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Medical Microbiology Medical MicrobiologyThe BacterialStructuresKey laboratory of Medical Molecular VirologyMinistry of Education and Ministry of HealthShanghai Medical College of Fudan University /H t th t tb k it i t b k f b i How to use the textbook, it is not a easy book for beginner.Attendingthe lectures, and on time!Follow the lecture notes and keywords, read the textbook, and add more information to you notebook;If there any questions, just ask;You may catchme in the lab after 4:00 pmat weekday or Youmaycatchme in the lab after 4:00 pm at weekday or weekends morning (54237524) ; [email protected]: at the end of lecture ppt or on elearning Homework: at the end of lecture ppt. or on elearningStudents: hand out on elearning in 2 weeks after lecture (deadline set on the elearning) email not acceptable (deadline set on the elearning), email not acceptableTeacher: Feedback in 2 weeks (after the deadline for handing the homework)2handing the homework)Key Words Key Words Key Words Key WordsCell wallProkaryoticEukaryoticCell wallPeptidoglycan (murein, mucopeptide)Eubacteria (Bacteria)Archaebacteria (Archaea)ChOuter membrane(LPS )Cell membraneChromosomeGram stainingCell membraneSpheroplast/protoplastL form Gram stainingGram negative Gram positiveFlagella (Chemotaxis)Pili (fimbriae) Pili (fimbriae)Capsule (slime layer, glycocalyx)Spore (resistant)EUKARYOTES-fungi -plantsPROKARYOTES-animalsBACTERIA BACTERIAARCHAEA ARCHAEAE b t "T " b t i Eubacter-"True" bacteria-human pathogens-clinical or environmentalChapter 3 p 41 clinical or environmental-one kingdomArchaea Chapter 3, p.41Classification of BacteriaTable 3-1 Taxonomic Rank4-Environmental organisms Characteristic Prokaryotic EukaryoticDrugtargets?Size (diameter) 0 1 2 0m 10 100 m homework Size (diameter) 0.1-2.0 m 10-100 m homeworkNucleus Nucleoid, no nucleoli, no membraneNucleusno nucleoli, no membraneOrganelles AbsentPresent Glycocalyx Capsule or slime layer In some cell Glycocalyx Capsule or slime layer In some cellCell wall Usually present-peptidoglycanMostno-celluose /chitin peptidoglycan celluose /chitinPlasma membraneLack cholesterol cholesterolRibosome 70S : 30S (16S rRNA) 50S (5S & 23S rRNA)80S40S/60S70S(mitochondria)S=sedimentation coefficientChromosome Single circular Haploid Diploid Chromosome Single circularHaploid DiploidCell division Binary fission MitosisS l R N H i t l t f f M i i5Sexual Rec. No, Horizontal transfer of DNA MeiosisEukaryotic cell Prokaryotic cellGram +C ll llR h d l iNucleoidC ll bFlagellumCell wallRough endoplasmic reticulumNucleoidNucleusCell membraneGram -PiliCytoplasmGranuleCapsule6Mitochondriay pCell (inner) membrane Outer membraneRibosomesCell wallSize of Bacteria Average bacteria 0.5 - 2.0 um in (- microsopes)-- RBC is 7.5 um in diam. Surface Area to Volume is 3:1-- Typical Eukaryote Cell SA/Vol is 0.3:1 Nutirtion enters through SA, quickly reaches all parts of bacteria-- Eukaroytes need structures & organellesLight microscope: Bright field microscope Lightmicroscope:Brightfield microscope100xobjective lens Phased contrastmicroscope10x ocular lensDark-field microscopeFluorescencemicroscopeElectron microscopeScan electron microscopeBacteriaare transparentScan electron microscopeConfocal microscopepChapter 2, p. 9Shapes of BacteriaBacteria are classified by shape into 3 groups:Spiral:Spirilla, SpirillumRod-shaped:bacilli, bacillusRound:Cocci, coccusShapes of Bacteria p Coccus Chain = Streptoccus Cluster = Staphylococcus p y Diplopcoccus Bacillus Chain = Streptobacillus Coccobacillus Vibrio = curved Spirillum Spirillum SpirocheteB ill BacillusSpiral bacteriumVibrioSpirillumHelicobateriumBacterial Structures Cell WallLi l h id -Lipopolysaccharides-Teichoic Acids Cytoplasm-Inclusions Ribosomes Nucleoid-Chromosome & Plasmids Capsule Flagella PiliChapter 2, p. 21 Sporesp , pCell WallThe Cell Wall- outside of cell membraneHans Christian Grammembrane- rigid, protecting cell from osmotic lysis y1884Gram stainingGram PositiveGram NegativeGrams Serendipitous Stain, still forms the basis forThe Cell WallI am aware that as yet it is very still forms the basis for identification of bacteriadefective and imperfect Hans Christian Gramp. 36PeptidoglycanThe Cell WallG+Bacteria (~90%) G-Bacteria(~10%)PeptidoglycanThe Cell Wall Peptidoglycan Polymer (amino acids + sugars) Unique to bacteria Unique to bacteria Sugars; NAG & NAM (backbone) N-acetylglucosamine N-acetylglucosamine N-acetylmuramic acid(The same in all bacterial species) (The same in all bacterial species) Tetrapeptide (vary fromspecies) Tetrapeptide (vary from species)-D form of Amino acids (not L form)Hard to break down Dform Hard to break down D form Pentapeptide (vary from species)Amino acids cross link NAG &NAMFig. 2-15p23- Amino acids cross link NAG & NAMp.23The Cell WallPeptidoglycanMore than 40 sheetsin Gram positive bacteriaOnly 1 2 sheets Only 1-2 sheetsIn Gram negative bacteriaGram+Peptidoglycan p g y17The Cell WallGram-PeptidoglycanFig. 2-15p.23G-PeptidoglycanG+Peptidoglycan19Peptidoglycan recognition protein, PGRPThe Cell WallE. coliDAPPeptidoglycan synthesis The Cell WallBacitracin BacitracinBactoprenol phosphate transports NAM and NAG across the cell membrane in the synthesis of peptidoglycan the cell membrane in the synthesis of peptidoglycanFigure 6-19p.88GRAM POSITIVE CELL WALLSpecial components: Teichoicacid TA (p.22)Teichoicacid (WTAwall associated)Lipoteichoic acid (LTA, membrane associated)-Negatively chargedS f ti tt h t f b t i t i l llLipoteichoic acidPeptidoglycan-teichoic acid -Surface antigen, attachment of bacteria to animal cellsPeptidoglycanCytoplasmic membrane22CytoplasmFigure 2-16 p.24GRAM NEGATIVE CELL WALLSpecial components: outer membraneLPSlipoprotein Outer Membrane(Major permeability barrier)LipopolysaccharidePorinB li t i Braun lipoproteinPeptidoglycan PeptidoglycanInner (cytoplasmic) membranePeriplasmic binding proteinPermease23Cytoplasm Figure 2-17 p.25Fi 2 17 25 Figure 2-17 p.25Outer MembraneGram negative bacteria major permeability barrier major permeability barrier space between inner and outer membranei l i periplasmic spacestore degradative enzymes Grampositive bacteria : no periplasmic space Gram positive bacteria : no periplasmic spaceCell Wall Summary Unique to bacteria Determine shape of bacteria Determine shape of bacteria (L form bacterias shape ?)Strength prevents osmotic rupture Strength prevents osmotic rupture G+ -peptidoglycan +TA G- -out membrane (LPS) + peptidoglycan Some antibiotics effect directly: So e a bo cs e ec d ec y lysozymePenicillin Penicillin27Wall-less forms (bacteria) Result from action of:- Lysozyme lytic for cell wally y y- antibiotics block peptidoglycan biosynthesis In osmotically protective media (isotonic)- spheroplasts (with outer membrane)protoplasts (no outer membrane)G-G+- protoplasts (no outer membrane) If wall-less bacteria can grow and divideGg L forms bacteria chronic infectionInduced by antibiotic (penicillin)i t t t tibi ti t t t -resistant to antibiotic treatment-reversion (to normal with wall)-relapses of the overt infection drug resistant -relapses of the overt infection drug resistant29Cell Membrane Bilayer Phospholipid Water can penetrate Flexible Flexible Not strong, ruptures easilyO ti t d b t l Osmotic pressure created by cytoplasmCytoplasm 80%Water 20%Salts Proteins) 80% Water, 20% Salts-Proteins) Osmotic Shock important Inclusion bodygranules for identification of bacteria -granules for identification of bacteria Chromosome Plasmids No organelles (Mitochondria Golgi etc ) No organelles (Mitochondria, Golgi, etc.)Nuclear material Chromosomecircular Haploid circular, Haploid Advantages of 1N DNA over 2N DNAmore efficient grows quicker -more efficient, grows quicker-mutations allow adaptation to environment quicker Plasmids PlasmidsExtra-chromosomal DNAI d d li i Independent replicationmultiple copy numberhorizontal transfercoding - pathogenesis factors32- antibiotic resistance factors-superbugRibosome Ribosome Protein synthesis70S :Protein synthesis Targets of antibiotics70S : 30S (16S rRNA) 50S (5S & 23S rRNA) ( )StreptomycinErythromycinp yCapsules and slime layers Capsules and slime layers Envelope outside cell wall Envelope outside cell wall Well defined: capsuleN t d fi d li l l l Not defined: slime layer or glycocalyx -polysaccharide on external surface Usually polysaccharide (Table 2-1, p30), but often lost during in vitro culture Protective in vivo-adhere bacteria to surfaceS t d l f t th S. mutans and enamel of teeth-prevent phagocytosiscomplement cant penetrate sugars34complement cant penetrate sugarsFl ll Flagella Some bacteria are motile- movement So e bacte a a e ot e o e e t Arrangement basis for classification Monotrichous; 1 flagella Monotrichous; 1 flagella Lophotrichous; tuft at one end Amphitrichous; both ends Amphitrichous; both ends Peritrichous; all around bacteriaChapter 4FLAGELLA Locomotory organelles- flagella oco oto y o ga e es age a Swarming occurs with some bacteria-Spread across Petri Dish Spread across Petri DishProteus species most evident Sense environment Senseenvironment Chemotaxis-respond to food/poison -respond to food/poisonor unfriendly environmentsProteus Flagellag embedded in cell membrane project as strand Flagellin (protein) subunits move cell by propeller like action E Coli with flagella E. Coli with flagellaShigela no flagella 38Pili (fimbriae) Short protein appendages (hair like) smaller than flagella pilins protein-vary Adhere bacteria to host epithelium E. coli has numerous typesK88, K99, F41, etc. Anti-pili antibodies to block adherence F-pilus (Fertility factor) p ( y )-Used in conjugation (sex conjugation )-Exchange of genetic information g g Flotation, increase boyancy Pellicle (scumon water) Pellicle (scum on water) More oxygen on surfaceF Pilus for Conjugation F-Pilus for ConjugationEndospores (spores)Dormant cell (non-reproductive structure )-a thick celled structure formed inside the cell,-encloses all the nuclear materials and some cytoplasmLocation important in classificationCentral, Subterminal, TerminalSporulation (Fig. 2-28)Contain calcium dipicolinate in coreResistant to adverse conditions Resistant to adverse conditions Heat, irradiation, cold, organic solvents, extremely dry Boiling >1 hr still viable gSterilization, autoclaveallows the bacteria to survive for many yearsTh i bl b t i l th t h b f d th t 40 illi There are viable bacterial spores that have been found that are 40 million years old on Earth-Steinn Sigurdsson Endospores Produced when starvedunder unfavorable conditionBacillus anthracis form spores in O2p2anthrax-corpse - no necropsy Dormant -germination vegetative form (reproductive) Dormantgermination vegetative form (reproductive) Activation conditions ?(home work, -related with medical practice, Clostridium (home work,related with medical practice, Clostridium tetani -tetenus )- Bacillus stearothermophilus -spores Bacillus stearothermophilus sporesUsed for quality control of heat sterilization equipmentBacillus anthracis spores (Bacillus and Clostridium) - Bacillus anthracis - spores (Bacillus and Clostridium)Used in biological warfareBacillus anthracis G+Aerobic spore-forming bacteria e ob c spo e o g bacte a(form spores in O2)2011 911 k 2011 911attackStarting1week after theg9/11/2001 attack, letters containing anthrax t t di ffi were sent to media offices and to Senators Tom Daschle and Patrick Leahy and Patrick Leahy Summary : Bacterial Structures Summary : Bacterial StructuresCapsules and slime layersEndospores (spores)47Review questions1. In p38: question 2, 5, 6, 7, and give the explains2 Summary the differences between the cell wall of 2. Summary the differences between the cell wall of G+bacteria and G-bacteria (related with medical practice)3. Why penicillin and lysozame have less effect on G-bacteria? bacte a4. Spore germination activation conditions (related with medical practice, Clostridium tetani -tetenus )5. Explain which bacterial structure can be as an antibiotics target.49The Cell WallI G iti b t i th l t l i l t t i i t d b In Gram-positive bacteria, the purple crystal violet stain is trapped by the layer of peptidoglycan which forms the outer layer of the cell.In Gram-negative bacteria the outer membrane of lipopolysaccharides Gram-negative bacteria, the outer membrane of lipopolysaccharides prevents the stain from reaching the peptidoglycan layer. The outer membrane is then permeabilized by ethanol treatment, and the pinkp y , psafranin counterstain is trapped by the peptidoglycan layer.51Peptidoglycan Peptidoglycan synthesisand antibioticsTeichoicacid:Polysaccharides of glycerol phosphate or ribitol phosphate linked via phosphodiester bonds p p p p53