Phase variation in Helicobacter pylori lipopolysaccharide due to changes in the lengths of poly(C) tracts in alpha3-fucosyltransferase genes.

The lipopolysaccharide (LPS) of Helicobacter pylori expresses the Lewis x (Lex) and/or Ley antigen. We have shown previously that H. pylori LPS displays phase variation whereby an Lex-positive strain yields variants with different LPS serotypes, for example, Lex plus Ley or nonfucosylated polylactosamine. H. pylori has two alpha3-fucosyltransferase genes that both contain poly(C) tracts. We now demonstrate that these tracts can shorten or lengthen randomly, which results in reversible frameshifting and inactivation of the gene products. We provide genetic and serological evidence that this mechanism causes H. pylori LPS phase variation and demonstrate that the on or off status of alpha3-fucosyltransferase genes determines the LPS serotypes of phase variants and clinical isolates. The role of the alpha3-fucosyltransferase gene products in determining the LPS serotype was confirmed by structural-chemical analysis of alpha3-fucosyltransferase knockout mutants. The data also show that the two alpha3-fucosyltransferase genes code for enzymes with different fine specificities, and we propose the names futA and futB to designate the orthologs of the H. pylori 26695 alpha3-fucosyltransferase genes HP0379 and HP0651, respectively. The data also show that the alpha3-fucosylation precedes alpha2-fucosylation [corrected], an order of events opposite to that which prevails in mammals. Finally, the data provide an understanding at the molecular level of the mechanisms underlying LPS diversity in H. pylori, which may play an important role in adaptation to the host.

Phase variation in Helicobacter pylori lipopolysaccharide.

Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigen Lewis x (Le(x)) in a polymeric form. Le(x) is beta-D-galactose-(1-4)-[alpha-L-fucose-(1-3)]-beta-D-acetylglucosamine. Schematically the LPS structure is (Le(x))n-core-lipid A. In this report, we show that Le(x) expression is not a stable trait but that LPS displays a high frequency (0.2 to 0.5%) of phase variation, resulting in the presence of several LPS variants in one bacterial cell population. One type of phase variation implied the loss of alpha1,3-linked fucose, resulting in variants that expressed nonsubstituted polylactosamines (also called the i antigen), i.e., Le(x) minus fucose; LPS: (lactosamine)n-core-lipid A. The switch of Le(x) to i antigen was reversible. A second group of variants arose by loss of polymeric main chain which resulted in expression of monomeric Le(y); LPS: (Le(y))-core-lipid A. A third group of variants arose by acquisition of alpha1,2-linked fucose which hence expressed Le(x) plus Le(y); LPS: (Le(y))(Le(x))n-core-lipid A. The second and third group of variants switched back to the parental phenotype [(Le(x))-core-lipid A] in lower frequencies. Part of the variation can be ascribed to altered expression levels of glycosyltransferase levels as assessed by assaying the activities of galactosyl-, fucosyl-, and N-acetylglucosaminyltransferases. Clearly phase variation increases the heterogeneity of H. pylori, and this process may be involved in generating the very closely related yet genetically slightly different strains that have been isolated from one patient.

Mechanisms of resistance to piperacillin of Enterobacter cloacae strains differ by antibiotic prescription policy.

During a study of piperacillin resistance among aerobic Gram-negative bacteria, 18 resistant strains of Enterobacter cloacae were obtained from a General Hospital in Rotterdam and 13 from a University Hospital in Amsterdam. The patterns of antibiotic susceptibilities were different: the Amsterdam strains were generally resistant to penicillins, the third generation cephalosporins and temocillin, whereas the Rotterdam strains were more often sensitive to the third generation cephalosporins and temocillin but more resistant to penicillins. Isoelectric focusing and substrate profiles showed the presence of chromosomal Class 1 beta-lactamase in ten of the Amsterdam strains: in three strains a plasmid mediated TEM-1 enzyme was detected. In contrast 15 of the 18 Rotterdam strains possessed a plasmid mediated beta-lactamase, ten of which were TEM-2. Eight of the ten strains with the TEM-2 enzyme harboured a transferable plasmid coding for resistance to piperacillin. Endonuclease analysis of plasmid DNA from these eight strains revealed an identical pattern in seven strains. Different selective pressures were operative in each hospital. In Amsterdam the general use of cefotaxim and piperacillin favoured emergence of strains with derepressed chromosomal Class 1 beta-lactamase, whereas in Rotterdam the use of cefuroxime favoured the spread of a plasmid, encoding TEM-2 beta-lactamase.

Typing of Helicobacter pylori with monoclonal antibodies against Lewis antigens in lipopolysaccharide.

Recently, it has been shown that the lipopolysaccharide (LPS) O antigen of Helicobacter pylori contains Lewis x (Lex), Lewis y (Ley), or both Lex and Ley antigens. We applied a serotyping method for H. pylori by an enzyme-linked immunosorbent assay with monoclonal antibodies (MAbs) specific for these antigens and the related fucosylated H type 1 (H1) antigen. The selected MAbs recognized the Lex and/or Ley structures in the LPS of H. pylori. The agreement between the results of biochemical compositional analysis and the serological data validated our serotyping system. A total of 152 strains from different geographic origins (The Netherlands, Canada, Poland, Italy, and People's Republic of China) were examined for typeability based on the presence of Lewis antigens. One hundred twenty-nine (84.9%) strains were typeable, and 12 different serotyping patterns were observed; 80.9% of the strains contained Lex and/or Le(y) antigens, and 18.4% reacted with the MAb against the related H1 antigen either alone or in combination with the Lex and/or Ley antigen. Our results show that the Lex and Ley antigens are frequently encountered in the LPS of H. pylori strains from various geographic origins. This typing method is an easy-to-perform technique, which can be used for strain differentiation in epidemiological studies of H. pylori infections.

Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity.

Helicobacter pylori is involved in gastritis, gastric and duodenal ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. Earlier studies already suggested a role for autoimmune phenomena in H. pylori-linked disease. We now report that lipopolysaccharides (LPS) of H. pylori express Lewis y, Lewis x, and H type I blood group structures similar to those commonly occurring in gastric mucosa. Immunization of mice and rabbits with H. pylori cells or purified LPS induced an anti-Lewis x or y or anti-H type I response, yielding antibodies that bound human and murine gastric glandular tissue, granulocytes, adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma cells. Experimental oral infections in mice or natural infection in humans yielded anti-Lewis antibodies also. The beta chain of gastric (H+,K+)-ATPase, the parietal cell proton pump involved in acid secretion, contained Lewis y epitopes; gastric mucin contained Lewis x and y antigenic determinants. Growth in mice of a hybridoma that secretes H. pylori-induced anti-Lewis y monoclonal antibodies resulted in histopathological evidence of gastritis, which indicates a direct pathogenic role for anti-Lewis antibodies. In conclusion, our observations demonstrate that molecular mimicry between H. pylori LPS and the host, based on Lewis antigens, and provide understanding of an autoimmune mechanism for H. pylori-associated type B gastritis.

In vitro susceptibility of Helicobacter pylori to several antimicrobial combinations.

Synergy between metronidazole and its hydroxymetabolite and between each compound and amoxicillin or tetracycline-HCl was determined against Helicobacter pylori. Metronidazole plus its hydroxymetabolite and either compound combined with amoxicillin showed synergism against all 10 strains of H. pylori tested. Metronidazole plus tetracycline-HCl or the hydroxymetabolite plus tetracycline-HCl acted synergistically against seven and six strains, respectively, acted additively against three strains, and had no additional effect against one strain. These results may help to explain the in vivo efficacies of metronidazole combinations in the treatment of H. pylori-associated gastritis.