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.

Adherence by Helicobacter pylori increases the risk of gastric disease. Here, we report that more than 95% of strains that bind fucosylated blood group antigen bind A, B, and O antigens (generalists), whereas 60% of adherent South American Amerindian strains bind blood group O antigens best (specialists). This specialization coincides with the unique predominance of blood group O in these Amerindians. Strains differed about 1500-fold in binding affinities, and diversifying selection was evident in babA sequences. We propose that cycles of selection for increased and decreased bacterial adherence contribute to babA diversity and that these cycles have led to gradual replacement of generalist binding by specialist binding in blood group O-dominant human populations.

Helicobacter pylori toxin, VacA, damages the gastric epithelium by erosion and loosening of tight junctions. Here we report that VacA also interferes with T cell activation by two different mechanisms. Formation of anion-specific channels by VacA prevents calcium influx from the extracellular milieu. The transcription factor NF-AT thus fails to translocate to the nucleus and activate key cytokine genes. A second, channel-independent mechanism involves activation of intracellular signaling through the mitogen-activated protein kinases MKK3/6 and p38 and the Rac-specific nucleotide exchange factor, Vav. As a consequence of aberrant Rac activation, disordered actin polymerization is stimulated. The resulting defects in T cell activation may help H. pylori to prevent an effective immune response leading to chronic colonization of its gastric niche.

Four G adhesins, cloned from uropathogenic Escherichia coli strains, were examined for binding to glycolipids and various eukaryotic cells. PapGAD110 and PapGIA2 showed virtually identical binding patterns to Gal alpha 1‐4Gal‐containing glycolipids, while PapGJ96 differed slightly and PrsGJ96 markedly with respect to the effect of neighbouring groups on the binding. Their hemagglutination patterns confirmed the existence of three receptor‐binding specificities. While the PapG adhesins bound to uroepithelial cells from man (T24) but not to those from the dog (MDCK II), the reverse was true of PrsG. These binding patterns were largely explained by the absence or presence of appropriate glycolipid isoreceptors, although the inability of the PapG adhesins to bind MDCK II cells was attributed to an inappropriate presentation of their receptor epitopes. The high prevalence of PrsG‐like specificities observed among wild‐type dog uropathogenic E. coli isolates, together with the determined isoreceptor composition of human and dog kidney target tissues, suggest variation in receptor specificity as a mechanism for shifting host specificity, and that this variation has evolved in response to the topography of the host cellular receptors. The receptor‐binding half proposed for the predicted amino acid sequences of the four G adhesins and the corresponding adhesin of one of the dog E. coli isolates varied considerably among the three receptor‐binding groups of adhesins, but only little within each group.

Nonobstructive acute pyelonephritis in humans is most often caused by P-fimbriated Escherichia cofl. P-fimbriae are heteropolymeric fibers carrying a Gal(al-4)Gal-specific PapG adhesin at its distal end. The pyelonephritic strain DS17 expresses P-fimbriae from a single gene cluster. A mutant strain, DS17-8, which expresses P-fimbriae lacking the PapG adhesin, was constructed by allelic replacement introducing a 1-bp deletion early in the papG gene. In cynomolgus monkeys, DS17 and DS17-8 were equally able to cause bladder infection, whereas only the wild-type strain DS17 could cause pyelonephritis as monitored by bacteriological, functional, and histopathological criteria. Since DS17, but not DS17-8, adheres to renal tissue, these data underscore the critical role of microbial adherence to host tissues in infectious disease and strongly suggest that the PapG tip adhesin of P-fimbriae is essential in the pathogenesis of human kidney infection.

Helicobacter pylori is a gram-negative bacterium which causes chronic gastritis and is associated with peptic ulcer disease, gastric carcinoma, and gastric lymphoma. The bacterium is characterized by potent urease activity, thought to be located on the outer membrane, which is essential for survival at low pH. The purpose of the present study was to investigate mechanisms whereby urease and HspB, a GroEL homolog, become surface associated in vitro. Urease, HspB, and catalase were located almost exclusively within the cytoplasm in fresh log-phase cultures assessed by cryo-immunoelectron microscopy. In contrast, significant amounts of surface-associated antigen were observed in older or subcultured preparations concomitantly with the appearance of significant amounts of extracellular antigen, amorphous debris, and membrane fragments. By use of a variety of biochemical methods, a significant fraction of urease and HspB was associated with the outer membrane in subcultured preparations of H. pylori. Taken together, these results strongly suggest that H. pylori cells undergo spontaneous autolysis during culture and that urease and HspB become surface associated only concomitant with bacterial autolysis. By comparing enzyme sensitivity to flurofamide (a potent, poorly diffusible urease inhibitor) in whole cells with that in deliberately lysed cells, we show that both extracellular and intracellular urease molecules are active enzymatically. Autolysis of H. pylori is an important phenomenon to recognize since it likely exerts significant effects on the behavior of H. pylori. Furthermore, the surface properties of H. pylori must be unique in promoting adsorption of cytoplasmic proteins.

Pili of Neisseria gonorrhoeae are correlated with increased bacterial attachment to epithelial cells and undergo both phase and antigenic variation. Phase variation of gonococcal pili can be brought about by recombination events in the pilin structural gene, pilE, or by the on/off switch in expression of PilC, a pilus biogenesis protein for which two loci exist. We have studied the binding to epithelial cell lines and to fixed tissue sections of N. gonorrhoeae MS11 derivatives and mutants carrying structurally defined PilE and PilC proteins. In situ binding studies of N. gonorrhoeae to formalin-fixed tissue sections resulted in a binding pattern similar to that obtained using viable epithelial cell lines of different origin. Piliated gonococcal clones, containing different pilE sequences, varied dramatically from one another in their efficiencies at binding to corneal and conjunctival tissue, but bound equally well to cervical and endometrial tissues. Further, the binding data suggested that PilC expression by itself, i.e. without pili, cannot confer bacterial binding and that expression of either PilC1 or PilC2 does not confer different binding properties to the bacterial cells. Possible receptors for piliated gonococci were expressed in human tissues, such as cervix, endometrium, cornea, intestine, stomach, mid-brain and meninges, but not in human kidney. Pretreatment of the target tissues with Proteinase K decreased the gonococcal binding dramatically, whereas pretreatment with neuraminidase and meta-periodate, which cleave carbon-carbon linkages between vicinal hydroxyl groups in carbohydrates, did not affect attachment of gonococci. These data argue that pilus-dependent attachment of N. gonorrhoeae to human tissue may be mediated by a eukaryotic receptor having protein characteristics, and that the pilus subunit sequence may play an important role in the interaction with human cornea.

Some strains ofHelicobacterpylori are known to produce an extracellular cytotoxin that causes vacuolization in various mammalian cells. In this study, we found that concentrated culture supernatants from four Helicobacter strains isolated from patients infected with the bacterium, but having normal gastric mucosa, lacked cytotoxic activity. We also show that a higher percentage of strains isolated from patients with polymorphonuclear leukocyte infiltration of gastric mucosa are toxin positive (78%) versus those isolated from patients lacking such infiltration (33%). In addition to examining the relationship between pathology and cytotoxic activity, we used the previously published N-terminal sequence of the protein to clone and characterize vacA, the structural gene encoding the cytotoxin. Briefly, three oligonucleotides capable of encoding the first nine amino acids corresponding to the sense strand and four oligonucleotides corresponding to the noncoding strand of the last seven known amino acids of the cytotoxin protein were made. They were used in all 12 possible combinations in 12 different PCR reactions, with DNA from a cytotoxin-positive strain as template. In four combinations, the expected 69-bp fragment was seen. The sequence of this 69-bp fragment confirmed that it encoded the known N-terminal sequence of the cytotoxin. This gene is capable of encoding a 136-kDa protein with a 33-amino-acid signal peptide, whereas the purified cytotoxin is only 87 kDa, suggesting processing in the C-terminal region of the protein. A single copy of the vacA gene encodes the cytotoxin in H. pylon. Consequently, the insertion of a kanamycin resistance marker in the vacA gene produced an isogenic mutant lacking the cytotoxic activity. This mutant provides genetic evidence that vacA encodes the cytotoxin. Sequence analysis of the DNA adjacent to the vacA gene demonstrated that this gene is next to a putative cysteinyl tRNA synthetase gene. From the sequence arrangement, we predict that there are no other genes transcribed together with vacA. We also show that five of seven cytotoxin-negative strains examined still carry the sequences encoding it whereas the other two have suffered a deletion of the vacA gene. We further show that in at least one cytotoxin-negative but vacA-positive strain (MO19), there are variations in the length of the vacA gene that could explain the cytotoxin-negative phenotype in this strain.