Ms Asma Ul Husna
Department of Microbiology and Immunology
University of Melbourne
I’ve generated a series of single, double and triple deletion mutants in methionine biosynthetic genes and high-affinity transporter MetNIQ by site-directed mutagenesis. All these biosynthetic and high affinity transporter mutants were characterized in M9 minimal media, HeLa cells and Raw macrophages for growth and intracellular survival and then were tested for virulence in mice. I’ve shown that de novo methionine synthesis is not essential for bacterial virulence in mice. High affinity transporter alone is also not essential bacterial virulence. Mutants deficient in biosynthesis and high affinity transporter both become attenuated in murine host. I made a deletion in an enzyme named Pfs which codes for S-adenosylhomocysteine/Methylthioadenosine Nucleosidase and found that Pfs deletion mutant is severely attenuated in mice which says that Pfs is essential for Salmonella Typhimurium virulence in mice. To explore the reason, S-adenosyl homocysteine hydrolase, an enzyme from Legionella pneumophila was expressed in Salmonella Typhimurium Pfs mutant and it reveals that the accumulation of S-adenosyl homocysteine is inhibitory for S-adenosyl methionine dependent methyltransferases. So, the immediate removal of S-adenosyl homocysteine is necessary.
Techniques/ExpertiseCell culture: Culture of HeLa cells and raw macrophages, assay for intracellular survival and replication of Salmonella strains
Molecular biology: Recombinant DNA techniques, transfection, transduction, overexpression of genes, gene deletion by site directed mutagenesis, nucleic acid (DNA, RNA, plasmid) extraction; protein purification and quantification, primer design, PCR, qPCR; Multiplex PCR, Electrophoresis (SDS- PAGE, agarose gel), western-blot, ELISA.
Immunology: Flow Cytometry.
Microbiology: Clinical and environmental sample collection and handling, Media preparation, Microscopy, Biochemical test (Simmon’s Citrate test, Motility Indole Urease test, Kligler’s Iron agar and Lysine decarboxylase test), Antimicrobial susceptibility testing, Plaque assay, Bacteriophage assay.
Animal work: Intravenous