Siderophore-independent acquisition of transferrin-bound iron by Haemophilus influenzae type b

Abstract

The specificity by which Huemophilus species acquired iron from transferrin (TF) was investigated. In a plate bioassay H. injYuenzse used iron bound to human, bovine and rabbit TFs but not mouse, rat, dog, horse, guinea-pig, pig or ovo-TFs or human and bovine lactoferrins. In contrast, H. pleuropneumoniae used iron only from pig TF whilst H. parainfienzae was unable to utilize iron bound to any of the human or animal TFs tested. The inhibition of growth imposed on H. injhenzae type b strain Eagan by the addition of the synthetic iron chelator EDDA to the culture medium was reversed by 30% iron-saturated human TF added directly to the medium but not when the TF was contained inside a dialysis bag. Dot-blotting of whole cells revealed that human TF bound to the surface of bacteria cultured in iron-restricted but not in iron-plentiful media. Incubation of whole bacterial cells in the presence of the proteolytic enzyme trypsin also abolished TF-binding activity, suggesting that the TF receptor was a protein. In competition dot blotting experiments, human and bovine but not rabbit, dog, mouse or guinea-pig TFs blocked the binding of a horseradish peroxid-human TF conjugate. SDS-PAGE and Western blotting of outer membranes revealed the presence of a TF-binding protein of approximately 72 kDa. These results suggest that the acquisition of TF-bound iron by H. injYuenzae type b probably involves a direct interaction with an outer-membrane protein which shows some TF-species specificity.

Characterization of the Haemophilus influenzae tehB gene and its role in virulence

“…Unlike most other bacterial species, H. influenzae has an absolute requirement for haem as it is unable to synthesize the protoporphyrin ring (Evans et al, 1974). Thus, it has evolved a variety of mechanisms to obtain this essential nutrient from human sources (Stull, 1987;Morton & Williams, 1989, 1990Morton et al, 2006bMorton et al, , 2008. Expression of many of the H. influenzae iron/ haem (Fe/Hm) acquisition systems is regulated in response to environmental levels of iron and/or haem.…”

Evolution of the ferric enterobactin receptor in gram-negative bacteria

“…tein has been cloned from the latter two bacteria (64a, 67a). The effects of iron stress have been studied in many gramnegative bacteria, including Escherichia (12,14,36,48,53), Haemophilus (49,50), Klebsiella (33,42), Neisseria (4,58,73), Pasteurella (16,31,55,66), Pseudomonas (60), Proteus (18), Salmonella (17,20,75), Serratia (46), Shigella (57), Vibrio (2,15,37,69), and Yersinia (8,9,61) spp. These bacteria synthesize numerous iron-regulated membrane proteins (IRMP) whose biochemical functions are for the most part uncharacterized.…”

Immunogenic outer-membrane proteins of Haemophilus influenzae type b in infection

“…The response of Hib to Fe-restriction may, in part, be strain-specific. With Eagan, the same OMP profile in silver-stained gels was found in Fe-restricted and in Fe-replete conditions (Morton & Williams, 1990), although other strains have been shown to have inducible OMPs visible with Coomassie blue (Williams & Brown, 1986) or silver staining (Pidcock et al, 1988) under conditions of Ferestriction. Variations in strain, media source, growth phase and the Fe chelator used make comparison between studies difficult.…”

“…Hib strain Eagan (Anderson et al, 1972) was grown to exponential phase (ODd9,-, = 0.27) in Brain Heart Infusion (BHI) broth supplemented with 2 pg ml-I protoporphyrin IX and 2pgm.I-' NAD at 37°C on an orbital shaker (200 r.p.m.). Overnight Fe-restricted cultures were obtained by growth in supplemented BHI broth plus 100 p&+thylenediaminedi-(ohydr0xy)phenylacetic acid (Morton & Williams, 1990), the latter compound having been freed from contaminating Fe by the method of Rogers (1973). Eagan was used because of the considerable data that has accumulated on this organism, leading to the suggestion that it should be adopted as a reference type b strain for investigations of Hib pathogenicity (Kroll & Moxon, 1989).…”