To cause disease in a mammalian host, most pathogenic bacteria require iron for their growth and survival. Since most of the host iron is sequestered as heme moieties in hemoglobin, bacteria have developed various mechanisms to acquire this iron. The Gram-positive spore-forming anaerobe, Clostridium perfringens, causes a wide range of diseases, including gas gangrene in humans. Our study showed that C. perfringens is able to utilize human hemoglobin and ferric chloride but not human transferrin or lactoferrin as the sole iron source for its in vitro growth. We identified a heme acquisition system, known as the Isd system, which encodes two NEAT proteins (IsdA and IsdB), a putative sortase (Srt) and a putative ABC transporter complex. We postulated that IsdA and IsdB were hemoprotein receptors since NEAT domains are conserved regions involved in heme binding. Both proteins also contained potential C-terminal cell wall sorting signals, which may be recognized by Srt for cell wall anchoring. We overexpressed IsdA, IsdB and Srt as His-tagged fusion proteins, purified the resultant proteins from E. coli lysates by metal chelation chromatography and generated specific polyclonal antibodies. His-IsdA and His-IsdB eluted from a Talon column with a red colour, indicative of bound heme, which was confirmed by absorbance spectroscopy and mass spectrometry. Western immunoblotting provided evidence that IsdA, IsdB and Srt were cell envelope proteins. In addition, isdA-isdB and srt mutants were constructed using allelic exchange and Targetron technology, respectively. Growth studies of the wild-type strain and its isogenic isdAB or srt mutants indicated that in vitro these genes were not essential for hemoglobin or ferric chloride utilization. Based on these results, we postulate that the Isd system mediates heme uptake and may be compensated by one or more of functionally redundant heme/iron uptake systems to support the in vitro growth of C. perfringens.
*joint senior authors