Interaction of Bartonella bacilliformis with human erythrocyte membrane proteins

Intracellular invasion is an important aspect of Carrión's disease caused by Bartonella bacilliformis. Both the hematic and tissue phases of the disease involve the initial attachment of the organism to erythrocytes and endothelial cells, respectively. Using two different approaches, preliminary evidence is provided that B. bacilliformis interacts with multiple surface-exposed proteins on human erythrocytes. Utilizing Western blot analysis, it was demonstrated that the organism binds several biotinylated erythrocyte proteins with approximate molecular masses of 230, 210, 100, 83 and 44 kDa. There was enhanced Bartonella binding to the 44 kDa protein and binding to a 25 kDa protein following exposure of intact red cells to trypsin. Moreover, there was a complete abrogation of binding to these proteins following exposure of erythrocytes to sodium metaperiodate oxidation, indicating the significance of carbohydrate moieties in the interactions of Bartonella with the erythrocyte. In a second approach, similar binding proteins or putative receptors were identified when Bartonella was co-incubated with isolated membrane proteins from red cell ghosts. A comparison of the molecular weights of these putative receptors with known erythrocyte proteins and their immunoreactivity to specific antisera suggested that the 230 and 210 kDa proteins are the alpha and beta subunits of spectrin; the 100 and 83 kDa proteins are band 3 protein and glycophorin A, respectively; and the 44 and 25 kDa proteins are the respective dimeric and monomeric forms of glycophorin B. Consistent with this notion was the binding of Bartonella to purified preparations of alpha and beta spectrin and glycophorin A/B.     

Entry of Bartonella bacilliformis into erythrocytes.

Bartonella bacilliformis, which causes the human diseases Oroya fever and verruga peruana, binds to human erythrocytes in vitro and produces substantial and long-lasting deformations in erythrocyte membranes, including cone-shaped depressions, trenches, and deep invaginations. The deforming force is probably provided by the polar flagella of these highly motile bacteria. Deep invaginations containing bacteria are commonly seen, and membrane fusion at the necks of the invaginations leads to the formation of intracellular vacuoles containing bacteria. Fluorescent compounds present externally render the vacuoles fluorescent and, occasionally, lightly fluorescent cells are seen, suggesting that the vacuoles sometimes rupture to admit the bacteria to the cytoplasm. Vacuoles present in fluorescent erythrocytes prepared by preloading the erythrocytes with fluorescent compounds are seen as dark areas from which the fluorescent marker is excluded. Entry of the bacteria appears to be the result of a process of forced endocytosis.     

Predominant outer membrane antigens of Bartonella henselae

A hallmark of Bartonella henselae is persistent bacteremia in cats despite the presence of a vigorous host immune response. To understand better the long-term survival of B. henselae in cats, we examined the feline humoral immune response to B. henselae outer membrane (OM) proteins in naturally and experimentally infected cats. Initially, a panel of sera (n = 42) collected throughout North America from naturally infected cats was used to probe B. henselae total membranes to detect commonly recognized antigens. Twelve antigens reacted with sera from at least 85% of cats, and five were recognized by sera from all cats. To localize these antigens further, OMs were purified on discontinuous sucrose density step gradients. Each membrane fraction (OM, hybrid or inner membrane [IM]) contained less than 1% of the total malate dehydrogenase activity (soluble marker), indicating very little contamination by cytoplasmic proteins. FtsI, an integral IM cell division protein, was used to identify the low-density fraction (rho = 1.13 g/cm3) as putative IM (<5% of the total FtsI localized to the high-density fraction) while lipopolysaccharide (LPS) and Pap31, a homolog of the Bartonella quintana heme-binding protein A (HbpA), defined the high-density fraction (rho = 1.20 g/cm3) as putative OM. Additionally, little evidence of cross-contamination between the IM and OM was evident by two-dimensional gel electrophoresis. When purified OMs were probed with feline sera, antigenic proteins profiles were very similar to those observed with total membranes, indicating that many, but not all, of the immunoreactive proteins detected in the initial immunoblots were OM components. Interestingly, two-dimensional immunoblots indicated that B. henselae LPS and members of the Hbp family of proteins did not appear to stimulate an humoral response in any infected cats. Seven proteins were recognized by at least 70% of sera tested, but only three were recognized by all sera. Nanospray-tandem mass spectrometry was used to identify OM components, including the immunodominant OM proteins. Recognition of the nonimmunogenic nature of the major OM components, such as LPS, and identification of the predominant immunogens should elucidate the mechanisms by which B. henselae establishes persistent bacteremic infections within cats. Additionally, the common antigens may serve as potential feline vaccine candidates to eliminate the pathogen from its animal reservoir.     

Bartonella bacilliformis: colonial types and erythrocyte adherence.

Bartonella bacilliformis was cultivated on a solid medium, and two bartonella colonial morphologies were differentiated and designated colony types T1 and T2. Although both T1 and T2 bartonellae adhered to human erythrocytes in vitro, approximately twice as many T2 bartonellae adhered as did T1. Maximum adherence required bartonella energy, most likely proton motive force-dependent motility. Bartonellae did not penetrate or lyse erythrocytes in vitro. Bartonellae adhered poorly to alpha- or beta-glucosidase-treated erythrocytes, but pronase or subtilisin treatment of erythrocytes stimulated adherence. This indicates that bartonellae probably adhere to an erythrocyte glycolipid moiety.     

Identification and characterization of Campylobacter jejuni outer membrane proteins

Outer membrane proteins from isolates of Campylobacter jejuni were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sarcosinate-insoluble membrane preparations were outer membrane enriched based on increased ketodeoxyoctonate concentrations, the presence of surface-exposed 125I-labeled proteins that were hydrophobic, and similarity to membrane vesicle (bleb) sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. Most isolates contained a single major band with molecular weight of 41,000 to 45,000. Profiles of C. jejuni and Campylobacter coli isolates were indistinguishable, but either could be easily differentiated from Campylobacter fetus and Campylobacter faecalis. The profiles were stable for strains under a variety of growth, incubation and passage conditions. We classified 110 isolates from patients with sporadic campylobacter enteritis into nine subtypes based on differences in outer membrane sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. Two categories accounted for 76% of the isolates. Complete concordance was observed in subtypes of strains obtained from epidemiologically related cases. Thus, comparison of the major outer membrane proteins of C. jejuni is a useful technique for investigating the transmission of this organism and may provide a basis for immunological characterization of the outer membrane proteins.     

Identification of Bartonella bacilliformis genotypes and their relevance to epidemiological investigations of human bartonellosis

Genotypic diversity among 26 isolates of Bartonella bacilliformis obtained from different areas of Peru, and at different times, was assessed by comparison of DNA sequences derived from 16S-23S ribosomal DNA intergenic spacer regions (ISR) and a citrate synthase gene (gltA) fragment and by amplified fragment length polymorphism (AFLP) analysis. gltA comparison divided the isolates into two groups, whereas ISR comparison revealed six sequences. AFLP analysis using a selective primer delineated five profiles that correlated well with those obtained by sequence comparison. Combination of all three data sets divided the isolates into six genotypes. One of these genotypes was common to isolates collected from a large area in western Peru that corresponded to the region of endemicity for bartonellosis; however, isolates belonging to two other genotypes were also found within this region. Two of these genotypes were found in isolates isolated more than 35 years apart. The remaining three genotypes were each specifically associated with three outbreaks of bartonellosis that have recently occurred in areas where the disease had not previously been recognized. Demonstration of the unique nature of these isolates indicates that the outbreaks with which they were associated did not result from the introduction of disease by individuals who acquired their infection in the recognized region of endemicity. The sources of these outbreaks remain unknown. A consensus approach to bacterial typing using comparative sequence analysis of multiple genetic loci and the pan-genomic sampling of AFLP appears to offer a well-supported assessment of B. bacilliformis diversity, and the genotypic differences identified appear to have epidemiological significance.     

Identification of polymorphic outer membrane proteins of Chlamydia psittaci 6BC

The genomes of Chlamydia spp. encode a family of putative outer membrane proteins, referred to as polymorphic outer membrane proteins (POMPs), which may play a role in the avoidance of host immune defenses. We analyzed avian strain 6BC of Chlamydia psittaci by polyacrylamide gel electrophoresis for the expression of POMPs. At least six putative POMPs were identified on the basis of their size (90 to 110 kDa) and labeling with an outer membrane-specific probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Three of the putative POMPs reacted with antiserum raised against a recombinant ovine C. psittaci strain POMP, and two possessed surface-exposed, trypsin-sensitive sites. The POMPs were dependent on disulfide bonds for their maintenance in sodium lauryl sarcosine- and sodium dodecyl sulfate-insoluble complexes but did not appear to be interpeptide disulfide bond cross-linked. The putative POMPs were found to be synthesized during the late phase of the chlamydial developmental cycle, cotemporally with the cysteine-rich doublet periplasmic proteins.     

Adhesion to and invasion of cultured human cells by Bartonella bacilliformis.

Bartonella bacilliformis was tested for its ability to adhere to and invade tissue culture cell monolayers. The parasite was able to efficiently bind and penetrate human dermal fibroblasts, human laryngeal epithelium, and human umbilical vein endothelial cells. Exposure of the organism to immune serum prepared against a crude Bartonella extract containing cell wall and membranous material resulted in decreased ability of the parasite to invade host cells. There was also an overall reduction in the invasiveness of bartonellae and total host cell association when human laryngeal epithelial cells and human umbilical vein endothelial cells were preexposed to cytochalasin D, indicating an active involvement of host cells in the uptake of bartonellae. Transmission electron microscopy revealed the presence of bartonellae inside and outside intracellular vacuoles. These data suggest that a surface-associated factor is involved in the invasion process and that internalization of the parasite by host cells involves a microfilament-dependent process similar to phagocytosis.     

Molecular characterization and proposal of a neotype strain for Bartonella bacilliformis.

Bartonella bacilliformis, the etiologic agent of bartonellosis, was characterized biochemically and by DNA hybridization, guanine-plus-cytosine content, genome size, and 16S rRNA sequencing. DNAs from the two strains in our collection exhibited 97% relatedness in hydroxyapatite reactions done at 55 degrees C (optimal reassociation criterion) and 100% relatedness in reactions done at 70 degrees C (stringent reassociation criterion). There was no evidence of divergence within the related sequences. B. bacilliformis DNA showed no relatedness to the cat scratch disease bacillus or to a strain of a second species in the same genus as the cat scratch disease bacillus in hybridization reactions done at 65 degrees C. The guanine-plus-cytosine contents of DNAs from the two B. bacilliformis strains were 39 and 40 mol%. Time course reassociation, done by determining spectrophotometrically the time required for one-half of the denatured DNA to form duplexes, indicated that B. bacilliformis has a genome size of approximately 4 x 10(8). The 16S rRNA sequence analysis indicated that B. bacilliformis is in the alpha-2 subgroup of the purple bacteria, class Proteobacteria, and that its closest relatives are Rochalimaea quintana and Brucella abortus. Strain KC583 (= Herrer 020/F12,63 = ATCC 35685) is proposed as the type strain of B. bacilliformis.     

Identification of immunogenic outer membrane proteins of Pasteurella multocida 3:A in rabbits.

Four groups of protective rabbit immune sera were used to identify Pasteurella multocida outer membrane immunogens by a radioimmunoprecipitation procedure and Western blot (immunoblot) analysis. These are rabbit hyperimmune sera against KSCN extract of P. multocida (group 1) and rabbit immune sera against the KSCN extract of P. multocida (group 2), the outer membrane of P. multocida (group 3), and live P. multocida cells (group 4). Rabbits mounted an antibody response to 18 proteins found in the outer membrane of P. multocida, and the major antibody activities were directed to the 27,000-molecular-weight outer membrane protein (27K protein), as well as the 37.5K, 49.5K, 58.7K, and 64.4K outer membrane proteins. These outer membrane immunogens appear to be exposed on the cell surface and accessible to antibodies, since adsorption of these immune sera with intact P. multocida cells resulted in a significant reduction of antibody activities directed against these proteins, especially the 37.5K protein. Antibodies eluted from immune serum-P. multocida cell complexes were reactive to the 37.5K immunogen, confirming that this protein is exposed on cell surface and accessible to antibodies. Western blot analyses with group 1, 3, and 4 immune sera confirmed that the 27K, 37.5K, 49.5K, 58.7K, and 64.4K proteins are the major outer membrane immunogens of P. multocida in rabbits. Lung lavages of immunized rabbits also contained similar antibody activities directed against several outer membrane proteins, with major activities against the 37.5K and 64.4K proteins.     

Identification of Campylobacter jejuni and C. coli by gel electrophoresis of the outer membrane proteins.

Analysis of the electrophoretic profiles of the outer membrane proteins could be used to differentiate Campylobacter jejuni (16 strains) from Campylobacter coli (10 strains). This observation was confirmed by the study of DNA homology obtained by a quantitative filter hybridization method. The hippurate hydrolysis test gave a poor correlation with the results of differentiation obtained by DNA homology studies and outer membrane protein profile.     

Structural analysis of chlamydial major outer membrane proteins.

The primary structure and surface exposure of the major outer membrane protein (MOMP) isolated from 14C intrinsically or 125I extrinsically radiolabeled Chlamydia trachomatis serotypes D/UW-3, G/UW-57, H/UW-4, I/UW-12, and L2/434 and the Chlamydia psittaci meningopneumonitis strain were analyzed by two different peptide-mapping techniques. Radiolabeled proteins were digested with either Staphylococcus aureus V8 protease, the patterns of peptide fragments produced being displayed by sodium dodecyl sulfate gel electrophoresis, or alpha-chymotrypsin, the peptides being analyzed after separation by high-voltage electrophoresis and thin-layer chromatography. The comparative structural data obtained from these two different techniques were remarkably similar. From these data, the following points could be made. (i) MOMPs are structurally heterogeneous between members of chlamydial species; the C. psittaci MOMP was clearly distinct from each of the C. trachomatis MOMPs. (ii) Considerable structural homology occurs among MOMPs from different C. trachomatis serotypes; however, distinct differences in the primary structure of each C. trachomatis MOMP were evident. (iii) These observed differences were most obvious in peptide maps of MOMPs isolated from chlamydiae that had been surface labeled by lactoperoxidase-mediated radioiodination. The surface-exposed portions of the MOMPs from serotypes L2 and D were very similar. In contrast, those from serotypes G, H, and I were quite different. These structural data are in agreement with the serospecificities described for these proteins.     

Isolation and characterization of Bartonella bacilliformis from an expatriate Ecuadorian

Carrion's disease is typically biphasic with acute febrile illness characterized by bacteremia and severe hemolytic anemia (Oroya fever), followed by benign, chronic cutaneous lesions (verruga peruana). The causative agent, Bartonella bacilliformis, is endemic in specific regions of Peru and Ecuador. We describe atypical infection in an expatriate patient who presented with acute splenomegaly and anemia 3 years after visiting Ecuador. Initial serology and PCR of the patient's blood and serum were negative for Bartonella henselae, Bartonella quintana, and B. bacilliformis. Histology of splenic biopsy was suggestive of bacillary angiomatosis, but immunohistochemistry ruled out B. henselae and B. quintana. Bacilli (isolate EC-01) were subsequently cultured from the patient's blood and analyzed using multilocus sequence typing, protein gel electrophoresis with Western blotting, and an immunofluorescence assay (IFA) against a panel of sera from patients with Oroya fever in Peru. The EC-01 nucleotide sequences (gltA and internal transcribed spacer) and protein band banding pattern were most similar to a subset of B. bacilliformis isolates from the region of Caraz, Ancash, in Peru, where B. bacilliformis is endemic. By IFA, the patient's serum reacted strongly to two out of the three Peruvian B. bacilliformis isolates tested, and EC-01 antigen reacted with 13/20 Oroya fever sera. Bacilliary angiomatosis-like lesions were also detected in the spleen of the patient, who was inapparently infected with B. bacilliformis and who presumably acquired infection in a region of Ecuador where B. bacilliformis was not thought to be endemic. This study suggests that the range of B. bacilliformis may be expanding from areas of endemicity in Ecuador and that infection may present as atypical clinical disease.     

Bartonella quintana variably expressed outer membrane proteins mediate vascular endothelial growth factor secretion but not host cell adherence

Bartonella quintana causes trench fever, endocarditis, and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis in humans. Little is known about the interaction of this pathogen with host cells. We attempted to elucidate the interaction of B. quintana with human macrophages (THP-1) and epithelial cells (HeLa 229). Remarkably, only B. quintana strain JK-31 induced secretion of vascular endothelial growth factor (VEGF) from THP-1 and HeLa 229 cells upon infection similar to the secretion induced by B. henselae Marseille, whereas other strains (B. quintana 2-D70, B. quintana Toulouse, and B. quintana Munich) did not induce such secretion. Immunofluorescence testing and electron microscopy revealed that the B. quintana strains unable to induce VEGF secretion did not express the variable outer membrane proteins (Vomps) on their surfaces. Surprisingly, the increase in VEGF secretion mediated by B. quintana JK-31 was not paralleled by elevated host cell adherence rates compared with the rates for Vomp-negative B. quintana strains. Our results suggest that the Vomps play a leading role in the angiogenic reprogramming of host cells by B. quintana but not in the adherence to host cells.     

Identification and strain differentiation of Vibrio cholerae by using polyclonal antibodies against outer membrane proteins.

Cholera is caused only by O1 and O139 Vibrio cholerae strains. For diagnosis, 3 working days are needed for bacterial isolation from human feces and for biochemical characterization. Here we describe the purification of bacterial outer membrane proteins (OMP) from V. cholerae O1 Ogawa, O1 Inaba, and O139 strains, as well as the production of specific antisera and their use for fecal Vibrio antigen detection. Anti-OMP antisera showed very high reactivity and specificity by enzyme-linked immunosorbent assay (ELISA) and dot-ELISA. An inmunodiagnostic assay for V. cholerae detection was developed; this assay avoids preenrichment and costly equipment and can be used for epidemiological surveillance and clinical diagnosis of cases, considering that prompt and specific identification of bacteria is mandatory in cholera.     

Disulfide-bonded outer membrane proteins in the genus Legionella.

Staphylococcus aureus has been classified into at least eight different capsular types by using polyclonal rabbit antisera specific for their associated capsular polysaccharides. We produced and characterized monoclonal antibodies reactive with two serologically distinct capsular types, types 5 and 8, which account for more than 70% of all S. aureus bacteremias. These type-specific, monoclonal antibodies reacted with S. aureus clinical isolates possessing the homologous capsular type and exhibited no cross-reactivity against S. aureus clinical isolates possessing the heterologous capsular type, nontypeable S. aureus clinical isolates, Staphylococcus epidermidis clinical isolates, or a variety of gram-negative organisms. The anti-type 8 monoclonal antibodies also reacted with purified capsular polysaccharide derived from the prototype type 8 S. aureus strain.     

Bartonella bacilliformis stimulates endothelial cells in vitro and is angiogenic in vivo.

Bartonellosis, a biphasic disease caused by motile intracellular bacteria, produces in its tissue phase a characteristic dermal eruption (Verruga peruana) resulting from a pronounced endothelial cell proliferation. Bacteria are found in the interstitium and within the cytoplasm of endothelial cells (Rocha-Lima inclusion). The aim of this study was to determine if Bartonella bacilliformis produce a substance(s) that might be responsible for the vascular proliferation seen in the Verruga. This was assessed in an in vitro system using human endothelial cells and measuring proliferation as well as production of tissue type plasminogen activator after exposure to the endothelial cultures to B. bacilliformis extracts. Our results indicate that B. bacilliformis possess an activity that stimulates endothelial cell proliferation up to three times that of control. The factor(s) is specific for endothelial cells, heat sensitive, larger than 12 to 14 kd, not enhanced by heparin, has no affinity for heparin, and is precipitated by 45% ammonium sulfate. In addition, the B. bacilliformis extracts stimulate production of t-PA antigen in a concentration-dependent fashion. This activity is also heat sensitive and not lost after dialysis (12 to 14 kd). B. bacilliformis extracts, however, do not increase the production of plasminogen activator inhibitor. It was also determined that B. bacilliformis extracts stimulate the formation of new blood vessels in an in vivo model for angiogenesis. These results describe a bacterial factor(s) that stimulates two important steps in the development of new blood vessels in vitro, as well as the formation of new blood vessels in vivo. Determining the mechanism of action, combined with a complete characterization of this factor(s), may help in understanding the pathogenesis not only of the Verruga and angiogenesis in general but also the recently described Cat-Scratch-associated epithelioid hemangiomas in patients with AIDS and Kaposi sarcoma.     

Identification and occurrence of Vibrio cholerae flagellar core proteins in isolated outer membrane

Sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of outer membranes from a flagellated and an isogenic nonflagellated strain of Vibrio cholerae (classical, Inaba) suggested that two proteins were absent from the nonflagellated strain. Immunoblot examination of such preparations demonstrated that two proteins, present only in outer membrane from the flagellated strain, were associated with flagella. Analysis of purified flagellar cores from strains CA401 and N16961 (El Tor, Inaba) by electron microscopy, sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis, and immunoblotting showed that these two proteins, with apparent molecular weights of 47,000 and 49,000, composed the flagellar core. Antiserum specific for flagellar core proteins did not agglutinate or inhibit the motility of intact V. cholerae. These latter findings suggested that, for intact cells, the flagellar core proteins are not accessible to antibody.     

Characterization of the outer membrane proteins of Bordetella avium.

The outer membrane proteins of Bordetella avium were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sarkosyl-insoluble outer membrane protein-enriched profiles from 50 virulent B. avium isolates, containing major 21,000- and 37,000-molecular-weight proteins (21K and 37K proteins, respectively) and at least 13 less intensely stained proteins with molecular weights ranging from 13,500 to 143,000, were very similar. The 21K, 27K, 31K, and 37K outer membrane proteins were shown to be associated noncovalently with the underlying peptidoglycan layer. It was necessary to treat cell envelopes with 2% sodium dodecyl sulfate and at temperatures in excess of 60 degrees C for 15 min to release these proteins. Exposure of proteins on the cell surface of B. avium was assessed by labeling with 125I followed by electrophoresis. As many as 13 bands were present in profiles from labeled whole cells. Of the surface-labeled bands, eight corresponded to bands in a radiolabeled outer membrane preparation. The outer membrane protein profile of B. avium was compared with profiles from other Bordetella spp., including 20 B. avium-like and 16 B. bronchiseptica strains isolated from turkeys. The outer membrane protein profile of B. avium was distinctly different from those of the other bordetella. The effect of variations in the growth medium on the expression of outer membrane proteins of B. avium was examined. Expression of 22K, 26K, 56K, and 73K proteins was decreased or eliminated by addition of 50 mM MgSO4 to the medium.