Identification Hydrogen Sulfide Metabolism Genes Of Mycobacterium And Its Underlying Function

Tuberculosis（TB）is caused by Mycobacterium tuberculosis,which is spread when a patient with tuberculosis vents the bacteria into the air.The disease usually affects the lungs,but it also can extend to affect other parts of the body,causing extra-pulmonary tuberculosis.In the past 2000 years,tuberculosis was responsible for more mortality than any other plagues such as the Black Death,the Spanish flu and COVID-19.Millions of people have died in these deadly pandemics,but their toll pales in comparison with that of tuberculosis（TB）.In the past 2000 years,tuberculosis has caused more than 1 billion deaths,and there are still 1.5 million deaths worldwide each year.However,how and when tuberculosis got to be so deadly has long been a mystery.Now,by tracking the evolution of the genetic mutations that make people more susceptible to the disease,researchers have been able to disclose the rise and fall of tuberculosis over the past 10,000 years and demonstrate how tuberculosis has reshaped people’s immune systems.Lluis Quintana-Murci,a population geneticist at the Pasteur Institute and the French Academy,said:“We are all descendants of patients who survived the past epidemic.At present,tuberculosis is still the world’s largest infectious killer.According to the latest statistics from the World Health Organization,in 2019,about 10 million people suffered from tuberculosis,and 1.4 million people died,including 208,000 people who were infected with HIV.It is one of the top ten globally and the leading causes of death in the world and the main cause of death from a single source of infection.Simultaneously,Multi-drug resistance,extensive drug resistance,and latent infections are exacerbating the severity of the situation.Hydrogen sulfide（H₂S）plays a vital role in maintaining cell physiology and limiting tissue damage.Its abnormality leads to a variety of diseases.Three H₂S generating enzymes have been characterized in mammals:cystathionine b-synthase（CBS）,cystathionineγ-lyase（CSE）,and 3-mercaptopyruvate sulfurtransferase（3-MST）.These enzymes are found in different locations in the eyeball of mammals and their abnormal expression or accumulation of substrates and intermediates can change the level of H₂S by multiple orders of magnitude,ultimately leading to abnormal structure or function in the eye.Werner syndrome,an autosomal recessive genetic disease characterized by accelerated aging caused by mutations in Werner protein.In the cell culture model of Werner syndrome,the use of hydrogen sulfide donors can reverse abnormalities related to the disease and protein aggregation.The normal total plasma Hcy concentration in the human body is 5-10μM,but in hyperhomocysteinemia（HHcy）,the plasma total Hcy level increases（>15μM）,one of the main reasons is the abnormal function of the CBS gene.Increasing the flux of the transsulfur pathway delays aging and prolong life.The above examples are closely related to H₂S levels under physiological conditions.Many prokaryotes also produce hydrogen sulfide（H₂S）gas in their natural environment.However,the physiological and biochemical functions of this gas in bacteria still need further research.In 2011,Evgeny Nudler et al.knocked out the key H₂S synthase cystathionineβ-synthase（CBS）,cystathionineλ-lyase（CSE）and 3-mercaptopyruvate sulfurtransferase（3-MST）in Pseudomonas aeruginosa,Staphylococcus aureus and Escherichia coli,revealing that endogenous H₂S can reduce the oxidative stress exerted by antibiotics,thereby protecting bacteria against the killing effect of antibiotics.In 2020,Adrie JC Steyn et al.confirmed that H₂S can stimulate the respiration and pathogenesis of Mycobacterium tuberculosis from the perspective of exogenous H₂S.They showed that Mycobacterium tuberculosis can derive hydrogen sulfide from the host to promote its persistence and survival.In addition,they also found that exogenous H₂S can regulate the expression of mycobacterium tuberculosis copper metabolism-related genes,Dos regulatory factors,cell wall and cell processes,and PE＿PGRS family genes.In 2019,Paul R.F.Cordero et al.discovered that Mycobacterium tuberculosis can use CO from the host to meet the nutrients it needs during dormancy,and thus it can stay in the human body for a long time.Therefore,H₂S and CO may have a lot of overlap in their physiological functions.In order to further study the physiological effects of H₂S on mycobacteria,we estimated the possible key genes sse B（Rv2291）,CBS（Rv1077）,sse A（Rv3283）,Cys A2（Rv2291）,sse B（Rv1077）,sse A（Rv3283）,Cys A2（Rv0815c）and Cys A3（Rv3117）.Through homology comparison,we identified the homologous genes of sse B（Rv2291）and CBS（Rv1077）in Mycobacterium smegmatis as MSMEG＿5270 and MSMEG＿3238,respectively.Subsequently,we knocked out MSMEG＿5270 and MSMEG＿3238 in Mycobacterium smegmatis,and complemented Rv1077 or Rv2291to the knockout strain.Through experiments,we found that deletion of MSMEG＿5270（ΔMs＿5270）reduced the production of H₂S,while knocking out MSMEG＿3238（ΔMs＿3238）promoted the production of H₂S.Therefore,we conclude that MSMEG＿5270 promotes the production of H₂S.On the contrary,MSMEG＿3238 has the function of metabolizing H₂S.Furthermore,antibiotic experiments,we found that the knockout of MSMEG＿5270 hampered the resistance of bacteria to a variety of antibiotics（such as Norfloxacin,Ofloxacin,Gatifloxacin,Rifampicin,Gentamicin,and Vancomycin）.However,it promoted the tolerance to Isoniazid and Ethionamide.We estimated that H₂S may promote the production of MSH（Mycothiol）,thus promoting the bactericidal ability of Isoniazid and Ethionamide.Interestingly,knocking out MSMEG＿3238 resulted in a large amount of H₂S in mycobacteria.Generally speaking,its survival ability should be stronger under the action of multiple antibiotics.However,my result is that knocking out MSMEG＿3238 caused the bacteria to be susceptible to multiple antibiotics（such as Norfloxacin,Ofloxacin,Gatifloxacin,Rifampicin,Gentamicin,Vancomycin,Isoniazid,and Ethionamide）.Further experiments revealed that knocking out MSMEG＿3238 results in eliciting the production of reactive oxygen species（ROS）to be produced in the bacteria,which impedesΔMs＿3238 to survive under antibiotics stress.Paradoxically,when the antibiotic concentration is too high,knocking out MSMEG＿3238 increases the resistance of mycobacteria to antibiotics.For example,when we use 60MIC norfloxacin to treat wild-type Mycobacterium smegmatis（WT）orΔMs＿3238,ΔMs＿3238 survival ability was significantly enhanced.By RT-PCR analysis of several key genes of the Dos system,it was found that the expression of these genes inΔMs＿3238 was significantly up-regulated.These results show that endogenous hydrogen sulfide can indeed activate the Dos system,however its mechanism needs to be further studied.In addition,we also found that hydrogen sulfide can promote the respiration of mycobacteria（such as Mycobacterium smegmatis or Mycobacterium abscessus）,because by measuring the growth curve of knockout strains or exogenous supplementation of hydrogen sulfide donor（Na SH）,both show H₂S can promote the growth of mycobacteria.By infecting macrophages（THP-1）,we found that H₂S can promote the survival of mycobacteria in macrophages.Phagocytosis is an important way for bacteria to enter macrophages.It is controlled by the nature of the host receptors and the ligands exposed on the bacteria.Phagocytosis is the primary defense mechanism for macrophages to isolate bacteria from phagosomes.Through experiments,we found that H₂S can affect the phagocytosis of macrophages,and this effect may be attributed to the ability of H₂S to reshape the mycobacterial cell wall,thereby affecting the ligand exposed on the bacteria.In 2020,Adrie J.C.Steyn et al.revealed that exogenous H₂S affects multiple metabolic pathways of Mycobacterium tuberculosis,including the PE＿PGRS family.The ability of Mycobacterium tuberculosis to escape the host’s immune system through a variety of mechanisms is one of the convoluted factors that make it has become a successful pathogen.Deciphered whole genome sequencing of Mycobacterium tuberculosis revealed precise information,including the important PE/PPE protein family.The proteins of the PE/PPE family are named based on the conserved PE（proline-glutamic acid）and PPE（proline-proline-glutamic acid）domains at the N-terminus.Their distribution in the genome of tuberculosis bacteria is fascinating,mostly in clusters.The PE subfamily is derived from the conserved P（Pro）-E（Glu）domain at its N-terminus.The family contains 100 members and the N-terminus domain consists of 110 conserved amino acid residues.However,the length of the C-terminal sequence is different,and some are PGRS,which is a repetitive sequence rich in GC nucleotides.The PE subfamily contains 61 PE＿PGRS genes and 37 PE genes according to the differences in the domains.The PPE subfamily distinguished by the N-terminal Pro（P）-Pro（P）-Glu（E）sequence contains 69 members,but according to the difference in the C-terminal domain,it can be divided into three subclasses,including PPE＿SVP,PPE＿PPW and PPE＿MPTR subfamily.The unique sequences of these proteins imply their special physiological functions during Mycobacterium tuberculosis infection.PE/PPE family proteins are often arranged in pairs on the genome and are closely related to the ESX secretion system.Bioinformatics analysis shows that the evolution of PE/PPE genes is related to the ESX secretion system.Studies have reported that some PE/PPE proteins are secreted into the mycobacterial cell wall through the ESX-5 secretion system.The PE/PPE family plays a key role in the pathogenic pathway of tuberculosis.Most of them exist exclusively in pathogenic mycobacteria,which may be virulence factors.It is estimated that its function may be related to the antigen presentation of Mycobacterium tuberculosis.Antigenic variation is related to the function of regulating the immune effect of macrophages.According to reports,during the process of host cell infection by tuberculosis,most of the PPE family proteins are up-regulated and secreted on the cell surface,playing an important role in the interaction between Mycobacterium tuberculosis and the host.Therefore,the study of proteins in this family has great inspiration for finding drug targets against Mycobacterium tuberculosis.These characteristics stimulated our desire to explore the genes of this family.Through literature and databases,we focus on the functional research of PE＿PGRS59 and PE＿PGRS18.A study found that the single nucleotide deletion of the PE＿PGRS59（Rv3595c）homologue in H37Ra resulted in the formation of two PE＿PGRS proteins in H37Ra,namely MRA＿3634 and MRA＿3635.Both MRA＿3635 and Rv3595c are relatively stable and have the same spherical domain.In addition,compared with Rv3595c,MRA＿3635 and MRA＿3634 have stronger hydrophobicity and more phosphorylation sites,which indicates that Rv3595c may be a key virulence factor.Analyzing its sequence,PE＿PGRS59,a member of the PE＿PGRS family of Mycobacterium tuberculosis,contains four calcium-binding motifs.Therefore,we constructed an overexpression strain of PE＿PGRS59 in Mycobacterium smegmatis for further study.Our results show that PE＿PGRS59 promotes the production of endogenous H₂S of Mycobacterium smegmatis and enhances the ability of Mycobacterium smegmatis to resist multiple stress stresses,including SDS,low p H,antibiotics,H₂O₂,macrophage killing.We also revealed that PE＿PGRS59 activates the ERK,JNK,and NF-KB signal pathways,and promotes the production of tumor necrosis factor-α（TNFα）,interleukin（IL）-6,IL-10 and IL-1β.By using Lyso Tracker dye,we found that PE＿PGRS59inhibits the autophagy process of macrophages.Using FITC/PI dyes,it was found that PE＿PGRS59 inhibited the apoptosis of macrophages,which was dependent on caspase.In general,we revealed that PE＿PGRS59 inhibited the autophagy and apoptosis of macrophages,aggravated inflammation and cell necrosis,and promoted the survival of Mycobacterium smegmatis.PE＿PGRS18 belongs to another member of the PE＿PGRS family.Srivastava et al.found that PE＿PGRS18 as up-regulated three folds in the lungs of infected mice,which is very likely to become a potential drug target.In order to illustrate the function of PE＿PGRS18,we also expressed PE＿PGRS18 in Mycobacterium smegmatis,and found that PE＿PGRS18 promoted the production of endogenous H₂S in mycobacteria.Unlike Ms＿PE＿PGRS59,Ms＿PE＿PGRS18 can significantly reduce the production of IL-6,IL-1β,and IL-10 after infecting macrophages for 24 hours.However,both of them inhibited the apoptosis of macrophages and promoted intracellular survival.These results reflect the diversity and similarity of PE＿PGRS family gene functions.In general,these results mainly emphasize the importance of PE＿PGRS18 and PE＿PGRS59 for the virulence of Mycobacterium tuberculosis,which can manipulate the inflammatory response and dictate the fate of host cells.These studies will help understand the pathogenesis of Mycobacterium tuberculosis and promote the development of drug targets.