Methods in infection biology Flashcards
Models to study infectious diseases
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
Axenic culture of microorganisms - def., pro/contra, measures
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
Co-cultivation with host cell?
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)
Organoids
Organ-like structures in vivo. (Diff. of pSC in presence of growth factors)
Organ explants
vs organoids
Parts of organ/tissue obtained by surgery (biopsy).
Even more closely resembles in vivo situation, but not as long lived
Infection of avertebrate hosts with human pathogens (examples)
C. elegans (small, fast development, infection through diet, transparent bodies!) , D. melanogaster
Not considered animal experiment, no approval necessary
Embryonated chicken egg model
vertebrate model, no approval
Monitor survival, histology etc
Experimental infection of laboratory animals, in vivo measures?
mostly rodents, variety of models (caution: diff courses of infection in diff mouse strains)
measures: LD50, ID50, Kaplan-Meier survival curve
Examples for tissue microbiology
Bioluminescence imaging BLI (measure of infection: luciferase-expressing pathogens)
In vivo fluorescence microscopy (isolation of organ)
Approaches to identify microbial virulence factors
- Traditional biochemical and genetic approaches
- Approaches to identify virulence genes expressed in vivo
- Genomic approaches
Biochmemical identification of VFs. (how, ex, cons)
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
Molecular genetic approaches of VF identification (3)
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
VF id by transposon mutagenesis ini bacteria
Mobile DNA sequence.
Introduce transposon into invasive bacteria (random integration into genome) > find colony that is no longer invasive > clone gene interrupted by transposon
CRISPR/Cas system
sgRNA (tracrRNA+cRNA)+Cas9, optional repair template
Signature-tagged mutagenesis
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»_space; this gene is important for survival of bacterial strain in vivo
Genomic substractive hybridization
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)
Microarrays
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)
NGS
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
Approaches to identify host pathogenicity factors
transgenic animal models
genomic approaches
genomic approaches to identify host pathogenicity factors
microarray analysis
RNA interference (silencing of mRNA to identify host genes required for infection)
CRISPR/Cas (KO of gene > identification of resistance genes)
