The Structure of the CS1 Pilus of Enterotoxigenic Escherichia coli Reveals Structural Polymorphism
20
Citation
52
Reference
10
Related Paper
Citation Trend
Abstract:
Enterotoxigenic Escherichia coli (ETEC) is a bacterial pathogen that causes diarrhea in children and travelers in developing countries. ETEC adheres to host epithelial cells in the small intestine via a variety of different pili. The CS1 pilus is a prototype for a family of related pili, including the CFA/I pili, present on ETEC and other Gram-negative bacterial pathogens. These pili are assembled by an outer membrane usher protein that catalyzes subunit polymerization via donor strand complementation, in which the N terminus of each incoming pilin subunit fits into a hydrophobic groove in the terminal subunit, completing a β-sheet in the Ig fold. Here we determined a crystal structure of the CS1 major pilin subunit, CooA, to a 1.6-Å resolution. CooA is a globular protein with an Ig fold and is similar in structure to the CFA/I major pilin CfaB. We determined three distinct negative-stain electron microscopic reconstructions of the CS1 pilus and generated pseudoatomic-resolution pilus structures using the CooA crystal structure. CS1 pili adopt multiple structural states with differences in subunit orientations and packing. We propose that the structural perturbations are accommodated by flexibility in the N-terminal donor strand of CooA and by plasticity in interactions between exposed flexible loops on adjacent subunits. Our results suggest that CS1 and other pili of this class are extensible filaments that can be stretched in response to mechanical stress encountered during colonization.Keywords:
Pilin
Fimbriae Proteins
Enterotoxigenic Escherichia coli
Enterotoxigenic Escherichia coli (ETEC) is a bacterial pathogen that causes diarrhea in children and travelers in developing countries. ETEC adheres to host epithelial cells in the small intestine via a variety of different pili. The CS1 pilus is a prototype for a family of related pili, including the CFA/I pili, present on ETEC and other Gram-negative bacterial pathogens. These pili are assembled by an outer membrane usher protein that catalyzes subunit polymerization via donor strand complementation, in which the N terminus of each incoming pilin subunit fits into a hydrophobic groove in the terminal subunit, completing a β-sheet in the Ig fold. Here we determined a crystal structure of the CS1 major pilin subunit, CooA, to a 1.6-Å resolution. CooA is a globular protein with an Ig fold and is similar in structure to the CFA/I major pilin CfaB. We determined three distinct negative-stain electron microscopic reconstructions of the CS1 pilus and generated pseudoatomic-resolution pilus structures using the CooA crystal structure. CS1 pili adopt multiple structural states with differences in subunit orientations and packing. We propose that the structural perturbations are accommodated by flexibility in the N-terminal donor strand of CooA and by plasticity in interactions between exposed flexible loops on adjacent subunits. Our results suggest that CS1 and other pili of this class are extensible filaments that can be stretched in response to mechanical stress encountered during colonization.
Pilin
Fimbriae Proteins
Enterotoxigenic Escherichia coli
Cite
Citations (20)
Pilin
Acinetobacter baumannii
Fimbriae Proteins
Cite
Citations (64)
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 PilEΔ1–28 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 PilXNm and PilVNm, 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 PilXNm and PilVNm, likely connects a priming subcomplex to the major pilin, promoting efficient assembly of T4aP. 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 PilEΔ1–28 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 PilXNm and PilVNm, 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 PilXNm and PilVNm, likely connects a priming subcomplex to the major pilin, promoting efficient assembly of T4aP.
Pilin
Fimbriae Proteins
Priming (agriculture)
mCherry
Cite
Citations (26)
Pili of Neisseria gonorrhoeae undergo both phase and structural variation. Phase variation of gonococcal pili can be caused by a RecA-independent on/off switch in PilC, a protein involved in pilus biogenesis. We show here that spontaneous nonpiliated PilC- derivatives as well as PilC- insertional mutants have also acquired sequence alterations in pilE relative to the pilE gene of the piliated MS11mk(P+)-u parent, so that the pilin produced is processed to soluble S-pilin and can be released into the medium. It is proposed that pilin alterations are selected for in PilC- bacteria if the parental nonassembled pilin is toxic to the cells--i.e., is not degradable to S-pilin at rates sufficient to allow viability of the cells. Toxicity is indicated by the extreme instability of certain unassembled pilin sequences and by the low frequency of nonpiliated, pilin+, PilC- variants that emerge from piliated recA- cells. The presence of a point mutation changing leucine-39 to phenylalanine at the cleavage site for S-pilin in one nonpiliated, PilC-, recA- variant relative to its piliated parent is a further argument for a selective mechanism of structural diversity of the gonococcal pilin.
Pilin
Fimbriae Proteins
Neisseria gonorrhoeae
Phase variation
Cite
Citations (51)
Pilin
Fimbriae Proteins
Cite
Citations (20)
PilA, the major pilin subunit of Pseudomonas aeruginosa type IV pili (T4P), is a principal structural component. PilA has a conserved C-terminal disulfide-bonded loop (DSL) that has been implicated as the pilus adhesinotope. Structural studies have suggested that DSL is involved in intersubunit interactions within the pilus fiber. PilA mutants with single-residue substitutions, insertions, or deletions in the DSL were tested for pilin stability, pilus assembly, and T4P function. Mutation of either Cys residue of the DSL resulted in pilins that were unable to assemble into fibers. Ala replacements of the intervening residues had a range of effects on assembly or function, as measured by changes in surface pilus expression and twitching motility. Modification of the C-terminal P-X-X-C type II beta-turn motif, which is one of the few highly conserved features in pilins across various species, caused profound defects in assembly and twitching motility. Expression of pilins with suspected assembly defects in a pilA pilT double mutant unable to retract T4P allowed us to verify which subunits were physically unable to assemble. Use of two different PilA antibodies showed that the DSL may be an immunodominant epitope in intact pili compared with pilin monomers. Sequence diversity of the type IVa pilins likely reflects an evolutionary compromise between retention of function and antigenic variation. The consequences of DSL sequence changes should be evaluated in the intact protein since it is technically feasible to generate DSL-mimetic peptides with mutations that will not appear in the natural repertoire due to their deleterious effects on assembly.
Pilin
Fimbriae Proteins
Cite
Citations (44)
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 3(10)-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 3(10)-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.
Pilin
Fimbriae Proteins
Enterotoxigenic Escherichia coli
Cite
Citations (24)
Summary Adherence to host tissues mediated by pili is pivotal in the establishment of infection by many bacterial pathogens. Corynebacterium diphtheriae assembles on its surface three distinct pilus structures. The function and the mechanism of how various pili mediate adherence, however, have remained poorly understood. Here we show that the SpaA‐type pilus is sufficient for the specific adherence of corynebacteria to human pharyngeal epithelial cells. The deletion of the spaA gene, which encodes the major pilin forming the pilus shaft, abolishes pilus assembly but not adherence to pharyngeal cells. In contrast, adherence is greatly diminished when either minor pilin SpaB or SpaC is absent. Antibodies directed against either SpaB or SpaC block bacterial adherence. Consistent with a direct role of the minor pilins, latex beads coated with SpaB or SpaC protein bind specifically to pharyngeal cells. Therefore, tissue tropism of corynebacteria for pharyngeal cells is governed by specific minor pilins. Importantly, immunoelectron microscopy and immunofluorescence studies reveal clusters of minor pilins that are anchored to cell surface in the absence of a pilus shaft. Thus, the minor pilins may also be cell wall anchored in addition to their incorporation into pilus structures that could facilitate tight binding to host cells during bacterial infection.
Pilin
Fimbriae Proteins
Corynebacterium diphtheriae
Cite
Citations (165)
Pilin
Fimbriae Proteins
Neisseria gonorrhoeae
Neisseria
Cite
Citations (63)