Bacterial adhesins: structural studies reveal chaperone function and pilus biogenesis

During the past year, remarkable progress has been made in understanding how periplasmic chaperones fold and protect protein modules that are destined for assembly into adhesive pili in Gram-negative bacteria. The first two three-dimensional structures of complexes of periplasmic chaperones with substrate pilus subunits have revealed much about the structural basis for chaperone-mediated folding and aggregation prevention, and have provided insight into the structure of adhesive pili.     

Trichomonas vaginalis: The adhesins AP51 and AP65 bind heme and hemoglobin

Trichomonas vaginalis is the cause of human trichomoniasis, the most common non-viral sexually transmitted disease worldwide. Although acquisition of iron by binding to host hemoglobin through distinct receptor(s) has been described, no specific heme- or hemoglobin-binding site has been reported in this parasite.To determine the presence of hemoglobin-binding protein(s), membrane proteins were subjected to hemoglobin-affinity chromatography. Eluted proteins were analysed by SDS-PAGE. Two protein bands of 48 and 63 kDa were detected. Competition assay with an excess amount of hemoglobin or hemin in hemoglobin-affinity chromatography could block the 63- and 48-kDa bands, respectively.Further analysis by mass spectrometry indicated that the 48- and 63-kDa proteins had identity with two T. vaginalis adhesins: AP51 and AP65, respectively. This study confirms the existence of multifunctional proteins in T. vaginalis, and suggested that AP51 and AP65, besides serving as adhesion molecules, could also act as heme- and hemoglobin-binding proteins.     

The role of surface carbohydrates on the interaction of microconidia of Trichophyton mentagrophytes with epithelial cells

The presence of carbohydrate-binding adhesins on the microconidia of Trichophyton mentagrophytes surface and their role on cellular interactions were investigated. Flow cytometry showed that this fungus recognizes the sugars mannose and galactose. The binding was inhibited by the addition of methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside, and showed higher fluorescence intensity at 37 degrees C than 28 degrees C. Trypsin treatment and heating of the cells reduced the binding, suggesting a (glyco) protein nature of the microconidia adhesin. The interaction of the fungus to Chinese hamster ovary epithelial cells and its glycosylation-deficient mutants demonstrated a higher adhesion index in Lec1 and Lec2 mutants, which express mannose and galactose, respectively, as the terminal carbohydrate on the cell surface. Endocytosed fungi were shown preferentially in Lec2 cells. Addition of the carbohydrates methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside to the interaction medium, pretreatment of Lec1 and Lec2 cells with lectins Concanavalina A and Arachis hypogaea and pretreatment with sodium periodate decreased the adhesion and the endocytic index. Examination of thin section by transmission electron microscopy showed that after fungal ingestion by Lec2 cells the fungi are enclosed in a 'loose'-type vacuole while the other cells are found within a 'tight'-type membrane-bound cytoplasmic vacuole. Our results suggest the occurrence of carbohydrate-specific adhesins on microconidia surface that recognize mannose and galactose. This may have a role in the adhesion process during the infectious process of dermatophytosis.     

Tight Conformational Coupling between the Domains of the Enterotoxigenic Escherichia coli Fimbrial Adhesin CfaE Regulates Binding State Transition

CfaE, the tip adhesin of enterotoxigenic Escherichia coli colonization factor antigen I fimbriae, initiates binding of this enteropathogen to the small intestine. It comprises stacked β-sandwich adhesin (AD) and pilin (PD) domains, with the putative receptor-binding pocket at one pole and an equatorial interdomain interface. CfaE binding to erythrocytes is enhanced by application of moderate shear stress. A G168D replacement along the AD facing the CfaE interdomain region was previously shown to decrease the dependence on shear by increasing binding at lower shear forces. To elucidate the structural basis for this functional change, we studied the properties of CfaE G168D (with a self-complemented donor strand) and solved its crystal structure at 2.6 Å resolution. Compared with native CfaE, CfaE G168D showed a downward shift in peak erythrocyte binding under shear stress and greater binding under static conditions. The thermal melting transition of CfaE G168D occurred 10 °C below that of CfaE. Compared with CfaE, the atomic structure of CfaE G168D revealed a 36% reduction in the buried surface area at the interdomain interface. Despite the location of this single modification in the AD, CfaE G168D exhibited structural derangements only in the adjoining PD compared with CfaE. In molecular dynamics simulations, the G168D mutation was associated with weakened interdomain interactions under tensile force. Taken together, these findings indicate that the AD and PD of CfaE are conformationally tightly coupled and support the hypothesis that opening of the interface plays a critical modulatory role in the allosteric activation of CfaE.     

Investigation of the Secretory Pathway in Trichomonas vaginalis Argues against a Moonlighting Function of Hydrogenosomal Enzymes

The enzymes pyruvate ferredoxin oxidoreductase (PFO), malic enzyme (ME), and the α‐ and β‐subunits of succinyl‐CoA synthetase (SCS) catalyze key steps of energy metabolism in Trichomonas vaginalis hydrogenosomes. These proteins have also been characterized as the adhesins AP120 (PFO), AP65 (ME), AP33, and AP51 (α‐ and β‐SCS), which are localized on the cell surface and mediate the T. vaginalis cytoadherence. However, the mechanisms that facilitate the targeting of these proteins to the cell surface via the secretory pathway and/or to hydrogenosomes are not known. Here we adapted an in vivo biotinylation system to perform highly sensitive tracing of protein trafficking in T. vaginalis. We showed that α‐ and β‐SCS are biotinylated in the cytosol and imported exclusively into the hydrogenosomes. Neither α‐ nor β‐SCS is biotinylated in the endoplasmic reticulum and delivered to the cell surface via the secretory pathway. In contrast, two surface proteins, tetratricopeptide domain‐containing membrane‐associated protein and tetraspanin family surface protein, as well as soluble‐secreted β‐amylase‐1 are biotinylated in the endoplasmic reticulum and delivered through the secretory pathway to their final destinations. Taken together, these results demonstrate that the α‐ and β‐SCS subunits are targeted only to the hydrogenosomes, which argues against their putative moonlighting function.     

Identification of glycoprotein receptors within the human salivary proteome for the lectin‐like BabA and SabA adhesins of Helicobacter pylori by fluorescence‐based 2‐D bacterial overlay

Because gastric infection by Helicobacter pylori takes place via the oral route, possible interactions of this bacterium with human salivary proteins could occur. By using modified 1‐ and 2‐D bacterial overlay, binding of H. pylori adhesins BabA and SabA to the whole range of salivary proteins was explored. Bound salivary receptor molecules were identified by MALDI‐MS and by comparison to previously established proteome maps of whole and glandular salivas. By use of adhesin‐deficient mutants, binding of H. pylori to MUC7 and gp‐340 could be linked to the SabA and BabA adhesins, respectively, whereas binding to MUC5B was associated with both adhesins. Binding of H. pylori to the proline‐rich glycoprotein was newly detected and assigned to BabA adhesin whereas the SabA adhesin was found to mediate binding to newly detected receptor molecules, including carbonic anhydrase VI, secretory component, heavy chain of secretory IgA1, parotid secretory protein and zinc‐α₂‐glycoprotein. Some of these salivary glycoproteins are known to act as scavenger molecules or are involved in innate immunity whereas others might come to modify the pathogenetic properties of this organism. In general, this 2‐D bacterial overlay technique represents a useful supplement in adhesion studies of bacteria with complex protein mixtures.     

Cell envelope characteristics of Helicobacter pylori: their role in adherence to mucosal surfaces and virulence

Abstract Helicobacter pylori colonises the gastric mucosa of humans and causes both antral gastritis and duodenal ulcer disease. Exactly how H. pylori causes disease is not known but several pathogenic determinants have been proposed for the organism. These include adhesins, cytotoxins and a range of different enzymes including urease, catalase and superoxide dismutase. Surface molecules of H. pylori such as flagella, lipopolysaccharide, the urease enzyme and outer membrane proteins are putative adhesin molecules. While phosphatidylethanolamine and the Lewis^b blood group antigen have been proposed as receptor molecules for the organism the exact mechanism by which H. pylori adheres to the gastric mucosa has still to be identified. Characterisation of the adhesins of H. pylori could lead to the development of adhesin analogues for use in the inhibition of colonisation and improved therapy for ulcer disease. In vivo studies with isogenic mutants which are incapable of adhering to the gastric mucosa would greatly clarify the significance of adherence. Such mutants could possibly be useful as a vaccine against infection with wild-type organisms.