Effects Of PPE19 Protein On Mycobacterium Smegmatis Biological Characteristics And Its Survival In Macrophages

Backgrounds:Tuberculosis（TB）is caused by Mycobacterium tuberculosis（MTB）,which has been symbiotic with human for thousands of years.Since the 1950s,with the emergence of isoniazid（INH）,rifampicin（RFP）,ofloxacin,aminosalicylic acid and other first-line and second-line anti tuberculosis drugs,we had made great progress in the treatment of tuberculosis,However,the prevalence of multiple drug resistance（MDR）and extensive drug resistance（XDR）tuberculosis has made the epidemic situation of tuberculosis rebound and spread more widely.So up to now,tuberculosis is still a global public health problem,and it is urgent for people to develop anti tuberculosis drugs for new targets.Since ancient times,China has been good at using all kinds of herbs for disease treatment.In recent years,with the innovation of science and technology,the natural products isolated from animals and plants become an important source of drugs.Euphorbia fischeriana Steud is a plant of Euphorbiaceae,which is distributed in Northeast China.It has been proved that it has anti-cancer and anti-inflammatory effects.In our preliminary studies,dozens of small molecular compounds were isolated from the roots of Euphorbia fischeriana Steud,a herbaceous plant,and the compound 37 with specific inhibitory effect on mycobacterial strains was screened.After Mtb H37Ra strain was treated by compound 37,transcriptome data showed that many genes encoding PE/PPE protein family were expressed differentially genes.The results of q PCR showed that the m RNA of PPE19 decreased significantly with the increase of the concentration of compound 37,suggesting that PPE19 protein might be related to the antibacterial mechanism of compound 37.PE/PPE family genes cover 10%of Mycobacterium tuberculosis genome and encode168 proteins.Among them,69 proteins encode proline-glutamate named PE and 99proteins encode proline-proline-glutamate named PPE.Most of the proteins in this family are found in pathogenic mycobacteria,which may be related to the virulence of mycobacteria.PPE19 protein encoded by ppe19（Rv1361c）belongs to the SVP subfamily of PPE family,which is less investigated.It is known that ppe19（Rv1361c）gene is regulated by sig B,and it is confirmed that PPE19 protein can make Mycobacterium smegmatis show stronger resistance under acid pressure,oxidative pressure and surface activity pressure.However,its effect on biofilm and its role in macrophages have not been described in detail.In order to investigate the biological function of PPE19 protein and the antibacterial mechanism of compound 37,we used Mycobacterium smegmatis,which is nonpathogenic and grows rapidly,to construct a recombinant strain MS/p VV2-His Halotag-ppe19 with overexpressed PPE19 protein.The effects of PPE19 protein on the growth rate of Mycobacterium smegmatis,biofilm formation and the sensitivity to compound 37 in vitro were detected by determing absorbance at 595 nm,crystal violet staining and resazurin staining.CFU method and ELISA method were used to detect the effect of PPE19 protein on the survival rate of M.smegmatis in macrophages and production of inflammatory factors from macrophages.Objectives:1.To clarify the effect of overexpressed PPE19 protein on the biological characteristics of M.smegmatis;2.To explore whether PPE19 protein plays a role in inhibition of compound 37 from Euphorbia fischeriana Steud on the growth of mycobacteria;3.To investigate whether PPE19 protein is involved in the virulence of mycobacteria.Results:1.PPE19 protein promoted the formation of M.smegmatis biofilm.PPE19 was cloned and expressed in M.smegmatis.The absorbance of the bacterial culture at 595 nm,OD₅₉₅ was determined by a microtiter plate reader.There was no significant difference between the two groups.Therefore,we confirmed that PPE19 protein did not affect the growth of M.smegmatis.The results by Crystal violet method showed that PPE19 protein promoted the formation of M.smegmatis biofilm.2.PPE19 protein played a role in inhibition of compound 37 from Euphorbia fischeriana Steud on growth of mycobacteria.The results showed that overexpression of PPE19 protein reduced the sensitivity of M.smegmatis to compound 37,since more compound 37 was needed to achieve the same antibacterial effect.It is suggested that PPE19 was involved in the antibacterial activity of#37,but we need more further study to makesure whether it is a direct target.3.PPE19 protein was involved in the virulence of mycobacteria.The CFU results showed that the overexpression of PPE19 protein enhanced the invasion of M.smegmatis to RAW264.7macrophages and the viability of M.smegmatis in RAW264.7 macrophages.However,the ELISA results indicated that PPE19 promoted the secretion of only TNF-α,but did not alter expression of other cytokines,IL-1βand IL-6.Conclusions:1.The recombinant strain MS/p VV2-His Halotag-ppe19 was successfully constructed,and PPE19 protein was located in the cell wall.2.PPE19 protein did not have significant effect on the growth of M.smegmatis,but promoted the formation of M.smegmatis biofilm and reduced the sensitivity of M.smegmatis to compound 37 from Euphorbia fischeriana Steud.3.PPE19 protein enhanced the survival of M.smegmatis in macrophages,promoted the secretion of TNF-α,but had no effect on the secretion of IL-1βand IL-6.Future studies:1.To find and identify the proteins in RAW264.7 macrophage,which interact with PPE19 protein by Pull down experiment and mass spectrometry.2.To investigate the signal transduction pathways mediated by PPE19 protein to promote the survival of M.smegmatis in macrophages.3.To analyze the changes of humoral and cellular immune responses in mice infected with recombinant M.smegmatis.