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MOLECULAR AND CELLULAR PATHOGENESIS

Binding of Heme-Hemopexin Complexes by Soluble HxuA Protein Allows Utilization of This Complexed Heme byHaemophilus influenzae

Leslie D. Cope, Sharon E. Thomas, Zbynek Hrkal, Eric J. Hansen
Leslie D. Cope
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9048, 1 and
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Sharon E. Thomas
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9048, 1 and
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Zbynek Hrkal
Department of Cellular Biochemistry, Institute of Haemotology and Blood Transfusion, 128 20 Prague-2, Czech Republic 2
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Eric J. Hansen
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9048, 1 and
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DOI: 
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  • Fig. 1.
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    Fig. 1.

    Detection of the 100-kDa HxuA protein in CS from wild-type and mutant Hib strains. Proteins present in CS from the wild-type Hib strain DL42 (lanes A, C, and E) and from the HibhxuA mutant strain DL42.61 (lanes B, D, and F) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and either stained with Coomassie blue (panel 1) or transferred to nitrocellulose for Western blot analysis with rat antiserum raised against CS from Hib strain DL26 (panel 2) or with the Hib DL42 HxuA-specific monoclonal antibody 4C11 (panel 3). The arrowhead to the left of lane A indicates the position of the HxuA protein. Molecular mass position markers (in kilodaltons) are present on the left side of the figure.

  • Fig. 2.
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    Fig. 2.

    Effect of exogenous Hib HxuA on the ability of wild-type and mutant Hib strains to utilize heme-hemopexin. Heme-starved cells of the wild-type strain DL42 (A) and the hxuA mutant DL42.61 (B) were inoculated into BHI-NAD broth (basal medium) supplemented with the following compounds at the concentrations listed in the text: no supplement (negative control) (circles), heme (Hm) (positive control) (squares), heme-hemopexin (HmHx) (asterisks), Hib DL42 CS (i.e., HxuA) (diamonds), and Hib DL42 CS (i.e., HxuA) and heme-hemopexin (triangles). Growth was monitored by plating samples from each culture on BHIs agar plates at timed intervals. The data points in each graph are the means of results from two separate experiments; error bars are included.

  • Fig. 3.
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    Fig. 3.

    Effect of exogenous NTHI HxuA on the ability of wild-type and mutant Hib strains to utilize heme-hemopexin. Heme-starved cells of the Hib hxuA mutant DL42.61 (A) and the wild-type Hib strain 760705 (B) were inoculated into BHI-NAD broth (basal medium) supplemented with the following compounds: no supplement (negative control) (circles), heme (Hm) (positive control) (squares), heme-hemopexin (HmHx) (asterisks), NTHI N182 CS (i.e., HxuA) (diamonds), and NTHI N182 CS (i.e., HxuA) and heme-hemopexin (triangles). Growth was monitored by plating samples from each culture on BHIs agar plates at timed intervals.

Tables

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  • Table 1.

    Bacterial strains and plasmids used in this study

    Strain or plasmidDescription or phenotypeReference(s) or source
    H. influenzae strains
     DL26Wild-type Hib strain that expresses the HxuA protein and can utilize heme-hemopexin 16
     DL42Wild-type Hib strain that expresses the HxuA protein and can utilize heme-hemopexin 4, 5
     DL42.107dDL42 mutant with a NotI linker inserted into the hxuA structural gene; this strain does not express HxuA and cannot utilize heme-hemopexin 17
     DL42.61DL42.107d with a cat cartridge inserted into the NotI site within the hxuAgene 5
     760705Wild-type Hib strain that does not express the HxuA protein and does not utilize heme-hemopexin 4, 47
     N182Wild-type NTHI strain that expresses the HxuA protein and can utilize heme-hemopexin 4
    E. colistrains
     RR1Host strain for recombinant plasmids 38
     RR1(pBR322)Recombinant strain that does not release HxuA into the CS38
     RR1(pHX1-6)Recombinant strain that releases HxuA into the CSThis study
    Plasmids
     pBR322Cloning vector, Ampr Tetr 38
     pHX1-6pBR322 with a 13.6-kb insert of H. influenzae DL42 chromosomal DNA containing the hxuCBAgene cluster 5, 17
  • Table 2.

    RIP-based detection of the binding of soluble HxuA to heme-hemopexin complexes

    Source of soluble CS proteins125I-heme-hemopexin immuno- precipitated (cpm) by:
    Antiserum to CS proteinsaControl serumb
    Hib DL42206,89221,892
    Hib DL42.107d15,20821,880
    E. coliRR1(pBR322)7,90620,306
    E. coliRR1(pHX1-6)177,31219,766
    • ↵a Rat antiserum raised against soluble CS proteins from Hib strain DL26 (16). The HxuA proteins expressed by Hib strains DL26 and DL42 are antigenically cross-reactive.

    • ↵b Control serum from unimmunized rats.

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Binding of Heme-Hemopexin Complexes by Soluble HxuA Protein Allows Utilization of This Complexed Heme byHaemophilus influenzae
Leslie D. Cope, Sharon E. Thomas, Zbynek Hrkal, Eric J. Hansen
Infection and Immunity Sep 1998, 66 (9) 4511-4516; DOI:

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Binding of Heme-Hemopexin Complexes by Soluble HxuA Protein Allows Utilization of This Complexed Heme byHaemophilus influenzae
Leslie D. Cope, Sharon E. Thomas, Zbynek Hrkal, Eric J. Hansen
Infection and Immunity Sep 1998, 66 (9) 4511-4516; DOI:
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