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Query: UNIPROT:O75191 (H. influenzae)
 4,961 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The specificity by which Haemophilus species acquired iron from transferrin (TF) was investigated. In a plate bioassay H. influenzae 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. parainfluenzae was unable to utilize iron bound to any of the human or animal TFs tested. The inhibition of growth imposed on H. influenzae 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 peroxidase--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. influenzae type b probably involves a direct interaction with an outer-membrane protein which shows some TF-species specificity.
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PMID:Siderophore-independent acquisition of transferrin-bound iron by Haemophilus influenzae type b. 214 16

The expression of human transferrin and lactoferrin binding activity in Haemophilus influenzae, detected by a binding assay using human transferrin or lactoferrin conjugated to peroxidase, was regulated by the level of available iron in the medium. Transferrin binding activity was present in all H. influenzae isolates tested but not detected in other Haemophilus species or in species of Pasteurella or Actinobacillus. Lactoferrin binding activity was only detected in 1/15 H. influenzae isolates tested. The transferrin and lactoferrin receptors were shown to be specific for the respective human proteins by means of a competition binding assay. Competition binding assays also showed that iron-loaded transferrin was more effective at blocking the transferrin receptor than apotransferrin, but no differences in receptor blocking were observed between iron-loaded lactoferrin and apolactoferrin.
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PMID:Characterization of the human transferrin and lactoferrin receptors in Haemophilus influenzae. 284 24

A new reagent for the detection of penicillin-binding proteins (PBPs) was developed. An N-hydroxysuccinimide ester of biotin was used to tag beta-lactam antibiotics with free side chain amino groups such as ampicillin (BIO-AMP), 6-aminopenicillanic acid (BIO-APA), and 7-aminocephalosporanic acid (BIO-ACA). Bacterial PBPs from cells or isolated cytoplasmic membranes of Escherichia coli, Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae were labeled with BIO-AMP, subjected to electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels, and transferred onto nitrocellulose membranes. Electrophoretic PBP profiles were detected on blots, using colorimetric or chemiluminescence systems, on the basis of the interaction of BIO-AMP-PBP complexes and a streptavidin-peroxidase conjugate. The chemiluminescent reaction permitted a high sensitivity of detection, and PBP profiles could be determined within seconds. All PBP profiles were similar to those obtained with a traditional PBP labeling technique with 125I-labeled penicillin V, except that an additional unidentified PBP (approximately 55,000 Da) was labeled with BIO-AMP in E. coli and H. influenzae. Differences in the intensities of labeling for specific PBPs were observed between the chemiluminescent and radioactive labeling agents and were attributed to the differences in their affinities for PBPs. Similarly, BIO-AMP, BIO-APA, and BIO-ACA produced different PBP profiles. We also investigated the use of BIO-AMP in PBP purification. BIO-AMP-PBP complexes from a mixture of H. influenzae proteins were allowed to bind to avidin immobilized on an agarose support in a microcentrifuge tube. After several washes in the presence of salts, PBPs were eluted by boiling and treatment with SDS. The eluted proteins were separated by electrophoresis on SDS-polyacrylamide gels, and biotinylated proteins were identified on blots by a chemiluminescence reaction. Biotinylation of beta-lactams is rapid, safe, and inexpensive. Our results demonstrate the feasibility of using biotinylated beta-lactams as nonradioactive reagents for the study of PBPs and for the purification of these proteins.
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PMID:Use of biotinylated beta-lactams and chemiluminescence for study and purification of penicillin-binding proteins in bacteria. 806 79

The absolute requirement for elemental iron and the porphyrin nucleus for growth of Haemophilus influenzae led us to investigate the role of iron and hemin in regulation of expression of the H. influenzae transferrin receptor. H. influenzae type b strain H1689 was grown in brain heart infusion broth supplemented with beta-NAD and either 10 or 0.1 microgram of hemin ml-1. Transferrin-binding ability was determined with a dot blot assay using human transferrin-horseradish peroxidase conjugate. Cells grown in media with 0.1 microgram of hemin ml-1 bound transferrin, but organisms grown in media with 10 micrograms ml-1 did not. In hemin-restricted media, transferrin binding occurred despite addition of up to 10 mM ferric nitrate, ferric citrate, or ferric PPi, whereas addition of 10 micrograms of hemoglobin ml-1 repressed expression. The breadth of species distribution of this mode of regulation was determined with strains previously characterized by multilocus enzyme electrophoresis. When grown in hemin-restricted media, 24 of 28 type b strains and 52 of 57 serologically nontypeable strains exhibited transferrin binding, although none did so in hemin- and iron-sufficient media. Strain H1689 and serologically nontypeable strain HI1423 grown in heat-inactivated pooled normal human serum, human cerebrospinal fluid, or human breast milk exhibited transferrin binding. Growth in these fluids with 10 micrograms of added hemin ml-1 abolished transferrin binding, whereas addition of 10 mM ferric nitrate did not. These data suggest that the transferrin receptor of H. influenzae is regulated by levels of hemin but not elemental iron alone and that this property is widely distributed among several major cloned families in the species.
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PMID:Expression of the Haemophilus influenzae transferrin receptor is repressible by hemin but not elemental iron alone. 840 90

Nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide, the major component of H. influenzae endotoxin, was localized in the middle and inner ear subsequent to the resolution of experimental otitis media induced by this pathogen. A monoclonal antibody specific for the lipooligosaccharide of this strain was used to probe sections of middle and inner ear tissue and visualized by means of the avidin-biotin peroxidase complex technique. Sixteen to seventeen days post inoculation with either viable or formalin-inactivated NTHi, endotoxin could be localized in both the middle and inner ear at a time when the middle ear was culture negative. Our data demonstrate that endotoxin shed by NTHi during otitis media penetrates the inner ear and binds to both tissue components and inflammatory cells in both the middle and inner ear.
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PMID:Localization of nontypeable Haemophilus influenzae endotoxin in the middle and inner ear during experimental otitis media. 1047

Since they are equipped with several strategies by which they evade the antimicrobial defense of host macrophages, it is surprising that members of the genus Haemophilus appear to be deficient in common antioxidant systems that are well established to protect prokaryotes against oxidative stress. Among others, no genetic evidence for glutathione (gamma-Glu-Cys-Gly) (GSH) biosynthesis or for alkyl hydroperoxide reduction (e.g., the Ahp system characteristic or enteric bacteria) is apparent from the Haemophilus influenzae Rd genome sequence, suggesting that the organism relies on alternative systems to maintain redox homeostasis or to reduce small alkyl hydroperoxides. In this report we address this apparent paradox for the nontypeable H. influenzae type strain NCTC 8143. Instead of biosynthesis, we could show that this strain acquires GSH by importing the thiol tripeptide from the growth medium. Although such GSH accumulation had no effect on growth rates, the presence of cellular GSH protected against methylglyoxal, tert-butyl hydroperoxide (t-BuOOH), and S-nitrosoglutathione toxicity and regulated the activity of certain antioxidant enzymes. H. influenzae NCTC 8143 extracts were shown to contain GSH-dependent peroxidase activity with t-BuOOH as the peroxide substrate. The GSH-mediated protection against t-BuOOH stress is most probably catalyzed by the product of open reading frame HI0572 (Prx/Grx), which we isolated from a genomic DNA fragment that confers wild-type resistance to t-BuOOH toxicity in the Ahp-negative Escherichia coli strain TA4315 and that introduces GSH-dependent alkyl hydroperoxide reductase activity into naturally GSH peroxidase-negative E. coli. Finally, we demonstrated that cysteine is an essential amino acid for growth and that cystine, GSH, glutathione amide, and cysteinylglycine can be catabolized in order to complement cysteine deficiency.
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PMID:Exogenous glutathione completes the defense against oxidative stress in Haemophilus influenzae. 1259 74

While belonging to the same family of antioxidant enzymes, members of the peroxiredoxins do not necessarily employ one and the same method for their reduction. Most representatives become reduced with the aid of thioredoxin, whereas some members use AhpF, tryparedoxin, or cyclophilin A. Recent research on a new peroxiredoxin isoform (type C) from Populus trichocarpa has shown that these particular types may also use glutaredoxin instead of thioredoxin. This finding is supported by the occurrence of chimeric proteins composed of a peroxiredoxin and glutaredoxin region. A gene encoding such a fusion protein is enclosed in the Haemophilus influenzae Rd genome. We expressed the H. influenzae protein, denoted here as PGdx, in Escherichia coli and purified the recombinant enzyme. In vitro assays demonstrate that PGdx, in the presence of dithiothreitol or glutathione, is able to protect supercoiled DNA against the metal ion-catalyzed oxidation-system. Enzymatic assays did, indeed, characterize PGdx as a peroxidase, requiring the glutathione redox cycle for the reduction of hydrogen peroxide (k(cat)/K(m) 5.01 x 10(6) s(-1) m(-1)) as well as the small organic hydroperoxide tert-butylhydroperoxide (k(cat)/K(m) 5.67 x 10(4) s(-1) m(-1)). Enzymatic activity as function of the glutathione concentration deviated from normal Michaelis-Menten kinetics, giving a sigmoidal pattern with an apparent Hill coefficient of 2.9. Besides the formation of a disulfide-linked PGdx dimer, it was also shown by mass spectrometric analysis that cysteine 49, which is equivalent to the active site cysteine of the peroxiredoxins, undergoes glutathionylation during purification under nonreducing conditions. Based on these results, we propose a model for the catalytic mechanism.
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PMID:Purification and characterization of a chimeric enzyme from Haemophilus influenzae Rd that exhibits glutathione-dependent peroxidase activity. 1260 54

The chimeric peroxidase PGdx of Haemophilus influenzae Rd belongs to a recently identified family of thiol peroxidases capable of reducing hydrogen peroxide as well as alkylhydroperoxides by means of glutathione redox cycling. In the present study, we constructed a H. influenzae Rd strain, deficient in its PGdx encoding gene (open reading frame HI0572). The mutant was shown by disk inhibition and liquid culture growth assays to exhibit increased susceptibility to organic hydroperoxides. The hampered growth was restored by complementing the interrupted gene on the genome with a replicating plasmid bearing an intact copy of the gene, hereby rejecting the possible influences of polar effects. Elevated levels of hydrogen peroxide scavenging activity, due to the catalase HktE, were measured in the absence of a functional pgdx gene rendering the mutant more resilient against hydrogen peroxide. On the other hand, after initiation of the stationary phase, aerobic cultures of the pgdx mutant were practically devoid of living cells, whereas wild-type counterparts retained viability. This observed feature was alleviated by complementation with the functional gene or with the addition of catalase.
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PMID:Physiological characterization of Haemophilus influenzae Rd deficient in its glutathione-dependent peroxidase PGdx. 1470 67

Recently, novel hybrid thiol peroxidase (TPx) proteins fused with a glutaredoxin (Grx) were found from some pathogenic bacteria, cyanobacteria, and anaerobic sulfur-oxidizing phototroph. The phylogenic tree analysis that was constructed from the aligned sequences showed two major branches. Haemophilus influenzae TPx.Grx was grouped in one branch as a 1-Cys subfamily of the thiol-specific antioxident protein/AhpC family. Most TPx.Grx proteins, including Vibrio cholerae TPx.Grx, were grouped in the 2-Cys subfamily. To explain the existence of two subgroups in novel hybrid TPx proteins, we have compared the kinetics given by V. cholerae TPx.Grx, H. influenzae TPx.Grx, their separated TPx domains, and a set of mutants devoid of the redox-active cysteines. The kinetic study described here demonstrates clearly that V. cholerae TPx.Grx is a 2-Cys TPx subfamily. For the first time, we also demonstrate the lipid peroxidase activity of V. cholerae TPx.Grx fusion and suggest the in vivo function of 2-Cys TPx.Grx fusion serving as a lipid peroxidase.
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PMID:Vibrio cholerae thiol peroxidase-glutaredoxin fusion is a 2-Cys TSA/AhpC subfamily acting as a lipid hydroperoxide reductase. 1470 41

In 1995, The Institute for Genomic Research completed the genomic sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. This sequence, though extremely useful in understanding the basic biology of H. influenzae, has yet to provide significant insight into our understanding of disease caused by nontypeable H. influenzae (NTHI), because serotype d strains are not generally pathogens. In contrast, NTHI strains are frequently mucosal pathogens and are the primary pathogens of chronic otitis media as well as a significant cause of acute otitis media in children. Thus, it is of great importance to further understand their biology. We used a DNA-based microarray approach to identify genes present in a clinical isolate of NTHI that were absent from strain Rd. We also sequenced the genome of a second NTHI isolate from a child with chronic otitis media to threefold coverage and then used an array of bioinformatics tools to identify genes present in this NTHI strain but absent from strain Rd. These methods were complementary in approach and results. We identified, in both strains, homologues of H. influenzae lav, an autotransported protein of unknown function; tnaA, which encodes tryptophanase; as well as a homologue of Pasteurella multocida tsaA, which encodes an alkyl peroxidase that may play a role in protection against reactive oxygen species. We also identified a number of putative restriction-modification systems, bacteriophage genes and transposon-related genes. These data provide new insight that complements and extends our ongoing analysis of NTHI virulence determinants.
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PMID:Partial analysis of the genomes of two nontypeable Haemophilus influenzae otitis media isolates. 1510 13


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