Modulation of Neisseria gonorrhoeae susceptibility to vertebrate antibacterial peptides due to a member of the resistance/nodulation/division efflux pump family

Abstract

We have previously described the antibacterial capacity of protegrin-1 (PG-1), a cysteine-rich, cationic peptide from porcine leukocytes, against Neisseria gonorrhoeae. We now report genetic and biochemical evidence that gonococcal susceptibility to the lethal action of PG-1 and other structurally unrelated antibacterial peptides, including a peptide (LL-37) that is expressed constitutively by human granulocytes and testis and inducibly by keratinocytes, is modulated by an energy-dependent efflux system termed mtr. These results indicate that such efflux systems may enable mucosal pathogens like gonococci to resist endogenous antimicrobial peptides that are thought to act during infection.

Figure 1

The primary amino acid sequence in single letter code of the antibacterial peptides used in this study. The location and linkage of the intramolecular disulfide bonds are shown above or below each relevant sequence. HNP 2, human defensin 2; NP 2, rabbit defensin 2, PG-1, protegrin-1; PC-8, synthetic, linearized variant of PG-1; TP-1, tachyplesin-1; LL-37, antibacterial peptide derived from CAP18 (5). Note that the C-terminal arginine residue of PG-1 and PC-8 is amidated (9).

Figure 2

The susceptibility of isogenic strains FA19 and KH14 to different concentrations of PG-1 (A) or a fixed concentration (B) of PG-1. The results (reported as log₁₀ units of activity) with strain FA19 are shown in the solid circles (A) or hatched bar (B), whereas results with strain KH14 are shown in the open circles (A) or shaded bar (B). (A) The data are from a single experiment. (B) The data are from triplicate experiments.

Figure 3

The susceptibility of strain FA19 to a sublethal amount of PG-1 (1 μg/ml) in the presence or absence of CCCP (50 μM) and/or glucose (20 mM). The conditions used are described in the text and under each bar. In control experiments, it was determined that incubation of strain FA19 with PG-1 (1 μg/ml) and 20 mM of glucose did not increase or decrease the bactericidal capacity of PG-1 (data not shown). All assays were performed in triplicate and the results are from four independent experiments with the data presented as log₁₀ units of bactericidal activity ± standard deviation.

Figure 4

(A) The accumulation of 0.25 μg/ml of [¹²⁵I]PG-1 by strain FA19 in the absence of CCCP (•—•), after CCCP addition (○—○) or after glucose addition to the CCCP-treated culture (○- - -○); the time points of these additions are shown. (B) The accumulation of [¹²⁵I]PG-1 (0.25 μg/ml) by isogenic strains FA19 (•—•) and KH14 (○—○). All values were calculated as nanomoles of PG-1 accumulated per 10⁷ cfu.