lecture 2 Flashcards
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
in between what do bacterial transposons move within and between?
chromosomes and plasmids
what are 3 different kinds of bacterial transposons?
- Insertion Sequences (IS elements)
- Composite Transposons (tn5 elements)
- Tn3 elements
what are the steps for insertional mutagenesis?
- transposons integrates randomly
- single insertion event
- insertion site marked by Tn
- can be cloned out or adjacent DNA sequenced easily
- in vitro transposition
What are some features of IS elements?
are the simplest bacterial transposons, contain only genes whose products are involved in transposition, inverted terminal repeats are found at the ends, and some IS elements encode for transposase
what does the insertion of an IS element cause?
target site duplication, which is a duplication of sequences at the insertion site
what are the steps of target site duplication?
- two strands of the target DNA are cleaved at different sites
- the IS element is inserted into the gap created by staggered cleavage of the target DNA
- DNA synthesis fills in the gaps on each side of the IS element, producing a direct duplication of the target site
what are two different ways to cut DNA by restriction enzymes?
blunt ends and over hanging ends (sticky ends)
when does a homologous recombination occur?
when a particular IS element is found on both a plasmid and a chromosome
what are some characteristics of composite transposons?
they are a bacterial cut and paste transposons, denoted by the symbol Tn, are created when two IS elements insert near each other, and the two IS elements in composite transposons flank a region that contains one or more genes for antibiotic resistance
what are some features of Tn3 elements?
they are larger than the IS elements, contain genes that are not required for transposition, have simple inverted repeats at each end, and produce target site duplication when they transpose
what is an example of a replicative transposon?
Tn3
what are the steps in the the transposition of Tn3?
- the transposase encoded by Tn3 catalyzes the formation of a cointegrate between the donor and recipient plasmids.
- during this process Tn3 is replicated so there is a copy of the element at each junction in the cointegrate
- Resolvase produced by the tnpR gene resolves the cointegrate by mediating recombination between the two Tn3 elements
- Donor and recipient plasmids separate, each with a copy of Tn3
what are the steps to IVET?
- infect animal with library of IVET fusions
- only purA+ bacteria survive (those with fusions driven by active promoters)
- retrieve survivors
- place on Lac indicitor media
- Lac (-) colony indicating vivo induced fusion so you keep, Lac (+) you discard
what are the steps for differential fluorescence induction?
- make random promoter library fused to GFP integrated into chromosome
- infect cells and release bacteria
- FACS sort for high GFP
- Re-screen for low GFP in absence of host cells
what has STM been widely used for?
on gram positive, negative bacteria, on TB, fungi, and lots of housekeeping genes
What are some genomic and proteomic approaches to idenitfy virulence factors?
microarrays, proteomics and metagenomics
how are microarrays used for virulence factor identification?
genes present in pathogenic strains but absent from non-pathogens, genes expressed during infection
how are proteomics used for virulence factor identification?
proteins present in pathogenic strains but absent from non-pathogens, proteins expressed during infection
how are metagenomics used for virulence identification?
species or genes present in disease states and at site of pathology and absent in healthy controls
