Effect of Multiple Mutations in the Hemoglobin- and Hemoglobin-Haptoglobin-Binding Proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae Type b

Abstract

Haemophilus influenzae requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in H. influenzae HI689. Insertional mutation of hgpA andhgpB, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approximately 120 kDa from thehgpA hgpB double mutant. We have cloned and sequenced the gene encoding this third hemoglobin/hemoglobin-haptoglobin binding protein and designate it hgpC. Insertional mutation ofhgpC did not affect the ability of the strain to utilize either hemoglobin or hemoglobin-haptoglobin. An hgpA hgpB hgpC triple mutant constructed by insertional mutagenesis showed a reduced ability to use the hemoglobin-haptoglobin complex but was unaltered in the ability to use hemoglobin. A second class of mutants was constructed in which the entire structural gene of each of the three proteins was deleted. The hgpA hgpB hgpCcomplete-deletion triple mutant was unable to utilize the hemoglobin-haptoglobin complex and showed a reduced ability to use hemoglobin. We have identified three hemoglobin/hemoglobin-haptoglobin-binding proteins in Haemophilus influenzae. Any one of the three proteins is sufficient to support growth with hemoglobin-haptoglobin as the heme source, and expression of at least one of the three is essential for hemoglobin-haptoglobin utilization. Although the three proteins play a role in hemoglobin utilization, an additional hemoglobin acquisition mechanism(s) exists.

Iron and heme utilization inPorphyromonas gingivalis

“…The ability of P. gingivalis to bind hemoglobin and/or hemin is also attributed to proteins other than HmuR and Kgp ( Table 1). The probability that multiple proteins comprise the heme/ hemoglobin-binding machinery of P. gingivalis correlates well with the involvement of several hemoglobinbinding proteins reported for other Gram-negative microorganisms [205,243,244], and we cannot rule out the engagement in iron/heme utilization of additional P. gingivalis proteins that have recently been identified [e.g. 20,160,161,163,183].…”

The role of variable DNA tandem repeats in bacterial adaptation

“…As such, pathogens are able to extract the iron from heme groups of iron-binding proteins via surface receptors. In H. influenzae, a family of hemoglobin-binding and hemoglobin-haptoglobin-binding proteins (Hgp) is known to mediate heme scavenging (Ren et al, 1998;Jin et al, 1999;Morton et al, 1999). Individual strains of H. influenzae have 1-4 hgp genes, of which knockout analysis has confirmed that they are indispensable virulence determinants in invasive disease (Seale et al, 2006).…”

Continuous Microevolution Accelerates Disease Progression during Sequential Episodes of Infection

“…Despite the essential requirement for heme acquisition from molecules like hemoglobin, few studies have investigated which systems are utilized during infection. Prior studies using complete gene knockouts of all three hemoglobin-haptoglobin encoding genes resulted in decreased utilization of hemoglobin and hemoglobin-haptoglobin for a type B strain of Haemophilus under laboratory growth conditions (Morton et al, 1999). Furthermore, inactivation of all three hemoglobin/haptoglobin binding proteins had minimal effect on the pathogenesis of NTHI during septicemia.…”