Richard Sherburne

Molecular genetic basis for the variable expression of Lewis Y antigen in Helicobacter pylori: analysis of the alpha (1,2) fucosyltransferase gene.

Helicobacter pylori lipopolysaccharides (LPS) express human oncofetal antigens Lewis X and Lewis Y. The synthesis of Lewis Y involves the actions of α(1,3) and α(1,2) fucosyltransferases (FucTs). Here, we report the molecular cloning and characterization of genes encoding H. pyloriα(1,2) FucT (Hp fucT2 ) from various H. pylori strains. We constructed Hp fucT2 knock‐out mutants and demonstrated the loss of Lewis Y production in these mutants by enzyme‐linked immunosorbent assay (ELISA) and immunoelectron microscopy. The Hp fucT2 gene contains a hypermutable sequence [poly (C) and TAA repeats], which provides a possibility of frequent shifting into and out of coding frame by a polymerase slippage mechanism. Thus, the Hp fucT2 gene displays two major genotypes, consisting of either a single full‐length open reading frame (ORF; as in the strain UA802) or truncated ORFs (as in the strain 26695). In vitro expression of Hp fucT2 genes demonstrated that both types of the gene have the potential to produce the full‐length protein. The production of the full‐length protein by the 26695 fucT2 gene could be attributed to translational −1 frameshifting, as a perfect translation frameshift cassette resembling that of the Escherichia coli dnaX gene is present. Examination of the strain UA1174 revealed that its fucT2 gene has a frameshifted ORF at the DNA level, which cannot be compensated by translation frameshifting, accounting for its Lewis Y off phenotype. In another strain, UA1218, the fucT2 gene is apparently turned off because of the loss of its promoter. Based on these data, we proposed a model for the variable expression of Lewis Y by H. pylori, in which regulation at the level of replication slippage (mutation), transcription and translation of the fucT2 gene may all be involved.

Lack of correlation between Lewis antigen expression by Helicobacter pylori and gastric epithelial cells in infected patients

BACKGROUND & AIMS Lewis antigens are expressed by both human gastric epithelial tissue and Helicobacter pylori. We examined Lewis antigens expressed by gastric epithelium and by H. pylori isolated from the corresponding biopsy tissue. METHODS H. pylori Lewis expression was determined by enzyme immunoassays, and immunoelectron microscopy was used to confirm the Lewis antigens on some H. pylori cells and in some biopsy specimens. Histopathology using identical monoclonal antibodies specific for Lewis A, B, X, and Y antigens was used to detect these antigens in 24 gastric biopsy specimens. RESULTS We identified Lewis Y in 100%, Lewis X and Lewis B in 95.8%, and Lewis A in 87.5% of biopsy specimens. In H. pylori, 87.5% expressed Lewis Y, 79.2% Lewis X, and 4.2% (one strain) Lewis B. No Lewis A was detected. Antibody specific for Lewis X labeled the bacteria and associated adhesion pedestal. The cagA gene was present in 92% of strains. CONCLUSIONS There was no direct relationship between Lewis antigen expression by H. pylori and gastric epithelial cells in infected patients. Expression of Lewis X and Lewis Y by H. pylori suggests the possibility of their requirement for establishment and/or maintenance of infection. An immunoelectron micrograph of H. pylori interaction with the gastric epithelial adhesion pedestal suggests a tentative role for Lewis X in the adhesion process.

The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F‐like plasmids

The effects of defined mutations In the lipopolysaccharide (LPS) and the outer membrane protein OmpA of the recipient cell on mating‐pair formation in liquid media by the transfer systems of the F‐Iike plasmids pOX38 (F), ColB2 and R100‐1 were investigated. Transfer of all three plasmids was affected differently by mutations in the rfa (LPS) locus of the recipient cell, the F plasmid being most sensitive to mutations that affected rfaP gene expression which is responslbie for the addition of pyrophosphorylethanolamine (PPEA) to heptose I of the inner core of the LPS. CoIB2 transfer was more strongly affected by mutations in the heptose II‐heptose III region of the LPS (rfaF) whereas R100‐1 was not strongly affected by any of the rfa mutations tested. ompA but not rfa mutations further decreased the mating efficiency of an F plasmid carrying a mutation in the mating‐pair stabilization protein TraN. An F derivative with a chloramphenicol acetyltransferase (CAT) cassette interrupting the traA pilin gene was constructed and pilin genes from F‐like plasmids (F, ColB2, R100‐1) were used to complement this mutation. Unexpectediy, the results suggested that the differences in the pilin sequences were not responsible for recognizing specific groups in the LPS, OmpA or the TraT surface exclusion protein. Other corroborating evidence is presented suggesting the presence of an adhesin at the F pilus tip.

Structure and location of tellurium deposited in Escherichia coli cells harboring tellurite resistance plasmids

The plasmids RP4Ter and pHH1508a, which belong to the P and HII incompatibility groups, respectively, confer resistance to potassium tellurite (K2TeO3) on Escherichia coli. The genes for tellurite resistance were cloned from each plasmid onto the vector pUC8 to create pDT1366 and pD1364, respectively. Unstained, unfixed bacteria carrying these plasmids contained black intracellular deposits when grown on media containing tellurite. Thin sections of these bacteria fixed with glutaraldehyde were prepared and examined by electron microscopy. The black deposits were located inside the cell and were frequently associated with the inner membrane of the bacterium. Bacteria containing pDT1366 or pDT1364, and therefore a higher gene dosage of the Ter determinant, contained more black deposits, but had a decreased resistance, as measured by the minimum inhibitory concentration using the agar dilution method. Using the technique of electron spectroscopic imaging, the black intracellular deposits were shown to contain predominantly reduced metallic tellurium, and significant amounts of oxygen or carbon, thereby confirming earlier results using X-ray diffraction analysis of whole cells.

Binding and invasion of HeLa and MRC-5 cells by Streptococcus agalactiae

The interactions of group B streptococci (GBS) with HeLa cells (an epithelial cell line) and MRC-5 cells (a fibroblastic cell line) were explored. A host-cell invasion assay using GBS strains from all serotypes revealed that GBS invaded HeLa cells to a greater extent than MRC-5 cells. One strain, a serotype V (NCS13), was highly invasive against HeLa cells. All strains were poorly invasive against MRC-5 cells. Further characterization of the binding of NCS13 to HeLa and MRC-5 cell surfaces showed that the lack of recoverable c.f.u. from MRC-5 cells was due to a lack of binding of NCS13 to the MRC-5 cell surface in comparison to HeLa cells. Although fibronectin had been reported to bind to GBS, fibronectin assays showed 2.7-fold more fibronectin on the MRC-5 cell surface in comparison to HeLa cells, suggesting that other extracellular matrix proteins besides fibronectin may be involved in GBS binding. Scanning electron microscopy of NCS13 and HeLa cells over a 6 h time period showed increased numbers of NCS13 on the HeLa cell surface over time until cell death at 6 h. Direct contact of the HeLa cell surface by NCS13 was found to be necessary for cell death to occur. Further scanning electron microscopy studies found that, once GBS are bound to the HeLa cell surface, HeLa cell microvilli entwine the bacteria, which then enter the HeLa cell in a polar fashion. Cytoskeletal actin is involved, as this process is disrupted by cytochalasin D, and recruitment of actin is visible at the site of adherent chains of GBS. Also, the host-cell signalling enzyme, PI 3-kinase, is involved in the GBS internalization process, since the PI 3-kinase inhibitor, wortmannin, inhibited NCS13 invasion of HeLa cells in a dose-dependent manner.

Characterization of an immunodominant Giardia lamblia protein antigen related to alpha giardin

The trophozoites ofGiardia lamblia possess several protein antigens, predominant among them a protein of ∼32,000 Da. In the present study, we used monospecific antibodies that recognize this protein to demonstrate its presence on a variety ofG. lamblia isolates from human and animal sources. Immune electron microscopy was used to localize 32-kDa antigen on the trophozoite membrane and disk. Immunofluorescent assays employing monospecific antibodies confirmed the presence of 32-kDa antigen on the membrane and disk and its absence on flagella or nuclei. The N-terminal 17 amino acids of the 32-kDa antigen are identical to α-1-giardin, a protein component of microribbons on the ventral disk. These results suggest that the 32-kDa immunodominant trophozoite antigen is α-1-giardin.

Antigenic Mimicry Between Helicobacter pylori and Gastric Mucosa: Failure to Implicate Heat-Shock Protein Hsp60 Using Immunoelectron Microscopy

Background. Helicobacter pylori infection induces autoantibodies that cross‐react with human gastric mucosa from infected individuals. Candidates for the antigens responsible for molecular mimicry causing autoreactivity include the heat‐shock protein HspB (Hsp60, sometimes called Hsp54) or Lewis x and Lewis y carbohydrate antigens.

Bacteriophages for incompatibility group H plasmids: Morphological and growth characteristics

Two independently isolated temperature-sensitive bacteriophage that are specific for enterobacterial hosts harboring HI and HII plasmids were characterized to determine if any identifiable differences existed between them. The traits examined included adsorption pattern of phage to H pili, bacteriophage size, sensitivity to chloroform, RNA strandedness, reaction with F-specific antiphage serum, virion protein pattern, temperature range of lytic ability, and plaque morphology. No differences between the phages were observed for any of the features analyzed. Ecological questions on the origin and maintenance of temperature-sensitive phages are discussed.

Molecular organization and expression of the tetracycline resistance (TetB1) determinant specified by IncHI1 plasmids

R27, a plasmid of the HI1 incompatibility group, carries a tetracycline resistance (TcR) determinant previously designated class B1. A 4.5 kbHindIII fragment of R27 specifying TcR was cloned into pACYC177 and the recombinant plasmid designated pDT1539. The minimal inhibitory concentration (MIC) of tetracycline for R27 and pDT1539 was 64 μ/ml, whereas the MIC for the TetB determinant from Tn10 was 128 μg/ml. The restriction maps of the TetB and TetB1 determinants appeared identical, and DNA-DNA heteroduplex analysis of the 4.5 kbHindIII fragment from R27 and pRT11 derived from Tn10, which contained thetetR andtetB genes, was consistent with extended homology between the two DNA molecules. The TetB and TetB1 polypeptides produced in anEscherichia coli in vitro transcription/translation system had the same apparent molecular size of 36 kilodaltons. Thus, apart from the minor variation in MIC levels, there appeared to be little difference between the TetB and TetB1 determinants. DNA homology was demonstrated between a TetB1 probe and ten other incompatibility group HI1 plasmids from various geographic sources. Moreover, all specified a level of tetracycline resistance similar to that of R27. These data are consistent with clonal spread of a progenitor IncHI1 plasmid carrying the TetB1 determinant.

Characterization of maxi- and mini-derivatives of the IncHI1 plasmid R27

R27, an IncHI1 plasmid of 182 kilobases (kb), which was originally isolated fromSalmonella typhimurium, was found to contain two copies of IS1 in direct orientation. Deletion derivatives constructed in vitro were of two types: a maxi-derivative of 110 kb (pDT1047) with a single complete copy of IS1, and mini-derivatives of 5–6 kb which contained less than a complete copy of IS1. The IncHI1 miniplasmids also contained a portion of the tetracycline resistance determinant from R27 and a replication region related to the RepFIA replicon. Electron microscopic homoduplex studies demonstrated the presence of two other inverted repeat sequences within the miniplasmid that were unrelated to IS1.