what is the expansion for Koch’s original postulates?
Therapeutic or preventative measures can eliminate disease
what are the molecular versions of Koch’s postulates?
- virulence genes are associated with the bacteria that cause disease but are absent or inactive in strains that fail to cause disease
- disruption of virulence gene in virulent strain leads to avirulence
- introduction of cloned gene into avirulent strain gives virulence
- redundant virulence factors, gene may not be expressed in the avirulent strain
what are the two ways Koch’s postulates helped identify new pathogens associated with disease?
- metagenomics: bulk sequencing from the environment to define new species, new genes, and/or new pathways
- Nucleic acid Based identification (microarrary): putative pathogen sequence is present during disease and at sites of disease, nucleic acid sequence of pathogen is absent or reduced in healthy controls, and dose response correlation
what are some quantification points of virulence when using animal models?
- a good animal model recapitulates the major features of human disease
- end points (LD50, time to death, organ burden)
- need for surrogate end points
- FDA’s two animal rule
what are some problems with animal models?
- ability of animal model to closely mimic human disease
- ethics
- costs
- difficult to carry out genetic screens
What is the one issue with tissue culture models?
they can never completely replace animals
why is arabidopsis thaliana a good model?
first plant to have its entire genome sequenced, changes in the plant are easily observed, life cycle is short about 6 weeks
why is caenorhabditis elegans a good model?
- easy to maintain in the lab
- can be frozen and thawed
- complete cell lineage of the species has been determined
- is the simplest multicellular eukaryote
- very simple nervous system
- easy to disrupt function of certain genes
- susceptible to several pathogens
- can be fed genetically modified bacteria which can express the genes of interest
what makes dictyostelium amoebae a good model?
- been used as a model in molecular biology, genetics, cell communication, differentiation, and programmed cell death
- entire genome has been sequenced
why is drosophila melanogaster a good model?
- small and easy to grow in the lab
- short generation time and produce offspring in great numbers
- only has four pairs of chromosomes including one sex chromosome
- easy to manipulate genetically
why are zebrafish good models?
- embryos develop rapidly and externally
- drugs may be administered by adding directly to the tank
- useful in the study of microbial toxins
what are molecular approaches to study virulence factors?
- secreted proteins or abundant surface molecules- easy to purify
- highly antigenic proteins- use serum antibodies as probes
- stage specific genes/genes expressed only in the host- microarrays, proteomics, promoter traps
- gene products required for survival of the pathogen in cells or in animals- signature tagged mutagenesis
- other gene products that are unique to pathogens- comparison of genomes
how genomic islands defined?
defined by specific niches rather than genetic composition
what are ways to find virulence factors biochemically?
- isolate proteins, fractionate and study in the appropriate detection system
- sequence the protein, go back to DNA to find a gene
- has been widely used in the early molecular biology era
- tedious “fishing expedition”
what are ways to find virulence factors through molecular biology?
- make a library of genome fragments
- use cloning vector to insert the fragments into and transfer the vector into the appropriate host
- select clones expressing the virulence factor in the appropriate test system
What are the steps in cloning a DNA library in a plasmid vector?
- digest both genomic DNA and plasmid vector with same restriction enzyme
- mix digested plasmid and genomic DNA. Join fragments with DNA ligase
- Transform into E. coli
- Plate on media containing appropriate antibiotic
what are the steps for complementation screen?
- make library from wild type organism
- transform library into a new non pathogenic host or mutant host
- select for “+” transformants
- recover transforming fragment
What are some microbial genetic tools to manipulate DNA?
- use of cloning and expression vectors
- targeted mutations to the chromosome
- gene knockouts (random or directed)
what are some scenarios that occur when a foreign DNA is introduced into a cell?
- replicated autonomously with the vector
- gene replacement by homologous integration
- gene duplication
- ectopic integration
What is a transposable element?
also known as transposon, they are specific sequence of DNA, found in the genomes of many kinds of organisms, and are structurally and functionally diverse
what are the three types of transposition?
- cut and paste
- replicative transposition
- retrotransposition
what is the cut and paste transposon?
it is excised from one genomic position and inserted into another by an enzyme (transposase), which is usually encoded by the transposon itself
what is the replicative transposon?
copied during the process of transposition
what is a retrotransposon?
produces RNA molecules that are reverse transcribed into DNA molecules, these DNA molecules are subsequently inserted into new genomic positions
