N-Terminal amino acid sequence of pilin isolated from Pseudomonas aeruginosa.

The amino-terminal amino acid sequence of the pili protein from Pseudomonas aeruginosa K pili is presented. The sequence is compared with those reported by others for pilin obtained from Neisseria gonorrhoeae and Moraxella nonlique-faciens. All three sequences are highly homologous, contain only two hydrophilic residues in the first 22 positions, and contain an unusual amino acid, N-monomethylphenylalanine, at the amino terminus.     

Inter-strain homology of pilin gene sequences in Neisseria meningitidis isolates that express markedly different antigenic pilus types

Neisseria meningitidis isolates examined in this study elaborated one of two pilus types that were antigenically markedly different. Each pilus type reacted either with SM1, a monoclonal antibody that recognizes an epitope common to all gonococcal pili, or with a polyclonal antiserum raised against meningococcal pili that did not react with SM1, but not both. Total genomic DNA from all N. meningitidis isolates analysed, irrespective of pilus type, contained at least one region with extensive homology to a gonococcal pilE probe. Different N. meningitidis strains possessed one of several configurations of genomic pilE-homologous segments. Chromosomal rearrangement of pilE-homologous sequences was associated with P+ to P- pilus phase transition in the strains examined. The arrangement of pilE-homologous segments in total genomic DNA from N. meningitidis isolated from the blood and cerebro-spinal fluid of the same patient was apparently identical.     

Nucleotide sequence of the structural gene for class I pilin from Neisseria meningitidis: homologies with the pilE locus of Neisseria gonorrhoeae

The nucleotide sequence has been determined for the expressed pilin (pilE) locus of Neisseria meningitidis strain C311 which produces class I pili that are antigenically and structurally similar to those of gonococci. The deduced amino acid sequence of the N. meningitidis pilE translation product contains a 7 amino acid N-terminal pre-pilin leader sequence which is identical to that found in gonococcal pilin and which is characteristic of N-methylphenylalanine pili in general. The succeeding N-terminal 53 amino acids are identical to those found in the equivalent position in antigenically variant gonococcal pilins and confirm direct peptide sequencing of the amino-terminus of at least one type of meningococcal pilin. Other regions that are conserved in variant pilin polypeptides from Neisseria gonorrhoeae are conserved at the amino acid level in the class I meningococcal pilin but the coding DNA contains numerous base substitutions when compared with the equivalent gonococcal pil sequence. Sequences extending downstream for about 140 bp on the 3' side of the coding region for both pilin genes are only about 85% homologous.     

PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC-deficient gonococci

Type 4 pili produced by the pathogenic Neisseria species constitute primary determinants for the adherence to host tissues. In addition to the major pilin subunit (PilE), neisserial pili contain the variable PilC proteins represented by two variant gene copies in most pathogenic Neisseria isolates. Based upon structural differences in the conserved regions of PilE, two pilus classes can be distinguished in Neisseria meningitidis . For class I pili found in both Neisseria gonorrhoeae and N. meningitidis , PilC proteins have been implicated in pilus assembly, natural transformation competence and adherence to epithelial cells. In this study, we used primers specific for the pilC2 gene of N. gonorrhoeae strain MS11 to amplify, by the polymerase chain reaction, and clone a homologous pilC gene from N. meningitidis strain A1493 which produces class II pili. This gene was sequenced and the deduced amino acid sequence showed 75.4% and 73.8% identity with the gonococcal PilC1 and PilC2, respectively. These values match the identity value of 74.1% calculated for the two N. gonorrhoeae MS11 PilC proteins, indicating a horizontal relationship between the N. gonorrhoeae and N. meningitidis pilC genes. We provide evidence that PilC functions in meningococcal class II pilus assembly and adherence. Furthermore, expression of the cloned N. meningitidis pilC gene in a gonococcal pilC1,2 mutant restores pilus assembly, adherence to ME-180 epithelial cells, and transformation competence to the wild-type level. Thus, PilC proteins exhibit indistinguishable functions in the context of class I and class II pili.     

Immunological heterogeneity of pili of Neisseria gonorrhoeae.

Sera of rabbits immunized with pili or formalized cultures of Neisseria gonorrhoeae contained pili antibodies. The reaction between pili and specific gamma globulin was followed by direct visual observation with an electron microscope. Using pili from 31 strains and antisera against three strains, only a few crossreactions between pili were observed. From this it is concluded that gonococcal pili are antigenically heterogeneous. However, serum raised with a T3 culture (with no detectable pili) contained antibodies against pili of the homologous T2 strain. It is proposed that the pilus antigen may exist in a form different from the typical pilus in gonococci.     

Purification of the Escherichia coli type 1 pilin and minor pilus proteins and partial characterization of the adhesin protein.

Type 1 pili of Escherichia coli contain three integral minor proteins with apparent molecular weights (Mr) of 28,000 (28K protein), 16,500, and 14,500 attached to rods composed of Mr-17,000 pilin subunits (Hanson and Brinton, Nature [London] 322:265-268). We describe here an improvement on our earlier method of pilus purification, which gives higher yields and higher purity. Also reported are methods allowing fractionation of intact type 1 pili into rods of pure pilin and free minor proteins, as well as fractionation of the 28K tip adhesion protein from the 16.5K and 14.5K proteins. We have determined the amino acid composition and amino-terminal sequence of the adhesion protein. This sequence shows limited homology with the amino-terminal sequences of several E. coli pilins, including type 1.     

Characterization of the major iron-regulated protein of Neisseria gonorrhoeae and Neissereria meningitidis

The major iron-regulated protein (MIRP) was purified, from both Neisseria gonorrhoeae and N. meningitidis by selective extraction with cetyltrimethylammonium bromide followed by ion-exchange and moleculair-seive chromatography. Solutions of the purified proteins had a characteristic pink color. The overall amino acid composition of these proteins was similar, although differences were noted in the number of serine, threonine, and lysine residues. Nevertheless, the N-terminal amino acid sequence was identical through 47 residues for both the meningococcal and gonococcal MIRP. Plasma emission spectrophotometry revealed that the meningococcal 37K protein contained ca. 1 mole Fe/mole protein.     

Identification of epitopes recognized by monoclonal antibodies SM1 and SM2 which react with all pili of Neisseria gonorrhoeae but which differentiate between two structural classes of pili expressed by Neisseria meningitidis and the distribution of their encoding sequences in the genomes of Neisseria spp

The pili expressed by all isolates of Neisseria gonorrhoeae react with two monoclonal antibodies, SM1 and SM2. In contrast, although many isolates of Neisseria meningitidis also express pili (class I) which react with antibodies SM1 and SM2, a proportion express pili (class II) which fail to react. In order to define the epitopes recognized by these antibodies, a series of overlapping peptides corresponding to the amino acid sequence of conserved regions of gonococcal pili have been synthesized. The minimum epitope recognized by antibody SM1 was found to comprise a linear peptide EYYLN, corresponding to residues 49-53 of mature pilin. In contrast, antibody SM2 reacted with a number of peptides from around the cysteine residue (Cys 1) at position 120, suggesting that an extended region may contribute to a conformational epitope recognized by this antibody in the native protein. The identification of the two epitopes defines structural differences between the classes of pili expressed by meningococci. In order to determine the distribution of pilin gene sequences in Neisseria we used as hybridization probes an oligonucleotide (PS1) with the sequence 5'-GAGTATTACCTGAATCA-3' which spans the coding region for the SM1 epitope, and a fragment of the 3' end of the gonococcal pilE gene which contains conserved sequences flanking the two Cys codons and encodes the SM2 epitope. All strains of N. gonorrhoeae and N. meningitidis tested, regardless of piliation phenotype, harboured DNA sequences homologous to those encoding the carboxy-terminus of meningococcal class I pilin. Furthermore, all gonococci and all meningococci producing class I pili hybridized with oligonucleotide probe PS1. Non-reverting non-piliated derivatives of previously class I pilus-producing strains showed reduced hybridization signals with this probe, but nevertheless retained sequences homologous to the coding sequence for the SM1 epitope. However, meningococci producing class II pili could be divided into two groups on the basis of their reaction with the PS1 probe: half the strains tested failed to react, which is consistent with our previous analysis of silent class I pilin sequences; the remainder reacted (relatively weakly) with the probe, suggesting that the silent pil sequences in these strains extend further towards the 5' end of the pilin gene than in strains studied previously. Some strains of Neisseria lactamica reacted weakly with both types of probe but failed to produce SM1-reactive pili. In contrast, isolates of Neisseria flava, Neisseria pharyngis, Neisseria sicca and a series of unrelated bacteria failed to react with both SM1 antibody and the DNA probes. This confirms that possession of 'gonococcal' pilin sequences is limited to the pathogenic neisseriae.     

Amino acid sequences of pilins from serologically distinct strains ofBacteroides nodosus

Amino acid sequences of pilin from a strain of Bacteroides nodosus from serogroup B (234) and serogroup C (217) were determined. The amino-terminal N-methylphenlalanine residue of both proteins was followed by a hydrophobic sequence of 30 residues closely related to the N-terminal sequence of other pili having an amino-terminal residue of N-methylphenylalanine. These data lend support to the hypothesis that in pilins of this type, the amino-terminal sequence functions as a transport signal necessary for pilin to reach its external environment, as well as promoting intersubunit interactions for muintenance of the structural integrity of the pilus. Two hydrophilic hypervariable regions can be discerned across the pilin sequences, indicating possible locations of antigenic domains.     

Amino acid sequences of pilins from serologically distinct strains ofBacteroides nodosus

Amino acid sequences of pilin from a strain of Bacteroides nodosus from serogroup B (234) and serogroup C (217) were determined. The amino-terminal N-methylphenlalanine residue of both proteins was followed by a hydrophobic sequence of 30 residues closely related to the N-terminal sequence of other pili having an amino-terminal residue of N-methylphenylalanine. These data lend support to the hypothesis that in pilins of this type, the amino-terminal sequence functions as a transport signal necessary for pilin to reach its external environment, as well as promoting intersubunit interactions for muintenance of the structural integrity of the pilus. Two hydrophilic hypervariable regions can be discerned across the pilin sequences, indicating possible locations of antigenic domains.     

The preparation and properties of gonococcal pili.

Pili have been isolated from Neisseria gonorrhoeae by controlled homogenization followed by selective disaggregation in sucrose and purification by CsCl density gradient centrifugation. Pili from six gonococcal strains had buoyant densities of 1-30 to 1-31 g ml-1 on CsCl. The pili were immunologically distinct when tested with rabbit antisera to purified pili. The amino acid composition of pilin from strains P9 and 201 was very similar, consisting of 208 and 212 amino acid residues respectively giving molecular weights of 22 600 and 22352. The pili contained a high proportion (46%) of non-polar amino acids. Further analysis of strain P9 pili revealed the presence of 1 to 2 phosphate groups and 1 to 2 hexose groups per pilin subunit; no amino sugars were detected. Pili from strain P9 were resolved into two bands by equilibrium density gradient centrifugation or column isoelectric focusing, suggesting the presence of more than one kind of pilus.     

Structure and antigenic properties of the tip-located P pilus proteins of uropathogenic Escherichia coli.

Pyelonephritogenic Escherichia coli frequently expresses pili which bind to Gal alpha (1-4)Gal receptors present on the uroepithelium. Binding of these pili is mediated by a pilus-associated adhesin, PapG, and not by the major subunit which constitutes the bulk of the pilus structure. The adhesin and two pilinlike proteins, PapE and PapF, are present in only a few copies each at the pilus tip. Surface exposure of both PapF and PapG is required to achieve receptor-specific binding. The nucleotide sequences for the genes encoding the tip-associated proteins PapE, PapF, and PapG were determined for two E. coli clones expressing P pili of serotypes F11 and F7(2) and compared with the corresponding sequences established for proteins of F13 pili. Specific antisera were used to study the cross-reactivity between the F13 tip proteins and the equivalent proteins in F11 and F7(2) pili. We present data showing that, like the major pilus subunit, PapE varies its structure and antigenic properties among pili of different serotypes. In contrast, the PapF protein was highly conserved, and PapF-specific antisera raised against serotype F13 cross-reacted with the PapF proteins of both F11 and F7(2) serotypes. The PapG adhesin protein from F11 and F7(2) pili differed by only five amino acids out of 316 residues. However, the F13 adhesin showed only 45% amino acid homology with the other two variants.     

Pilus biogenesis and epithelial cell adherence of Neisseria gonorrhoeae pilC double knock-out mutants

The phase-variable PilC proteins of pathogenic Neisseria species have recently been implicated in both assembly and cellular adherence functions of the type 4 pili of these pathogens. We describe here the cloning of full-length pilC1 and pilC2 genes and the complete sequencing of the pilC2 gene of Neisseria gonorrhoeae MS11. Sequential inactivation of both genes by gene replacement in piliated (P⁺) variants of N. gonorrhoeae MS11 led initially to a non-piliated (P⁻) phenotype; however, spontaneous P⁺ variants could be derived from some pilC1,2 double mutants which produced morphologically intact pili. Purified pili from pilC1,2 mutants revealed no detectable PilC protein. Instead, a novel protein about 70 kDa in size appeared in the pili preparations of P⁺ mutants; this protein exhibited no immunological cross-reactivity with PilC1 or PilC2. We propose that this novel factor replaces the function of PilC in pilus biogenesis. Using isogenic N. gonorrhoeae strains which produce identical PilE (pilin) proteins we demonstrate that pili associated with the 70 kDa protein do not confer gonococcal adherence to human epithelial cells, in contrast to pili assembled in the presence of PilC1 or PilC2.     

N-terminal amino acid sequences and amino acid compositions of the Spirochaeta aurantia flagellar filament polypeptides.

The amino-terminal sequences and amino acid compositions of the three major and two minor polypeptides constituting the filaments of Spirochaeta aurantia periplasmic flagella were determined. The amino-terminal sequence of the major 37.5-kDa outer layer polypeptide is identical to the sequence downstream of the proposed signal peptide of the protein encoded by the S. aurantia flaA gene. However, the amino acid composition of the 37.5-kDa polypeptide is not in agreement with that inferred from the sequence of flaA. The 34- and 31.5-kDa major filament core polypeptides and the 33- and 32-kDa minor core polypeptides show a striking similarity to each other, and the amino-terminal sequences of these core polypeptides show extensive identity with homologous proteins from members of other genera of spirochetes. An additional 36-kDa minor polypeptide that occurs occasionally in preparations of S. aurantia periplasmic flagella appears to be mixed with the 37.5-kDa outer layer polypeptide or a degradation product of this polypeptide.     

Molecular cloning and analysis of genes for sialic acid synthesis in Neisseria meningitidis group B and purification of the meningococcal CMP-NeuNAc synthetase enzyme.

The gene encoding for the CMP-NeuNAc synthetase enzyme of Neisseria meningitidis group B was cloned by complementation of a mutant of Escherichia coli defective for this enzyme. The gene (neuA) was isolated on a 4.1-kb fragment of meningococcal chromosomal DNA. Determination of the nucleotide sequence of this fragment revealed the presence of three genes, termed neuA, neuB, and neuC, organized in a single operon. The presence of a truncated ctrA gene at one end of the cloned DNA and a truncated gene encoding for the meningococcal sialyltransferase at the other confirmed that the cloned DNA corresponded to region A and part of region C of the meningococcal capsule gene cluster. The predicted amino acid sequence of the meningococcal NeuA protein was 57% homologous to that of NeuA, the CMP-NeuNAc synthetase encoded by E. coli K1. The predicted molecular mass of meningococcal NeuA protein was 24.8 kDa, which was 6 kDa larger than that formerly predicted (U. Edwards and M. Frosch, FEMS Microbiol. Lett. 96:161-166, 1992). Purification of the recombinant meningococcal NeuA protein together with determination of the N-terminal amino acid sequence confirmed that this 24.8-kDa protein was indeed the meningococcal CMP-NeuNAc synthetase. The predicted amino acid sequences of the two other encoded proteins were homologous to those of the NeuC and NeuB proteins of E. coli K1, two proteins involved in the synthesis of NeuNAc. These results indicate that common steps exist in the biosynthesis of NeuNAc in these two microorganisms.     

The identification of cryptic rhamnose biosynthesis genes in Neisseria gonorrhoeae and their relationship to lipopolysaccharide biosynthesis.

Neisseria gonorrhoeae synthesizes a rough lipopolysaccharide that does not contain any of the repetitive units characteristic of the smooth lipopolysaccharide of members of the family Enterobacteriaceae. Three gonococcal homologs of Salmonella serovar typhimurium genes involved in the synthesis of the rhamnose component of the repetitive subunits have been isolated. Gonococcal homologs for rfbB, rfbA, and rfbD were found downstream of the galE gene in a region of the chromosome which shows overall homology with the meningococcal capsule gene complex region D. Sequence alignment demonstrated that the gonococcal gene products have 69, 65, and 54% amino acid identity with the Salmonella proteins RfbB, RfbA, and RfbD. The gonococcal RfbB and RfbA amino acid sequences share even more identical residues (73 and 65%, respectively) with the amino acid sequences derived from Escherichia coli genes o355 and o292, respectively. These genes are clustered with the genes involved in the biosynthesis of enterobacterial common antigen, and o355 is listed in the GenBank and Swiss Protein data banks as rffE (encoding UDP-GlcNAc-2-epimerase). However, complementation studies demonstrated that o355 does not encode the enzyme UDP-GlcNAc-2-epimerase. Gonococcal strains constructed with null mutations in the rfbBAD genes were unchanged in lipopolysaccharide phenotype and in the synthesis of gonococcal carbohydrate-containing C antigen. We were unable to detect any changes in gonococcal phenotype with respect to lipopolysaccharide sialylation, monoclonal-antibody binding, serum sensitivity, or interaction with eukaryotic cells in vitro. We conclude that the absence of a homolog for rfbC precludes the existence of a functional dTDP-rhamnose biosynthesis pathway in the gonococcal strains examined and that these genes are only maintained in N. gonorrhoeae either because of the presence of the galE gene or because of another as yet unrecognized function.     

Assembly of CS1 pili: the role of specific residues of the major pilin, CooA

CS1 pili are important virulence factors of enterotoxigenic Escherichia coli strains associated with human diarrheal disease. They are the prototype for a family of pili that share extensive sequence similarity among their structural and assembly proteins. Only four linked genes, cooB, cooA, cooC, and cooD, are required to produce CS1 pili in E. coli K-12. To identify amino acids important for the function of the major pilin CooA, we used alanine substitution mutagenesis targeting conserved residues in the N and C termini of the protein. To test function, we examined cooA mutants for the ability to agglutinate bovine erythrocytes. Each hemagglutination-negative (HA(-)) cooA mutant was examined to identify its assembly pathway defect. CooA has been shown to be degraded in the absence of CooB (K. Voegele, H. Sakellaris, and J. R. Scott, Proc. Natl. Acad. Sci. USA 94:13257-13261, 1997). We found several HA(-) cooA mutants that produced no detectable CooA, suggesting that recognition by CooB is mediated by residues in both the N and C termini of CooA. In addition, we found that alanine substitution for some of the conserved residues in the C-terminal motif "AGxYxG(x(6))T," which is found in all subunits of this pilus family, had no effect on pilus formation. However, alanine substitution for some of the alternating hydrophobic residues within this motif prevented CooA from interacting with CooD, which serves as both the tip adhesin and nucleation protein for pilus formation. Thus, it appears that some, but not all, of the residues in both the N and C termini of CooA play a critical role in the intermolecular interactions of the major pilin with the other structural and assembly proteins. We anticipate that the results obtained here for CS1 pili in enterotoxigenic E. coli will help develop an understanding of the pilus assembly pathway used by CS1 family members in several important human pathogens.     

Evidence of two polygalacturonases produced by a mycorrhizal ericoid fungus during its saprophytic growth

Abstract The NH₂-terminal amino sequence through the first 20 amino acids was obtained for transferrin-binding protein (TBP)1 from three strains of Neisseria meningitidis . These were identical except for a glutamine to a glycine substitution at residue 6 in one case. The sequences of the NH₂-terminal 20 amino acids of TBP2 from the same three strains were also determined; one TBP2 had a M _r of 68 000 and the other two of 78 000. Sequences were identical up to residue 13 in all three proteins. Peptides based on the NH₂-terminal sequences of TBP1 and 2 were synthesized, linked to Keyhole Limpet haemocyanin and used to raise antibodies in rabbits. Anti-peptide antibodies cross-reacted on immunoblotting with the respective TBPs from all meningococcal strains tested, as well as with those from N. gonorrhoeae suggesting that the NH₂-terminals of these proteins are well conserved in the Neisseria . Neither anti-peptide serum reacted with the analogous TBP1 and 2 from Haemophilus influenzae , although common epitopes have previously been shown to exist.