Adhesive Property of Toxin-Coregulated Pilus of Vibrio cholerae O1

The adhesive property of toxin-coregulated pilus (TCP) to the human intestine (jejunum), and whether or not TCP mediates the adhesion of Vibrio cholerae 395 organisms to the intestinal epithelium were investigated using visually proving methods. The purified TCP did not agglutinate human erythrocytes nor adhere to the surface of human intestinal epithelium. V. cholerae 395 adhered to the epithelium, but the adhesion was not inhibited by blocking the pili with the Fab fraction of anti-TCP IgG. The organisms adhered to the intestine treated with purified TCP in advance, as well as to the intact intestine. These findings suggest that TCP is not involved in the adhesion of these organisms to the intestinal epithelium.     

Pili of Aeromonas hydrophila: purification, characterization, and biological role

Aeromonas hydrophila (Ae6) has 2 morphologically distinctive kinds of pili. One appeared rigid, channeled, and straight with a diameter of 9 nm (Ae6-R pili). The other looked flexible, wavy, and having helical structure with a diameter of 7 nm (Ae6-W pili). Ae6-R pili were purified and characterized. The pili consisted of a subunit protein with a molecular weight of 18 kDa as estimated by SDS-PAGE, and contained 42.3% hydrophobic amino acids and one cysteine residue. The pilus was solubilized to 18 kDa subunit protein by 2-mercaptoethanol, dithiothreitol, hydrochloric acid, or heating at 120 C for 5 min. The organism Ae6 was strongly adhesive to rabbit intestines as well as human intestines, and agglutinated erythrocytes. Anti-pili antibody (Fab fraction) did not block the adhesion. Purified Ae6-R pili did not adhere to the intestine or to the erythrocytes. However, the anti-pili Fab inhibited pellicle formation of the organisms cultured in broth, and also inhibited salt agglutination with ammonium sulfate. From these results, Ae6-R pili are not likely a colonization factor but probably play a role in the autoaggregation of the organisms.     

Production of pili on Vibrio parahaemolyticus

Electron microscopic examination showed that all strains of Vibrio parahaemolyticus examined had pili on their surface when the organism was grown on marine agar at 28 degrees C for 6-12 h. The pili were morphologically stable on heat treatment at 60 degrees C for 10 min, but both the lateral and polar flagella possessed by this organism were labile. No immunological cross-reactivity between pili of enterotoxigenic Escherichia coli and Vibrio cholerae non-01 and those of V. parahaemolyticus was observed.     

Sequence Analyses of Type IV Pili from Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus

Bacterial surface structures called pili have been studied extensively for their role as possible colonization factors. Most sequenced Vibrio genomes predict a variety of pili genes in these organisms, including several types of type IV pili. In particular, the mannose-sensitive hemagglutinin (MSHA) and the PilA pili, also known as the chitin-regulated pilus (ChiRP), are type IVa pili commonly found in Vibrio genomes and have been shown to play a role in the colonization of Vibrio species in the environment and/or host tissue. Here, we report sequence comparisons of two type IVa pilin subunit genes, mshA and pilA, and their corresponding amino acid sequences, for several strains from the three main human pathogenic Vibrio species, V. cholerae, V. parahaemolyticus, and V. vulnificus. We identified specific groupings of these two genes in V. cholerae, whereas V. parahaemolyticus and V. vulnificus strains had no apparent allelic clusters, and these genes were strikingly divergent. These results were compared with other genes from the MSHA and PilA operons as well as another Vibrio pili from the type IVb group, the toxin co-regulated pilus (TCP) from V. cholerae. Our data suggest that a selective pressure exists to cause these strains to vary their MSHA and PilA pilin subunits. Interestingly, V. cholerae strains possessing TCP have the same allele for both mshA and pilA. In contrast, V. cholerae isolates without TCP have polymorphisms in their mshA and pilA sequences similar to what was observed for both V. parahaemolyticus and V. vulnificus. This data suggests a possible linkage between host interactions and maintaining a highly conserved type IV pili sequence in V. cholerae. Although the mechanism underlying this intriguing diversity has yet to be elucidated, our analyses are an important first step towards gaining insights into the various aspects of Vibrio ecology.     

Piliation of Lactobacillus rhamnosus GG Promotes Adhesion,Phagocytosis, and Cytokine Modulation in Macrophages

Recently, spaCBA-encoded pili on the cell surface of Lactobacillus rhamnosus GG were identified to be key molecules for binding to human intestinal mucus and Caco-2 intestinal epithelial cells. Here, we investigated the role of the SpaCBA pilus of L. rhamnosus GG in the interaction with macrophages in vitro by comparing the wild type with surface mutants. Our results show that SpaCBA pili play a significant role in the capacity for adhesion to macrophages and also promote bacterial uptake by these phagocytic cells. Interestingly, our data suggest that SpaCBA pili also mediate anti-inflammatory effects by induction of interleukin-10 (IL-10) mRNA and reduction of interleukin-6 (IL-6) mRNA in a murine RAW 264.7 macrophage cell line. These pili appear to mediate these effects indirectly by promoting close contact with the macrophages, facilitating the exertion of anti-inflammatory effects by other surface molecules via yet unknown mechanisms. Blockage of complement receptor 3 (CR3), previously identified to be a receptor for streptococcal pili, significantly decreased the uptake of pilus-expressing strains in RAW 264.7 cells, while the expression of IL-10 and IL-6 mRNA by these macrophages was not affected by this blocking. On the other hand, blockage of Toll-like receptor 2 (TLR2) significantly reduced the expression of IL-6 mRNA irrespective of the presence of pili.     

NorM of vibrio parahaemolyticus is an Na(+)-driven multidrug efflux pump

NorM of Vibrio parahaemolyticus apparently is a new type of multidrug efflux protein, with no significant sequence similarity to any known transport proteins. Based on the following experimental results, we conclude that NorM is an Na(+)-driven Na(+)/drug antiporter. (i) Energy-dependent ethidium efflux from cells possessing NorM was observed in the presence of Na(+) but not of K(+). (ii) An artificially imposed, inwardly directed Na(+) gradient elicited ethidium efflux from cells. (iii) The addition of ethidium to cells loaded with Na(+) elicited Na(+) efflux. Thus, NorM is an Na(+)/drug antiporting multidrug efflux pump, the first to be found in the biological world. Judging from the similarity of the NorM sequence to those of putative proteins in sequence databases, it seems that Na(+)/drug antiporters are present not only in V. parahaemolyticus but also in a wide range of other organisms.     

Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells

Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a spaCBA pilus knockout mutant in comparison with the wild type and other adhesin mutants. The SpaCBA pilus of L. rhamnosus GG showed to be key for efficient adherence to the Caco-2 intestinal epithelial cell (IEC) line and biofilm formation. Moreover, the spaCBA mutant induces an elevated level of interleukin-8 (IL-8) mRNA in Caco-2 cells compared to the wild type, possibly involving an interaction of lipoteichoic acid with Toll-like receptor 2. In contrast, an L. rhamnosus GG mutant without exopolysaccharides but with an increased exposure of pili leads to the reduced expression of IL-8. Using Transwells to partition bacteria from Caco-2 cells, IL-8 induction is blocked completely regardless of whether wild-type or mutant L. rhamnosus GG cells are used. Taken together, our data suggest that L. rhamnosus GG SpaCBA pili, while promoting strong adhesive interactions with IECs, have a functional role in balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid.     

The three-dimensional structure of CFA/I adhesion pili: traveler's diarrhea bacteria hang on by a spring

To survive the harsh environment of a churning intestinal tract, bacteria attach to the host epithelium via thin fibers called pili (or fimbriae). Enterotoxigenic Escherichia coli bacteria expressing colonization factor antigen I (CFA/I) pili and related pili are the most common known bacterial cause of diarrheal disease, including traveler's diarrhea. CFA/I pili, assembled via the alternate chaperone pathway, are essential for binding and colonization of the small bowel by these pathogenic bacteria. Herein, we elucidate unique structural features of CFA/I pili that appear to optimize their function as bacterial tethers in the intestinal tract. Using transmission electron microscopy of negatively stained samples in combination with iterative three-dimensional helical reconstruction methods for image processing, we determined the structure of the CFA/I pilus filament. Our results indicate that strong end-to-end protein interactions and weak interactions between the coils of a sturdy spring-like helix provide the combination of strength, stability, and flexibility required to sustain bacterial adhesion and incite intestinal disease. We propose that CFA/I pili behave like a spring to maintain attachment to the gut lining during vortex mixing and downward flow of the intestinal contents, thereby persisting long enough for these bacteria to colonize the host epithelium and cause enteric disease.     

蒙脱石对细菌黏附Caco-2细胞的影响

采用Caco-2细胞培养模型,观察两歧双歧杆菌、嗜酸乳杆菌、嗜水气单胞菌、副溶血弧菌、大肠杆菌、鼠伤寒沙门菌的黏附率,并在培养液中加入蒙脱石,计算蒙脱石对细菌黏附的阻断率,探讨蒙脱石对上述细菌黏附作用的影响。结果表明:所试菌与Caco-2细胞均有不同程度的黏附作用;蒙脱石对细菌黏附Caco-2细胞均有不同程度的阻断作用,对病原菌黏附Caco-2细胞的阻断作用要明显大于其对益生菌的阻断效果,其中对大肠杆菌、鼠伤寒沙门菌、嗜水气单胞菌、副溶血弧菌黏附的阻断率分别为54.22%、48.41%、60.53%、50.64%,而对两歧双歧杆菌、嗜酸乳杆菌黏附的阻断率分别为25.64%和21.49%。结果提示蒙脱石可有效阻断病原菌黏附,从而防治肠道细菌感染和细菌移位。     

A Survey of the Occurrence of Vibrio parahaemolyticus and V. alginolyticus on Mussels and Oysters and in Estuarine Waters in the Netherlands

S ummary : In a survey on the occurrence of Vibrio parahaemolyticus and V. alginolyticus on mussels and oysters and in estuarine waters in The Netherlands it appeared necessary to use at least 2 different enrichment procedures as well as isolation methods in parallel to obtain reliable results. A consistent identification of V. parahaemolyticus on biochemical grounds remains a difficult task; further research in this area is definitely required. The ligated intestinal segment technique gave inconsistent results; injection of organisms into the yolk sac of fertilized hen eggs seems to be a more reliable diagnostic procedure. Of 288 samples of mussels examined 2·4% were V. parahaemolyticus positive, 80 samples of oysters were all negative and amongst 64 water samples 4·7% were positive. V. alginolyticus was isolated in similar percentages of samples of mussels and water, but oysters were positive to an extent of 6·8%.     

A filamentous phage of Vibrio parahaemolyticus O3:K6 isolated in Laos.

A filamentous phage, 'lvpf5,' of Vibrio parahaemolyticus O3:K6 strain LVP5 was isolated and characterized. The host range was not restricted to serotype O3:K6, but 7 of 99 V. parahaemolyticus strains with a variety of serotypes were susceptible to the phage. The phage was inactivated by heating at 80 C for 10 min and by treating with chloroform. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phage exhibited a 3.8 kDa protein. The amino-terminal amino acid sequence of the coat protein was determined as AEGGAADPFEAIDLLGVATL. The phage genome consisted of a single-stranded DNA molecule. The activity of the phages was inhibited by anti-Na2 pili antibody.     

Pili mediate specific adhesion of Streptococcus pyogenes to human tonsil and skin

Very little is known about the biological functions of pili that have recently been found to be expressed by important Gram-positive pathogens such as Corynebacterium diphtheriae, Streptococcus agalacticae, S. pneumoniae and S. pyogenes. Using various ex vivo tissue and cellular models, here we show that pili mediate adhesion of serotype M1 S. pyogenes strain SF370 to both human tonsil epithelium and primary human keratinocytes, which represent the two main sites of infection by this human-specific pathogen. Mutants lacking minor pilus subunits retained the ability to express cell-surface pili, but these were functionally defective. In contrast to above, pili were not required for S. pyogenes adhesion to either immortalized HEp-2 or A549 cells, highlighting an important limitation of these extensively used adhesion/invasion models. Adhering bacteria were internalized very effectively by both HEp-2 and A549 cells, but not by tonsil epithelium or primary keratinocytes. While pili acted as the primary adhesin, the surface M1 protein clearly enhanced adhesion to tonsil, but surprisingly, had the opposite effect on adhesion to keratinocytes. These studies provide clear evidence that S. pyogenes pili display an adhesive specificity for clinically relevant human tissues and are likely to play a critical role in the initial stages of infection.     

The phenomenon of pilus formation in the interaction of Yersinia pestis with macrophages in experimental animals

According to the data of the enzyme immunoassay, adhesion pili were expressed by Y. pestis cells EB76 after their cultivation in media with pH similar to that of phagolysosomes. The expression of adhesion pili was found to occur 18 hours after the contact of apiliate cells EB76 with a monolayer of native, but not inactivated (at 56 degrees C for 30 minutes) macrophages of the peritoneal exudate of white mice and guinea pigs. Purified adhesion pili possessed cytotoxic action and inhibited the digestive activity of macrophages with respect to Y. pestis. The formation of pili in interaction with macrophages and the pronounced effect of the preparations of purified pili on the function of phagocytes make it possible to regard the formation of pili as an important determinant of virulence.     

The role of common and type-specific pilus antigenic domains in adhesion and virulence of gonococci for human epithelial cells

A panel of monoclonal antibodies with varying pilus specificities ranging from cross-reacting to type-specific has been used to investigate the role of conserved and variable antigenic domains in the adhesion of gonococci to human epithelial cells. The binding of 125I-labelled alpha pili from strain P9 to buccal epithelial cells was inhibited by three type-specific but not by two cross-reacting antibodies. Four type-specific antibodies inhibited the binding of gamma pili while the two cross-reacting antibodies were again without effect. The virulence of the variants P9-2 (alpha pili) and P9-35 (gamma pili) for Chang conjunctiva epithelial cells was similarly reduced only in the presence of relevant type-specific antibodies. These results indicate the importance of variable antigenic domains in the adhesion of gonococci to human epithelial cells.     

Immune-enhanced phagocytosis of Neisseria gonorrhoeae by macrophages: characterization of the major antigens to which opsonins are directed

antisera were prepared in rabbits against whole organisms of colony type 1 Neisseria gonorrhoeae strains F62 and B (fron gonococcal urethritis) and 7122 (a strain typical of those associated with disseminated gonococcal infection), and against purified outer membrane components from the same strains including pili and principal outer membrane protein. Antibody levels to pili, principal outer membrane protein and lipopolysaccharide were determined using a quantitative enzyme-linked immunosorbent assay. Each antiserum was heat-inactivated and tested for opsonic for its homologous strain, and this immune-enhanced phagocytosis was decreased by adsorption with homologous purified outer membrane components: pili greater than lipopolysaccharide greater than principal outer membrane protein. Opsonic activity was approximately equal for antiserum to purified pili and antiserum to the whole organisms for each of the three strains, and purified antibody to pili was highly opsonic. The F(ab')2 fragments of antibody to pili were not opsonic, indicating a role for the Fc receptor on the phagocyte membrane in immune-enhanced phagocytosis of gonococci.     

Catch-Bond Behavior of Bacteria Binding by Slip Bonds

It is shown that multipili-adhering bacteria expressing helix-like pili binding by slip bonds can show catch-bond behavior. When exposed to an external force, such bacteria can mediate adhesion to their hosts by either of two limiting means: sequential or simultaneous pili force exposure (referring to when the pili mediate force in a sequential or simultaneous manner, respectively). As the force is increased, the pili can transition from sequential to simultaneous pili force exposure. Since the latter mode of adhesion gives rise to a significantly longer bacterial adhesion lifetime than the former, this results in a prolongation of the lifetime, which shows up as a catch-bond behavior. The properties and conditions of this effect were theoretically investigated and assessed in some detail for dual-pili-adhering bacteria, by both analytical means and simulations. The results indicate that the adhesion lifetime of such bacteria can be prolonged by more than an order of magnitude. This implies that the adhesion properties of multibinding systems cannot be directly conveyed to the individual adhesion-receptor bonds.     

Purification and characterization of the CS2 pili of colonization factor antigen II produced by human enterotoxigenic Escherichia coli

A subtype (CS2) of the colonization factor antigen II (CFA/II) of human enterotoxigenic Escherichia coli (ETEC) was studied. Analysis revealed that CS2-possessing ETEC was predominant among isolates from traveller's diarrhea at Osaka, Japan. TH61 pili produced by a clinical strain (TH61) were purified as a native form to homogeneity by zone electrophoresis and successive column chromatographies on Sepharose 4B and Phenyl-Sepharose CL-4B. It was demonstrated by immunogold staining technique and bacterial agglutination test that antisera against the purified pili of strain TH61 recognized pili of both strain TH61 and strain #C91f, a control strain possessing only CS2 pili. This suggests that TH61 pili purified in this study are CS2 pili. Subunit (pilin) of the purified pili has a molecular weight of about 16,000. Strains bearing CS2 could attach to human jejunal epithelial cells, and this attachment was inhibited by pretreating the enterocytes with purified pili. These indicate that CS2 pili are a factor responsible for attachment of ETEC bearing CS2 to human intestinal cells.     

Exploring the genome sequence of Bifidobacterium bifidum S17 for potential players in host-microbe interactions

Bifidobacterium bifidum S17 is a promising probiotic candidate, which displays potent anti-inflammatory activity both in vitro and in vivo. Furthermore, tight adhesion to intestinal epithelial cells (IECs) has been shown for this strain and is considered to be an important prerequisite for host colonization and consequently the anti-inflammatory properties. By analyzing the recently sequenced genome at least 9 surface proteins and various gene clusters encoding for putative pili were identified. Most of these proteins are expressed in vitro, with higher expression during exponential growth phase. Increased expression of the putative adhesins in exponential growth phase was associated with higher adhesion of B. bifidum S17 to Caco-2 cells.