微生物学美国IndianaUniversityPurdueUniversity授课03.ppt

Lecture Three微生物学美国IndianaUniversityPurdueUniversity授课03 Still waters run deep.流静水深流静水深,人静心深人静心深 Where there is life,there is hope。有生命必有希望。有生命必有希望Lecture ThreeChoosing an Animal ModelPathogen may not affect animal at all -OR-may give different symptomsGiven disease may have a number of animal models,none of which fully satisfies characteristics of diseaseBIOL 5332Lecture ThreeChoosing an Animal ModelOne model may show certain aspects of disease,but not anotherDifferent models may rely on different routes of introducing pathogen;e.g.:Bordetella pertussisIntracranialInterperitonealRespiratory aspirationBIOL 5333Lecture ThreeChoosing an Animal ModelIdeally,want model to:Use same route as human diseaseDisplay same symptomsDisplay same virulenceAlternative:cell culture,organ cultureBIOL 5334Lecture ThreeCell Culture/Organ CultureDifficultCell-lines often tumor lines that are genetically and physiologically different(immortalmany mutations)Removed from effects of other organs,hormonesCells grown in artificial media differ from in vivoCell lines may not express same Ag on surface as when in animalBIOL 5335Lecture ThreeStudying Pathogenic OrganismsLook at phylogeny to find closely related organisms;for example:S.typhimurium vs.S.typhiOne may respond more easily than the other to variety of genetic techniquesBIOL 5336Lecture ThreeStudying Pathogenic OrganismsLook at other,similar members of the same genus;for example:M.smegnatis vs.M.tuberculosisM.smegnatis is faster-growing;methods may be applicable to M.tuberculosisBIOL 5337Lecture ThreeStudying Pathogenic OrganismsApproaches for identifying virulence factor and proving its importance in causing disease:BiochemicalGeneticImmunologicalBest to combine approachesBIOL 5338Lecture ThreeBiochemical/ImmunologicalPurify molecule and study in vitroYields detailed information aboutCofactors General physical propertiesBIOL 5339Lecture ThreeBiochemical/ImmunologicalTwo limitations:Molecule must be assayable;most applicable if know product and functionMeasurements on isolated molecules may not accurately reflect function in intact bacteriumProve function in vivo;have to take either genetic or immunological approachBIOL 53310Lecture ThreeImmunologicalDetermine whether Ab to bacterial product are protective in infected animalsPossible problem:Ab to bacterial surface molecules might prevent infection by opsonizing or enhancing complement action rather than inactivating virulence factorBIOL 53311Lecture ThreeImmunologicalUsed to ascertain that putative virulence factor is being produced in animal during infectionBIOL 53312Lecture ThreeGeneticSequence wild-type gene and compare to othersSequence identity and similarity infers functionHybridize to related species and make mutations in gene that encodes virulence factorBIOL 53313Lecture ThreeGeneticTest mutants for changes in virulence-OR-Introduce cloned genes back into avirulent mutants;is virulence restored?-OR-Identify potential virulence genes by regulation;are they co-regulated?BIOL 53314Lecture ThreeGeneticIn Vivo Experimental Technique(IVET)Identify in vivo-induced(ivi)genes that are highly expressed in animal tissues,but not in laboratory mediaLimitation of techniques(see slide 14):Each requires some understanding of lab conditions to get virulence gene expressionBIOL 53315Lecture ThreeGeneticStrengths of genetic approach:Starts with function of known importanceIsolating mutants with this function affected can lead to discovering new virulence factors that previously had no assayAlso,connection between genes and some aspect of virulence is established from the beginningBIOL 53316Lecture ThreeGeneticLimitations of genetic approach:Difficult to determine specific function of virulence genesExample:loss of ability to invade kidney cellLoss of regulatory protein needed for activation?Loss of invasin structural gene?Loss of genes needed for processing,localizing?Function having some indirect effect?BIOL 53317Lecture ThreeGeneticLimitations of genetic approach:Variety and interest of mutant from a given selection or screening depends on cleverness and specificity of the procedureBIOL 53318Lecture ThreeWild TypeSequence wild-type or mutated gene:Sometimes find unexpected relationshipsUseful only if match known gene sequenceUse one organisms DNA as a probe and hybridize with DNA from related organismIf pathogenic strain contains genetic material that is absent from non-pathogenic strain,that material may encode genes that confer pathogenicityBIOL 53319Lecture ThreeWild TypeExample:E.coli and S.typhimuriumChromosomal maps very similarS.typhimurium has DNA sequences that E.coli does notS.typhimurium is pathogen and normal E.coli is not;therefore,the differing sequences may be virulence genesBIOL 53320Lecture ThreeWild TypeExperimental technique:(see Nester 10.13Colony Hybridization)Recombinant plasmids containing S.typhimurium-specific sequences identified on filter blots as not hybridizing to probe made from entire E.coli chromosomeBIOL 53321Lecture ThreeWild TypeResults:6.4 kb region maps to minute 60 on chromosome,and deletions abolish ability of S.typhimurium to enter epithelial cellsSimilar analyses revealed other genesBIOL 53322Lecture ThreeMutantCloned genes introduced into avirulent mutants or E.coliWorks for E.coli only if foreign gene can be expressed in E.coli;most cannot:May not have accessory genes needed(e.g.,capsule)May not have necessary regulatory sequencesBIOL 53323Lecture ThreeMutantExample:Ordinary E.coli strains dont adhere to or invade tissue culture monolayersPotential adhesins and invasins can be identified by screening for clones containing DNA sequences that enable E.coli to adhere or invade monolayersBIOL 53324Lecture ThreeMutantLimitations:Standard cloning techniques isolate only small portions of genome(30 kb)Approach works best if only one or a few genes are required for trait to be expressedGene must be expressed in E.coliApproach most successful when foreign organism is closely related to E.coliBIOL 53325Lecture ThreeMutantsConstruct and test mutants for changes in virulenceCommon method for obtaining mutants is to mutagenize with transposonsScreen for loss of virulenceBIOL 53326Lecture ThreeMutantAdvantages:Every selected colony has selectable phenotypeMost disrupt a geneTransposon serves as marker to locate gene;useful for cloningCan be used to detect genes not expressed in E.coli or not closely linked to other virulence genesBIOL 53327Lecture ThreeMutantLimitations:Carrying transcriptional terminatorsIf transposon inserts into first gene in operon,eliminates transcription for that gene and other genes as well;therefore,insertions are polarAvirulent phenotype could be due to loss of expression of downstream geneWill not work with essential genes,because organism will not survive to form colonyBIOL 53328Lecture ThreeMutantGroisman and HeffronPilot studyScreened 400 random transposon mutants for virulence in miceResults:2%of mutations increased IP 50%lethal dose(LD50)by 10,0006%increased oral LD50BIOL 53329Lecture ThreeMutantIf S.typhimurium has 3,000 genes,results of this pilot study would suggest that 60 to 180 genes play a role in pathogenesis.Further examinationmust consider:Definition of virulence geneDefects found among avirulent mutantsBIOL 53330Lecture ThreeMutantsFurther examination:Difficult to identify mutants with weak effect on LD50Not ideal,because Salmonella pathogenesis varies in severityMany different properties affect infection processBIOL 53331Lecture ThreeMutantsCertain virulence factors decrease LD50 100-fold in mice while others,like motility,may not affect LD50 but are important in other modelsBIOL 53332Lecture ThreeMutantTherefore,using one infection model and specific definition of virulence,study probably underestimated number of virulence genesHowever,may also have overestimated if you eliminate housekeeping genes,such as recA,that have other functionsBIOL 53333Lecture ThreeMutantCan make a case that housekeeping genes contribute,as do other genes concerned with bacterial physiologyBIOL 53334Lecture ThreeMutantIdentifying virulence genes by regulationVirulence genes are frequently in operons and regulons controled by same proteinsIf one gene found,other genes may also be foundApproach uses transcriptional fusionsBIOL 53335Lecture ThreeMutantIntroduction by plasmid(suicide vector)Common way to introduce transposon into chromosomeAlso could be done with intact or inactivated cloned geneBIOL 53336Lecture ThreeLecture ThreeQuestions?Comments?Assignments.BIOL 53337