A Haemophilus ducreyi strain lacking the yfeABCD iron transport system is virulent in human volunteers

Abstract

Haemophilus ducreyi causes the genital ulcer disease chancroid and painful cutaneous ulcers in children who live in the tropics. To acquire heme from the host, H. ducreyi expresses a TonB-dependent hemoglobin receptor, HgbA, which is necessary and sufficient for H. ducreyi to progress to the pustular stage of disease in a controlled human infection model. HgbA transports hemoglobin across the outer membrane; how heme is transported across the cytoplasmic membrane is unclear. In previous studies, transcripts encoding the YfeABCD heme transporter were upregulated in experimental lesions caused by H. ducreyi in human volunteers, suggesting the latter may have a role in virulence. Here we constructed a double deletion mutant, 35000HPΔyfeABΔyfeCD, which exhibited growth defects relative to its parent 35000HP in media containing human hemoglobin as an iron source. Five human volunteers were inoculated at three sites on the skin overlying the deltoid with each strain. The results of the trial showed that papules formed at 100% (95% CI, 71.5, 100) at both 35000HP and 35000HPΔyfeABΔyfeCD-inoculated sites (P = 1.0). Pustules formed at 60% (95% CI, 25.9, 94.1) at parent-inoculated sites and 53% (95% CI, 18.3, 88.4) at mutant-inoculated sites (P = 0.79). Thus, the ABC transporter encoded by yfeAB and yfeCD was dispensable for H. ducreyi virulence in humans. In the absence of YfeABCD, H. ducreyi likely utilizes other periplasmic binding proteins and ABC-transporters such as HbpA, SapABCDF, and DppBCDF to shuttle heme from the periplasm into the cytoplasm, underscoring the importance of redundancy of such systems in gram-negative pathogens.

Keywords: Haemophilus ducreyi; human infection; iron transport.

Fig 1

yfeAB and yfeCD are members of distinct operons. (A) RT-PCR of gene junctions between HD1814, HD1815, HD1816 (yfeA), HD1817 (yfeB), and HD1818 indicated that there are four genes in the operon that contain yfeAB. (B) RT-PCR of the gene junctions between HD1027, HD1026, HD1025 (yfeC), HD 1024 (yfeD), HD1023, HS1022, and HD1021 indicated that there are seven genes in the operon that contains yfeCD. RT+, reactions containing reverse transcriptase; RT−, reactions lacking reverse transcriptase; gDNA, genomic DNA control. The presence of dnaE transcripts (right panels) was used as quality control for the RNA in each experiment. The gels are representative of three independent experiments.

Fig 2

Growth kinetics of 35000HP (circles) and 35000HPΔyfeABΔyfeCD (squares) in brain heart infusion broth supplemented with human hemoglobin at final concentrations of 5 (open symbols) and 50 (closed symbols) µg/mL. Growth was measured by monitoring optical density (OD₆₆₀) (A) or colony forming units (CFU) (B) over time in three independent experiments. For clarity, only the upper components of error bars representing SDs are shown.

Fig 3

35000HP (circles) and 35000HPΔyfeABΔyfeCD (squares) were grown aerobically in human challenge media containing 50 µg/mL of bovine hemin for 10 h. Growth was measured by optical density (OD₆₆₀) in three independent experiments.