Neisseria gonorrhoeae heme biosynthetic mutants utilize heme and hemoglobin as a heme source but fail to grow within epithelial cells

Abstract

Many bacterial pathogens, including pathogenic neisseriae, can use heme as an iron source for growth. To study heme utilization by Neisseria gonorrhoeae, two heme biosynthetic mutants were constructed, one with a mutation in hemH (the gene encoding ferrochelatase) and one with a mutation in hemA (the gene encoding gamma-glutamyl tRNA reductase). The hemH mutant failed to grow without an exogenous supply of heme or hemoglobin, whereas the hemA mutant failed to grow unless heme, hemoglobin, or heme precursors were present. Growth of the mutants with hemoglobin required expression of the hemoglobin receptor (HpuAB) and was TonB dependent. However, growth with heme required neither HpuAB nor TonB. An fbpA mutant grew normally when either heme or hemoglobin was present in the medium. The heme biosynthetic mutants showed reduced intracellular survival, compared to the parent strain, within A-431 endocervical epithelial cell cultures. These studies demonstrate that in addition to synthesizing their own heme, N. gonorrhoeae strains are able to internalize and utilize exogenous heme independently of FbpA but appear unable to obtain heme from within epithelial cells for growth.

FIG. 1

Construction of pUNCH1304, pUNCH1306, and pUNCH1324. A single line indicates a PCR product, and a box represents either the antibiotic cassette or plasmid vector. Only part of the plasmid vector is shown. The open reading frame (ORF) and direction of transcription for the relevant gonococcal gene, interrupted by insertion of the antibiotic cassette, are indicated in gray. The PCR fragment for hemH contains only a portion of the hemH gene. The restriction sites used to linearize the plasmid and insert the antibiotic cassette are shown in parentheses.

FIG. 2

Ethidium bromide-stained 0.7% agarose gel showing the PCR products from plasmid and gonococcal chromosomal DNA following electrophoresis. Lanes: 1, FA1090; 2, pUNCH1304; 3, FA6973 (FA1090 hemH::CAT); 4, FA1090; 5, pUNCH1306; 6, F6974 (FA1090 hemA::Ω); 7, FA1090; 8, pUNCH1324; 9, FA6978 (FA1090 fbpA::Ω). Oligonucleotide primers for PCR were HEMH1 and HEMH2 for lanes 1 to 3, HEMA1 and HEMA2 for lanes 4 to 6, and FBP1 and FBP2 for lanes 7 to 9. DNA molecular size markers are shown in kilobases.

FIG. 3

Growth phenotypes of FA1090, FA6973 (FA1090 hemH::CAT), FA6974 (FA1090 hemA::Ω), and FA6978 (FA1090 fbpA::Ω) around 0.6-cm wells cut in GCB agar with 50 μM Desferral. The wells were filled with 60 μl of one of the following: heme, 1 mg/ml (Hm); hemoglobin, 10 mg/ml (Hg); transferrin, 25 mg/ml (Tf); iron citrate, 10 mM (FeCit); and iron chloride, 10 mM (FeCl₃). All the plates were incubated at 37°C for 24 h in 5% CO₂.

FIG. 4

Growth phenotypes of FA6973 (FA1090 hemH::CAT) and FA6974 (FA1090 hemA::Ω) around 0.6-cm wells cut in GCB agar both with and without 50 μM Desferral (DF). The wells contain 60 μl of either 10-mg/ml hemoglobin (Hg) or 10 mM ALA. The plates were incubated at 37°C for 24 h in 5% CO₂.

FIG. 5

Growth of MS11(Δ), FA6976 (MS11 hemA::Ω) (□), and FA6977 (MS11 hemH::CAT) (○) in GCB broth with and without additional heme. (A) Strains grown in GCB broth without heme; (B) strains grown in GCB broth with 1 to 3 μM heme. The results are based on three independent experiments, and the vertical bars represent one standard deviation. Similar results were obtained with FA6973 (FA1090 hemH::CAT) and FA6974 (FA1090 hemA::Ω), respectively (data not shown).

FIG. 6

Western blot of gonococcal whole-cell lysates from FA1090, FA6978 (FA1090 fbpA::Ω), and FA6979 (FA1090 fbpA::aphA-3) probed with polyclonal antibody against FbpA. Lanes: 1, FA1090 (iron replete); 2, FA1090 (iron limited); 3, FA6979 (iron replete); 4, FA6979 (iron limited); 5, FA6978 (iron replete); 6, FA6978 (iron limited).

FIG. 7

Intracellular growth and survival of MS11 (Δ), FA6976 (MS11 hemA::Ω) (□) and FA6977 (MS11 hemH::CAT) (○) inside epithelial cell line A-431. Gentamicin was added 12 h postinoculation to allow time for attachment and invasion. Time refers to the time after addition of gentamicin. The results are standardized to give a ratio by dividing the number of surviving colonies (CFU_s) at various time points between 2 and 10 h after addition of the gentamicin by the number of initial colonies (CFU_i) at 2 h after addition of the gentamicin. Thus, CFU_s/CFU_i at 2 h gives a ratio of 1 for all three strains. Results are based on three independent experiments, each with three wells per strain, and the vertical bars represent one standard deviation.