Lecture Dos

Question 1

Summarize Koch’s Postulates and describe exceptions

Answer 1

1. 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)
2. 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)
3. microbes can cause disease when inoculted in animal or man
   
   • gonococcus has no animal model

Question 2

what is the expanision of Koch’s original postuate

Answer 2

therapeutic or prevetative meausres can eliminate disease

Question 3

Modern version of Koch’s Postulates for defining Virulence factors

Answer 3

• 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

Question 4

How does updated Koch’s Postulates help identify new pathogens associated with diesease?

Answer 4

• 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_

Question 5

Describe the quantification of virulence using animal models

Answer 5

• 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

Question 6

what are the problems with animal models?

Answer 6

• 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

Question 7

describe the disadvantages of tissue culture models

Answer 7

• 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
  *

Question 8

benefits to tissue culture models

Answer 8

cheaper, simpler, controlled environments

Question 9

Simple Eukaryotic host

Question 10

Describe Arabidopsis thaliana model

Answer 10

• 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

Question 11

describe how arabidopsis is a genetically tractable host….with the example given in class about Pseudomonas aeruginosa

Answer 11

• 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

Question 12

Describe the Caenorhabditis elegans as a model organism

Answer 12

• 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

Question 13

Describe Dictyostelium amoebae

Answer 13

• 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

Question 14

Describe drosophila melanogaster (fruit fly)

Answer 14

• 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

Question 15

Describe Zebrafish as a model

Answer 15

• 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

Question 16

What are the typical properties of virulence factors (VF) and the molecular approaches (MA) to study them?

Answer 16

• VF = secreted proteins or abundand surface molecules
  
  • MA = easy to purify (cholera toxin, diptheira toxin)
• highly antigenic proteins
  
  • use serum anitbodies as probes (animals exposed to them make antibodies)
• stage-spefic genes/genes expressed only in the host
  
  • microarrays, proteomics, promoter traps (gene expressed only in host after the bacteria get in)
• gene products requre for survival of pathogen in cells or in animals
  
  • signature-tagged mutagenesis
• other gene products that are unique to pathogens
  
  • comparison of geonomes (ex. Candida vs saccharomyces genome)

Question 17

describe the evolution of bacteria through genomic island

Answer 17

• horizontal gene transfer…..can transfer DNA sequences that go into genomic island that carry genes of specific function
• genomic island can increase bacteria fitness and allow them to surive better –> fitness island can be specialized futher in the environment or the host
  
  • ecological island (environmental adaptation)
  • saphophytic island (saprophytic interaction)
  • symbiosis island (symbiosis)
  • pathogencity island (parasitism)

Question 18

picture of the evolution of bacteria

Answer 18

Question 19

What is the Biochemical Approaches to finding a virulence factor?

Answer 19

1. isolate proteins, fractionate and study in the appropriate detection system
2. sequence the protein, go back to DNA to find a gene

has been widely used in the early molecular biology era…. tedious way

Question 20

molecular biology approaches to find a virulence factor

Answer 20

1. make a library of genome fragments to find a gene
2. use cloning vector to insert the frangments into and transfer the vector into the appropriate host
3. select clones expressing the virulence factor in the appropriate test system

Question 21

Describe the process of cloning a DNA librabry in a plasmid vector

Answer 21

1. digest both genomic DNA and plasmid vector with same restriction enzyme
2. mix digested plasmid and genomic DNA….join fragments with DNA ligase
3. transforma into E.coli
4. plate on media containng appropriate antibiotic

Question 22

What’s Complementation screenL gain of function phenotype?

Answer 22

1. make library from wild type organism
2. transform library into new non-pathogenic host or a mutant host
3. select for “+” transofrmants
4. recover transforming fragment

Question 23

Nefarious Misus

Answer 23

skipped because I couldn’t handle it rn

Question 24

How to use microbial genetic tools to manipulate DNA

Answer 24

• Use of cloning and expression vectors (for protein factors)
  
  • Modification of DNA (endonuclease digestion, ligation, amplification, cloning)
  • introduction of DNA (transformation, transduction, conjugation, electroporation, gene gun, etc.)
  • requires genetic markers (drug resistant, enzymes–beta-gal, fluorescent protein genes- GFP)
  • broad host range plasmids
• targeted mutation to the chromosome
• gene knockouts (random or directed)