A mutant form of the Neisseria gonorrhoeae pilus secretin protein PilQ allows increased entry of heme and antimicrobial compounds

Abstract

A spontaneous point mutation in pilQ (pilQ1) resulted in phenotypic suppression of a hemoglobin (Hb) receptor mutant (hpuAB mutant), allowing gonococci to grow on Hb as the sole source of iron. PilQ, formerly designated OMP-MC, is a member of the secretin family of proteins located in the outer membrane and is required for pilus biogenesis. The pilQ1 mutant also showed decreased piliation and transformation efficiency. Insertional inactivation of pilQ1 resulted in the loss of the Hb utilization phenotype and decreased entry of free heme. Despite the ability of the pilQ1 mutant to use Hb for iron acquisition and porphyrin, there was no demonstrable binding of Hb to the cell surface. The pilQ1 mutant was more sensitive to the toxic effect of free heme in growth medium and hypersensitive to the detergent Triton X-100 and multiple antibiotics. Double mutation in pilQ1 and tonB had no effect on these phenotypes, but a double pilQ1 pilT mutant showed a reduction in Hb-dependent growth and decreased sensitivity to heme and various antimicrobial agents. Insertional inactivation of wild-type pilQ also resulted in reduced entry of heme, Triton X-100, and some antibiotics. These results show that PilQ forms a channel that allows entry of heme and certain antimicrobial compounds and that a gain-of function point mutation in pilQ results in TonB-independent, PilT-dependent increase of entry.

FIG. 1.

Representative growth curves of the ΔhpuA hpuB::cat pilQ1 point mutant, FA7168, and HpuAB phase on FA1090 in liquid cultures, as measured by the OD₆₀₀ in the Klett colorimeter. Gonococci were grown in liquid cultures of CDM-Des supplemented with Hb (1 μM) or Hb and HSA (16 μM). Symbols: •, FA1090 in Hb; ○, FA1090 in Hb-HSA; ▪, FA7168 in Hb; □, FA7168 in Hb-HSA.

FIG. 2.

Free heme supported growth of hpuAB mutants and their pilQ inactivation mutants. GCB-Des plates were spread with cell suspensions of respective strains: FA7167, ΔhpuA hpuB::cat pilQ⁺ parent; FA7319, pilQ::Ω transformant of FA7167; FA7317, pilQ1 transformant of FA7167; FA7320, pilQ1::Ω transformant of FA7317. Wells were cut into the agar and loaded with 50 μl of heme at 0.1 or 0.05 μg/μl. Images were obtained after 48 h of incubation. At 5.0 μg of heme per well, FA7167 grew in a wide growth ring, whereas the pilQ1 mutant, FA7317, showed a thin growth ring with a wide zone of growth inhibition. Decreasing the amount of heme decreased the growth inhibition zones. Insertional inactivation of pilQ abolished heme toxicity.

FIG. 3.

Effects of pilQ inactivation on pilQ1 mutant's sensitivity to antimicrobial agents. Filter paper disks placed on the surface of FA7168 (ΔhpuA hpuB::cat pilQ1)- and FA7320 (ΔhpuA hpuB::cat pilQ1::Ω)-coated plates were loaded with AMP (1 μg per disk, top left), ERY (0.25 μg, top right), RIF (0.05 μg, bottom left), and TX-100 (5 μl of a 1:100 dilution, bottom right). Images were obtained after 24 h of incubation.

FIG. 4.

Bar graph illustrating transformation efficiencies (A) and electron micrographs comparing the piliation (B) of RM11.2recA6 (wild type), FA7332 (ΔhpuA hpuB::cat), and FA7333 (ΔhpuA hpuB::cat pilQ1). The piliation of these three strains is given as the percentage of cells expressing pili, as visualized by immunogold electron microscopy with anti-pilin sera, and the percent piliation is indicated on each electron micrograph. The bar in each electron micrograph is 500 nm. Two grids for each strain were examined. The numbers of cells examined were 216, 346, and 558, respectively. All three strains exhibited predominantly bundled pili. FA7333 showed decreased piliation as well as decreased transformation efficiency. The asterisk in panel A indicates a significant difference by using the Student t test (P < 0.05) compared to RM11.2 recA6 and FA7332.

FIG. 5.

Effects of pilE deletion and pilT inactivation on Hb (50 μl at 10 μg/μl) and heme (50 μl at 0.1 μg/μl) supported growth of a pilQ1 mutant, FA7333 (ΔhpuA hpuB::cat pilQ1, Hb⁺). FA7338 is the ΔpilE transformant and FA7340 is the pilT::erm transformant of FA7333. Images were obtained after 48 h of incubation. The pilQ1 pilT double mutant gave only isolated large colonies around the Hb well. The zone of growth inhibition around the heme well was wider in the pilQ1 ΔpilE double mutant but was reduced in the pilQ1 pilT double mutant compared to that of FA7333.