Influence of the combination and phase variation status of the haemoglobin receptors HmbR and HpuAB on meningococcal virulence

Abstract

Neisseria meningitidis can utilize haem, haemoglobin and haemoglobin-haptoglobin complexes as sources of iron via two TonB-dependent phase variable haemoglobin receptors, HmbR and HpuAB. HmbR is over-represented in disease isolates, suggesting a link between haemoglobin acquisition and meningococcal disease. This study compared the distribution of HpuAB and phase variation (PV) status of both receptors in disease and carriage isolates. Meningococcal disease (n = 214) and carriage (n = 305) isolates representative of multiple clonal complexes (CCs) were investigated for the distribution, polyG tract lengths and ON/OFF status of both haemoglobin receptors, and for the deletion mechanism for HpuAB. Strains with both receptors or only hmbR were present at similar frequencies among meningococcal disease isolates as compared with carriage isolates. However, >90 % of isolates from the three CCs CC5, CC8 and CC11 with the highest disease to carriage ratios contained both receptors. Strains with an hpuAB-only phenotype were under-represented among disease isolates, suggesting selection against this receptor during systemic disease, possibly due to the receptor having a high level of immunogenicity or being inefficient in acquisition of iron during systemic spread. Absence of hpuAB resulted from either complete deletion or replacement by an insertion element. In an examination of PV status, one or both receptors were found in an ON state in 91 % of disease and 71 % of carriage isolates. We suggest that expression of a haemoglobin receptor, either HmbR or HpuAB, is of major importance for systemic spread of meningococci, and that the presence of both receptors contributes to virulence in some strains.

Fig. 1.

Variations in the size of the hpuAB locus among N. meningitidis isolates. The hpuAB locus was amplified with primers specific for flanking genes. Amplicons were grouped into five classes based on size, and representative samples are shown. Lanes: 1, size standards; 2, Hpu1, hpu+ strain (Z2491); 3, Hpu2, hpu+ strain (Z6414); 4, ΔHpu3, hpu− strain (Z4686); 5, ΔHpu4, hpu− strain (Z6427); 6, ΔHpu5, hpu− strain (Z4685).

Fig. 2.

Genetic arrangement of the hpuAB locus in N. meningitidis isolates. Directional arrows represent ORFs. Repetitive elements and the IS element are represented by different symbols. An absence of symbols indicates a region that is deleted in a particular clone. Note that the figure is not drawn to scale but is a representation of the various elements and genes present in this locus.

Fig. 3.

PV status of haemoglobin receptors in meningococcal disease and carriage isolates. PV status for the hmbR and hpuAB (hpu) genes was determined based on the length of the repeat tract. This tract is located within the reading frame such that alterations in these tracts switch the genes ON and OFF. Disease (n = 90) and carriage (n = 103) isolates were separated into three groups based on presence/absence of the receptors: both receptors present (n = 47 and 41 for disease and carriage, respectively); hpuAB only (n = 10 and 54); and hmbR only (n = 33 and 13). Strains with both receptors were separated into four groups depending on the ON/OFF status of the receptors. Strains with a single receptor will exhibit either an ON or OFF phenotype and so fall into two groups. Black bars, disease isolates; grey bars, carriage isolates.

Fig. 4.

Phenotypic analysis of hmbR and hpuAB phase variants. An hpuAB OFF variant of strain N54 (this strain lacks the hmbR gene) and an hmbR ON variant of strain MC58 (lacks the hpuAB genes) were tested for growth with haemoglobin or transferrin as the sole iron source. Suspensions of meningococcal strains were seeded onto MHA plates containing 40 µM desferal. Disks were inoculated with 10 µl of either human haemoglobin (10 µg ml⁻¹) or transferrin (50 mg ml⁻¹), or were uninoculated. Growth was recorded after overnight growth. (a) N54, with haemoglobin; (b) N54, with transferrin; (c) N54, no iron; (d) MC58, with haemoglobin; (e) MC58, with transferrin; (f) MC58, no iron.

Fig. 5.

Tract distribution of hmbR and hpuAB in meningococcal disease and carriage isolates. Lengths of the polyG tracts of hmbR and hpuAB were determined by amplification with specific primers and by DNA sequencing of the products. White bars, hpuAB in disease isolates; black bars, hpuAB in carriage isolates; light-grey bars, hmbR in disease isolates; dark-grey bars, hmbR in carriage isolates. An ON PV state is produced by 9G, 12G, 15G and 18G for HmbR, and by 7G, 10G, 13G, 16G and 19G for HpuAB.