Utilization of Host Iron Sources by Corynebacterium diphtheriae: Multiple Hemoglobin-Binding Proteins Are Essential for the Use of Iron from the Hemoglobin-Haptoglobin Complex

Abstract

The use of hemin iron by Corynebacterium diphtheriae requires the DtxR-and iron-regulated ABC hemin transporter HmuTUV and the secreted Hb-binding protein HtaA. We recently described two surface anchored proteins, ChtA and ChtC, which also bind hemin and Hb. ChtA and ChtC share structural similarities to HtaA; however, a function for ChtA and ChtC was not determined. In this study, we identified additional host iron sources that are utilized by C. diphtheriae. We show that several C. diphtheriae strains use the hemoglobin-haptoglobin (Hb-Hp) complex as an iron source. We report that an htaA deletion mutant of C. diphtheriae strain 1737 is unable to use the Hb-Hp complex as an iron source, and we further demonstrate that a chtA-chtC double mutant is also unable to use Hb-Hp iron. Single-deletion mutants of chtA or chtC use Hb-Hp iron in a manner similar to that of the wild type. These findings suggest that both HtaA and either ChtA or ChtC are essential for the use of Hb-Hp iron. Enzyme-linked immunosorbent assay (ELISA) studies show that HtaA binds the Hb-Hp complex, and the substitution of a conserved tyrosine (Y361) for alanine in HtaA results in significantly reduced binding. C. diphtheriae was also able to use human serum albumin (HSA) and myoglobin (Mb) but not hemopexin as iron sources. These studies identify a biological function for the ChtA and ChtC proteins and demonstrate that the use of the Hb-Hp complex as an iron source by C. diphtheriae requires multiple iron-regulated surface components. Corynebacterium diphtheriae is a Gram-positive bacterial pathogen that is associated with infections of the upper respiratory tract or skin in humans. In respiratory diphtheria, C. diphtheriae colonizes the nasopharynx or adjacent regions, where it produces the iron-and DtxR-regulated diphtheria toxin, an exotoxin that is responsible for the severe symptoms associated with infection by this organism (1, 2). Relatively few studies have identified factors that are critical for the survival and adherence of C. diphtheriae in the human host. Recent in vitro studies reported that specific pilus structures are associated with adherence of C. diphtheriae to various epithelial cells (3), and factors involved in iron acquisition are essential for the growth of C. diphtheriae under iron-depleted conditions (4, 5). Numerous factors in bacteria that are expressed under low-iron conditions are involved in the transport and metabolism of iron, and many bacterial pathogens encode systems that facilitate the utilization of host iron sources, including transferrin, lactoferrin, and various heme proteins such as hemoglobin (Hb), hemopexin (Hpx), human serum albumin (HSA), and myoglobin (Mb) (6-8). Another significant source of host iron is the Hb-haptoglobin (Hp) complex. The release of Hb into serum following cell lysis results in the formation of Hb dimers that are quickly bound by Hp, forming a tightly bound Hb-Hp complex (9). The Hb-Hp complex has been proposed to be a major source of host iron for certain bacterial pathogens ...

Heme Synthesis and Acquisition in Bacterial Pathogens

“…It appears that these heme-binding proteins serve redundant functions, and as such, a mutant lacking both HtaB and ChtB is deficient at utilizing Hb as an iron source [152]. Recently, it has been shown that ChtA and ChtC are both capable of binding Hp–Hb for heme extraction, and acquisition of heme from Hp–Hb requires HtaA [153]. The current model (Fig.…”

“…The current model (Fig. 2) for Hp–Hb heme acquisition involves binding of Hp–Hb by a combination of HtaA and ChtA or ChtC, heme extraction either actively or passively, and transfer to HtaB, HmuT, and HmuUV [153]. …”

The ChrSA and HrrSA Two-Component Systems Are Required for Transcriptional Regulation of the hemA Promoter in Corynebacterium diphtheriae

“…C. diphtheriae, in particular, acquires heme iron by utilizing the HmuTUV and HtaA heme transport system (9)(10)(11). Upon entering the cytosol, the heme moiety is degraded by the heme oxygenase HmuO, causing the release of the heme-associated iron (12,13).…”

Characterization of the second conserved domain in the heme uptake protein HtaA from Corynebacterium diphtheriae

“…Analysis of the full genome sequence [2] is consistent with a number of iron uptake systems in this organism. The iron and heme transporter systems are predicted to be coordinately expressed with the toxin [4–7]. This, along with the iron requirement for virulence, suggests that iron acquisition is a key component for pathogenicity.…”

“…The hmu gene cluster comprises two heme-binding surface-anchored proteins, HtaA and HtaB, along with a heme binding lipoprotein (HmuT) and an ABC-type heme transporter (HmuUV) [7, 36–39] (Fig. 1).…”