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1、Lecture 10微生物学美国IndianaUniversityPurdueUniversity授课10Stillwatersrundeep.流静水深流静水深,人静心深人静心深Wherethereislife,thereishope。有生命必有希望。有生命必有希望Lecture 10StaphylococciImportant human pathogenCauses both relatively minor and serious diseasesOne of the hardiest of the non-sporeforming bacteriaCan exist on dry su
2、rfaces for a long periodRelatively heat-resistant; temperature range of 18 - 40 CBIOL 5332Lecture 10StaphylococciMorphologyGram+ grape-like cluster, but in clinical specimens, can be a single cocci or diplococciGeneral physiological characteristicsNonmotileFacultatively anaerobicCatalase +Grows in m
3、edia containing 10% NaClBIOL 5333Lecture 10StaphylococciRelationship to disease (only 3 important)S. aureuscauses a number of diseasesS. epidermidispresent in normal flora (normally benign, except when introduced via catheters, etc.)S. saprophyticuscauses uninary tract infectionsBIOL 5334Lecture 10S
4、taphylococciMicrobial physiology and structureCapsule may not be found growing on media, but it is usually present in vivoTeichoic acids are phosphate containing polysaccharides bound to both peptidoglycan and cytoplasmic membraneSpecies specificPoor immunogens, but when bound to peptidoglycan, get
5、an antibody responseBIOL 5335Lecture 10Pathogenesis of S. aureusFeatures typical of staphylococci infections:Initial lesion is normally mild and localizedResults in a boilnormally, it is self-limitingCan result in systemic infectionBIOL 5336Lecture 10Pathogenesis of S. aureusStage I: encounterhumans
6、 are major reservoir for S. aureusColonize nose and are found in about 30% of individualsTransiently found on skin, oropharynx, and fecesTransmitted via:Hand contactAerosols from pneumonia patientsBIOL 5337Lecture 10Pathogenesis of S. aureusStage I, continuedCertain occupations are more prone to col
7、onizationPhysicians, nurses, hospital workersCertain classes of patients are more prone to colonizationDiabetics, hemodialysis patients, and drug abusersBIOL 5338Lecture 10Pathogenesis of S. aureusStage II: entrynot normally through unbroken skinCan enter if large numbers have accumulated through po
8、or hygieneBIOL 5339Lecture 10Pathogenesis of S. aureusStage III: spread and multiplicationSurvival depends onNumber of organismsSite involvedSpeed with which inflammatory response is mountedImmunological competence of hostIf inoculum is small and host immunologically competent: infection normally de
9、featedBIOL 53310Lecture 10Pathogenesis of S. aureusStage IV: damageLocal infection leads to formation of abscess (collection of pus)In skin, boils or furunclesInterconnected abscesses are called carbunclesMay also spread in subcutaneous or submucosal tissuescellulitisBIOL 53311Lecture 10Pathogenesis
10、 of S. aureusStage IV, continuedDevelopmentinvolves both host and bacterial factorsAcute inflammatory reactionProportion of bacteria survive and are capable of lysing neutrophils that engulfed themOutpouring of lysosomal enzymes that damage surrounding tissuesInflammatory area surrounded by fibrin c
11、lotBIOL 53312Lecture 10Virulence Factors of S. aureusStage IV, continuedVirulence factorsmost designed to avoid phagocytosis or survive once ingestedWall componentsSurrounded by capsule: not as effective as pneumococcus or meningococcusCell wall murein activates complement by alternative pathwayTeic
12、hoic acid also activates and involved in adherenceProtein A interferes with opsonization by binding with Fc region of Abcomplement activated primary pathwayBIOL 53313Lecture 10Virulence Factors of S. aureusStage IV, continuedSecretion of enzymesCatalasehydrogen peroxide to water and oxygen (all stap
13、hylococci produce)Coagulasemakes fibrin clot (wbc penetrate badly; only S. aureus)Hylauronidasedegrades connective tissues (facilitates spread; 90% of S. aureus strains)Fibrinolysin (staphylokinase)dissolve fibrin clots (virtually all S. aureus)BIOL 53314Lecture 10Virulence Factors of S. aureusStage
14、 IV, continuedSecretion of enzymesLipasesrequired for invasion into cutaneous and subcutaneous tissues (found in all S. aureus and 30% of others)Nucleaseheat stable (role is uncertain; S. aureus)PenicillinaseBIOL 53315Lecture 10Virulence Factors of S. aureusStage IV, continuedSecretion of toxinsCyto
15、lytic (membrane-damaging by pores)Alpha, beta, (sphingomyelinase C), delta, gamma, leukocidin (cannot lyse red blood cells)Others lyse rbc and leukocytes (referred to previously as hemolysins)Cause lysis of neutrophils leading to massive lysosomal enzyme secretionBIOL 53316Lecture 10Virulence Factor
16、s of S. aureusStage IV, continuedSecretion of toxinsExfoliative toxin (scalded skin syndrome) extrachromosomalToxic Shock Syndrome toxin-1 (enterotoxin F)exotoxin secreted during growthSome produce enterotoxin B instead (role not clear)BIOL 53317Lecture 10Virulence Factors of S. aureusStage IV, cont
17、inuedSecretion of toxinsEnterotoxins (A-E)found in both S. aureus and S. epidermidisResistant to hydrolysis by gastric and jejunal enzymesStable to heating at 100C for 30 minutesMechanism of toxin activity not understood; no satisfactory animal modelStimulate intestinal peristalsis and have CNS effe
18、ct; intense vomitingBIOL 53318Lecture 10Pathogenesis of S. aureusTreatmentAntibioticsTypes:Methicillin, oxacillin, nafcillin, and dicloxacillin (semisynthetic penicillins resistant to -lactam hydrolysis)Majority of patients can be treated, but 10-15% S. aureus and 40% coagulase-negative staphylococc
19、i are resistant; treat with vancomycinBIOL 53319Lecture 10Pathogenesis of S. aureusTreatmentAntibioticsResistance:Plasmid-borne (hydrolysis of -lactam ring)Chromosomalchange in structure of penicillin-binding proteinsBIOL 53320Lecture 10StreptococciFermentative (oxygen tolerant) Gram+ cocci in chain
20、sSensitive to penicillinsHuman reservoirpassed from person to personBIOL 53321Lecture 10StreptococciProperties of Lancefield Groups (CHO antigens on wallsee handout)Group A: cross-reaction can lead to:Rheumatic feverGlomerulonephritisBIOL 53322Lecture 10StreptococciRecent Group A Streptococcus virul
21、ence factorsM-like proteinsbind IgM IgG (protease inhibitor) and alpha2 macroglobulinF proteinadherence to epithelial cellsC5a peptidasedegrades C5A pyrogenic exotoxins; previously called erythrogenic toxinsBIOL 53323Lecture 10Staph and Strep ToxinsS. aureus toxic shock TSST-1S. pyogenes toxic shock
22、 TSSL-1S. pyogenes scarlet feverSPE-1 (children, not adults; immunity)BIOL 53324Lecture 10Staph and Strep ToxinsS. aureus : Toxic Shock SyndromeFever, diffuse rashExfoliation of skin on palms and soles of feetNormally doesnt compete well in relatively anaerobic vaginal areaBIOL 53325Lecture 10Staph
23、and Strep ToxinsS. aureus : Toxic Shock SyndromeSuper-absorbent tampon:Created aerobic pocketsRemoved Mgproducing toxinAfter removed tampon, cases declined; did not disappearStill associated with wounds, rare nasal surgeryBIOL 53326Lecture 10Staph and Strep ToxinsS. pyogenes: Toxic Shock-Like Syndro
24、meSkin or wound infection - bloodstreamDeath rate 30%; over 10-fold higher than TSSTSeen in immunocompromised peopleAlso other infections occurred: soft tissue infection with influenza symptomsHigh fatality rate because rapid development of shock and multiple organ failureBIOL 53327Lecture 10Staph a
25、nd Strep ToxinsS. pyogenes: Toxic Shock-Like SyndromeFeatures in common with scarlet feverOccur in healthy peopleBoth associated with high fatality rateProduce same exotoxin: streptococcal pyrogenic exotoxin (Spe)Similar in mechanism to TSST-1BIOL 53328Lecture 10Staph and Strep ToxinsComparing TSLS-
26、1 and TSST-1:Rash, fever, shock, multiple organ failure; resemble endotoxin septic shockBoth toxins superantigensSame mechanisms of actionLimited similarity at amino acid sequence levelBIOL 53329Lecture 10Staph and Strep ToxinsTSLS-1 related to erythrogenic toxin (scarlet fever; SpeA)Serotypes:Spe A
27、TSLS or invasive S. progenesSpe BSpe CSome strains dont produce Spe A, so Spe B or Spe C also has roleBIOL 53330Lecture 10Staph and Strep ToxinsHow do TSST-1 and SPE cause shock and multiple organ failure?Hypotheses not mutually exclusiveBIOL 53331Lecture 10Shock and Organ FailureFirst Hypothesis: s
28、ame as LPS triggering release cytokines IL1,TNFConsistent with role as superantigenPromote association between macrophage and helper T cellsproliferation of T cells producing high IL2 levelSecondarily produce IL1 TNFInject TSST-1 into rabbits; elevated levels IL1 TNFBIOL 53332Lecture 10Shock and Org
29、an FailureSecond hypothesis: increase bodys sensitivity to LPS; consistent with:Acts synergistically with LPS to amplify toxic effects in vitro and in animalsConceivablelow levels leaching into blood due to lysis of resident microfloraNormally no effectPresence of Spe or TSST-1 causes an effectBIOL
30、53333Lecture 10Shock and Organ FailureEvidence to support role for LPS in TSST and TSLSInjecting TSST-1 or Spe is lethal to rabbitsInjecting exfolatin and concanavalin A not lethal to rabbits Both elicit T cell proliferation, but dont enhance sensitivity to LPSBIOL 53334Lecture 10Shock and Organ Fai
31、lureEvidence to support role for LPS in TSST and TSLSTSST-1 not lethal to gnotobiotic animalsWouldnt expect leakage, but still T cell responseTherefore, both suggest T cell proliferation not as important as synergy of LPSNot conclusive; difficult to prove same level T cell stimulation, proliferation
32、 occurred in all casesBIOL 53335Lecture 10Shock and Organ FailureThird hypothesis:TSST-1 can act directly on endothelial cellsDamage causes malfunction in circulatory system, which creates hypotensionData: swelling associated with massive leakage of fluid from capillaries is marked symptom of both T
33、SST and TSLSCould also be result of action of blood vessels by cytokines, coagulation, or complement cascadeBIOL 53336Lecture 10Staph and Strep ToxinsMortality of S. pyogenes vs. S. aureusTSLS higher than TSSTSLS strains enter bloodstreamTSS, only the toxin circulatesS. pyogenes known to be invasive
34、; killed by PMNs and macrophage if ingestedBIOL 53337Lecture 10S. pyogenes InvasivenessStrategies for evading phagocytosis; (1):M protein binds H factor better than factor BLeads to degradation of C3bTherefore, prevents opsonization by C3b and formation of C3 convertaseBIOL 53338Lecture 10S. pyogene
35、s InvasivenessStrategies for evading phagocytosisData supporting:M mutants yield more susceptible to phagocytosis; less virulent than wild typeAb against M protective80 serotypes of M; possibly evades host antibodies by changing serotype; however, no data to support this hypothesisBIOL 53339Lecture
36、10S. pyogenes InvasivenessStrategies for evading phagocytosis; (2):Protease cleaves C5aChemoattractant stimulates oxidative burstSome activation of complement could occur in spite of M protein becauseLysis releases wall components that activate complementStreptococci could protect themselves- C5a pe
37、ptidaseData supporting:C5a mutants less virulent that wild type in animalsBIOL 53340Lecture 10S. pyogenes InvasivenessStrategies for evading phagocytosis; (3):M like proteinsSequence and structural similarity to MCOOH embedded; NH2 exposedMost similar to M and each other at carboxy endThese proteins
38、 bind Fc portion of IgG and IgABIOL 53341Lecture 10S. pyogenes InvasivenessStrategies for evading phagocytosisM like proteins; possible rolesCoat with host proteinless likely detected as invader by complement and immune systemAdherence for body cells that contain Ab on surfaceAlso can bind host prot
39、ease inhibitor such as 2 macroglogulinHost uses protease inhibitor to protect against proteases released by phagocytesBIOL 53342Lecture 10S. pyogenes InvasivenessStrategies for evading phagocytosis; (4):F proteinbinds fibronectinAdherence of bacteria to tissuesEvasion of immune systemSummary of inva
40、sivenessNo direct evidence M-like proteins involved in virulenceFound in impetigo strains, not always in severe invasive strainsNeed mutant studies to answer questionsBIOL 53343Lecture 10S. pyogenes VirulenceRegulation of S. pyogenes virulence genesExpression M, C5a peptidase, and some M-like protei
41、ns; regulated at transcriptional levelResponds to CO2 levelsIncreased CO2 causes increased productionBIOL 53344Lecture 10S. pyogenes VirulenceRegulation of S. pyogenes virulence genesRegulatory genemry transcriptional activator; sequence analysis shows it is part of two-component systemSensornot fou
42、ndActivatorAlso known that speA gene on temperate phageBIOL 53345Lecture 10S. pyogenes PathogenesisTreatment and preventionTSST, TSLS are medical emergenciesSurgical debridement of wounds prevents further production of toxinAntibiotics; penicillinToxic effects TSST-1 countered by intravenous rehydra
43、tion; counter hypotensionBIOL 53346Lecture 10Streptococcal TreatmentPreventionVaccine possibleTarget against MPossible problems# serotypes, but severe invasive disease caused by fewAB against M cross-reacts with heartBIOL 53347Lecture 10Streptococcal Sequellae HypothesesFirst: Autoimmune theoryEpito
44、pes that cross react with epitopes on cardiac myosin and sarcolemmal membrane proteinsThus, T cells or antibodies could attack tissueInflammatory response damages heart valvesBIOL 53348Lecture 10Streptococcal Sequellae HypothesesGlomerulonephritisHigh levels Ab to streptococcal Ag circulating in blo
45、od stream causes AgAb complexes to accumulate in kidneyInflammatory response attacks kidney interfering with kidney functionBIOL 53349Lecture 10Streptococcal Sequellae HypothesesData supportingAg-Ab complexes visible in people with glomerulonepheritisglomeruliDecrease in C3 and other complement comp
46、onents also seen; supports hypothesis that inflammatory response is occurringSecond: Toxins cause sequellaeBIOL 53350Lecture 10Streptococcal Sequellae HypothesesMain argument againstTime lag between initial infection and development of rheumatic fever (RF; several weeks) or glomerulonephritis (10 da
47、ys)Normally, if due to toxin, within a weekCandidates for toxin most likely to cause glomerulonephritis: streptococcal O, streptokinase, or SpeBIOL 53351Lecture 10Glomerulonephritis HypothesesStreptococcal O cytotoxinMechanism and aa sequence similarity to pneumolysinPore-forming toxinInjected into
48、lab animals; damages heartTherefore, may have role in RFAlso, very immunogenic; maybe Ab damageBIOL 53352Lecture 10Glomerulonephritis HypothesesStreptokinasePlasminogenplasminTherefore causes symptoms similar to glomerulonephritis in animalsInteresting, but not provenBIOL 53353Lecture 10Rheumatic Fe
49、ver HypothesesSpe RF strains produce Spe; others dontEnhances cardiotoxicity caused by Streptococcal O in animalsHavent explained long time lagBIOL 53354Lecture 10Rheumatic Fever HypothesesMysterious feature of RF unexplainedTreated with antibiotics for as late as 9 days after symptoms, still protec
50、ted against RFAfter 9 days, toxic products should be circulating and immune response underwayRecurrence of diseaseNormally, infection results from different strainSome result from same strainRF symptoms take as long to develop as in originalBIOL 53355Lecture 10Rheumatic Fever HypothesesMysterious fe
51、ature of RF unexplainedIf caused by autoimmune response, would expect faster responsePossible explanation: previously exposed produces primed immune systemBIOL 53356Lecture 10Streptococcal SequellaeTreatment and preventionStrep throat self-limitingTreat with antibiotics and prevent RF and glomerulon
52、ephritisOnly S. pyogenes strains cause RF or GNot all people colonized actually contract RF or GBecause of the seriousness, treat any strain with antibioticsBIOL 53357Lecture 10Streptococcal SequellaeTestsBlood agarHemolysisBacitracin sensitivityRapid test and culture test both yield high number of
53、false negativesFor patients who are allergic to penicillin, use erythromycinBIOL 53358Lecture 10Streptococcal SequellaePrevious damageEven heart murmurDentists recommend prophylactic penicillin before dental workKill bacteria escaping into blood stream from mouth (oral bacteria are susceptible to pe
54、nicillin)Reduces chance of colonizationBIOL 53359Lecture 10Staphylococcal EnterotoxinsNormal enterotoxins cause diarrheaWater loss from small intestine mucosaCause c-GMP or c-AMP levels in mucosal cells to riseBIOL 53360Lecture 10Staphylococcal EnterotoxinsStaph toxins operate in a different mode; h
55、ypotheses include:1) Stimulates vagus nerve endings in stomach lining that control emetic (vomiting) responseIf hypothesis correct, its a neurotoxin, not enterotoxin2) SuperantigenIL2Administering IL2 to human volunteers produces many of the same symptoms (nausea, vomiting, fever, malaise)BIOL 53361
56、Lecture 10Staphylococcal EnterotoxinsNeither hypothesis conclusively provenCould be a combination of bothSeven serotypes: SEA, SEB, SEC1, SEC2, SEC3, SED, SEE30 kDa proteins share considerable aa similarity and single internal S-SBIOL 53362Lecture 10Staphylococcal EnterotoxinsSE closely related to S
57、peGenes coding for SEC1 and Spe A have 60% nucleotide identitySE produced in different amounts by different strainsentA produces much less toxin than entBDifferences in promoter strengthBIOL 53363Lecture 10Staphylococcal EnterotoxinsSEB on integrated plasmidSEA on lysogenized phageOne strain: entB, entC, plasmid-borne SED: entD on 27.6 kb plasmidBIOL 53364Lecture 10Lecture 10Questions?Comments?Assignments.BIOL 53365
