Saulino, E. Snapshots of usher-mediated protein secretion and ordered pilus assembly. Natl Acad. USA 97 , — Strom, M. Structure—function and biogenesis of the type IV pili. Mulvey, M. Induction and evasion of host defenses by type 1-piliated uropathogenic Escherichia coli.
Science , — Directly showed immune evasion by piliated bacteria. Chen, I. DNA transport during transformation. Mattick, J. Type IV pili and twitching motility. Amano, A. Molecular interaction of Porphyromonas gingivalis with host cells: implication for the microbial pathogenesis of periodontal disease. Sung, M. The solution structure of PapGII from uropathogenic Escherichia coli and its recognition of glycolipid receptors.
EMBO Rep. Schweizer, F. Yanagawa, R. Electron microscopy of fine structure of Corynebacterium renale with special reference to pili. Jpn J. The first electron-microscopy study to show the presence of pili in a Corynebacterium species. Some properties of the pili of Corynebacterium renale. Cisar, J. Mutants of Actinomyces viscosus T14V lacking type 1, type 2, or both types of fimbriae.
Yeung, M. Synthesis and function of Actinomyces naeslundii T14V type 1 fimbriae require the expression of additional fimbria-associated genes. Identification of a gene involved in assembly of Actinomyces naeslundii T14V type 2 fimbriae. Ton-That, H. Assembly of pili on the surface of Corynebacterium diphtheriae. Gaspar, A. Assembly of distinct pilus structures on the surface of Corynebacterium diphtheriae.
Levesque, C. Streptococcus salivarius fimbriae are composed of a glycoprotein containing a repeated motif assembled into a filamentous nondissociable structure. Weerkamp, A. Negative staining and immunoelectron microscopy of adhesion-deficient mutants of Streptococcus salivarius reveal that the adhesive protein antigens are separate classes of cell surface fibril.
Fives-Taylor, P. Surface properties of Streptococcus sanguis FW mutants nonadherent to saliva-coated hydroxyapatite. Mora, M. Group A Streptococcus produce pilus-like structures containing protective antigens and Lancefield T antigens. USA , — Lauer, P. Genome analysis reveals pili in Group B Streptococcus. Science , Rosini, R. Identification of novel genomic islands coding for antigenic pilus-like structures in Streptococcus agalactiae.
Barocchi, M. A pneumococcal pilus influences virulence and host inflammatory responses. Maione, D. Identification of a universal group B Streptococcus vaccine by multiple genome screen. One of the first reports to provide direct evidence that pili from Gram-positive bacteria are protective antigens. McNab, R. Cell wall-anchored CshA polypeptide kilodaltons in Streptococcus gordonii forms surface fibrils that confer hydrophobic and adhesive properties.
Willcox, M. Surface structures, co-aggregation and adherence phenomena of Streptococcus oralis and related species. Microbios 59 , 19—29 Jameson, M. Polypeptides associated with tufts of cell-surface fibrils in an oral Streptococcus.
Microbiology , — Yamaguchi, T. Isolation and some properties of fimbriae of oral Streptococcus intermedius. Assembly of pili in Gram-positive bacteria. Trends Microbiol. An excellent review of the pilus-assembly process in Gram-positive bacteria. Sortases and pilin elements involved in pilus assembly of Corynebacterium diphtheriae. Navarre, W. Surface proteins of Gram-positive bacteria and mechanisms of their targeting to the cell wall envelope.
Protein sorting to the cell wall envelope of Gram-positive bacteria. Acta , — Tettelin, H. Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae : implications for the microbial 'pan-genome'.
Bessen, D. Genomic localization of a T serotype locus to a recombinatorial zone encoding extracellular matrix-binding proteins in Streptococcus pyogenes. Podbielski, A. Characterization of nra , a global negative regulator gene in group A streptococci. Kreikemeyer, B. Group A streptococcal RofA-type global regulators exhibit a strain-specific genomic presence and regulation pattern.
Nakata, M. MsmR, a specific positive regulator of the Streptococcus pyogenes FCT pathogenicity region and cytolysin-mediated translocation system genes. Hava, D. Transcriptional regulation in the Streptococcus pneumoniae rlrA pathogenicity islet by RlrA. Hemsley, C. MgrA, an orthologue of Mga, acts as a transcriptional repressor of the genes within the rlrA pathogenicity islet in Streptococcus pneumoniae. Gibbons, R. Human salivary acidic proline-rich proteins and statherin promote the attachment of Actinomyces viscosus LY7 to apatitic surfaces.
Whittaker, C. Mechanisms of adhesion by oral bacteria. Patti, J. Microbial adhesins recognizing extracellular matrix macromolecules. Cell Biol. Schwarz-Linek, U. Streptococcus pyogenes collagen type I-binding Cpa surface protein. Expression profile, binding characteristics, biological functions, and potential clinical impact.
Kirn, T. Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio cholerae.
Ochiai, K. Effect of co-aggregation on the pathogenicity of oral bacteria. Paranjpye, R. A Vibrio vulnificus type IV pilin contributes to biofilm formation, adherence to epithelial cells, and virulence. Jr et al. One of the first reports to describe the use of pili from Gram-negative bacteria for the development of a vaccine.
Google Scholar. Johnson, S. Human immunization with Pgh gonococcal pilus results in cross-reactive antibody to the cyanogen bromide fragment-2 of pilin.
Morgan, R. Immunization of suckling pigs against enterotoxigenic Escherichia coli -induced diarrheal disease by vaccinating dams with purified or K99 pili: protection correlates with pilus homology of vaccine and challenge.
Dochez, R. Studies on the biology of Streptococcus. Antigenic relationship between strains of Streptococcus haemolyticus. Dramsi, S. Assembly and role of pili in group B streptococci. Download references. We acknowledge M. In particular, fibrinogen, fibronectin, laminin, lactoferrin and collagen I were selected, as these cellular compounds are known to be recruited by pathogenic agents [9] , [38] — [40].
Apart from overall pili adhesive properties special interest was drawn to a potential role of distinctly surface exposed RrgA and RrgC. For this purpose, serial diluted samples of recombinant proteins RrgA, RrgB and RrgC, as well as native purified pili and a pilus negative control were added to well plates coated with the selected ECM components.
Binding was detected using polyclonal sera raised against the single recombinant pilus subunits and quantified by enzyme-linked immunosorbent assay ELISA. In addition, RrgA binding was observed for lactoferrin and fibrinogen whereas no binding was detected to vitronectin coated plates data not shown. Bovine serum albumin BSA was used as negative control in all the assays. Binding studies performed using purified pili Figure 8 , lane B showed binding to ECM components clearly distinguishable from the Streptococcus pneumoniae delta pilus negative control.
BSA and delta pilus mock preparation served as negative controls. Points represent the means error bars, standard errors of the means of measurements made in triplicate.
Pili are considered important key players in bacterial pathogenesis and disease [10] , [27]. To date structural information of the native pilus in Gram-positive bacteria is lacking, therefore the elucidation of their structure and function are of great interest. Our approach consisted in obtaining native purified pili from a pathogenic strain of Streptococcus pneumoniae to study pilus structure and function.
Special emphasis was drawn on the overall structural principle of the native pilus and the role of the individual structural proteins RrgA, RrgB and RrgC. As opposed to pili attached to the bacterial surface, isolated pili allow a broader spectrum of analyses and at the same time permit a comprehensive characterization of their structure in sufficient detail to describe the function at the quasi-molecular level.
We developed a multi step purification procedure to obtain native pilus material that allowed to perform the desired analyses. Interestingly, we observed pili of various morphologies: individual single pili, distinguishable into different classes by their diameter ranging from 9.
Whether or not this has a physiological role has yet to be evaluated. The established purification method allowed for the isolation of pure HMW material that showed pili morphotypes having the same features as those found for wild-type pili expressed on whole bacteria.
Structural analysis based on cryo-EM data of vitrified, purified single pili revealed that they are organised in coiled-coil superstructures made by at least two protofilaments.
The observed range in pilus diameters could either reflect a difference in the degree of packaging of the identical protofilaments into the pili superstructures or a higher number of protofilaments composing the larger pili.
The protofilaments of the thin pilus type are organized to form a rather compact superstructure. However no distinct internal cavity could be identified within the thin pilus structures. Preliminary results on the thick type of pilus suggest also a protofilament based structure. The picture of the individual pili that emerges from our analysis indicates that the Streptococcus pneumoniae pilus does not exist in a single structural state but rather in several structural states that are underlaying, among other things, the flexibility and elasticity of these polymers while keeping the same protein composition and proteins roles: RrgB forming the backbone, surface located clustered RrgA being the major ancillary protein involved in adhesion and RrgC as minor ancillary protein of still unknown role.
Additional biochemical analysis of isolated pili supports RrgB as the main pilus building block: Mass analysis of native pili revealed clear signals only for peptides that could be assigned to structural protein RrgB. Neither RrgA nor RrgC related signals could be identified, which is probably due to their minor abundance and the overall hindered protease digestibility of the isolated HMW pili. Similarly, the determination of the N-terminal amino acid sequence of the purified pili by Edman analysis, matched only with the sequence following the predicted signal sequence of RrgB.
The observation that purified pili show a free N-terminal part of RrgB starting exactly after the signal sequence may reflect properties of pilus biosynthesis and subsequently its structure. Purified pili of a Streptococcus pneumoniae delta RrgA background show a similar overall pili structure composed of protofilaments. Gram-positive and Gram-negative pili differ substantially in their assembly mechanisms Gram-negative pili: non-covalently linked protein subunits versus Gram-positive pili: covalently linked subunits , interestingly both types of bacterial pili share a common arrangement, the coiled-coil superstructure.
Our work supports that, also for Gram-positive bacteria, adhesive pili extending from the bacterial surface are the most appropriate structures to promote biological function like adherence to the host due to their structural arrangement leading to flexibility and elasticity.
Until now this could be only observed in Gram-negative bacteria like Haemophilus influenzae type b pili and Escherichia coli P-pili [41] or Actinobacillus actinomycetemcomitans [42]. Results by Kang et al. Further work will have to show whether similar design can also be found in other Gram-positive pili, like those of Streptococcus pneumoniae. Our results suggest that the coiled-coil arrangement of the protofilaments, forming the pneumococcal pili, might be an additional principle, other than isopeptide bond formation, to confer stability and flexibility to subtle surface structures in order to withstand mechanical rigors outside the cell.
Research on bacteria and therefore also the study of the pneumococcal pilus should be seen in the context of bacterial life cycles within specific ecological niches and e. Pneumococcal infection of the host occurs mainly via the mucosal route [1] , thus bacteria need to develop strategies to adhere and resist actions of the human immune system like mucosal clearance [43].
Studies performed using pilus negative mutants of T4 clearly demonstrate a positive correlation between bacterial virulence and colonization and the presence of the pilus [10]. We therefore wanted to study, if the structural data found for the isolated pili help us to better understand the functionality of pilus mediated pneumococcal behaviour within a host, and whether structural properties of the pneumococcal pilus could be function derived.
How does the pneumococcal pilus mediate interaction with its host? Our data suggest that pneumococcal pili are flexible protofilament-based structures composed of ancillary proteins RrgA and RrgC and the RrgB backbone Figure 9.
Recently, proteins of group B streptococcal pilus were found to facilitate the interaction with endothelial cells [44]. Our data elucidate the adhesive properties of RrgA to fibronectin, laminin and collagen, suggesting that the clusters containing RrgA are the adhesive regions of pili.
Interestingly, PapG, the adhesin of Escherichia coli P-pili, that binds to uroepithelial cells in its human host was also found to be located on the pilus surface, but only at the very distal end of the pilus fiber [46].
Streptococcus pneumoniae is a mucosal commensal, a mucosal pathogen and an invasive pathogen. Colonization of the nasopharynx by Streptococcus pneumoniae is a prerequisite for the development of pneumococcal disease and the result of a complex interplay between host and pathogen factors. Respiratory pathogens are known to release products which interfere with mucosal defences, causing epithelial disruption and cell death [47] , [48]. Streptococcus pneumoniae was seen to adhere in particular to damaged cells and extruded cells [47] , and bacteria were often found to be associated with damaged epithelium and exposed ECM [49].
Pathogen-ECM interactions have been found to be associated with adhesion and subsequent invasion of the pathogen [9]. Adhesive properties of pilus surface located ancillary protein RrgA to selected compounds of the ECM might therefore be part of the pilus mediated host-pathogen interplay. Flexibility of the pilus, as suggested by the protofilament-based structure, supports its functionality under in vivo conditions.
Interestingly, recent work done by Nelson et al. This together with data showing the impact of RrgA on pneumococcal virulence in mice [19] , [50] indicate that the polypeptide may function at more than one stage in the infection process.
R and L illustrate a possible right and left handed orientation of the thin pilus respectively. Outlines are not drawn to scale. In summary, this report presents support for the structural composition of the Streptococcus pneumoniae pilus as an oligomeric appendage with adhesive properties and future work will help to further improve our understanding of the structure and function of the pilus and its main components.
Streptococcus pneumoniae type 4 strain TIGR4 has been described [20]. Henriques-Normark Karolinska Institute, Stockholm. When appropriate, erythromycin Sigma-Aldrich as selection marker was used. Standard recombinant DNA techniques were used to construct all expression plasmids. Pooled fractions were dialysed overnight ON against 0. Streptococcus pneumoniae T4 was chosen as starting material as far as the bacteria belong to a clinical relevant serotype 4 isolate, the sequence of which is known [20] and it represents a well characterized pneumococcal strain.
Collected gradient fractions were tested for pilus material using anti-RrgB antibodies. Gel filtration resulted in separation of pilus containing material of different molecular weight. Protein identification was carried out by both automatic and manual comparison of experimentally generated monoisotopic values of peptides in the mass range of — Da with computer-generated fingerprints using MASCOT software running on proprietary databases.
Identification of the N-terminal amino acid sequence of HMW pili material by Edman degradation was performed according to standard conditions. Western analysis was done using standard protocols. Antibodies against recombinant HisTag-RrgA mouse; guinea pig , -RrgB mouse , and -RrgC mouse; rabbit were produced in our lab and tested for specificity. Recently published results indicate that glycosylation of proteins in Francisella is not limited to Tfp related proteins, but includes other surface proteins Balonova et al.
Genome information, as well as our own preliminary data, suggests that glycosylation of pilins is common to all subspecies Brotcke et al. It is also possible that the degree of modification at specific glycosylation sites may vary depending on growth conditions which are reflected in the ladder like appearance of PilA in Western blot analysis Forslund et al.
Posttranslational modification of Tfp has been shown to affect twitching motility and properties of the pilus by rendering the pilus fiber less hydrophobic and more stable Smedley et al. For P. This opens up the exciting possibility that glycosylation of Tfp may also be of relevance for tularemia either at the level of infection or at the level of transmission.
The F. When it comes to the gene clusters encoding Tfp there are specific differences at genetic level, where type A strains and F. One interesting functional difference between the subspecies is that Tfp can promote secretion of a subset of proteins in F.
While it remains a possibility that the in vitro conditions that promotes secretion may differ, it is still a possibility that evolution of the highly virulent subspecies included loss of Tfp-mediated secretion and that the key molecule in this development is the differences seen in the C-terminal part of PilA between F. When it comes to functional analysis and virulence, PilA has been found to be required for virulence in the human pathogenic subspecies Forslund et al.
In addition PilA has been shown to function as a pilin subunit when expressed in a heterologous Tfp expressing system in N. Several other pilin proteins that were evaluated in this assay were also expressed in N. Several studies have also revealed the presence of Tfp-like structures in various Francisella strains even if no one yet have been able to identify the major subunit in pili expressed by Francisella Gil et al. The only way to validate if different subspecies express different Tfp is to use biochemical or immunological approaches to identify the main component of the Tfp filaments in different strains.
However, it is fair to say that Tfp expressed by Francisella appear to be different in many aspects and properties compared to what is known from studies in other pathogens. Another significant finding regarding the role of PilA in virulence is that the attenuation of pilA mutants in the mouse infection model for tularemia is less pronounced the more virulent the strain is Forslund et al.
The interpretation and significance of these results is somewhat difficult as all these studies were conducted in the mouse infection model which fails to discriminate between strains that differ significantly in virulence for humans. This makes it difficult to assess if the differences in Tfp genes between type A and type B strains also results in differences in human virulence.
Our recent finding, that PilA and also other pilin proteins are glycosylated, opens other possibilities and raises new questions regarding the role of Tfp in tularemia. Glycosylation is known to influence the properties of Tfp by lowering hydrophobicity, increasing stability and motility. In addition to its role in virulence it is possible that Tfp glycosylation is important for survival and transmission of tularemia.
We are still far from understanding the role of Tfp in virulence and transmission of tularemia, but the identified highly conserved differences strongly suggest that the different subspecies may express Tfp with different properties with respect to filament subunits as well as ability to promote motility. Future work to resolve the role of Tfp in tularemia, need to include both infection models which better reflect the human infection as well as studies on how Tfp genes affect survival and transmission in different natural environments.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Aas, F. Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure.
Pubmed Abstract Pubmed Full Text. Alm, R. Identification of two genes with prepilin-like leader sequences involved in type 4 fimbrial biogenesis in Pseudomonas aeruginosa. Balonova, L. Multimethodological approach to identification of glycoproteins from the proteome of Francisella tularensis , an intracellular microorganism. Proteome Res. Piliation control mechanisms in Neisseria gonorrhoeae.
Broekhuijsen, M. Genome-wide DNA microarray analysis of Francisella tularensis strains demonstrates extensive genetic conservation within the species but identifies regions that are unique to the highly virulent F. Brotcke, A. Identification of MglA-regulated genes reveals novel virulence factors in Francisella tularensis. Catlin, B. Branhamella catarrhalis : an organism gaining respect as a pathogen.
Chakraborty, S. Type IV pili in Francisella tularensis : roles of pilF and pilT in fiber assembly, host cell adherence, and virulence. Comolli, J. Pseudomonas aeruginosa gene products PilT and PilU are required for cytotoxicity in vitro and virulence in a mouse model of acute pneumonia.
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Faast, R. Nucleotide sequence of the structural gene, tcpA, for a major pilin subunit of Vibrio cholerae. Gene 85, — Faridmoayer, A. Functional characterization of bacterial oligosaccharyltransferases involved in O-linked protein glycosylation. Forslund, A. Direct repeat-mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis.
The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensis. BMC Microbiol. Gil, H. Presence of pili on the surface of Francisella tularensis.
An inducible bundle-forming pilus of enteropathogenic Escherichia coli. Science , — CrossRef Full Text. Golovliov, I. Bacterial type IV pilins are similar in structure to the component flagellins of Archaeal flagella. Attachment of bacteria to host surfaces is required for colonization during infection or to initiate formation of a biofilm. A fimbria is a short pilus that is used to attach the bacterium to a surface.
Fimbriae are either located at the poles of a cell or are evenly spread over its entire surface. Mutant bacteria that lack fimbriae cannot adhere to their usual target surfaces and, thus, cannot cause diseases.
Some fimbriae can contain lectins. The lectins are necessary to adhere to target cells, because they can recognize oligosaccharide units on the surface of these target cells.
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