Heme Utilization by Nontypeable Haemophilus influenzae Is Essential and Dependent on Sap Transporter Function

Abstract

Bacterial strategies of innate immune evasion and essential metabolic functions are critical for commensalhost homeostasis. Previously, we showed that Sap translocator function is necessary for nontypeable Haemophilus influenzae (NTHI) behaviors that mediate diseases of the human airway. Antimicrobial peptide (AP) lethality is limited by binding mediated by the Sap complex. SapA shares homology with the dipeptide-binding protein (DppA) and the heme-binding lipoprotein (HbpA), both of which have previously been shown to bind the iron-containing compound heme, whose acquisition is essential for Haemophilus survival. Computational modeling revealed conserved SapA residues, similarly modeled to mediate heme binding in HbpA.Here, we directly demonstrate that SapA bound heme and was essential for heme utilization by ironstarved NTHI. Further, the Sap translocator permease mediated heme transport into the bacterial cytoplasm, thus defining a heretofore unknown mechanism of intracytoplasmic membrane heme transport in Haemophilus. Since we demonstrate multiple ligand specificity for the SapA-binding protein, we tested whether APs would compete with heme for SapA binding. We showed that human ␤-defensins 2 and 3, human cathelicidin LL-37, human neutrophil protein 1, and melittin displaced heme bound to SapA, thus supporting a hierarchy wherein immune evasion supercedes even the needed iron acquisition functions of the Sap system.

The Haemophilus influenzae Sap Transporter Mediates Bacterium-Epithelial Cell Homeostasis

“…Sap transporter function maintains NTHI heme iron homeostasis and persistence in the host, providing mechanisms to resist antimicrobial peptides, acquire heme, and influence NTHI biofilm formation (41)(42)(43)(44)(45). The host microenvironment at the mucosal surface challenges NTHI survival through nutrient (i.e., heme iron) limitation and bactericidal innate immune effectors (48); thus, we hypothesized that Sap transporter functions are critical for NTHI interaction with the host epithelial cells and establishment of colonization.…”

“…Similarly, the Sap transporter functions in the uptake of heme iron. First bound by the SapA periplasmic binding protein, heme iron is delivered for transport across the cytoplasmic membrane through the SapBC permease complex (44). In addition to the multifunctional roles of Sap transporter function in innate immune resistance and nutrient acquisition, Sap transporter function influences NTHI biofilm development and architecture.…”

“…We have previously demonstrated an essential role for the Sap transporter, a multifunctional inner membrane ABC transport complex, in resistance to antimicrobial peptide killing and the transport of the essential heme iron (41)(42)(43)(44)). Antimicrobial peptides are transported into the bacterial cytoplasm in a Sap-dependent manner and are subsequently degraded by cytoplasmic peptidase activity (45).…”

Ferric Uptake Regulator and Its Role in the Pathogenesis of Nontypeable Haemophilus influenzae

“…H. influenzae is unable to synthesize heme but can utilize ferrochelatase to catalyze the insertion of iron into protoporphyrin IX, the precursor of heme (67), or mediate heme uptake directly from the environment (68)(69)(70)(71). The role of Fur in iron homeostasis of H. influenzae has not been explicitly described.…”

Haemophilus influenzae stores and distributes hemin by using Protein E

“…In fact, the free iron concentration is only ≈10 −18 M within the human host (Bullen and Ward, 1988). However, freely available iron may occasionally arise during tissue injury, and hence free heme is released from erythrocytes upon intravascular hemolysis and inflammation caused by 4 polymorphonuclear neutrophils (Aruoma et al, 1988;Evans et al, 1994;Skaar, 2010 (Fournier et al, 2011;Jin et al, 1996;Jin et al, 1999;Khan et al, 2007;Mason et al, 2011). These proteins are located in the periplasm, but HxuA can also be secreted.…”