Antipilus Antibody Research Articles

Mucosal vaccination with pili from Group A Streptococcus expressed on Lactococcus lactis generates protective immune responses

The human pathogen Group A Streptococcus (GAS) produces pili that are involved in adhesion and colonisation of the host. These surface-exposed pili are immunogenic and therefore represent an attractive target for vaccine development. The pilus is encoded in the genomic region known as the fibronectin-collagen-T-antigen (FCT)-region, of which at least nine different types have been identified. In this study we investigate expressing two of the most common FCT-types (FCT-3 and FCT-4) in the food-grade bacteria Lactococcus lactis for use as a mucosal vaccine. We show that mucosally delivered L. lactis expressing GAS pili generates specific antibody responses in rabbits. Rabbit anti-pilus antibodies were shown to have both a neutralising effect on bacterial adhesion, and immunised rabbit antiserum was able to facilitate immune-mediated killing of bacteria via opsonophagocytosis. Furthermore, intranasal immunisation of mice improved clearance rates of GAS after nasopharyngeal challenge. These results demonstrate the potential for a novel, pilus-based vaccine to protect against GAS infections.

Identification of a Monoclonal Antibody Against Pneumococcal Pilus 1 Ancillary Protein Impairing Bacterial Adhesion to Human Epithelial Cells.

The adhesion of Streptococcus pneumoniae is a key step during colonization of human respiratory tract mucosae. Here we demonstrate that pneumococcal type I pilus significantly increases the adhesiveness of poorly adhering highly capsulated strains in vitro. Interestingly, preincubation of bacteria with antibodies against the major pilus backbone subunit (RrgB) or the adhesin component (RrgA) impaired pneumococcal association to human epithelial cells. Screening for anti-RrgA monoclonal antibodies specifically affecting the adhesive capacity of S. pneumoniae led to the identification of the monoclonal 11B9/61 antibody, which greatly reduced pilus-dependent cell contact. Proteomic-based epitope mapping of 11B9/61 monoclonal antibody revealed a well-exposed epitope on the D2 domain of RrgA as the target of this functional antibody. The data presented here confirm the importance of pilus I for S. pneumoniae pathogenesis and the potential use of antipilus antibodies to prevent bacterial colonization.

Expression of the Type 1 Pneumococcal Pilus Is Bistable and Negatively Regulated by the Structural Component RrgA

The pneumococcal type 1 pilus, which is present in 25 to 30% of clinical isolates, has been associated with increased adherence and inflammatory responses and is being evaluated as a potential vaccine candidate. Here we show that expression of the pilus is bistable as a result of the molecular interaction between the transcription activator RrlA and a structural component of the pilus called RrgA. Sampling various clinical pneumococcal isolates that harbor the type 1 pilus-encoding islet, we show that distinct populations of cells can be identified with either undetectable or prominent pilus expression. When these two populations are separated and regrown in liquid medium, they are phenotypically different: the nonexpressing population reverts to the previous bimodal distribution, whereas the expressing population retains the same high level of pilus expression. Controlled exogenous expression of the regulatory pilus gene rlrA in a strain from which the endogenous version has been deleted increases pilus expression steadily, suggesting that the bistable expression of the pilus observed in wild-type cells is dependent on the native rlrA promoter. Finally, we demonstrate that RrgA is a negative regulator of pilus expression and that this repression is likely mediated through direct interaction with RlrA. We conclude that type 1 pilus expression in pneumococcus exhibits a bistable phenotype, which is dependent upon the molecular interplay between the RlrA and RrgA proteins. We suggest that this flexibility in expression may assist adaptation to a range of immune conditions, such as evasion of antipilus antibodies, within potential hosts.

Intratracheal immunization with pili protein protects against mortality associated withPseudomonas aeruginosapneumonia in mice

We examined the protective effect of intratracheal immunization with Pseudomonas aeruginosa pili protein against respiratory infection caused by P. aeruginosa. Mice were immunized intratracheally or subcutaneously with purified pili protein or bovine serum albumin as a control. Intratracheally but not subcutaneously pili protein-immunized mice showed significant improvement of survival after intratracheal challenge with the PAO1 strain. Furthermore, bacterial cell counts in pili protein-immunized murine lungs were significantly decreased compared to controls at 18 h after the challenge. Antipili protein antibody titers in bronchoalveolar lavage fluid of intratracheally pili protein-immunized mice were higher than in bovine serum albumin immunized mice. However, antipili antibody titers were not increased in bronchoalveolar lavage fluid of subcutaneously pili protein-immunized mice, despite the high serum antipili antibody titers. Inoculation of P. aeruginosa induced immediate increases in interleukin-12 and interferon-gamma in bronchoalveolar lavage fluid of pili protein-immunized mice, reflecting an adequate and rapid immune response against P. aeruginosa respiratory tract infection. Our findings suggest that intratracheal pili protein immunization is effective against respiratory tract infection caused by P. aeruginosa in mice.

Paradoxical Emergence of Humerus Osteomyelitis During Treatment of Pulmonary and Lymph-node Tuberculosis

Bacterial pili have been shown to be an important virulence factor for urinary tract infections. In this report we relate the results of studies which evaluated the influence of antipili antibody on the susceptibility of rats to ascending pyelonephritis and on several antibody-mediated antibacterial mechanisms. Rats immunized with E. coli type 1 pili, and animals infected with E. coli developed antipili antibodies in their serum. Active or passive immunization of rats with pili protected the animals from ascending pyelonephritis. Antipili antibody did not mediate complement-dependent bacteriolysis, opsonophagocytosis or promote more rapid intravascular clearance of injected E. coli. Humoral immunity to pili did, however, effectively inhibit bacterial adherence to epithelial cells. These studies indicate that type 1 E. coli pili are immunogenic and that antipili antibodies afford protection from ascending pyelonephritis. They suggest further that a mechanism of protection is inhibition of bacterial adherence.

Interaction of Neisseria meningitidis with a polarized monolayer of epithelial cells.

An important step in the pathogenesis of Neisseria meningitidis is the crossing of two cellular barriers, one in the nasopharynx and one in the brain. To approach the mechanisms by which this bacterium can achieve these goals, we studied the interactions between N. meningitidis and a monolayer of polarized tight junction-forming T84 cells grown on filter units. A capsulated, piliated, Opa-, and Opc- N. meningitidis strain is shown to be capable of adhering to and crossing this monolayer several orders of magnitude more efficiently than an isogenic nonpiliated derivative. This bacterial interaction does not affect the barrier function of tight junctions, as assessed by (i) the absence of modification of the transepithelial resistance, (ii) the lack of increase of [3H]inulin penetration across the monolayer, and (iii) the absence of delocalization of ZO-1, a tight junction protein. Electron microscopy studies and confocal examinations demonstrated that N. meningitidis (i) induces cytoskeletal rearrangements with actin polymerization beneath adherent bacteria, (ii) is intimately attached to the apical membrane of the cells, and (iii) can be internalized inside cells. Immunofluorescent staining with antipilus antibodies showed evidence that meningococcal piliation was dramatically reduced at later time points of bacterial cell interaction compared to the early phase of this interaction. In addition, adhesive bacteria recovered from an infected monolayer are piliated, capsulated, Opa-, and Opc-, a phenotype similar to that of the parental strain. Taken together, these data demonstrate that following pilus-mediated adhesion, N. meningitidis is involved in an intimate attachment which requires a bacterial component different from Opa and Opc and that meningococci cross a monolayer of tight-junction-forming epithelial cells by using a transcellular pathway rather than a paracellular route.

Purification and characterization of a pilus of a Vibrio cholerae strain: a possible colonization factor.

A new flexible type of pilus was purified from Vibrio cholerae non-O1, non-0139 strain NAGV14 and characterized. The molecular mass of the pilin was estimated to be 20 kDa, and the antigenicity differed from that of known pili such as toxin-coregulated pili, mannose-sensitive hemagglutinating pili, V10 pili, and Al-1841 pili. The NAGV14 pilus was regarded as a colonization factor because the purified pili adhered to rabbit intestine and adhesion was inhibited by treating the organisms with the Fab fraction of an antipilus antibody. An intestinal receptor blockade using purified pili failed to inhibit adhesion of the organisms. The NAGV14 pili adhered to the surface of live V. cholerae. An antigen cross-reacting with the NAGV14 pili was widely and specifically distributed among V. cholerae strains irrespective of serotype and biotype. The amino acid sequence of the pilin was homologous with that of MshA. The NAGV14 pili did not agglutinate human and rabbit erythrocytes.

Aeromonas trota strains, which agglutinate with Vibrio cholerae O139 Bengal antiserum, possess a serologically distinct fimbrial colonization factor.

Pili of Aeromonas trota strain 1220, which agglutinates with Vibrio cholerae O139 Bengal antiserum, were purified and characterized. The molecular mass of the subunit protein was estimated to be 20 kDa and the pl was 5 center dot 4. The pili were immunologically unrelated to the other Aeromonas pili reported so far. However, the N-terminal amino acid sequence of the subunit pilin was similar to those of the pilins from other Aeromonas pili reported previously. Neither A. trota cells nor pili purified from strain 1220 agglutinated human and rabbit erythrocytes, but both adhered to the rabbit intestine. Bacterial cells pretreated with antipilus antibody (Fab portion) failed to adhere to the rabbit intestine. Moreover, bacteria did not adhere to the rabbit intestine pretreated with the purified pili. This pilus antigen was not detected in V. cholerae O139 Bengal and other Aeromonas spp. These findings suggest that the pilus of the A. trota strain is a novel colonization factor of Aeromonas spp.

Some P. aeruginosa pilus-binding proteins of human corneal epithelium are cytokeratins.

The purpose of this study was to determine whether any of the previously identified human corneal epithelial pilus-binding proteins were cytokeratins. Soluble human corneal epithelial proteins (hcep) were separated by one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis under reducing conditions and transferred to nitrocellulose membrane for Western blot analysis. To characterize pilus-binding hcep (major proteins of < 21, 38, 45, 66 and 97 kDa and minor proteins, including a 55 kDa protein), blots were immunostained using three anti-keratin antibodies, including Pruss monoclonal antibody (MAb), specific for all classes of IFs, AE5 MAb, specific for a 64 kDa cytokeratin, and J7 polyclonal antibody (PAb), specific for a 55 kDa cytokeratin. In addition, major pilus-binding proteins were cut from 1-D SDS gel, electroeluted, dot blotted onto nitrocellulose membrane, and similarly analyzed. In addition, to further test whether any pilus-binding proteins were cytokeratins, soluble hcep were immunoprecipitated by MAb XLR-3, a specific anti-pilus antibody, after incubation with bacterial pili. The immunoprecipitated proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane. The blotted immunoprecipitated proteins were immunostained using Pruss MAb and MAb XLR-3. Immunoblots using Pruss MAb showed immunostaining of hcep of approximate molecular weights 45, 48, 55, 62 and 66 kDa. Other immunoblot analysis using AE5 MAb allowed identification of a 66 and a 45 kDa protein in both 1-D and dot blot analysis of eluted hcep. J7 PAb specifically immunostained a 55 kDa protein. In 2-D gel immunoblots, three 55 kDa proteins were immunostained by J7. Three proteins of molecular weights 45, 55 and 66 kDa, isolated after incubation of hcep and pili by immunoprecipitation with MAb XLR-3, also were positively immunostained by the Pruss MAb. Two of the previously identified major pilus-binding proteins of 45 and 66 kDa are cytokeratins.. Additionally, the 55 kDa minor pilus-binding protein is also a cytokeratin and appears to carry different electric charges. Novel approaches such as these provide new insight into the pathogenesis of P. aeruginosa infection in human cornea, and may lead to improved prevention and treatment of bacterial induced corneal disease.

Cell-associated hemagglutinin of classicalVibrio choleraeO1 with reference to intestinal adhesion

Vibrio cholerae O1 86B3 biovar cholerae has at least two types of cell-associated hemagglutinin. One is cell wall-associated and L-fucose sensitive, whereas the other is pili-associated and D-mannose sensitive. A pilus rich variant of 86B3 and a poorly piliated parent strain adhered equally to the rabbit intestine. This adhesion was inhibited by L-fucose but not by D-mannose. A Fab fraction prepared from anti-pilus antibody did not inhibit the adhesion. These results suggest that not the pili but a colonization factor located in the outer membrane of the organisms plays a role in intestinal adhesion.

Immune recognition of polar pili from Pseudomonas aeruginosa O

The B- and T-cell antigenic sites on type O pili from Pseudomonas aeruginosa were determined by using an antipilus antibody competition assay and PAO-immune T-cell blasts in proliferation studies. The citraconylated tryptic digest III region (residues 54 to 120) was determined to be an immunodominant site for both T and B cells on the pilin molecule.

Purification and characterization of Aeromonas sobria Ae24 pili: a possible new colonization factor

Pili of Aeromonas sobria Ae24 were purified and characterized. The molecular mass of the pilin was estimated to be about 19 kDa by SDS-PAGE. The Ae24 pili were electrophoretically distinguishable from previously reported Aeromonas hydrophila Ae6 W pili and A. sobria Ae1 pili, although all three had indistinguishable morphology and shared a high degree of homology in the N-terminal amino acid sequences. Strain Ae24 and its purified pili adhered to rabbit intestine and agglutinated human and rabbit erythrocytes. Hemagglutination was inhibited by D-galactose and D-mannose, but not by L-fucose. Organisms pretreated with Fab fraction of the antipilus antibody failed to adhere to the intestine. Organisms did not adhere to intestine pretreated with the purified pili. These findings suggest that the pili are a colonization factor of A. sobria Ae24 for the rabbit intestine, and that the receptor is galactose- and mannose-containing structure.

Characterization of Aeromonas sobria TAP13 pili: a possible new colonization factor.

Pili of Aeromonas sobria TAP13 were purified and characterized. The molecular mass of the pilin was estimated to be about 23 kDa by SDS-PAGE. The TAP13 pili were immunologically different from A. sobria Ae1 pili and A. hydrophila Ae6 W pili as previously reported, nevertheless all three had indistinguishable morphology and shared a high degree of homology in their N-terminal amino acid sequences. Strain TAP13 and its purified pili did not agglutinate human, rabbit or sheep erythrocytes. However, they adhered to rabbit intestine. Organisms pretreated with the Fab fraction of an antipilus antibody failed to adhere to rabbit intestine, and organisms did not adhere to intestine pretreated with purified pili. These results suggest that the pili are a colonization factor of A. sobria TAP13 for the rabbit intestine.

The role of pili in colonization of the rabbit intestine by Vibrio parahaemolyticus Na2.

Vibrio parahaemolyticus Na2 and its pili were studied in relation to intestinal colonization. The isolated pili were adhesive to the intestinal epithelium. The adhesion of the organisms was blocked by masking the epithelial receptor with the purified pili, or by treating the organisms with anti-pilus antibody (Fab fraction). These results suggest that the pili of V. parahaemolyticus Na2 play an important role in the adhesion of the organisms to the rabbit intestine.

Binding of nonmucoid Pseudomonas aeruginosa to normal human intestinal mucin and respiratory mucin from patients with cystic fibrosis.

Lung infections due to Pseudomonas aeruginosa and Pseudomonas cepacia are common in patients with cystic fibrosis. Initial colonization is due to nonmucoid P. aeruginosa, while later mucoid variants emerge and are associated with chronic infection. P. cepacia colonization tends to be more prevalent in older patients. The present study was conducted to discover whether highly purified mucins (from cystic fibrosis sputum and control intestinal secretions) exhibited specific binding of nonmucoid P. aeruginosa. In vitro solid phase microtiter binding assays (with or without a blocking agent) as well as solution phase assays were conducted. Bacteria bound to both mucins via bacterial pili, but no differences in binding capacity were noted between the mucins. Unlike P. cepacia (described in the accompanying manuscript) there was also no preferential binding of P. aeruginosa to mucins versus bovine serum albumin, casein, gelatin, or a host of structurally unrelated proteins and glycoproteins. Carbohydrate hapten inhibition studies did not suggest the existence of specific mucin carbohydrate receptors for P. aeruginosa. In solid phase assays a low concentration (0.05 M) of tetramethylurea abolished P. aeruginosa bacterial binding to both mucins as well as to BSA, whereas in solution phase assays mucin binding to bacteria was not completely disrupted by tetramethylurea. Specific monoclonal antipilus antibodies did not inhibit binding to a greater extent than did Fab fragments of normal mouse IgG. Binding of strains PAO1 and PAK (and isolated PAK pili) to buccal epithelial cells was not influenced by the presence of mucin in binding assay mixtures. Our findings do not support the widely held notion that specific mucin receptors are responsible for the attachment of P. aeruginosa pili, nor do they support the idea that there is a competitive interference by mucins of bacterial binding to respiratory cells. In patients with cystic fibrosis, it would seem unlikely therefore that initial colonization of the lungs by P. aeruginosa is due to a 'selective tropism' of these bacteria for respiratory mucin.

Purification and characterization of Aeromonas sobria pili, a possible colonization factor

Pili of Aeromonas sobria Ae1 were purified and characterized. The molecular mass of the pilin was estimated to be about 23 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Ae1 pili were electrophoretically and immunologically distinguishable from the W pili of A. hydrophila Ae6, although the two pili were morphologically indistinguishable. The N-terminal amino acid sequences of the two pilins were identical in the first 10 residues. Strain Ae1 and its purified pili adhered to human and rabbit intestines and agglutinated human and rabbit erythrocytes. Hemagglutination was inhibited by D-galactose and D-mannose, but not by L-fucose. Organisms pretreated with the Fab fraction of the antipilus antibody failed to adhere to the intestines. Organisms did not adhere to intestines pretreated with the purified pili. These findings suggest that the pili are a colonization factor of A. sobria Ae1.

Pili of an Aeromonas hydrophila Strain as a Possible Colonization Factor

Aeromonas hydrophila Ae6 had 2 morphologically distinctive kinds of pili. One appeared rigid and straight with a diameter of 9 nm (R-pili). The other appeared wavy and flexible with a diameter of 7 nm (W-pili). W-pili were very few on the cell as compared with R-pili. In this study, W-pili were purified and characterized. The pili consisted of a subunit protein with a molecular weight of 21 kDa as estimated by SDS-PAGE. There was no immunological cross-reaction between W-pili and other cellular components. The strain Ae6 and its purified W-pili adhered to human and rabbit intestine and agglutinated human and rabbit erythrocytes. Organisms pretreated with the Fab fraction of anti-pilus antibody failed to adhere to the intestine. Pretreatment of intestine with purified W-pili blocked adherence of the organisms to the intestine. These results suggest that the W-pili are the colonization factor of A. hydrophila Ae6.

Pili of Vibrio cholerae non-O1

Pili of Vibrio cholerae non-O1 strain S7 were purified and characterized. The pili of S7 were morphologically, electrophoretically, and immunologically (as far as polyclonal antibody was used) indistinguishable from the 16-kilodalton pili of V. cholerae O1 strain 82P7. The purified pili and organisms had D-mannose- and L-fucose-resistant hemagglutinin. The hemagglutinating activity of the purified pili was inhibited by the Fab fraction of antipilus antibody, but the hemagglutinating activity of live organisms was not inhibited completely. The purified pili or Fab fraction of antipilus antibody did not inhibit the adhesion of V. cholerae non-O1 to rabbit intestines. Therefore, the pili were not regarded as a colonization factor of V. cholerae non-O1. A total of 148 V. cholerae non-O1 and O1 clinical isolates were screened for the presence of S7 pili by using an agglutination test with anti-S7 pilus serum; 12 of 49 V. cholerae non-O1 strains and 25 of 99 V. cholerae O1 strains were positive for agglutination. These agglutination reactions were not correlated with adhesion of the organisms to intestines.

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.

Pili of a Vibrio parahaemolyticus strain as a possible colonization factor

Pili of Vibrio parahaemolyticus were purified from a Kanagawa phenomenon-positive strain (Ha7) that belongs to serogroup O2:K3 and is adhesive to rabbit intestine. The organisms treated with the Fab fraction of antipilus antibody failed to adhere to the intestine. Purified pili had the ability to adhere to the intestine, but the pretreatment of the intestine with purified pili did not allow adherence of the organisms to the intestine. These results suggest that pili of this V. parahaemolyticus strain play an important role in colonization.