Antimicrobial peptides: structure, function and resistance
Abstract
Higher eukaryotes produce a vast range of antimicrobial peptides (AMPs) that play
important roles in their defence against microbial infection. Beta defensins are small
(3-5 kDa), cationic peptides that display broad, potent antimicrobial activity against a
range of microbes and also act as chemoattractants of important immunomodulatory
cells. To generate highly pure peptides for structural and functional studies, we
developed a method to prepare recombinant human beta defensin-2 (HBD2). The
HBD2 gene was synthesised by recursive PCR with codons optimised for expression
in Escherichia coli. HBD2 was expressed as an insoluble fusion to a His-tagged
ketosteroid isomerase. After cleavage from the fusion with cyanogen bromide, 1H
NMR spectroscopy and mass spectrometry confirmed that the oxidised HBD2 was
folded and possessed the correct b-defensin disulfide bond topology. The recombinant
HBD2 was active against E. coli, P. aeruginosa, S. aureus and C. albicans and was
also a chemoattractant against HEK293 cells expressing the chemokine receptor
CCR6. 15N-labelled HBD2 was also prepared and was highly suitable for future
structural studies. Since defensins are thought to interact with bacterial membranes we
also tested the recombinant HBD2 in biophysical studies (surface plasmon resonance,
SPR, Biacore). We observed different binding to artificial model membranes
containing either E. coli Kdo2-lipid A or phospholipids.
Bacterial resistance to AMPs has been linked to the covalent modification of
the outer membrane lipid A by 4-amino-4-deoxy-L-arabinose (L-Ara4N). This
neutralises the charge of the LPS, thereby decreasing the electrostatic attraction of
cationic peptides to the bacterial membrane. The pathogen Burkholderia cenocepacia
displays extremely high resistance to AMPs and other antibiotics and the Ara4N
pathway appears to be essential. To explore this further we expressed recombinant
forms of two enzymes (ArnB and ArnG) from the B. cenocepacia Ara4N pathway.
Purified ArnB is a pyridoxal 5’-phosphate (PLP)-dependent transaminase and we
tested its ability to bind amino acid substrates. We investigated the binding of
inhibitors L- and D-cycloserine to ArnB and tested their antibiotic activity against
Burkholderia strains. We also studied the B. cenocepacia ArnG – a proposed
membrane protein undecaprenyl-L-Ara4N flippase – and showed that the protein
behaved as a dimer by non-denaturing gel analysis. The B. cenocepacia ArnG failed
to complement E. coli knock-out strains encoding the equivalent flippase proteins
ArnE and ArnF, suggesting that ArnG is a Burkholderia-specific protein.