Detection of Heme-Binding Proteins in Epidemic Strains of Burkholderia cepacia

Transmissible Burkholderia cepacia genomovar IIIa strains bind and convert monomeric iron(III) protoporphyrin IX into the μ-oxo oligomeric form

“…Alternatively, it may facilitate cell-surface deposition of ferrihaems which can then act defensively against attack by hydrogen peroxide by virtue of their inherent catalase activity (Brown et al, 1970;Jones et al, 1973) as recently demonstrated for Porphyromonas gingivalis (Smalley et al, 2000). However, we have found that the ability of cells to bind iron(III) protoporphyrin IX is not restricted to HBP-positive genomovar IIIa isolates (Smalley et al, 2001). Moreover, it is thus not known how, or in what form iron protoporphyrin IX reacts with the cell surface.…”

“…Two B. cepacia genomovar IIIa strains, C5424 and BC7, expressing the putative 97 kDa outer-membrane HBP and two HBP-negative B. cepacia genomovar I strains, LMG 17997 and ATCC 25416, were chosen as isolates from the previously studied panel of organisms (Smalley et al, 2001). The bacteria were maintained by routine transfer on horse-blood agar, on Columbia agar (Oxoid) and on M9 Minimal Salts Medium agar (Sigma) containing 0?5 % (w/v) glucose.…”

“…The bacteria were maintained by routine transfer on horse-blood agar, on Columbia agar (Oxoid) and on M9 Minimal Salts Medium agar (Sigma) containing 0?5 % (w/v) glucose. We have found that Columbia agar contains low levels of iron protoporphyrin IX (Smalley et al, 2001) and therefore haem-cell interaction studies were performed on cells subcultured at least three times on M9 Minimal Salts Medium agar to deplete any exogenous haem derived from growth on blood or Columbia agar. Cells were then cultured as lawn growths on M9 agar for 3 days and harvested by gentle scraping with sterile plastic loops and collected into 0?14 M NaCl buffered at pH 7?5 with 0?1 M Tris/HCl (NaCl/Tris, pH 7?5), or at pH 6?5 with 0?2 M Na 2 HPO 4 /NaH 2 PO 4 (NaCl/phosphate, pH 6?5).…”

“…A survey of a previously well-characterized panel of strains belonging to the Burkholderia cepacia complex (Mahenthiralingam et al, 2000(Mahenthiralingam et al, , 2001 has revealed the possession of a putative 97 kDa haem-binding protein (HBP) by epidemic strains of genomovar IIIa (Smalley et al, 2001). Such isolates have been described as highly transmissible among cystic fibrosis (CF) patients (Mahenthiralingam et al, 2001) and can cause the socalled 'cepacia syndrome', a necrotizing pneumonia leading to a fatal clinical deterioration.…”

“…The exact role of the 97 kDa HBP in pathogenesis during lung infection with epidemic genomovar IIIa strains is not yet clear. The putative 97 kDa HBP is located in the outer membrane and can bind iron(III) protoporphyrin IX in both the m-oxo oligomeric ([Fe(III)PPIX] 2 O) and the monomeric forms [Fe(III) PPIX.OH] (Smalley et al, 2001). [The m-oxo oligomeric haem species is often referred to as the m-oxo dimer and the m-oxo bishaem of iron(III) protoporphyrin IX, but is more correctly termed the m-oxo oligomer, an appellation attributed to J. L. Hoard (see White, 1978).]…”