3 & 4. bacterial morphology and cell envelope structure

one hour, 4 questions

most common bacterial shapes

-rods

-cocci

-spiral

unique variations of rods

-long

-short

-thick

-thin

-rounded or pointed ends

-thicker at one end than the other

unique variations of cocci

-large

-small

-oval

unique variations of spiral

-fat

-thin

-loose spirals

-very tight spirals

bacteria form ___________ both in liquid and on solid medium

group associations

gram positive unique structures

-mesosome

-surface proteins

gram negative unique structures

-pili

-outer membrane

-porin proteins

-periplasmic space

gram stain steps

1. stain with crystal violet (purple)

2. modify with potassium iodide

3. decolorize with alcohol

   -gram positive remains purple

4. counterstain with safranin

   -gram-negative: pink

   -gram-positive: purple

acid-fast stain steps

1. stain with hot carbol-fuchsin (red)

2. decolorize with acid alcohol

   -acid-fast remain red

3. counterstain with methylene blue

   -acid-fast: red

   -others: blue

gram negative cell wall tetrapeptide

L-Ala-D-Glu-meso-DPA-D-Ala

gram positive cell wall tetrapeptide

L-Ala-D-Gln-L-Lys-D-Ala

D-alanine bond to DA (diamino acid)

peptide bond

function of peptidoglycan

prevents osmotic lysis

-cell bursting as result of osmotic pressure from a hypotonic environment

how can you cause bacteria to burst from osmotic lysis?

inhibit normal synthesis of peptidoglycan

for bacteria to increase their size 3 enzymes are used

-autolysins

-transglucosidase

-transpeptidase

autolysins

break the peptide cross links in the peptidoglycan

transglucosidase

insert and link new peptidoglycan monomers into peptidoglycan breaks

transpeptidase

rebuild the peptide cross-links between the rows and layers of peptidoglycan to make the wall strong

what enzyme is a target for beta-lactam antibiotics?

transpeptidase they prevent the patching

antibiotics that inhibit peptidoglycan synthesis

penicillins (b-lactam)

-cillin

cephalosporins (b-lactam)

-ceph or -cef

T/F beta-lactams attack all bacteria

FALSE

only growing bacteria

b-lactams: penicillins and cephalosporins bind to _________

transpeptidases

what is the MOA of beta-lactams?

-bind to transpeptidases

-prevent resealing the cell wall as new peptidoglycan monomers are added during bacterial cell growth

-binding blocks the transpeptidases from cross-linking the sugar chains and results in a weak cell wall and subsequent osmotic lysis of the bacterium

what kind of antibiotic is vancomycin?

glycopeptide

MOA of vancomycin

-forms H bond interactions with terminal D-alanyl-D-alanine moieties of the NAM-NAG peptides

-prevents crosslinking of peptidoglycan

how do you get bacterial resistance to penicillin?

-producing penicillinase that destroys penicillin

-producing b-lactamase that destroys b-lactam antibiotics

gram-positive stain

retain initial dye crystal violet

purple

common gram-positive bacteria

-strep pyogenes

-strep pneumoniae

-staph aureus

-enterococcus faecalis

-clostridium

gram-negative stain

decolorize pick up counterstain safranin

RED (pink)

common gram-negative bacteria

-salmonella

-shigella

-n gonnorrhoeae

-n meningitis

-h influenzae

-e coli

-klebsiella pneumoniaea

-proteus

-pseudomonas aeruginosa

acid-fast stains

resist decolorizatioon with an acid-alcohol mixture

retain initial dye carbolfuchsin

red

common acid-fast bacteria

myobacterium

structure of murein (aka peptidoglycan)

strand of alternating aminosugars: NAM and NAG

gram negative pentapeptide (comes off of NAM)

L-alanine

D-glutamic acid

meso diaminopimelic acid

d-alanine

gram positive pentapeptide (comes off of NAM)

L-alanine

D-glutamine

L-lysine

d-alanine

gram positive vs gram negative NAG-NAM

insert picture

vancomycin MOA

inhibits transglycosylation by binding D-Ala-D-Ala

fosfomycin MOA

inhibits formation of NAM

cycloserine MOA

blocks incorporation of D-Ala

in gram positive bacteria, what are the cross-links?

peptide bridge cross-links Lys to D-Ala

in gram negative bacteria, what are the cross-links?

DAP cross-links directly to D-Ala

where are peptidoglycan monomers synthesized?

cytosol

what do peptidoglycans use as a carrier?

bactoprenol

autolysins break 2 things

1. glycosidic bonds at the point of growth along the existing peptidoglycan

2. peptide cross-bridges that link the rows of sugars together

what is the role of bactoprenol and transglucosidase in peptidoglycan synthesis?

insert new monomers into breaks transglucosidases catalyze formation of glycosidic bonds between NAM-NAG of the monomers and NAG-NAM of the existing peptidoglycan

___________ reform peptide cross-links between the rows and layers of peptidoglycan, making the wall strong

transpeptidases

synthesis of peptidoglycan occurs in three phases

1. assembly

2. transport

3. polymerization

composition of gram positive cell wall

-broad, dense

-numerous interconnecting layers of peptidoglycan

-teichoic acids

-lipoteichoic acids attached to lipids of the cell membrane

teichoic acids

polymers of glycerol, phosphates, and ribitol

ONLY IN GRAM POSITIVE

lipoteichoic acids

outer surface studded with species specific proteins

induce septic shock

ONLY IN GRAM POSITIVE

composition of gram-positive envelope

cytoplasmic membrane

-phospholipid bilayer

-integral membrane proteins

-peripheral membrane proteins

capsule (some)

-protection against phagocytosis

surface antigens

-most exposed to host immune system

functions of gram-positive cell wall: peptidoglycan and teichoic acid

-bind pattern-recognition receptors on cells of host's immune system

-trigger innate immune response

-activate alternative complement pathway

-severe systemic infections —> release of high levels of gram-positive cell wall components —> excessive cytokine production

functions of gram-positive cell wall: surface proteins in the bacterial murein

-enzymes

-adhesins: adheres to host cells

-invasins: penetrate host cells

-resist phagocytic destruction

composition of gram-negative cell wall

-thin peptidoglycan wall: 2-3 layers, prevents osmotic lysis

-outer membrane

-periplasm: contains enzymes and binding proteins

structure & functions of gram-negative cell wall

-lipopolysaccharide

-membranes: phospholipid bilayer

-lipoprotein: anchors outer membrane in murein

-membrane proteins: porin, integral proteins, peripheral proteins

lipopolysaccharides (LPS; endotoxin) structure; located in outer layer of the outer membrane

lipid A: phospholipid portion embedded in the membrane; toxic

core polysaccharide: linked to aminoglucose phosphate

repeating O-antigen subunits: polysaccharide portion extending outward from bacterial surface

functions of outer membrane: semipermeable

helps retain certain enzymes and prevents some toxic substances from entering the cell

-penicillin G

-lysozyme

functions of outer membrane: LPS

adds strength to outer membrane recognized by immune system

activates innate immunity

functions as harmful endotoxin: excessive cytokine production

what does LPS activate?

-alternative complement pathway

-lectin pathway

(innate immunity)

OM surface proteins functions

-enzymes

-adhesins

-invasins

-resisting phagocytic destruction

_____________ disrupt bacterial membranes

topical antibiotics

-polymyxin A

-polymyxin B

what is polymyxin B more useful against: gram-positive or gram-negative?

gram-negative

OM is readily accessible

structure of an acid-fast cell wall

-glycolipids (mycolic acids)

-peptidoglycan layer linked to arabinogalactan, which is linked to mycolic acids

what inhibits mycolic acid synthesis and is first line treatment for TB?

isoniazid (INH)

glycocalyx structure

-capsule: polysaccharide and protein

-slime layer

-biofilm

T/F capsules are necessary for survival in the host and an important virulence factor

TRUE

-antiphagocytic

-poorly immunogenic

-promote adherence

-act as barrier to hydrophobic toxicants