Screening Of Autophagy-related Anti-mycobacterium Tuberculosis Peptides

BackgroundTuberculosis(TB)is leading cause of fatal infectious disease beyond the acquired immune deficiency syndrome(AIDS),causing by the infection of Mycobacterium tuberculosis(MTB)that mainly affected the lungs.In 2016,according the data from World Health Organization(WHO),about 10.4 million people acquired TB,and among them 1.67 million people died from this disease.Meanwhile,the emerging of drug resistant MTB still threathen human being’s health.Until 2016,more than 600,000 people infected by the rigsmpin resistant MTB and among them 490,000 people were infected by multi-drug resistant MTB(MDR-MTB),and 47%of the above cases were appeared in India,China and Russia.Therefore,TB still poses a serious threat to human health and exerts heavy burden on pubic health globally.Thus,it is urgent to concentrate on the mechanism of MTB infection,immune escape and clearance in order to find new and effective drugs and targets of treatment.In a general sense,we believe that the effective immune response against MTB infection is dependent on the T-cell mediated adaptive immune response,whereas the innate immune response complements the adaptive immune response by controlling the growth of latent MTB to jointly resist MTB infection.In this process,alveolar macrophages and lung epithelial cells together constitute the first defense barrier for MTB invasion.Studies have shown that type Ⅱ pneumocytes play an important role in host defense against a variety of invasives,including MTB infection.In addition to physical barriers,the lung epithelial cells can secrete various antimicrobial peptides(AMPs)that can antagonize the growth of pathogens.Antimicrobial peptides(AMPs)are a group of polypeptides consisted with 50-100 amino acids that existed in many tissues and cell types.AMPs are able to kill various microorganisms by inserting into membrane bilayers and forming pores and function as a key effector in innate immune system.Basically,there is three main families of antimicrobial peptides:defensins,cathelicidins and histatins.They exhibit antimicrobial activity against a variety of microbes,such as bacteria,fungi and some viruses,including MTB.It is reported MTB infection could stimulate human-corneal fibroblast and human endothelial cells secrete β-defensin 1(hBD1).In mice,mycobacterial infection could promote the expression of β-defensin 3(BD3)and β-defensin 4(BD4).It has been reported that hBDl and hBD3 act a crucial role in the epithelial barrier defense system during the bacterial invasion.In addition,there is relatively mature and extensive studies on the anti-TB function and mechanism of cathelicidins.It is currently known that the human cathelicidin gene(CAMP)encodes an inactive precursor protein which is about 18 KDa therefore named as hCAP18.In human neutrophils and other cells that express antibacterial peptides,the C-terminus of hCAP18 is released from the precursors by proteolytic hydrolysis,forming an active antimicrobial peptide consisting of 37 amino acids,named LL37.It can be proved from the existing studies that LL37 could inhibit the growth of intracellular MTB,and clinical studies have shown that LL37 is indeed highly expressed in TB patients and plays a role in anti-MTB infection.In the aspect of bactericidal mechanism,LL37 could alter the ultrastructure of MTB,impair the cell wall of cells and shrink the cytoplasm,thus inhibiting the growth of MTB in the infected cells.However,in the course of MTB infection,how the pulmonary epithelial cells exert antibacterial effects through the regulation of the production of active antimicrobial peptides needs further study.The production of AMPs is generally expressed as precursors and cleaved by protease to release the active peptides,and this process could be regulated in terms of transcriptional and post-transcriptional levels.As said above,LL37 is released from its precursor protein hCAP18 by protease hydrolysis process,and it has been discovered that serine protease cleavage 3 could cleave hCAP18 into LL37 to endow it antibacterial activity during the exocytosis process.Based on that,the process of the cleavage of AMPs’ precursors and the function of proteases may have a greater effect on the production of AMPs and thereby affecting their effects on against pathogen invasion,compared with the regulation of AMPs’ gene expression.In recent studies,it is revealed that autophagy might be a mechanism of the AMPs’ cleavage.Generally,autophagy has been identified as a physiological process of cells to maintain self-homeostasis by selectively encapsulating intracellular materials thus forming the autophagosomes,then the autophagosomes are fused with lysosomes and form autolysosomes to degrade the contents by acidification or hydrolase.However,it has been found that autophagosomes could selectively encapsulate some innocuous components in the cytoplasm mediated by autophagic adapter proteins and process them into short peptides with antimicrobial activity(ie antimicrobial peptides)to kill MTB.It is generally believed that autophagy can wrap phagosomes containing MTB and fused with lysosomes,then the wrapped MTB was degraded the MTB by proteolytic enzymes.But the mechanism that autophagy could cleave AMPs’precursors to active peptides to against MTB bring us new avenues for further research on anti-TB immune mechanisms and aspired us new TB treatment strategy.It has been already found that ribosomal protein rps30 could be degraded by autophagy mediated by autophagy adapter p62 to produce short peptides,which act in concert with lysosomal hydrolases to kill MTB in autophagosomes.But whether there is existed the others novel antimicrobial peptides produced by autophagy degradation is still unknown.In this study,we explored that human alveolar type II pneumocytes could expressed antimicrobial peptides hBDl and LL37 during MTB or MTB variant bo vis BCG infection,which indicating that this type of cells may exhibit anti-MTB activity via productions of AMPs.Then we found out the production of AMPs is closely related with the function of autophagy and lysosome,and the results revealed that the process of formation of autophsosomes and autolysosome is critical of productions ofAMPs.Also,we verified the role of autophagy adapter protein p62 in the expression of antimicrobial peptides.To clarify the above mechanism will help to further understand the mechanism of pulmonary epithelial cells as the first immune barrier against MTB infection,and have a great significance for the prevention and treatment of tuberculosis.Objective1.To find antimicrobial peptides that can be highly expressed in lung epithelial cells during MTB infection and reveal the function of lung epithelial cells as the first immune barrier against MTB;2.Screening the high expressed antimicrobial peptides during BCG infection in human alveolar type Ⅱ pneumocytes and confirm their role of anti-BCG infection through the autophagic production;Method1.The expression levels of hBDl and LL37 were detected by Real-time PCR and Western Blot after mycobacterial infection.2.Authophagy inhibitors and agonists were used to influence the signalling pathways involved in MTB-induced hBDl and LL37 expression.3.Western blot,Real-time PCR and immunofluorescence was used to measure the protein level of microtubule-associated protein light chain 3(LC3),a marker of autophagy and so on.Results1.The increased expression of antimicrobial peptides hBD1 and LL37 were detected in TB patients’ PBMCs and lung epithelial type Ⅱ cells;2.After BCG infection,the expression of antimicrobial peptides hBD1 and LL37 were increased in A549 cells;3.Both autophagy agonist rapamycin and starvation medium EBSS could induce the up-regulation of the antimicrobial peptides hBDl and LL37’s protein level,but not their mRNA level.Meanwhile,the stimulation of autophagy inhibitor 3-MA or ATG5 and 7 defective cells could down-regulated the protein level of hBDl and LL37 protein levels,but no change in mRNA levels;4.Suppressing lysosome function by Baf A1 or NH4C1 could effectively down-regulated the protein level of hBD1 and LL37 protein levels,but no change in mRNA levels;5.The protein levels of antimicrobial peptides hBDl and LL37 were down-regulated if p62 was silenced and CO-IP results exhibited that there is intercaiton between AMPs and p62;6.Using rapamycin to induce autophagy in A549 cells,the amounts of BCG could signifianctly decresased while silencing hBDl and/or LL37 during the process this phenomenon will disappear.ConclusionsIn this study,we observed the high expression of antimicrobial peptides LL37 and hBD1 in TB patients’ PBMCs and lung epithelial type Ⅱ,and BCG infection can also induce the high-level production of LL37 and hBD1.BCG infection can also induce antibacterial peptide hBDl and LL37 in human lung epithelial A549 cells,and these two antimicrobial peptides play a role in the anti-tuberculosis process of lung type II epithelial cells.Further in-depth investigations revealed that the level of autophagy in human lung epithelial A549 cells can affect the production of antimicrobial peptide-active peptides,and this effect is post-transcriptional.And in this processs,the autophagy adapter protein p62 plays a key role which could selectively the precusors of AMPs to autophagosomes for processing the acitive AMPs.We further found out that the antimicrobial activity of AMPs relyed on the formation of autophagosomes and autolysosomes.Furthermore,we found that the upregulation of autophagy in lung type Ⅱ epithelial cells can enhance the inhibitory effect on intracellular MTB growth,and the antimicrobial peptides hBDl and LL37 play a significant role in this process.The above conclusions suggest that the increased level of autophagy in lung type Ⅱ epithelial cells can up-regulate the levels of active peptides of antimicrobial peptides hBD1 and LL37 and can exert anti-tuberculosis function.This is a newly discovered mechanism of the body against MTB.