The Annlysis Of The Mechanism Of Heparin-binding Hemagglutiniu Inhibited The Autophagy Of The Alveolar Type2Epithelial Cells

Mycobacterium tuberculosis (MTb), was identified to cause tuberculosis byGerman bacteriologist in1882for the first time. Now MTB infects one third ofthe world’s population and kills an estimate1.7million people one yearaccording to WHO reports. The death rate ranks the second place in the singleinfectious diseases. It greatly affects the public health. The heparin-bindinghemagglutinin (HBHA) is one of the most important adhesin factor of MTb,which the molecular weight is28-kDa. It was mainly located on bacteria surface,capble of binding with heparin and vulcanized dextran. It has three struct uraldomains, but its specific function that in the process of bacterial adhesion andinvasion on the alveolar epithelial cells is unknown.The alveolar epithelial cell type is a group of alveolar protective cells withmultiple functions. It can synthesize and secret surface-active agents andcytokines, play an important role in the innate immune process. Our previous studies suggested that MTb could suppress the autophagy of A549cellsthrough inhibiting the expression of the LC3, then prevented the A549cells tokill the MTb.To further illustrate the mechanism of HBHA protein on inhibiting theexpression of LC3, and promoting the effect of bacteria.We used recombinantHBHA full-length fragments, C-terminal deletions and N-terminal deletions totreat the A549cells. The the expression of autophagy-related genes,the LDH andcytokine were observed.1. Purification of HBHA protein, and its effects on A549cellsThe recombinant proteins were extracted and purified, including thefull-length HBHA protein, the C-terminal deleted fragments and the N-terminaldeleted fragments. And the SDS-PAGE was used to confirm the protein.In this study, A549cells were treated with extracted the full-length HBHAprotein, the C-terminal deleted fragments and N-terminal deleted fragments,respectively. Then the expression of LC3and Atg5were verified usingWestern-blot analysis. The result showed that all three kinds of HBHAfragments could inhibit the expression of LC3, but not Atg5. It suggested thatHBHA protein could not influence formation of Atg5-Atg12complex, butinhibit the formation of LC3complex.To verify the relation between the MTb and the autophagy of A549cells, weused the BCG and protein to treated A549. After that the released LDH wasmeasured. The results displayed that LDH was significantly increased afteradding HBHA, proveing that A549cells lost the ability to kill the MTb byautophagy inhibition. To further verify the role of HBHA protein in MTbinfection of A549cells,we used BCG adetected the LDH released, The resultsshowed that LDH released from BCG joined anti-HBHA antibody group was significantly lower than BCG only. It proved that MTb could suppress the abilityof A549cells to kill bacteria through HBHA inhibiting autophagy.2. The role of HBHA protein in cell secretion of cytokinesWhile the mechanism of HBHA proteins inhibiting autophagy might berelated with innate immune system, the cytokines such as IL-1β, IL-6,IL-10、TNF-α and IFN-γ were measured after the A549cells was treated byrecombinant HBHA. The results showed that A549cells could secrete IL-1β,IL-6, IL-10、TNF–α and IFN-γ, five cell factors. The recombinant HBHAfull-length fragment could promote A549cells to secrete IL-1β,IL-6, TNF-αand IFN-γ,but not IL-10.Therefore, this research showed that HBHA protein could inhibit the LC3expression and mature through its alpha helix structure. And it could be blockedby anti-HBHA antibody. At the same time, HBHA could also cause the releaseof inflammatory factor IL-1β、IFN-γ. It suggested that HBHA was a veryimportant virulence factor involved in MTB infection, dissemination andinflammation.