Quantitative test for Bacteroides nodosus pilus protein by an agglutination-absorption test

A pili agglutination-absorption test (PAAT) was developed for the quantitative measurement of pilus protein in cultures of Bacteroides nodosus and to quantify pili yields during purifications. The test was calibrated by recording the amount of pilus protein required to absorb a measurable amount of anti-pili antibody from antiserum. The amount of anti-pilus antibody in absorbed and unabsorbed serum was specifically measured by a Bact. nodosus K-agglutination test. The PAAT could be calibrated using any combination of crude or pure pili preparations and specific anti-pili serum or non-specific anti-Bact. nodosus serum. This had advantages over the use of radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques for measurement of pili because they require the use of highly purified reagents for calibration. The minimum quantity of pilus protein measurable by PAAT was 0.1 microgram per test which was similar in sensitivity to that reported for RIA and ELISA. The reagents used in PAAT were stable for at least six months. The amount of pilus protein per bacterium as measured by PAAT was directly proportional to the average number of pili per bacterium as measured by electron microscopy. The test was Bact. nodosus serotype specific.     

Expression of multiple types of N-methyl Phe pili in Pseudomonas aeruginosa

The nature of pili synthesized by Pseudomonas aeruginosa when plasmid-borne genes of homologous pilins from Bacteroides nodosus are introduced as thermoregulated expression systems has been ascertained. Expression of B. nodosus pili inhibited the production of indigenous P. aeruginosa pili, and an organism harbouring pilin genes from two strains of B. nodosus produced two serologically distinct populations of pili on each cell. Simultaneous production of both indigenous and foreign pili was achieved by partial induction of expression. Homogeneity in pilus structure suggests either that there is an exclusive specificity of interaction between identical pilin subunits in pilus assembly, or that each pilus is produced from the translation products of a single messenger RNA molecule, with translation and pilus assembly closely coupled.     

Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species

Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.     

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.     

Electron microscopic studies of the insertion of pili in the newly isolated hydrogen-oxidizing bacterium 2K1]

Cells of the hydrogen-oxidizing bacterium 2K1 exhibited numerous pili of varying length, with a diameter of 7--8 nm as measured from negatively stained samples. At the basis of each pilus, a cell wall differentiation was observed appearing, in face-on-view, as a ring-like structure made up of subunits, and in side-on view as a hollow cylinder penetrating through the cell wall. It is assumed that these ring-like structures are in direct relation with the presence of the pili. As the number of ring-like structures per cell was higher than the number of pili per cell, rings without associated pili might either be sites of polymerizing pili, or "dead" pilus channel complexes out of function due to pilus break-down.     

Isolation of the gene encoding pilin of Bacteroides nodosus (strain 198), the causal organism of ovine footrot

The gene for pilin, the monomeric protein subunit from which the pilus of Bacteroides nodosus is constructed, has been isolated. Isolation was achieved by cloning the fragmented genome of B. nodosus in Escherichia coli RR1 using the plasmid vector pBR322. Pilin-producing colonies were identified by screening with a colony immunoassay using antiserum from a sheep immunized against purified pili from B. nodosus strain 198, and were further characterized by immunoblot analysis. Final confirmation of the presence of the pilin gene was by nucleotide sequence data which translated to the known pilin amino acid sequence.     

The human antibody response to uropathogenic Escherichia coli: a review

Urinary tract infections caused by Escherichia coli are associated with a local and systemic antibody response. We have studied the serum and urine antibody responses to Escherichia coli in men and women with pyelonephritis, cystitis, and asymptomatic bacteriuria. Protein immunoblots consistently demonstrated serum antibody response to lipopolysaccharide (LPS). Anti-LPS antibody titres rose significantly and progressively when comparing acute with convalescent sera in those who have had their first urinary infection. For those with repeated infections, high titre LPS antibodies were present and did not change significantly between acute and convalescent sera. Antibody responses to the major outer membrane proteins were present but did not differ significantly when compared with normal human serum. A specific anti-P pilus antibody response was demonstrated by immunoblotting. Anti-P pilus antibody was quantitated using ELISA and the titres were found to be very low. Three other techniques were also used to demonstrate the presence of serum antibody. Antibody was detectable by immunofluorescence, but the antigenic specificity of the antibody was more difficult to ascertain. Immunoprecipitation was more specific for determining the nature of the antibody response. Lastly, immunoelectron microscopy was valuable in demonstrating antipilus and antiflagellar antibodies. Immunoelectron microscopy and immunoblotting provided evidence that human antiserum to P pili was modestly cross-reactive and could bind heterologous P pili. These studies indicated that the major antibody response in humans occurs after pyelonephritis and is directed against LPS. An anti-P pilus response is frequently present and is cross-reactive to some extent with other P pili.     

Integrity of Escherichia coli P pili during biogenesis: properties and role of PapJ

The papJ gene of uropathogenic Escherichia coli is required to maintain the integrity of Gal alpha (1-4)Gal-binding P pili. Electron microscopy and ELISA have established that strains carrying the papJ1 mutant allele have a large amount of pilus antigen free of the cells. In contrast to the whole pili released by strains unable to produce the PapH pilus anchor, the free papJ1 pili consist of variably sized segments that appear to result from internal breakages to the pilus. The DNA sequence of papJ is presented and its gene product identified as an 18kD periplasmic protein that possesses homology with nucleotide-binding proteins. PapJ may function as a 'molecular chaperone' directly or indirectly establishing the correct assembly of PapA subunits in the P pilus.     

2型猪链球菌菌毛结构亚蛋白tSSU0473的原核表达与免疫学特性研究

目的：探究2型猪链球菌（S.suis2）强毒株05ZYH33的srtF基因簇编码的菌毛结构亚蛋白SSU0473的细菌定位及其免疫保护效能。方法：原核表达截短的SSU0473（tSSU0473）,并以亲和层析法纯化目的蛋白,Western印迹检测tSSU0473蛋白的免疫原性,ELISA法检测多抗血清的效价及IgG亚型,小鼠试验测试重组蛋白的免疫保护效能,免疫电镜观测tSSU0473蛋白的细菌定位。结果：在原核系统中表达了tSSU0473蛋白;ELISA结果显示重组蛋白能够刺激小鼠产生高效价的免疫抗体;动物试验表明tSSU0473蛋白免疫小鼠可抵御致死剂量病原体的攻击,显示出较好的免疫保护作用;免疫电镜检测显示tSSU0473蛋白定位于细菌表面。结论：菌毛亚蛋白tSSU0473是S.suis2膜表面蛋白,具有良好的免疫原性和免疫保护性,可作为S.suis2亚单位疫苗的候选分子。研究结果为系统揭示S.suis2的菌毛生物学结构与功能奠定了基础。     

Uptake of extracellular DNA: Competence induced pili in natural transformation of Streptococcus pneumoniae

Transport of DNA across bacterial membranes involves complex DNA uptake systems. In Gram‐positive bacteria, the DNA uptake machinery shares fundamental similarities with type IV pili and type II secretion systems. Although dedicated pilus structures, such as type IV pili in Gram‐negative bacteria, are necessary for efficient DNA uptake, the role of similar structures in Gram‐positive bacteria is just beginning to emerge. Recently two essentially very different pilus structures composed of the same major pilin protein ComGC were proposed to be involved in transformation of the Gram‐positive bacterium Streptococcus pneumoniae – one is a long, thin, type IV pilus‐like fiber with DNA binding capacity and the other one is a pilus structure that was thicker, much shorter and not able to bind DNA. Here we discuss how competence induced pili, either by pilus retraction or by a transient pilus‐related opening in the cell wall, may mediate DNA uptake in S. pneumoniae.     

Pilus Biogenesis in Lactococcus lactis: Molecular Characterization and Role in Aggregation and Biofilm Formation

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used.     

Major antigenic determinants of F and ColB2 pili.

F-like conjugative pili are expressed by plasmids with closely related transfer systems. They are tubular filaments that are composed of repeating pilin subunits arranged in a helical array. Both F and ColB2 pilin have nearly identical protein sequences, and both contain an acetylated amino-terminal alanine residue. However, they differ by a few amino acid residues at their amino termini. Rabbit antisera raised against purified F and ColB2 pili are immunologically cross-reactive by only 25%, as measured by a competition enzyme-linked immunosorbent assay (ELISA). A tryptic peptide corresponding to the first 15 amino acid residues of ColB2 pilin was isolated and found to remove nearly 80% of ColB2 pilus-directed rabbit antibodies. The corresponding tryptic peptide from F pilin, which reacted with anti-F pilus antibodies to remove 80%, was less than 20% reactive with anti-ColB2 pilus antiserum. Cleavage of these peptides with cyanogen bromide (at a methionine residue approximately in the middle of the peptide) did not affect the antigenicity of these peptides. Synthetic N alpha-acetylated peptides corresponding to the first eight amino acids of F pilin (Ac-Ala-Gly-Ser-Ser-Gly-Gln-Asp-Leu-COOH) and the first six amino acids of ColB2 pilin (Ac-Ala-Gln-Gly-Gln-Asp-Leu-COOH) were prepared and tested by competition ELISA with homologous and heterologous anti-pilus antisera. The F peptide F(1-8) inhibited the interaction of F pili and anti-F pilus antiserum to 80%, while the ColB2 peptide ColB2(1-6) inhibited anti-ColB2 pilus antiserum reacting with ColB2 pili by greater than 60%. The two peptides F(1-8) and ColB2(1-6) were inactive by competition ELISAs with heterologous antisera. These results suggest that the major antigenic determinant of both F and ColB2 pili is at the amino terminus of the pilin subunit and that 80% of antibodies raised against these pili are specific for this region of the pilin molecule.     

Localizing the subunit pool for the temporally regulated polar pili of Caulobacter crescentus

The pili of the stalked bacterium Caulobacter crescentus are assembled at a specific time in the life cycle at one pole of the cell and are composed of the monomer protein, pilin. A previous study demonstrated that the onset of pilin synthesis occurs well before pili appear on the surface, suggesting that pilin accumulates within the cell. In the present study, an electron microscope immunocytochemistry assay was used to determine the subcellular location of this unassembled pilin and its fate during pilus assembly and cell division. Populations of synchronously growing cells were embedded in epoxy resin at selected times during the cell cycle. Ultrathin sections were treated with pilin-specific antibody, followed by protein A coupled to colloidal gold. It was determined that the cellular location for unassembled pilin was the cell cytoplasm. All cell membranes and regions of nuclear material were poorly labeled. Quantitation demonstrated that label density increased during the period of pilin synthesis and declined during the period of pilus assembly and maintenance. The pilin pool was not unequally segregated at division; e.g., to the daughter cell that is elaborating pili. Mutants which have simultaneously lost the ability to produce flagella, pili, and other polar organelles, possibly due to alterations in the specialized region of polar organelle assembly, were also examined by the immunocytochemistry technique. There was no significant difference in the pilin pool size relative to the wild type, indicating that pilin synthesis continues in the absence of a functioning assembly site. This pattern of synthesis and assembly for the pilus is significantly different from that of the polar flagellum which is produced at the same time and location on the cell surface. These findings are discussed in relation to the hypothesized organization center at the cell pole which may have a major role in directing the assembly of all the polar structures.     

Location of the antigenic determinants of conjugative F-like pili

The amino terminus of the pilin protein constitutes the major epitope of F-like conjugative pili studied to date (F, ColB2, R1-19, R100-1, and pED208). Anti-pED208 pilus antibodies were passed through a CNBr-Sepharose affinity column linked to bovine serum albumin which was conjugated to a synthetic peptide, AcP(1-12), containing the major epitope at the amino terminus of pED208 pilin. This allowed the separation of two classes of antibodies; one was specific for the amino terminus and bound to the column, while the other, which recognizes a second epitope on the pilus, did not bind to the column. In addition, antibodies were raised against two amino-terminal peptide-bovine serum albumin conjugates [AcP(1-8) and AcP(1-12)] to ensure a source of pure, high-titer antibodies directed against the amino terminus. The location of these antibodies on intact pili was assayed by immunoelectron microscopy with a protein A-gold technique. The amino terminus-specific antibodies did not bind to the sides of the pili but appeared to be associated with the pilus tip. In addition, these antibodies were found to bind to the vesicle-like structure at the base of the pilus. The anti-pilus antibodies not specific for the amino terminus (unbound immunoglobulin G) were found to bind to the sides of the pilus. Anti-F and anti-ColB2 pilus antibodies bound to the sides of F, ColB2, and R1-19 pili, which have only their secondary epitope in common. The carboxyl-terminal lysine of R1-19 pilin prevents the absorption of anti-F plus antiserum but not anti-ColB2 pilus antiserum to the sides of the pilus, presumably by interfering with the recognition of this secondary epitope.     

Preparation and characterization of detoxified lipopolysaccharide-protein conjugates

Alkaline treatment of Pseudomonas aeruginosa type 5 lipopolysaccharide (LPS) resulted in reduced toxicity as measured by both the Limulus amoebocyte assay and the rabbit pyrogenicity test. Chemical analysis of the deacylated LPS (D-LPS) revealed that ester-linked fatty acids were removed while the amide-linked fatty acids remained intact. The neutral and amino sugar compositions for native LPS and D-LPS were identical within experimental error. Antigenic determinants for complement-dependent human opsonic antibody were retained under these deacylation conditions. To enhance its immunogenicity, D-LPS was covalently coupled to Pseudomonas pili and the 1,4-diaminobutyl derivatives of Pseudomonas exotoxin A and tetanus toxoid. Quantitative amino sugar analyses revealed that 2.6 and 3.2 mol of D-LPS were covalently bound to aminobutyl Pseudomonas exotoxin A and aminobutyl tetanus toxoid, respectively. Gel electrophoresis data indicated at least 1 mol of D-LPS covalently bound per pilus subunit protein. Initial immunologic data indicated that antibody against D-LPS could be induced when the D-LPS is covalently attached to protein carriers.     

PapD, a periplasmic transport protein in P-pilus biogenesis.

The product of the papD gene of uropathogenic Escherichia coli is required for the biogenesis of digalactoside-binding P pili. Mutations within papD result in complete degradation of the major pilus subunit, PapA, and of the pilinlike proteins PapE and PapF and also cause partial breakdown of the PapG adhesin. The papD gene was sequenced, and the gene product was purified from the periplasm. The deduced amino acid sequence and the N-terminal sequence obtained from the purified protein revealed that PapD is a basic and hydrophilic peripheral protein. A periplasmic complex between PapD and PapE was purified from cells that overproduced and accumulated these proteins in the periplasm. Antibodies raised against this complex reacted with purified wild-type P pili but not with pili purified from a papE mutant. In contrast, anti-PapD serum did not react with purified pili or with the culture fluid of piliated cells. However, this serum was able to specifically precipitate the PapE protein from periplasmic extracts, confirming that PapD and PapE were associated as a complex. It is suggested that PapD functions in P-pilus biogenesis as a periplasmic transport protein. Probably PapD forms complexes with pilus subunits at the outer surface of the inner membrane and transports them in a stable configuration across the periplasmic space before delivering them to the site(s) of pilus polymerization.     

Human buccal epithelial cell receptors of Pseudomonas aeruginosa: identification of glycoproteins with pilus binding activity

Adherence of Pseudomonas aeruginosa to a patient's epithelial surface is thought to be an important first step in the infection process. Pseudomonas aeruginosa is capable of attaching to epithelial cells via its pili, yet little is known about the epithelial receptors of this adhesin. Using nitrocellulose replicas of polyacrylamide gels of solubilized human buccal epithelial cells (BECs), glycoproteins (Mz: 82,000, and four bands between 40,000 and 50,000) that bound purified pili from P. aeruginosa strain K (PAK) were identified by immunoblotting with a pilus-specific monoclonal antibody that does not affect pilus binding to BECs (PK3B). All pilus-binding glycoproteins were surface localized, as determined by surface radioiodination of intact BECs. Binding of pili to all of the glycoproteins was inhibited by Fab fragments of monoclonal antibody PK99H, which inhibits PAK pili binding to BECs by binding to or near the binding domain of the pilus, but not by Fab fragments of monoclonal antibody PK41C, which binds to PAK pilin but does not inhibit pili binding to BECs, demonstrating that pilus binding to these glycoproteins is likely via the same region of the pilus that binds to intact BECs. Periodate oxidation of the blot eliminated pili binding to all glycoproteins, indicating that a carbohydrate moiety is an important determinant for pilus-binding activity. However, not all of the glycoproteins exhibited the same degree of sensitivity to periodate oxidation. Furthermore, monosaccharide inhibition of pilus binding to BECs implicated L-fucose and N-acetylneuraminic acid as receptor moieties.     

Defective F pili and other characteristics of Flac and Hfr Escherichia coli mutants resistant to bacteriophage R17.

Mutants resistant to the donor-specific bacteriophage R17 were isolated from Hfr and Flac-containing strains of Escherichia coli K-12. Thirty-five mutants were examined for the presence of F pili by electron microscopy. The pilus morphology was studied, as were the abilities of the cells to retract their pili and to synthesize new pili. Measurements were made of the efficiency of the conjugal deoxyribonucleic acid transfer and of M13 and R17 phage infection. All mutants had noticeable defects in pilus production, structure, or function. Mutants were found which produced unusually long pili, displayed wide variations in the number of pili per cell, and were deficient in pilus retraction and synthesis. Evidence is presented that there may be two pathways of pilus retraction.