Antimicrobials and Drug Efflux

Question 1

outline the cell wall structure of gram-negative bacteria

Answer 1

thin layer of peptidoglycan in periplasmic space between inner and outer membranes

Question 2

outline the structure of gram positive bacteria

Answer 2

only have a single lipid membrane surrounded by a cell wall composed of a thick layer of peptidoglycan and lipoteichoic acid, anchored to the cell membrane via diacylglycerol

Question 3

outline the cell wall of mycobacteria

Answer 3

thin layer of peptidoglycan and arabinogalactin and a thick layer of mycolic acids - this cell wall surrounds a single lipid membrane

Question 4

what are the ways in which bacteria become drug resistant

Answer 4

decreased influx
efflux pumps
drug inactivation
resistance mutations
membrane/cell wall

Question 5

function of transpeptidases

Answer 5

responsible for cross-linking peptidoglycan strands in bacterial cell walls

Question 6

how does penicillin/beta-lactams perform their function

Answer 6

binds and inhibits transpeptidase
leads to weakened cells that cannot resist osmotic pressure

Question 7

how are beta-lactams able to bind to transpeptidase

Answer 7

the beta-lactam ring is structurally similar to the D-Ala-D-Ala portion of peptidoglycan precursor

Question 8

when are beta-lactams most effective

Answer 8

against dividing bacteria which need transpeptidase to synthesise new cell walls

Question 9

what type of bacteria can beta-lactams target

Answer 9

gram+
and some gram-

Question 10

how does the use of penicillin lead to antibacterial resistance

Answer 10

bacteria develop beta-lactamase that degrade the beta-lactam ring
inhibiting its transpeptidase binding capabilities

Question 11

what is ciprofloxacin

Answer 11

a flouroquinolone

Question 12

what does ciprofloxacin bind to

Answer 12

DNA gyrase
topoisomerase IV

Question 13

function of DNA gyrase and how does ciprofloxacin affect it

Answer 13

cleaves DNA backbone
ligases the DNA - ATP dependent
done by introducing negative supercoils
ciprofloxacin - prevents it from re-ligating

Question 14

how can bacteria become resistant to ciprofloxacin

Answer 14

mutations in the gyrAB and parCE genes to reduce affinity to DNA gyrase and topoisomerase IV
overexpression of efflux pumps to reduce ciprofloxacin M

Question 15

how does ciprofloxacin inhibit topoisomerase IV

Answer 15

it intercalates into DNA at the nicks introduced by topoisomerases

Question 16

what is erythromycin

Answer 16

a macrolide
ribosome 50s subunit inhibitor - protein synthesis inhibitor

Question 17

how do macrolides inhibit protein synthesis

Answer 17

bind to the exit tunnel of 50s ribosome subunits

Question 18

how do bacteria gain resistance against macrolides

Answer 18

methylation of ribosomes to reduce macrolide affinity

Question 19

function of tetracycline

Answer 19

prevents protein synthesis by binding to the 30s subunit
inhibits the tRNA’s binding to the acceptor side of the mRNA

Question 20

how do bacteria develop resistance to tetracycline

Answer 20

efflux pumps
changing the 30s ribosome subunit binding sites

Question 21

what is polymyxin B

Answer 21

a group of peptides effective against gram- bacteria

Question 22

what is the mechanism of action of polymyxin B

Answer 22

they act as cationic detergents that bind to negatively charged LPS on the outer membrane
results in disruption of the outer membrane
outer membrane disruption allows polymyxin to disrupt inner membrane
increases cell permeability
leads to cell death

Question 23

why have polymyxin’s been historically restricted to topical use (like creams n stuff)

Answer 23

they are very toxic when given systemically

Question 24

how do gram- bacteria in particular develop resistance to polymyxin B

Answer 24

they modify their LPS by substituting anionic groups with cationic
formation of capsules to protect their membrane
over-expression of OprH - a protein that increases outer membrane stability by binding to LPS

Question 25

how do bacterial MDR efflux pumps work

Answer 25

utilise energy from the proton/sodium motive force to catalyse substrate efflux

Question 26

how do the ABC efflux family export substrates

Answer 26

using ATP energy from hydrolysis

Question 27

what is the major pro of efflux pumps

Answer 27

they can export many classes of drugs - very promiscuous
allow bacteria to exist in many extreme environments

Question 28

what does the distal binding pocket in RND efflux pumps contain that allow it to export hydrophobic molecules

Answer 28

contains many residues that are
hydrophobic
charged and uncharged

Question 29

what is a major factor for the level of promiscuousity for RND efflux pumps

Answer 29

various channels for drug entry
3 channels that can detect whether the drug is polar/non-polar and a mix in between

Question 30

why is hard to design an efflux pump inhibitor

Answer 30

because of the multiple pockets and promiscuousness of RND efflux pumps, the inhibitors usually just become substrates themselves and become exported

Question 31

how can EPI’s be used to inhibit RND efflux pumps

Answer 31

bind to accessory proteins of the AcrAB-TolC complex

Question 32

how can NSC 60339 act as a EPI

Answer 32

binds to the cleft in between the lipoyl and alpha-beta barrel domains in AcrAB
acts as wedge that stops conformational change in the RND pump

Question 33

how can synthetic peptides act as EPI

Answer 33

aimed to destabilise the AcrB trimerisation interface
they do work but that is not how they work and we don’t know how

Question 34

how can you test the efficacy of an EPI

Answer 34

nile red is a hydrophobic molecule that binds to membranes
in hydrophobic environments it is fluorescent
if effluxed into aqueous environment it loses fluorescence