Download 8/26/08 Transcript I

Fundamentals I 8/26/08 10-11:00
Microbial Polysaccharides- #1
 Read right off slide- overview of today’s lecture.
Classification of Bacteria by Gram Staining- #2
 120 years ago Christian Gram was studying bacteria. In order to see bacteria in the
microscope you must stain them.
 Used crystal violet
 Traditional Gram stain:
1. Make smear of bacteria
2. Then heat fix by holding it over a flame or adding some acetone.
3. Then stain the bacteria and it all turns dark purple or blue
4. Then add a mordant which helps fix the stain- use iodine alcohol solution.
5. Then allow alcohol to run over the stained bacteria which washes the stain out of
about half the bacteria- decolorization step.
6. Counterstain with safranin- pinkish color
 Example picture: Streptococci- Gram positive (blue)- retain dye
E.coli- Gram negative (pink)-lost blue dye
 Gram found that most bacteria could be divided into Gram positive or Gram negativereflects the fundamental difference in the cell wall of bacteria.
Gram-positive bacteria- #3
 Have a single cell membrane (lipid bilayer) and a thick peptidoglycan layer (PDG)
 PDG layers differ between bacteria
 Lipid bilayer is symmetrical
 See diagram
Gram-negative bacteria- #4
 Two membrane layers and a thin layer of PDG between two membranes.
 Outer membrane very different- phospholipid layer is the inner leaflet, outer leaflet
contains LPS (lipopolysaccharide)
 LPS causes severe toxic reactions even in nanogram amounts.
Slide#5
 Another picture-Same message as above
Peptidoglycan Structure- #6
 Bigger in Gram positive, smaller in Gram negative.
 Know the basic structure- see picture on slide.
 N-Acetylglucosamine with a lactic acid linked is called NAM (N-Acetylmuramic
acid)
 Disaccharide repeated over and over
 Amide linked to NAM is an alanine
 Isoglutamate liked differently than glutamic acid in a protein. Isoglutamate- linked to
gamma carboxyl group
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Lysine and D-alanine- in some organisms (esp. gram positives)- can get crosslinks
between these two.
PDG- long polysaccharide structure linked with the peptides which are then
crosslinked directly or to an inner peptide bridge.
S. aureus has 5 glycines in a row in the inner peptide bridge.
Imagine PDG forms a mesh bag around bacteria because intercellular pressure is
estimated to be 5 atm, so it must be held together tightly- PDG allows this
Slide#7
 N-acetyl unique to bacteria, not found in human cells.
Slide#8
 Pictures show gram positive and gram negative peptidoglycan
 S. aureus has pentaglycine crosslinks
 NAM and NAG repeating
 Some gram positives such as anthrax don’t have interpeptide bridges- they are
directly crosslinked.
 Gram positive and Gram negative very similar.
 Must be highly crosslinked or bacteria could not survive the internal pressure.
Transpeptidase Reaction- #9
 PDG cross-linked using the transpeptidation reaction.
 D-glutamic acid- never see D- amino acids in proteins, but bacteria have D-amino
acids in PDG
 See diagram (S. aureus)
 Causes linkage of amino group to the first D-alanine, and cleaves the terminal one
Biosynthesis of Peptidoglycan- #10 and #11
 Glucosamine-1-phosphate reacts with UTP to form UDP-N-Acetylglucosamine.
 When attach sugars, must activate them first
 Don’t worry about details-just main idea.
 See diagram- read straight off it.
 Build up fully crosslinked PDG
Lysozyme-#12
 Cleaves PDG
 Fleming- Scottish microbiologist- made several discoveries
 Stories behind these may be myths
 He had a bad cold and allowed his nose to drip in one of his plates. When he came
back the next morning, the bacteria did not grow where nasal secretions were. He
thought it was a way to treat disease- called lysozyme
 Killed much bacteria except human pathogens. Thought that is why human
pathogens because our secretion can not kill them
 All human pathogens resistant to lysozyme in our secretions.
Penicillin- #13 and #14
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About 6 years later Fleming made another discovery.
He found that one of his plates was contaminated with a mold and discovered clearing
of bacteria from yellow substance secreted by mold
Had trouble making large quantities, but did kill human pathogens. However nothing
happened for about 10 years until World War II
Look at penicillin molecule- 5 membered ring with sulfur as well as a 4 membered
ring with a carbonyl group and nitrogen- cyclic amide (lactam)- Beta- lactam
Can mill corn-Wet-milling method- one of the bi-products is corn steep liqour and
penicillin grew in this so could make tons of penicillin
Penicillin- #15 and #16
 How does penicillin work? Already shown this in different form
 During crosslinking, the terminal amino group of glycine in the crossbridge reacts
with the D-ala unit splitting off one D-alanine and making a covalent bond.
 2 step process- get an Acyl enzyme intermediate:
1. Peptide sidechain with 2 D-ala react with an enzyme splitting off the D-ala and get
acyl enzyme intermediate.
2. Acyl enzyme intermediate reacts with the glycine crossbridge and you get the cross
linked PDG.
 Penicillin fools the enzyme, transpeptidase, and you get penicillin enzyme complex.
 Open up beta-lactam ring, very stable linkage, so can not do the next step and transfer
it to the pentaglycine chain.
 One molecule of penicillin totally destroys a huge enzyme
 So can not create crosslinks, so bacteria swells up and pops.
 Very effective antibiotic until resistance developed 2-3 years later.
Penicillin Resistance- #17
 Bacteria resistant to penicillin made an enzyme called beta-lactamase.
 Splits beta-lactam between the nitrogen and carbon making the compound inactiveno antibiotic activity.
 Most Staph infecting patients today are penicillin resistant
 Vancomycin still an important drug for treating S. aureus - binds to the second of the
D-ala on the PDG- another way of interfering with the crosslinking of bacteria.
 MRSA- methicillin resistant S. aureus- resistant to methicillin that used to be very
effective
 Problem: Bacteria keep getting resistance to new antibiotics.
 Almost every year a drug company stops antibiotic research because not very much
money off it compared to every day medications such as blood pressure medications.
Teichoic and Lipoteichoic Acids- #18
 On the surface of Gram positive bacteria
 Phosphate groups attached to an aldetol (glycerol or ribitol)
 Sometimes sharp branches of amino acids which play a role in the attachment of the
bacteria to mucosal surfaces.
 Lipoteichoic acids linked to phospholipids inserted in the membranes
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Group-Specific Polysaccharides of -Hemolytic Streptococci- Slide#19
 Rebecca Lancefield- “patron saint” of streptococci- responsible for grouping strep
 Her classifications are the most widely used
1. Group A- strep throat
 -Hemolytic streptococci- bacteria secrete an enzyme which lyse red blood cells- If
you have an agar plate with sheep blood plate and you streak, then get green look
 Cause lots of diseases in animals and humans
 Flesh-eating bacteria also an example
 Jim Henson also died of Group A strep
2. Group C strep
 Get from eating unpasteurized cheese
 Also affects horses.
3. Group E strep- infects pigs
 Lancefield developed a system for distinguishing between groups:
 Make acid extract to bacteria- extract group specific polysaccharide
 Capillary tubes to suck up some of the neutralized acid extract and put it in some
rabbit antiserum and look at junction to see if there is a precipitate- called capillary
precipitation.
 Basis for the test is the Group A strep will make a particular polysaccharide which
has the backbone of Rhamnose alternating, and every second one has a NAG
 Group C is similar but it has a disaccharide of 2 NAG’s
 Group E has a glucose
 Detection of the polysaccharides is the basis for grouping strep
 1/3 of dry weight of bacteria may be the group specific polysaccharide
 There are also type specific polysaccharides
Serotype-Specific Polysaccharides of Group B Streptococci- #20
 Can have 8 different type specific polysaccharides
 Ib identical to the oligosaccharide in human milk- kills thousands of human babiesthe oligosaccharide may interfere with the adherence in the bacteria to the GI tract.
Pneumococcal Polysaccharides- #21
 Capsular polysaccharide
 Streptococcus pneumoniae
 Read off slide
 Bacteremia- bacteria growing in blood
 Otitis media- middle ear infection
 Identified immunologically 1 of 90 different polysaccharides on the surface.
Pneumococcal Vaccines
 23 valent vaccine that has been administered for the last 20 years, but do not work
well on children or adults because they do not make antibodies to polysaccharides
very well
 Do not learn structures
 Take home message: Polysaccharides have repeating units of 4-5 sugars. Repeating
units may repeat 300 times to make polysaccharide.
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Identifying structures chemically usually takes a few years, but you can do them very
quick immunologically. This is very useful for epidemiological studies, so can find
out where patient got infection.
Capsular Polysaccharide of Haemophilus influenzae type B (Hib)- #23
 Haemophilus influenzae- H flu- Gram negative but an honorary gram positive due to
its teichoic acid
 Teichoic acid is a ribose linked to a ribitol to a phosphate which is repeated
 15 years ago pediatrician saw about 50 cases of H flu a year, 5 would die and half of
the ones that survived would have permanent problems
 Then H flu vaccines became available. First it was just the polysaccharides, but then
discovered if you link the polysaccharides to a protein carrier then enhance
immunogenicity of polysaccharide- so it was much more protective.
Haemophilus influenzae- #24
 Chart from an experiment in Finland, but same thing happened as described above.
 Dropped to about 1 case per year after vaccine; these children were not vaccinated
 Conjugate vaccine- carbohydrate linked to protein
Lipopolysaccharides- #25
 LPS- endotoxins or pyrogens (fever causing)
 Gram negative bacteria have these unusual glycolipids called LPS- which have very
potent biological effects
 Lipid A in the membrane
 Outer layer is asymmetrical- saccharide core and o-antigen
 O-antigen is a long polysaccharide
 Different parts of LPS have different roles
Some Biological Properties of Lipopolysaccharides- #26
 Don’t have to know list
 Bad effects if get into blood!
Drugs that are injected and medical instruments must be “pyrogen-free”- #27
 Pyrogen free- no endotoxin there
 Anything that goes into patient must be pyrogen free
 Can eat gram negative, but do not want to get in blood
 Tiny amounts of endotoxins cause problems
 Sterile is not good enough! Need to autoclave and clean because endotoxin may still
be there after autoclaving.
Detection of Endotoxin- #28
 Need very sensitive way
 Utilizes the blood of horseshoe crabs (East Coast beaches of 3 continents)- related to
scorpions
 Horseshoe crab blood is called hemolymph (blue). Oxygen carrying pigment called
hemocyanin. In blood there are amebocytes.
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Draw blood out of horseshoe crab
Spin down blood and add distilled water.
Amebo cells will then lyse and companies will sell amebocyte lysate to people.
Lysate will gel if you add any endotoxin.- How people detect endotoxin.
Lipid A- #29
 Responsible for the toxic properties- the pyrogen
 Glucosamine units with amide linked fatty acids, mostly 14C fatty acids.
 Fatty acid chains will have a hydroxyl group with is esterified to another carboxyl
group of a fatty acid.
 See structure of lipid A
*Recording of the lecture stopped here but he pretty much read directly off the rest of the
slides.*
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