Lecture Dos Flashcards
1
Q
Summarize Koch’s Postulates and describe exceptions
A
- Microbes should be in diseased tissue but not in healthy
- exception: some pathogens are colonizers that only cause disease sometimes like opportunistic disease (ex. Staph. aureus, Helicobacter pylori)
- microbes can be isolated from diseased tissues as pure culture
- some organisms are non-cultruable (T. pallidum- syphilis)
- disease may require multiple organism (Hep D)
- microbes can cause disease when inoculted in animal or man
- gonococcus has no animal model
2
Q
what is the expanision of Koch’s original postuate
A
therapeutic or prevetative meausres can eliminate disease
3
Q
Modern version of Koch’s Postulates for defining Virulence factors
A
- Virulence genes are associated with the bacteria that cause disease but are absent or inactive in strainss 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
4
Q
How does updated Koch’s Postulates help identify new pathogens associated with diesease?
A
- Metagenomics: bulk sequencing from the environment (16s rRNA or random) to define new species, new genes and/or new pathways…look at all gneomes of bacteria
- nucleic acid-based identification (microarray, etc.)
- putative pathogen sequence is present during disease and at sites of disease
- nucleic acid sequence of pathogen is absent or reduced in healthy controls
- dose-response correlation (higher number of bacteria = higher infection_
5
Q
Describe the quantification of virulence using animal models
A
- a good animal model recapitulates the major features of human disease
- inbread animals often required for uniformity
- doses of inoculation are often different for people
- end points
- LD50… observing how long it take the animal to die
- time to death
- organ burden… collect organ of interest and quantify bacteria in that organ
- need for surrogate end points
- meausre a specific effect that many correleate well with an actual in vivo endpoint (ex. temp change = death)
- FDA’s two animal rule
- need to show adequate protection against infection in 2 animal species (1 non-rodent)
- the agent is safe for use in humans
6
Q
what are the problems with animal models?
A
- ability of animal model to closely mimic human disease
- different physiology and pathophysiology of diseas… susceptibility to different strains or not at all
- ethics
- cost (genetic screen is very expensive)
- difficult to carry out genetic screens
7
Q
describe the disadvantages of tissue culture models
A
- high throughput screens
- immortalized cells are different from primary cells
- multiple cell types usually co-exist in vivo
- poorly differentiated or non-polarized
- different environmental conditions from in vivo
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8
Q
benefits to tissue culture models
A
cheaper, simpler, controlled environments
9
Q
Simple Eukaryotic host
A
10
Q
Describe Arabidopsis thaliana model
A
- related to cabbage and mustard…first plant to have its entire genome sequenced
- changes are easily observe = useful model
- short life cycle… about 6 weeks from germination to seed maturation
11
Q
describe how arabidopsis is a genetically tractable host….with the example given in class about Pseudomonas aeruginosa
A
- Pseudomonas aeruginosa PA14 infects plants and animals l…….mutants that failed to damage leaves are screened and those mutants show reduced virulence in mouse models…
- so the selected mutant that show no disease symptoms are meausred on arabidopsis and then tested in mice
12
Q
Describe the Caenorhabditis elegans as a model organism
A
- C. elegans….worms– can be frozen and thawed and come back to life & very easy to maintain a population in the lab
- the complete cell lineage of the species has been determined
- C. elegans are the simplest multicellular eukaryotes that have only 1031 cells—–during development, 131 are killed through apoptosis
- one of the simplest organisms with a nervous system (comprising of 302 neurons whose pattern of connectivity has been completely mapped out
- easy to disrupt/manipulte the function of specific genes in nematode by RNA interference (RNAi)….nematode can be soaked in/injected with a solution of dsRNA
- susceptible to several pathogens
- worms can be fed on genetically-modified bacteria which express the gene of interest
13
Q
Describe Dictyostelium amoebae
A
- cellular slime moulds — that normally feed on bacteria
- used as a model organism in molecular biology and genetics and in studies of cell communication, differentiation and programmed cell death
- grow as seperate, independent cells but interation to form multicellular structures when challenged by adverse conditions like starvation
- entire genome has been sequenced
14
Q
Describe drosophila melanogaster (fruit fly)
A
- small, cheap, and easty to grow
- short generation time (~2 weeks) and has the ability to produce large amount of offspring (>800 eggs in a day)
- only has 4 pairs of chromosomes – 1 sex chromosome pair
- entire genome sequenced – sophisticated genetic maipulation of the fly genome has been developed = short gestation time and ability to produce high numbers at low cost
- used to study mechanisms underfly different aspects of genetics, neurobiological disorders, immunity, diabetes and cancer
15
Q
Describe Zebrafish as a model
A
- common & useful model organism for studying vertebrate development and gene function
- not amendable to sophisticated genetic maipulation
- zebrafish embyros develop rapidly within 3 days
- The embryos are large, robust and transparent and develop externally
- drugs are administed directly to the tank
- useful in study the microbial toxins
18
Q
picture of the evolution of bacteria
A
21
Q
Describe the process of cloning a DNA librabry in a plasmid vector
A
- digest both genomic DNA and plasmid vector with same restriction enzyme
- mix digested plasmid and genomic DNA….join fragments with DNA ligase
- transforma into E.coli
- plate on media containng appropriate antibiotic
22
Q
What’s Complementation screenL gain of function phenotype?
A
- make library from wild type organism
- transform library into new non-pathogenic host or a mutant host
- select for “+” transofrmants
- recover transforming fragment
