Publications (19)121.77 Total impact
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Article: Adjuvant activity of free Bordetella pertussis filamentous haemagglutinin delivered by mucosal routes.
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ABSTRACT: The development of safe and potent mucosal adjuvants remains a major objective in vaccinology. The potential usefulness of filamentous haemagglutinin (FHA) of Bordetella pertussis as an adjuvant was assessed in a mouse model. The glutathione-S-transferase of Schistosoma mansoni (Sm28GST) was used for intranasal administration, while the gut-resistant keyhole limpet haemocyanin (KLH) was administrated by the oral route. For both antigens, coadministration with FHA increased antigen-specific immunoglobulin titres. This adjuvant effect did not require chemical cross-linking or direct interaction between FHA and the antigen tested. FHA also behaved as an adjuvant by the subcutaneous route, indicating that its adjuvanticity is not restricted to binding to mucosal surfaces. The FHA-induced adjuvanticity was also observed in mice with high anti-FHA antibody titres as a result of antipertussis vaccination, indicating that pre-existing anti-FHA antibodies do not impair FHA adjuvanticity. No mRNA coding for proinflammatory cytokines was induced in the lungs after intranasal FHA administration. However, an increase in the levels of mRNAs coding for B7-1, transforming growth factor (TGF)-beta and major histocompatibility complex (MHC)-II was detected in the lungs after FHA administration. Although the molecular mechanisms of the FHA-induced adjuvanticity remain to be elucidated, the data presented here indicate that this adhesin, already assessed for human use as a pertussis vaccine constituent, represents a promising adjuvant to improve the humoral immune response when given by mucosal routes.Scandinavian Journal of Immunology 12/2003; 58(5):503-10. · 2.23 Impact Factor -
Article: Immuno-crossreactivity of an anti-Pichia anomala killer toxin monoclonal antibody with a Williopsis saturnus var. mrakii killer toxin.
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ABSTRACT: A monoclonal antibody (mAbKT4), produced against the Pichia anomala ATCC 96603 killer toxin (PaKT) was used to detect the toxin (WmKT) produced by Williopsis saturnus var. mrakii MUCL 41968 which inhibits the growth of a PaKT-sensitive P. anomala strain MUCL 41969. Immunofluorescence studies revealed that mAbKT4 specifically labels the surface of P. anomala and W. saturnus var. mrakii, suggesting that both taxa secrete a killer toxin bearing a common epitope. Immunoblot analyses of concentrated supernatants from P. anomala and W. saturnus var. mrakii cultures showed that in both taxa mAbKT4 reacts with high molecular weight secreted proteins ranging 85-200 kDa. However, immunoblot experiments showed that the molecular weights of PaKT and WmKT are quite different, indicating that the two toxins are related but not identical molecules.Medical Mycology 11/2001; 39(5):395-400. · 2.46 Impact Factor -
Article: The heparin-binding haemagglutinin of M. tuberculosis is required for extrapulmonary dissemination.
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ABSTRACT: Tuberculosis remains the world's leading cause of death due to a single infectious agent, Mycobacterium tuberculosis, with 3 million deaths and 10 million new cases per year. The infection initiates in the lungs and can then spread rapidly to other tissues. The availability of the entire M. tuberculosis genome sequence and advances in gene disruption technologies have led to the identification of several mycobacterial determinants involved in virulence. However, no virulence factor specifically involved in the extrapulmonary dissemination of M. tuberculosis has been identified to date. Here we show that the disruption of the M. tuberculosis or Mycobacterium bovis Bacille Calmette-Guérin (BCG) hbhA gene encoding the heparin-binding haemagglutinin adhesin (HBHA) markedly affects mycobacterial interactions with epithelial cells, but not with macrophage-like cells. When nasally administered to mice, the mutant strains were severely impaired in spleen colonization, but not in lung colonization. Coating wild-type mycobacteria with anti-HBHA antibodies also impaired dissemination after intranasal infection. These results provide evidence that adhesins such as HBHA are required for extrapulmonary dissemination, and that interactions with non-phagocytic cells have an important role in the pathogenesis of tuberculosis. They also suggest that antibody responses to HBHA may add to immune protection against tuberculosis.Nature 08/2001; 412(6843):190-4. · 36.28 Impact Factor -
Article: Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin-binding haemagglutinin.
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ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.Molecular Microbiology 02/2001; 39(1):89-99. · 5.01 Impact Factor -
Article: Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin.
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ABSTRACT: The mycobacterial adhesin heparin-binding hemagglutinin (HBHA) contains several lysine-rich repeats at its carboxyl-terminal end. Using truncated recombinant HBHA forms and hybrid proteins containing HBHA repeats grafted onto the Escherichia coli maltose-binding protein (MBP), we found that these repeats are responsible for heparin binding. Immunofluorescence microscopy studies revealed that their deletion abrogates binding of HBHA to human pneumocytes. Conversely, when fused to MBP, the HBHA repeats confer pneumocyte adherence properties to the hybrid protein. Treatment of pneumocytes with glycosaminoglycan-degrading enzymes showed that HBHA binding depends on the presence of heparan sulfate chains on the cell surface. The epitope of a monoclonal antibody that inhibits mycobacterial adherence to epithelial cells was mapped within the lysine-rich repeats, confirming their involvement in mycobacterial adherence to epithelial cells. Surface plasmon resonance analyses showed that recombinant HBHA binds to immobilized heparin with fast association kinetics (k(a) = 5.62 (+/- 0.10) x 10(5) m(-1) s(-1)), whereas the dissociation kinetics were slower (k(d) = 0.015 (+/- 0.002) s(-1)), yielding a K(D) value of 26 nm. Similar analyses with grafted MBP indicated similar kinetic constants, indicating that the carboxyl-terminal repeats contain the entire heparin-binding site of HBHA. The molecular characterization of the interactions of HBHA with epithelial glycosaminoglycans should help to better understand mycobacterial adherence within the lungs and may ultimately lead to new approaches for therapy or immunoprophylaxis.Journal of Biological Chemistry 06/2000; 275(19):14273-80. · 4.77 Impact Factor -
Article: Molecular characterization of the mycobacterial heparin-binding hemagglutinin, a mycobacterial adhesin.
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ABSTRACT: Although it generally is accepted that the interaction of Mycobacterium tuberculosis with alveolar macrophages is a key step in the pathogenesis of tuberculosis, interactions with other cell types, especially epithelial cells, also may be important. In this study we describe the molecular characterization of a mycobacterial heparin-binding hemagglutinin (HBHA), a protein that functions as an adhesin for epithelial cells. The structural gene was cloned from M. tuberculosis and bacillus Calmette-Guérin, and the sequence was found to be identical between the two species. The calculated Mr was smaller than the observed Mr when analyzed by SDS/PAGE. This difference can be attributed to the Lys/Pro-rich repeats that occur at the C-terminal end of the protein and to a putative carbohydrate moiety. Glycosylation of HBHA appears to protect the protein from proteolytic degradation, which results in the removal of the C-terminal Lys/Pro-rich region responsible for binding of HBHA to sulfated carbohydrates. Evidence suggests that glycosylation is also important for HBHA-mediated hemagglutination and for certain immunologic properties of the protein. Finally, the absence of a signal peptide in the coding region of HBHA raises the possibility that this protein is not secreted via the general secretion pathway.Proceedings of the National Academy of Sciences 11/1998; 95(21):12625-30. · 9.68 Impact Factor -
Article: Immunodominant domains present on the Bordetella pertussis vaccine component filamentous hemagglutinin.
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ABSTRACT: To identify immunologically important domains on filamentous hemagglutinin (FHA), a Bordetella pertussis protein included in new acellular pertussis vaccines (ACPVs), a series of monoclonal antibodies, sera from infants vaccinated with ACPVs or whole cell pertussis vaccine (WCPV), and sera from patients with pertussis were analyzed by immunoblots containing FHA fragments and recombinant FHA proteins. Immunodominant domains located at the COOH-terminus of FHA (type I domain) and near the NH2-terminus (type II domain) were defined by the reactivity with monoclonal antibodies. The sera from patients with pertussis and sera from infants vaccinated with WCPV or with 6 different investigational ACPVs specifically recognized well-defined regions within the type I and type II domains. Identification of these prominent immunologic epitopes on FHA should be useful for the construction of more well-defined pertussis vaccines and for the interpretation of human serologic responses, which may correlate with efficacy of pertussis vaccines.The Journal of Infectious Diseases 07/1997; 175(6):1423-31. · 6.41 Impact Factor -
Article: Identification of a heparin-binding hemagglutinin present in mycobacteria.
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ABSTRACT: Adherence to mammalian host tissues is an important virulence trait in microbial pathogenesis, yet little is known about the adherence mechanisms of mycobacteria. Here, we show that binding of mycobacteria to epithelial cells but not to macrophages can be specifically inhibited by sulfated carbohydrates. Using heparin-Sepharose chromatography, a 28-kD heparin-binding protein was purified from culture supernatants and cell extracts of Mycobacterium bovis and Mycobacterium tuberculosis. This protein, designated heparin-binding hemagglutinin (HBHA), promotes the agglutination of rabbit erythrocytes, which is specifically inhibited by sulfated carbohydrates. HBHA also induce mycobacterial aggregation, suggesting that it can mediate bacteria-bacteria interactions as well. Hemagglutination, mycobacterial aggregation, as well as attachment to epithelial cells are specifically inhibited in the presence of anti-HBHA antibodies. Immunoelectron microscopy using anti-HBHA monoclonal antibodies revealed that the protein is surface exposed, consistent with a role in adherence. Immunoblot analyses using antigen-specific antibodies indicated that HBHA is different from the fibronectin-binding proteins of the antigen 85 complex and p55, and comparison of the NH2-terminal amino acid sequence of purified HBHA with the protein sequence data bases did not reveal any significant similarity with other known proteins. Sera from tuberculosis patients but not from healthy individuals were found to recognize HBHA, indicating its immunogenicity in humans during mycobacterial infections. Identification of putative mycobacterial adhesins, such as the one described in this report, may provide the basis for the development of new therapeutic and prophylactic strategies against mycobacterial diseases.Journal of Experimental Medicine 10/1996; 184(3):993-1001. · 13.85 Impact Factor -
Article: Distinct roles of the N-terminal and C-terminal precursor domains in the biogenesis of the Bordetella pertussis filamentous hemagglutinin.
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ABSTRACT: The 220-kDa Bordetella pertussis filamentous hemagglutinin (FHA) is the major exported protein found in culture supernatants. The structural gene of FHA has a coding potential for a 367-kDa protein, and the mature form constitutes the N-terminal 60% of the 367-kDa precursor. The C-terminal domain of the precursor was found to be important for the high-level secretion of full-length FHA but not of truncated analogs (80 kDa or less). The secretion of full-length and truncated FHA polypeptides requires the presence of the approximately 100-amino-acid N-terminal domain and the outer membrane protein FhaC, homologous to the N-terminal domains of the Serratia marcescens and Proteus mirabilis hemolysins and their accessory proteins, respectively. By analogy to these hemolysins, it is likely that the N-terminal domain of the FHA precursor interacts, directly or indirectly, with the accessory protein during FHA biogenesis. However, immunogenicity and antigenicity studies suggest that the N-terminal domain of FHA is masked by its C-terminal domain and therefore should not be available for its interactions with FhaC. These observations suggest a model in which the C-terminal domain of the FHA precursor may play a role as an intramolecular chaperone to prevent premature folding of the protein. Both heparin binding and hemagglutination are expressed by the N-terminal half of FHA, indicating that this domain contains important functional regions of the molecule.Journal of Bacteriology 03/1996; 178(4):1053-60. · 3.83 Impact Factor -
Article: Amino-terminal maturation of the Bordetella pertussis filamentous haemagglutinin.
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ABSTRACT: The 220 kDa filamentous haemagglutinin (FHA) is a major adhesin of Bordetella pertussis and is produced from a large precursor designated FhaB. Although partly surface associated, it is also very efficiently secreted into the extracellular milieu. Its secretion depends on the outer membrane accessory protein FhaC. An 80 kDa N-terminal derivative of FHA, named Fha44, can also be very efficiently secreted in a FhaC-dependent manner, indicating that all necessary secretion signals are localized in the N-terminal region of FhaB. A comparison of predicted and apparent sizes of FHA derivatives, in addition to immunoblot analyses of cell-associated and secreted FHA polypeptides, indicated that FhaB undergoes N-terminal maturation by the cleavage of an 8-9 kDa segment. However, phenotypic analyses of translational lacZ and phoA fusions showed that this segment does not function as a typical signal peptide. Co-expression of the Fha44-encoding gene with fhaC also did not allow for secretion of Fha44 in Escherichia coli. High levels of secretion could, however, be observed when the OmpA signal peptide was fused to the N-terminal end of Fha44. Regardless of the OmpA signal peptide-Fha44 fusion point, the E. coli-secreted Fha44 had the same M(r) as that secreted by B. pertussis, indicating that the N-terminal proteolytic maturation does not require a B. pertussis-specific factor. Similar to FHA, the B. pertussis-secreted Fha44 contains an as yet uncharacterized modification at its N-terminus. This modification did not occur in E. coli and is therefore not required for secretion. The N-terminus of Fha44 secreted by E. coli was determined and found to correspond to the 72nd residue after the first in-frame methionine of FhaB. The N-terminal modification was also found not to be required for haemagglutination or interaction with sulphated glycoconjugates.Molecular Microbiology 02/1996; 19(1):65-78. · 5.01 Impact Factor -
Article: Sulfated glycoconjugate receptors for the Bordetella pertussis adhesin filamentous hemagglutinin (FHA) and mapping of the heparin-binding domain on FHA.
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ABSTRACT: Filamentous hemagglutinin (FHA) is a major adhesin present on the surface of the gram-negative respiratory pathogen Bordetella pertussis. A number of binding mechanisms have been described for the interaction of FHA with eukaryotic cells. We have focused on its function as a sulfated polysaccharide-binding protein and on identifying potential receptors for FHA on the epithelial cell surface. Using a thin-layer overlay technique, we found that FHA binds specifically to sulfated glycolipids but not to gangliosides or other neutral glycolipids. These results suggest that epithelial cell surface sulfated glycolipids function as receptors for FHA. Further studies demonstrated that a Chinese hamster ovary (CHO) cell strain deficient in glycosaminoglycan expression exhibits greatly diminished attachment to FHA. By FHA-Affi-Gel chromatography, a putative receptor for FHA that has characteristics consistent with a heparan sulfate proteoglycan was isolated from epithelial cell extracts. In addition, by using recombinant FHA fusion proteins, a specific glycosaminoglycan-binding domain located near the N terminus of the FHA molecule was identified. Our results indicate that the B. pertussis adhesin FHA may utilize sulfated glycolipids and proteoglycans commonly found on the surface of human cells and tissues to initiate infection.Infection and Immunity 12/1994; 62(11):5010-9. · 4.16 Impact Factor -
Article: Surface-associated filamentous hemagglutinin induces autoagglutination of Bordetella pertussis.
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ABSTRACT: Filamentous hemagglutinin (FHA) is a major adhesin produced by Bordetella pertussis, the etiologic agent of whooping cough. FHA has been shown to be surface associated but is also secreted by virulent bacteria. Microscopic observations of lungs of mice infected with B. pertussis showed that the bacteria grow as clusters within the alveolar lumen. When B. pertussis was cultivated in vitro with chemically defined medium, bacteria grew as aggregates, mimicking growth observed in vivo. This aggregation was abolished by the addition of cyclodextrin (CDX) to the growth medium and depended on the production of FHA, because a mutant lacking the FHA structural gene failed to form aggregates in a CDX-free medium. Western blot (immunoblot) analyses revealed that, in the absence of CDX, FHA was attached to the bacterial surface and was not efficiently released into the growth medium. Hydrophobic chromatography of FHA showed that CDX drastically reduced the hydrophobicity of FHA, suggesting a direct binding of CDX to FHA, which was further supported by the partial protection of FHA from trypsin digestion in the presence of CDX. In addition, free FHA can interact in a CDX-inhibitable manner with solid phase-immobilized FHA. It can therefore be postulated that the B. pertussis aggregates are most likely due to direct FHA-FHA interaction.Infection and Immunity 11/1994; 62(10):4261-9. · 4.16 Impact Factor -
Article: The modular architecture of bacterial response regulators. Insights into the activation mechanism of the BvgA transactivator of Bordetella pertussis.
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ABSTRACT: Control of virulence factor expression in Bordetella pertussis is mediated by the products of the bvg operon. The BvgS membrane protein responds to certain environmental cues by activating the BvgA protein, which in turn modulates the expression of the target virulence factor genes. The BvgA and BvgS proteins are members of a large family of sensory transduction proteins called the two-component systems. We show that BvgA fusion proteins can activate transcription of a reporter gene containing the bvg promoter in Escherichia coli, and that this activity correlates with its ability to interact specifically with a recognition sequence in cognate promoters. Using homologies between BvgA and other bacterial response regulators as a guide, two BvgA truncation mutants were constructed and their transactivation and DNA-binding capacities were examined. We discovered that (1) DNA-binding activity is localized to the C-terminal half of BvgA, (2) sequence-specific DNA-binding is necessary, but not sufficient for transactivation, and (3) DNA-binding requires the last 20 amino acid residues at its carboxy terminus. A BvgA fusion protein lacking the receiver domain is inactive in transcriptional activation, but retains sequence-specific DNA-binding activity and forms multimeric complexes. We show that BvgA is able to utilize acetyl phosphate as a phosphoryl group donor and the instability of the covalent linkage at extremes of pH is consistent with an acyl phosphate group. Furthermore, the in vitro phosphorylated form of BvgA exhibits an enhanced capacity for binding DNA target sites, while a dephosphorylated form exhibits a limited capacity to bind these sites. We discuss the implications that these observations have on the mechanism by which BvgA is activated to a transcriptionally competent state.Journal of Molecular Biology 09/1994; 241(3):363-77. · 4.00 Impact Factor -
Article: Heparin-inhibitable lectin activity of the filamentous hemagglutinin adhesin of Bordetella pertussis.
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ABSTRACT: Bordetella pertussis, the etiologic agent of whooping cough, produces an outer membrane-associated filamentous hemagglutinin (FHA) which is the major adhesin of this organism. FHA exhibits a lectin-like activity for heparin and dextran sulfate. By using in vitro adherence assays to cultured epithelial cells, the attachment of B. pertussis was reduced in the presence of sulfated polysaccharides such as heparin and dextran sulfate but not in the presence of dextran, indicating the crucial role of polysaccharide sulfation. In addition, inhibition of cellular sulfation by chlorate treatment of the cells resulted in a reduction of B. pertussis adherence, suggesting that epithelial cell surface-exposed sulfated glycoconjugates may serve as receptors for the microorganism. B. pertussis mutant strains deficient in FHA production expressed residual adherence that was no longer inhibited by sulfated polysaccharides. In addition, purified FHA displayed heparin-inhibitable binding to epithelial cells. Mapping experiments of the heparin-binding site of FHA indicated that this site is different from the RGD site and the recently proposed carbohydrate-binding site involved in the interaction of FHA with lactosylceramide. This result demonstrates that FHA contains at least three different binding sites, a feature unusual for bacterial adhesions but similar to features of eukaryotic adhesins and extracellular matrix proteins.Infection and Immunity 04/1994; 62(3):769-78. · 4.16 Impact Factor -
Article: The filamentous haemagglutinin, a multifaceted adhesion produced by virulent Bordetella spp.
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ABSTRACT: Filamentous haemagglutinin (FHA) is the major attachment factor produced by virulent Bordetella spp. Similar to the other virulence factors, its production is tightly regulated by a two-component system in response to environmental changes. Although of impressive size (c. 220 kDa), it is very efficiently released into the culture supernatant of Bordetella pertussis. Its biogenesis involves complex processing of a larger precursor with a calculated molecular mass of 370 kDa. Export of FHA into the culture medium depends on an outer membrane protein homologous to haemolysin accessory proteins. Purified extracellular FHA is able to increase the adherence of other pathogens to the host, which may contribute to super-infection in whooping cough. Although FHA- mutants colonize lungs as efficiently as the wild-type parent strains, immune responses against FHA appear to protect against colonization. Unlike many other adhesins, FHA expresses at least three different attachment activities, one specific for the CR3 integrins of macrophages, one involving a carbohydrate-binding site, specific for interactions with cilia, and a heparin-binding activity that may be important for interaction of B. pertussis with epithelial cells or extracellular matrices.Molecular Microbiology 09/1993; 9(4):653-60. · 5.01 Impact Factor -
Article: Identification and purification of transferrin- and lactoferrin-binding proteins of Bordetella pertussis and Bordetella bronchiseptica.
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ABSTRACT: Bordetella pertussis and Bordetella bronchiseptica were both able to grow in iron-deficient medium when supplemented with iron-saturated human lactoferrin or transferrin but not with human apotransferrin. Direct contact between the transferrins and the Bordetella cells did not appear to be required for growth but considerably improved the growth of the organisms. Analysis of B. pertussis and B. bronchiseptica whole-cell lysates from cultures carried out in iron-deficient or iron-replete media revealed iron-repressible proteins (IRPs) of 27 kDa in B. pertussis and of 30, 32, 73.5, and 79.5 kDa in B. bronchiseptica. Iron-inducible proteins of 16, 23.5, 36.5, and 92.5 kDa and of 17, 23.5, 70, 84, and 91 kDa were also identified in B. pertussis and B. bronchiseptica, respectively. By use of affinity chromatography with iron-saturated human lactoferrin or transferrin as ligands, the 27- and 32-kDa IRPs from B. pertussis and B. bronchiseptica, respectively, were specifically isolated. By using iron-chelated affinity columns, we showed that these proteins exhibit an affinity for iron. Cell fractionation experiments indicated that both of these proteins are probably associated with the outer membrane. Growth of the organisms under modulating conditions showed that the production of these IRPs is not under the genetic transcriptional control of vir or bvg, the general virulence regulon in Bordetella spp.Infection and Immunity 12/1991; 59(11):3982-8. · 4.16 Impact Factor -
Article: Interaction of the Bordetella pertussis filamentous hemagglutinin with heparin.
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ABSTRACT: Heparin, a glycosaminoglycan synthesized in connective tissue-mast cells, appeared to inhibit the hemagglutination of rabbit erythrocytes induced by the filamentous hemagglutinin (FHA), a major adhesin of Bordetella pertussis. This inhibition suggested an interaction of heparin with the FHA region responsible for the hemagglutination activity. FHA-heparin interactions may play a role in bacterial attachment and persistence in the lungs during human pertussis. To confirm a direct FHA-heparin interaction, heparin was used as ligand in an affinity chromatography procedure. This technique allowed to purify FHA directly from the bacterial culture medium in a single-step using heparin-Sepharose CL-6B or Zetaffinity heparin 60 disks. The purified FHA was highly immunoreactive with anti-FHA monoclonal antibodies and showed no signs of degradation after 15 successive cycles of freezing-thawing. The described purification method is simple, and suitable for the rapid preparation of FHA.FEMS Microbiology Letters 03/1991; 62(1):59-64. · 2.04 Impact Factor -
Article: Affinity purification of plasminogen by radial-flow affinity chromatography.
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ABSTRACT: A method for the purification of plasminogen using immobilized L-lysine on a membrane, the whole system being constructed in a radial flow cartridge, is described. Human plasma was applied to the cartridge at 20 ml/min. The results showed that under the chromatographic conditions chosen, in a single pass, greater than 85% recovery of plasminogen was attained with a 110-fold increase in specific activity.Journal of Chromatography 03/1991; 539(2):531-3. · 4.53 Impact Factor -
Article: Characterization of a Williopsis saturnus var. mrakii high molecular weight secreted killer toxin with broad-spectrum antimicrobial activity
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ABSTRACT: Williopsis saturnus var. mrakii MUCL 41968 secretes a killer toxin (WmKT), which is active against a wide range of pathogens. From the W. saturnus var. mrakii MUCL 41968 culture supernatant a protein of 85 kDa with killer activity was purified to homogeneity. The purified protein was demonstrated to be a killer toxin since it displays the toxin activity and cross-reacts with mAbKT4, a monoclonal antibody that blocks WmKT activity. Its partial amino acid sequencing revealed that WmKT might be related to yeast SUN proteins, but not to other killer toxins described. Immunofluorescence studies using polyclonal antibodies raised against purified WmKT revealed that it acts by binding to the cell surface of sensitive strains. We showed that WmKT is inactive against mutant strains of Saccharomyces cerevisiae deficient in the synthesis of beta-glucans, indicating that these polysaccharides constitute the target of the toxin. WmKT was demonstrated to induce rapid lethal cell permeation, since strong propidium iodide labelling was shown for sensitive strains treated with the killer toxin. These findings indicate that WmKT is a novel killer toxin whose molecular characterization may lead to the development of new wide-spectrum antimicrobial compounds.J Antimicrob Chemother. 49(6):961-71.
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Institutions
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1996–1998
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Institut national de la santé et de la recherche médicale
Paris, Ile-de-France, France
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1991–1996
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Institut de Biologie de Lille
Lille, Nord-Pas-de-Calais, France -
Institut de Génétique Moléculaire de Montpellier
Montpellier, Languedoc-Roussillon, France
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