Point mutations in HpuB enable gonococcal HpuA deletion mutants to grow on hemoglobin

Abstract

Neisseria gonorrhoeae ordinarily requires both HpuA and HpuB to use hemoglobin (Hb) as a source of iron for growth. Deletion of HpuA resulted in reduced Hb binding and failure of growth on Hb. We identified rare Hb-utilizing colonies (Hb(+)) from an hpuA deletion mutant of FA1090, which fell into two phenotypic classes. One class of the Hb(+) revertants required expression of both TonB and HpuB for growth on Hb, while the other class required neither TonB nor HpuB. All TonB/HpuB-dependent mutants had single amino acid alterations in HpuB, which occurred in clusters, particularly near the C terminus. The point mutations in HpuB did not restore normal Hb binding. Human serum albumin inhibited Hb-dependent growth of HpuB point mutants lacking HpuA but did not inhibit growth when expression of HpuA was restored. Thus, HpuB point mutants internalized heme in the absence of HpuA despite reduced binding of Hb. HpuA facilitated Hb binding and was important in allowing use of heme from Hb for growth.

FIG. 1.

hpuA/B genotype of key strains constructed and/or used in this study. The open arrow indicates that the gene is not expressed. The solid arrow indicates that the gene is expressed.

FIG. 2.

Illustration of point mutation sites found in FA7170 and ΔhpuA hpuB* mutants derived from FA7185 and FA7186. The arrows indicate alterations of amino acids (aa), with amino acids of the wild type on the top row and those of the mutants on the bottom row. The # indicates sites with multiple occurrences. The corresponding sites of mutation in hpuB are bp 650, 770, 1526, 1780, 1838, 2146, 2199, 2181, 2344, 2369, and 2378 from the beginning of coding sequence. The shaded area is the signal sequence, and the boxes are seven regions of homology derived by Lewis et al. (16) from their peptide alignment of meningococcal HpuB- and TonB-dependent outer membrane proteins described by Cornelissen et al. (9).

FIG. 3.

Western blot analysis of whole-cell lysates prepared from the indicated strains grown under iron-stressed condition. FA7185H1 and FA7185H2 were Hb⁺ revertants of FA7185. FA7186H1 and FA7186H2 were revertants of FA7186. FA7186 H1 was an hgbX mutant, while the other three were hpuB* mutants. The top panel was probed with rabbit antiserum raised against the HpuB N-terminal peptide, and the bottom panel was probed with rabbit antiserum raised against the HpuA C-terminal peptide. The positions of size standards are shown on the left (in kilodaltons).

FIG. 4.

Plate assays testing the ability of indicated ΔhpuA mutants for growth on Hb and heme (Hm). Wells were cut into GCB/Des agar and loaded with Hb (60 μl at 10 μg/μl) and heme (60 μl at 0.1 μg/μl). All tested strains grew on heme. The ΔhpuA hpuB⁺ strain FA7169 could not grow on Hb, but the ΔhpuA hpuB* mutants could.

FIG. 5.

Growth of ΔhpuA hpuB* mutants and hpuA⁺ hpuB* mutants in medium supplemented with 1 μM Hb (A) and 1 μM Hb plus HSA at 16 μM (B). FA1090 Hb⁺ is the wild-type parent (bull;). FA6929 is an hpuA⁺ hpuB* mutant (○). The hpuA⁺ hpuB* strains FA7242 (□) and FA7243 (▵) were derivatives of the ΔhpuA hpuB* mutants FA7170 (▪) and FA7185H14 (▴), respectively.

FIG. 6.

Growth of the ΔhpuA hpuB* mutant and its ΔhpuA hpuB⁺ parent in medium supplemented with 0.5 μM heme. FA1090 Hb⁺ is the wild-type parent (bull;). FA7167 is the ΔhpuA hpuB::cat negative control (○). FA7169 (▪) is the hpuB⁺ parent of FA7170 (□). Results presented in this graph were also true for FA7186H16 and its parent FA7186. Similar results were observed in two other experiments.

FIG. 7.

Results from radioimmunoassays of Hb binding of ΔhpuA mutants. FA7169, FA7185, and FA7186H1 are HpuB⁺, while the rest are HpuB*. Hb binding to each mutant was significantly less than that of the wild type (P < 0.01, paired Student’s t test). # indicates that FA7185H14 bound more Hb than its parent FA7185 (P < 0.01, Student’s t test). The other FA7185 derivatives and FA7170 did not bind significantly more Hb than their HpuB⁺ parents. Since experiments were done over a period of 4 weeks using different batches of iodinated anti-human Hb antibody, binding data for each tested strain are presented as a percentage of that of the corresponding wild-type control (FA1090 Hb⁺). Bars indicate standard deviations. The number at the base of each column represents the number of determinations.