Lessons 4-7 Flashcards
Concepts introduced in lectures/ journal clubs 4-7
Mouse Models
- Infant mice have immature immune systems
- Irradiated mice are immunocompromised because their immune cells have been destroyed
- Nude mice are genetically defective in the ability to produce T cells
- Severe combined immunodeficiency (SCID) mice are genetically defective in the ability to produce functional B cells and T cells. Good for studying innate immunity
- Transgenic mice are genetically defective for example in specific immune cells or immunity genes - often more susceptible to infection
- Gnotobiotic (germ-free) animals are raised in a germ-free environment. Because of the absence of resident microbiota, they have severely underdeveloped mucosa-associated lymphoid tissues (MALT).
- Specific-pathogen-free (SPF) animals are raise in an environment free of particular pathogens but are exposed to other microbes (including potential pathogens). Naive against particular pathogen, ensure experimental exposure is primary exposure.
ID of VF: Biochemical Approach
Purify virulence factor, and use it to reproduce symptoms associated with infection
ID of VF: Molecular Genetic Approach
Clone virulence factor gene into avirulent strain -> demonstrate acquired virulence.
ID of VF: Transposon mutagenesis
Introduce transposon with selectable marker into pathogen to generate collection of mutants. Use marker to see which mutated genes result in loss of virulence
ID of VF: Reporter Fusions
Reporter gene expressed only when vf would be. See if coincides with virulence.
- β-Galactosidase converts X-Gal to deep blue dye. X-Gal supplied through media/agar.
- GFP - conjugated double bonds
- Luciferase - bacterial operon produces substrate.
ID of VF: Signature Tagged Mutagenesis
Same as transposon mutagenesis, but is used to find genes that are expressed in vivo. A library of barcoded transposon-mutated mutants is created, where one copy of the library is passed through the model (mouse). The control copy and the model-processed copies are compared on micro-arrays containing probes to see which mutants did not survive in host.
ID of VF: In vivo expression technology
- Not very specific, many genes that are detected are housekeeping genes.
- Bacterial genomic DNA digested, fragment containing desired promoter (Is this directed?) integrated into plasmid containing purA lacZY cassette. Homology-directed recombination leads to the integration of the cassette into the genome, and the doubling of the promoter, so that the cassette is expressed when the promoter’s natural downstream genes are. purA allows the ΔpurA bacterium to survive in the host. Extraction from the host and plating on GalX medium, allows to see which genes are still active outside the host. Theoretically looking for clones that do not turn blue (i.e. gene expressed only in host).
ID of VF: Genomic Subtractive Hybridization
- Id genes present in virulent but not avirulent strains.
- DNA from both strains digested hybridized, but avirulent strain fragments labeled with biotin and purified with streptavidin. Remaining fragments only in virulent strain.
ID of VF: Selective capture of transcribed sequences
mRNA from experimental condition -> cDNA. Hybridized to gDNA, which is bound to biotin and captured with streptavidin. Allows the easy isolation of expressed sequences from a particular organism and separation from other genomes.
ID of VF: In-vivo induced antigen technology
- Isolate antibodies from pooled patient (i.e. multiple) serum and present to in-vitro grown bacteria. Label remaining free antibodies and see which ones bind in-vivo grown bacteria.
- Advantage: Patient serum is readily available, can detect response differences between strains, chronic or acute infections, reduced variability from pooled serum.
- Limitations: Does not detect cellular immune responses. Construction of genomic expression library in E. coli may lead to variable expression resulting from codon bias, restriction site limitations.
ID of VF: microarray technology
Uses chips that contain DNA oligonucleotides corresponding to all genes in the genome. Identifies differences in relative transcript amounts between experimental conditions
Competitive Index
The ratio of fitness between two strains
Trans vs. Cis complementation
Trans-complementation means complementing an allele on a plasmid (transient), whereas cis complementation integrates that allele into the genomic DNA.
Complementing allows to confirm the causal nature of a gene-phenotype relationship.
Francisella tularensis
- Very highly infectious bacterium (ID about 10 bacteria). Multiple routes of infection, natural reservoir in rodents. Non-motile, gram -
- Facultative intracellular pathogen, infects macrophages
Journal Club:
- encounters a bottleneck for systemic spread after s.c. infection, had to inject i.p. Negatively selected genes: capsule, some stress response genes. Some mutants are hypercytotoxic, some v.f.’s suppress macrophage death by limiting the secretion of inflammatory cytokines.
Early symptoms of viral infection and their causes
- malaise (tnf-α)
- fever (IL-1)
- fatigue
Remember: viral disease = sum of cytopathic effects of virus + tissue damage from host’s anti-viral immune response
Possible mechanisms of measles-induced immunosuppression
- lymphocyte apoptosis
- impair lymphocyte proliferative responses
- induction of immune-suppressive cytokines (IL-4, IL-10)
- down-regulation of IL-12, preventing differentiation on T cells.
- impair differentiation and antigen presentation by dendritic cells
Respiratory Syncytial virus
- distributed globally
- causes yearly outbreaks
- is common in young infants
- is highly contagious
- wide range of symptoms. common cold -> life threatening lower respiratory infection (pneumonia) in infants, 70% mortality in infants with congenital heart defect
- transmitted by aerosol
Immunosuppressive viruses
- measles virus
- HIV
- pox virus
myeloid cells
Cells from common myeloid progenitor. Includes monocytes, macrophages, dendritic cells.
