The Effect And Mechanism Of5-aminolevulinic Acid Photodynamic Therapy On Mycobacterium Abscessus

Worldwide infections caused by non-tuberculosis mycobacteria are gradually increasing year by year,among which Mycobacterium abscessus is one of the most common one,resulting in serious respiratory,skin and mucosal infections in human beings.In recent years,M.abscessus has become a major emerging pathogen and one of the most antibiotic-resistant non-tuberculosis mycobacteria.The multidrug resistance and the inherent resistance of M.abscessus have brought great challenges to clinical treatment.Therefore,there is an urgent need to better understand the pathogenesis and find new therapies against M.abscessus infection.Photodynamic therapy is a new treatment modality that does not induce drug resistance.Since the dawn of photodynamic therapy at the beginning of the last century,scientists have discovered that the combination of photosensitizers and light can kill a variety of microorganisms.5-aminolevulinic acid photodynamic therapy（ALA-PDT）has been widely utilized in clinical treatment of various skin tumors.However,whether ALA-PDT is feasible in anti-non-tuberculosis mycobacteria treatment,especially for M.abscessus infection,still requires a large number of experimental data.ALA-PDT could effectively inactivate Mycobacterium fortuitum,Mycobacterium marine,and Mycobacterium smegmatis in vitro.Therefore,we speculate that ALA-PDT can also effectively inactivate be a new clinical treatment the non-tuberculous mycobacterial-based infections as well.In order to evaluate the extent of killing of M.abscessus by ALA-PDT and its underlying molecular mechanism,we first conducted a series of bactericidal experiments in vitro to evaluate their survival.We then focused on the effect and mechanism of ALA-PDT on M.abscessus infection in vivo using a mouse model.The main research contents include:（1）Mechanism underlying the killing of M.abscessus by ALA-PDT in vitro1)ALA-PDT induced the death of M.abscessus by promoting the production of reactive oxygen speciesIn order to study the level of killing of M.abscessus by ALA-PDT,we first established a bactericidal model and found that ALA-PDT could significantly kill M.abscessus in vitro.Further studies showed that ALA-PDT promoted the ferroptosis-like death of M.abscessus,in which the production of reactive oxygen species was the main factor.To explore alternative molecular mechanisms,we performed transcriptome sequencing analysis to M.abscessus treated with ALA-PDT or not.The results demonstrated that ALA-PDT might induce bacterial ferroptosis-like death by regulating bacterial iron metabolism,in which MAB＿4773,encoding a heme oxygenase,involved as the core factor.By constructing MAB＿4773 overexpression strain,we found that MAB＿4773 boosted the production of Fe²⁺in bacteria to affect bacterial iron metabolism.Collectively,ALA-PDT might increase the production of Fe²⁺by promoting the expression of MAB＿4773,which resulted in bacterial ferroptosis-like death.To identify the mechanism in which ALA-PDT promoted the generation of reactive oxygen species in M.abscessus,we determined the level of cell membrane integrity and permeability,and the extent of killing by antibiotics.The results demonstrated that killing by ALA-PDT was due to an increase in the production of reactive oxygen species,promoting an increase cell permeability due to loss of of cell membrane integrity,contributing to the role of antibiotics on M.abscessus inactivation.2)ALA-DPT damaged the M.abscessus persistent infection by upregulating membrane glycopeptide lipidsThe surface morphology of M.abscessus can affect its ability to sustain infection.Although ALA-PDT could promote bacterial ferroptosis-like death and the killing effect of antibiotics,the effect on the surface morphology of M.abscessus and its ability to sustain infection still remained ambiguous.We examined the morphology of the single colony and the intracellular survival in macrophages.The results showed that ALA-PDT could make the single colony morphology of M.abscessus smoother and the ability of forming persistent infection decreased.Further studies showed that ALA-PDT promoted the production of glycopeptide lipids on the surface of M.abscessus.In conclusion,ALA-PDT promoted the smoothness of bacterial surface and reduce persistent infection by increasing the production of glycopeptide lipids on bacterial surface.Furthermore,Overexpression MAB＿4773 could also increase the smoothness of M.abscessus surface,suggesting that MAB＿4773 may also be involved in the reduction of ALA-PDT on the M.abscessus persistent infection ability by ALA-PDT.3)ALA-PDT killing M.abscessus intracellular by activating autophagy by promoting the production of reactive oxygen speciesM.abscessus is a typical intracellular bacterium,which can resist the phagosome defense mechanism and reproduce within host cells.To further understand the bactericidal mechanism of ALA-PDT,we performed intracellular survival assay on M.abscessus-infected macrophages and found that ALA-PDT assisted the killing effect of intracellular M.abscessus.To investigate the cause of this killing effect,we performed transcriptome analysis on infected macrophages treated with ALA-PDT.The results found that ALA-PDT up-regulated the expression of genes related to the autophagy pathway.By detecting the expression of autophagosomes and related proteins,we discovered that ALA-PDT induced the generation of autophagosomes and the expression of autophagy-associated proteins.Autophagy inhibitor treatment further confirmed that ALA-PDT killed intracellular M.abscessus by activating autophagy.Further experiments on reactive oxygen species proved that ALA-PDT activated autophagy and kill intracellular M.abscessus by promoting the production of reactive oxygen species.To identify the specific molecules involved in killing intracellular M.abscessus by ALA-PDT,transcriptome sequencing analysis was performed on macrophages treated with ALA-PDT.The results showed that EP300 played an essential role in macrophages treated with ALA-PDT.EP300 encodes an acetyltransferase.We found that ALA-PDT reduced EP300 level in macrophages.Further detection of the acetylation level of macrophages treated with ALA-PDT confirmed that ALA-PDT significantly altered the intracellular acetylation level,indicating that ALA-PDT might affect the epigenetic characteristics of cells.The addition of EP300 activator further demonstrated that ALA-PDT reduced the expression of EP300 and affect the epigenetic modification of cells,thereby promoting autophagy and annihilating the survival of M.abscessus.（2）Mechanism of ALA-PDT treating M.abscessus skin and soft tissue infection in miceWe demonstrated that ALA-PDT could kill M.abscessus in vitro and investigated its mechanism.To further discuss the feasibility of ALA-PDT utilization clinically and the potent mechanism,we established a mouse model with M.abscessus skin and soft tissue infection and found that ALA-PDT significantly reduced the size of skin and soft tissue abscesses.In vivo fluorescence imaging and bacterial colony forming unit assay proved the anti-M.abscessus effect of ALA-PDT in infected mouse skin soft tissue.In order to explore the effect of ALA-PDT on mouse skin and soft tissue and its specific molecular mechanism,we extracted mouse skin and soft tissue after ALA-PDT treatment for histopathological observation,and found that ALA-PDT treatment significantly changed the immune response in skin and soft tissue abscesses.RT-q PCR,Western-blotting and immunofluorescence experiments further evaluate the effect of ALA-PDT on the expression of cytokines such as IL1βand IL6 and autophagy-related protein LC3,which indicated that ALA-PDT remarkably changed the expression of inflammatory factors and autophagy-related proteins in skin and soft tissue abscesses.Collectively,ALA-PDT significantly dampened the survival of M.abscessus in mouse skin and soft tissue by regulating the immune and autophagy responses.In conclusion,this study investigated the effect of ALA-PDT against M.abscessus and its molecular mechanism both in vitro and in vivo.This study shed lights on the new anti-M.abscessus treatments and provided basis for further clinical application of ALA-PDT against M.abscessus infection.