Methods in infection biology

Question 1

Models to study infectious diseases

Answer 1

axenic in vitro culture,
co-cultivation with single type of host cell,
co-cultivation in complex cell cultures,
organoids,
ex vivo cultivation of organs,
experimental infection of avertebrates,
embryonated egg model,
experimental infection of laboratory animals,
experimental infection of non-human primates

Question 2

Axenic culture of microorganisms - def., pro/contra, measures

Answer 2

Culture of single microorganism without any host cells.
Test for antibiotic resistance, necessary nutrients, cheap.
But: virulence/pathogenicity can’t be determined.
CFU: colony-forming units

Question 3

Co-cultivation with host cell?

Answer 3

Defined in vitro cultures of primary and permanent mammalian cells.
Easy genetic manipulation of host cells, infection in vitro, low complexity, robust systems.

PFU: plaque-forming units (intracellular pathogens that lyse host cells)

Question 4

Organoids

Answer 4

Organ-like structures in vivo. (Diff. of pSC in presence of growth factors)

Question 5

Organ explants

vs organoids

Answer 5

Parts of organ/tissue obtained by surgery (biopsy).

Even more closely resembles in vivo situation, but not as long lived

Question 6

Infection of avertebrate hosts with human pathogens (examples)

Answer 6

C. elegans (small, fast development, infection through diet, transparent bodies!) , D. melanogaster

Not considered animal experiment, no approval necessary

Question 7

Embryonated chicken egg model

Answer 7

vertebrate model, no approval

Monitor survival, histology etc

Question 8

Experimental infection of laboratory animals, in vivo measures?

Answer 8

mostly rodents, variety of models (caution: diff courses of infection in diff mouse strains)
measures: LD50, ID50, Kaplan-Meier survival curve

Question 9

Examples for tissue microbiology

Answer 9

Bioluminescence imaging BLI (measure of infection: luciferase-expressing pathogens)
In vivo fluorescence microscopy (isolation of organ)

Question 10

Approaches to identify microbial virulence factors

Answer 10

1. Traditional biochemical and genetic approaches
2. Approaches to identify virulence genes expressed in vivo
3. Genomic approaches

Question 11

Biochmemical identification of VFs. (how, ex, cons)

Answer 11

isolation and purification of VF from culture of pathogenic bacteria > confirmation of toxicity > id by MS

diphteria toxin, cholera toxin

impurity of molecule, wrong model, sequence info not available

Question 12

Molecular genetic approaches of VF identification (3)

Answer 12

A) Cloning of VF gene, expression, isolation of protein
2) Loss of function: deletion of gene in pathogenic microorganism, complementation of deficient strain by re-introducing gene
C) Gain of function: Expression of suspected VF in avirulent bacterium

Question 13

VF id by transposon mutagenesis ini bacteria

Answer 13

Mobile DNA sequence.
Introduce transposon into invasive bacteria (random integration into genome) > find colony that is no longer invasive > clone gene interrupted by transposon

Question 14

CRISPR/Cas system

Answer 14

sgRNA (tracrRNA+cRNA)+Cas9, optional repair template

Question 15

Signature-tagged mutagenesis

Answer 15

add distinct tags to transposon to generate library > introduce transposons into bacterial strain > random insertion of transposons into bacterial genome (ideally: all genes are hit) > pool mutants > inject mice > isolation of bacteria that survived from mice > compare input with output pool > transposon that is missing dna be used to identify the adjacent gene&raquo_space; this gene is important for survival of bacterial strain in vivo

Question 16

Genomic substractive hybridization

Answer 16

Tag genomes of two bacterial strains > add linkers to ends of genes, from one strain (conjugated with biotin) > hybridization of genomes > matching ones will join > biotin present > removed by streptavidin beads

identification of distinctive genes for that species (left over, no biotin)

Question 17

Microarrays

Answer 17

Comparison of DNA/RNA/proteins of strains

Two strains > RNA extraction > fluorescent marker (red/green) > microarray: either one, both or none bind > distinct color (red/green/yellow)

Question 18

NGS

Answer 18

Isolation of genomic DNA > cut DNA > add linkers > input library onto flow cell > in situ PCR > sequencing: add nucleotides (conjugated with different colors) > image > alignment of sequence reads to reference genome

Question 19

Approaches to identify host pathogenicity factors

Answer 19

transgenic animal models

genomic approaches

Question 20

genomic approaches to identify host pathogenicity factors

Answer 20

microarray analysis

RNA interference (silencing of mRNA to identify host genes required for infection)

CRISPR/Cas (KO of gene > identification of resistance genes)