Analysis Of Biofilm-related Genes In Mycobacterium Tuberculosis By Constructing And Screening H37Ra Mutant Library

Tuberculosis(TB)is an infectious disease mainly caused by pathogenic Mycobacterium tuberculosis(MTB).When MTB invades the organism,it rapidly spreads through the respiratory and digestive tracts,then infects other tissues and organs such as the lung,kidney,and nerves,and finally causes active tuberculosis;or,hides in the body for a long time and causes latent tuberculosis infection(LTBI).Each year,TB causes more than 10 million new cases and 1.5 million deaths(range,1.4-1.6 million)and covers all age groups and all regions,which has caused great concern at home and abroad.Due to the abuse of multiple antibiotics,the global movement of the population and the prevalence of HIV,it has become a new crisis in the diagnosis and treatment of TB.At present,many laboratories around the world are devoted to analyzing the comprehensive pathogenesis of MTB and the survival mechanism in the organism from the genome aspect.They aim to provide new ideas and techniques for the diagnosis and treatment of tuberculosis.In this study,we constructed a transposon mutant library in M.tuberculosis H37Ra,screened biofilm mutants,and mapped the transposon insertion site by next generation sequencing.Besides,we analyzed the characteristic of biofilm-related genes and their coding products by bioinformatics.This study is to establish a foundation of the molecular mechanism research of MTB for our laboratory,and to provide a model for screening MTB biofilm mutants and get novel mutant material,which helps to explain biofilm-related mechanism of MTB.1 Construction of H37Ra transposon mutant libraryThe construction of H37Ra transposon mutant library aims to provide a means for our laboratory to study gene function of slow-growing mycobacteria.To get phages ?MycoMarT7,we amplified a large number of phages using Mycobacterium smegmatis,and select phages with temperature-sensitive property,which can form plaques only at 30? not 37?.Continuously,the temperature-sensitive phage was refined to increase the titer to 1×1011 pfu/ml.For transduction,the phage stock added to H37Ra.After incubation,remove suspension and plate different dilutions on 7H10 medium with kanamycin.These plates cultured at 37? for several weeks until colonies appeared.Finally,select the optimal primers and identify kanamycin resistant gene by PCR.This standardized process for a transposon mutant library in Mycobacterium tuberculosis is suitable for our laboratory.By this process,we constructed a library of 35,800 mutants.The transduction efficiency was up to 7.96×10-62 Screening and analysis of biofilm-related genesBiofilms are of great significance for MTB in many significant aspects.Here,biofilm formation ability was determined as the screening direction of H37Ra transposon mutant library.After trying different media,culture methods and biofilm detection methods,we chose a practical means to select H37Ra biofilm mutants.Select 7H9 liquid medium supplemented with little Tween-80,seal and culture for three weeks,loosen the cap and shake slightly at the end of third week,and then culture in a micro-oxygen environment.Directly observe and select mutants that cannot form biofilm and form defective biofilm.Next,extract the whole genome by the CTAB method and map the transposon insertion site by NGS.Until now,we have screened 891 mutants,among which 28 mutants have special biofilm-forming capabilities,mapping 21 mutant sites and preliminarily analyzing 10 biofilm formation-related genes.We made a homologous comparison between transposon insertion sites in H37Ra genome and genes in H37Rv genome.Finally,we analysed biofilm-related genes using various databases and tried to demonstrate the link between MTB genotype and phenotype of biofilm formation.These genes are related to intermediary metabolism and respiration,cell wall,lipid metabolism and conserved hypotheticals,which can contribute the anti-stress survival of MTB.By this screening and analysis,we also provided valuable mutant materials for subsequent research of mycobacteria biofilm.Finally,we constructed rMS::pALACE-Rv3099c to verify preliminarily that Rv3099c gene can play a role in biofilm growth.