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MICB 308 > Studying Virulence Factors 1 > Flashcards

Studying Virulence Factors 1 Flashcards

1
Q

what is the purpose of the techniques for studying virulence factors

A
  • they are used to investigate whether something is actually a virulence factor
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2
Q

Koch’s Postulate: First Postulate

A
  • the microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms
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3
Q

Koch’s Postulates: Second Postulate

A
  • the microorganism must be isolated from a diseased organism and grown in pure culture
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4
Q

Koch’s Postulate: Third Postulate

A
  • the cultured microorganism should cause disease when introduced into a healthy host
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5
Q

Koch’s Postulates: Fourth Postulate

A
  • microorganism must be re-isolated from the inoculated diseased experimental host and identified as being identical to the specific causative agent
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6
Q

Molecular Version of Koch’s Postulates: First Postulate

A
  • gene for virulence should be present in the strain of bacteria that cause disease and absent in avirulent strains
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7
Q

Molecular Version of Koch’s Postulates: Second Postulate

A
  • (i) knocking out or disruption the gene should reduce virulence, and (ii) introduction of the cloned gene into an avirulent strain should render the avirulent strain virulent
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8
Q

Molecular Version of Koch’s Postulates: Third Postulate

A
  • expression of the gene should be demonstrated in human or a relevant model
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9
Q

Molecular Version of Koch’s Postulates: Fourth Postulate

A
  • antibodies or a cell-mediated immune response to a virulence factor should be protective
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10
Q

Considerations of Models (3)

A
  • relevant experimental system should be chosen as the model
  • the assay that is used only defines the virulence of that assay
  • if the system is flawed, it cannot be extrapolated to humans or other hosts
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11
Q

what should we consider before choosing bacterial strains (5)

A
  • representative strains
  • clinical isolates that can be sequences and be tested with genetic tools
  • prototypical wild type strains
  • maintenance of virulence factors that may be metabolically expensive
  • appropriate mutants
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12
Q

how can we encourage the maintenance of strain virulence?

A
  • freezing and taking small amounts to study instead of breeding the bacteria for long periods of time
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13
Q

what mutants can be used for a study (3)

A
  • spontaneous mutants
  • randomly generated mutants
  • directed mutants
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14
Q

spontaneous mutants

A
  • unstable genetic mutants, capable of converting back
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15
Q

direct mutants

A
  • more stable mutants
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16
Q

what should we consider when choosing hosts for models (3)

A
  • the infection model should be similar; aerosol infection should use an aerosol model
  • tissue distribution of pathology should be similar in both hosts
  • identify closely related pathogens that infect different species
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17
Q

advantages of rodent host models (2)

A
  • variability of experiment is smaller due to inbreeding
  • knockouts and transgenic animals exist or can be made easily
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18
Q

disadvantages of rodent host models (4)

A
  • lack of variability
  • mice can have different microbiota depending on source
  • mice have different physiologies, habits, and genetics compared to humans
  • human pathogens may not be able to infect rodent model by same route of infections
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19
Q

cell lines (2)

A
  • simple model compared to whole organisms
  • generally use epithelial cells, fibroblasts, or monocytes
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20
Q

cell lines: advantages (2)

A
  • simple, more controlled, and cheaper than using whole organisms
  • often immortalized by using tumour cells
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21
Q

cell lines: disadvantages (3)

A
  • repeated culturing of cell lines can change their properties
  • genes expressed in an organ may not be expressed in tissue culture
  • cell lines are usually not polarized, while cells in organs are
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22
Q

polarization of intestinal cells (2)

A
  • cell receptors are asymmetrical
  • apical side (lumen) and basolateral size (tissue/blood) express differential receptors
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23
Q

organoids (2)

A
  • new host model that has great potential as it aims to replicate actual organization of human organs
  • derived from human stem cells
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24
Q

frequently used host models (7)

A
  • rodents
  • worms (C. elegans)
  • frogs
  • yeast
  • slime mold
  • bees
  • flies (drosophila)
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25
Q

other models commonly used to study virulence (6)

A
  • worms (C. elegans)
  • frogs
  • yeast
  • slime mold
  • bees
  • flies
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26
Q

what are two categories of techniques used to study virulence factors (2)

A
  • biochemical; developed first
  • genetic
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27
Q

supernatant

A
  • liquid lying above a solid residue after crystallization, precipitation, centrifugation, or other process
28
Q

what is the basis for biochemical techniques (2)

A
  • to purify and identify the virulence factor (toxins, adhesins, etc)
  • relies on having functional assay for the virulence trait
29
Q

what are the disadvantages of using biochemical techniques (3)

A
  • purified molecule may be missing co-factor that was removed during purification
  • molecule may not have same function in the test tube compared to the cell
  • does not tell us if our purified protein is the sole contributor to disease/virulence
30
Q

what virulence factors work well in biochemical techniques (2)

A
  • protein-based macromolecules
  • factors that can be targeted for hydrophobicity, charge, mass, ligand binding, etc
31
Q

what are some examples of biochemical techniques (6)

A
  • centrifugation
  • ion exchange
  • size exclusion
  • immunoprecipitation
  • ligand binding
  • Ni2+ affinity/6xHis
32
Q

biochemical techniques: centrifugation (2)

A
  • harvest bacteria by centrifugation
  • separate supernatant into fractions and test for toxicity in animal model/cultured cell assay
33
Q

biochemical techniques: ion exchange (2)

A
  • use of positively or negatively charged beads in a column
  • proteins attracted to the beads will remain in the column, while those repelled with leaves through the bottom of the column
34
Q

biochemical techniques: size exclusion (2)

A
  • use of beads with small aqueous channels in a column
  • large molecules pass quickly, but small molecules are slow as they spend more time in the channels
35
Q

biochemical techniques: immunoprecipitation (4)

A
  • use of antibodies attached to beads in a column
  • proteins are loaded in pH 7 buffer; proteins recognized by antibody bond to beads and other proteins remain in solution
  • column washed with pH 7 buffer to remove proteins in solution
  • column washed with pH 3 buffer to disrupt protein-antibody bonds and elute proteins
36
Q

biochemical techniques: ligand binding (4)

A
  • use of affinity bead with ligand attached in a column
  • proteins are loaded; proteins recognized by ligand bond to beads and other proteins remain in solution
  • column washed with buffer to remove proteins in solution
  • column washed with low pH buffer or soluble ligand to elute proteins
37
Q

biochemical techniques: Ni2+ affinity/6xHis (4)

A
  • use of NTA-coated agarose bead coordinate Ni2+ ions in a column
  • tagged and untagged proteins are loaded; tagged proteins bind to Ni2+ and other proteins remain in solution
  • column washed with buffer to remove proteins in solution
  • column washed with low pH buffer or imidazole (binds Ni2+) to elute proteins
38
Q

how would you prove that a putative virulence factor (purified protein) caused disease (3)

A
  • isolate toxin and investigate effects in other cells
  • sequence toxin and knockout toxin to see if disease occurs or investigate for its presence in avirulent strain
  • identify immune response in model to show that virulence factor grants immunity
39
Q

molecular genetic techniques (2)

A
  • gain of function experiments
  • loss of function experiments
40
Q

gain of function techniques (2)

A
  • look for putative virulence factors by doing gain of function in an avirulent strain
  • observing for restoration of virulence
41
Q

loss of function techniques (2)

A
  • knock out genes and look for reduction in virulence
  • can be done through directed mutants or transposon mutants
42
Q

gene expression experiments (3)

A
  • reporter assays - promoter traps
  • hybridization-based experiments
  • sequencing (RNA-seq)
43
Q

advantages of genetic approaches (3)

A
  • allows us to find novel genes by screening mutants
  • connection with virulence is established at the beginning of the experiment
  • relatively biased
44
Q

disadvantages of genetic approaches (2)

A
  • usefulness of the information (results) depends on the screen
  • you may not be able to identify the function of the gene product
45
Q

disadvantages of gain of function experiments (3)

A
  • there may be other accessory factors or genes involved
  • many different restriction enzymes may need to be used
  • virulence may not be expressed in an avirulent strain
46
Q

GoF Salmonella invasion experiment: what are the steps to create the library (5)

A
  1. isolate virulent Salmonella DNA
  2. cut DNA with a restriction enzyme
  3. run on gel to separate DNA fragments according to size
  4. DNA between 1000-3000bp are isolated from the gel
  5. these DNA are cloned into plasmids
  6. plasmids are transformed into avirulent E. coli to create a library
47
Q

GoF Salmonella invasion experiment: why is DNA between 1000-3000bp isolated only

A
  • average size of genes, so scientists are hoping to find intact genes in this region
48
Q

GoF Salmonella invasion experiment: what kind of assay is used (2)

A
  • an invasion assay
  • the gentamycin protection assay
49
Q

GoF Salmonella invasion experiment: what are the steps to test the library against an assay (6)

A
  1. library of E. coli clones is used to infect tissue cells
  2. cells are washed and incubated
  3. gentamycin is added; kills extracellular bacteria, but not intracellular bacteria
  4. wash to remove gentamycin
  5. lyse the host cells to release the surviving bacteria
  6. plate surviving bacteria on lab media
50
Q

GoF Salmonella invasion experiment: what is gentamycin (2)

A
  • an antibiotic
  • does not penetrate host cells
51
Q

GoF Salmonella invasion experiment: what are the steps to identify the possible virulence factor gene (3)

A
  1. plasmids are isolated from each colony
  2. the inserted Salmonella DNA is sequenced using Sanger’s Sequencing
  3. the sequence is ran through databases, such as BLAST, to identify the gene
52
Q

Sanger Sequencing steps (3)

A
  1. DNA sequence for chain terminator PCR
  2. size separation by gel electrophoresis
  3. gel analysis of determination of gene sequence
53
Q

Illumina Sequencing steps (4)

A
  1. sample prep
  2. clustering
  3. sequencing
  4. data analysis
54
Q

sanger sequencing: templates

A
  • usually one template
55
Q

sanger sequencing: output

A
  • one sequence
56
Q

sanger sequencing: typical usage (3)

A
  • plasmids
  • PCR products
  • gDNA sequence
57
Q

illumina sequencing: templates

A
  • many barcoded templates
58
Q

illumina sequencing: output (2)

A
  • millions of barcoded sequencing
  • cluster = one sequence
59
Q

illumina sequencing: usage

A
  • can multiplex (combine) experiments
60
Q

transposon mutagenesis (3)

A
  • mobile genetic elements
  • “random” mutagenesis approach
  • all contain an antibiotic resistance marker
61
Q

loss of function experiments (4)

A
  • uses transposon mutagenesis
  • virulent bacteria is “infected” in transposon
  • plates are selected with appropriate antibiotic
  • collection of mutants is the library
62
Q

what are the characteristics of the bacteria in a loss of function experiment library (2)

A
  • mostly random mutants
  • Tn is in their chromosome
63
Q

what are some disadvantages of loss of function experiments (3)

A
  • only generates mutants in non-essential genes
  • transposons have transcriptional terminators within them
  • assays can sometimes be laborious
64
Q

loss of function experiments: how do the terminators within Tn affect the experiment (2)

A
  • bacterial mRNA is polycistronic
  • can lead to the “polar” effect
65
Q

how can you identify a mutated gene without sequencing the entire genome (7)

A
  1. insert Tn into bacterial chromosomes to create the random mutations
  2. use restriction enzymes to randomly cut the chromosome
  3. clone the fragments into E. coli
  4. subject E. coli to antibiotics; the surviving E. coli contain a mutated gene
  5. identify insertion site of transposon using prime from plasmid and primer from Tn
  6. use Sanger’s sequencing to identify gene disrupted by Tn
  7. BLAST against the database
66
Q

why do we use two primers for Sanger’s Sequencing in plasmids (2)

A
  • this method has a limit of ~800bp
  • running sequencing both ways allows scientist to obtain more length of the desired DNA

MICB 308 (22 decks)

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