Screening Peptide Inhibitors Using Phage Peptide Library with Isocitrate Lyase in Mycobacterium tuberculosis as Target

When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to stop persistent infections by interdicting the glyoxylate bypass in which the isocitrate lyase（ICL） is the key rate-limiting enzyme and a persistence factor.ICL is the target of anti-TB（TB：tubercular） drugs,which could screen ICL out and effectively inhibit the activity of ICL in Mycobacterium tuberculosis,and because of this,anti-TB drugs can be used to kill persistent Mycobacterium tuberculosis.In this study,the ICL gene of the Mycobacterium tuberculosis H37Rv was cloned successfully and recombinant protein with bioactivity was obtained through the enzyme characteristic appraisal.The specific activity of the recombined ICL is 24μmol·mg-1·min-1.The recombined ICL protein was used as the target,and phages which can specifically combine to ICL were screened in the phage 7 peptide library.According to the results of the ELISA and DNA sequence detection,eventually three 7-peptide chains were synthesized.Then the peptide chains were reacted with ICL,respectively,to detect their inhibitory effects on ICL.The results show that all the three 7-peptide chains possessed varying inhibitory effects on the activity of ICL.This study provided lead compounds for the research and development of new peptide anti-TB drugs.

Isocitrate lyase from Mycobacterium tuberculosis promotes survival of Mycobacterium smegmatis within macrophage by suppressing cell apoptosis

Background Isocitrate lyase （ICL） was previously demonstrated to play a pivotal role in the intracellular metabolism of Mycobacterium tuberculosis （MTB）. Presently several lines of evidence suggest that ICL from MTB （MTB-ICL） may play some roles in the interaction between MTB and host macrophage. However, there has been no research on the interaction between MTB-ICL and host macrophage.Methods MTB-icl and M. smegmatis （MS）-icl genes were amplified by polymerase chain reaction （PCR） and cloned into the E. coli-mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmids pMTB-icl and pMS-icl. Following transformation into MS by electroporation, the expression of pMTB-icl and pMS-icl was verified by reverse transcriptase （RT）-PCR. The expression of recombinant plasmids derived from pUV15 when rMS was phagocytized by macrophage was also verified via fluorescence microscope. Ms 1-2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were used to infect RAW264.7 cells and the survival of intracellular MS was monitored by bacterial culture at 0, 24 and 48 hours after infection. The culture supernatants from macrophage infected by Ms 1-2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were collected and the interferon （IFN）-y and nitric oxide （NO） concentrations were measured by ELISA or by Griess assay, respectively. The apoptosis of macrophage was assayed by the in situ TUNEL technique.Results RT-PCR showed that both pMTB-icl and pMS-icl could be expressed in MS. Fluorescence microscopic observation showed that recombinant plasmids derived from pUV15 （pUV15-iG） could also be expressed in MS when MS were phagocytized by macrophage. Bacterial culture data demonstrated that rMS-pMTB-icl exhibited significantly increased intracellular survival in the murine macrophage cell line RAW264.7 compared with Ms 1-2c, rMS-pUV15 and rMS-pMS-icl. This increased intracellular survival was not accompanied by the upregulation of IFN-y and NO in host macrophage. But a lower apoptosis rate of macrophages infected with rMS-pMTB-icl was observed when compared with macrophages infected with other strains of MS.Conclusions MTB-ICL could promote the intracellular survival of MS. Suppressing the apoptosis of host macrophage may be one of the important mechanisms involved in this increased intracellular survival.

An in vitro and in silico study of anti-dermatophytic activity of gossypol from fruits of Thespesia populnea(L.)Sol.ex Correa

Objective:To isolate,purify,and characterize gossypol from the fruits of Thespesia populnea(L)Sol.ex Correa,test its antidermatophytic activity,identify its targets on the dermatophyte,and confirm the binding of gossypol with the fungal target by molecular docking study.Methods:Gossypol from Thespesia populnea was characterized by high performance liquid chromatography,liquid chromatographmass spectrometry,Fourier transform infrared spectroscopy,and nuclear magnetic resonance.The anti-dermatophytic activity of gossypol was tested against four different dermatophytes,viz.Trichophyton mentagrophytes,Trichophyton rubrum,Microsporum canis,and Microsporum gypseum.Trichophyton mentagrophytes was selected for further studies.The inhibitory mode of action of gossypol on Trichophyton mentagrophytes was determined by analyzing the modulation of gene expression in various pathways of the dermatophyte.Results:Gossypol inhibited all the dermatophytes.The minimum inhibitory concentrations were 12.5μg/mL for Trichophyton mentagrophytes and Microsporum canis and 25μg/mL for Trichophyton rubrum and Microsporum gypseum.The minimum fungicidal concentrations were 50μg/mL for Trichophyton mentagrophytes,100μg/mL for Microsporum canis and Trichophyton rubrum,and 200μg/mL for Microsporum gypseum.Gossypol inhibited the mRNA expression of metalloprotease(MEP4)and isocitrate lyase(ICL).The binding of gossypol with the enzymes was confirmed by molecular docking studies.The best docking poses were found and the low binding energies were recorded with the two target enzymes.Conclusions:Gossypol is a potential antifungal agent and can be further explored as an anti-dermatophytic drug.