Mycobacterium Von Willebrand Factor Type A Protein MSMEG₃641 Is Involved In Biofilm Formation And Intracellular Survival

Tuberculosis?TB?caused by Mycobacterium tuberculosis infection remains one of the top 10 causes of death and ranks top one from a single infectious agent?above HIV/AIDS?.In 2018,there is an estimated 10 million TB incidence.Mycobacterium tuberculosis,in addition to infecting the human lung,can also infect other parts.Worldwide,around a quarter of people are infected with M.tuberculosis and are therefore at risk for tuberculosis.In addition,the emergence of drug-resistant tuberculosis has brought great trouble to the treatment of clinical tuberculosis.In 2018,there were approximately 50 million new cases of rifampin-resistant tuberculosis,78% of which were multidrug-resistant tuberculosis and China accounts for about 14%.Bacterial biofilm is the complex structure formed by bacteria encapsulated by extracellular polymeric substances?EPS?.EPS is mainly composed of polysaccharides,proteins,and DNA.The formation of biofilm can make bacteria obtain drug resistance by reducing metabolic activity,forming retained bacteria,and preventing antibacterial agents from entering the bacteria.The formation of biofilms is regulated by a variety of metabolisms in bacteria.Among them,cell wall-related components are closely involved in the formation of biofilms,such as the synthesis of peptidoglycans,glycopeptide lipids,fatty acids and pilus of bacteria.The quorum sensing system,toxin-antitoxin and numerous transcriptional regulatory factors can regulate biofilm formation.Interfering with various stages of bacterial biofilm formation and destroying important components on bacterial cell walls can affect biofilm formation.Von Willebrand factor was first discovered in coagulation proteins.Proteins containing von Willebrand factor type A?vWA?domains are involved in multiple physiological pathways,such as the membrane system formation,cell migration,differentiation,adhesion,signal transduction,tumor malignant transformation,etc.The binding of vWA to the ligand is mainly mediated by metal ion-dependent adhesion sites.The eukaryotic vWA domain is homologous to prokaryotes,but in prokaryotes,the vWA domain has been rarely studied.Pil Y1 containing vWA domain can induce the virulence of Pseudomonas aeruginosa through inductive surface contact,and Enterococcus faecium pilin can change the virulence of bacteria through vWA domain.So the study of this domains is particularly important.Changes of cell wall components can often cause changes in bacterial colony morphology,and cell wall components are closely related to biofilm,bacterial persistence,and bacterial resistance.Our goal is to screen and study unreported key factors that can affect the formation of bacterial biofilms and bacterial resistance,with a view to providing new insights into the formation of biofilms and bacterial resistance.M.smegmatis is a good model strain for studying mycobacteria.In this paper,we used the transposon insertion mutation library established by M.smegmatis to screen for strains with changed colony morphology,and successfully obtained a strain Strains with altered colony morphology.Through PCR and sequencing identification,we determined that the transposon insertion site of the mutant strain was located between nucleotides 208 and 209 of the gene MSMEG₃641;through conservative domain analysis,we found that MSMEG₃641 contains a conserved vWA domain,and contains metal ion-dependent binding sites;by transmembrane domain analysis,we found that the protein has a strong transmembrane domain;at the same time,through in vitro biofilm culture,we demonstrated that the biofilm formation ability of the mutant strain was significantly reduced;through bacterial migration experiments,we found that the slide ability of the mutant strain was significantly weakened.Subcellular localization experiments suggested that the protein is located on the surface of bacteria.Cell permeability tests assay have shown that mutations in this protein can significantly increase bacteria's permeability to the fluorescent dye ethidium bromide;by measuring the optical density of the bacterial culture with or without TWEEN 80,we found that the protein mutation reduces the bacteria-bacteria aggregation.Through the experiment of norfloxacin-induced persistent bacteria formation,we identified that mutations in this protein caused bacteria to be more sensitive to norfloxacin and reduced the ability of bacteria to form persisters.By infecting THP-1 induced macrophages,we found that the mutant strain's ability to infect macrophages was significantly reduced,and its ability to survive in macrophages was greatly decreased.At the same time,through fatty acid sensitivity experiments,we speculated that the protein might affect the fatty acid metabolism of bacteria through the glyoxylate cycle,resulting in structural changes in the cell wall.In summary,we found that M.smegmatis vWA protein MSMEG₃641 can change the structure of bacterial cell walls and affect the formation of bacterial persistence and the survival in macrophages.This study not only broadened the scope of vWA protein research in prokaryotes and provided new insights into bacterial biofilm formation,but also provided new ideas for mechanisms of bacterial resistance and the development of antibacterial drugs.