurium and EHEC infection activated innate immune responses.The innate immune responses in EHEC-infected cGAS^-/-or STING^-/-PMs were significantly attenuated compared to WT PMs,indicating that the EHEC infection-induced innate immune responses were partially dependent on the cGAS-STING signaling pathway.Manganese treatment enhanced bacteria infection-elicited innate immune responses,while this was not detected in cGAS^-/-and STING^-/-PMs,indicating that manganese-enhanced S.Typhimurium and EHEC induced innate immune responses via the cGAS-TING pathway.In addition,manganese reduced the intracellular survival rate of bacteria.All the results indicated that manganese could enhance the innate immune responses of host cells by activating the cGAS-STING pathway and then help cells to defend against bacterial infections.（3）To further explore the role of bacterial manganese transport system during S.Typhimurium and EHEC infection,we used S.Typhimurium WT,S.TyphimuriumΔmnt HΔsit A,EHEC WT and EHECΔmnt H strain to infect PMs and RAW264.7.The results showed that the bacterial manganese transport system inhibited the innate immune response of host cells.However,such observations were attenuated in STING^-/-PMs,indicating that the manganese transport system inhibits the innate immune response induced by EHEC and S.Typhimurium infection via the cGAS-STING pathway.In conclusion,we clarified the manganese transport and stress resistance function of manganese transport protein（Mnt H and Sit ABCD）in EHEC and S.Typhimurium.We also found that during EHEC and S.Typhimurium infection,manganese could enhance antimicrobial innate immune responses by activating the cGAS-STING pathway.Finally,we demonstrated that the manganese transport system could inhibit innate immune response through the cGAS-STING pathway.