Exocytosis ofToxoplasma gondii dense granules into the parasitophorous vacuole after host cell invasion

Tachyzoites ofToxoplasma gondii have been shown to exocytose the contents of dense granules into the parasitophorous vacuole after host cell invasion. A monoclonal antibody specific for a 27-kDa protein was used to locate the dense granules by immunoelectron microscopy. The same antibody also reacted with the tubular network found in the parasitophorous vacuole, which confirmed that the dense granules were exocytosed by tachyzoites.     

A 46000 dalton Plasmodium falciparum merozoite surface glycoprotein not related to the 185000–195000 dalton schizont precursor molecule: isolation and characterization

A 46000 dalton glycoprotein was isolated by extraction of freshly harvested P. falciparum merozoites (FCB1 strain), followed by gel electrophoresis of the extract and electroelution. The antigen is present in the late ring, trophozoite, schizont, and segmenter stages and is localized on the merozoite surface at the end of schizogony. It is not related to the 185000–195000 dalton schizont antigen. An antiserum against the 46000 dalton antigen inhibits invasion of erythrocytes by merozoites. The isolated antigen is identical to the antigen against which monoclonal antibody (mcab) 13.4 is directed.     

Identification, localization, and distribution of the PilT protein in Neisseria gonorrhoeae.

A monoclonal antibody (MAb) directed against a highly conserved protein of Neisseria gonorrhoeae with a molecular size of 40 kDa was isolated and characterized. The protein antigen detected by this MAb was detected by enzyme-linked immunosorbent assay and immunoblotting in all strains of N. gonorrhoeae tested across a wide range of serovars. The 40-kDa protein was found to be expressed at relatively low levels and localized to both the cytosolic and cytoplasmic membrane fractions. Screening of a lambda gt11 expression library derived from gonococcal genomic DNA with the anti-40-kDa MAb and DNA sequence analysis suggested that the 40-kDa protein and the product of the gonococcal pilT gene were identical. Immunoblotting analysis of gonococcal mutants carrying defined mutations in the pilT gene confirmed that the 40-kDa protein was indeed PilT. The N-terminal sequence derived by microsequencing of the protein purified from gonococci led to the correction of the previously published pilT gene sequence. Sequencing of the pilT gene from three different strains revealed an extremely high degree of conservation at both the amino acid and DNA levels.     

Purification and characterization of a pilin specific for Brazilian purpuric fever-associated Haemophilus influenzae biogroup aegyptius (H. aegyptius) strains.

Brazilian purpuric fever (BPF) is a recently described fatal pediatric disease caused by systemic infection with Haemophilus influenzae biogroup aegyptius. Previous studies have shown that all H. influenzae biogroup aegyptius strains isolated from BPF cases and case contacts share several unique phenotypic and genotypic characteristics that differentiate them from other H. influenzae biogroup aegyptius strains isolated from conjunctivitis cases in Brazil. One key characteristic of this BPF clone is reactivity in a BPF-specific monoclonal antibody enzyme-linked immunosorbent assay. We have purified and partially characterized a pilin, referred to as the 25-kilodalton (kDa) protein. Aggregates of this protein contain a heat-labile epitope which is recognized by a monoclonal antibody used in the BPF-specific enzyme-linked immunosorbent assay. The protein has a molecular weight of approximately 25,000, is insoluble in most detergents, and fractionates with outer membrane vesicles after LiCl extraction. Biochemical analysis of the 25-kDa protein shows it to have an amino acid composition similar but not identical to that of the H. influenzae type b pilin. The sequence of 20 N-terminal amino acids of the 25-kDa protein shows almost complete homology with the N terminus of the H. influenzae type b pilin and the types 1 and P pilins of Escherichia coli. Transmission electron microscopic analysis of the purified protein shows the presence of filamentous structures similar in morphology to those of H. influenzae pili. Reactivity between the 25-kDa protein and the BPF-specific monoclonal antibody is demonstrated by Western blotting (immunoblotting) and colloidal gold-enhanced immunoelectron microscopy. Hemadsorption analysis shows that expression of this protein is associated with increases in piliated cells and enhanced binding of these cells to human erythrocytes. These studies indicate that expression of the 25-kDa protein is a characteristic unique to the BPF clone and suggest that this protein plays a role in the pathogenesis of BPF.     

The nature of breast dense core granules: chromogranin reactivity

Ultrastructural immunoreactivity for chromogranin with the LK2H10 antibody was examined in nine cases of breast carcinoma and one example of normal resting breast. These tissues were selected for study on the basis of argyrophilia or LK2H10 immunostaining by light microscopy. In two cases, positive reactivity with the chromogranin antibody was seen in breast neuroendocrine-like dense core granules. In a further six cases chromogranin reactivity was not seen in similar granules that showed the neuroendocrine properties of ultrastructural argyrophilia and uranaffinity. Inability to exhibit the full complement of neuroendocrine characteristics in breast carcinomas contrasts with the facility to demonstrate them in tissues of usual neuroendocrine differentiation. Furthermore, in two mucoid carcinomas and one example of normal resting breast ultrastructural cytoplasmic LK2H10 reactivity was evident, which was not localized to dense core granules, although these were present in two of the cases. Our findings demonstrate the heterogeneity of breast dense core granules and discourage acceptance of such characteristics as evidence of histogenesis of these carcinomas from a neuroendocrine cell type in breast tissue.     

Proteins of the Plasmodium falciparum two transmembrane Maurer’s cleft protein family, PfMC-2TM, and the 130?kDa Maurer’s cleft protein define different domains of the infected erythrocyte intramembranous network

Plasmodium falciparum Maurer’s clefts participate in the transport of macromolecules within the cytoplasm, including the transport of virulence proteins to the erythrocyte membrane surface. We identified a family of genes PfMC-2TM encoding transmembrane proteins located within the intramembranous network of the infected erythrocyte using monoclonal antibody SP1C1. The distribution of the PfMC-2TM protein family within domains of the network was investigated by colocalization and confocal microscopy studies using monoclonal antibody SP1C1 specific for PFMC-2TM and monoclonal antibody SP1A6 specific for the130 kDa Maurer’s cleft protein. Peptide-specific antibodies were prepared against six peptides from different domains of PfMC-2TM and used with the Mabs, as well as known antibodies specific to Maurer’s clefts proteins (ring-expressed protein and membrane-associated histidine-rich protein 1), the erythrocyte membrane protein 1 (PfEMP-1), and serine-rich antigen in colocalization studies. We show that PfMC-2TM is located in the Maurer’s clefts throughout the intracellular blood stage, and immunoelectron microscopy shows domains of PfMC-2TM localized in the parasitophorous vacuole and parasitophorous vacuole membrane. The distribution of the 130 kDa Maurer’s cleft protein changes from within the parasite to the clefts during intracellular development as the parasite matures from young trophozoite to segmented schizont.     

GroEL Heat Shock Protein of Haemophilus ducreyi: Association with Cell Surface and Capacity To Bind to Eukaryotic Cells

The Haemophilus ducreyi homolog of GroEL, a 58.5-kDa heat shock protein (Hsp), is a dominant protein produced not only in response to heat stress but also under in vitro growth conditions. Extracellular localization of the 58.5-kDa Hsp was investigated by whole-cell enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy and in supernatants of washed bacteria by immunoblotting with a Haemophilus ducreyi GroEL-specific mouse monoclonal antibody (BB11). To investigate binding of the Hsp to eukaryotic cells, the 58.5-kDa Hsp was purified by ion-exchange and size exclusion chromatography; incubated with HEp-2 cells, HeLa cells, and human fibroblasts; and then analyzed by immunoblotting. Direct involvement of the 58.5-kDa Hsp in the adherence of H. ducreyi to HEp-2 cells was investigated by using an inhibition assay. An epitope of the 58.5-kDa Hsp was detected by whole-cell ELISA on all of the strains tested, suggesting that it is associated with the cell surface. This was also supported by immunoelectron microscopy results. In supernatants of washed bacteria, the 58.5-kDa Hsp was detected by immunoblotting after 10 h of cultivation. The 58.5-kDa Hsp bound to the eukaryotic cells tested but exerted only limited (about 20%) inhibition of H. ducreyi adherence to HEp-2 cells. These results demonstrate that the 58.5-kDa Hsp of H. ducreyi is associated with the bacterial surface, binds to eukaryotic cells, and partially influences H. ducreyi adherence to HEp-2 cells, indicating possible involvement of the 58.5-kDa Hsp in the attachment of bacteria to host cells and to each other.     

Formation of subepithelial dense deposits in rats induced by a monoclonal antibody against the glomerular cell surface antigen

We developed a monoclonal antibody, H5H3, of IgG1 subclass by hybridization technique using spleen cells of mice immunized with plasma membrane fraction of isolated rat glomeruli. H5H3 recognized main bands at about 220 kD by immuno-overlay technique and bound to the glomerulus as well as brush border of proximal tubules by indirect immunofluorescence (IF) microscopy on normal rat kidney frozen sections. By immunoelectron microscopy (IEM) it bound to the surface of mainly glomerular epithelial cell and weakly to the endothelial cell. After injection to Wistar rats it remained granularly in the glomerulus for more than 2 weeks seen by IF. When rats were preimmunized with murine IgG 4 days before the injection of H5H3, mouse IgG, rat IgG and C3 were strongly visible granularly in the glomerulus in 14 days by IF. Numerous dense deposits were formed at subepithelial area seen by transmission electron microscopy. Perfusion experiment of H5H3 into rat left kidney showed granular distribution of mouse IgG in 48 h, indicating that the reaction occurred in situ. H5H3 bound diffusely in fine granular pattern on the surface of cultured glomerular epithelial cells (GEC) studied by IF and IEM. Antigenic redistribution occurred on GEC after incubation of H5H3 at 37 C. These results suggested the required conditions to form subepithelial immune dense deposits, namely that H5H3 after reaction with antigen could stay for long time in the glomerulus; that H5H3 became an antigen in autologous phase to induce large immune complexes; and H5H3 could induce antigenic modulation.     

A new blood stage antigen of Plasmodium falciparum transported to the erythrocyte surface

On screening a lambda gt11 library from Plasmodium falciparum genomic DNA with an antiserum against the 41-kDa protein band, which confers protective immunity to monkeys, two strongly reacting clones were isolated. One of the clones codes for parts of the P. falciparum 41-kDa aldolase, while the other (41-2) codes for parts of an unknown antigen; this was analyzed further. The 41-2 insert was used to identify two genomic fragments which carry the entire gene. The 41-2 gene codes for 184 amino acids specifying a 29-kDa schizont protein as estimated by Western blot analysis. The protein contains no repetitive sequences, and is characterized by a signal sequence and by two additional hydrophobic segments which could function as membrane anchor sequences. Computer analysis of the protein sequence predicted a dominant epitope in the N-terminal part which was confirmed by immunoreactivity data. The 41-2 protein could be localized in the schizont membrane, associated with membranous structures in the erythrocytic cytoplasm and with the erythrocyte membrane.     

Characterization of an immunoreactive species-specific 19-kilodalton outer membrane protein from Helicobacter pylori by using a monoclonal antibody.

Immunoblotting experiments on hyperimmune rabbit serum and sera from patients with Helicobacter pylori gastritis showed a consistent antibody response to a 19-kDa outer membrane protein antigen. A monoclonal antibody, designated HP 40, which reacted by Western immunoblotting with this protein was produced. It was shared by all H. pylori strains tested (D 273, NCTC 11637, and 24 wild strains) but not by the thermophilic Campylobacter species, Campylobacter fetus, Helicobacter mustellae, or Helicobacter fennelliae. Immunogold staining suggested that the 19-kDa antigen was exposed on the outer surface of the bacteria. Its functional role and effectiveness as a serological diagnostic tool are under study.     

Characterization of a cryptic gene pair from Neisseria gonorrhoeae that is common to pathogenic Neisseria species.

A pair of genes, each of which produces in Escherichia coli a 20-kDa, periplasmically localized protein that cross-reacts with anti-rpoN monoclonal antibody, was isolated from Neisseria gonorrhoeae. Homologs of the two genes were detected in pathogenic Neisseria species but not in commensal species. These genes are designated cnp1 and cnp2 (cryptic neisserial protein).     

Isolation and characterization of cDNA clones encoding a 32-kDa dense-granule antigen of Sarcocystis muris (Apicomplexa)

A monoclonal antibody (2F4) directed against a 32-kDa dense-granule antigen of Sarcocystis muris cyst merozoites (bradyzoites) was used to screen a lambda ZAP cDNA expression library. A clone with an insert of 1.4 kb in length (DG 32/1) was isolated. A fusion protein derived from bacteria harbouring the recombinant plasmid DG 32/1 reacted with monoclonal antibody (mAb) 2F4. Southern blot hybridization suggests that the gene is present as a single copy. On Northern blots, a single mRNA species of 1.8 kb was detected by a cDNA-derived probe. In addition, we isolated a full-length clone (DG 32/PH1) by screening the cDNA library with a non-radioactive-labelled cDNA probe. The nucleotide sequence of DG 32/PH1 comprises 1.57 kb. It contains an open reading frame of 882 bp with a coding capacity of approximately 32 kDa. The hypothetical polypeptide consists of a putative N-terminal signal peptide and the mature protein sequence. The occurrence of an N-terminal signal sequence is consistent with the observation that the 32-kDa protein of S.␣muris is secreted from the dense granules. Received: 12 December 1997 / Accepted: 15 January 1998     

Identification of a virulence-associated antigen of Toxoplasma gondii by use of a mouse monoclonal antibody.

A monoclonal antibody generated against the mouse-lethal RH strain of Toxoplasma gondii was developed. Tachyzoites of virulent and avirulent T. gondii isolates grown in permanent macrophage cell cultures were examined for differences in reactivity with this antibody. Virulence of these Toxoplasma isolates was quantified by injecting different numbers of tachyzoites into NMRI mice and observing the animals for signs of infection or death. The monoclonal antibody identified a 23-kDa antigen expressed by the mouse-lethal strains BK and RH, whereas this antigen was not detected in low-mouse-virulent strains, which were all clinical isolates from Europe. Using Western blot (immunoblot), immunofluorescence, and immunoelectron microscopy, we localized the 23-kDa antigen to the membrane compartment. From these results, we suggest that this 23-kDa antigen is a marker of strain virulence upon which a virulence classification of T. gondii may be based.     

Serum antibody responses to Helicobacter pylori and the cagA marker in patients with mucosa-associated lymphoid tissue lymphoma.

The lymphoma of the mucosa-associated lymphoid tissue (MALT) of the stomach has been linked to Helicobacter pylori infection, but the mechanisms involved in B-cell proliferation remain elusive. In a search for putative H. pylori-specific monoclonal immunoglobulin production, an H. pylori strain was isolated from 10 patients with MALT lymphoma and used to detect the specific serum antibody response to the homologous strain by immunoblotting. Moreover, the antigenicity of the different strains was compared by using each of the 10 sera. We found that the different strains induced highly variable patterns of systemic immunoglobulin G antibody response, although several bacterial antigens, such as the 60-kDa urease B, were often recognized by the different sera. The cagA marker was detected in the strains by PCR with specific primers and by dot blot analysis, and the CagA protein was found in the sera of 4 of the 10 patients by immunoblotting. In conclusion, MALT lymphoma patients, like other patients with H. pylori gastritis, exhibit a polymorphic systemic antibody response, despite an apparently similar antigenic profile. The CagA marker of pathogenicity is not associated with this disease.     

Production and characterization of monoclonal antibodies against a major membrane protein of Mycobacterium avium subsp. paratuberculosis

The Mycobacterium avium subsp. paratuberculosis 35-kDa major membrane protein (MMP) encoded by MAP2121c is an important membrane antigen recognized in cattle with Johne's disease. In this study, purified recombinant MMP was used to produce two stable monoclonal antibodies, termed 8G2 and 13E1, which were characterized by immunoblotting, epitope mapping, and immunofluorescence microscopy.     

Immunochemical characterization of a novel secretory protein (defined by monoclonal antibody HISL-19) of peptide hormone producing cells which is distinct from chromogranin A, B, and C

In this study we have investigated the biochemical properties as well as the subcellular localization of a 67 kD (35/32 kD dimer) polypeptide detected by a monoclonal antibody (HISL-19), which was generated after immunization of BALB/c mice with human islet cell preparations. This protein is expressed by neuronal and peptide hormone producing cells and shares many biochemical and molecular key features with the chromogranin proteins. As demonstrated by one-dimensional and two-dimensional gel electrophoresis, immunoblotting, monoclonal antibody HISL-19 immunoaffinity chromatography, and immunoelectron microscopy, it is a water-soluble, acidic protein stored within secretory granules of peptide hormone-producing cells, and it is released in detectable amounts into the serum of patients bearing neuroendocrine carcinomas. Differences of the HISL-19 protein and chromogranins A, B, and C are indicated by their different tissue distribution, the discrepancy of their apparent molecular weights in sodium dodecyl sulfate gels and isoelectric points, and by the lack of cross-reactivity of their specific antibodies. The protein detected by monoclonal antibody HISL-19 represents therefore a novel component of the soluble compartments of neurosecretory granules, which is distinct from chromogranin A, B, and C.     

27 kDa extracellular matrix protein revealed by a monoclonal antibody raised against rat testis

In search of unique components of the seminiferous tubule extracellular matrix, monoclonal antibodies were raised against an isolated seminiferous tubule extracellular matrix, and the monoclonal antibody 12G11 was cloned. By immunofluorescence microscopy in eight kinds of rat tissues (testis, lung, liver, small intestine, cardiac muscle, skeletal muscle, kidney, and brain), 12G11 antigen existed only in the testis. Immunoelectron microscopy revealed that the antigen is localized in the basement membrane of the seminiferous tubule and in the basement membranes of myoid cells. For a biochemical analysis, eight kinds of rat extracellular matrices were isolated and solubilized with 8 M urea and 2% beta-mercaptoethanol. Immunoblot analysis of these samples in 0.8% agarose gel also showed that the antigen was specific for the testis, and in a two high-molecular weight aggregates. These aggregates seemed to contain type IV collagen and laminin chains. The antigen of 12G11 antibody was shown to be 27 kDa by 10% SDS-PAGE followed by immunoblotting. From these data, the existence of a testis specific 27 kDa basement membrane protein, which associate with type IV collagen and laminin, was suggested.     

Antigenic cross reactivity between p195 and a distinct protein of 100 kDa in Plasmodium falciparum

A monoclonal antibody raised against merozoites of Plasmodium falciparum clone T9/96 was shown to react with an extremely strain specific epitope on a 195 kDa protein synthesized only by late trophozoites and schizonts. This protein was shown to exhibit all of the characteristics attributed to the molecule known variously as merozoite surface protein precursor, polymorphic schizont antigen and p195. The monoclonal antibody also identified a cross-reactive epitope on a distinct protein of 100 kDa in ring stage parasites which was shown to be synthesized throughout the asexual cycle and was not a processing product of p195. One-dimensional peptide mapping studies suggested that these two proteins share a degree of common sequence or structure.     

A merozoite-specific 22-kDa rhoptry protein of the coccidium Eimeria nieschulzi (Sporozoa,Coccidia) is exocytosed in the parasitophorous vacuole upon host cell invasion

A monoclonal antibody (Mab D12A4) was used to follow the genesis and fate of rhoptries from early first-generation merogony through second-generation merozoites of the rat coccidium Eimeria nieschulzi. The epitope recognized by Mab D12A4 belongs to a 22-kDa protein which can be localized first in developing meronts 25 h post-infection. Early rhoptries appear as distinct granules in the cytoplasm of developing meronts and elongate into mature organelles before merozoite release. The 22-kDa protein is found in the parasitophorous vacuole after host cell invasion. Western blotting and immunofluorescence showed that the 22-kDa rhoptry protein is expressed in schizonts and merozoites but not in sporozoites. Received: 9 August 1997 / Accepted: 14 October 1997