Pathogen Lab: Adaptations to host environment and gene deletions
C. neoformans = fungal pathogen infecting individuals with compromised immune systems.
Requires high concentrations of glucose available in the environment, normally only 2% is available in the host environment as an adaptive defense of the host (that's us).
It will resort to the use of other pathways to make use of other carbon sources: ethanol, acetate, lactic acid. Enzymes involved in glucomutagenesis (conversion of alternative sources to glucose) may be key in their pathogenesis. So, when we delete a gene encoding one of these enzymes, a phenotype should appear at least in glucose deprived conditions. If not, we may have deleted the wrong gene (in which case we would have to make another single mutant) or the strain has homologous genes (more than one gene that will do the job and make that enzyme). Then we'll have to make a double mutant: some bug with both genes deleted.
Heat shock proteins: upregulated in high temperatures for the protection of the cell.
They tend to build up in various stressful conditions. The host has adaptations to prevent the invasion and growth of pathogens, so these may be important pathogenic adaptations as well.
Yesterday I used the Biolistic Gun (just think of a high pressure contraption that looks like a coffee maker or an easy bake oven, not so much a gun) to delete genes in wild type strains with intact genomes, and pre-existing mutants missing one of the above mentioned genes. I spread some cells on sorbitol/sugar plates and shot them. I shot them with DNA on gold beads, special DNA constructs the research associate made with PCR. The constructs have DNA that code for nothing but can be inserted into that specific gene by recombination. In addition, they have a gene sequence encoding a specific antibiotic resistance. This resistance, allows us to select for mutants with the gene construct successfully inserted into their genome on antibiotic plates. Because they're resistant, they'll grow. Next week is phenotyping and genetic screening if transformants grow on the antibiotic plates.
Requires high concentrations of glucose available in the environment, normally only 2% is available in the host environment as an adaptive defense of the host (that's us).
It will resort to the use of other pathways to make use of other carbon sources: ethanol, acetate, lactic acid. Enzymes involved in glucomutagenesis (conversion of alternative sources to glucose) may be key in their pathogenesis. So, when we delete a gene encoding one of these enzymes, a phenotype should appear at least in glucose deprived conditions. If not, we may have deleted the wrong gene (in which case we would have to make another single mutant) or the strain has homologous genes (more than one gene that will do the job and make that enzyme). Then we'll have to make a double mutant: some bug with both genes deleted.
Heat shock proteins: upregulated in high temperatures for the protection of the cell.
They tend to build up in various stressful conditions. The host has adaptations to prevent the invasion and growth of pathogens, so these may be important pathogenic adaptations as well.
Yesterday I used the Biolistic Gun (just think of a high pressure contraption that looks like a coffee maker or an easy bake oven, not so much a gun) to delete genes in wild type strains with intact genomes, and pre-existing mutants missing one of the above mentioned genes. I spread some cells on sorbitol/sugar plates and shot them. I shot them with DNA on gold beads, special DNA constructs the research associate made with PCR. The constructs have DNA that code for nothing but can be inserted into that specific gene by recombination. In addition, they have a gene sequence encoding a specific antibiotic resistance. This resistance, allows us to select for mutants with the gene construct successfully inserted into their genome on antibiotic plates. Because they're resistant, they'll grow. Next week is phenotyping and genetic screening if transformants grow on the antibiotic plates.