Cloning of a functional Salmonella SPI-1 type III secretion system and development of a method to create mutations and epitope fusions in the cloned genes.
Bacterial type III secretion systems have significant potential to be harnessed for beneficial purposes including vaccine development, anti-cancer therapies, strategies to counteract harmful bacteria-host interactions, and evolutionary studies. The ability to clone and manipulate type III secretion systems would allow researchers to perform novel experiments that would progress the biotechnological development of the potentially positive uses of these systems. Here, we report the cloning of the entire Salmonella pathogenicity island 1 (SPI-1) type III secretion system on a single DNA fragment that is contained on a self-transmissible plasmid vector for convenient transfer to alternate hosts. We demonstrate that the cloned SPI-1 type III system is functional for secretion and translocation via complementation of an S. typhimurium Delta SPI-1 strain. We also present a convenient method to construct mutations and epitope fusions in the cloned type III genes and demonstrate that the engineered substrate protein fusions are recognized by the cloned type III system. We transferred the cloned SPI-1 type III system into bacterial strains of different genera and found that there is a SPI-1 gene expression defect in these strains. The results describe a novel strategy for cloning and manipulation of bacterial secretion system gene clusters and provide a foundation for future studies to develop the beneficial uses of cloned type III secretion systems.
Wilson
James W
JW
Program in Molecular Pathogenesis and Immunity, Department of Microbiology and Immunology, Tulane University Health Sciences Center, 1430 Tulane Avenue, Room 5728, New Orleans, LA 70112, USA. jwilson4@tulane.edu
Nickerson
Cheryl A
CA
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D000939:Epitopes; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; C094578:Salmonella invasion protein C; C118287:SipA protein, Salmonella; C419942:Spi1 protein, Salmonella