英文版-Indiana-University-Purdue-University-Fort-Wayne微生物学授课讲义-lecture-04课件.ppt

1、Lecture 4BIOL 5331Strategies for StudyingMicrobial PathogenesisBIOL 533Lecture 4Medical MicrobiologyLecture 4BIOL 5332Reporter Gene Fusions Operon fusion:transcriptional Promoter from foreign gene Translational sequence from reporter geneLecture 4BIOL 5333Reporter Gene Fusions Gene fusion:translatio

2、nal Promoter and translational signal from foreign gene Initiation signal/+NH2 coding sequence Translational signal also from reporter gene Initiation sequence and part of NH2 sequence of reporter gene missing,but COOH region still present Two coding sequences must be in frameLecture 4BIOL 5334Techn

3、ique Use transposon vector containing lac fusion Carries promoterless lacZ gene and selectable antibiotic marker Selection for antibiotic marker generates set of random insertions in chromosome Colonies carrying transposon screened for expression under conditions testedLecture 4BIOL 5335Foreign Gene

4、 Expression Assay for production of gal Higher temperature+iron etc.Locate unknown geneLecture 4BIOL 5336lacZ Fusion Used to find regulatory genes that control expression of virulence genelacZ fusion strain is mutagenized(chemical or another transposon)Resulting colonies screened for aberrant regula

5、tionLecture 4BIOL 5337lacZ Fusion Aberrant regulation:Colonies are not Lac+in presence of inducing conditions Loss of activator Colonies are Lac+under both inducing and non-inducing conditions Loss of repressorLecture 4BIOL 5338lacZ Fusion Gene tagged by transposon can be cloned Segment of DNA adjac

6、ent to transposon:Can be used as hybridization probe to clone wild-type locus Can also be cloned by complementation Cloned DNA can be sequencedLecture 4BIOL 5339phoA Gene Fusions Virulence genes are small subset of total genes,so random mutagenesis will affect many more non-virulence genes Way to op

7、timize number of virulence gene mutations takes advantage of fact that most virulence genes are on bacterial surface or excreted into mediumLecture 4BIOL 53310phoA Gene Fusions Translational protein fusion Alkaline phosphatase is expressed only in periplasm Foreign gene product has to be expressed o

8、utside of cytoplasm In vector,signal sequence mutations block export of alkaline phosphatase and render it inactiveLecture 4BIOL 53311phoA Gene Fusions Export and activity restored by fusing in frame a restriction fragment to truncated phoA gene;i.e.,lacking signal sequences No AUG start codon No ri

9、bosome binding site Restriction fragment has portions of NH2-terminal genes encoding signal sequences of heterologous proteins,such as OmpF or LamBLecture 4BIOL 53312TnphoA Transposon Created TnphoA that randomly inserts phoA into target genes Hybrid proteins display alkaline phosphatase activity on

10、ly if target gene encodes a periplasmic outer membrane or extracellular proteinLecture 4BIOL 53313TnphoA Transposon Use TnphoA:Study topology of inner membrane proteins gal fusions expressed only in cytoplasm Identify genes that encode export proteins Exported proteins represent major class of virul

11、ence factors Number of additional delivery systems Remember analysis of Groisman and HeffronLecture 4BIOL 53314Analysis of Virulence Study TnphoA mutants of S.typhimurium identified as Pho+on L-agar plates Tested for virulence by oral infection of BALB/C mice Found 15/150 Pho+mutants were avirulent

12、9/15 were defective in LPS biosynthesis None had insertions in virulence plasmidLecture 4BIOL 53315Analysis of Virulence Conclusion:same caveats as apply to normal transposon mutagenesis:But use of TnphoA limits identification of random insertions to those affecting cell envelope factorsLecture 4BIO

13、L 53316Analysis of Virulence Disadvantage:Limits selection to genes expressed in S.typhimurium on L-agar Genes expressed at low levels or repressed on L-agar plates would be missedLecture 4BIOL 53317Analysis of Virulence Expression problem may explain why 9/15 Pho+avirulent mutant strains were LPS r

14、ather than being defective in other virulence genesLecture 4BIOL 53318Analysis of Virulence TnphoA narrows,but doesnt solve all detection problems While disrupting gene,the TnphoA mutations produce alkaline phosphatase hybrid proteins Could be phenotype not due to loss of gene,but presence of delete

15、rious hybrid protein;Therefore,to be sure mutation in virulence gene,must conduct other genetic analysesLecture 4BIOL 53319In Vivo Expression TechnologyMahan et.al.,1993.Science 259:686-688 Based on gene that,when mutated,causes organism to be attenuated Used Salmonella typhimurium purA lacZY both l

16、ack promoter (in suicide plasmid)Lecture 4BIOL 53320In Vivo Expression TechnologySau3A digested chromosomal DNAelectroporated Salmonella PurA strain Injected into mice twice After each 3 daysharvest spleen Plate on artificial media to detect Lac strainsLecture 4BIOL 53321In Vivo Expression Technolog

17、y Based solely on fact that ivi genes are highly expressed in animal tissues,but are not expressed in lab media Selection strategy:Bacterial strain with mutation that attenuated growth in vivo Complemented by operon fusions to same attenuated geneLecture 4BIOL 53322In Vivo Expression Technology Crit

18、eria for IVET system:Fusions in single chromosomal copy No plasmid complications Fusions constructed without disruption of gene Loss of gene could be lethal to bacterium Monitored both in animal and on lab mediaLecture 4BIOL 53323pIVET Vector pIVET1 plasmid Suicide vector Replication dependent on Pi

19、 replication protein Salmonella does not produce Promoterless purA gene and lacZ operon Can monitor level or transcription of entire operon Unique cloning site 5 to purA gene Clone random fragments of DNAbla gene allows plasmid to be selectedLecture 4BIOL 53324pIVET Vector Construction of clones Clo

20、ne Sau3A fragments into vector Put fragment pool into PurA deletion strain of Salmonella(Pi replication protein)Selection for ApR plasmid:integrates at homologous Salmonella gene No natural lacZ gene Deleted purA geneLecture 4BIOL 53325pIVET Vector Clones of interest Product of plasmid integration g

21、enerates a duplication One promoter drives purA lacZ while other drives wild-type copy of virulence gene Integrate at unknown gene because purA and lacZ are from E.coli;not enough homology to SalmonellaLecture 4BIOL 53326Properties of Clones To survive and propagate in host:purA lacZ fusion has to b

22、e expressed at sufficient level to overcome deletion of purA If gene of interest codes for virulence factor,then expression also required for infection Monitor expression in animal and lab by measuring gal from strains that survive three days and are recovered from spleenLecture 4BIOL 53327Analysis

23、of Virulence To identify genes code for virulence,junctions of fusions cloned and sequenced Prepared inactivated genes Any effect on pathogenesis?Transposon insertions in 3/5 genes BALB/C mice infected orally have increased LD50(200 to 20,000 fold)Therefore,IVET selects for virulence Also,mice immun

24、ized with 1 mutant were protected when challenged with wild-typeLecture 4BIOL 53328Identity of Genes Results:Most genes found so far are housekeeping genes,perhaps because of screening conditions Screened only for expression of genes on aerobic media,but most body parts are anaerobic Crude approach;

25、at best,identifies a set of genes that are expressed at some point of infectionLecture 4BIOL 53329Identity of GenespheST himA operon encodes 2 su phenylalanyl t-RNA synthetase and 1 su host integration factor(IHF)Antisense Rfb O-antigen synthesis O-antigen synthesis mutants attenuated-orally,but vir

26、ulent IP route Mutations that increase LD50 do affect virulenceLecture 4BIOL 53330Pros and Cons Detailed analysis of genes and gene products expressed in animal necessary to determine where and when during infection the gene was expressed More serious problemfocuses on transcriptional regulationLect

27、ure 4BIOL 53331Pros and Cons Genes that are regulated or activated post-transcriptionally will be missed Also,many genes expressed on lab media may be important in vivo If some aspect of in vitro growth condition is similar or identical to in vivo condition(e.g.,37C),certain genes may be discardedLe

28、cture 4BIOL 53332Research Strategy To get maximal variability in genes selected,use different:Reporter genes Infection modelsin vitro growth conditions Likely that infection by different routes or hosts characterized by unique H-P interactions lead to different genesLecture 4BIOL 53333Analysis of Vi

29、rulenceHeithoff et.al.,1999(Mahans group)Previously found over 100 different genes 25%unidentified All affect virulence Looked at regulation of genes in intestine,liver,and spleen,plus cultured murine macrophageLecture 4BIOL 53334Analysis of Virulence Regulation in response to Mg+2,pH,and Fe proceed

30、ed same as in vitro at approximately the same levels Not true in spleen;has to be different signals Think that response to Mg+2 and pH correlates with assuming macrophage existenceLecture 4BIOL 53335Uses Expected to provide Ag for vaccine Genes expressed only in vivo Assist in construction live-atte

31、nuated vaccines(inactivated genes)Also can establish in vivo regulated expression of heterologous Ag in live vaccinesLecture 4BIOL 53336Uses Identification of ivi genes allows detection of changes in metabolism,gene regulation,and cell surface properties during growth in animal tissues Allows new antimicrobial targets to be identifiedLecture 4BIOL 53337Lecture 4 Questions?Comments?Assignments.