Strategies for Studying  Microbial Pathogenesis BIOL 533 Lecture 3 Medical Microbiology
Choosing an Animal Model <ul><li>Pathogen may not affect animal at all   -OR- may give different symptoms </li></ul><ul><l...
Choosing an Animal Model <ul><li>One model may show certain aspects of disease, but not another </li></ul><ul><li>Differen...
Choosing an Animal Model <ul><li>Ideally, want model to: </li></ul><ul><ul><li>Use same route as human disease </li></ul><...
Cell Culture/Organ Culture <ul><li>Difficult </li></ul><ul><li>Cell-lines often  tumor  lines that are genetically and phy...
Studying Pathogenic Organisms <ul><li>Look at phylogeny to find closely related organisms; for example: </li></ul><ul><ul>...
Studying Pathogenic Organisms <ul><li>Look at other, similar members of the same genus; for example: </li></ul><ul><ul><li...
Studying Pathogenic Organisms <ul><li>Approaches for identifying virulence factor and proving its importance in causing di...
Biochemical/Immunological <ul><li>Purify molecule and study  in vitro </li></ul><ul><li>Yields detailed information about ...
Biochemical/Immunological <ul><li>Two limitations: </li></ul><ul><ul><li>Molecule must be assayable; most applicable if kn...
Immunological <ul><li>Determine whether Ab to bacterial product are protective in infected animals </li></ul><ul><li>Possi...
Immunological <ul><li>Used to ascertain that putative virulence factor is being produced in animal during infection </li><...
Genetic <ul><li>Sequence wild-type gene and compare to others </li></ul><ul><ul><li>Sequence identity and similarity infer...
Genetic <ul><li>Test mutants for changes in virulence </li></ul><ul><li>-OR- </li></ul><ul><li>Introduce  cloned genes bac...
Genetic <ul><li>In Vivo Experimental Technique (IVET) </li></ul><ul><li>Identify  in vivo -induced ( ivi ) genes that are ...
Genetic <ul><li>Strengths of genetic approach: </li></ul><ul><ul><li>Starts with function of known importance </li></ul></...
Genetic <ul><li>Limitations of genetic approach: </li></ul><ul><ul><li>Difficult to determine specific function of virulen...
Genetic <ul><li>Limitations of genetic approach: </li></ul><ul><ul><li>Variety and interest of mutant from a given selecti...
Wild Type <ul><li>Sequence wild-type or mutated gene: </li></ul><ul><ul><li>Sometimes find unexpected relationships </li><...
Wild Type <ul><li>Example:  E. coli  and  S. typhimurium </li></ul><ul><ul><li>Chromosomal maps very similar </li></ul></u...
Wild Type <ul><li>Experimental technique: (see Nester 10.13—Colony Hybridization) </li></ul><ul><li>Recombinant plasmids c...
Wild Type <ul><li>Results: </li></ul><ul><ul><li>6.4 kb region maps to minute 60 on chromosome, and deletions abolish abil...
Mutant <ul><li>Cloned genes introduced into avirulent mutants or  E. coli </li></ul><ul><li>Works for  E. coli   only  if ...
Mutant <ul><li>Example: </li></ul><ul><ul><li>Ordinary  E. coli  strains don’t adhere to or invade tissue culture monolaye...
Mutant <ul><li>Limitations: </li></ul><ul><ul><li>Standard cloning techniques isolate only small portions of genome (<30 k...
Mutants <ul><li>Construct and test mutants for changes in virulence </li></ul><ul><ul><li>Common method for obtaining muta...
Mutant <ul><li>Advantages: </li></ul><ul><ul><li>Every selected colony has selectable phenotype </li></ul></ul><ul><ul><li...
Mutant <ul><li>Limitations: </li></ul><ul><ul><li>Carrying transcriptional terminators </li></ul></ul><ul><ul><ul><li>If t...
Mutant <ul><li>Groisman and Heffron </li></ul><ul><ul><li>Pilot study </li></ul></ul><ul><ul><li>Screened 400 random trans...
Mutant <ul><li>If  S. typhimurium  has 3,000 genes, results of this pilot study would suggest that  60 to 180 genes  play ...
Mutants <ul><li>Further examination: </li></ul><ul><ul><li>Difficult to identify mutants with weak effect on LD 50 </li></...
Mutants <ul><li>Certain virulence factors decrease LD 50  <100-fold in mice while others, like motility, may not affect LD...
Mutant <ul><li>Therefore, using one infection model and specific definition of virulence, study probably  underestimated  ...
Mutant <ul><li>Can make a case that housekeeping genes contribute, as do other genes concerned with bacterial physiology <...
Mutant <ul><li>Identifying virulence genes by regulation </li></ul><ul><ul><li>Virulence genes are frequently in operons a...
Mutant <ul><li>Introduction by plasmid (suicide vector) </li></ul><ul><ul><li>Common way to introduce transposon into chro...
Lecture Three <ul><li>Questions? </li></ul><ul><li>Comments? </li></ul><ul><li>Assignments... </li></ul>
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Strategies for Studying Microbial Pathogenesis

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Strategies for Studying Microbial Pathogenesis

  1. 1. Strategies for Studying Microbial Pathogenesis BIOL 533 Lecture 3 Medical Microbiology
  2. 2. Choosing an Animal Model <ul><li>Pathogen may not affect animal at all -OR- may give different symptoms </li></ul><ul><li>Given disease may have a number of animal models, none of which fully satisfies characteristics of disease </li></ul>
  3. 3. Choosing an Animal Model <ul><li>One model may show certain aspects of disease, but not another </li></ul><ul><li>Different models may rely on different routes of introducing pathogen; e.g.: </li></ul><ul><ul><li>Bordetella pertussis </li></ul></ul><ul><ul><ul><li>Intracranial </li></ul></ul></ul><ul><ul><ul><li>Interperitoneal </li></ul></ul></ul><ul><ul><ul><li>Respiratory aspiration </li></ul></ul></ul>
  4. 4. Choosing an Animal Model <ul><li>Ideally, want model to: </li></ul><ul><ul><li>Use same route as human disease </li></ul></ul><ul><ul><li>Display same symptoms </li></ul></ul><ul><ul><li>Display same virulence </li></ul></ul><ul><li>Alternative: cell culture, organ culture </li></ul>
  5. 5. Cell Culture/Organ Culture <ul><li>Difficult </li></ul><ul><li>Cell-lines often tumor lines that are genetically and physiologically different (immortal—many mutations) </li></ul><ul><li>Removed from effects of other organs, hormones </li></ul><ul><li>Cells grown in artificial media differ from in vivo </li></ul><ul><li>Cell lines may not express same Ag on surface as when in animal </li></ul>
  6. 6. Studying Pathogenic Organisms <ul><li>Look at phylogeny to find closely related organisms; for example: </li></ul><ul><ul><li>S. typhimurium vs. </li></ul></ul><ul><ul><li>S. typhi </li></ul></ul><ul><li>One may respond more easily than the other to variety of genetic techniques </li></ul>
  7. 7. Studying Pathogenic Organisms <ul><li>Look at other, similar members of the same genus; for example: </li></ul><ul><ul><li>M. smegnatis vs. </li></ul></ul><ul><ul><li>M. tuberculosis </li></ul></ul><ul><li>M. smegnatis is faster-growing; methods may be applicable to M. tuberculosis </li></ul>
  8. 8. Studying Pathogenic Organisms <ul><li>Approaches for identifying virulence factor and proving its importance in causing disease: </li></ul><ul><ul><li>Biochemical </li></ul></ul><ul><ul><li>Genetic </li></ul></ul><ul><ul><li>Immunological </li></ul></ul><ul><li>Best to combine approaches </li></ul>
  9. 9. Biochemical/Immunological <ul><li>Purify molecule and study in vitro </li></ul><ul><li>Yields detailed information about </li></ul><ul><ul><li>Cofactors </li></ul></ul><ul><ul><li>General physical properties </li></ul></ul>
  10. 10. Biochemical/Immunological <ul><li>Two limitations: </li></ul><ul><ul><li>Molecule must be assayable; most applicable if know product and function </li></ul></ul><ul><ul><li>Measurements on isolated molecules may not accurately reflect function in intact bacterium </li></ul></ul><ul><ul><ul><li>Prove function in vivo ; have to take either genetic or immunological approach </li></ul></ul></ul>
  11. 11. Immunological <ul><li>Determine whether Ab to bacterial product are protective in infected animals </li></ul><ul><li>Possible problem: </li></ul><ul><ul><li>Ab to bacterial surface molecules might prevent infection by opsonizing or enhancing complement action rather than inactivating virulence factor </li></ul></ul>
  12. 12. Immunological <ul><li>Used to ascertain that putative virulence factor is being produced in animal during infection </li></ul>
  13. 13. Genetic <ul><li>Sequence wild-type gene and compare to others </li></ul><ul><ul><li>Sequence identity and similarity infers function </li></ul></ul><ul><li>Hybridize to related species and make mutations in gene that encodes virulence factor </li></ul>
  14. 14. Genetic <ul><li>Test mutants for changes in virulence </li></ul><ul><li>-OR- </li></ul><ul><li>Introduce cloned genes back into avirulent mutants; is virulence restored? </li></ul><ul><li>-OR- </li></ul><ul><li>Identify potential virulence genes by regulation; are they co-regulated? </li></ul>
  15. 15. Genetic <ul><li>In Vivo Experimental Technique (IVET) </li></ul><ul><li>Identify in vivo -induced ( ivi ) genes that are highly expressed in animal tissues, but not in laboratory media </li></ul><ul><li>Limitation of techniques (see slide 14): </li></ul><ul><ul><li>Each requires some understanding of lab conditions to get virulence gene expression </li></ul></ul>
  16. 16. Genetic <ul><li>Strengths of genetic approach: </li></ul><ul><ul><li>Starts with function of known importance </li></ul></ul><ul><ul><li>Isolating mutants with this function affected can lead to discovering new virulence factors that previously had no assay </li></ul></ul><ul><ul><li>Also, connection between genes and some aspect of virulence is established from the beginning </li></ul></ul>
  17. 17. Genetic <ul><li>Limitations of genetic approach: </li></ul><ul><ul><li>Difficult to determine specific function of virulence genes </li></ul></ul><ul><ul><li>Example: loss of ability to invade kidney cell </li></ul></ul><ul><ul><ul><li>Loss of regulatory protein needed for activation? </li></ul></ul></ul><ul><ul><ul><li>Loss of invasin structural gene? </li></ul></ul></ul><ul><ul><ul><li>Loss of genes needed for processing, localizing? </li></ul></ul></ul><ul><ul><ul><li>Function having some indirect effect? </li></ul></ul></ul>
  18. 18. Genetic <ul><li>Limitations of genetic approach: </li></ul><ul><ul><li>Variety and interest of mutant from a given selection or screening depends on cleverness and specificity of the procedure </li></ul></ul>
  19. 19. Wild Type <ul><li>Sequence wild-type or mutated gene: </li></ul><ul><ul><li>Sometimes find unexpected relationships </li></ul></ul><ul><ul><li>Useful only if match known gene sequence </li></ul></ul><ul><li>Use one organism’s DNA as a probe and hybridize with DNA from related organism </li></ul><ul><ul><li>If pathogenic strain contains genetic material that is absent from non-pathogenic strain, that material may encode genes that confer pathogenicity </li></ul></ul>
  20. 20. Wild Type <ul><li>Example: E. coli and S. typhimurium </li></ul><ul><ul><li>Chromosomal maps very similar </li></ul></ul><ul><ul><li>S. typhimurium has DNA sequences that E. coli does not </li></ul></ul><ul><ul><li>S. typhimurium is pathogen and normal E. coli is not; therefore, the differing sequences may be virulence genes </li></ul></ul>
  21. 21. Wild Type <ul><li>Experimental technique: (see Nester 10.13—Colony Hybridization) </li></ul><ul><li>Recombinant plasmids containing S. typhimurium -specific sequences identified on filter blots as not hybridizing to probe made from entire E. coli chromosome </li></ul>
  22. 22. Wild Type <ul><li>Results: </li></ul><ul><ul><li>6.4 kb region maps to minute 60 on chromosome, and deletions abolish ability of S. typhimurium to enter epithelial cells </li></ul></ul><ul><ul><li>Similar analyses revealed other genes </li></ul></ul>
  23. 23. Mutant <ul><li>Cloned genes introduced into avirulent mutants or E. coli </li></ul><ul><li>Works for E. coli only if foreign gene can be expressed in E. coli ; most cannot: </li></ul><ul><ul><li>May not have accessory genes needed (e.g., capsule) </li></ul></ul><ul><ul><li>May not have necessary regulatory sequences </li></ul></ul>
  24. 24. Mutant <ul><li>Example: </li></ul><ul><ul><li>Ordinary E. coli strains don’t adhere to or invade tissue culture monolayers </li></ul></ul><ul><ul><li>Potential adhesins and invasins can be identified by screening for clones containing DNA sequences that enable E. coli to adhere or invade monolayers </li></ul></ul>
  25. 25. Mutant <ul><li>Limitations: </li></ul><ul><ul><li>Standard cloning techniques isolate only small portions of genome (<30 kb) </li></ul></ul><ul><ul><li>Approach works best if only one or a few genes are required for trait to be expressed </li></ul></ul><ul><ul><li>Gene must be expressed in E. coli </li></ul></ul><ul><ul><li>Approach most successful when foreign organism is closely related to E. coli </li></ul></ul>
  26. 26. Mutants <ul><li>Construct and test mutants for changes in virulence </li></ul><ul><ul><li>Common method for obtaining mutants is to mutagenize with transposons </li></ul></ul><ul><ul><li>Screen for loss of virulence </li></ul></ul>
  27. 27. Mutant <ul><li>Advantages: </li></ul><ul><ul><li>Every selected colony has selectable phenotype </li></ul></ul><ul><ul><li>Most disrupt a gene </li></ul></ul><ul><ul><li>Transposon serves as marker to locate gene; useful for cloning </li></ul></ul><ul><ul><li>Can be used to detect genes not expressed in E. coli or not closely linked to other virulence genes </li></ul></ul>
  28. 28. Mutant <ul><li>Limitations: </li></ul><ul><ul><li>Carrying transcriptional terminators </li></ul></ul><ul><ul><ul><li>If transposon inserts into first gene in operon, eliminates transcription for that gene and other genes as well; therefore, insertions are polar </li></ul></ul></ul><ul><ul><ul><li>Avirulent phenotype could be due to loss of expression of downstream gene </li></ul></ul></ul><ul><ul><li>Will not work with essential genes, because organism will not survive to form colony </li></ul></ul>
  29. 29. Mutant <ul><li>Groisman and Heffron </li></ul><ul><ul><li>Pilot study </li></ul></ul><ul><ul><li>Screened 400 random transposon mutants for virulence in mice </li></ul></ul><ul><ul><li>Results: </li></ul></ul><ul><ul><ul><li>2% of mutations increased IP 50% lethal dose (LD 50 ) by  10,000 </li></ul></ul></ul><ul><ul><ul><li>6% increased oral LD 50 </li></ul></ul></ul>
  30. 30. Mutant <ul><li>If S. typhimurium has 3,000 genes, results of this pilot study would suggest that 60 to 180 genes play a role in pathogenesis. </li></ul><ul><li>Further examination—must consider: </li></ul><ul><ul><li>Definition of virulence gene </li></ul></ul><ul><ul><li>Defects found among avirulent mutants </li></ul></ul>
  31. 31. Mutants <ul><li>Further examination: </li></ul><ul><ul><li>Difficult to identify mutants with weak effect on LD 50 </li></ul></ul><ul><ul><li>Not ideal, because Salmonella pathogenesis varies in severity </li></ul></ul><ul><ul><li>Many different properties affect infection process </li></ul></ul>
  32. 32. Mutants <ul><li>Certain virulence factors decrease LD 50 <100-fold in mice while others, like motility, may not affect LD 50 but are important in other models </li></ul>
  33. 33. Mutant <ul><li>Therefore, using one infection model and specific definition of virulence, study probably underestimated number of virulence genes </li></ul><ul><li>However, may also have over estimated if you eliminate housekeeping genes, such as rec A, that have other functions </li></ul>
  34. 34. Mutant <ul><li>Can make a case that housekeeping genes contribute, as do other genes concerned with bacterial physiology </li></ul>
  35. 35. Mutant <ul><li>Identifying virulence genes by regulation </li></ul><ul><ul><li>Virulence genes are frequently in operons and regulons controled by same proteins </li></ul></ul><ul><ul><li>If one gene found, other genes may also be found </li></ul></ul><ul><ul><li>Approach uses transcriptional fusions </li></ul></ul>
  36. 36. Mutant <ul><li>Introduction by plasmid (suicide vector) </li></ul><ul><ul><li>Common way to introduce transposon into chromosome </li></ul></ul><ul><ul><li>Also could be done with intact or inactivated cloned gene </li></ul></ul>
  37. 37. Lecture Three <ul><li>Questions? </li></ul><ul><li>Comments? </li></ul><ul><li>Assignments... </li></ul>

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