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Molecular mechanisms used by Staphylococcus aureus to access iron from human haemoglobin

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Dickson, CF (2014) Molecular mechanisms used by Staphylococcus aureus to access iron from human haemoglobin. PhD thesis, University of Tasmania.

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Abstract

Iron is used as a co-factor in a range of biological reactions and is an essential nutrient
across all domains of life. During infection, pathogens must acquire iron from their host
environment. The tetrameric oxygen transport protein, haemoglobin A (Hb), represents
the largest pool of mammalian iron, accounting for ~ 65 % of iron in a healthy individual.
Genes encoding for Hb receptors are found in the genomes of Gram-positive and Gramnegative
pathogens and their deletion results in attenuated virulence, suggesting that Hb is
an important source of iron during host colonization. As yet, the mechanisms by which
these receptors interact with Hb and capture the haem co-factor (containing the iron) are
largely unknown.
Staphylococcus aureus is a Gram-positive human pathogen and is a significant health
burden in both community and health care settings. S. aureus expresses an elaborate
system of proteins known as the iron regulated surface determinant (Isd) system, which is
specialized to strip iron from Hb. Two related Hb receptors have been identified in the
staphylococcal genome, IsdB and IsdH. IsdB and IsdH bind to Hb on the bacterial cell
surface, actively remove the haem cofactor and relay it to the down stream haem binding
proteins IsdA and IsdC in the cell wall. IsdB and IsdH are multi-domain proteins, with
separate domains dedicated to Hb binding and haem binding. Recent structural studies
have revealed how Hb and haem are bound by the isolated domains, however, as the
isolated domains do not liberate the haem cofactor from Hb these studies provide limited
insight into the receptor mechanism. The aim of this project was to characterize the
structure and function of the intact Hb receptors and reveal the molecular mechanism by
which they capture the haem from Hb and relay it through the Isd pathway. Small angle X-ray scattering studies were used to show that a conserved three-domain
region of IsdB and IsdH, which includes one Hb-binding and one haem-binding domain,
adopts a conserved bi-lobed structure. Two X-ray crystal structures of IsdB and IsdH at
4.2 Å and 3.7 Å resolution, respectively, indicate that this architecture is maintained upon
binding to Hb. Each Hb globin chain is bound independently by one receptor
molecule and functional studies confirmed that haem is captured from all four globin
haem pockets, suggesting a mechanism where the receptors can access all the globin
haem groups regardless of the Hb oligomeric state. The Hb-binding domains of the full-length receptors bind to a surface of Hb that is distant
from the globin haem coordination site. The haem-binding domain is positioned via a
scaffolding linker domain directly adjacent to the globin haem pocket suggesting that a
second set of specific interactions is involved in liberating the haem cofactor. As this
interface is not well resolved in the low-resolution structures, rational mutagenesis was
used to alter the Hb-binding properties of IsdH. A version of IsdH that bound through
only the subunit of Hb yielded crystals that diffracted to 2.5 Å resolution. The higher
resolution structure revealed specific contacts on the haem transfer interface, and a
conformational change in the globin haem pocket, which begin to illustrate how the haem
cofactor is extracted from the globin.
To investigate the mechanism of haem relay through the Isd pathway, haem transfer
assays were designed. IsdH and IsdB captured haem from Hb with similar efficiency.
However, IsdB showed superior activity in transferring haem to IsdA and IsdC. Haem
capture from Hb required the specific recognition of Hb though the Hb-targeting domain,
but, the speed of haem relay to IsdA/C was inversely related Hb-binding affinity
suggesting that haem relay activity relies on a weak interaction with the Hb molecule.
Using site directed mutagenesis, Hb binding affinity was attenuated or enhanced in a
range of IsdB constructs. The activity of these mutants in haem relay experiments was
consistent with the hypothesis that Hb binding affinity is fine tuned to allow for a specific
interaction while maximizing the transfer speed of the haem cargo. Hb utilization is a common strategy employed by pathogenic bacteria to enhance growth
and survival during infection. This work has contributed to our understanding of how the
human pathogen, S. aureus, interacts with and utilizes host Hb, and is the first study to
characterize the molecular mechanism by which the haem ligand is captured from Hb by
a bacterial receptor.

Item Type: Thesis (PhD)
Keywords: Haemoglobin, Staphylococcus aureus, protein crystallography, Isd system, haem import
Copyright Holders: Copyright the Author
Copyright Information:

Copyright 2014 the Author

Date Deposited: 12 Jun 2015 06:19
Last Modified: 15 Sep 2017 01:06
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