The heme sensing response regulator HssR in Staphylococcus aureus but not the homologous RR23 in Listeria monocytogenes modulates susceptibility to the antimicrobial peptide plectasin

Abstract

Background: Host defence peptides (HDPs), also known as antimicrobial peptides (AMPs), have emerged as potential new therapeutics and their antimicrobial spectrum covers a wide range of target organisms. However, the mode of action and the genetics behind the bacterial response to HDPs is incompletely understood and such knowledge is required to evaluate their potential as antimicrobial therapeutics. Plectasin is a recently discovered HDP active against Gram-positive bacteria with the human pathogen, Staphylococcus aureus (S. aureus) being highly susceptible and the food borne pathogen, Listeria monocytogenes (L. monocytogenes) being less sensitive. In the present study we aimed to use transposon mutagenesis to determine the genetic basis for S. aureus and L. monocytogenes susceptibility to plectasin.

Results: In order to identify genes that provide susceptibility to plectasin we constructed bacterial transposon mutant libraries of S. aureus NCTC8325-4 and L. monocytogenes 4446 and screened for increased resistance to the peptide. No resistant mutants arose when L. monocytogenes was screened on plates containing 5 and 10 fold Minimal Inhibitory Concentration (MIC) of plectasin. However, in S. aureus, four mutants with insertion in the heme response regulator (hssR) were 2-4 fold more resistant to plectasin as compared to the wild type. The hssR mutation also enhanced resistance to the plectasin-like defensin eurocin, but not to other classes of HDPs or to other stressors tested. Addition of plectasin did not influence the expression of hssR or hrtA, a gene regulated by HssR. The genome of L. monocytogenes LO28 encodes a putative HssR homologue, RR23 (in L. monocytogenes EGD-e lmo2583) with 48% identity to the S. aureus HssR, but a mutation in the rr23 gene did not change the susceptibility of L. monocytogenes to plectasin.

Conclusions: S. aureus HssR, but not the homologue RR23 from L. monocytogenes, provides susceptibility to the defensins plectasin and eurocin. Our data suggest that a functional difference between response regulators HssR and RR23 is responsible for the difference in plectasin susceptibility observed between S. aureus and L. monocytogenes.

Figure 1

Measurement of ATP leakage from Staphylococcus aureus after treatment with plectasin (A), eurocin (B), protamine (C), and novicidin (D). Measurement of intracellular (IC) and extracellular (EC) ATP after treatment with plectasin (500 μg/ml), eurocin (500 μg/ml), protamine (1,000 μg/ml), novicidin (1,000 μg/ml), or peptide dilution buffer. Treatment with the two defensins does not lead to leakage of intracellular ATP, whereas treatment with protamine and novicidin lead to leakage of ATP. Representative results from S. aureus are shown as treatment of S. aureus and L. monocytogenes resulted in similar leakage profiles. The experiment shown is representative of two independent experiments.

Figure 2

Growth of S. aureus 8325-4 (A) and 15981 (B) wild types and hssR mutants in the presence of plectasin. Plectasin (35 μg/ml) inhibited the growth of S. aureus 8325-4 and 15981 wild-types but hardly affected the growth of the 8325-4 hssR::bursa transposon mutant, the transduced 8235-4 hssR mutant or the 15981 ΔTCS15 (hssRS) mutant. Complementation of 8325-4 hssR::bursa (8325-4 hssR::bursa/pRMC2-hssRS) affected the growth slightly, but addition of plectasin inhibited the growth to a level comparable to wild type. The experiment shown is representative of three independent experiments.

Figure 3

Kinetics of bacterial killing in vitro. S. aureus 8325-4 wild type, 8325-4 hssR::bursa and 8325-4 hssR::bursa/p RMC2-hssRS were incubated in the presence of 1XMIC. The colony counts are shown as representative of three independent experiments. CFU, colony-forming units.

Figure 4

Growth of Staphylococcus aureus wild type and hssR mutants in the presence of hemin and plectasin. The growth of the S. aureus 8325-4 wild type is only affected by plectasin (35 μg/ml) and not hemin (4 μM). On the contrary, the 8325-4 hssR mutants do not grow in the presence of hemin, regardless of the presence or absence of plectasin, confirming the heme-sensitive phenotype of hssR mutants. The experiment shown is representative of three independent experiments.