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Alex BIOL 320 > Experimental Approaches to Studying Host-bacterium Interactions > Flashcards

Experimental Approaches to Studying Host-bacterium Interactions Flashcards

1
Q

Describe each of the four ways to identify potential bacterial pathogens from clinical samples.

A
  1. Microscopy
    - Gram stains: presence of bacteria in normally sterile body fluid, staining properties and morphology of the bacteria that can help with species identification or the selection of antibiotics for the patient, diagnosis
    - Antibody-based identification: use specific antibodies and direct microscopy
  2. Cultivation and identification
    - Non-selective, selective, differential media
    - Blood culture
    - Use multiplex PCR to identify
  3. Detection of specific antibodies to infection
    - ELISA
    - Lateral flow immunoassays
  4. Detection of pathogen-specific macromolecules
    - DNA - PCR
    - ELISA and lateral flow immunoassays
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2
Q

Describe the four organismal Koch’s postulates and their limitations.

A
  1. Association
  2. Isolation
  3. Demonstration
  4. Re-isolation
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3
Q

Explain the significance of Koch’s postulates.

A
  • Provide a scientific procedure/framework to design experiments and determine whether there is a cause-effect relationship between a suspected bacterium and a clinical disease
  • Help eliminate bias in one’s thinking
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4
Q

Explain why Koch’s postulates, with all their problems, are still as relevant today as they were when Koch first proposed them.

A

Give us a framework for how to approach the question of whether a pathogen is the cause of a disease

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5
Q

Explain why Treponema pallidum that can’t be cultured has been accepted as the causative bacterial agent for syphilis.

A
  • It was found in patients with syphilis
  • T. pallidum-specific antibodies have been found in patients with syphilis
  • Penicillin clear syphilis
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6
Q

Explain why it is critically important to choose the right experimental system to define/study virulence or host defense.

A

(this is a guess)
- Ensure accurate results
- Take into account any ethical considerations
- Use resources efficiently
- Reproducibility

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7
Q

Define clinical isolates and laboratory strains of a bacterial pathogen.

A
  • Clinical isolates: bacterial strains isolated from patients
  • Laboratory strains: bacterial strains cultured in a laboratory setting
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8
Q

Explain why isolates may not be ideal for studying host-bacterium interactions.

A

Clinical isolates do not account for the genetic background of the host or the environment in which it was obtained (i.e. the host’s immune system could be reacting to something else in the environment)

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9
Q

Discuss advantages and limitations of using laboratory strains.

A
  • Advantages
    • Known genetic background
    • Available experimental tools
  • Disadvantages
    • Possibly less pathogenic or attenuated
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10
Q

Define substitute species and describe its use.

A

Bacterial species that is used in place of another bacterial species when studying bacterial infections/host defense
- Useful for safety (actual bacteria may be too dangerous/pathogenic)
- Makes studies possible

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11
Q

List properties of a good animal model.

A
  • Same route of infection
  • Same tissue tropism or distribution
  • Same symptoms
  • Similar defense mechanisms
  • More virulent strains should produce a severer disease
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12
Q

Define inbred and outbred mice.

A
  • Inbred mice: breeding progeny from each generation together
  • Outbred mice: breeding mice of different genetic backgrounds
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13
Q

Explain the advantages and limitations of using inbred mice.

A

Advantages
- Eliminates heterozygosity
- Less genetic variation (more consistent data)

Limitations
- More susceptible to disease

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14
Q

Give one application for immunocompromised mice.

A

Cancer research

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15
Q

Give one application for knockout mice.

A

To study the functions of certain genes

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16
Q

Give one application for transgenic mice.

A

To create animal models of human disease (ex. Alzheimer’s disease) and drug development

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17
Q

Give one application for germ free mice.

A

To study the relationship between the normal microbiota and the immune system

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18
Q

Describe advantages and limitations associated with cultured mammalian cells for studying host-pathogen interactions.

A

Advantages
- Easily maintained under lab conditions
- Uniformity in the cell type
- Easily controlled
- Readily available
- Genetical manipulability

Disadvantages
- Loss of cell-cell contact
- Lack of extracellular matrix
- Lack of proper stimuli
- Lack of orientation

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19
Q

Exaplin why in some cases, bacterial adhesion observed to occur with an intact hose tissue is absent in cultured cells that are derived from the the same host tissue.

A
  • Cultured cells may not express the receptor
  • Binding requires the extracellular matrix
  • Accessory molecules required for binding may be absent from the cultured cells
20
Q

Define ID50.

A

Infective dose for infecting 50% of experimental animals

21
Q

Define LD50.

A

Lethal dose for killing 50% of experimental animals

22
Q

Define infection load.

A

Quantity/concentration of an infectious agent in a host at one time

23
Q

Define survival curve.

A

A graph showing the proportion of a population living at a given time after contracting a serious disease

24
Q

Discuss why two strains of a bacterial pathogen may differ significantly in their ID50 and LD50.

A
  • Genetic variation between strains
  • Host-pathogen interactions
  • Antibiotic resistance
25
Q

Describe the type of disease that would have a low ID50 but a high LD50.

A
  • Low ID50 –> does not take a lot of bacteria to infect host
  • High LD50 –> needs a lot of bacteria to kill host

This disease would be very infectious but not deadly. The host would likely be able to survive the disease long enough to pass it on to another host. (Ex. common cold, flu)

26
Q

Explain whether it is possible to have a disease that has a high ID50 but a low LD50.

A

Possible but not common
- Would need a lot of bacteria to infect but not a lot to kill
-

27
Q

Describe the type of disease that should be measured by LD50 but not by ID50.

A

Diseases whose toxicity is more important than its infectiousness (non-communicable diseases)

28
Q

Reading assignment: Describe the experimental procedure to establish a survival response (survival curve) to a bacterial infection (Fig. 3).

A
  • Used wild-type and mutant mice that were immunocompromised and complement deficient
  • Infected them with the bacteria
  • Waited to see how how much of that type of mouse survived after a certain amount of time
29
Q

Reading assignment: Explain the purpose of the experiments in Fig. 3 and describe the results.

A
  • Purpose: Determine the role of various complement components in mouse resistance to bacterial infection and compared the suvival of wile-type mice to that of complement deficient mice
  • Results: complement deficient mice hella died compared to wild-type
30
Q

Describe molecular Koch’s postulates and their significance.

A

Significance: guidelines for establishing a gene as a virulence factor (not proposed by Koch)

  1. The gene or its product should be found only in the strains of bacteria that cause the disease.
  2. The gene should be isolated by cloning.
  3. The gene-associated virulence should be demonstrated in gene-disrupted or knock-out mutants and in gene-reconstituted isolates.
    - Disrupt (or knockout) the gene to obtain mutants
    - Demonstrate loss or reduction of virulence in the mutants, compared to the wild type
    - Demonstrate gain or restoration of virulence in the gene-reconstituted isolates
  4. The gene is expressed by the bacteria during the infection of their host.
31
Q

Describe the use of avirulent bacteria to identify virulence genes by complementation (Fig. 4.1).

A

Avirulent strains for gain of virulence/function (a complementation approach)
1. Isolate DNA of interest and cut into fragments (genomic library)
2. Clone DNA into plasmids so they can be put into E. coli (genomic library)
3. Screen invasive E. coli
4. Sequence invase E. coli
5. Mutate gene and reintroduce to Y. tuberculosis (mutants fail to invade)
6. Identify protein by SDS-PAGE

32
Q

Identify the steps shown in Fig. 4.1 that introduce DNA fragments from Y. pseudotuberculosis to E. coli.

A

Steps 1-3

33
Q

Define transposon mutagenesis.

A

Disruption of a functional gene by the insertion of a transposon (loss of virulence/function)

34
Q

Describe the use of transposon mutagenesis to identify virulence genes in virulent bacteria (Fig. 4.4 and 4.6).

A
  1. Transposon inserted into bacterial genome using a suicide plasmid
  2. Selection of kanamycin-resistant bacteria
  3. Infect mice with that bacteria
  4. Recover bacteria from spleen
  5. Find colonies that are no longer invasive (not recovered from mice)
35
Q

Define suicide plasmid and explain why the use of a suicide plasmid is necessary for transposon mutagenesis.

A
  • Plasmid that can’t replicate in the recipient bacteria
  • Necessary for transposon mutagenesis because they allow scientists to introduce genes into bacteria in a controlled manner
36
Q

Describe in vivo expression technology and explain the significance of identifying genes that are expressed during bacterial infection.

A
  • Method of identifying genes that bacteria express when they’re in a host
  • If you identify the genes expressed during bacterial infection, you can use them as a target for new treatments
37
Q

Explain the purpose of the promoter-less reporter gene purA in the purA-lacZY system.

A

To serve as a negative control to help identify the genes that are active during infection

38
Q

Explain the purpose of the promoter-less reporter gene lacZY in the purA-lacZY system.

A

To serve as a control that checks for non-specific activity of the lacZY reporter genes and help researchers distinguish genuine promoter-driven expression from unintended transcription

39
Q

Define in vivo-induced antigen technology and describe its significance.

A
  • Method for identifying proteins expressed by bacteria during infection but not during in vitro growth
  • Significance:
40
Q

Use Fig. 4.9 to describes the steps in in vivo-induced antigen technology to identify in
vivo-induced antigen in Mycobacterium tuberculosis.

A
  • Humans infected with M. tuberculosis produce antibodies reactive with infection-specific (in vivo) and infection-non-specific antigens
  • Removal of infection-non-specific antibodies with antigens produced under in vitro conditions by M. tuberculosis and E. coli
  • Expression of all or part of every M. tuberculosis protein
    • Bacterial phage lambda-based expression system with IPTG
    • Infect E. coli
    • Transfer of expressed proteins from plagues onto a membrane
  • Identify infection-specific antigens by the absorbed serum
41
Q

Identify the step shown in Fig. 4.1 that is to satisfy Molecular Koch’s postulate 3.

A

Step 6

42
Q

Define isogenic mutant.

A

Genetically identical to its wild-type parent except the gene(s) of interest

43
Q

Define knockout mutant.

A

The gene of interest is no longer functional

44
Q

Define reconstituted isolate.

A

Addition of the wild-type gene to the knockout mutant

45
Q

Explain why a wild-type strain, its knockout mutant, and reconstituted isolate are needed to define a virulence gene.

A
  • Wild-type: has virulence gene, probably invasive
  • Knockout mutant: the gene you think is the virulence gene is no longer functional, shouldn’t be invasive
  • Reconstituted isolate: wild-type gene added back to knockout mutant, should be invasive
  • Basically proves that the gene you fucked up is the virulence gene
46
Q

Use Fig. 4.1 to identify additional experimental approaches to studying bacterial virulence
factors.

A
  • Genetic
  • Biochemical
  • Cellular and molecular
  • Immunological

Alex BIOL 320 (5 decks)

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