Purification and characterization of a hemoglobin-binding outer membrane protein of Prevotella intermedia

Guan, Su-Min; Nagata, Hideki; Maeda, Kengo; Kuboniwa, Masae; Minamino, Naoto; Shizukuishi, Satoshi

doi:10.1016/j.femsle.2004.05.008

Cited by 3 publications

(1 citation statement)

References 26 publications

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“…Both binding to the outer membrane and degradation of haemoglobin mediated by enzymes in the growth supernatant of P. intermedia is enhanced at acid pHs [10, 30]. In addition, pigment production during growth on blood agar is associated with a drop in pH to around 5.8 [3].…”

Section: Resultsmentioning

confidence: 99%

Role of the cysteine protease interpain A of Prevotella intermedia in breakdown and release of haem from haemoglobin

Byrne

Wawrzonek

Jaworska

et al. 2009

Biochemical Journal

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SUMMARY The Gram-negative oral anaerobe Prevotella intermedia forms an iron(III) protoporphyrin IX pigment from haemoglobin. The microorganism expresses a 90 kDa cysteine protease, Interpain A (InpA), a homologue of Streptococcus pyogenes streptopain (SpeB). The role of InpA in haemoglobin breakdown and haem release was investigated. At pH 7.5, InpA mediated oxidation of oxyhaemoglobin to hydroxymethaemoglobin (in which the haem iron is oxidised to the Fe(III) state and which carries OH− as the sixth co-ordinate ligand) by limited proteolysis of globin chains as indicated by SDS-PAGE and MALDI-TOF analysis. Prolonged incubation at pH 7.5, did not result in further haemoglobin protein breakdown, but in the formation of a haemoglobin haemichrome (where the haem Fe atom is co-ordinated by another amino acid ligand in addition to the proximal histidine) stable to degradation by InpA. InpA-mediated haem release from hydroxymethaemoglobin-agarose was minimal compared with trypsin at pH 7.5. At pH 6.0, InpA increased oxidation at a rate greater than auto-oxidation, producing aquomethaemoglobin (with H2O as sixth co-ordinate ligand), and resulted in its complete breakdown and haem loss. Aquo-methaemoglobin proteolysis and haem release was prevented by blocking haem dissociation by ligation with azide, whilst InpA proteolysis of haem-free globin was rapid even at pH 7.5. Both oxidation of oxyhaemoglobin and breakdown of methaemoglobin by InpA were inhibited by the cysteine-protease inhibitor E64. In summary we conclude that InpA may play a central role in haem acquisition by mediating oxyhaemoglobin oxidation, and by degrading aquomethaemoglobin in which haem-globin affinity is weakened under acidic conditions.

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Section: Resultsmentioning

confidence: 99%

Role of the cysteine protease interpain A of Prevotella intermedia in breakdown and release of haem from haemoglobin

Byrne

Wawrzonek

Jaworska

et al. 2009

Biochemical Journal

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Hemoglobin binding activity and hemoglobin-binding protein of prevotella nigrescens

et al. 2010

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Prevotella nigrescens, lacking siderophores was found to bind to the hemoproteins. The binding was observed also in the envelope which was prepared by sonication of the cell. The binding occurred in the pH-dependent manner; the binding was observed below neutral pHs of the incubation mixtures but only slightly observed in the neutral and alkaline pHs. Furthermore, hemoglobin bound to the envelope was dissociated at high pHs buffers. Maximum amounts of hemoglobin bound to 1 mg envelope was 51.2 μg. Kd for the reaction at pH 5.0 was 2.1 × 10-10M (210 pM). From the dot blot assay, hemoglobin could bind to a protein solubilized from the envelope by a detergent, referred to as hemoglobin-binding protein (HbBP), then it was purified by the sequential procedures of ion exchange chromatography, affinity chromatography and isoelectric focusing. Molecular weight and isoelectric point of the HbBP were 46 kDa and 6.1, respectively.

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Biofilms of black tooth stains: PCR analysis reveals presence of Streptococcus mutans

et al. 2012

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This study investigated the presence of the black-pigmented bacteria Prevotella nigrescens and Prevotella intermedia, the non-black-pigmented bacteria Actinomyces spp and particularly the cariogenic pathogen Streptococcus mutans in the dental biofilms of patients with or without black extrinsic tooth stains, using the multiplex polymerase chain reaction (PCR) technique. Analysis of the dental biofilms of patients with (n=26) or without (n=26) black tooth stains was performed using duplex PCR for the 16S ribosomal RNA gene (P. nigrescens, P. intermedia, Actinomyces spp) and glucosyltransferase-I gene for S. mutans. P. nigrescens and S. mutans were the most frequent bacteria detected in both groups. The least frequently detected were P. intermedia and Actinomyces spp. The similar bacterial composition of dental biofilms of black tooth stains and healthy tooth surfaces indicates that black tooth stains are not free of cariogenic bacteria.

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Purification and characterization of a hemoglobin-binding outer membrane protein of Prevotella intermedia

Cited by 3 publications

References 26 publications

Role of the cysteine protease interpain A of Prevotella intermedia in breakdown and release of haem from haemoglobin

Role of the cysteine protease interpain A of Prevotella intermedia in breakdown and release of haem from haemoglobin

Hemoglobin binding activity and hemoglobin-binding protein of prevotella nigrescens

Biofilms of black tooth stains: PCR analysis reveals presence of Streptococcus mutans

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