Pili Of Gram-positive Bacteria Research Articles

Detection of Cell Wall-Anchoring Machinery by Immunogold-Labeling Thin-Section Electron Microscopy.

Cell wall anchoring of surface proteins and pili in Gram-positive bacteria is mediated by sortase - a highly conserved transpeptidase enzyme. Early studies have demonstrated the membrane-associated nature of this enzyme in close proximity with its cognate substrates, using immunogold-labeling thin-section electron microscopy. Here, we provide a detail protocol of this methodology, including specimen preparation, ultrathin sectioning, and immunogold-labeling electron microscopic procedures, with an experimental model of sortase enzymes from Actinomyces oris. In principle, this protocol can be employed for any bacterial ultrathin-section samples to detect subcellular localization of proteins and organelles by immuno-electron microscopy.

X-ray structures of Clostridium perfringens sortase C with C-terminal cell wall sorting motif of LPST demonstrate role of subsite for substrate-binding and structural variations of catalytic site

Pili of Gram-positive bacteria are flexible rod proteins covalently attached to the bacterial cell wall, that play important roles in the initial adhesion of bacterial cells to host tissues and bacterial colonization. Pili are formed by the polymerization of major and minor pilins, catalyzed by class C sortase (SrtC), a family of cysteine transpeptidases. The Gram-positive bacterium Clostridium perfringens has a major pilin (CppA), a minor pilin (CppB), and SrtC (CpSrtC). CpSrtC recognizes the C-terminal cell wall sorting signal motifs with five amino acid residues, LPSTG of CppA and LPETG of CppB, for the polymerization of pili. Here, we report biochemical analysis to detect the formation of Clostridium perfringens pili in vivo, and the X-ray structure of a novel intermolecular substrate-enzyme complex of CpSrtC with a sequence of LPST at the C-terminal site. The results showed that CpSrtC has a subsite for substrate-binding to aid polymerization of pili, and that the catalytic site has structural variations, giving insights into the enzyme catalytic reaction mechanism and affinities for the C-terminal cell wall sorting signal motif sequences.

New Paradigms of Pilus Assembly Mechanisms in Gram-Positive Actinobacteria.

Adhesive pili in Gram-positive bacteria represent a variety of extracellular multiprotein polymers that mediate bacterial colonization of specific host tissues and associated pathogenesis. Pili are assembled in two distinct but coupled steps, an orderly crosslinking of pilin monomers and subsequent anchoring of the polymer to peptidoglycan, catalyzed by two transpeptidase enzymes - the pilus-specific sortase and the housekeeping sortase. Here, we review this biphasic assembly mechanism based on studies of two prototypical models, the heterotrimeric pili in Corynebacterium diphtheriae and the heterodimeric pili in Actinomyces oris, highlighting some newly emerged basic paradigms. The disparate mechanisms of protein ligation mediated by the pilus-specific sortase and the spatial positioning of adhesive pili on the cell surface modulated by the housekeeping sortase are among the notable highlights.

High Force Magnetic Tweezers Reveal That Bacterial Adhesion Pili Act as Megadalton-scale Schock Absorbers

Gram positive pathogens colonize the oral cavity with their adhesive pili. These organelles are exposed to large mechanical perturbations, such as mastication or teeth brushing, which constantly challenge the bacterial adhesion. To survive these events, the pilus proteins contain intramolecular isopeptide bonds that confer them high mechanical stability. In the dental plaque pathogen Actinomyces oris, two strategically located isopeptide bonds in the 2nd and the 3rd domain of the pilus protein FimA prevent the mechanical unfolding of this protein. The only structure that can be stretched under force is the 40 residue sequence trapped between both isopeptide bonds, the isopeptide-delimited loop (IDL) motif. Our previous AFM force spectroscopy experiments unveiled the large forces (>600 pN) required to stretch FimA's IDL; however, the low force resolution of AFM prevented us from studying the IDL folding at low forces. Herein, we develop a high force magnetic tweezers force spectroscopy assay to expose FimA's IDL to forces ranging from 4 to 280 pN, allowing us to cover the IDL (un)folding dynamics with nm and sub-pN resolution. Our approach permits us to detect the IDL extension in a few seconds at forces >200 pN, and to monitor IDL folding at forces <15 pN. After IDL folding, FimA mechanical stability increases along time, suggesting a time-dependent maturation of the interfacial contacts established between FimA's 2nd and 3rd domains. Stretching-relaxation experiments reveal that a pilus composed of 150 FimA subunits would be able to dissipate as heat up to ∼4·105 zJ, acting as a megaDalton-scale shock-absorber. Given the ubiquitous presence of this protein motif among the pili of Gram positive bacteria, IDL pharmaceutical targeting could become a strategy to weaken the adhesion resistance of these pathogens.

Structures of major pilins in Clostridium perfringens demonstrate dynamic conformational change.

Pili in Gram-positive bacteria are flexible rod proteins associated with the bacterial cell surface, and they play important roles in the initial adhesion to host tissues and colonization. The pilus shaft is formed by the covalent polymerization of major pilins, catalyzed by sortases, a family of cysteine transpeptidases. Here, X-ray structures of the major pilins from Clostridium perfringens strains 13 and SM101 and of sortase from strain SM101 are presented with biochemical analysis to detect the formation of pili in vivo. The major pilin from strain 13 adopts an elongated structure to form noncovalently linked polymeric chains in the crystal, yielding a practical model of the pilus fiber structure. The major pilin from strain SM101 adopts a novel bent structure and associates to form a left-handed twist like an antiparallel double helix in the crystal, which is likely to promote bacterial cell-cell interactions. A modeling study showed that pilin with a bent structure interacts favorably with sortase. The major pilin from strain SM101 was considered to be in an equilibrium state between an elongated and a bent structure through dynamic conformational change, which may be involved in pili-mediated colonization and sortase-mediated polymerization of pili.

Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae

Type IV pili are important virulence factors on the surface of many pathogenic bacteria and have been implicated in a wide range of diverse functions, including attachment, twitching motility, biofilm formation, and horizontal gene transfer. The respiratory pathogen Streptococcus pneumoniae deploys type IV pili to take up DNA during transformation. These “competence pili” are composed of the major pilin protein ComGC and exclusively assembled during bacterial competence, but their biogenesis remains unclear. Here, we report the high resolution NMR structure of N-terminal truncated ComGC revealing a highly flexible and structurally divergent type IV pilin. It consists of only three α-helical segments forming a well-defined electronegative cavity and confined electronegative and hydrophobic patches. The structure is particularly flexible between the first and second α-helix with the first helical part exhibiting slightly slower dynamics than the rest of the pilin, suggesting that the first helix is involved in forming the pilus structure core and that parts of helices two and three are primarily surface-exposed. Taken together, our results provide the first structure of a type IV pilin protein involved in the formation of competence-induced pili in Gram-positive bacteria and corroborate the remarkable structural diversity among type IV pilin proteins.

Motility and adhesion through type IV pili in Gram-positive bacteria.

Type IV pili are hair-like bacterial surface appendages that play a role in diverse processes such as cellular adhesion, colonization, twitching motility, biofilm formation, and horizontal gene transfer. These extracellular fibers are composed exclusively or primarily of many copies of one or more pilin proteins, tightly packed in a helix so that the highly hydrophobic amino-terminus of the pilin is buried in the pilus core. Type IV pili have been characterized extensively in Gram-negative bacteria, and recent advances in high-throughput genomic sequencing have revealed that they are also widespread in Gram-positive bacteria. Here, we review the current state of knowledge of type IV pilus systems in Gram-positive bacterial species and discuss them in the broader context of eubacterial type IV pili.

Pili of Gram-positive bacteria: roles in host colonization

In the last decade, pili, which are encoded within pathogenicity islands, have been found in many Gram-positive bacteria, including the major streptococcal and enterococcal pathogens. These long proteinaceous polymers extending from the bacterial surface are constituted of covalently linked pilin subunits, which play major roles in adhesion and host colonization. They are also involved in biofilm formation, a characteristic life-style of the bacteria constituting the oral flora. Pili are highly immunogenic structures that are under the selective pressure of host immune responses. Indeed, pilus expression was found to be heterogeneous in several bacteria with the co-existence of two subpopulations expressing various levels of pili. The molecular mechanisms underlying this complex regulation are poorly characterized except for Streptococcus pneumoniae. In this review, we will discuss the roles of Gram-positive bacteria pili in adhesion to host extracellular matrix proteins, tissue tropism, biofilm formation, modulation of innate immune responses and their contribution to virulence, and in a second part the regulation of their expression. This overview should help to understand the rise of pili as an intensive field of investigation and pinpoints the areas that need further study.

Genetic analysis and morphological identification of pilus-like structures in members of the genus Bifidobacterium

BackgroundCell surface pili in Gram positive bacteria have been reported to orchestrate the colonization of host tissues, evasion of immunity and the development of biofilms. So far, little if any information is available on the presence of pilus-like structures in human gut commensals like bifidobacteria.Results and discussionIn this report, Atomic Force Microscopy (AFM) of various bifidobacterial strains belonging to Bifidobacterium bifidum, Bifidobacterium longum subsp. longum, Bifidobacterium dentium, Bifidobacterium adolescentis and Bifidobacterium animalis subsp. lactis revealed the existence of appendages resembling pilus-like structures. Interestingly, these microorganisms harbour two to six predicted pilus gene clusters in their genome, with each organized in an operon encompassing the major pilin subunit-encoding gene (designated fimA or fimP) together with one or two minor pilin subunit-encoding genes (designated as fimB and/or fimQ), and a gene encoding a sortase enzyme (strA). Quantitative Real Time (qRT)-PCR analysis and RT-PCR experiments revealed a polycistronic mRNA, encompassing the fimA/P and fimB/Q genes, which are differentially expressed upon cultivation of bifidobacteria on various glycans.

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.

Immunoprotective activities of a Streptococcus suis pilus subunit in murine models of infection

Pili of gram-positive bacteria are key virulence factors and their subunits are considered excellent vaccine candidates. Streptococcus suis is an emerging zoonotic agent that can cause epidemics of life-threatening infections in humans, but the functional role or immunoprotective potential of its pilus components have not been studied yet. Using a selective proteomics approach, we have identified a surface protein of serotype 2 S. suis showing features of an ancillary pilus subunit, as evidenced by bioinformatics analysis, immunoblot and immunoelectron microscopy. Immunization with recombinant fragments of this protein, designated herein as PAPI-2b, markedly protected mice from systemic S. suis infection.

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 microm 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.

Cell Wall Anchor Structure of BcpA Pili in Bacillus anthracis

Assembly of pili in Gram-positive bacteria and their attachment to the cell wall envelope are mediated by sortases. In Bacillus cereus and its close relative Bacillus anthracis, the major pilin protein BcpA is cleaved between the threonine and the glycine of its C-terminal LPXTG motif sorting signal by the pilin-specific sortase D. The resulting acyl enzyme intermediate is relieved by the nucleophilic attack of the side-chain amino group of lysine within the YPKN motif of another BcpA subunit. Cell wall anchoring of assembled BcpA pili requires sortase A, which also cleaves the LPXTG sorting signal of BcpA between its threonine and glycine residues. We show here that sortases A and D require only the C-terminal sorting signal of BcpA for substrate cleavage. Unlike sortase D, which accepts the YPKN motif as a nucleophile, sortase A forms an amide bond between the BcpA C-terminal carboxyl group of threonine and the side-chain amino group of diaminopimelic acid within the cell wall peptidoglycan of bacilli. These results represent the first demonstration of a cell wall anchor structure for pili, which are deposited by sortase A into the envelope of many different microbes.

Pili in Gram-negative and Gram-positive bacteria — structure, assembly and their role in disease

Many bacterial species possess long filamentous structures known as pili or fimbriae extending from their surfaces. Despite the diversity in pilus structure and biogenesis, pili in Gram-negative bacteria are typically formed by non-covalent homopolymerization of major pilus subunit proteins (pilins), which generates the pilus shaft. Additional pilins may be added to the fiber and often function as host cell adhesins. Some pili are also involved in biofilm formation, phage transduction, DNA uptake and a special form of bacterial cell movement, known as 'twitching motility'. In contrast, the more recently discovered pili in Gram-positive bacteria are formed by covalent polymerization of pilin subunits in a process that requires a dedicated sortase enzyme. Minor pilins are added to the fiber and play a major role in host cell colonization.This review gives an overview of the structure, assembly and function of the best-characterized pili of both Gram-negative and Gram-positive bacteria.

The molecular switch that activates the cell wall anchoring step of pilus assembly in gram-positive bacteria

Cell surface pili in gram-positive bacteria orchestrate the colonization of host tissues, evasion of immunity, and the development of biofilms. Recent work revealed that pilus assembly is a biphasic process wherein pilus polymerization is catalyzed by a pilus-specific sortase followed by cell wall anchoring of the pilus that is promoted by the housekeeping sortase. Here, we present molecular genetic and biochemical studies of a heterotrimeric pilus in Corynebacterium diphtheriae, uncovering the molecular switch that terminates pilus polymerization in favor of cell wall anchoring. The prototype pilus contains a major pilin (SpaA) forming the shaft, a tip pilin (SpaC), and another minor pilin (SpaB). Cells lacking SpaB form pilus fibers, but they are largely secreted in the medium, a phenotype also observed when cells lack the housekeeping sortase. Furthermore, the average pilus length is greatly increased in the absence of SpaB. Remarkably, a SpaB mutant that lacks the cell wall sorting signal but contains a critical lysine residue is incorporated in the pilus. However, the resulting pili fail to anchor to the cell wall. We propose that a specific minor pilin acts as the terminal subunit in pilus assembly. Cell wall anchoring ensues when the pilus polymer assembled on the pilus-specific sortase is transferred to the minor pilin presented by the housekeeping sortase via lysine-mediated transpeptidation.

Pili with strong attachments: Gram‐positive bacteria do it differently

Bacteria attach to their appropriate environmental niche by using adhesins. To maximize their contact with the environment, adhesins are often present on the ends of long hairlike structures called pili. Recently, attention has focused on pili of Gram-positive bacteria because they may be vaccine candidates in important human pathogens. These pili differ from the well-studied pili of Gram-negative bacteria because their subunits are covalently linked, they do not require specific chaperones for assembly, and the tip protein (likely to be the adhesin) is not required to initiate formation of the pilus structure. In Gram-positive bacteria, the genes for pili occur in clusters, which may constitute mobile genetic elements. These clusters include the transpeptidase(s) of the sortase family that is/are required for polymerization of the subunit proteins. However, efficient covalent attachment of the completed pilus structure to the cell wall is accomplished, in cases where this has been studied, by the 'housekeeping' sortase, which is responsible for attachment to the peptidoglycan of most surface proteins containing cell wall sorting signals. This enzyme is encoded elsewhere on the genome. Because pili of Gram-positive bacteria have not been extensively investigated yet, we hope that this MicroReview will help to pinpoint the areas most in need of further study.