Phase Variation of Hemoglobin Utilization in Neisseria gonorrhoeae

Abstract

Most Neisseria gonorrhoeae isolates are unable to use human hemoglobin as the sole source of iron for growth (Hgb−), but a minor population is able to do so (Hgb+). This minor population grows luxuriously on hemoglobin, expresses two outer membrane proteins of 42 kDa (HpuA) and 89 kDa (HpuB), and binds hemoglobin under iron-stressed conditions. In addition to the previously reported HpuB, we identified and characterized HpuA, which is encoded by the gene hpuA, located immediately upstream of hpuB. Expression of both proteins was found to be controlled at the translational level by frameshift mutations in a run of guanine residues within thehpuA sequence encoding the mature HpuA protein. The “on-phase” hemoglobin-utilizing variants contained 10 G’s, while the “off-phase” variants contained 9 G’s. InsertionalhpuB mutants of FA19 Hgb+ and FA1090 Hgb+ no longer expressed HpuB but still produced HpuA. A polar insertional mutation of the upstream hpuA gene in FA1090 Hgb+ eliminated production of both HpuA and HpuB, whereas a nonpolar insertional mutant expressed HpuB only. Insertional mutagenesis of either hpuA or hpuB or both substantially decreased the hemoglobin binding ability of the FA1090 Hgb+ variant and prevented growth on hemoglobin plates. Therefore, both HpuA and HpuB were required for the utilization of hemoglobin for growth.

Interactions of haemoglobin with the Neisseria meningitidis receptor HpuAB: the role of TonB and an intact proton motive force

“…Thus, HpuB alone may bind free Hb, albeit with a very low affinity, as significant binding of [ 125 I]-Hb was not detected in liquid phase binding assays. Overall, our findings are consistent with the results of Chen et al (1998), who showed that an HpuA -B + strain of N. gonorrhoeae showed impaired, but detectable, Hb binding in a solid phase dot-blot assay. Nonetheless, although HpuB itself may interact with Hb, optimal binding of this ligand and acquisition of haem-Fe by HpuAB of both pathogenic Neisseria species require both receptor proteins acting in concert.…”

“…In the structurally analogous Tf and Lf receptors, the lipoprotein is surface exposed and is not required for receptor function, playing an accessory role in ligand specificity and binding (Cornelissen and Sparling, 1996;Bonnah and Schryvers, 1998;Lewis et al, 1998b;Pettersson et al, 1998). In contrast, isogenic single mutants of meningococcal and gonococcal HpuAB suggested that both receptor components are required for the binding and utilization of Hb or HbHp (Chen et al, 1998;Lewis et al, 1999). The precise roles of HpuA and HpuB in this process have not yet been described in detail.…”

Iron acquisition systems in the pathogenic Neisseria

“…It is interesting that gonococci primarily use the HpuAB receptor because of a premature stop codon in the hmbR gene (Chen et al, 1996;Stojiljkovic et al, 1996), perhaps reflecting the prevalence of Hb-Hp as an iron source under these conditions. Phase variation of expression of HpuAB, resulting from slipped-strand mispairing in a poly(G) tract in the hpuA gene (Chen et al, 1998), also seems appropriate considering the episodic nature of the supply of haem iron.…”

Role of CCAA Nucleotide Repeats in Regulation of Hemoglobin and Hemoglobin-Haptoglobin Binding Protein Genes of Haemophilus influenzae

“…It is unclear what advantage might be gained by the regulation of hemoglobin binding proteins by both phase variation and heme levels. It has been proposed that phase variation of a hemoglobin binding protein in gonococci might enable efficient utilization of menstrual hemoglobin (2). Such a mechanism may modulate expression of proteins in different host sites, depending on the prevalent heme source, enabling the infecting organism to adapt to specific host microenvironments.…”