Helicobacter pylori outer membrane proteins and gastroduodenal disease.

BACKGROUND AND AIMS: A number of Helicobacter pylori outer membrane proteins (OMPs) undergo phase variations. This study examined the relation between OMP phase variations and clinical outcome. METHODS: Expression of H pylori BabA, BabB, SabA, and OipA proteins was determined by immunoblot. Multiple regression analysis was performed to determine the relation among OMP expression, clinical outcome, and mucosal histology. RESULTS: H pylori were cultured from 200 patients (80 with gastritis, 80 with duodenal ulcer (DU), and 40 with gastric cancer). The most reliable results were obtained using cultures from single colonies of low passage number. Stability of expression with passage varied with OipA > BabA > BabB > SabA. OipA positive status was significantly associated with the presence of DU and gastric cancer, high H pylori density, and severe neutrophil infiltration. SabA positive status was associated with gastric cancer, intestinal metaplasia, and corpus atrophy, and negatively associated with DU and neutrophil infiltration. The Sydney system underestimated the prevalence of intestinal metaplasia/atrophy compared with systems using proximal and distal corpus biopsies. SabA expression dramatically decreased following exposure of H pylori to pH 5.0 for two hours. CONCLUSIONS: SabA expression frequently switched on or off, suggesting that SabA expression can rapidly respond to changing conditions in the stomach or in different regions of the stomach. SabA positive status was inversely related to the ability of the stomach to secrete acid, suggesting that its expression may be regulated by changes in acid secretion and/or in antigens expressed by the atrophic mucosa.

Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging.

The bacterium Helicobacter pylori is the causative agent for peptic ulcer disease. Bacterial adherence to the human gastric epithelial lining is mediated by the fucosylated Lewis b (Leb) histo-blood group antigen. The Leb-binding adhesin, BabA, was purified by receptor activity-directed affinity tagging. The bacterial Leb-binding phenotype was associated with the presence of the cag pathogenicity island among clinical isolates of H. pylori. A vaccine strategy based on the BabA adhesin might serve as a means to target the virulent type I strains of H. pylori.

Expression of two csg operons is required for production of fibronectin- and congo red-binding curli polymers in Escherichia coli K-12.

Two divergently transcribed operons in Escherichia coli required for the expression of fibronectin- and Congo red-binding curli polymers were identified and characterized by transposon mutagenesis, sequencing and transcriptional analyses, as well as for their ability to produce the curli subunit protein. The csgBA operon encodes CsgA, the major subunit protein of the fibre, and CsgB, a protein with sequence homology to CsgA. A non-polar csgB mutant is unaffected in its production of CsgA, but the subunit protein is not assembled into insoluble fibre polymers. A third open reading frame, orfC, positioned downstream of csgA may affect some functional property of curli since an insertion in this putative gene abolishes the autoagglutinating ability typical of curliated cells without affecting the production of the fibre. The promoter for the oppositely transcribed csgDEFG operon was identified by primer extension and shown, like the csgBA promoter, to be dependent upon the alternate stationary phase-specific sigma factor sigma s in wild-type cells, but not in mutants lacking the nucleoid associated protein H-NS. Insertions in csgD abolish completely trancription from the csgBA promoter. Therefore, any regulatory effect on the csgBA promoter might be secondary to events controlling the csgDEFG promoter and/or activation of CsgD. Insertions in csgE, csgF and csgG abolish curli formation but allow CsgA expression suggesting that one or more of these gene products are involved in secretion/assembly of the CsgA subunit protein. No amino acid sequence homologies were found between the CsgE, CsgF and CsgG proteins and secretion/assembly proteins for other known bacterial fibres, suggesting that the formation of curli follows a novel pathway.

Sigma S-dependent growth-phase induction of the csgBA promoter in Escherichia coli can be achieved in vivo by sigma 70 in the absence of the nucleoid-associated protein H-NS.

The stationary-phase-specific sigma factor sigma S (RpoS/KatF) is required for Escherichia coli to induce expression of fibronectin-binding curli organelles upon reaching stationary phase. We show that the csgA gene which encodes the curlin subunit protein belongs to a dicistronic operon, csgBA. The transcriptional start site of csgBA was determined and an AT-rich up-stream activating sequence (UAS) required for transcriptional activation was identified. The pcsgBA promoter is not specific for sigma S since the same promoter sequence can be used by E sigma 70 in vivo in a strain lacking nucleoid-associated protein H-NS and sigma S. Transcription remained growth-phase induced and dependent upon the UAS in such a double mutant. Furthermore, we demonstrate that an additional operon, hdeAB, which is also dependent upon sigma S for transcription, can be transcribed by E sigma 70 in vivo in the absence of H-NS by utilizing the phdeAB promoter. Two other genes known to be under the control of sigma S for expression, bolA and katE, remained transcriptionally silent in the absence of H-NS. It is suggested that a subset of E. coli promoters can be recognized by both E sigma S and E sigma 70 in vivo but H-NS interacting with these sequences prevents formation of successful transcription-initiation complexes with E sigma 70.

Environmental regulation of curli production in Escherichia coli.

Curli are novel surface organelles on E. coli that mediate binding to soluble matrix proteins. The expression of curli is affected by environmental factors, such as temperature, osmolarity, and growth conditions. Curli formation is regulated at the level of transcription, in that the csgA gene can be transcriptionally activated by the cytosolic Crl protein or transcriptionally relieved by a mutation in hns. The expression of curli is also dependent on functional RpoS. E. coli--expressing curli bind to human skin tissue, provided they are precoated with soluble fibronectin, suggesting that curli may act as a colonization factor in host-microbe interactions. Fibronectin is a multifunctional extracellular matrix and plasma protein involved in cell adhesion and cell spreading. It also interacts with a variety of microorganisms, and thus the role of fibronectin in mediating binding of curliated E. coli is of great interest. An investigation of the epitopes of both the fibronectin molecule and the curlin subunit protein involved in the binding of E. coli to tissue will give us more insight into the initial colonization of host surfaces by bacteria.

The RpoS sigma factor relieves H-NS-mediated transcriptional repression of csgA, the subunit gene of fibronectin-binding curli in Escherichia coli.

Curli encoded by the curlin subunit gene, csgA, are fibronectin- and laminin-binding fibres expressed by many natural Escherichia coli and E. coli K-12 strains in response to low temperature, low osmolarity and stationary-phase growth conditions. Curli expression is dependent on RpoS, a sigma factor that controls many stationary phase-inducible genes. Many commonly used K-12 strains carry an amber mutation in rpoS. Strains able to form curli carry an amber suppressor whereas curli-negative E. coli K-12 strains, in general, are sup0. Introduction of SupD, SupE, or supF suppressors into sup0 strains resulted in expression of temperature-regulated curli. In curli-deficient, RpoS- E. coli K-12 strains, csgA is transcriptionally activated by mutations in hns, which encodes the histone-like protein H-NS. Curli expression, fibronectin binding, and csgA transcription remain temperature- and osmoregulated in such double mutants. Our data suggest that RpoS+ strains, and hence curli-proficient strains of E. coli K-12, are relieved for the transcriptional repression mediated by the H-NS protein upon accumulating RpoS as cells reach stationary phase.

The Crl protein activates cryptic genes for curli formation and fibronectin binding in Escherichia coli HB101.

Curli are thin, coiled, temperature-regulated fibres on fibronectin-binding Escherichia coli. The subunit protein of curli was highly homologous at its amino terminus to SEF-17, the subunit protein of thin, aggregative fimbriae of Salmonella enteritidis 27655 strain 3b, suggesting that these fibres form a novel class of surface organelles on enterobacteria. E. coli HB101 is non-curliated and unable to bind soluble, iodinated fibronectin. The phenotypically cryptic curlin subunit gene, csgA, in HB101 is transcriptionally activated by expressing the cytoplasmic Crl on a multicopy plasmid. Transcriptional activation of csgA by Crl was observed after growth at 26 degrees C but not at 37 degrees C, even though crl transcription was not thermoregulated. A deletion of the 39 carboxy-terminal residues abolished Crl activity, whereas a deletion of 10 residues at the C-terminus did not, implying that a region between residue 93 and 122 in the 132-amino-acid-residue large Crl protein is required for activating curli expression in E. coli HB101. crl is a normal housekeeping gene in E. coli and it is suggested that its gene product may either be a DNA-binding protein affecting chromatin structure as has been suggested for histone-like protein H1 or interact with specific regulatory protein(s) controlling transcription of genes required for curli formation and fibronectin binding.

The impact of consonant clusters on preschool children's phonemic awareness: a comparison between readers and nonreaders.

Preschool children's ability to segment and blend real words and nonsense words, with and without consonant clusters was investigated in two experiments. In the first experiment, preschool children's ability to segment real words into phonemes was examined. Readers performed better than nonreaders on a phoneme counting task and words containing consonant clusters were harder to segment compared to words without consonant clusters. In experiment two, the ability to segment and blend nonsense words was investigated. Nonreaders had significantly more difficulty with nonsense words compared to readers in both a phoneme synthesis and a phoneme analysis task. A two-way interaction between reading level and word type showed that nonsense words containing consonant clusters were particularly difficult for nonreaders. The results were discussed in relation to theories suggesting that syllables consist of an onset and a rime.