Micro- and nanostructure of the adhesive material secreted by the tube feet of the sea star Asterias rubens

To attach to underwater surfaces, sea stars rely on adhesive secretions produced by specialised organs, the tube feet. Adhesion is temporary and tube feet can also voluntarily become detached. The adhesive material is produced by two types of adhesive secretory cells located in the epidermis of the tube foot disc, and is deposited between the disc surface and the substratum. After detachment, this material remains on the substratum as a footprint. Using LM, SEM, and AFM, we described the fine structure of footprints deposited on various substrata by individuals of Asterias rubens. Ultrastructure of the adhesive layer of attached tube feet was also investigated using TEM. Whatever the method used, the adhesive material appeared as made up of globular nanostructures forming a meshwork deposited on a thin homogeneous film. This appearance did not differ according to whether the footprints were fixed or not, and whether they were observed hydrated or dry. TEM observations suggest that type 2 adhesive cells would be responsible for the release of the material constituting the homogeneous film whereas type 1 adhesive cells would produce the material forming the meshwork. This reticulated pattern would originate from the arrangement of the adhesive cell secretory pores on the disc surface.     

The use of sortase-mediated ligation for the immobilisation of bacterial adhesins onto fluorescence-labelled microspheres: a novel approach to analyse bacterial adhesion to host cells

Commensal and pathogenic bacteria express adhesive proteins on their cell surface, which are important for colonisation of the host. In Gram-positive bacteria, these adhesins are often covalently anchored to the cell wall by a sortase enzyme. A recent bioinformatic study has revealed a total of 860 predicted cell wall-anchored proteins in 94 completely sequenced genomes. The interaction of adhesins with host cells can be analysed with the use of adhesin-coated microbeads. Here we show that sortase-mediated ligation can be used for the site-specific immobilisation of adhesins to red-fluorescence microspheres. This coupling method allows for the native orientation of the adhesins on the beads. Furthermore, the high substrate specificity of the sortase enzyme allows the use of only partially purified recombinant proteins, which reduces preparation time and costs, and also prevents coupling of any contaminants that might interfere with cell binding.     

The pathogenicity factors of bacteria in the genus Acinetobacter]

Eighty Acinetobacter strains, isolated in Togliatti from patients with purulent inflammatory diseases, were studied to determine their pathogenicity factors. Out of these 80 strains, 32.5% were found to have enterotoxigenic activity and 46.2%, adhesive activity. They were related to adhesins of the human type and to adhesins of the sheep, rabbit, swine and guinea pig types. But the most important phenomenon established in this study was the combination of different pathogenicity factors detected in Acinetobacter bacteria. Analysis of the combination of pathogenicity factors revealed that 7.5% of Acinetobacter strains had adhesive and enterotoxigenic activity, 15.3% of these strains combined adhesive and hemolytic activity and 1.2% of them were found to be enterotoxigenic and hemolytic. Only 5.0% of Acinetobacter strains were found to carry all there pathogenicity factors simultaneously.     

Urinary tract infections of Escherichia coli strains of chaperone-usher system

Urinary tract infections are a very serious health and economic problem affecting millions of people each year worldwide. The most common etiologic agent of this type of bacterial infections, involving the upper and lower urinary tract, are E. coli strains representing approximately 80% of cases. Uropathogenic E. coli strains produce several urovirulence factors which can be divided into two main types, surface virulence factors and exported virulence factors. Surface-exposed structures include mainly extracellular adhesive organelles such as fimbriae/pili necessary in adhesion, invasion, biofilm formation and cytokine induction. Among the surface-exposed polymeric adhesive structures there are three most invasive groups, type 1 pili, type P pili and Dr family of adhesins which are bioassembled via the conserved, among Gram-negative bacteria, chaperone-usher secretion system. Type 1 and P-piliated E. coli cause cystitis and pyelonephritis. The Dr family of adhesins recognizing DAF receptor is responsible for cystitis, pyelonephritis (especially in pregnant women) and diarrhoea (in infants). In addition, Dr-positive E. coli strains carry the risk of recurrent urinary tract infections. Pyelonephritis in pregnant women leads to a series of complications such as bacteremia, urosepsis, acute respiratory distress syndrome and even death. In the era of increasing drug resistance of bacteria, the development of vaccines, drugs termed pilicides and inhibitors of adhesion may be a promising tool in the fight against urogenital infections.     

Pili and the interaction of Aeromonas species with humanperipheral blood polymorphonuclear cells

Abstract The interaction of differentiallu piliated Aeromonas strains expressing pili of two broadly different morphologic types (short, rigid (S/R) and/or long, wavy (L/W)) with human peripheral blood mononuclear leukocytes (PMN) was investigated to determine whether host defense cells might exert a selective pressure on pili expression in vivo accounting for the different pili phenotypes of clinical and environmental strains. A majority of Aeromonas veronii biotype sobria strains from water (6/6) and faeces (8/11) readily associated with PMN (>60% PMN with adherent and/or internalised bacteria), irrespective of their degree, or predominant type, of piliation. Rigid pili of Aeromonas species did not promote interaction with PMN. However, the majority (55%) of strains which interacted well with PMN were adherent to HEp-2 cells. Interactio with PMN is unlikely to be the reason few S/R pili are seen on faecal strains, but it may be a selective pressure on L/W adhesive pili, or other OMP adhesins, resulting in the shedding of strains which have lost critical adhesins.     

Rhomboid 4 (ROM4) Affects the Processing of Surface Adhesins and Facilitates Host Cell Invasion by Toxoplasma gondii

Host cell attachment by Toxoplasma gondii is dependent on polarized secretion of apical adhesins released from the micronemes. Subsequent translocation of these adhesive complexes by an actin-myosin motor powers motility and host cell invasion. Invasion and motility are also accompanied by shedding of surface adhesins by intramembrane proteolysis. Several previous studies have implicated rhomboid proteases in this step; however, their precise roles in vivo have not been elucidated. Using a conditional knockout strategy, we demonstrate that TgROM4 participates in processing of surface adhesins including MIC2, AMA1, and MIC3. Suppression of TgROM4 led to decreased release of the adhesin MIC2 into the supernatant and concomitantly increased the surface expression of this and a subset of other adhesins. Suppression of TgROM4 resulted in disruption of normal gliding, with the majority of parasites twirling on their posterior ends. Parasites lacking TgROM4 bound better to host cells, but lost the ability to apically orient and consequently most failed to generate a moving junction; hence, invasion was severely impaired. Our findings indicate that TgROM4 is involved in shedding of micronemal proteins from the cell surface. Down regulation of TgROM4 disrupts the normal apical-posterior gradient of adhesins that is important for efficient cell motility and invasion of host cells by T. gondii.     

Thin pilus PilV adhesins of plasmid R64 recognize specific structures of the lipopolysaccharide molecules of recipient cells

IncI1 plasmid R64 encodes a type IV pilus called a thin pilus, which includes PilV adhesins. Seven different sequences for the C-terminal segments of PilV adhesins can be produced by shufflon DNA rearrangement. The expression of the seven PilV adhesins determines the recipient specificity in liquid matings of plasmid R64. Salmonella enterica serovar Typhimurium LT2 was recognized by the PilVA' and PilVB' adhesins, while Escherichia coli K-12 was recognized by the PilVA', PilVC, and PilVC' adhesins. Lipopolysaccharide (LPS) on the surfaces of recipient cells was previously shown to be the specific receptor for the seven PilV adhesins. To identify the specific receptor structures of LPS for various PilV adhesins, R64 liquid matings were carried out with recipient cells consisting of various S. enterica serovar Typhimurium LT2 and E. coli K-12 waa mutants and their derivatives carrying various waa genes of different origins. From the mating experiments, including inhibition experiments, we propose that the GlcNAc(alpha1-2)Glc and Glc(alpha1-2)Gal structures of the LPS core of S. enterica serovar Typhimurium LT2 function as receptors for the PilVB' and PilVC' adhesins, respectively, while the PilVC' receptor in the wild-type LT2 LPS core may be masked. We further propose that the GlcNAc(beta1-7)Hep and Glc(alpha1-2)Glc structures of the LPS core of E. coli K-12 function as receptors for the PilVC and PilVC' adhesins, respectively.     

Absence of surface-associated microorganisms in adult oysters (Crassostrea gigas).

Healthy, actively feeding intertidal oysters were removed from an estuarine environment (Pipeclay Lagoon, Tasmania). The epithelial surfaces of various organs of the mantle cavity and alimentary tract were explored by scanning and transmission electron microscopy. All epithelial tissues examined were ciliated, and nearly all were partly covered with secreted mucus. However, microorganisms were seen rarely in the adhesive mucus and never attached to the epithelium. Electron microscopy also failed to demonstrate a surface microflora in emersed oysters which had been incubated at 5 to 25 degrees C for 6 or 24 h. The absence of an internal surface microflora did not vary on a seasonal basis. In laboratory experiments, oysters were allowed to filter feed from seawater containing diverse types of marine bacteria at concentrations of 10(3) to 10(7)/mL. However, no surface microflora could be found within actively feeding oysters or in emersed animals incubated at 20 degrees C for 6 or 24 h. In contrast, surface-associated microorganisms were detected readily by scanning electron microscopy on the external shell of healthy oysters and on various internal tissues in spoiled oysters. It is suggested that the major mechanisms restricting microbial growth within oysters are ciliary movement and mucus secretion.     

Helicobacter pylori adhesins: review and perspectives

It is highly unlikely that chronic infection with H. pylori could occur in the absence of adhesin–host cell interactions. Also, there is no evidence that any of the serious outcomes of H. pylori infection such as gastric and duodenal ulcers, gastric cancer or mucosa‐associated lymphoid tissue (MALT) lymphoma could occur without prior colonization of the gastric epithelium mediated by H. pylori adhesins. H. pylori is highly adaptable, as evidenced by the fact that it can occupy a single host for decades. An important facet of this adaptability is its ability to physically interact with various types of host cells and also with host mucins and extracellular matrix proteins using a number of different adhesins displaying a variety of unique receptor specificities. Thus it is highly unlikely that any one particular H. pylori adhesin will ever be proven responsible for a particular outcome such as duodenal ulcer, MALT lymphoma, or adenocarcinoma. Also, while the search for additional H. pylori adhesins should and certainly will continue, we suggest that the scope of this effort should be expanded to include investigations into the patterns of expression and interaction between individual outer membrane proteins. Which of the numerous H. pylori outer membrane proteins (OMPs) actually function as adhesins (i.e., have receptor‐binding sites) and which OMPs are simply necessary for optimal display of the adhesive OMPs? There are many other important questions about H. pylori adhesins waiting to be answered. For example, which adhesins are responsible for loose adherence to host cells and which adhesins are responsible for intimate, or membrane‐to‐membrane, adherence, and do these adhesins normally work in concert or in a sequential fashion? Also, is a specific type of adhesin necessary for type IV protein translocation into host cells and, if so, is adhesin expression coregulated with the effector protein export?     

occurrence of mannose- resistant adhesins MRHA in E.Coli strains isolated from children with diarrhea]

The study was aimed at determination of the frequency of occurrence of mannose-resistant adhesins in E. coli strains isolated from children with diarrhoea. It was also of interest whether their presence is associated with the serological type or other virulence factors. The material used in this study consisted of 1022 strains of E. coli (EPEC, ETEC and EIEC) and 3431 isolates from sick children and 960 from healthy children (non-EPEC-ETEC-EIEC). Enterotoxigenicity and entero-invasiveness of strains was evaluated by biological tests performed on animals and in tissue culture. Production of MRHA adhesins was determined by the test of mannose-resistant active hemagglutination, and of colonization factors antigens CFA by application of agglutination and agar gel immunodiffusion tests. Most frequently MRHA adhesins were produced by ETEC strains-80% of strains. All of them appeared to be a colonization factor antigen CFA/I. EPEC strains produced various MRHA adhesins only by 12.6% of strains. Production of MRHA adhesins by EIEC strains was not detected. Frequency of occurrence of MRHA adhesins in E. coli strains which were non-EPEC-ETEC-EIEC was dependent from the isolation source. MRHA adhesins were most frequently found in strains isolated from sporadic cases of light diarrhoea in ambulatory treated children (49%), much less among isolates from children hospitalized because of severe diarrhoea (33%), and from healthy children in 9% of isolates only. These results may indicate the potential role of MRHA adhesins in pathogenesis of diarrhoea in children.     

Trimeric autotransporters require trimerization of the passenger domain for stability and adhesive activity

In recent years, structural studies have identified a number of bacterial, viral, and eukaryotic adhesive proteins that have a trimeric architecture. The prototype examples in bacteria are the Haemophilus influenzae Hia adhesin and the Yersinia enterocolitica YadA adhesin. Both Hia and YadA are members of the trimeric-autotransporter subfamily and are characterized by an internal passenger domain that harbors adhesive activity and a short C-terminal translocator domain that inserts into the outer membrane and facilitates delivery of the passenger domain to the bacterial surface. In this study, we examined the relationship between trimerization of the Hia and YadA passenger domains and the capacity for adhesive activity. We found that subunit-subunit interactions and stable trimerization are essential for native folding and stability and ultimately for full-level adhesive activity. These results raise the possibility that disruption of the trimeric architecture of trimeric autotransporters, and possibly other trimeric adhesins, may be an effective strategy to eliminate adhesive activity.     

The characteristics of Corynebacterium diphtheriae adhesins

The study of the properties of C. diphtheriae adhesins revealed the absence of their thermostability, which suggested the protein nature of these adhesins. C. diphtheriae also showed pronounced mannose-resistant hemagglutinating activity, as this activity remained unchanged even in the presence of d-mannose. The comparison of the adhesive activity of C. diphtheriae with their phage and corycine sensitivity revealed essential differences between these macroorganisms in the degree of their adhesiveness. The phage lysability and corycine sensitivity of C. diphtheriae strains, determined by the properties of their surface structure, correlated with the degree of their activity.     

Significance of hydrobiont persistent properties for symbiotic interactions

Significance of symbiotic relations formed by associative symbiosis type for autochthonous and allochthonous microflora of natural water bodies is shown. Generality of symbiotic interaction mechanisms of symbionts in limnetic and halophilous communities provided by secreted factors of natural resistance from the side of the host, and by factors of persistence from the side of symbionts is proven based on a set of examples. Features of operation of lysozyme-antilysozyme, histon-antihiston, hydrogen peroxide-catalase functional systems in symbiotic interactions of autotrophic and heterotrophic components of hydrobiocenosis with dominant and associative microflora are presented. Associative microflora of allochthonous origin was shown to actively use the ecologically formed system of interaction between hydrobionts that facilitates survival of these microorganisms and preservation of their persistent potential, and as a result leads to biocenosis disorders. The knowledge obtained open new possibilities and perspectives of research of sanitary and ecological aspects of vital activity of aquatic biocenoses.     

Radiolabeling of and macromolecular syntheses in Neisseria gonorrhoeae types 1 and 4.

Type 1 and type 4 cells of Neisseria gonorrhoeae were grown in a liquid medium that maintained colony type for several hours. Organisms from both types of colonies incorporated labeled glucose, as well as nucleic acid and protein precursors, into specific cellular fractions during growth in the medium. Ribonucleic acid and protein were synthesized actively by the bacteria, with incorporation of label chased by the addition of unlabeled precursors. The method can be used to label type 1 and type 4 cells of gonococci with specific isotopes for studies of metabolic and biochemical changes.     

Radiolabeling of and macromolecular syntheses in Neisseria gonorrhoeae types 1 and 4.

Type 1 and type 4 cells of Neisseria gonorrhoeae were grown in a liquid medium that maintained colony type for several hours. Organisms from both types of colonies incorporated labeled glucose, as well as nucleic acid and protein precursors, into specific cellular fractions during growth in the medium. Ribonucleic acid and protein were synthesized actively by the bacteria, with incorporation of label chased by the addition of unlabeled precursors. The method can be used to label type 1 and type 4 cells of gonococci with specific isotopes for studies of metabolic and biochemical changes.     

The correction of dysbiotic disorders of the vaginal microflora by using a preparation made from highly adhesive lactobacteria

The effectiveness of a new bacterial preparation obtained from highly adhesive lactobacteria and intended for the correction of dysbiotic disturbances of vaginal microflora was studied in the treatment of 60 pregnant women with dysbacteriosis of the maternal passages. 30 pregnant women were simultaneously treated by the vaginal application of Lactobacterin. The study showed that the use of the preparation of highly adhesive lactobacteria caused the pronounced and stable correction of the microflora of the maternal passages. This correction was manifested by the domination of lactic acid bacterial flora and a decrease in the number of opportunistic microorganisms.     

Heterogeneity of Bacillus anthracis strains in terms of their adhesive capacity

The homogeneity of colonies of two B. anthracis vaccine strains in R- and RS- forms (100 colonies of each strain) in terms of their adhesive capacity was studied. B. anthracis strain 228/8 showed more heterogeneous composition than B. anthracis strain 71/12, moderately and highly adhesive colonies prevailing in both forms and nonadhesive colonies being absent. The prevalence of highly adhesive clones was established in the RS- form of B. anthracis strain 72/12 in comparison with R- form. By the average value of the adhesion index the RS- form colonies of this strain were classified as highly adhesive, while the colonies in the R- form were characterized as moderately adhesive.     

Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier

Members of the Dr family of adhesins of Escherichia coli recognize as a receptor the Dr(a) blood-group antigen present on the complement regulatory and signalling molecule, decay-accelerating factor (DAF). One member of this family, the Dr haemagglutinin, also binds to a second receptor, type IV collagen. Structure/function information regarding these adhesins has been limited and domains directly involved in the interaction with DAF have not been determined. We devised a strategy to identify amino acids in the Dr haemagglutinin that are specifically involved in the interaction with DAF. The gene encoding the adhesive subunit, draE, was subjected to random mutagenesis and used to complement a strain defective for its expression. The resulting mutants were enriched and screened to obtain those that do not bind to DAF, but retain binding to type IV collagen. Individual amino acid changes at positions 10, 63, 65, 75, 77, 79 and 131 of the mature DraE sequence significantly reduced the ability of the DraE adhesin to bind DAF, but not collagen. Over half of the mutants obtained had substitutions within amino acids 63-81. Analysis of predicted structures of DraE suggest that these proximal residues may cluster to form a binding domain for DAF.     

Type IV pili of pathogenic Neisseriae elicit cortical plaque formation in epithelial cells

The pathogenic Neisseriae Neisseria meningitidis and Neisseria gonorrhoeae, initiate colonization by attaching to host cells using type IV pili. Subsequent adhesive interactions are mediated through the binding of other bacterial adhesins, in particular the Opa family of outer membrane proteins. Here, we have shown that pilus-mediated adhesion to host cells by either meningococci or gonococci triggers the rapid, localized formation of dramatic cortical plaques in host epithelial cells. Cortical plaques are enriched in both components of the cortical cytoskeleton and a subset of integral membrane proteins. These include: CD44v3, a heparan sulphate proteoglycan that may serve as an Opa receptor; EGFR, a receptor tyrosine kinase; CD44 and ICAM-1, adhesion molecules known to mediate inflammatory responses; f-actin; and ezrin, a component that tethers membrane components to the actin cytoskeleton. Genetic analyses reveal that cortical plaque formation is highly adhesin specific. Both pilE and pilC null mutants fail to induce cortical plaques, indicating that neisserial type IV pili are required for cortical plaque induction. Mutations in pilT, a gene required for pilus-mediated twitching motility, confer a partial defect in cortical plaque formation. In contrast to type IV pili, many other neisserial surface structures are not involved in cortical plaque induction, including Opa, Opc, glycolipid GgO4-binding adhesins, polysialic acid capsule or a particular lipooligosaccharide variant. Furthermore, it is shown that type IV pili allow gonococci to overcome the inhibitory effect of heparin, a soluble receptor analogue, on gonococcal invasion of Chang and A431 epithelial cells. These and other observations strongly suggest that type IV pili play an active role in initiating neisserial infection of the mucosal surface in vivo. The functions of type IV pili and other neisserial adhesins are discussed in the specific context of the mucosal microenvironment, and a multistep model for neisserial colonization of mucosal epithelia is proposed.