Hal Is a Bacillus anthracis Heme Acquisition Protein

Abstract

The metal iron is a limiting nutrient for bacteria during infection. Bacillus anthracis, the causative agent of anthrax and a potential weapon of bioterrorism, grows rapidly in mammalian hosts, which suggests that it efficiently attains iron during infection. Recent studies have uncovered both heme (isd) and siderophore-mediated (asb) iron transport pathways in this pathogen. Whereas deletion of the asb genes results in reduced virulence, the loss of three surface components from isd had no effect, thereby leaving open the question of what additional factors in B. anthracis are responsible for iron uptake from the most abundant iron source for mammals, heme. Here, we describe the first functional characterization of bas0520, a gene recently implicated in anthrax disease progression. bas0520 encodes a single near-iron transporter (NEAT) domain and several leucine-rich repeats. The NEAT domain binds heme, despite lacking a stabilizing tyrosine common to the NEAT superfamily of hemoproteins. The NEAT domain also binds hemoglobin and can acquire heme from hemoglobin in solution. Finally, deletion of bas0520 resulted in bacilli unable to grow efficiently on heme or hemoglobin as an iron source and yielded the most significant phenotype relative to that for other putative heme uptake systems, a result that suggests that this protein plays a prominent role in the replication of B. anthracis in hematogenous environments. Thus, we have assigned the name of Hal (heme-acquisition leucine-rich repeat protein) to BAS0520. These studies advance our understanding of heme acquisition by this dangerous pathogen and justify efforts to determine the mechanistic function of this novel protein for vaccine or inhibitor development.

Progress toward the Development of a NEAT Protein Vaccine for Anthrax Disease

“…As mentioned above, a version of the NEAT protein IsdB (V710) was evaluated in phase I and II clinical trials as a vaccine from S. aureus, but the trials were suddenly halted due to toxicity concerns (63). Because Hal is important in heme-iron utilization in B. anthracis (53), is homologous at the amino acid level to the other four NEATs from bacilli (55), and is a component of the NEAT cocktail used here, we investigated its structure and the molecular determinants of its activity to gain a better understanding of how to develop a safer NEAT-based vaccine.…”

“…BslK is an S-layer protein that binds heme and can also transfer heme to IsdC (52). Finally, Hal is necessary for growth on hemoglobin and heme and is also thought to be attached to the bacillus cell wall (53). Two of these NEATs, IsdX2 and IsdC, were identified as being highly antigenic by a functional genomic-serologic screen, and Hal is immunoreactive with B. anthracis antisera (58).…”

“…Secreted and surfacelocalized NEAT-containing proteins mediate the acquisition and import of heme from host heme sources such as hemoglobin (47)(48)(49)(50)(51). B. anthracis encodes five proteins that contain one or more NEAT domains: IsdC, IsdX1, IsdX2, BslK, and Hal (49,(52)(53)(54)(55). IsdX1 and IsdX2 are secreted into the culture medium and actively acquire heme from hemoglobin (49,56).…”

Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

“…The released hemoglobin might serve as a source of two essential nutrients. First, under conditions of low iron, secreted hemophores harboring near-iron transporter (NEAT) domains work to actively extract heme from free hemoglobin, thereby helping satiate a need for iron (63)(64)(65). Second, we propose that the InhA1 secreted by B. anthracis could efficiently cleave hemoglobin, releasing peptides (Fig.…”

Global Metabolomic Analysis of a Mammalian Host Infected with Bacillus anthracis

“…Perfusion efficiencies were analyzed as a proxy for the amount of whole blood still present in tissue vasculature after perfusions by determining the average optical density (OD) at 403 nm per gram of perfused tissues in comparison to nonperfused tissues, a value that is directly proportional to the amount of hemoglobin present (see Fig. S3 in the supplemental material) (30).…”