Identification of a heparin-binding hemagglutinin present in mycobacteria.

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.

The modular architecture of bacterial response regulators. Insights into the activation mechanism of the BvgA transactivator of Bordetella pertussis.

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.

Stimulation of protein accumulation in HeLa cells by inhibitors of DNA replication. Ferritin.

Incubation of HeLa cells for 24 h with either hydroxyurea (HU), aphidicolin (APHI), thymidine (T) or butyrate (BU), substances used to inhibit replication and accumulate cells at the G1/S interphase, followed by the elimination of the inhibitor and the addition of iron to the growth medium, results in an immediate (HU, APHI, T) or slightly delayed (BU) increased accumulation (18-24-fold higher than the basal level) of ferritin. Under the same experimental circumstances, 5-azacytidine is without effect. As a result of the action of these inhibitors on the structure of DNA, it is proposed that ferritin genes remain accessible to RNA polymerase allowing the accumulation in the cytoplasm of mature ferritin mRNA ready to be mobilized by iron for the production of ferritin molecules.

One-step purification of mouse monoclonal antibodies by mass ion exchange chromatography on Zetaprep.

A rapid one-step purification procedure was developed to isolate mouse monoclonal antibodies present in the growth medium of hybridoma cultures. The procedure, based on a sulfopropyl mass ion exchange chromatography, yields considerable amounts of purified monoclonal antibody. The immunoglobulins are essentially free of transferrin and albumin, as determined by SDS-polyacrylamide gel electrophoresis.

Interaction of the Bordetella pertussis filamentous hemagglutinin with heparin.

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.

Molecular cloning of a gene encoding a Chlamydia psittaci 57-kDa protein that shares antigenic determinants with ca. 60-kDa proteins present in many gram-negative bacteria.

In order to develop reagents to study the immune response of guinea pigs to infection by Chlamydia psittaci guinea pig inclusion conjunctivitis strain (GPIC), we constructed a plasmid clone bank with C. psittaci DNA. One of the recombinant clones isolated produced large amounts of a 57-kilodalton (kDa) protein that was immunoreactive with sera from GPIC infected guinea pigs. While investigating this recombinant protein, we discovered that all the Gram-negative bacteria analyzed so far have immunoreactive proteins of similar size. This protein seems to be a 'common antigen' already described in various Gram-negative bacteria.

The filamentous haemagglutinin, a multifaceted adhesion produced by virulent Bordetella spp.

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.

Affinity purification of plasminogen by radial-flow affinity chromatography.

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.

Identification and purification of transferrin- and lactoferrin-binding proteins of Bordetella pertussis and Bordetella bronchiseptica.

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.

Microbeads and anchorage-dependent eukaryotic cells: the beginning of a new era in biotechnology.

Modern methods for the mass cultivation of anchorage-dependent mammalian cells started with the advent of microcarrier technology. Largely for reasons pertaining to their mode of preparation and ease of cultivation, 150-230 microns microbeads have been overwhelmingly adopted and the technology around them developed. To meet high biomass, macroporous microbeads have been developed. Also, the chemistry of the microsupport has been adapted in order to afford better protection of fragile cells to mechanical wear while simultaneously reorienting their differentiation towards the sought aims (production of cytokines, enzymes etc. ...). Future progress depends upon solutions being brought to problems inherent to this new technology (maintenance of steady state conditions of growth etc. ...) as well as to requirements arising from animal cell culture in general (biosensors, bioreactor's design etc. ...). Besides such technical implementations, biology at large is also expected to benefit from the advent of microcarriers in fields as diverse as the preparation of metaphasic chromosomes in bulk, toxicity testing, organ reconstitution following cell transplantation etc.

Bordetella pertussis TonB, a Bvg-independent virulence determinant.

In gram-negative bacteria, high-affinity iron uptake requires the TonB/ExbB/ExbD envelope complex to release iron chelates from their specific outer membrane receptors into the periplasm. Based on sequence similarities, the Bordetella pertussis tonB exbB exbD locus was identified on a cloned DNA fragment. The tight organization of the three genes suggests that they are cotranscribed. A putative Fur-binding sequence located upstream from tonB was detected in a Fur titration assay, indicating that the tonB exbB exbD operon may be Fur-repressed in high-iron growth conditions. Putative structural genes of the beta-subunit of the histone-like protein HU and of a new two-component regulatory system were identified upstream from tonB and downstream from exbD, respectively. A B. pertussis DeltatonB exbB::Km(r) mutant was constructed by allelic exchange and characterized. The mutant was impaired for growth in low-iron medium in vitro and could not use ferrichrome, desferal, or hemin as iron sources. Levels of production of the major bacterial toxins and adhesins were similar in the TonB(+)/TonB(-) pair. The DeltatonB exbB mutant was still responsive to chemical modulators of virulence; thus, the BvgA/BvgS two-component system is not TonB dependent. Nevertheless, in vivo in the mouse respiratory infection model, the colonization ability of the mutant was reduced compared to the parental strain.

Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin.

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.

Distinct roles of the N-terminal and C-terminal precursor domains in the biogenesis of the Bordetella pertussis filamentous hemagglutinin.

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.

Surface-associated filamentous hemagglutinin induces autoagglutination of Bordetella pertussis.

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.

Sulfated glycoconjugate receptors for the Bordetella pertussis adhesin filamentous hemagglutinin (FHA) and mapping of the heparin-binding domain on FHA.

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.

Amino-terminal maturation of the Bordetella pertussis filamentous haemagglutinin.

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.

Fastin situ determination of the biomass of anchorage-dependent cells.

The lengthy and cumbersome protocol used to establish the growth kinetics characteristics of anchorage-dependent cells (ADC's)in situ (i.e. while the cells adhere on their microsupport) by Aperture Impedance Pulse Spectroscopy (AIPS) can be replaced by an accelerated procedure. This we have named Turbo AIPS whereby the same results can be obtained without actually performing the manipulations leading to the determination of the biomass.

Genital antibody responses in mice after intranasal infection with an attenuated candidate vector strain of Bordetella pertussis.

Intranasal administration of live attenuated Bordetella pertussis, from which the pertussis toxin gene has been deleted, has previously been shown to give rise to high levels of serum immunoglobulin G (IgG) antibodies against both the protective antigen filamentous hemagglutinin (FHA) and heterologous antigens genetically fused to FHA. Here, we extend these results by demonstrating that anti-FHA IgA and IgG antibodies are also produced in the genital tract of mice, both in the vagina and in the uterus, after a single intranasal administration of B. pertussis. By comparing the immune responses induced after infection with wild-type virulent B. pertussis with that induced by infection with an attenuated pertussis toxin-deficient strain, we conclude that pertussis toxin produced by the virulent bacteria does not modify antibody production to FHA in the genital tract of B. pertussis-infected mice. The intranasal infection with either the attenuated or the virulent B. pertussis strain also led to the development of immunologic memory that could be efficiently boosted with purified FHA administered either intranasally or intravaginally to give rise to a significant increase in the levels of specific IgA and IgG produced locally in the genital tract, as well as of specific antibodies in the serum. These observations suggest that attenuated B. pertussis could be a promising vector for intranasal administration to induce antibody responses against antigens from sexually transmitted pathogens fused to FHA.

Molecular characterization of the mycobacterial heparin-binding hemagglutinin, a mycobacterial adhesin.

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.