| 1. |
- Janson, Håkan, et al.
(författare)
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Limited diversity of the protein D gene (hpd) among encapsulated and nonencapsulated Haemophilus influenzae strains
- 1993
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Ingår i: Infection and Immunity. - 1098-5522. ; 61:11, s. 4546-4552
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Tidskriftsartikel (refereegranskat)abstract
- Protein D is a surface-exposed lipoprotein of the gram-negative bacterium Haemophilus influenzae with affinity for human immunoglobulin D myeloma protein. The gene encoding protein D (hpd) in a serotype b strain of H. influenzae was cloned. Escherichia coli carrying the hpd gene bound human myeloma immunoglobulin D. Nucleotide sequence analysis identified an 1,092-bp open reading frame that was more than 99% identical to the hpd gene from a nontypeable H. influenzae strain. In the deduced amino acid sequences for protein D, only 2 of 364 amino acid residues differed. The restriction fragment length polymorphism of the hpd region in different strains was analyzed by Southern blot analyses of PstI- or EcoRI-digested genomic DNA from 100 H. influenzae strains. The analysis was performed by using isolated fragments of the cloned hpd gene, originating from the nontypeable H. influenzae 772, as probes. All strains tested had DNA sequences with a high degree of homology to the hpd probes. The analysis also showed that restriction endonuclease sites within the gene were more conserved than sites adjacent to the hpd gene. An interesting difference between type b strains and unencapsulated strains was observed. The majority of type b strains seem to have a 1.4-kbp DNA fragment upstream of the hpd gene that is absent in nontypeable strains. On the basis of the high degree of conservation of the hpd gene among H. influenzae strains, we conclude that protein D is a possible vaccine candidate.
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| 2. |
- Janson, Håkan, et al.
(författare)
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Protein D, an immunoglobulin D-binding protein of Haemophilus influenzae: cloning, nucleotide sequence, and expression in Escherichia coli
- 1991
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Ingår i: Infection and Immunity. - 1098-5522. ; 59:1, s. 119-125
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Tidskriftsartikel (refereegranskat)abstract
- The gene for protein D, a membrane-associated protein with specific affinity for human immunoglobulin D, was cloned from a nontypeable strain of Haemophilus influenzae. The gene was expressed in Escherichia coli from an endogenous promoter, and the gene product has an apparent molecular weight equal to that of H. influenzae protein D (42,000). The complete nucleotide sequence of the gene for protein D was determined, and the deduced amino acid sequence of 364 residues includes a putative signal sequence of 18 amino acids containing a consensus sequence, Leu-Ala-Gly-Cys, for bacterial lipoproteins. The sequence of protein D shows no similarity to those of other immunoglobulin-binding proteins. Protein D is the first example of immunoglobulin receptors from gram-negative bacteria that has been cloned and sequenced.
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| 3. |
- Janson, Håkan, et al.
(författare)
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Protein D, the glycerophosphodiester phosphodiesterase from Haemophilus influenzae with affinity for human immunoglobulin D, influences virulence in a rat otitis model
- 1994
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 62:11, s. 4848-4854
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Tidskriftsartikel (refereegranskat)abstract
- A mutant lacking the ability to express the surface-exposed lipoprotein protein D was constructed by linker insertion and deletion mutagenesis of a cloned DNA insert containing the protein D structural gene from a nontypeable Haemophilus influenzae strain (NTHi). An isogenic NTHi mutant was isolated after transformation of genetically competent bacteria. The transformant was unreactive to a protein D-specific monoclonal antibody in a colony immunoassay. In addition, the mutant lacked the ability to synthesize detectable levels of protein D by protein staining, immunoblot methods, glycerophosphodiester phosphodiesterase activity, and binding studies of radiolabelled immunoglobulin D. The isogenic protein D-deficient mutant was compared with its parental strain for its ability to induce experimental otitis media in rats challenged with bacteria. An approximately 100-times-higher concentration of the mutant compared with that of the wild-type strain was required in order to cause otitis among all rats challenged with that given dose. The protein D mutant exhibited a generation time that was equal to that of the wild-type strain in complex broth medium. No difference in lipopolysaccharide expression was found between the mutant and the parental strain. These results suggest that protein D may influence the pathogenesis of NTHi in the upper respiratory tract.
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| 4. |
- Janson, Håkan, et al.
(författare)
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Protein D, the immunoglobulin D-binding protein of Haemophilus influenzae, is a lipoprotein
- 1992
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Ingår i: Infection and Immunity. - 1098-5522. ; 60:4, s. 1336-1342
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Tidskriftsartikel (refereegranskat)abstract
- Protein D is an immunoglobulin D-binding membrane protein exposed on the surface of the gram-negative bacterium Haemophilus influenzae. Results reported here indicate that protein D is a lipoprotein. The protein is apparently synthesized as a precursor with an 18-residue-long signal sequence modified by the covalent attachment of both ester-linked and amide-linked palmitate to the cysteine residue, which becomes the amino terminus after cleavage of the signal sequence. Globomycin inhibited maturation of protein D in H. influenzae, implying that protein D is exported through the lipoprotein export pathway. A mutant expressing a protein D lacking the cysteine residue was constructed by oligonucleotide site-directed mutagenesis. The mutated protein D molecule was not acylated and partitioned in the aqueous phase after Triton X-114 extraction of intact bacteria, unlike native and recombinant protein D, which partitioned in the detergent phase. The nonacylated protein D molecule was localized to the periplasmic space of Escherichia coli. The hydrophilic protein D molecule will be used in investigations concerning its ability to function as a vaccine component.
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| 5. |
- Jonsson, Maria, et al.
(författare)
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Heterogeneity of outer membrane proteins in Borrelia burgdorferi : comparison of osp operons of three isolates of different geographic origins.
- 1992
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 60:5, s. 1845-53
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Tidskriftsartikel (refereegranskat)abstract
- Biochemical and immunochemical studies of the outer membrane proteins of Borrelia burgdorferi have shown that the OspA and OspB proteins from strains of different geographic origins may differ considerably in their reactivities with monoclonal antibodies and in their apparent molecular weights. To further characterize this variation in Osp proteins between strains, the osp operons and deduced translation products from two strains, one from Sweden (ACAI) and one from eastern Russia (Ip90), were studied. Polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses confirmed differences between ACAI, Ip90, and the North American strain B31 in their Osp proteins. The sequences of the ospA and ospB genes of ACAI and Ip90 were compared with that of the previously studied osp operon of B31 (S. Bergström, V. G. Bundoc, and A. G. Barbour, Mol. Microbiol. 3:479-486, 1989). The osp genes of ACAI and Ip90, like the corresponding genes of B31, were found on plasmids with apparent sizes of about 50 kb and are cotranscribed as a single unit. Pairwise comparisons of the nucleotide sequences revealed that the ospA genes of ACAI and Ip90 were 85 and 86% identical, respectively, to the ospA gene of strain B31 and 86% identical to each other. The ospB sequences of these two strains were 79% identical to the ospB gene of B31 and 81% identical to each other. There was significantly greater similarity between the ospA genes of the three different strains than there was between the ospA and ospB genes within each strain. These studies suggest that the duplication of osp genes in B. burgdorferi occurred before the geographical dispersion of strains represented by ACAI, Ip90, and B31.
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| 6. |
- Katouli, Mohammad, et al.
(författare)
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Composition and diversity of intestinal coliform flora influence bacterial translocation in rats after hemorrhagic stress
- 1994
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 2:11, s. 4768-4774
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Tidskriftsartikel (refereegranskat)abstract
- Coliform bacteria are the most frequently reported bacteria to translocate afterhemorrhage. We investigated the correlation between composition and diversity of the cecal coliform flora and the degree of translocation in a rat model of hemorrhagic stress. Two groups of nine rats each were bled to 60 and 50 mm Hg mean arterial blood pressure, respectively. A sham-operated group without bleeding (n = 9) and a noninstrumented group (n = 6) served as controls. From each rat, 40 coliform isolates from the cecum and up to 16 from positive mesenteric lymph node (MLN) cultures were tested with an automated biochemical fingerprinting method. The phenotypic diversity of coliforms in each cecal sample was calculated as Simpson's diversity index (DI), and similarities between bacterial types in different samples were calculated as population similarity coefficients. Three rats in the sham-operated group and seven in each of the bled groups showed bacterial translocation. Of the different biochemical phenotypes (BPTs) found in the cecum of bled rats (mean, 6.5 BPTs), only a few were detected in MLNs (mean, 1.9 BPTs per MLN), with Escherichia coli being the dominant species. The translocating E. coli strains were mainly of two BPTs. Rats showing no translocation either did not carry these strains or had a high diversity of coliforms in the cecum. Furthermore, translocation of these coliform types was independent of their proportion in the cecum. In bled rats, the diversityof coliforms (mean DI, 0.53) was significantly higher than that in control groups (mean DI, 0.30; P = 0.004), suggesting that hemorrhage stimulates an increase in diversity of cecal coliforms. Rats with similar coliform flora and subjected to the same treatment showed similar patterns of translocation. Our results suggest that the composition of the coliformflora is an important factor in translocation and that certain coliform strains have the ability to translocate and survive in MLNs more easily than others.
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| 7. |
- Majeed, Meytham, et al.
(författare)
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Roles of Ca2+ and F-actin in intracellular aggregation of Chlamydia trachomatis in eucaryotic cells
- 1993
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 61:4, s. 1406-1414
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Tidskriftsartikel (refereegranskat)abstract
- The effect of intracellular free Ca2+ ([Ca2+]i) on the intracellular aggregation of Chlamydia trachomatis serovars L2 and E in McCoy and HeLa cells is investigated. Loading the cells with the Ca2+ chelator MAPT/AM (1,2-bis-5-methyl-amino-phenoxylethane-N,N-n'-tetra-acetoxymethyl acetate), thereby decreasing the [Ca2+]i from 67 to 19 nM, decreased the number of cells with a local aggregation of chlamydiae in a dose-dependent manner. Neither the attachment nor the uptake of elementary bodies (EBs) was, however, affected after depletion of Ca2+ from the cells. There was no significant difference in the level of measured [Ca2+]i between infected and uninfected cells. Reducing the [Ca2+]i also significantly inhibited chlamydial inclusion formation. Differences in the organization of the actin filament network were observed in response to [Ca2+]i depletion. In Ca(2+)-depleted cells, where few EB aggregates were formed, few local accumulations of F-actin were observed in the cytosol. These results suggest that the aggregation of EBs in eucaryotic cells requires a normal homeostasis of intracellular Ca2+. By affecting F-actin reorganization and putatively certain Ca(2+)-binding proteins, [Ca2+]i plays a vital role in the infectious process of chlamydiae.
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| 8. |
- Sadziene, Ariadna, et al.
(författare)
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A bactericidal antibody to Borrelia burgdorferi is directed against a variable region of the OspB protein
- 1994
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Ingår i: Infection and Immunity. - : American Society for Microbiology (ASM). - 0019-9567 .- 1098-5522. ; 62:5, s. 2037-2045
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Tidskriftsartikel (refereegranskat)abstract
- Borrelia burgdorferi, an agent of Lyme disease, is killed by some monoclonal antibodies in the absence of complement or phagocytes. In the present study, the bactericidal action of monoclonal antibodies against B. burgdorferi and B. hermsii, a cause of relapsing fever, was further characterized. H6831, an antibody recognizing the OspB proteins of some B. burgdorferi strains, and H4825, an antibody specific for one serotype of B. hermsii, were purified, and Fab fragments of the antibodies were prepared. In time-kill studies, more than 99.9% of strain B31 B. burgdorferi cells were killed after 30 min of exposure to H6831 Fab fragments. The MBC of the Fab fragments was 10 micrograms/ml. Electron microscopy revealed that the bactericidal Fab fragments produced numerous blebs and cell lysis of the borrelias for which they were specific. To identify the epitope for H6831, the OspB sequences of H6831-susceptible and -resistant strains and mutants were determined. The deduced OspB proteins of all H6831-resistant strains and mutants differed from the strain B31 OspB at residue 253. Murine antisera raised against a 21-mer synthetic peptide representing the region around residue 253 were specific for strain B31 by Western blot (immunoblot) and growth inhibition assays. Furthermore, the antipeptide serum inhibited the binding of H6831 to whole borrelias. These findings indicated that the linear component of the bactericidal antibody's epitope was located at or near residue 253.
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