scholarly journals Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacteriumStreptococcus sanguinis

2018 ◽  
Author(s):  
Jamie-Lee Berry ◽  
Ishwori Gurung ◽  
Jan Haug Anonsen ◽  
Ingrid Spielman ◽  
Elliot Harper ◽  
...  
Keyword(s):  
Structural Analysis ◽  
3D Structure ◽  
Type Iv Pili ◽  
Gram Positive ◽  
Post Translational Modifications ◽  
Type Iv ◽  
C Terminus ◽  
Evolutionary Relatedness ◽  
Bona Fide ◽  
Prepilin Peptidase

AbstractType IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model,Streptococcus sanguinis. In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology inS. sanguinis. We found that the two major pilins, PilE1 and PilE2, (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments; (ii) display only one of the post-translational modifications frequently found in pilins,i.e. a methylated N-terminus; (iii) are found in the same hetero-polymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved by NMR, revealed a classical pilin fold with a highly unusual flexible C-terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff thanbona fideTfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show thatS. sanguinisTfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines.

Type IV pili‐dependent gliding motility in the Gram‐positive pathogenClostridium perfringensand other Clostridia

2006 ◽  
Vol 62 (3) ◽  
pp. 680-694 ◽  
Author(s):  
John J. Varga ◽  
Van Nguyen ◽  
David K. O'Brien ◽  
Katherine Rodgers ◽  
Richard A. Walker ◽  
...  
Keyword(s):  
Type Iv Pili ◽  
Gliding Motility ◽  
Gram Positive ◽  
Type Iv

scholarly journals Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacteriumStreptococcus sanguinis

2019 ◽  
Vol 294 (17) ◽  
pp. 6796-6808 ◽  
Author(s):  
Jamie-Lee Berry ◽  
Ishwori Gurung ◽  
Jan Haug Anonsen ◽  
Ingrid Spielman ◽  
Elliot Harper ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Type Iv Pilus ◽  
Gram Positive ◽  
Type Iv

scholarly journals Novel Class of Mutations of pilS Mutants, Encoding Plasmid R64 Type IV Prepilin: Interface of PilS-PilV Interactions

2007 ◽  
Vol 190 (4) ◽  
pp. 1202-1208 ◽  
Author(s):  
Eriko Shimoda ◽  
Tatsuya Muto ◽  
Takayuki Horiuchi ◽  
Nobuhisa Furuya ◽  
Teruya Komano
Keyword(s):  
Genetic Analysis ◽  
Type Iv Pili ◽  
Class I ◽  
Minor Components ◽  
Class Iii ◽  
Class V ◽  
Type Iv ◽  
Prepilin Peptidase ◽  
Class Iv ◽  
Plasmid R64

ABSTRACT The type IV pili of plasmid R64 belonging to the type IVB group are required only for liquid mating. They consist of the major and minor components PilS pilin and PilV adhesin, respectively. PilS pilin is first synthesized as a 22-kDa prepilin from the pilS gene and is then processed to a 19-kDa mature pilin by PilU prepilin peptidase. In a previous genetic analysis, we identified four classes of the pilS mutants (T. Horiuchi and T. Komano, J. Bacteriol. 180:4613-4620, 1998). The products of the class I pilS mutants were not processed by prepilin peptidase; the products of the class II mutants were not secreted; in the class III mutants type IV pili with reduced activities in liquid mating were produced; and in the class IV mutants type IV pili with normal activities were produced. Here, we describe a novel class, class V, of pilS mutants. Mutations in the pilS gene at Gly-56 or Tyr-57 produced type IV pili lacking PilV adhesin, which were inactive in liquid mating. Residues 56 and 57 of PilS pilin are suggested to function as an interface of PilS-PilV interactions.

scholarly journals FppA, a Novel Pseudomonas aeruginosa Prepilin Peptidase Involved in Assembly of Type IVb Pili

2006 ◽  
Vol 188 (13) ◽  
pp. 4851-4860 ◽  
Author(s):  
Sophie de Bentzmann ◽  
Marianne Aurouze ◽  
Geneviève Ball ◽  
Alain Filloux
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Type Iv Pili ◽  
Reading Frame ◽  
Type Iv ◽  
Bacterial Aggregation ◽  
Epithelial Cell Surface ◽  
Prepilin Peptidase ◽  
Pilus Assembly

ABSTRACT Several subclasses of type IV pili have been described according to the characteristics of the structural prepilin subunit. Whereas molecular mechanisms of type IVa pilus assembly have been well documented for Pseudomonas aeruginosa and involve the PilD prepilin peptidase, no type IVb pili have been described in this microorganism. One subclass of type IVb prepilins has been identified as the Flp prepilin subfamily. Long and bundled Flp pili involved in tight adherence have been identified in Actinobacillus actinomycetemcomitans, for which assembly was due to a dedicated machinery encoded by the tad-rcp locus. A similar flp-tad-rcp locus containing flp, tad, and rcp gene homologues was identified in the P. aeruginosa genome. The function of these genes has been investigated, which revealed their involvement in the formation of extracellular Flp appendages. We also identified a gene (designated by open reading frame PA4295) outside the flp-tad-rcp locus, that we named fppA, encoding a novel prepilin peptidase. This is the second enzyme of this kind found in P. aeruginosa; however, it appears to be truncated and is similar to the C-terminal domain of the previously characterized PilD peptidase. In this study, we show that FppA is responsible for the maturation of the Flp prepilin and belongs to the aspartic acid protease family. We also demonstrate that FppA is required for the assembly of cell surface appendages that we called Flp pili. Finally, we observed an Flp-dependent bacterial aggregation process on the epithelial cell surface and an increased biofilm phenotype linked to Flp pilus assembly.

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

2016 ◽  
Vol 44 (6) ◽  
pp. 1659-1666 ◽  
Author(s):  
Kurt H. Piepenbrink ◽  
Eric J. Sundberg
Keyword(s):  
Bacterial Species ◽  
Cellular Adhesion ◽  
Type Iv Pili ◽  
Twitching Motility ◽  
Gram Positive ◽  
Bacterial Surface ◽  
Gram Positive Bacteria ◽  
Type Iv ◽  
Current State ◽  
Highly Hydrophobic

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.

scholarly journals Cyclic Di-GMP Binding by an Assembly ATPase (PilB2) and Control of Type IV Pilin Polymerization in the Gram-Positive Pathogen Clostridium perfringens

2017 ◽  
Vol 199 (10) ◽  
Author(s):  
William A. Hendrick ◽  
Mona W. Orr ◽  
Samantha R. Murray ◽  
Vincent T. Lee ◽  
Stephen B. Melville
Keyword(s):  
Clostridium Perfringens ◽  
Biochemical Characterization ◽  
Core Protein ◽  
Type Iv Pili ◽  
Host Cells ◽  
Dependent Manner ◽  
Gram Positive ◽  
Content Type ◽  
Type Iv

ABSTRACT The Gram-positive pathogen Clostridium perfringens possesses type IV pili (TFP), which are extracellular fibers that are polymerized from a pool of pilin monomers in the cytoplasmic membrane. Two proteins that are essential for pilus functions are an assembly ATPase (PilB) and an inner membrane core protein (PilC). Two homologues each of PilB and PilC are present in C. perfringens, called PilB1/PilB2 and PilC1/PilC2, respectively, along with four pilin proteins, PilA1 to PilA4. The gene encoding PilA2, which is considered the major pilin based on previous studies, is immediately downstream of the pilB2 and pilC2 genes. Purified PilB2 had ATPase activity, bound zinc, formed hexamers even in the absence of ATP, and bound the second messenger molecule cyclic di-GMP (c-di-GMP). Circular dichroism spectroscopy of purified PilC2 indicated that it retained its predicted degree of alpha-helical secondary structure. Even though no direct interactions between PilB2 and PilC2 could be detected in vivo or in vitro even in the presence of c-di-GMP, high levels of expression of a diguanylate cyclase from C. perfringens (CPE1788) stimulated polymerization of PilA2 in a PilB2- and PilC2-dependent manner. These results suggest that PilB2 activity is controlled by c-di-GMP levels in vivo but that PilB2-PilC2 interactions are either transitory or of low affinity, in contrast to results reported previously from in vivo studies of the PilB1/PilC1 pair in which PilC1 was needed for polar localization of PilB1. This is the first biochemical characterization of a c-di-GMP-dependent assembly ATPase from a Gram-positive bacterium. IMPORTANCE Type IV pili (TFP) are protein fibers involved in important bacterial functions, including motility, adherence to surfaces and host cells, and natural transformation. All clostridia whose genomes have been sequenced show evidence of the presence of TFP. The genetically tractable species Clostridium perfringens was used to study proteins involved in polymerizing the pilin, PilA2, into a pilus. The assembly ATPase PilB2 and its cognate membrane protein partner, PilC2, were purified. PilB2 bound the intracellular signal molecule c-di-GMP. Increased levels of intracellular c-di-GMP led to increased polymerization of PilA2, indicating that Gram-positive bacteria use this molecule to regulate pilus synthesis. These findings provide valuable information for understanding how pathogenic clostridia regulate TFP to cause human diseases.

scholarly journals Type IV competence pili in Streptococcus pneumoniae are highly dynamic structures that retract to promote DNA uptake

2021 ◽  
Author(s):  
Trinh Lam ◽  
Courtney K. Ellison ◽  
Ankur B. Dalia ◽  
David T. Eddington ◽  
Donald A. Morrison
Keyword(s):  
Cell Surface ◽  
Active Role ◽  
Type Iv Pili ◽  
Dna Uptake ◽  
Gram Positive ◽  
Dynamic Activity ◽  
Positive Type ◽  
Type Iv ◽  
Labeling Approach ◽  
Dynamic Structures

SUMMARYThe competence pili of transformable Gram-positive species form a subset of the diverse and widespread class of extracellular filamentous organelles known as type IV pili (T4P). In Gram-negative bacteria, T4P act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether T4P in Gram-positive species exhibit this same dynamic activity, and their mechanism of action for DNA uptake remains unclear. They are hypothesized to either (1) passively form transient cavities in the cell wall to facilitate DNA passage, (2) act as static adhesins to enrich DNA near the cell surface for subsequent uptake by membrane-embedded transporters, or (3) play an active role in translocating bound DNA via their dynamic activity. Here, using a recently described pilus labeling approach, we demonstrate that pneumococcal competence pili are highly dynamic structures that rapidly extend and retract from the cell surface. By labeling ComGC with bulky adducts, we further demonstrate that pilus retraction is essential for natural transformation. Together, our results indicate that Gram-positive type IV competence pili are dynamic and retractile structures that play an active role in DNA uptake.Short summaryCompetent pneumococci kill non-competent cells on contact. Retractable DNA-binding fibers in the class of type IV pili may provide a key tool for retrieving DNA segments from cell wreckage for internalization and recombination.

scholarly journals Identification of Surprisingly Diverse Type IV Pili, across a Broad Range of Gram-Positive Bacteria

PLoS ONE ◽  
2011 ◽  
Vol 6 (12) ◽  
pp. e28919 ◽  
Author(s):  
Saheed Imam ◽  
Zhongqiang Chen ◽  
David S. Roos ◽  
Mechthild Pohlschröder
Keyword(s):  
Type Iv Pili ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Type Iv

scholarly journals Pseudomonas aeruginosa TfpW is a multifunctional D-Araf glycosyltransferase and oligosaccharyltransferase

2020 ◽  
Author(s):  
Anne D. Villela ◽  
Hanjeong Harvey ◽  
Katherine Graham ◽  
Lori L. Burrows
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Fluorescence Microscopy ◽  
Group Iv ◽  
Type Iv Pili ◽  
Central Portion ◽  
E Coli ◽  
Type Iv ◽  
C Terminus ◽  
Two Hybrid

ABSTRACTTfpW is an oligosaccharyltransferase that modifies the subunits of type IV pili from group IV strains of Pseudomonas aeruginosa with oligomers of α-1,5-linked-D-arabinofuranose (D-Araf). Besides its oligosaccharyltransferase activity, TfpW may be responsible for periplasmic translocation and polymerization of D-Araf. Here we investigated these potential roles of TfpW in Pa5196 pilin glycosylation. Topology studies confirmed the periplasmic location of loop 1 and the large C-terminus domain, however the central portion of TfpW had an indeterminate configuration. Reconstitution of the Pa5196 pilin glycosylation system by providing pilA, tfpW +/- tfpX and the D-Araf biosynthesis genes PsPA7_6246-6249 showed that TfpW is sufficient for glycan polymerization and transfer to pilins in P. aeruginosa PAO1, while TfpX is also necessary in Escherichia coli. In addition to PsPA7_6246, DprE1 (PsPA7_6248) and DprE2 (PsPA7_6249), the GtrA-like component PsPA7_6247 was required for pilin glycosylation in E. coli versus PAO1. In a PAO1 ΔarnE/F mutant, loss of PsPA7_6247 expression decreased the level of pilin glycosylation, suggesting that arnE/F may play a role in pilin glycosylation when PsPA7_6247 is absent. Bacterial two-hybrid studies showed interactions of TfpW with itself, TfpX, PsPA7_6247 and DprE2, suggesting the formation of a complex that enables efficient pilin glycosylation. Fluorescence microscopy of E. coli and Pa5196ΔdprE1 expressing a DprE1-sGFP fusion showed that the protein is expressed in the cytoplasm, supporting our model that includes cytoplasmic biosynthesis of the lipid carrier-linked D-Araf precursor prior to its periplasmic translocation. Together these data suggest that TfpW may be the first example of a trifunctional flippase, glycosyltransferase, and oligosaccharyltransferase.

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