Molecular architecture of Streptococcus pneumoniae TIGR4 pili

Although the pili of Gram‐positive bacteria are putative virulence factors, little is known about their structure. Here we describe the molecular architecture of pilus‐1 of Streptococcus pneumoniae, which is a major cause of morbidity and mortality worldwide. One major (RrgB) and two minor components (RrgA and RrgC) assemble into the pilus. Results from TEM and scanning transmission EM show that the native pili are approximately 6 nm wide, flexible filaments that can be over 1 μm long. They are formed by a single string of RrgB monomers and have a polarity defined by nose‐like protrusions. These protrusions correlate to the shape of monomeric RrgB–His, which like RrgA–His and RrgC–His has an elongated, multi‐domain structure. RrgA and RrgC are only present at the opposite ends of the pilus shaft, compatible with their putative roles as adhesin and anchor to the cell wall surface, respectively. Our structural analyses provide the first direct experimental evidence that the native S. pneumoniae pilus shaft is composed exclusively of covalently linked monomeric RrgB subunits oriented head‐to‐tail.     

Structural Basis of Pilus Anchoring by the Ancillary Pilin RrgC of Streptococcus pneumoniae

Pili are surface-attached, fibrous virulence factors that play key roles in the pathogenesis process of a number of bacterial agents. Streptococcus pneumoniae is a causative agent of pneumonia and meningitis, and the appearance of drug-resistance organisms has made its treatment challenging, especially in developing countries. Pneumococcus-expressed pili are composed of three structural proteins: RrgB, which forms the polymerized backbone, RrgA, the tip-associated adhesin, and RrgC, which presumably associates the pilus with the bacterial cell wall. Despite the fact that the structures of both RrgA and RrgB were known previously, structural information for RrgC was still lacking, impeding the analysis of a complete model of pilus architecture. Here, we report the structure of RrgC to 1.85 Å and reveal that it is a three-domain molecule stabilized by two intradomain isopeptide bonds. RrgC does not depend on pilus-specific sortases to become attached to the cell wall; instead, it binds the preformed pilus to the peptidoglycan by employing the catalytic activity of SrtA. A comprehensive model of the type 1 pilus from S. pneumoniae is also presented.     

Pneumococcal pili are composed of protofilaments exposing adhesive clusters of Rrg A

Pili have been identified on the cell surface of Streptococcus pneumoniae, a major cause of morbidity and mortality worldwide. In contrast to Gram-negative bacteria, little is known about the structure of native pili in Gram-positive species and their role in pathogenicity. Triple immunoelectron microscopy of the elongated structure showed that purified pili contained RrgB as the major compound, followed by clustered RrgA and individual RrgC molecules on the pilus surface. The arrangement of gold particles displayed a uniform distribution of anti-RrgB antibodies along the whole pilus, forming a backbone structure. Antibodies against RrgA were found along the filament as particulate aggregates of 2-3 units, often co-localised with single RrgC subunits. Structural analysis using cryo electron microscopy and data obtained from freeze drying/metal shadowing technique showed that pili are oligomeric appendages formed by at least two protofilaments arranged in a coiled-coil, compact superstructure of various diameters. Using extracellular matrix proteins in an enzyme-linked immunosorbent assay, ancillary RrgA was identified as the major adhesin of the pilus. Combining the structural and functional data, a model emerges where the pilus RrgB backbone serves as a carrier for surface located adhesive clusters of RrgA that facilitates the interaction with the host.     

The full-length Streptococcus pneumoniae major pilin RrgB crystallizes in a fibre-like structure, which presents the D1 isopeptide bond and provides details on the mechanism of pilus polymerization

RrgB is the major pilin which forms the pneumococcal pilus backbone. We report the high-resolution crystal structure of the full-length form of RrgB containing the IPQTG sorting motif. The RrgB fold is organized into four distinct domains, D1-D4, each of which is stabilized by an isopeptide bond. Crystal packing revealed a head-to-tail organization involving the interaction of the IPQTG motif into the D1 domain of two successive RrgB monomers. This fibrillar assembly, which fits into the electron microscopy density map of the native pilus, probably induces the formation of the D1 isopeptide bond as observed for the first time in the present study, since neither in published structures nor in soluble RrgB produced in Escherichia coli or in Streptococcus pneumoniae is the D1 bond present. Experiments performed in live bacteria confirmed that the intermolecular bond linking the RrgB subunits takes place between the IPQTG motif of one RrgB subunit and the Lys183 pilin motif residue of an adjacent RrgB subunit. In addition, we present data indicating that the D1 isopeptide bond is involved in RrgB stabilization. In conclusion, the crystal RrgB fibre is a compelling model for deciphering the molecular details required to generate the pneumococcal pilus.     

Structure of the full-length major pilin from Streptococcus pneumoniae: implications for isopeptide bond formation in gram-positive bacterial pili

The surface of the pneumococcal cell is adorned with virulence factors including pili. The major pilin RrgB, which forms the pilus shaft on pathogenic Streptococcus pneumoniae, comprises four immunoglobulin (Ig)-like domains, each with a common CnaB topology. The three C-terminal domains are each stabilized by internal Lys-Asn isopeptide bonds, formed autocatalytically with the aid of an essential Glu residue. The structure and orientation of the crucial N-terminal domain, which provides the covalent linkage to the next pilin subunit in the shaft, however, remain incompletely characterised. We report the crystal structure of full length RrgB, solved by X-ray crystallography at 2.8 Å resolution. The N-terminal (D1) domain makes few contacts with the rest of the RrgB structure, and has higher B-factors. This may explain why D1 is readily lost by proteolysis, as are the N-terminal domains of many major pilins. D1 is also found to have a triad of Lys, Asn and Glu residues in the same topological positions as in the other domains, yet mass spectrometry and the crystal structure show that no internal isopeptide bond is formed. We show that this is because β-strand G of D1, which carries the Asn residue, diverges from β-strand A, carrying the Lys residue, such that these residues are too far apart for bond formation. Strand G also carries the YPKN motif that provides the essential Lys residue for the sortase-mediated intermolecular linkages along the pilus shaft. Interaction with the sortase and formation of the intermolecular linkage could result in a change in the orientation of this strand, explaining why isopeptide bond formation in the N-terminal domains of some major pilins appears to take place only upon assembly of the pili.     

Two Crystal Structures of Pneumococcal Pilus Sortase C Provide Novel Insights into Catalysis and Substrate Specificity

The respiratory tract pathogen Streptococcus pneumoniae is a primary cause of morbidity and mortality worldwide. Pili enhance initial adhesion as well as the capacity of pneumococci to cause pneumonia and bacteremia. Pilus-associated sortases (SrtB, SrtC, and SrtD) are involved in the biogenesis of pneumococcal pili, composed of repeating units of RrgB that create the stalk to which the RrgA adhesin and the preferential pilus tip subunit RrgC are covalently associated. Using single sortase-expressing strains, we demonstrate that both pilin-polymerizing sortases SrtB and SrtC can covalently link pili to the peptidoglycan cell wall, a property shared with the non-pilus-polymerizing enzyme SrtD and the housekeeping sortase SrtA. Comparative analysis of the crystal structures of S. pneumoniae SrtC and SrtB revealed structural differences explaining the incapacity of SrtC, but not of SrtB, to incorporate RrgC into the pilus. Accordingly, site-directed mutagenesis of Thr¹⁶⁰ in SrtB to an arginine as in SrtC (Arg¹⁶⁰) partially converted its substrate specificity into that of SrtC. Solving two crystal structures for SrtC suggests that an opening of a flexible lid and a concomitant cysteine rotation are important for catalysis and the activation of the catalytic cysteine of pilus-associated sortases.     

Roles of the sortases of Streptococcus pneumoniae in assembly of the RlrA pilus

Pili have been observed on the surface of several gram-positive bacteria, including Streptococcus pneumoniae. The S. pneumoniae strain TIGR4 pilus is composed of three structural subunit proteins encoded in the rlrA pathogenicity islet, RrgA, RrgB, and RrgC. RrgB comprises the pilus backbone, RrgA is observed at intervals along surface pili, while RrgC is found in a loosely defined relationship with RrgA. We investigated the incorporation of each subunit into pili and the reliance of such placement on each of the other subunits. Both accessory subunits RrgA and RrgC are present in similar quantities in pili of all sizes. However, neither protein is required for the polymerization of RrgB, suggesting a nonessential role for RrgA and RrgC in the initiation of pilus assembly. Additionally, the rlrA islet encodes three sortases, SrtC-1, SrtC-2, and SrtC-3 (formerly SrtB, SrtC, and SrtD), which are divergent in sequence from the housekeeping sortase, SrtA. We determined the contributions of these four sortases to pilus assembly and found that SrtA is dispensable for pilus assembly and localization to the cell wall. Instead, SrtC-1, SrtC-2, and SrtC-3 are responsible for pilus assembly and exhibit functional redundancy with respect to backbone assembly and cell wall localization. A level of specificity and coordination among the class C sortases was revealed by the finding that SrtC-1 and SrtC-3 are required for the incorporation of the accessory subunits and by showing a deleterious effect on pilus assembly upon alteration of the cell wall sorting signals of the accessory subunit proteins.     

Evidence for the Sialylation of PilA,the PI-2a Pilus-Associated Adhesin of Streptococcus agalactiae Strain NEM316

Streptococcus agalactiae (or Group B Streptococcus, GBS) is a commensal bacterium present in the intestinal and urinary tracts of approximately 30% of humans. We and others previously showed that the PI-2a pilus polymers, made of the backbone pilin PilB, the tip adhesin PilA and the cell wall anchor protein PilC, promote adhesion to host epithelia and biofilm formation. Affinity-purified PI-2a pili from GBS strain NEM316 were recognized by N-acetylneuraminic acid (NeuNAc, also known as sialic acid) specific lectins such as Elderberry Bark Lectin (EBL) suggesting that pili are sialylated. Glycan profiling with twenty different lectins combined with monosaccharide composition by HPLC suggested that affinity-purified PI-2a pili are modified by N-glycosylation and decorated with sialic acid attached to terminal galactose. Analysis of various relevant mutants in the PI-2a pilus operon by flow-cytometry and electron microscopy analyses pointed to PilA as the pilus subunit modified by glycosylation. Double labeling using PilB antibody and EBL lectin, which specifically recognizes N-acetylneuraminic acid attached to galactose in α-2, 6, revealed a characteristic binding of EBL at the tip of the pilus structures, highly reminiscent of PilA localization. Expression of a secreted form of PilA using an inducible promoter showed that this recombinant PilA binds specifically to EBL lectin when produced in the native GBS context. In silico search for potentially glycosylated asparagine residues in PilA sequence pointed to N427 and N597, which appear conserved and exposed in the close homolog RrgA from S. pneumoniae, as likely candidates. Conversion of these two asparagyl residues to glutamyl resulted in a higher instability of PilA. Our results provide the first evidence that the tip PilA adhesin can be glycosylated, and suggest that this modification is critical for PilA stability and may potentially influence interactions with the host.     

Structural analysis of the Saf pilus by electron microscopy and image processing

Bacterial pili are important virulence factors involved in host cell attachment and/or biofilm formation, key steps in establishing and maintaining successful infection. Here we studied Salmonella atypical fimbriae (or Saf pili), formed by the conserved chaperone/usher pathway. In contrast to the well-established quaternary structure of typical/FGS-chaperone assembled, rod-shaped, chaperone/usher pili, little is known about the supramolecular organisation in atypical/FGL-chaperone assembled fimbriae. In our study, we have used negative stain electron microscopy and single-particle image analysis to determine the three-dimensional structure of the Salmonella typhimurium Saf pilus. Our results show atypical/FGL-chaperone assembled fimbriae are composed of highly flexible linear multi-subunit fibres that are formed by globular subunits connected to each other by short links giving a “beads on a string”-like appearance. Quantitative fitting of the atomic structure of the SafA pilus subunit into the electron density maps, in combination with linker modelling and energy minimisation, has enabled analysis of subunit arrangement and intersubunit interactions in the Saf pilus. Short intersubunit linker regions provide the molecular basis for flexibility of the Saf pilus by acting as molecular hinges allowing a large range of movement between consecutive subunits in the fibre.     

Structural and functional characterization of the Streptococcus pneumoniae RrgB pilus backbone D1 domain

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.     

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.     

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.     

A Structural Snapshot of Type II Pilus Formation in Streptococcus pneumoniae

Pili are fibrous appendages expressed on the surface of a vast number of bacterial species, and their role in surface adhesion is important for processes such as infection, colonization, andbiofilm formation. The human pathogen Streptococcus pneumoniae expresses two different types of pili, PI-1 and PI-2, both of which require the concerted action of structural proteins and sortases for their polymerization. The type PI-1 streptococcal pilus is a complex, well studied structure, but the PI-2 type, present in a number of invasive pneumococcal serotypes, has to date remained less well understood. The PI-2 pilus consists of repeated units of a single protein, PitB, whose covalent association is catalyzed by cognate sortase SrtG-1 and partner protein SipA. Here we report the high resolution crystal structures of PitB and SrtG1 and use molecular modeling to visualize a “trapped” 1:1 complex between the two molecules. X-ray crystallography and electron microscopy reveal that the pneumococcal PI-2 backbone fiber is formed by PitB monomers associated in head-to-tail fashion and that short, flexible fibers can be formed even in the absence of coadjuvant proteins. These observations, obtained with a simple pilus biosynthetic system, are likely to be applicable to other fiber formation processes in a variety of Gram-positive organisms.     

The minor pilin subunit Sgp2 is necessary for assembly of the pilus encoded by the srtG cluster of Streptococcus suis

Gram-positive pili are composed of covalently bound pilin subunits whose assembly is mediated via a pilus-specific sortase(s). Major subunits constitute the pilus backbone and are therefore essential for pilus formation. Minor subunits are also incorporated into the pilus, but they are considered to be dispensable for backbone formation. The srtG cluster is one of the putative pilus gene clusters identified in the major swine pathogen Streptococcus suis. It consists of one sortase gene (srtG) and two putative pilin subunit genes (sgp1 and sgp2). In this study, by constructing mutants for each of the genes in the cluster and by both immunoblotting and immunogold electron microscopic analysis with antibodies against Sgp1 and Sgp2, we found that the srtG cluster mediates the expression of pilus-like structures in S. suis strain 89/1591. In this pilus, Sgp1 forms the backbone, whereas Sgp2 is incorporated as the minor subunit. In accordance with the current model of pilus assembly by Gram-positive organisms, the major subunit Sgp1 was indispensable for backbone formation and the cognate sortase SrtG mediated the polymerization of both subunits. However, unlike other well-characterized Gram-positive bacterial pili, the minor subunit Sgp2 was required for polymerization of the major subunit Sgp1. Because Sgp2 homologues are encoded in several other Gram-positive bacterial pilus gene clusters, in some types of pili, minor pilin subunits may contribute to backbone formation by a novel mechanism.     

Crystal Structure of the Minor Pilin CofB,the Initiator of CFA/III Pilus Assembly in Enterotoxigenic Escherichia coli

Type IV pili are extracellular polymers of the major pilin subunit. These subunits are held together in the pilus filament by hydrophobic interactions among their N-terminal α-helices, which also anchor the pilin subunits in the inner membrane prior to pilus assembly. Type IV pilus assembly involves a conserved group of proteins that span the envelope of Gram-negative bacteria. Among these is a set of minor pilins, so named because they share their hydrophobic N-terminal polymerization/membrane anchor segment with the major pilins but are much less abundant. Minor pilins influence pilus assembly and retraction, but their precise functions are not well defined. The Type IV pilus systems of enterotoxigenic Escherichia coli and Vibrio cholerae are among the simplest of Type IV pilus systems and possess only a single minor pilin. Here we show that the enterotoxigenic E. coli minor pilins CofB and LngB are required for assembly of their respective Type IV pili, CFA/III and Longus. Low levels of the minor pilins are optimal for pilus assembly, and CofB can be detected in the pilus fraction. We solved the 2.0 Å crystal structure of N-terminally truncated CofB, revealing a pilin-like protein with an extended C-terminal region composed of two discrete domains connected by flexible linkers. The C-terminal region is required for CofB to initiate pilus assembly. We propose a model for CofB-initiated pilus assembly with implications for understanding filament growth in more complex Type IV pilus systems as well as the related Type II secretion system.     

RrgA is a pilus-associated adhesin in Streptococcus pneumoniae

Adherence to host cells is important in microbial colonization of a mucosal surface, and Streptococcus pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits, RrgA, RrgB and RrgC. We sought to determine which subunit(s) confers adherence. Bacteria deficient in RrgA are significantly less adherent than wild-type organisms, while overexpression of RrgA enhances adherence. Recombinant monomeric RrgA binds to respiratory cells, as does RrgC with less affinity, and pre-incubation of epithelial cells with RrgA reduces adherence of wild-type piliated pneumococci. Non-adherent RrgA-negative, RrgB- and RrgC-positive organisms produce pili, suggesting that pilus-mediated adherence is due to expression of RrgA, rather than the pilus backbone itself. In contrast, RrgA-positive strains with disrupted rrgB and rrgC genes exhibit wild-type adherence despite failure to produce pili by Western blot or immunoelectron microscopy. The density of bacteria colonizing the upper respiratory tract of mice inoculated with piliated RrgA-negative pneumococci was significantly less compared with wild-type; in contrast, non-piliated pneumococci expressing non-polymeric RrgA had similar numbers of bacteria in the nasopharynx as piliated wild-type bacteria. These data suggest that RrgA is central in pilus-mediated adherence and disease, even in the absence of polymeric pilus production.     

Structural characterization of CFA/III and Longus type IVb pili from enterotoxigenic Escherichia coli

The type IV pili are helical filaments found on many Gram-negative pathogenic bacteria, with multiple diverse roles in pathogenesis, including microcolony formation, adhesion, and twitching motility. Many pathogenic enterotoxigenic Escherichia coli (ETEC) isolates express one of two type IV pili belonging to the type IVb subclass: CFA/III or Longus. Here we show a direct correlation between CFA/III expression and ETEC aggregation, suggesting that these pili, like the Vibrio cholerae toxin-coregulated pili (TCP), mediate microcolony formation. We report a 1.26-Å resolution crystal structure of CofA, the major pilin subunit from CFA/III. CofA is very similar in structure to V. cholerae TcpA but possesses a 10-amino-acid insertion that replaces part of the α2-helix with an irregular loop containing a 3₁₀-helix. Homology modeling suggests a very similar structure for the Longus LngA pilin. A model for the CFA/III pilus filament was generated using the TCP electron microscopy reconstruction as a template. The unique 3₁₀-helix insert fits perfectly within the gap between CofA globular domains. This insert, together with differences in surface-exposed residues, produces a filament that is smoother and more negatively charged than TCP. To explore the specificity of the type IV pilus assembly apparatus, CofA was expressed heterologously in V. cholerae by replacing the tcpA gene with that of cofA within the tcp operon. Although CofA was synthesized and processed by V. cholerae, no CFA/III filaments were detected, suggesting that the components of the type IVb pilus assembly system are highly specific to their pilin substrates.     

Pilus backbone protein PitB of Streptococcus pneumoniae contains stabilizing intramolecular isopeptide bonds

Streptococcus pneumoniae type 2 pili are recently identified fimbrial structures extending from the bacterial surface and formed by polymers of the structural protein PitB. Intramolecular isopeptide bonds are a characteristic of the related pilus backbone protein Spy0128 of group A streptococci. Based on the identification of conserved residues in PitB, we predicted two intramolecular isopeptide bonds in PitB. Using a combination of tandem mass spectrometry and Edman sequencing, we show that these bonds were formed between Lys₆₃-Asn₂₁₄ and Lys₂₄₃-Asn₃₇₂ in PitB. Mutant proteins lacking the intramolecular isopeptide bonds retained the proteolytic stability observed with the wild type protein. However, absence of these bonds substantially decreased the melting temperature of the PitB-derivatives, indicating a stabilizing function of these bonds in PitB of the pneumococcal type 2 pilus.     

Isopeptide bonds of the major pilin protein BcpA influence pilus structure and bundle formation on the surface of Bacillus cereus

Bacillus cereus strains elaborate pili on their surface using a mechanism of sortase-mediated cross-linking of major and minor pilus components. Here we used a combination of electron microscopy and atomic force microscopy to visualize these structures. Pili occur as single, double or higher order assemblies of filaments formed from monomers of the major pilin, BcpA, capped by the minor pilin, BcpB. Previous studies demonstrated that within assembled pili, four domains of BcpA - CNA(1), CNA(2), XNA and CNA(3) - each acquire intramolecular lysine-asparagine isopeptide bonds formed via catalytic glutamic acid or aspartic acid residues. Here we showed that mutants unable to form the intramolecular isopeptide bonds in the CNA(2) or CNA(3) domains retain the ability to form pilus bundles. A mutant lacking the CNA(1) isopeptide bond assembled deformed pilin subunits that failed to associate as bundles. X-ray crystallography revealed that the BcpA variant Asp(312) Ala, lacking an aspartyl catalyst, did not generate the isopeptide bond within the jelly-roll structure of XNA. The Asp(312) Ala mutant was also unable to form bundles and promoted the assembly of deformed pili. Thus, structural integrity of the CNA(1) and XNA domains are determinants for the association of pili into higher order bundle structures and determine native pilus structure.