Describe PG synthesis
- first steps happen in cytoplasm
- 5 amino acids attached to NAM
- NAM attached to undecaprenol —> Lipid I
- NAG attached to NAM in lipid I –> Lipid II
Describe PG synthesis mentioning undecaprenyl
undecaprenyl helps hydrophilic species cross hydrophobic core of cytoplasmic membrane into the periplasmic space. also tethers them on the surface of the cell keeps them in place for the enzymes that need to recognize them.
NAM attached through pyrophosphate group to undecaprenyl (called lipid I). NAG sugar is added (forms lipid II).
once lipid II is assembled, it is flipped into the periplasm. PBPs incorporate lipid II into the pre-existing layer of PG. PBPs have transpeptidase activity and glycosyltransferase activity.
is undecaprenyl the same thing as a flippase?
No, but they do the same thing (undecaprenyl acts as a substitute for it)
what does cycloserine do?
inhibits alanine racemase and D-ala-D-ala ligase, meaning it cant produce lipid I or lipid II
What does bacitracin do?
it interferes with the recycling of undecaprenyl (binds to it when its on the outer leaflet of the cytoplasmic membrane preventing dephosphorylation), meaning we can’t produce more lipid II. and BYE BYE BACTERIAL CELL (no PG)
what does vancomycin do?
binds to the pentapeptide, wrapping around D-ala-D-ala terminus very tightly, preventing PBP transpeptidase domain from forming crosslinks and therefore killing cells (e.g. osmotic lysis)
What do b-lactams do?
interfere with the active site of transpeptidase domain of PBP (competitive inhibition) preventing it from making cross links
Which one is vancomycin more effective for and why: gram negative, gram positive
what is a better alternative of antibiotics for the other one?
It is better at targeting gram positives because the gram + PG is very exposed (harder to target gram - because vancomycin is very bulky so hard to cross the outer membrane).
Penicillins are better for gram-negatives because they are smaller in size, and therefore have an easier time entering the cell (eg through porins) to target gram negatives
what is the type V secretion system?
can transport through cytoplasm with sec (unfolded proteins, uses ATP) or tat (folded proteins, uses secondary active transport such as PMF)
once it reaches the periplasm, it can be transported through the B-barrel protein thats already there
describe T1SS
- single step
- protein may have a signal peptide that directs it to an ABC transporter
- travels through cytoplasmic membrane with ABC transporter (uses ATP)
- goes directly from ABC transported into a fusion protein that connects it to the outer membrane B-barrel protein (hence 1 step)
describe T3SS
- very commonly used for pathogenic bacteria
- goes directly from cytoplasm of bacterial cell to eukaryotic cytoplasm
- continous channel
- similarities in structure to flagella
effectors:
- host cell effectors can manipulate the cytoskeleton (eg salmonella, tir or enterohemmorhagic E.coli) in a way that bacterial cells can invade our cells
- effectors also interfere with the immune response (NF-kB)
describe the function of sortases, and what they are normally used for. (describe 2 functions)
sortases are transpeptidases, make peptide bonds between secreted proteins and peptide chain of lipid II. this transfers the secreted protein to the lipid II molecule. this is then incorporated into the PG by PBPs.
sortases can also build gram + pili, which attach to PG layer. pilus subunits are attached to the sortase and sortase polymerizes them, until the entire pilus has been synthesized. housekeeping sortase can then attach this to lipid II from where it is attached with transpeptidase domain into the PG layer
describe the function of sortases, and what they are normally used for in gram positives
- in gram negatives, substances have a hard time passing through the outer membrane by themselves so sortases are not needed as much for this function (they’ll stay in the cell)
- however, gram + lack outer membrane so they have to make sure to attach the substance to the surface, which is done by sortases.
- unfolded protein travels through sec through cytoplasmic membrane, folding once it reaches the periplasmic space
- proteins have special cell wall signal that tethers them to the phospholipids in the cytoplasmic membrane
- some proteins have LPXpg motif
- sortase enzymes are constantly looking out for this motif, and once they discover a protein that has this they make a covalent bond
describe the formation of LPS
Lpt pathway (how LPS is transported):
- sugars that go into o-antigen synthesized in cytoplasm, and undecaprenyl transports them into periplasm
- lipid A very hydrophobic and doesn’t want to cross next membrane. but LPS molecules slide along the Lpt pathway, and travel through B-barrel protein to the outside
do gram positives produce LPS?
NO
What type of pili are used for twitching motility?
Type IV pili
What type of pili are used for swarming motility?
NONE. It uses flagella (also requires surfactants)
describe one method of capsule assembly in gram-negs (wzx-wzy)
wzx-wzy-dependent pathway
- subunits assembled in cytoplasm
- flipped across membrane by Wzx (flippase)
- polymerized by Wzy (polymerase)
- translocated to surface through Wza channel
- incorporated on cell surface
desribe another method of capsule assembly in gram negatives
- some dont use undecaprenol
- assemble full polysaccharide in cytoplasm with GTs, transfer to lipid
- polysaccharide and lipid transported to surface by ABC transporter
list the types of pili in gram negatives
type IV secretion pili (sex pili): conjugation
type IV pili: twitching motility
type III secretion systems
chaperone-usher (CU) pili (most common)
describe P pilus assembly
- P pilus subunits exported to periplasm by sec (unfolded)
- papD binds to subunits and helps them fold (chaperone)
- papD delivers subunits to papC
- papC forms base of pilus, and translocates subunits to the surface (usher)
- papA (major pilin) proteins added to base by papC. mutants lacking papA dont make pili but are adherent
- papH keeps pilus attached to the cell and BLOCKS papA addition
Descibe the role of papA and papH in pilus length
PapH > PapA = shorter pili
PapA > PapH = longer pili
too much papH would mean really short pili
Describe the assembly of gram + pili
- subunits transported by sec
- pilus subunits contain cell wall sorting signal (eg LPXTG motif) which sortase cuts to form a complex with the subunit
- sortase then forms covalent bonds between this subunit and others
- housekeeping sortase transfers the pilus to lipid II, by which it is incorporated into PG by PBPs
