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  • 1
    Online Resource
    Online Resource
    Evidence for possible involvement of an elastolytic serine protease in aspergillosis
    American Society for Microbiology ; 1993
    In:  Infection and Immunity Vol. 61, No. 6 ( 1993-06), p. 2357-2368
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 61, No. 6 ( 1993-06), p. 2357-2368
    Abstract: A number of isolates of Aspergillus fumigatus obtained from the hospital environment produced extracellular elastolytic activity. This activity was found to be catalyzed by a single 33-kDa protein which was purified and characterized to be a serine protease. A. fumigatus, when grown on the insoluble structural material obtained from murine and bovine lung, produced the same extracellular 33-kDa elastolytic protease, indicating that this enzyme is likely to be produced when the organism infects the lung. Polymerase chain reaction with an oligonucleotide primer based on the N-terminal amino acid sequence of the elastolytic enzyme yielded a cDNA which was cloned and sequenced. The active serine motif showed more similarity to subtilisin than to mammalian elastase. The amino acid sequence showed 80% identity to the alkaline protease from Aspergillus oryzae. Screening of hospital isolates of Aspergillus flavus showed great variation in the production of elastolytic activity and a much lower level of activity than that produced by A. fumigatus. The elastolytic protease from A. flavus was shown to be a serine protease susceptible to modification and inactivation by active serine and histidine-directed reagents. This protease cross-reacted with the antibodies prepared against the elastolytic protease from A. fumigatus. Immunogold localization of the elastolytic enzyme showed that A. fumigatus germinating and penetrating into the lungs of neutropenic mice secreted the elastolytic protease. An elastase-deficient mutant generated from a highly virulent isolate of A. fumigatus caused drastically reduced mortality when nasally introduced into the lung of neutropenic mice. All of the evidence suggests that extracellular elastolytic protease is a significant virulence factor in invasive aspergillosis.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/iai.61.6.2357-2368.1993
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1993
    detail.hit.zdb_id: 1483247-1
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  • 2
    Online Resource
    Online Resource
    Role of fimbriae expressed by nontypeable Haemophilus influenzae in pathogenesis of and protection against otitis media and relatedness of the fimbrin subunit to outer membrane protein A
    American Society for Microbiology ; 1994
    In:  Infection and Immunity Vol. 62, No. 5 ( 1994-05), p. 2002-2020
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 62, No. 5 ( 1994-05), p. 2002-2020
    Abstract: Nontypeable Haemophilus influenzae is a primary pathogen in both acute otitis media (OM) and chronic OM, yet the pathogenesis of this disease is not fully understood. Although fimbriae have been observed on all clinical OM isolates examined to date, their role in pathogenesis remains unclear. Therefore, the gene which codes for the fimbrial subunit protein (fimbrin) in nontypeable H. influenzae 1128 was isolated, cloned, and sequenced. The nucleotide sequence of the fimbrin gene was found to contain an open reading frame of 1,077 bp which would encode a mature fimbrin protein consisting of 338 amino acid with a calculated molecular mass of 36.4 kDa. The translated amino acid sequence was found to be homologous with various OmpA proteins of other gram-negative bacteria, and algorithmic analysis predicted that this protein is organized as a coiled coil. To directly test whether fimbriae are involved in pathogenesis, the fimbrin gene was disrupted, and the biological consequences of disruption were absence of both expression of the fimbrial appendage and the specific immunogold labeling thereof with antisera directed against isolated fimbrial protein, reduced adherence to human oropharyngeal cells in vitro, augmented clearance from the tympanum post-transbullar inoculation, and significantly reduced induction of OM post-intranasal inoculation in a chinchilla model compared with the fimbriated parent strain. We additionally find that either passive immunization or active immunization against isolated fimbrial protein confers partial protection against transbullar challenge. A Western blot (immunoblot) indicated a degree of serological relatedness among fimbrin proteins of 15 nontypeable and type b isolates. These data suggest that fimbrin could be useful as a component of a vaccine to protect against OM.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/iai.62.5.2002-2020.1994
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1994
    detail.hit.zdb_id: 1483247-1
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  • 3
    Online Resource
    Online Resource
    Molecular cloning of the cDNA and gene for an elastinolytic aspartic proteinase from Aspergillus fumigatus and evidence of its secretion by the fungus during invasion of the host lung
    American Society for Microbiology ; 1995
    In:  Infection and Immunity Vol. 63, No. 10 ( 1995-10), p. 3796-3803
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 63, No. 10 ( 1995-10), p. 3796-3803
    Abstract: Hydrolysis of structural proteins in the lung by extracellular proteinases secreted by Aspergillus fumigatus is thought to play a significant role in invasive aspergillosis. This fungus was found previously to secrete an elastinolytic serine proteinase and a metalloproteinase. We report that A. fumigatus also secretes an aspartic proteinase (aspergillopepsin F) that can catalyze hydrolysis of the major structural proteins of basement membrane, elastin, collagen, and laminin. The pH optimum for the enzymatic activity was 5.0 with elastin-Congo red as the substrate, and the activity was not significantly inhibited by pepstatin A, diazoacetyl norleucine methylester, and 1,2-epoxy-3-(p-nitrophenoxy) propane. The cDNA and gene encoding this aspartic proteinase were cloned and sequenced. The open reading frame, interrupted by three introns, would encode a protein of 393 amino acids composed of a putative 21-amino-acid signal peptide and a 49-amino-acid propeptide preceding the 323-amino-acid mature protein. The amino acid sequence of A. fumigatus aspartic proteinase has 70, 66, and 67% homology to the sequences of those from Aspergillus oryzae, Aspergillus awamori, and Aspergillus saitoi, respectively. The active-site motif (DTG) and the catalytic aspartic residues characteristic of aspartic proteinases are found in the presently described enzyme, indicating that it belongs to a family of aspartic proteinases. Polyclonal antibodies were produced in rabbits against both the mature and precursor forms of the aspartic proteinase expressed in Escherichia coli. Immunoblotting with both antibodies detected a 39-kDa mature enzyme in the culture supernatant of A. fumigatus. The aspartic proteinase activity was inhibited by the antibodies, suggesting that the aspartic proteinase in the culture supernatant corresponds to the product of the cloned gene. Immunogold electron microscopy showed that the aspartic proteinase was secreted by A. fumigatus invading neutropenic mouse lung and its secretion was directed toward the germ tubes of penetrating hyphae.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/iai.63.10.3796-3803.1995
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1995
    detail.hit.zdb_id: 1483247-1
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  • 4
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    Online Resource
    Cloning and Sequence Analysis of a Pilin-Like Gene from an Otitis Media Isolate of Nontypeable Haemophilus influenzae
    Oxford University Press (OUP) ; 1992
    In:  Journal of Infectious Diseases Vol. 165, No. Supplement 1 ( 1992-06-01), p. S201-S203
    Details
    In: Journal of Infectious Diseases, Oxford University Press (OUP), Vol. 165, No. Supplement 1 ( 1992-06-01), p. S201-S203
    Type of Medium: Online Resource
    ISSN: 0022-1899 , 1537-6613
    URL: Article
    DOI: 10.1093/infdis/165-Supplement_1-S201
    RVK:
    XA 10000
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 1992
    detail.hit.zdb_id: 1473843-0
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  • 5
    Online Resource
    Online Resource
    Monocyte Chemotactic Protein-1 Receptor CCR2B Is a Glycoprotein That Has Tyrosine Sulfation in a Conserved Extracellular N-Terminal Region
    The American Association of Immunologists ; 2000
    In:  The Journal of Immunology Vol. 165, No. 9 ( 2000-11-01), p. 5295-5303
    Details
    In: The Journal of Immunology, The American Association of Immunologists, Vol. 165, No. 9 ( 2000-11-01), p. 5295-5303
    Abstract: Monocyte chemotactic protein-1 (MCP-1) binding to its receptor, CCR2B, plays an important role in a variety of diseases involving infection, inflammation, and/or injury. In our effort to understand the molecular basis of this interaction and its biological consequences, we recognized a conserved hexad of amino acids at the N-terminal extracellular domain of several chemokine receptors, including CCR2B. Human embryonic kidney 293 cells expressing Flag-tagged CCR2B containing site-directed mutations in this region, 21–26, including a consensus tyrosine sulfation site were used to determine MCP-1 binding and its biological consequences. The results showed that several of these amino acids are important for MCP-1 binding and consequent lamellipodium formation, chemotaxis, and signal transduction involving adenylate cyclase inhibition and Ca2+ influx into cytoplasm. Mutations that prevented adenylate cyclase inhibition and Ca2+ influx did not significantly inhibit lamellipodium formation and chemotaxis, suggesting that these signaling events are not involved in chemotaxis. CCR2B was found to be sulfated at Tyr26; this sulfation was abolished by the substitution of Tyr with Ala and severely reduced by substitution of Asp25, a part of the consensus sulfation site. The expressed CCR2B was found to be N-glycosylated, as N-glycosidase F treatment of the receptor or growth of the cells in tunicamycin reduced the receptor size to the same level, from 50 to 45 kDa. Thus, CCR2B is the first member of the CC chemokine receptor family shown to be a glycoprotein that is sulfated at the N-terminal Tyr. These post-translational modifications probably have significant biological functions.
    Type of Medium: Online Resource
    ISSN: 0022-1767 , 1550-6606
    URL: Article
    DOI: 10.4049/jimmunol.165.9.5295
    RVK:
    XA 54100
    RVK:
    XA 10000
    Language: English
    Publisher: The American Association of Immunologists
    Publication Date: 2000
    detail.hit.zdb_id: 1475085-5
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  • 6
    Online Resource
    Online Resource
    Isolation, characterization, and cloning of cDNA and the gene for an elastinolytic serine proteinase from Aspergillus flavus
    American Society for Microbiology ; 1994
    In:  Infection and Immunity Vol. 62, No. 1 ( 1994-01), p. 79-85
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 62, No. 1 ( 1994-01), p. 79-85
    Abstract: An elastinolytic serine proteinase produced by Aspergillus flavus 28 that was isolated from a patient who died of aspergillosis has been purified and characterized. The enzyme was inhibited by the serine proteinase inhibitors phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. The metal-chelating agents EDTA and EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] did not severely inhibit the enzyme. A cDNA and a 2.95-kb segment of genomic DNA containing the proteinase gene were sequenced. The open reading frame that would code for a protein containing 403 amino acids was interrupted by three introns. The mature protein lacks 121 N-terminal amino acids including a putative 21-amino-acid signal peptide. The purified mature protein showed a molecular mass of 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas that calculated from the deduced protein sequence was 30 kDa. This elastinolytic serine proteinase of A. flavus has 83 and 82% sequence homology to the similar proteinases from A. fumigatus and A. oryzae. The catalytic properties and the sequence homology around the putative catalytic amino acids suggest that this enzyme belongs to the serine proteinases of the subtilisin family.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/iai.62.1.79-85.1994
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1994
    detail.hit.zdb_id: 1483247-1
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  • 7
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    Online Resource
    Source of methylmalonyl-coenzyme A for erythromycin synthesis: methylmalonyl-coenzyme A mutase from Streptomyces erythreus
    American Society for Microbiology ; 1984
    In:  Antimicrobial Agents and Chemotherapy Vol. 25, No. 2 ( 1984-02), p. 173-178
    Details
    In: Antimicrobial Agents and Chemotherapy, American Society for Microbiology, Vol. 25, No. 2 ( 1984-02), p. 173-178
    Abstract: Streptomyces erythreus produces erythromycin, presumably from methylmalonyl-coenzyme A (CoA), which could be generated by the isomerization of succinyl-CoA. In S. erythreus cultures, [1,4-14C,2,3-3H]succinate was incorporated into erythromycin with a doubling of the 3H/14C ratio. This result is consistent with the hypothesis that succinyl-CoA is isomerized to methylmalonyl-CoA before incorporation into the macrocyclic lactone of erythromycin. The presence of methylmalonyl-CoA mutase, which catalyzes this isomerization, was demonstrated in cell-free extracts prepared from this organism. Consistent with the suggested role for this enzyme, methylmalonyl-CoA mutase activity increased over 12-fold at the time of the most rapid antibiotic production, and the activity level drastically declined when the antibiotic production ceased. The mutase was partially purified from this organism with DEAE-cellulose, ammonium sulfate precipitation, and affinity chromatography on a B12-coenzyme Sepharose column. The enzyme was stimulated 2.5-fold by the addition of B12-coenzyme. The enzyme showed a typical Michaelis-Menten type substrate saturation patterns, with KmS of 0.31 mM and 0.09 microM for methylmalonyl-CoA and B12-coenzyme, respectively, and a V of 0.5 mumol/min per mg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a major band with a molecular weight of 63,000. The properties of this enzyme appear to be fairly similar to those of the mutase previously obtained from other sources.
    Type of Medium: Online Resource
    ISSN: 0066-4804 , 1098-6596
    URL: Article
    DOI: 10.1128/AAC.25.2.173
    RVK:
    XA 24552
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1984
    detail.hit.zdb_id: 1496156-8
    SSG: 12
    SSG: 15,3
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  • 8
    Online Resource
    Online Resource
    Molecular cloning and sequencing of the cDNA and gene for a novel elastinolytic metalloproteinase from Aspergillus fumigatus and its expression in Escherichia coli
    American Society for Microbiology ; 1994
    In:  Infection and Immunity Vol. 62, No. 10 ( 1994-10), p. 4208-4218
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 62, No. 10 ( 1994-10), p. 4208-4218
    Abstract: An extracellular elastinolytic metalloproteinase, purified from Aspergillus fumigatus isolated from an aspergillosis and patient/and an internal peptide derived from it were subjected to N-terminal sequencing. Oligonucleotide primers based on these sequences were used to PCR amplify a segment of the metalloproteinase cDNA, which was used as a probe to isolate the cDNA and gene for this enzyme. The gene sequence matched exactly with the cDNA sequence except for the four introns that interrupted the open reading frame. According to the deduced amino acid sequence, the metalloproteinase has a signal sequence and 227 additional amino acids preceding the sequence for the mature protein of 389 amino acids with a calculated molecular mass of 42 kDa, which is close to the size of the purified mature fungal proteinase. This sequence contains segments that matched both the N terminus of the mature protein and the internal peptide. A. fumigatus metalloproteinase contains some of the conserved zinc-binding and active-site motifs characteristic of metalloproteinases but shows no overall homology with known metalloproteinases. The cDNA of the mature protein when introduced into Escherichia coli directed the expression of a protein with a size, N-terminal sequence, and immunological cross-reactivity identical to those of the native fungal enzyme. Although the enzyme in the inclusion bodies could not be renatured, expression at 30 degrees C yielded soluble enzyme that showed chromatographic behavior identical to that of the native fungal enzyme and catalyzed hydrolysis of elastin. The metalloproteinase gene described here was not found in Aspergillus flavus.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/iai.62.10.4208-4218.1994
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1994
    detail.hit.zdb_id: 1483247-1
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  • 9
    Online Resource
    Online Resource
    Purification and characterization of an elastinolytic metalloprotease from Aspergillus fumigatus and immunoelectron microscopic evidence of secretion of this enzyme by the fungus invading the murine lung
    American Society for Microbiology ; 1994
    In:  Infection and Immunity Vol. 62, No. 6 ( 1994-06), p. 2149-2157
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 62, No. 6 ( 1994-06), p. 2149-2157
    Abstract: Extracellular proteases have been suggested to be virulence factors in invasive aspergillosis. Since serine protease gene-disrupted mutants retain virulence, other proteases are suspected to be also involved in the degradation of lung structural material. An elastinolytic neutral metalloprotease was purified 320-fold from the extracellular fluid of Aspergillus fumigatus grown on elastin by affinity chromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-75. The molecular mass was determined to be 43 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No carbohydrate was attached to this metalloprotease, and its first 22 N-terminal amino acids did not show any homology with the known metalloproteases. The enzyme was completely inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by inhibitors specific for serine, aspartate, and cysteine proteases. Zn2+ and, to a lesser extent, Co2+ reversed the inhibition caused by 1,10-phenanthroline. The protease hydrolyzed the peptide bonds His-Leu, Ala-Leu, Tyr-Leu, Gly-Phe, and Phe-Phe in the B chain of insulin. Synthetic substrate Abz-Ala-Ala-Phe-Phe-pNA could be used for the fluorimetric assay of the A. fumigatus metalloprotease. This enzyme had maximum activity in the pH range 7.5 to 8.0 and at 60 degrees C. It retained 50% of the protease activity when held at 60 degrees C for 1 h. Zn2+ and Co2+ at 1 mM did not inhibit the protease activity. The metalloprotease was able to hydrolyze elastin, and its elastinolytic activity was comparable to that of the serine protease from this organism. The presence of Zn2+ in the culture medium stimulated the metalloprotease production. Rabbit antibodies prepared against the enzyme severely inhibited the enzyme activity. Immunogold electron microscopy revealed that A. fumigatus invading neutropenic mouse lungs secretes this metalloprotease.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/iai.62.6.2149-2157.1994
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1994
    detail.hit.zdb_id: 1483247-1
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