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Biochemistry and immunology of Gram‐positive bacterial heme acquisition systems
Author(s) -
Shipelskiy Yan,
Wang Zhiguo,
Rosinski Justin,
Buckles Tom,
Mahale Dilip,
Dragosjlvic Steve,
Newton Salete M.,
Klebba Phillip E.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.554.10
Subject(s) - heme , polyclonal antibodies , microbiology and biotechnology , antiserum , biology , monoclonal antibody , biochemistry , antibody , chemistry , immunology , enzyme
Gram‐positive pathogenic bacteria scavenge heme from their hosts during infection. Six heme binding proteins exist in L. monocytogenes (Hbp1 and 2) and S. aureus, (IsdA, B, C and H) that contain NEAT‐domains which presumably interact with heme during its uptake. We cloned the structural genes, purified and characterized the corresponding proteins to understand their specificities, affinities and interactions. We used affinity purified, 6H‐tagged proteins to generate murine polyclonal and monoclonal antibodies. The resulting high‐titer antisera showed specificity for the original immunogens, as well as cross‐reactivity to other NEAT‐domain containing orthologous and paralogous proteins. For example, anti‐Hbp2 recognized Hbp1 as well as IsdH by both ELISA and western immunoblot. These sera inhibited the uptake of hemin (Hn) and hemoglobin (Hb) by L. monocytogenes and S. aureus , and retarded the pathogenesis of these strains in a mouse sepsis model system. Anti‐Hbp1 and 2 sera were cross‐reactive with IsdH and attenuated the virulence of L. monocytogenes . Cross‐reactivity between NEAT‐domain containing proteins suggests that antibodies generated against the iron acquisition system of one Gram positive organism may provide immunity to infection by other Gram positive organisms, likely because the sera inhibit heme acquisition. Lastly, we introduced cysteine at key exposed residues, and labeled the Cys sulfhydryls with extrinsic fluorophores to evaluate localization of these proteins in the bacterial cell wall. This research was supported in part by Trellis Biosciences, S. San Francisco, CA.