The Recombinant Expression Of HBHA In Mycobacterium Smegmatis And Its Role In Inhibition Of Autophagy Of AECâ…¡

Tuberculosis(TB), is the first killer in infectious diseases in the world. It has infectedabout1/3population in the world[1]. In2010, there were8.8million new cases oftuberculosis. Patients died of TB with negative HIV reached up to1.1million, andparients died of co-infection TB and HIV reached up to350,000[2]. More austerity is thatChina has1.3million new TB patients annually. In which100,000patients are MDR-TB[3]. China becomes a big burden country for TB.Mycobacterium tuberculosis (MTB),being primary pathogen of TB,MTB spreadthrough the air into the lungs, will be phagocytosised by macrophages. The organism’sown immune system may quickly eliminate MTB normally. But week immune system willlead to active TB, and MTB will be very difficult to be eliminated[4], Whether the MTBcould successfully infect host cell, depends on the amount of bacteria, the virulence ofmycobacterial and immune status of the host. Thus, through regulating and maintainingintracellular signaling mechanisms, immune system of the host playes a very importantrole in defence of MTB infection[5].MTB infection is not only closely related with macrophages[6-9], but also alveolar epithelial cells in lung[10-14]. Alveolar epithelial cells typeⅡ (AEC Ⅱ) is a group of cellswith lung protective activity. It can synthesis and secret a lot of surfactant and cytokines[15], playing an important role in regulating natural immune process[16].So far, virulence factors of MTB are very limit, including ESAT6, CFP-10, Ag85,and Heparin-binding hemagglutinin protein (HBHA). HBHA is a major adhesin of MTB,28kDa protein, having an important role in invasion of AECII and macrophage. It mainlyexpressed on the surface of bacteria, can be combined with heparin and dextran sulfide[17].It has three functional domains, in which, C-terminal domain can mediate adhesionbetween MTB and host glycoconjugates in the tissue[18]. Our previous study found thatHBHA could inhibit autophagy in AECII. MTB is likely to take this helps in infectionand dissemination in AECII.To deeply prove whether the HBHA can inhibit autophagy to enhance MTB infection,Mycobacterium smegmatis (MS), homologous with the MTB with the fast-growing andnon-pathogenic mycobacteria, was used as model to express HBHA recombinant proteinin this study. Then the infection ability of the recombinant MS was identified.1.Construction and identification of recombinant MS1.1Construction of recombinant rMS-HBHABy using an E.coli-Mycobacterium shuttle expression vector pMV261,pMV261-HBHA was constructed and electrotransfected into MS. After comfirmation ofexpression of HBHA by PCR, the recombinant MS was named rMS-HBHA. ThenrMS-HBHA was induced at45°C for2hrs, and ultrasonic degradated. The reslult ofSDS-PAGE showed that a28kD protein could be specifically expressed in rMS. TheWestern-blot demanstrated that there was a specific bind band appearred at thecorresponding site, detected by the polyclonal antibodies against HBHA.1.2The growth characteristics observation of rMS-HBHA We inoculated rMS-HBHAand MS in culture to grow until OD600=0.6. Then103bacteria were applied to cultureagain. OD600was measured after4,12,24,48,72,96,120, and144hrs later, and growthcurve was made. The results showed that there were no significant change in the growthrate between MS and rMS-HBHA. 1.3Preliminary verification of interactions between rMS-HBHA and A549cells1.3.1Measurement of the viable bacteria in A549cells infected with rMS-HBHA:The rMS-HBHA and MS was introduced into A549cells culture with10:1MOI(bacteria: cell).1,10, and18hrs later,100μg/ml gentamicin was added for1hour to killextracellular viable organism. The cells were collected and coated on the7H10(OADC)solid culture dishes.3-4d later, MS and rMS-HBHA colonies were counted as CFU. Theresults showed that, the number of intracellular survival rMS-HBHA (6.3×106) wassignificantly more than the MS group (1×105).1.3.2The necrosis of A549cells infected with rMS-HBHAExperiment was divided into three groups: experimental group (rMS-HBHA+A549)、control group (MS+A549)、and no infection group. After A549cells were infected withMS and rMS-HBHA, supernatants were collected at4,6,10,14, and18hrs later. LDHmeasurements indicated that, rMS-HBHA group cytotoxicity was much higher（68%）than the MS group（40%）, P<0.05.2.The effect of rMS-HBHA on autophagy of AECIIWestern blot was performed to detect the expression level of themicrotubule-associated protein light chain3(LC3) in A549cells infected withrMS-HBHA and MS. And the LC3-II/LC3-I ratio was calculated to evaluate thematuration of autophagy. It was showed that rMS-HBHA could significantly inhibit theexpression of LC3-II and LC3-I in A549cells. And the LC3-II/LC3-I ratio of rMS-HBHAgroup (0.625) was significantly lower than the MS group (2.025), indicatingautophagosome formation of rMS-HBHA group was inhibited. In addition, confocal laserobserving autophagosome by MDC stain found that autophagosome reduced significantlyafter infected with rMS-HBHA-GFP or HBHA protein.These studies demanstrated that HBHA could improve MS infection throughinhibiting autophagy of AEC II.