The pili of Pseudomonas aeruginosa strains PAK and PAO bind specifically to the carbohydrate sequence βGalNAc(1–4)βGal found in glycosphingolipids asialo-GM1 and asialo-GM2

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. The pilus adhesin of P. aeruginosa strains PAK and PAO has been shown to bind to the glycolipid asialo-GM₁ (Lee et al., 1994 —accompanying article). PAK and PAO pili were examined for their abilities to bind to the synthetic βGalNAc(1–4)βGal (a minimal structural carbohydrate receptor sequence of asialo-GM₁ and asialo-GM₂ proposed by Krivan et al., 1988a) using solid-phase binding assays. Both pill specifically bound to βGalNAc(1–4)βGal. The binding of βGal-NAc(1–4)βGal-Biotin to the Immobilized PAK and PAO pili was inhibited by corresponding free pili. The receptor binding domain of the PAK pilus resides in the C-terminal disulphide-looped region (residues 128–144) of the pilin structural subunit (Irvin et al., 1989). Biotinylated synthetic peptides corresponding the C-terminal residues 128–144 of P. aeruginosa PAK and PAO pilin molecules were shown to bind to the βGalNAc(1–4)βGal-(bovine serum albumin (BSA)). The binding of biotinylated peptides to βGalNAc-(1–4)βGal-BSA was inhibited by PAK pili, Ac-KCTSDQDEOFIPKGCSK-OH (AcPAK(128–144)_ox-OH) and Ac-ACKSTQDPMFTPKGCDN-OH (AcPAO(128–144)_ox-OH) peptides. (In these peptides Ac denotes N^α -acetylation of the N-terminus, -OH means a peptide with a free a-carboxyl group at the C-terminus and the‘ox’denotes the oxidation of the sulphhydryl groups of Cys–129 and Cys–142.) Both acetylated peptides were also able to inhibit the binding of βGalNAc(1–4)βGal-biotin to the corresponding BSA-Peptide(128–144)_ox-OH conjugates. The βGlcNAc(1–3)βGal(1–4)βGlc-biotin conjugate was unable to specifically bind to either Immobilized PAK and PAO pili or the respective C-termlnal peptides. The data above demonstrated that the P. aeruginosa pili recognize asialo-GM₁ receptor analogue and that βGalNAc(1–4)βGal disaccharlde is sufficient for binding. Furthermore, the binding to βGalNAc(1–4)βGal was mediated by residues 128–144 of the pilin subunit.     

Crystal structure of Pseudomonas aeruginosa PAK pilin suggests a main-chain-dominated mode of receptor binding

Fibers of pilin monomers (pili) form the dominant adhesin of Pseudomonas aeruginosa, and they play an important role in infections by this opportunistic bacterial pathogen. Blocking adhesion is therefore a target for vaccine development. The receptor-binding site is located in a C-terminal disulphide-bonded loop of each pilin monomer, but functional binding sites are displayed only at the tip of the pilus. A factor complicating vaccination is that different bacterial strains produce distinct, and sometimes highly divergent, pilin variants. It is surprising that all strains still appear to bind a common receptor, asialo-GM1. Here, we present the 1.63 A crystal structure of pilin from P. aeruginosa strain PAK. The structure shows that the proposed receptor-binding site is formed by two beta-turns that create a surface dominated by main-chain atoms. Receptor specificity could therefore be maintained, whilst allowing side-chain variation, if the main-chain conformation is conserved. The location of the binding site relative to the proposed packing of the pilus fiber raises new issues and suggests that the current fiber model may have to be reconsidered. Finally, the structure of the C-terminal disulphide-bonded loop will provide the template for the structure-based design of a consensus sequence vaccine.     

Use of synthetic peptides to confirm that the Pseudomonas aeruginosa PAK pilus adhesin and the Candida albicans fimbrial adhesin possess a homologous receptor-binding domain

Pseudomonas aeruginosa PAK pili and Candida albicans fimbriae are adhesins present on the microbial cell surfaces which mediate binding to epithelial cell-surface receptors. The receptor-binding domain (adhesintope) of the PAK pilus adhesin has been shown previously to reside in the carboxy-terminal disulphide-bonded region of P. aeruginosa pilin (PAK128-144). The delineation of the C. albicans fimbrial adhesintope was investigated in these studies using synthetic peptides which correspond to the whole (PAK128-144) or part of (PAK134-140) adhesintope of the PAK pilus and their respective anti-peptide antisera and biotinylated PAK pili (Bt-PAK pili), fimbriae (Bt-fimbriae), P. aeruginosa whole cells (Bt- P. aeruginosa ) and C. albicans whole cells (Bt- C. albicans ). The results from these studies confirmed that a structurally conserved motif akin to the PAK(128-144) peptide sequence is present in C. albicans fimbrial adhesin and that the seven-amino-acid residue PAK(134-140) sequence plays an important role in forming the adhesintope for both P. aeruginosa PAK pilus and C. albicans fimbrial adhesins.     

The pilE gene product of Pseudomonas aeruginosa, required for pilus biogenesis, shares amino acid sequence identity with the N-termini of type 4 prepilin proteins

A new locus required for type 4 pilus biogenesis by Pseudomonas aeruginosa has been identified. A pilE mutant, designated MJ-6, was broadly resistant to pili-specific phages and unable to translocate across solid surfaces by the pilus-dependent mechanism of twitching motility (Twt⁻). Immunoblot analysis demonstrated that MJ-6 was devoid of pili (Pil⁻) but was unaffected in the production of unassembled pilin pools. Genetic studies aimed at localizing the pilE mutation on the P. aeruginosa PAO chromosome demonstrated a strong co-linkage between MJ-6 phage resistance and the proB marker located at 71 min. Cloning of the pilE gene was facilitated by the isolation and identification of a proB⁺-containing plasmid from a PAO1 cosmid library. Upon introduction of the PA01 proB⁺ cosmid clone into MJ-6, sensitivity to pili-specific phage, twitching motility and pilus production were restored. The nucleotide sequence of a 1 kb Eco RV-Clal fragment containing the pilE region revealed a single complete open reading frame with characteristic P. aeruginosa codon bias. PilE, a protein with a molecular weight of 15278, showed significant sequence identity to the pilin precursors of P. aeruginosa and to other type 4 prepilin proteins. The region of highest homology was localized to the N-terminal 40 amino acid residues. The putative PilE N-terminus contained a seven-residue basic leader sequence followed by a consensus cleavage site for prepilin pep-tidase and a largely hydrophobic region which contained tyrosine residues (Tyr-24 and Tyr-27) previously implicated in maintaining pilin subunit-subunit interactions. The requirement of PilE in pilus biogenesis was confirmed by demonstrating that chromosomal pilE insertion mutants were pilus- and twitching-motility deficient.     

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.     

The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa – a review

Pseudomonas aeruginosa (Pa) produces several surface-associated adherence factors or adhesins which promote attachment to epithelial cells and contribute to the virulence of this pathogen. Among them, the type-4 pilus accounts for about 90% of the adherence capability of Pa to human lung pneumocyte A549 cells. Furthermore, it is responsible for more than 90% of the virulence in AB.Y/SnJ mice. Pa type-4 pili display a tip-base differentiation with the adherence function located at the tip of the pilus. All Pa pili prototypes characterized so far contain an intrachain disulfide loop (DSL) of 12 to 17 semi-conserved amino acid residues at the C-terminus of pilin. In Pa, this DSL comprises the epithelial cell-binding domain. Despite little sequence homology, DSL-containing peptides of different pilin prototypes seemingly reveal striking structural similarities. Two β-turns within the loop and the disulfide bridge impose significant structural rigidity on the DSL pilin peptide, suggesting a conformationally conserved binding domain. Insertions of C-terminal pilin peptides with disrupted DSL displayed on the surface of bacterial S-layer mediate the same receptor binding characteristics as pili, indicating that a DSL is not essential in maintaining the functionality of the binding domain. Pa pili bind specifically to the carbohydrate moiety of the glycosphingolipids (GSL) asialo-G_M1 and asialo-G_M2 and, to a much weaker extent, to lactosyl ceramide and ceramide trihexoside. The disaccharide sequence GalNAcβ(1-4)Gal, common in both asialo-G_M1 and asialo-G_M2, likely represents the minimal structural receptor motif recognized by the pili. Pa pili also bind to surface-localized proteins of human epithelial cells and other cell types, suggesting that non-sialylated GSL and (glyco)proteins function as receptors of pili. In addition to the major pilus adhesin, exoenzyme S and, as recent studies indicate, flagella, are further protein adhesins of Pa with GSL receptor binding specificities similar to those of pili.     

Novel proteins that modulate type IV pilus retraction dynamics in Pseudomonas aeruginosa

Pseudomonas aeruginosa uses type IV pili to colonize various materials and for surface-associated twitching motility. We previously identified five phylogenetically distinct alleles of pilA in P. aeruginosa, four of which occur in genetic cassettes with specific accessory genes (J. V. Kus, E. Tullis, D. G. Cvitkovitch, and L. L. Burrows, Microbiology 150:1315-1326, 2004). Each of the five pilin alleles, with and without its associated pilin accessory gene, was used to complement a group II PAO1 pilA mutant. Expression of group I or IV pilA genes restored twitching motility to the same extent as the PAO1 group II pilin. In contrast, poor twitching resulted from complementation with group III or group V pilA genes but increased significantly when the cognate tfpY or tfpZ accessory genes were cointroduced. The enhanced motility was linked to an increase in recoverable surface pili and not to alterations in total pilin pools. Expression of the group III or V pilins in a PAO1 pilA-pilT double mutant yielded large amounts of surface pili, regardless of the presence of the accessory genes. Therefore, poor piliation in the absence of the TfpY and TfpZ accessory proteins results from a net increase in PilT-mediated retraction. Similar phenotypes were observed for tfpY single and tfpY-pilT double knockout mutants of group III strain PA14. A PilA_V-TfpY chimera produced few surface pili, showing that the accessory proteins are specific for their cognate pilin. The genetic linkage between specific pilin and accessory genes may be evolutionarily conserved because the accessory proteins increase pilus expression on the cell surface, thereby enhancing function.     

Structural and Functional Studies of the Pseudomonas aeruginosa Minor Pilin,PilE

Many bacterial pathogens, including Pseudomonas aeruginosa, use type IVa pili (T4aP) for attachment and twitching motility. T4aP are composed primarily of major pilin subunits, which are repeatedly assembled and disassembled to mediate function. A group of pilin-like proteins, the minor pilins FimU and PilVWXE, prime pilus assembly and are incorporated into the pilus. We showed previously that minor pilin PilE depends on the putative priming subcomplex PilVWX and the non-pilin protein PilY1 for incorporation into pili, and that with FimU, PilE may couple the priming subcomplex to the major pilin PilA, allowing for efficient pilus assembly. Here we provide further support for this model, showing interaction of PilE with other minor pilins and the major pilin. A 1.25 Å crystal structure of PilE_Δ1–28 shows a typical type IV pilin fold, demonstrating how it may be incorporated into the pilus. Despite limited sequence identity, PilE is structurally similar to Neisseria meningitidis minor pilins PilX_Nm and PilV_Nm, recently suggested via characterization of mCherry fusions to modulate pilus assembly from within the periplasm. A P. aeruginosa PilE-mCherry fusion failed to complement twitching motility or piliation of a pilE mutant. However, in a retraction-deficient strain where surface piliation depends solely on PilE, the fusion construct restored some surface piliation. PilE-mCherry was present in sheared surface fractions, suggesting that it was incorporated into pili. Together, these data provide evidence that PilE, the sole P. aeruginosa equivalent of PilX_Nm and PilV_Nm, likely connects a priming subcomplex to the major pilin, promoting efficient assembly of T4aP.     

Mapping the surface regions of Pseudomonas aeruginosa PAK pilin: the importance of the C-terminal region for adherence to human buccal epithelial cells

The adherence of non-mucoid Pseudomonas aeruginosa strains is believed to be mediated by the pilus, which consists of a single protein subunit of 15,000 Daltons called pilin. Ten antipeptide antisera were raised to map the surface regions of pilin from P. aeruginosa strain K (PAK). Only one of the antipeptide antisera to the eight predicted surface regions failed to react with PAK pili in direct ELISA. Five out of eight synthetic peptides representing the eight predicted surface regions reacted with anti-PAK pilus antiserum, indicating their surface exposure. Combining the antipeptide and antipilus antisera results, all eight predicted surface regions were demonstrated to be surface-exposed. The PAK 128-144-OH peptide produced the best binding antiserum to PAK pili. Only antipeptide Fab fragments directed against the disulphide bridged C-terminal region of PAK pilin blocked the adherence of pili to human buccal epithelial cells, which suggests that this region contains the receptor-binding domain of the PAK pilus.     

Evolutionary and functional diversity of the Pseudomonas type IVa pilin island

In Pseudomonas aeruginosa, most proteins involved in type IVa pilus (T4aP) biogenesis are highly conserved except for the major pilin PilA and the minor pilins involved in pilus assembly. Here we show that each of the five major pilin alleles is associated with a specific set of minor pilins, and unrelated strains with the same major pilin type have identical minor pilin genes. The sequences of the minor pilin genes of strains with group III and V pilins are identical, suggesting that these groups diverged recently through further evolution of the major pilin cluster. Both gene clusters are localized on a single ‘pilin island’ containing putative tRNA recombinational hotspots, and a similar organization of pilin genes was identified in other Pseudomonas species. To address the biological significance of group‐specific differences, cross‐complementation studies using group II (PAO1) and group III (PA14) minor pilins were performed. Heterologous minor pilins complemented twitching motility to various extents except in the case of PilX, which was non‐functional in non‐native backgrounds. A recombinant PA14 strain expressing the PAO1 minor pilins regained motility only upon co‐introduction of the PA14 pilX gene. Comparison of PilX and PilQ secretin sequences from group II, III and V genomes revealed discrete regions of sequence that co‐varied between groups. Our data suggest that changes in PilX sequence have led to compensatory changes in the PilQ secretin monomer such that heterologous PilX proteins are no longer able to promote opening of the secretin to allow pili to appear on the cell surface.     

DNA binding: a novel function of Pseudomonas aeruginosa type IV pili

The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.     

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 seroiogically 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.     

Assembly of mutant pilins in Pseudomonas aeruginosa: formation of pili composed of heterologous subunits.

Recently, we reported the degree of N-terminal processing within the cytoplasmic membranes of three mutant pilins from Pseudomonas aeruginosa PAK with respect to leader peptide removal and the methylation of the N-terminal phenylalanine (B. L. Pasloske and W. Paranchych, Mol. Microbiol. 2:489-495, 1988). The results of those experiments showed that the deletion of 4 or 8 amino acids within the highly conserved N terminus greatly inhibited leader peptide removal. On the other hand, the mutation of the glutamate at position 5 to a lysine permitted leader peptide cleavage but inhibited transmethylase activity. In this report, we have examined the effects of these mutant pilins upon pilus assembly in a P. aeruginosa PAO host with or without the chromosomally encoded pilin gene present. Pilins with deletions of 4 or 8 amino acids in the N-terminal region were not incorporated into pili. Interestingly, pilin subunits containing the glutamate-to-lysine mutation were incorporated into compound pili together with PAO wild-type subunits. However, the mutant pilins were unable to polymerize as a homopolymer. When wild-type PAK and PAO pilin subunits were expressed in the same bacterial strain, the pilin subunits assembled into homopolymeric pili containing one or the other type of subunit.     

Animal protection and structural studies of a consensus sequence vaccine targeting the receptor binding domain of the type IV pilus of Pseudomonas aeruginosa

One of the main obstacles in the development of a vaccine against Pseudomonas aeruginosa is the requirement that it is protective against a wide range of virulent strains. We have developed a synthetic-peptide consensus-sequence vaccine (Cs1) that targets the host receptor-binding domain (RBD) of the type IV pilus of P. aeruginosa. Here, we show that this vaccine provides increased protection against challenge by the four piliated strains that we have examined (PAK, PAO, KB7 and P1) in the A.BY/SnJ mouse model of acute P. aeruginosa infection. To further characterize the consensus sequence, we engineered Cs1 into the PAK monomeric pilin protein and determined the crystal structure of the chimeric Cs1 pilin to 1.35 Å resolution. The substitutions (T130K and E135P) used to create Cs1 do not disrupt the conserved backbone conformation of the pilin RBD. In fact, based on the Cs1 pilin structure, we hypothesize that the E135P substitution bolsters the conserved backbone conformation and may partially explain the immunological activity of Cs1. Structural analysis of Cs1, PAK and K122-4 pilins reveal substitutions of non-conserved residues in the RBD are compensated for by complementary changes in the rest of the pilin monomer. Thus, the interactions between the RBD and the rest of the pilin can either be mediated by polar interactions of a hydrogen bond network in some strains or by hydrophobic interactions in others. Both configurations maintain a conserved backbone conformation of the RBD. Thus, the backbone conformation is critical in our consensus-sequence vaccine design and that cross-reactivity of the antibody response may be modulated by the composition of exposed side-chains on the surface of the RBD. This structure will guide our future vaccine design by focusing our investigation on the four variable residue positions that are exposed on the RBD surface.     

Backbone dynamics of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pilin strain PAK from heteronuclear 1H-15N NMR spectroscopy

The backbone dynamics of a ¹⁵N-labeled recombinant PAK pilin peptide spanning residues 128–144 in the C-terminal receptor binding domain of Pseudomonas aeruginosa pilin protein strain PAK (Lys¹²⁸-Cys-Thr-Ser-Asp-Gln-Asp-Glu-Gln-Phe-Ile-Pro-Lys-Gly-Cys-Ser-Lys¹⁴⁴) were probed by measurements of ¹⁵N NMR relaxation. This PAK(128–144) sequence is a target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. The ¹⁵N longitudinal (T₁) and transverse (T₂) relaxation rates and the steady-state heteronuclear {¹H}-¹⁵N NOE were measured at three fields (7.04, 11.74 and 14.1 Tesla), five temperatures (5, 10, 15, 20, and 25°C ) and at pH 4.5 and 7.2. Relaxation data was analyzed using both the `model-free' formalism [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559 and 4559–4570] and the reduced spectral density mapping approach [Farrow, N.A., Szabo, A., Torchia, D.A. and Kay, L.E. (1995) J. Biomol. NMR, 6, 153–162]. The relaxation data, spectral densities and order parameters suggest that the type I and type II -turns spanning residues Asp¹³⁴-Glu-Gln-Phe¹³⁷ and Pro¹³⁹-Lys-Gly-Cys¹⁴², respectively, are the most ordered and structured regions of the peptide. The biological implications of these results will be discussed in relation to the role that backbone motions play in PAK pilin peptide immunogenicity, and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains.     

The Nanomechanical Properties of Lactococcus lactis Pili Are Conditioned by the Polymerized Backbone Pilin

Pili produced by Lactococcus lactis subsp. lactis are putative linear structures consisting of repetitive subunits of the major pilin PilB that forms the backbone, pilin PilA situated at the distal end of the pilus, and an anchoring pilin PilC that tethers the pilus to the peptidoglycan. We determined the nanomechanical properties of pili using optical-tweezers force spectroscopy. Single pili were exposed to optical forces that yielded force-versus-extension spectra fitted using the Worm-Like Chain model. Native pili subjected to a force of 0–200 pN exhibit an inextensible, but highly flexible ultrastructure, reflected by their short persistence length. We tested a panel of derived strains to understand the functional role of the different pilins. First, we found that both the major pilin PilB and sortase C organize the backbone into a full-length organelle and dictate the nanomechanical properties of the pili. Second, we found that both PilA tip pilin and PilC anchoring pilin were not essential for the nanomechanical properties of pili. However, PilC maintains the pilus on the bacterial surface and may play a crucial role in the adhesion- and biofilm-forming properties of L. lactis.     

Pseudomonas aeruginosa Type IV pilus expression in Neisseria gonorrhoeae: effects of pilin subunit composition on function and organelle dynamics

Type IV pili (TFP) play central roles in the expression of many phenotypes including motility, multicellular behavior, sensitivity to bacteriophages, natural genetic transformation, and adherence. In Neisseria gonorrhoeae, these properties require ancillary proteins that act in conjunction with TFP expression and influence organelle dynamics. Here, the intrinsic contributions of the pilin protein itself to TFP dynamics and associated phenotypes were examined by expressing the Pseudomonas aeruginosa PilA(PAK) pilin subunit in N. gonorrhoeae. We show here that, although PilA(PAK) pilin can be readily assembled into TFP in this background, steady-state levels of purifiable fibers are dramatically reduced relative those of endogenous pili. This defect is due to aberrant TFP dynamics as it is suppressed in the absence of the PilT pilus retraction ATPase. Functionally, PilA(PAK) pilin complements gonococcal adherence for human epithelial cells but only in a pilT background, and this property remains dependent on the coexpression of both the PilC adhesin and the PilV pilin-like protein. Since P. aeruginosa pilin only moderately supports neisserial sequence-specific transformation despite its assembly proficiency, these results together suggest that PilA(PAK) pilin functions suboptimally in this environment. This appears to be due to diminished compatibility with resident proteins essential for TFP function and dynamics. Despite this, PilA(PAK) pili support retractile force generation in this background equivalent to that reported for endogenous pili. Furthermore, PilA(PAK) pili are both necessary and sufficient for bacteriophage PO4 binding, although the strain remains phage resistant. Together, these findings have significant implications for TFP biology in both N. gonorrhoeae and P. aeruginosa.     

The Group A Streptococcus serotype M2 pilus plays a role in host cell adhesion and immune evasion

Group A Streptococcus (GAS), or Streptococcus pyogenes, is a human pathogen that causes diseases ranging from skin and soft tissue infections to severe invasive diseases, such as toxic shock syndrome. Each GAS strain carries a particular pilus type encoded in the variable f ibronectin‐binding, c ollagen‐binding, T antigen (FCT) genomic region. Here, we describe the functional analysis of the serotype M2 pilus encoded in the FCT‐6 region. We found that, in contrast to other investigated GAS pili, the ancillary pilin 1 lacks adhesive properties. Instead, the backbone pilin is important for host cell adhesion and binds several host factors, including fibronectin and fibrinogen. Using a panel of recombinant pilus proteins, GAS gene deletion mutants and Lactococcus lactis gain‐of‐function mutants we show that, unlike other GAS pili, the FCT‐6 pilus also contributes to immune evasion. This was demonstrated by a delay in blood clotting, increased intracellular survival of the bacteria in macrophages, higher bacterial survival rates in human whole blood and greater virulence in a Galleria mellonella infection model in the presence of fully assembled FCT‐6 pili.     

PilJ Localizes to Cell Poles and Is Required for Type IV Pilus Extension in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Twitching motility allows Pseudomonas aeruginosa to respond to stimuli by extending and retracting its type IV pili (TFP). PilJ is a protein necessary for this surface-associated twitching motility and bears high sequence identity with Escherichia coli methyl-accepting chemotaxis proteins (MCP). Here, we report that whereas wild-type P. aeruginosa PAO1 cells have extended pili at a single pole, pilJ mutant cells have shortened pili often at both poles despite normal levels of pilin accumulation, suggesting that PilJ is required for full TFP assembly/extension. Using yellow fluorescent protein fusions (pilJ-yfp), both plasmid born and in-frame chromosomal constructs, we determined that PilJ localizes to both poles of the cell. Overexpression of pilJ-yfp resulted in the protein accumulating between the poles. Paul DeLange and Tracy Collins contributed equally to this work.     

Antigen-antibody interactions: elucidation of the epitope and strain-specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K.

The C-terminal region of Pseudomonas aeruginosa strain K (PAK) pilin comprises both an epitope for the strain-specific monoclonal antibody PK99H, which blocks pilus-mediated adherence, and the adherence binding domain for buccal and tracheal epithelial cells. The PK99H epitope was located in sequence 134-140 (Asp-Glu-Gln-Phe-Ile-Pro-Lys) by using a single alanine replacement analysis on the 17-residue synthetic peptide corresponding to the PAK C-terminal sequence 128-144. Indeed, a 7-residue peptide corresponding to this sequence was shown to have a similar binding affinity to that of the native conformationally constrained (disulfide bridged) 17-residue peptide. This epitope was found to contain two critical residues (Phe137 and Lys140) and one nonessential residue (Gln136). Interestingly, the peptide, Phe-Ile-Pro-Lys, which constitutes the four most important side chains for antibody binding did not bind to PK99H. It was of interest to investigate the structural basis of the strain-specificity of PK99H utilizing naturally occurring pilin sequences. Therefore, all different residues found in the sequence corresponding to the PK99H epitope of the four other strains (PAO, CD4, K122-4, and KB7) were substituted one at a time in the PAK sequence and the changes in binding affinity of these analogs to the antibody PK99H were determined by competitive ELISA. The strain-specificity of PK99H for strains PAO, K122-4, and KB7 can be explained by the accumulated sequence changes in these strains, and at least two amino acid changes were required to explain the strain-specificity of PK99H. Similarly, cross-reactivity of PK99H with CD4 can be explained by the fact that there was only one side chain responsible for decreasing binding affinity compared to the PAK sequence.