Permeability & efflux pumps

what gram have a higher intrinsic resistance towards abx and why

gram- - bc they have a double membrane - hydrophilic abx (like beta lactams and aminoglycosides) are not able to pass the lipid bilayer of the outer membrane

can hydrophobic abx pass through the outer membrane? egs of them

yes they can! eg fluoroquinolines, macrolides and rifampicin

LPS long, descr

lipopolysaccharide, outer leaflet of the outer membrane, also known as endotoxin, major barrier to hydrophobic abx bc of the charged nature of the core structure

how can hydrophilic abx enter gram- bacteria

through porins

porins descr

span the membrane and are in contact w/ two compartments, cytoplasmic/inner membrane - transmembrane regions are highly hydrophobic, alpha helical structures
outer membrane - transmembrane beta sheet, plied into barrel (beta barrel)

are beta strans hydrophilic or hydrophobic?

amphipathic - alternating hydrophobic/hydrophilic residues

why structural differences between porins in outer and inner membrane

hydrophobic alpha helices would get stuck in the inner membrane, transport channel for OMPs (sec translocon) considers OMPs as soluble proteins - amphipathic strands

left vs right

left: general diffusion points
right: specific channels

general diffusion porins descr

hydrophilic molecules smaller than 600 Da are able to freely diffuse through them - ions, aa, small sugars, ALSO small abx like beta lactams and tetracycline

how can general diffusion porin functioning be modulated

physico chemical parameters (eg pH) can close the porin, ligand binding (eg polyamines produced by the bacterium or by the hosts) blocks the porin

general diffusion porins and abx resistance (2)

altering porin expression profiles - loss/severe reduction of porins, replacement of one/two major porins by another (maybe more selective) porin
altering of porin function - mutating porins reducing permeability

considerations for hydrophobic compounds

are able to bypass the LPS and easily diffuse through the outer membrane

considerations for hydrophilic compounds

are able to easily diffuse through general diffusion porins

where are efflux pumps found

in almost all bacterial species (gram- and gram+)

2 characteristics of efflux pumps

hightly efficient drug extrusion, broad substrate specificities

families of efflux pumps

5 of them
resistance nodulation division family (RND)
major facilitator superfamily (MFS)
ATP binding cassette (ABC) superfamily
small multidrug resistance (SMR) family - membrane of the drug/metabolite transporter (DMT) superfamily)
multidrug and toxic compound extrusion (MATE) family

which efflux families found in what gram

RND only found in gram-, others in both gram- and gram+

eg of a efflux pump family for a specific abx family

MATE family for aminoglycosides/fluoroquinolones and cationic drugs

what is energy for drug extrusion provided by?

proton/sodium gradient - proton motive force, ATP hydrolysis

what families of efflux pumps most clinically relevant for abx resistance in gram- and gram+

RND - most clinically relevant for gram- (eg e coli)
MFS - most clinically relevant for gram+ (eg s aureus)

3 diff conformations for MFS family efflux pumps

inward open, ligand-bound inward open, occluded

descr

functional rotating model - drug binding, drug extrusion and proton transport are alternated by the three subunits to allow efficient drug efflux

descr

activated AcrAB transporter triggers opening of TolC to allow diffusion of substrates across the outer membrane

physiological function of efflux pumps

involvement in detoxification - possibly also extrude various host-derived antimicrobial compounds, mutating efflux pumps reduced virulence of various pathogens, AcrAB-TolC mediates efflux of toxic bile salts - adaptation of e coli to intestinal tract
involvement in host invasion by regulating - quorum sensing, iron uptake

what is mechanism of resistance w/ efflux pumps

overexpression of efflux pumps

regulatory network of efflux pumps desccr

efflux pump expression is tightly regulated, w/o substrates expression remains very low, likely to avoid unwanted loss of metabolites

mechanism of upregulation to confer drug resistance (2 general, subpoints)

mutations in - local repressor gene, global regulatory gene, promoter region of efflux pump gene
insertion elements upstream of efflux pump gene

eg of a repressor of an efflux pump

MexZ - reprerssor of RND efflux pump MexXY, most frequently mutated gene in clinical isolates of P. aeruginosa; mutations in DNA binding domain of MexZ → MexZ cannot inhibit expression anymore → high expression of efflux pump in absence of any signal molecule

what is special ab cell envelope of mycobacteria

specific outer membrane composed of specific lipids (mainly mycolic acids, no LPS) - highly impermeable for hydrophilic molecules, only selective porins (no general diffusion porins)

problem of mycobacteria for antibacterial discovery

many abx do not enter the mycobacterial cell, also mycobacteria have efflux pumps to extrude abx

what can bacteria do w/ porins, what effect

can alter/lose them to gain resistance