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Heme utilization in Clostridium perfringens strain 13

thesis
posted on 16.02.2017 by Choo, Jocelyn
The acquisition of iron is important for the growth and survival of bacterial pathogens during an infection. The majority of iron in the host is complexed in the form of the heme-containing protein, hemoglobin. To successfully cause an infection in the mammalian host, many pathogenic bacteria have developed iron acquisition systems to utilize host iron. The Gram-positive anaerobic bacterium, C. perfringens, causes human diseases including food poisoning and the potentially fatal disease, gas gangrene. In this study, a derivative of C. perfringens strain 13, JIR325, was shown to be capable of utilizing human hemoglobin and ferric chloride for its growth in vitro. Two putative heme acquisition systems, designated as the Afu and Isd systems, were investigated. The Isd system comprised of eight genes that are co-transcribed and encoded two putative surface proteins containing NEAr-iron Transporter (NEAT) domains (IsdA and IsdB), a sortase (Srt) and a membrane transport complex. The NEAT domain is a conserved region found in Gram-positive pathogens and is implicated in heme binding. Many proteins containing NEAT domains are hemoprotein receptors, which are anchored to the bacterial cell surface by membrane-anchored transpeptidases, known as sortases. Western blot analysis of IsdA,IsdB and Srt showed an increase expression of these proteins in JIR325 under iron-limited conditions. Absorbance spectroscopy and LC-MS analysis indicated that both IsdA and IsdB binds to heme. Additionally, the localization of IsdA, IsdB and Srt to the cell envelope of JIR325 suggests that IsdA and IsdB are cell wall-anchored heme-binding proteins. Growth studies of isdAisdB (isdAB) and srt mutants indicated that the isd locus was not essential for hemoglobin utilization in JIR325. The isdAB and srt mutants were attenuated for virulence in a mouse myonecrosis model, although this phenotype could not be restored with the respective complemented strains. The Afu system consisted of three genes that were co-transcribed and potentially encoded a periplasmic binding protein (AfuA), a permease (AfuB) and an ATPase (AfuC). Upstream of the afu locus is the tlyC gene, which encoded a potential hemolysin. Hemolysins lyse red blood cells to mediate the release of intracellular heme-containing proteins. Analysis of a tlyC mutant for hemolysis on blood agar suggested that tlyC does not contribute to the hemolytic activity of JIR325, under the conditions tested. Growth studies of an afuB mutant suggested that the Afu system was not essential for hemoglobin utilization. The afuB mutant was defective in growth in the presence of ferric chloride, although this phenotype could not be complemented in trans. These studies provided evidence for functional redundancies of iron and heme acquisition systems, which suggest that iron is important to the survival of JIR325.

History

Principal supervisor

Julian Rood

Year of Award

2014

Department, School or Centre

Department of Microbiology

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences

Exports