Biology 216 - Lect 35 - Experimental Approaches to Pathos I. General - Koch's Postulates (etiology only); We discussed contributions of attachment, capsule, toxin.... Importance of dissecting pathogenesis: Define vaccine Ag; Places to intercede; Zinsser is result of numberous publication in I and I, J Infect Dis..... Alluded to approaches all semester, today systematic overview of exptl approaches to study pathos II. Animal Models - appropriate and relevant. Used to determine virulence (LD50) - show curve of live vs dose A. Considerations - limit number of variables 1. Match animals - inbred, sex, age, match to decrease variables. 2. Be consistent and report growth conditions: media, stage of growth, aeration (HA and piliation affected) 3. Choice of animal: SPF, Gnotobiotic, Nude mice (athymic), C3H/HeJ vs C3H/HeN (LPS non-responder) 4. IFN gamma deficient (gene replacement); 5. Severe combined immunodef (B&T) by irradiated, add human fetal thymus + liver -> AIDS model B. Examples of relevant models: 1. Ileal loop for enterotoxin; 2. Corneal infection - scratch mouse eye 3. Chronic rat lung - transtracheal into lung.; 4. Burn mouse 5. Hemagglutination - attachment (K88, GC), may not represent attachment C. Animal rights - cruelty to animals? Benefits derived? III. Four approaches to studying specific virulence factors. A. Survey or correlation studies - Does presence of factor correlate with specific disease? Ex. Schlievert screened S. aureus from TSS and other S. aureus infections for pyrogenic toxin. TSS isolates produce it, most other isolates don't; GC colony types Limitations: Correlation, not cause and effect; Isolates may vary inother factors as well. B. Biochemical purification: Test effect of pure factor (Ex. Anthrax LF, EF, PA); Do Ab to factor protect? MOA. Use prototypic strain (typical) that is representative of the species. Normally low yields, so need hyperproducer to get lots of factor for purification (V. cholerae 569B) Grow hyperproducer for optimal production of factor: Syncase for CT; Low Fe for DT. Need assay system to follow molecule through purification: Ex. hemolysin, protease. Run through biochemical separations: Differential salt out, gel filtration, ion exchange, affinity, immunoaffinity (purify small amount, Ex. purify pili attached to RBC, then inject rabbit) Give example of hemolysin through gel filtration; Test purity at steps of purification: SDS-PAGE What to do with pure factor: 1. Does it reproduce disease (Ex. Elastase -> corneal ulcer, TetTox); Not applicable w/capsule,pili.. 2. Do Ab to pure factor protect in animal model? (Try active or passive immunization) 3. Does pure factor enhance virulence of strain missing factor? 4. Determine MOA C. Genetic approaches - Generate isogenic mutants, then compare in animal model (LD50). 1. Generate mutants: Chemically (EMS mild so not pleotrophic); Transposon (suicide phage,ts vector) Insertional inactivation by Tn; Clone and site-specific mutate. 2. Screen for desired mutant - be creative since few are mutants (1/10(4)) Allow generations for mutation to segregate out before plating. (too long -> siblings) Ex. Elastase mutants - Screen on elastin media; PLC screen on BAP or Egg yolk; Use Ab to screen: Welek using specific Ab on plate; Immuno colony blot. 3. Check other factors to ensure only one factor mutated. 4. Compare LD50 of parent and mutant in animal model. Add back pure factor in animal model. 5. Look at virulence of revertants. D. In vivo expression - Work irrelevant if factor not produced in vivo. Test in vivo production of factor. 1. Check patient/animal serum for Ab. Ex. Anti-A titer good prognosis for Ps infection. 2. In vivo expression (Falkow) - Jellyfish fluor protein, clone bacterial DNA in front, In vivo Fl, none in vitro E. Information in Zinsser is from plethora of publication in I & I using all 4 approaches. IV. Contributions from Genomics A. Genome "mining" B. Microarray analysis of gene expression C. Pathogenicigy islands (horizontal transfer) 1. G + C 2. Codon usage 3. IS borders 4. Type III secretion delivery to host cell 5. Ex. Salmonella 5 islands VI. Significance of dissecting pathogenesis to this level; Why not stop at Koch's postulates. Helps in designing treatment and vaccine development 1. Ex. Antitoxin therapy for diphtheria and tetanus 2. Intercede in pathogenesis - Chlorpromazine and adenylate cyclase with cholera 3. Component vaccines - capsule (Hib), toxoids (DT), adhesin (FHA/PT pertussis) VII. Next - Use of all approaches with cholera and CT.