The ABC Transporter HrtAB Confers Resistance to Hemin Toxicity and Is Regulated in a Hemin-Dependent Manner by the ChrAS Two-Component System in Corynebacterium diphtheriae

Corynebacterium diphtheriae is a Gram-positive bacterium and a well-known human pathogen that causes the severe upper respiratory tract disease diphtheria. C. diphtheriae colonizes the upper respiratory tract and is rarely found outside this region during infection in humans. Much of the severe morbidity and mortality associated with diphtheria is due to the production and secretion of diphtheria toxin, which disseminates throughout the host (12). Numerous studies with C. diphtheriae have focused on the genetic regulation of diphtheria toxin, and it is known that transcription of the structural gene tox is repressed by iron and the diphtheria toxin repressor protein, DtxR, a global iron-dependent regulator (6, 10, 18, 37, 39). While it is well established that iron levels can serve as a signal to regulate expression of bacterial virulence factors, the ability to acquire iron as a nutrient source by pathogenic bacteria is also important for virulence (7, 9). Numerous bacterial pathogens, including C. diphtheriae, utilize various host iron sources for growth, including transferrin, lactoferrin, and heme sources such as hemoglobin (7, 26, 35). Bacteria have developed a variety of mechanisms for the uptake and utilization of iron, including siderophore uptake systems (9, 19, 50) and binding protein-dependent transporters that extract iron from various host sources (7, 50). Hemin is thought to be one of the primary host iron sources used by bacterial pathogens, and hemin iron transport and utilization systems in numerous bacterial species have been described (21, 45). Uptake and utilization of hemin as an iron source in C. diphtheriae involve the iron- and DtxR-regulated hmu hemin transport locus, which contains the hmuTUV genes, which encode an ABC hemin transporter, and the htaA, htaB, and htaC genes (1, 13, 18). HtaA and HtaB were recently shown to be surface-anchored hemin binding proteins, and mutations in htaA and in the hmuTUV genes result in reduced ability to use hemoglobin as an iron source, suggesting that factors encoded by these genes are important in hemin transport (1). The use of heme as an iron source in C. diphtheriae also involves the hmuO gene, which encodes a heme oxygenase that degrades intracellular heme, which results in the release of the heme-associated iron (31, 49). Transcription of the hmuO gene in C. diphtheriae is repressed in high-iron conditions by DtxR and is activated by heme or hemoglobin (32). Heme-dependent activation of hmuO is primarily mediated by the ChrA-ChrS (ChrAS) two component signal transduction system, which is composed of the ChrS sensor kinase and the response regulator ChrA (4, 33). The HrrS-HrrA (HrrAS) two-component system is also involved in the heme-dependent transcriptional activation of the hmuO promoter and contributes approximately 20% of the activity observed at this promoter (5). Moreover, HrrAS, as well as the ChrAS regulatory system, is involved in the heme-dependent repression of the hemA gene, the promoter-proximal gene in a heme biosynthetic operon. Mutations in either chrA or chrS result in reduced ability to activate expression of hmuO and in an increased sensitivity to hemin (4). The hemin sensitivity observed in the chrSA mutants was not due to the reduced expression of hmuO, since an hmuO mutant shows no increased sensitivity to hemin (4). It was proposed that the heightened hemin sensitivity observed in the chrSA mutants was likely due to reduced expression of an as yet identified gene(s) that is regulated by ChrAS (4). Recent studies with Staphylococcus aureus and Bacillus anthracis identified an ABC type transporter, designated HrtAB, which is proposed to protect these bacteria from the toxic effects of high levels of hemin (15, 40, 41, 42). Mutations in the S. aureus hrtA and hrtB genes result in an increased sensitivity to hemin (40). The HrtAB system, which is composed of an ATPase (HrtA) and a membrane permease (HrtB), is hypothesized to either directly export excess heme from the cytosol or remove toxic metabolites that accumulate in response to hemin exposure (40, 42). The mechanism by which the HrtAB system reduces heme stress has not been determined (40). The S. aureus hrtAB genes are found adjacent to and transcribed divergently from the recently described hssR-hssS genes, which encode a two-component signal transduction system that controls the expression of the hrtAB genes in a heme-dependent manner (40, 41, 42). In this study, we have identified a putative ABC type transporter in C. diphtheriae that is involved in protection from the toxic effects of hemin. We have designated this ABC transporter HrtAB to be consistent with previously described orthologous systems (15, 40, 41). A C. diphtheriae hrtAB deletion mutant exhibited an increased sensitivity to hemin; expression of the hrtAB genes is increased in the presence of heme or hemoglobin, and this regulation is mediated by the ChrAS two-component signal transduction system. Optimal expression from the hrtB promoter also requires DtxR. Mutations in the sensor region of ChrS were shown to affect heme-dependent activation of hrtAB transcription, demonstrating a direct association between detection of a heme-associated signal by ChrS and transcription of the hrtAB genes.