Fimbriae: Classification and Biochemistry

TOC

doi: 10.1128/ecosal.2.4.2.1

DAVID G. THANASSI,¹ SEAN-PAUL NUCCIO,² STEPHANE SHU KIN SO,¹ AND ANDREAS J. BÄUMLER^2*

[SECTION EDITORS: ANDREAS J. BÄUMLER AND DAVID LOW]

Posted August 23, 2007

Center for Infectious Diseases, Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794-5120,¹ and Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Ave., Davis, CA 95616-8645²

*Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, School of Medicine, 3146 Tupper Hall, University of California at Davis, One Shields Ave., Davis, CA 95616-8645. Phone: (530) 754-7225. Fax: (530) 754-7240. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it .

INTRODUCTION

Historical Overview

Proteinaceous appendages projecting from the surfaces of Escherichia coli or Salmonella enterica cells are traditionally classified as those required for bacterial locomotion, termed flagella (Latin for whip), and those functioning in adherence. Nonflagellar filaments on the surfaces of E. coli cells were first detected by electron microscopy in 1950 (105). In 1955, Duguid and coworkers described the presence of filamentous appendages that enabled E. coli to bind and agglutinate erythrocytes (hemagglutination) and coined the term fimbriae (Latin for thread or fiber) for these surface structures (68). Three years later, the expression of fimbriae by S. enterica was described (67). In 1959, Brinton introduced the term pili (Latin for hair) for nonflagellar appendages of E. coli (30). Surface appendages involved in the fertility of E. coli were first described in 1964 and were termed F pili (32). In 1975, Ottow suggested restricting the term fimbriae to surface structures involved in adherence while reserving the term pili for sexual appendages (205). However, Ottow’s nomenclature has not gained broad acceptance, and both terms are still in use to describe the same surface appendages.

General Properties

Fimbriae are generally involved in mediating attachment, either to host surfaces, to abiotic surfaces, or between bacterial cells. While fimbriae are usually present in large numbers, with 100 to 1,000 filaments protruding from the surface of an individual bacterial cell, this characteristic does not apply to all systems, as the expression of F pili leads to the formation of only one to three filaments per cell. Fimbrial filaments are between 2 and 8 nm in diameter (considerably thinner than flagellar filaments, which have a diameter of about 20 nm) and are composed of proteins, termed fimbrial subunits or pilins, which range in size from 7 to 30 kDa. Fimbriae may be composed of a single protein, termed the major fimbrial subunit, which forms the filament and functions in adherence. Alternatively, the major fimbrial subunit may form the filament and additional proteins, termed minor fimbrial subunits, may serve functions such as the formation of branch points for fimbrial assembly, the formation of a tip fibrillum, and/or the formation of a tip adhesin.

OVERVIEW OF CLASSIFICATION SCHEMES

Classification Based on Morphology and Function

In 1965, Brinton was able to distinguish six types of fimbriae of E. coli (31). One year later, Duguid and coworkers proposed a scheme that distinguishes seven types of fimbriae (types 1 to 6 and F) based on morphology and the ability to mediate hemagglutination (65). According to the classification by Duguid, type 1 fimbriae are defined as adhesins that mediate mannose-sensitive hemagglutination (63). Type 2 fimbriae are structures expressed by S. enterica serotype Gallinarum that do not mediate hemagglutination but are morphologically similar to type 1 fimbriae (67). Type 3 fimbriae mediate the mannose-resistant hemagglutination of erythrocytes treated with tannic acid (63), while type 4 fimbriae mediate the mannose-resistant hemagglutination of untanned erythrocytes (67). The designations type 5 fimbriae and type 6 fimbriae refer to surface structures detected in Pseudomonas and Klebsiella species. The final group, F fimbriae (F pili), comprises filaments expressed in F⁺ and Hfr strains of E. coli that function in conjugation (32). Subsequently, surface appendages of E. coli that are morphologically similar to type 1 fimbriae but that do not mediate hemagglutination were termed type 1C fimbriae (137).

More recently, the hemagglutination of human erythrocytes from different blood groups has been used to assign designations to E. coli fimbriae (Table 1). Examples include G fimbriae, which bind N-acetyl-D-glucosamine present on erythrocytes of the G blood group (223); P fimbriae of uropathogenic E. coli (UPEC), which bind the disaccharide Galα(1-4)Gal present on erythrocytes of the P blood group system (125, 148); M fimbriae, which bind erythrocytes of the M blood group (223); Dr fimbriae, which bind CD55 on Dr blood group erythrocytes (193, 194); and S fimbriae, which adhere to sialic acid-containing receptors and mediate neuraminidase-sensitive hemagglutination (141, 210).

Table 1Classification of *E. coli* fimbriae by hemagglutination

Genetic analyses of DNA regions involved in fimbrial biosynthesis illustrate that hemagglutination-based classification schemes are arbitrary. Type 1 fimbriae (as defined by Duguid) of S. enterica serotype Typhimurium are encoded by the fimAICDHF operon located on the chromosome at 15 centisomes (Fig. 1B). Although an orthologous gene cluster is present at the corresponding map position in S. enterica serotype Gallinarum, it encodes an adhesin classified by Duguid as a type 2 fimbria (Fig. 1A) (65). The inability of S. enterica serotype Gallinarum type 2 fimbriae to mediate mannose-sensitive hemagglutination is due to a single amino acid substitution in the FimH tip adhesin (134). A cryptic fimbrial gene cluster with homology to the S. enterica fimAICDHF operon, termed sfmACDHF, is present at the corresponding chromosomal location in E. coli K-12 (at 12 centisomes) (Fig. 1C) (23). However, the production of type 1 fimbriae by E. coli is encoded by the paralogous fimBEAICDFGH gene cluster, which is located at 98 centisomes (Fig. 1D). No orthologs of the E. coli fimBEAICDFGH genes are present at the corresponding map location in the S. enterica serotype Typhimurium chromosome (171). Thus, hemagglutination-based systems may assign fimbriae encoded by orthologous operons to different groups while at the same time placing fimbriae encoded by paralogous operons into the same group.

Fig. 1Operons encoding type 1 fimbriae and related fimbrial gene sequences. Type 1 fimbriae of S. enterica serotype Typhimurium are encoded by a gene cluster (B) that is orthologous to genes encoding type 2 fimbriae of S. enterica serotype Gallinarum (A) and a cryptic fimbrial operon (sfm) in E. coli (C). Type 1 fimbriae of E. coli are encoded by an operon (D) that is paralogous to the S. enterica serotype Typhimurium fim operon.

In a landmark review published in 1975, Ottow proposed an updated classification system that distinguishes six groups of fimbriae (205). The first group consists of peritrichously arranged fimbriae with adhesive properties, including the fimbrial types 1 to 4 described by Duguid. Group 2 comprises organelles involved in conjugation (F fimbriae, or F pili). Nonflagellar structures as described for Agrobacterium species form a third group. Group 4 includes polarly inserted fimbriae, originally described for Pseudomonas aeruginosa and Vibrio cholerae, which have the ability to promote bacterial motion (64, 297). Group 5 comprises polarly arranged, contractable tubules observed in star-forming soil bacteria (e.g., Agrobacterium and Rhizobium species), and group 6 refers to characteristic bundles of fimbriae expressed by the gram-positive bacterium Corynebacterium renale.

While the fimbrial classification schemes proposed by Duguid et al. (65) and Ottow (205) are of mostly historical interest, fragments of each nomenclature system have survived. The part of the nomenclature proposed by Duguid that has withstood the test of time is the designation type 1 fimbriae for adhesins mediating mannose-sensitive hemagglutination. The remnant of the nomenclature proposed by Ottow that remains in active use is the categorization group 4, a class of polar fimbriae mediating twitching motility, which has given rise to the commonly used designation type IV fimbriae. As only these two fragments of Duguid’s and Ottow’s classification schemes are presently in common use, fimbrial nomenclature is often confusing for scholars entering the field.

Classification Based on Serology

Gillies and Duguid were the first to investigate the serological relatedness of fimbriae by bacterial agglutination (86). This approach revealed that type 1 fimbriae of E. coli are serologically related to those of Shigella species but not to type 1 fimbriae of S. enterica serotype Typhimurium (66, 86). However, S. enterica serotype Typhimurium type 1 fimbriae are serologically related to type 2 fimbriae of S. enterica serotype Gallinarum (197). Thus, these early serological studies correctly predicted the genetic relatedness of fimbrial antigens that was later confirmed by sequence analysis of the corresponding biosynthesis genes (Fig. 1).

While serology has not been used to further classify fimbriae of S. enterica, this approach has been used extensively to differentiate fimbrial antigens of E. coli. Lawn and Meynell distinguished four serotypes of F fimbriae (F pili) among different F⁺E. coli strains (146). Over several years, Ørskov and Ørskov developed a serological classification scheme for E. coli fimbriae with designations of F1 through F17 (Table 2) (201). According to this nomenclature, type 1 fimbriae and type 1C fimbriae are assigned the antigen designations F1A and F1C, respectively. Colonization factor antigen I (CFA/I), CFA/2, K88 fimbriae, K99 fimbriae, and 987P fimbriae from enterotoxigenic E. coli (ETEC) are assigned to antigen designations F2, F3, F4, F5, and F6, respectively. Ørskov and Ørskov distinguished P fimbriae serologically into groups with antigen numbers F7 to F16. The gene clusters encoding these antigens are very similar (288). Antigen group F17 as defined by Ørskov and Ørskov includes fimbriae present in bovine ETEC (151), UPEC (167), and E. coli producing the cytotoxic necrotizing factor (159, 160). Antigen group F18 was later assigned to F107 fimbriae, 2134P pili, and colonization factor 8813 of E. coli isolates from cases of postweaning diarrhea in pigs (225). Other investigators used a similar nomenclature system but assigned F antigen designations that were not consecutive with those used by Ørskov and Ørskov. Sequence analysis of the gene encoding the major fimbrial subunit of P fimbriae, papA, led to the identification of two new serological variants, designated F40 and F48 (119). Adhesins of porcine ETEC isolates that are serologically related to P fimbriae are termed F165₁ (75). Fimbriae present in the bovine E. coli strain B41 were serologically distinguished from K88 fimbriae and assigned the designation F41 antigen (179). Subsequent serological studies identified the presence of fimbrial antigen F42 in bovine ETEC isolates (307).

Table 2Serological classification of *E. coli* fimbriae

A nomenclature that defines fimbriae present in human ETEC isolates serologically as CFA/I, CFA/II, CFA/III, and CFA/IV developed in parallel to the serological classification scheme established by Ørskov and Ørskov (55, 72, 73, 74, 138). CFA/II fimbriae of different ETEC isolates are heterogeneous and can be subdivided into coli surface antigen 1 (CS1), CS2, and CS3 (149, 183). Similarly, CFA/IV fimbriae are further subdivided into CS4, CS5, and CS6 (277). The term putative colonization factor is applied to adhesins, for which a role in colonization has yet to be determined (40). This nomenclature is still evolving, and the reader is referred to a recent review for a listing of all presently defined CS antigens (219).

Due to its high specificity, serological classification is not suited for assigning individual fimbriae to larger groups of related surface structures. Instead, serology generates lists of genetically distinct appendages that are expressed by E. coli strains in vitro.

Classification Based on Sequence of Major Fimbrial Subunit

Based on sequence comparisons of the major fimbrial subunits, Low and coworkers proposed a classification scheme that distinguishes six classes of fimbriae ( 155). The major subunits of the first class, which includes P fimbriae and type 1 fimbriae, contain two conserved cysteine residues that are spaced between 38 and 43 amino acids apart. In class 2 fimbriae, the conserved cysteine residues are spaced 31 amino acids apart, and this group includes F1845 fimbriae, Dr fimbriae, and the Afa adhesin. Major fimbrial subunits of K88 and F41 fimbriae do not contain conserved cysteine residues, and these fimbriae are grouped into class 3. Class 4 comprises type IV fimbriae, while fimbriae assembled by the alternate chaperone-usher pathway (CFA/I and CS1; see below) are grouped into class 5. Finally, class 6 includes curli fimbriae. The classification of fimbriae based on the sequences of the major fimbrial subunits results in subdivisions similar to those of the classification schemes discussed in the following section, which distinguish fimbriae based on their assembly mechanisms.

Classification Based on Assembly Mechanism

Fimbriae can be classified based on the mechanism by which these appendages are assembled on the bacterial surface. This classification has gained popularity because members of an assembly class can be readily identified by the sequence homology of their fimbrial biosynthesis genes. Generally, subunits of all E. coli or S. enterica fimbriae contain signal sequences that allow their transport into the periplasm. However, based on subsequent steps involved in fimbrial biosynthesis, four fundamentally different assembly mechanisms can be distinguished.

Fimbriae Assembled by a Chaperone- and Usher-Dependent Assembly Mechanism.

The first mechanism involves the binding of fimbrial subunits by a periplasmic chaperone and their subsequent transport to and assembly into filaments at the cell surface by an outer membrane usher protein. This transport mechanism defines the chaperone- and usher-dependent assembly class. All fimbriae listed in Table 1 and Table 2 are members of this assembly class.

An unrooted phenogram of usher amino acid sequences from E. coli and S. enterica is shown in Fig. 2. This comparison illustrates that the chaperone- and usher-dependent assembly class can be further subdivided into two families whose members show little sequence identity to those of the other family: a classical chaperone-usher family and an alternate chaperone-usher family. The alternate chaperone-usher family comprises fimbriae expressed by ETEC strains and S. enterica serotype Typhi, including CFA/I (CfaC), CFA/II CS1 (CooC), CFA/II CS2 (CotC), CFA/IV CS4 (CsaC), CS19 (CsdC), F17 (CsbC), and Typhi colonization factor (TcfC) (2, 3, 79, 83, 84, 95) (Fig. 2).

Fig. 2Subdivision of the chaperone- and usher-dependent assembly class based on usher amino acid sequence alignments. Members can be subdivided into two families: the classical chaperone-usher family and the alternate chaperone-usher family. The classical chaperone-usher family can be further subdivided into subfamilies 1 through 6. Usher amino acid sequences were aligned with MacVector 7.2 ClustalW to generate an unrooted phenogram. Pairwise distancing, neighbor joining, and plotting were performed with the PROTDIST, NEIGHBOR, and DRAWTREE programs, respectively, of the PHYLIP 3.65 software package with default settings. STy, S. enterica serotype Typhi; STm, S. enterica serotype Typhimurium; K12, E. coli K-12; EAEC, enteroaggregative E. coli; EHEC, enterohemorrhagic E. coli; REPEC, rabbit enteropathogenic E. coli.

The majority of known usher proteins of E. coli and S. enterica group into the classical chaperone-usher family. The classical chaperone-usher family contains the FGL chaperone subfamily, which has been defined based on shared structural features or chaperones (111, 311) and contains members of class 2 as defined by the similarity of major fimbrial subunits (155). The phylogenetic tree shown in Fig. 2 illustrates that usher proteins belonging to the FGL chaperone subfamily share a common ancestry. The FGL chaperone subfamily assembles fibrillar or nonfimbrial surface structures (111, 311). The remaining chaperones of the classical chaperone-usher family, which assemble fimbriae, have been assigned to the FGS chaperone subfamily (111). A sequence comparison of chaperone (26) or usher proteins (Fig. 2) shows that the FGS chaperone subfamily cannot be defined by a single node or branch of a tree.

Curli Fimbriae.

A second assembly pathway involves the secretion of fimbrial subunits across the outer membrane, followed by an extracellular assembly process that is initiated by a nucleator protein (97). Only one member of this class of fimbrial antigens is encoded in the genomes of E. coli and S. enterica strains, and it has been termed curli (199), thin curled fimbriae (92, 262), or thin aggregative fimbriae (47, 48). Stolpe and coworkers suggested that curli fimbriae of S. enterica represent type 3 fimbriae of the Duguid classification scheme (262). Curli biosynthesis genes of E. coli (csg) and S. enterica (agf) are highly conserved with regard to sequence, gene order, and chromosomal location (46, 96, 229) (Fig. 3). The major fimbrial subunit of curli, CsgA (AgfA), is recognized as a conserved pathogen-associated microbial pattern that stimulates the innate immune system of the host (15, 17, 285).

Fig. 3Conservation of the operon encoding curli production in E. coli and Salmonella serotypes. The operon encoding curli production in S. enterica serotype Typhimurium is shown at the top (229). Numbers below indicate the percentages of amino acid sequence identity of proteins encoded by individual S. enterica serotype Typhimurium agf genes to their counterparts in S. enterica serotype Enteritidis, S. enterica serotype Typhi, S. bongori, E. coli K-12, and E. coli O157:H7.

Type IV Fimbriae.

Type IV fimbriae comprise a class of polarly inserted appendages which have the ability to promote bacterial motion over moist surfaces by a process termed twitching motility (18, 29). The term type IV fimbriae originates from a nomenclature system proposed by Ottow in which this class of fimbriae was described as group 4 (205). Subsequent work has shown that type IV fimbriae are assembled by a mechanism that is unrelated to that of chaperone-usher-dependent fimbriae or curli fimbriae. The biosynthesis of type IV fimbriae requires the presence of a specialized prepilin peptidase (61, 104, 313), and the assembly of fimbrial subunits into filaments is initiated at the periplasmic face of the cytoplasmic membrane and involves ATP hydrolysis, which can energize the pilus retraction required for twitching motility (158, 174, 196, 302, 304, 305). Type IV fimbriae are found in S. enterica serotype Typhi, S. enterica serotype Typhimurium, Shiga toxin-producing E. coli, enteropathogenic E. coli (EPEC), and ETEC, where they are associated with plasmids or horizontally acquired DNA regions (Table 3).

Table 3Type IV fimbriae present in *E. coli* and *S. enterica*

Conjugative F Fimbriae.

The fourth assembly pathway for nonflagellar surface appendages present in E. coli and S. enterica is that of F fimbriae (F pili, or sex pili). F fimbriae are part of type IV secretion systems mediating the conjugative transfer of DNA. The mechanism of F fimbria assembly will not be further addressed in this chapter; readers are referred to the chapter on conjugation (Chapter Cytology).

FIMBRIAL REPERTOIRES IN E.COLI AND S.ENTERICA

Clinical isolates of ETEC and UPEC frequently express several fimbrial antigens upon laboratory culture that can be detected by electron microscopy and serological techniques (Table 4). ETEC isolates from human and livestock express different fimbrial repertoires, an observation that has given rise to the concept that fimbrial attachment to mucosal surfaces is involved in host adaptation. While most fimbrial antigens expressed by ETEC isolates are plasmid encoded, UPEC isolates express a distinct repertoire of fimbrial adhesins which are chromosomally encoded.

Table 4Fimbrial antigens commonly expressed in vitro by clinical isolates of ETEC and UPEC

Unlike ETEC and UPEC strains, which express multiple fimbriae in vitro, isolates of enterohemorrhagic E. coli, EPEC, or S. enterica express only a few surface structures upon laboratory culture. For instance, in some E. coli O157:H7 isolates, the expression of only type 1 fimbriae (70, 249), curli (132), and two fimbrial structures encoded by large plasmids (pO157 and pSFO157) (34, 128) has been detected by electron microscopy. However, whole-genome sequencing of two E. coli O157:H7 isolates revealed the presence of at least 16 fimbrial gene clusters (98, 215). The construction of transcriptional fusions with 13 of these gene clusters showed that most of the genes are not expressed in vitro (154), but analyses of cloned operons and of phenotypes resulting from mutational inactivation (71, 124, 153, 281, 282) have suggested that many of these loci encode functional fimbriae. Sequence analysis also shows that some O157:H7 isolates are unable to produce functional type 1 fimbriae (110, 228).

Similarly, the expression of only type 1 fimbriae and curli in S. enterica serotype Typhimurium has been demonstrated by electron microscopy (65, 67, 91, 92, 262), while the presence of 13 fimbrial gene clusters, termed fim, agf (csg), pef, lpf, saf, bcf, stb, stc, std, sth, stf, sti, and stj, was revealed by whole-genome sequencing (171). In most cases, fimbrial expression cannot be detected in vitro with antisera against major fimbrial subunits of S. enterica serotype Typhimurium (109), but findings from studies on fimbrial expression in vivo (108, 109) and on the role of fimbrial operons during the colonization of the mouse intestine (11, 12, 284, 296) suggest that most of the encoded adhesins are functional.

Genomic comparison shows that fimbrial gene sequences are among the most polymorphic regions in the genomes of E. coli and S. enterica. Comparisons of fimbrial repertoires of S. enterica serotypes Typhimurium (LT2), Typhi (CT18 and Ty2), and Paratyphi A (SARB64) illustrate that fimbrial gene sequences were lost or acquired frequently during the divergence of their lineages (Fig. 4). The strictly human adapted S. enterica serotypes Typhi and Paratyphi A carry stop codons or frameshift mutations in multiple fimbrial operons, which may reflect their host restriction or the decreased importance of intestinal persistence during systemic infections, such as typhoid fever (170, 209, 283). In E. coli, the tip adhesin subunits of fimbriae are under strong pressure selecting for functionally adaptive amino acid replacements (298).

Fig. 4Fimbrial gene sequences present in the genomes of S. enterica serotypes Typhimurium (LT2), Typhi (CT18 and Ty2), and Paratyphi A (SARB64) (10, 57, 65, 79, 82, 92, 170, 172, 209, 229, 262, 283, 284). Superscript letters indicate the following: a, pseudogenes are present in S. enterica serotype Typhi (CT18 and/or TY2); b, pseudogenes are present in S. enterica serotype Paratyphi A; c, pseudogenes are present in S. enterica serotypes Typhi and Paratyphi A; d, pseudogenes are present in S. enterica serotypes Typhi and Typhimurium.

BIOCHEMISTRY OF FIMBRIAE

Fimbriae Assembled by the Classical Chaperone-Usher Pathway

The chaperone-usher pathway is responsible for the assembly and secretion of a superfamily of virulence-associated surface structures by a variety of gram-negative bacteria (242, 275). The chaperone-usher pathway typically assembles rod-like fibers (pili or fimbriae) but may also assemble amorphous (afimbrial), capsule-like structures. In E. coli and Salmonella, this pathway assembles a diverse range of both fimbrial and afimbrial structures as described above. The fimbrial structures expressed by E. coli and Salmonella are rigid or flexible fibers ranging from 2 to 10 nm in diameter and generally 1 to 3 μm in length. The genetic loci coding for these structures are found both on the chromosome and on plasmids.

All fimbriae assembled by the chaperone-usher pathway are likely to share a common mechanism of biogenesis. The adhesive P and type 1 fimbriae of UPEC are prototypical structures assembled by the chaperone-usher pathway (1, 165). P and type 1 fimbriae are critical virulence determinants of UPEC, allowing binding in and colonization of the urinary tract. They are two of the best-characterized fimbrial systems, and this section will focus on the structures and biochemistry of these fimbriae as models. P and type 1 fimbriae of UPEC are encoded by the chromosomal pap and fim gene clusters, respectively. P fimbriae are critical for binding to Galα(1-4)Gal moieties present in the kidney and are associated with pyelonephritis (24, 156, 226, 259). Type 1 fimbriae mediate binding to mannosylated glycoproteins present in the bladder and to a number of other tissues in a mannose-sensitive manner and are associated with cystitis (7, 185, 257, 259).

Structures of Fimbriae.

P fimbriae are composite fibers comprised of two discrete subassemblies: a thin, flexible tip fibrillum and a rigid, helical rod (Fig. 5A and B) (36, 142). The tip fibrillum is a flexible, open helical fiber measuring 2 nm in diameter and approximately 40 nm in length (142 ). PapE (16 kDa) is the major component of the tip fibrillum and is present in approximately 5 copies per pilus. The adhesin, PapG (36 kDa), is present in a single copy at the distal end of the tip fibrillum and is joined to the PapE tip fiber by the PapF adaptor subunit (116, 142). Another adaptor protein, PapK, serves to link the tip fibrillum to the pilus rod (Fig. 5B) (116). The pilus rod is a homopolymer of over 1,000 repeating PapA (16.5-kDa) subunits wound into a tight, one-start-site, right-handed helix with an external diameter of 8.2 nm, a hollow axial channel of 2.5 nm diameter, and 3.28 subunits per helical turn (36, 181). The helical rod can convert under stress into a linear fiber similar to the tip fibrillum (36). This ability may allow the fimbriae to withstand shear forces caused by the flow of urine and thus maintain bacterial adhesion within the urinary tract. Finally, PapH terminates the pilus rod and may play a role in anchoring fimbriae to the outer membrane (8).

Fig. 5Examples of fimbriae assembled by different pathways. (A) Negatively stained electron micrograph of E. coli expressing P fimbriae. (B) Freeze-etch electron micrograph of a P pilus fiber showing the distal-tip fibrillum and the helical rod. To the right of the micrograph is a cartoon representation of the fiber and a topology diagram depicting the interaction of pilin subunits by donor strand exchange. Each subunit consists of an Ig-like fold and an N-terminal extension. The N-terminal extension completes the Ig fold of the preceding subunit. (C) Negatively stained electron micrograph of E. coli expressing CS1. (D) Negatively stained electron micrograph of E. coli expressing curli fimbriae. (E) Negatively stained electron micrograph of EPEC expressing BFP, indicated by the arrows, together with atomic-resolution models for the BFP fiber in ribbon and surface representations. In the ribbon representation, the bundlin subunits in each strand of the three-start helix are depicted in yellow, red, and blue. Each subunit consists of an extended N-terminal α-helix and a globular C-terminal head domain. In the surface representation, the hydrophobic N terminus that packs into the center of the fiber is depicted in gray, and the α-ß loop, the D-region, and the core of the C-terminal head domain are depicted in green, yellow, and blue, respectively.

Source: Panels A, B, C, D, and E were modified or reprinted from references 111a, 142 and 147a, 238, 9a, and 220, respectively, with permission of the publishers.

Type 1 fimbriae are encoded throughout the Enterobacteriaceae (1). Type 1 fimbriae are composite structures similar to P fimbriae but contain shorter, stubbier tip fibers (123). Type 1 fimbrial filament tips measure 10 to 19 nm in length and consist of the 29-kDa FimH adhesin and the FimF and FimG adaptor proteins (94, 123, 234, 243). Interestingly, the strength of FimH binding to its receptor is enhanced by shear forces such as those encountered in the urinary tract (278). The type 1 filament rod is composed of the major pilin subunit FimA (16 kDa) assembled into a one-start, right-handed helical cylinder with an external diameter of 6.9 nm, a hollow axial channel of 2.1 to 2.5 nm diameter, and 3.375 subunits per turn (31, 94).

Assembly Mechanism.

The chaperone-usher pathway takes its name from the components of its secretion machinery, which consists of a periplasmic chaperone (PapD for P fimbriae and FimC for type 1 fimbriae) that works in concert with an integral outer membrane usher protein (PapC for P fimbriae and FimD for type 1 fimbriae) (58, 122, 136, 143, 189). Both the pilins (pilus subunit proteins) and the assembly proteins are synthesized with typical N-terminal signal sequences for translocation across the inner membrane via the Sec general secretory pathway (163, 218). Once in the periplasm, the pilins must interact with the periplasmic chaperone for proper folding and the prevention of off-pathway interactions. In the absence of the chaperone, pilins misfold and form aggregates that are rapidly degraded by the DegP periplasmic protease (121, 206, 254). (The chaperone accelerates the rate of pilin folding, caps interactive surfaces of the pilins to prevent subunit-subunit interactions in the periplasm, and maintains the pilins in an assembly-competent state [8a, 215, 292]). In addition to the chaperone, the periplasmic disulfide isomerase DsbA is required for the proper formation of disulfide bonds in the pilins (9, 117).

Periplasmic chaperone-subunit complexes are next targeted towards the outer membrane usher for the assembly of pilins into the pilus fiber and the secretion of the fiber across the outer membrane to the cell surface. Pilus assembly is thought to take place at the periplasmic face of the usher, concomitant with the secretion of the fiber through the usher channel to the cell surface (276). The interaction of chaperone-subunit complexes with the usher facilitates the release of the chaperone from the pilin, thus allowing subunit-subunit interactions to take place. Fimbriae are built in a top-down order, with the adhesin incorporated first, followed by the assembly of the tip fibrillum and, lastly, the pilus rod. Each pilin specifically interacts with its appropriate neighbor subunit in the pilus, and the usher facilitates this organization by differentially recognizing chaperone-subunit complexes according to their final positions in the pilus (58, 244). The usher forms a dimeric complex containing channels ~2 nm in diameter (150), which is sufficient to allow the secretion of a linear fiber of folded pilus subunits (90, 142). However, a 2-nm channel is not large enough to accommodate the 7- to 8-nm-diameter helical pilus rod (94, 181). The rod is likely constrained to a linear form during passage through the usher, adopting its final helical conformation only upon reaching the cell surface (276).

Structures of Component Proteins and Physical Properties.

The molecular basis for many aspects of pilus assembly by the chaperone-usher pathway has been revealed by crystal structures of chaperone-subunit and subunit-subunit complexes (242). All pilus subunits fold with a single pilin domain, except the adhesin, which contains an N-terminal adhesin domain in addition to the pilin domain. Crystal structures of the PapDK chaperone-subunit complex of P fimbriae and the FimCH chaperone-adhesin complex of the type 1 system have revealed that the pilin domain is an immunoglobulin (Ig)-like fold (Fig. 5B) (43, 240). However, the pilin domain is missing the seventh (C-terminal) ß-strand present in canonical Ig folds. The lack of this ß-strand produces a deep groove along the surface of the subunit, exposing its hydrophobic core. The chaperone, which consists of two complete Ig domains oriented in a boomerang configuration (103), donates its G1 ß-strand and a portion of its F1-G1 loop to complete the Ig fold of the pilin in a reaction termed donor strand complementation. This interaction caps the subunit’s hydrophobic core. The subunit groove is also the site of subunit-subunit interactions. Thus, donor strand complementation couples the folding of subunits with the simultaneous capping of their interactive surfaces. The chaperone recognizes a highly conserved motif present in the C-terminal regions of all pilin subunits (258). This motif contains a series of alternating hydrophobic residues flanked by a glycine residue and a penultimate tyrosine. The G1 ß-strand donated by the chaperone also contains alternating hydrophobic residues, which form a ß-zipper interaction with the subunit motif (144, 258).

With the exception of the adhesin, all pilus subunits have a highly conserved N-terminal extension that participates in subunit-subunit interactions. This N-terminal extension contains a conserved motif of alternating hydrophobic residues, similar to the chaperone G1 ß-strand (258). During pilus assembly, this N-terminal extension displaces the G1 ß-strand donated by the chaperone in a mechanism termed donor strand exchange. Crystal structures of the PapDE chaperone-subunit complex and PapE in complex with the N-terminal extension peptide of PapK revealed an interesting conformational change in PapE upon the development of the donor strand exchange reaction (241). In donor strand complementation, the chaperone G1 ß-strand runs parallel to the PapE F1 strand, forming a noncanonical Ig fold that maintains subunits in an open, activated state. In contrast, the N-terminal extension donated by the subunit is inserted antiparallel to the F strand of the preceding subunit, thus completing the canonical Ig fold of the preceding subunit (241, 310). Thus, the pilus fiber consists of an array of complete Ig folds, with each subunit donating a strand to complete the Ig fold of its neighboring subunit (Fig. 5B). ATP is not available in the periplasm, and pilus biogenesis at the outer membrane does not require the transduction of energy from the inner membrane (117). The canonical Ig fold formed during subunit-subunit interaction and donor strand exchange represents a more compact, lower-energy state than the noncanonical Ig fold formed during donor strand complementation with the chaperone-subunit complex (241, 310). This topological transition from the high-energy chaperone-subunit complex to the low-energy subunit-subunit complex presumably provides the driving force for fiber formation and secretion at the usher.

For the assembly of the pilus rod, the linear fiber of strand-exchanged subunits must convert into its final helical form. Models of assembled pilus rods have been built by combining crystal structures of subunit proteins together with helical reconstructions from electron microscopy data (94, 181, 182). For P pili, two regions of PapA that promote the winding of the PapA linear fiber into the helical pilus rod were identified. PapA residues 106 to 109 form a surface involved in subunit-subunit interactions with the PapA monomer one turn down the helix (interactions between subunit n and n + 3); mutations of these residues prevent the assembly of the native pilus helix (182). In addition, PapA residues 12 to 20 form a protruding hinge region that appears to provide the flexibility required for the rotation of the PapA monomers to coil into the helix (181). Mutations of residues within this hinge region also prevent the formation of the native pilus helix.

The adhesin is distinct from the other pilus subunits in having an adhesin domain that replaces the N-terminal extension of the pilin domain. Structures of the complete FimH adhesin and the adhesin domain of PapG have been solved previously (43, 59, 110, 269). The adhesin domain of FimH folds with an elongated, jelly roll-like motif of 11 ß-strands, with the mannose-binding site located in a deep pocket at the tip of the domain (43, 110). There are three classes of PapG: class I binds to globotriasylceramide (GbO3); class II binds to GbO4, which consists of an N-acetyl-galactosamine (GalNAc) sugar added to GbO3; and class III binds to GbO5 (Forssman antigen), which consists of two GalNAc sugars added to GbO3 (267, 268). PapGII is associated mainly with pyelonephritis in humans, while PapGIII is associated predominantly with human cystitis. The PapGII adhesin domain folds with a large, elongated jelly roll motif consisting of an eight-stranded ß-sandwich (59, 269). The residues that interact with the glycolipid receptor are surface exposed and lie in a shallow pocket on one side of the PapGII adhesin domain. The flexibility of the P pilus tip fibrillum and the side-on orientation of the PapGII-binding site likely function to facilitate the docking of the adhesin onto the globoside moiety of the receptor, which is oriented parallel to the membrane surface (59). This organization is in contrast to that of type 1 fimbriae, which have a shorter and less flexible tip fibrillum, with the binding site located at the distal tip of the FimH adhesin. The structures of additional fimbrial adhesins (F17-G, GafD, DraE, and AfaE) have been solved previously (5, 38, 173, 216), revealing a common architecture consisting of an elongated jelly roll-like ß-barrel fold that may have evolved from the Ig fold present in the pilin domains. Despite this common architecture, the adhesins diverge greatly in sequence and use distinct mechanisms for binding to their receptors, reflecting their diverse functions within the host.

Fimbriae Assembled by the Alternate Chaperone-Usher Pathway

ETEC expresses a group of more than 20 serologically distinct fimbriae that are critical for its ability to adhere to and colonize the small intestine (85). ETEC is the leading cause of gastroenteritis (travelers’ diarrhea) and a major cause of diarrheal disease in underdeveloped nations, especially among children (25). CS1 fimbriae are the prototype for a subfamily of these colonization factors that includes CFA/I, CS2, CS4, CS14, CS17, and CS19 fimbriae (239). This subfamily of fimbriae has been designated class 5 on the basis of protein sequence (155), and CFA/I fimbriae belong to the F2 antigenic group (Table 2). In addition to ETEC fimbriae, the Tcf fimbriae of S. enterica serotype Typhi (79) belong to the CS1 family, as do the cable type II fimbriae expressed by Burkholderia cepacia (236). This section will focus on CS1 fimbriae, which are the best-studied members of this class of surface fibers. CS1 and related fimbriae do not show sequence homology to other types of fimbriae, but their assembly mechanism appears to be closely related to the chaperone-usher pathway and has been termed the alternate chaperone-usher pathway (Fig. 2). CS1 pili are encoded by the cooBACD operon present on the large pCoo plasmid in ETEC (214). The expression of the coo operon in ETEC requires the Rns transcriptional activator, which is encoded on a separate plasmid (39).

Structure of Fimbriae.

CS1 fimbriae are morphologically similar to P and type 1 pili, forming rigid, rodlike fibers 6 to 7 nm in diameter (Fig. 5C) (85, 149). However, they have a simpler architecture comprising only two structural components and no visible tip structure. CS1 fimbriae are composed of repeating copies of the major pilin subunit, CooA (15 kDa), which determines the serological type of the pilus. CooD (38 kDa) is a minor pilin present in 1 copy per pilus and located at the tip of the pilus fiber (237). Although the major CooA pilin was initially proposed to contain the binding activity of CS1 fimbriae (35, 166), studies by Sakellaris et al. (238) demonstrated that the tip-located CooD serves as the pilus adhesin. The receptor for CS1 fimbriae on intestinal epithelial cells is not known, but members of the CS1 family have been reported to bind sialic acid-containing glycolipids or glycoproteins, as well as asialogangliosides ( 85, 155).

Assembly Mechanism.

The assembly of CS1 fimbriae by the alternate chaperone-usher pathway is thought to closely resemble the assembly of P and type 1 fimbriae by the classical chaperone-usher pathway (239). The biogenesis of CS1 fimbriae requires the CooB periplasmic chaperone and the CooC outer membrane usher. The CooA and CooD pilins contain typical N-terminal signal sequences for translocation across the inner membrane via the Sec system. In the periplasm, CooA and CooD form intermolecular complexes with the CooB chaperone (237, 295). CooB is required for the stable expression of the pilin subunits in the periplasm. The CooB chaperone is also required for the full stability of the CooC usher, an activity that is not found in the classical chaperone-usher pathway (295). Periplasmic chaperone-subunit complexes next interact with the outer membrane CooC usher for the assembly of subunits into the pilus fiber and the secretion of the fiber to the cell surface (237). The chaperone is not part of the final pilus structure and presumably releases from the subunit at some point following interaction with the outer membrane usher. The CooD adhesin is located at the tip of the pilus and is required to initiate the assembly and secretion of CS1 fimbriae at the CooC usher (237). Thus, similar to P and type 1 fimbriae, CS1 fimbriae are assembled in a top-down order. The molecular details of CS1 fimbrial biogenesis remain to be elucidated, although a recent study demonstrated that subunit interactions in CFA/I fimbriae are governed by donor strand complementation as in the classical chaperone-usher pathway (217a).

Structures of Component Proteins and Physical Properties.

Crystal structures of the CS1 subunit proteins or assembly components are not available. The major subunits of CFA/I, CS1, CS2, CS4, CS14, CS17, and CS19 fimbriae show extensive homology and presumably fold into similar structures. CooA lacks the conserved N- and C-terminal sequence motifs that are important for the biogenesis of P and type 1-related fimbriae. In addition, CooA does not contain cysteine residues (214). However, highly conserved residues are present at both the N and C termini of CooA and related subunit proteins, and a conserved C-terminal motif consisting of the sequence Arg-Gly-X-Tyr-X-Gly-(X)₆-Thr is found at the C termini of both the major and minor subunits (239, 261). Starks and coworkers recently analyzed the functions of conserved N- and C-terminal residues of CooA (261). They found that residues at both termini are important for the assembly of functional CS1 fimbriae. A set of alternating hydrophobic residues at the N terminus is required for the assembly of adhesive fimbriae. These residues can form an interactive surface similar to the conserved N-terminal motif present in P and type 1 fimbrial subunits. Conserved alternating hydrophobic residues within the C-terminal motif (although not the invariable residues within the motif itself) were also identified to be important for the biogenesis of CS1 fimbriae. Interestingly, these residues are required for the interaction of CooA with the CooD pilus adhesin, rather than binding to the periplasmic chaperone as found for P and type 1 fimbriae. Residues identified within both the N- and C-terminal regions are required for stable protein expression in the periplasm and, thus, presumably for interaction with the periplasmic chaperone.

Similar to adhesins present in the Pap-related fimbriae, the CooD adhesin is about twice the size of the CooA major subunit protein. The mutation of residue Arg-181 in CooD into alanine abolishes the ability of CS1 fimbriae to agglutinate bovine erythrocytes (238). Similarly, the mutation of Arg-181 into alanine in the CFA/I fimbrial adhesin CfaE abolishes the agglutination of human type A red blood cells (238). Thus, the adhesive activity of CS1 and related fimbriae is conferred by the minor subunit, and Arg-181 forms a critical part of the binding site.

Curli Fimbriae

Both E. coli and Salmonella spp. express curli fimbriae (thin aggregative fimbriae) (46, 229), and genes coding for curli expression in other clinically important enteric bacteria have been detected previously (316). Curli are involved in colonizing inert surfaces, forming biofilms, and binding to numerous host proteins, including fibronectin, laminin, plasminogen, and human contact phase proteins (6, 13, 47, 199, 253, 293). In E. coli, curli are encoded by the chromosomal csg gene cluster, and in Salmonella, they are encoded by the agf gene cluster. The curli genes of Salmonella and E. coli show a high degree of conservation at the protein level, suggesting conserved roles in these two organisms (46, 229). This section will focus on the E. coli curli biogenesis system. Two operons are required for the production of curli in E. coli: csgBA and csgDEFG (96) (Fig. 3). CsgA and CsgB are secreted proteins that form the major structural subunit and nucleator for curli assembly, respectively. CsgD is a regulatory protein, and CsgE, CsgF, and CsgG are components of the assembly machinery. CsgD is a transcriptional activator that belongs to the LuxR/UhpA family and is essential for the transcription of the csg genes (96). Curli are expressed under specific growth conditions, including low temperatures, low salt concentrations, and stationary phase. The assembly of curli occurs on the bacterial surface by a distinct mechanism termed the extracellular nucleation-precipitation pathway.

Structure of Curli.

Curli fibers are thin, irregular, extremely aggregated, and curly (hence their name), measuring 6 to 8 nm in diameter and having various lengths (Fig. 5D) (41, 199). E. coli curli are composed primarily of multiple copies of the 15-kDa CsgA major subunit protein. CsgB is a minor fimbrial subunit and acts as a nucleator for CsgA assembly on the cell surface as described below. CsgB can also be detected along the length of curli fibers and may localize to branch points in the fibers (16, 299). Curli are amyloid-like structures and exhibit characteristic amyloid traits, such as binding to the dyes Congo red and thioflavin T (41). Curli fibers are highly stable and remarkably resistant to denaturation, requiring treatment with formic acid to dissociate the subunit proteins (41, 47, 198).

Assembly Mechanism.

Curli are unique among fimbriae expressed by gram-negative bacteria in that they are assembled on the cell surface. The CsgB protein acts at the cell surface to nucleate the CsgA polymer formed from a pool of soluble CsgA monomers (16, 97). In the absence of CsgB, CsgA monomers are secreted into the extracellular milieu. This soluble extracellular CsgA can act by interbacterial complementation to assemble curli fibers on the surfaces of neighboring cells if they express the CsgB nucleator (97). CsgE, CsgF, and CsgG are components of the curli assembly machinery; they are required for the assembly of the fibers but are not part of the final curli structure. CsgE may function as a periplasmic chaperone-like protein, and CsgF may work alone or together with CsgB to nucleate the product of the extracellular polymerization of CsgA (41). The lipoprotein CsgG is located in the outer membrane and is required for the stability and secretion of the CsgA and CsgB proteins (152). Recent work indicates that CsgG performs an usher-like function and provides the secretion channel across the outer membrane (227). CsgG forms an oligomeric complex in the outer membrane with an apparent central pore 2 nm in diameter (227). A 22-residue sequence at the mature N terminus of CsgA contains the targeting information for binding to CsgG (227). CsgG also forms stable complexes with the CsgE and CsgF periplasmic proteins. Thus, CsgG may function as a point of convergence for the assembly and the structural components of the curli biogenesis pathway. A major function of the CsgEFG assembly machinery may be to prevent the premature formation of CsgA fibers within the bacterial periplasm (41).

Structures of Component Proteins and Physical Properties.

As noted above, curli are amyloid fibers and exhibit characteristic amyloid traits (41). Circular dichroism analysis demonstrated that, whereas curli fibers are rich in ß-sheet structures, soluble CsgA monomers do not have significant ß-sheet secondary structures (41). Thus, the assembly of curli fibers involves a transition of the CsgA monomer into an amyloid-like structure. This conformational change in CsgA is likely triggered by interaction with the CsgB nucleator subunit on the bacterial surface (16). Following the interaction of CsgA with CsgB, the conformationally altered CsgA is likely able to act as a nucleator itself to trigger the incorporation of additional CsgA subunits, thus driving the assembly of the curli fiber. The CsgA and CsgB subunits are similar in size and show 49% sequence similarity and 30% identity (16). The structural basis for CsgA and CsgB interaction and the way in which CsgB triggers the conformational change in CsgA remain to be determined.

The analysis of the Salmonella CsgA ortholog AgfA identified two domains: a glycine-rich, protease-susceptible N-terminal domain (the first 22 residues of the mature subunit) and a protease-resistant, C-terminal core region (mature residues 23 to 131) comprising internal repeat sequences occurring 2, 5, and 10 times (49). As noted above, the corresponding N-terminal domain of CsgA functions as a targeting sequence for the CsgG outer membrane lipoprotein (227). The N-terminal region of CsgA does not form an integral part of the assembled fiber but is required for the stability and secretion of CsgA (49, 227). The C-terminal core domain of AgfA was predicted to fold into a parallel ß-helix structure that presumably provides the amyloid-forming building block of the curli fiber (49). AgfB, the Salmonella ortholog of CsgB, was found to have a similar two-domain structure, with the C-terminal domain containing repeated sequences and likely folding as a parallel ß-helix (299). Cherny and colleagues analyzed the repeat sequences present in CsgA and noted a hexapeptide motif (QFGGGN) that is similar to repeated sequences present in yeast and animal amyloid proteins (42). Peptides corresponding to this motif assemble into fibrillar structures. Furthermore, curli expression by E. coli is inhibited by the addition of peptides containing the repeated motif conjugated to amyloid-breaking elements (42). These results suggest that the repeated sequences present in curli subunits function in fiber formation.

Type IV Fimbriae

Type IV fimbriae are polarly localized fibers expressed by a variety of gram-negative bacteria, including E. coli and Salmonella (Table 3). Type IV fimbriae are involved in a number of functions, including adherence to host cells (60, 61), autoaggregation (18, 133, 208), biofilm formation (204), cellular invasion (315), horizontal gene transfer (248, 308), and twitching motility (37, 168). Many of these functions are important during pathogenesis, and type IV fimbriae are critical virulence factors (18, 76). Type IV fimbriae are distinguished from the classes of fimbriae described above by their ability to retract, which provides the basis for twitching motility (37, 168).

EPEC expresses type IV fimbriae termed bundle-forming pili (BFP) (87). The structure, function, and assembly mechanism of BFP have been actively studied, and this section will focus on BFP, along with the prototypical type IV fimbriae of P. aeruginosa and Neisseria gonorrhoeae. EPEC is the major causative agent of severe diarrhea among children in developing countries (188). EPEC bacteria typically form three-dimensional microcolonies on the surfaces of cells to which they adhere (87, 139). This characteristic, termed localized adherence, is mediated by BFP and is important for virulence. Several groups have proposed oligosaccharides as potential BFP receptors (52, 113, 118, 245, 291). However, although experimental evidence indicates that BFP act as an adhesin for host cells (279), it is still an open question whether BFP promote bacterial adherence by binding to a specific cell receptor or by some other mechanism (191). Type IV fimbriae in general may provide a mechanism for non-receptor-mediated adhesion, allowing bacterial colonization of a variety of surfaces (37). BFP biogenesis requires an operon containing 14 genes located on the EPEC adherence factor plasmid (256, 263). In contrast, the type IV fimbrial genes of P. aeruginosa and Neisseria occur in clusters or as individual genes at different locations on the chromosome (169, 280).

Structure of Fimbriae.

Type IV fimbriae are flexible, hair-like filaments 5 to 8 nm in diameter and up to several micrometers in length ( Fig. 5E). The fimbriae consist solely or predominantly of repeating subunits of the major pilin protein. X-ray fiber diffraction and electron microscopy analyses revealed that type IV pilins are arranged in a helical pattern in the pilus fiber (51, 80, 220). Type IV fimbriae can be categorized into type IVa and IVb subgroups based on the length of the leader sequence of the major pilin protein and the size of the mature pilin, as described below (50). Type IVa fimbriae are typically long, straight, and unbundled; type IVb fimbriae, including BFP, often form long, polar bundles and are associated with enteric pathogens such as E. coli and V. cholerae (87, 88, 100). Recent models indicate a common organization of all type IV fimbriae, with the subunits arranged into left-handed, three-start helices. The type IVa P. aeruginosa strain K fimbriae are modeled as having four subunits per turn, an outer diameter of ~58 Å, and a central channel of ~18 Å (50). In comparison, the type IVb toxin-coregulated pilus fimbriae of V. cholerae, comprising larger subunit proteins, are modeled as having six subunits per turn, an outer diameter of ~80 Å, and a central channel of ~10 Å (50). The proposed BFP structure closely matches the toxin-coregulated pilus model, with six subunits per turn and an outer diameter of ~75 Å (Fig. 5E) (220).

Assembly Mechanism.

The components required for the assembly and secretion of type IV fimbriae are homologous to the components of the type II secretion system (212, 233). In fact, type IV fimbriae can function in the secretion of soluble proteins (133), and type II secretion systems can be induced to assemble fibers resembling type IV fimbriae, termed pseudopili (69, 294). For the assembly of type IV fimbriae, the subunit proteins are first expressed in a prepilin form containing a short, basic, N-terminal signal sequence (265). The prepilin subunit is anchored in the inner membrane via its N terminus, with its globular C-terminal region exposed to the periplasm (312). The pilin subunit is then cleaved and N methylated by a bifunctional inner membrane prepilin peptidase (213, 266). DsbA acts in the periplasm to catalyze the formation of a disulfide bond in the pilin C terminus that is required for pilin stability (62, 312). Some type IV pilins are subjected to additional posttranslational modifications; for example, N. gonorrhoeae pilins are O glycosylated and phosphorylated (81, 164, 207). In the presence of the biogenesis machinery, the pilins polymerize from the inner membrane into the periplasm, and the fiber crosses to the cell surface through an outer membrane channel-forming protein termed the secretin (274, 303). The three-start helical structure proposed for type IV fimbriae suggests that assembly may occur by the simultaneous addition of pilin subunits at three independent sites (220).

In EPEC, 13 of the14 bfp genes are required for the assembly of functional BFP and at least 10 of the proteins can be cross-linked, implying the formation of a large complex (4, 112, 222). The bfpA gene encodes the major prepilin subunit, termed bundlin (61, 255), which is processed by the BfpP prepilin peptidase to form the mature, 19-kDa protein (313). BfpD and BfpF are cytoplasmic nucleotide-binding proteins (54, 256, 263). BfpD is believed to provide energy for pilus assembly and secretion, whereas BfpF, a homolog of the P. aeruginosa PilT protein required for twitching motility, is thought to energize pilus retraction (3, 18). BfpD is a member of the PulE superfamily of hexameric nucleotide-binding proteins that also participate in DNA uptake and the type II and type IV secretion systems (212). BfpE is an integral, polytopic cytoplasmic membrane protein that interacts with other components of the secretion machinery to form a subassembly complex (22). BfpE binds to the bitopic cytoplasmic membrane protein BfpC, and both proteins bind to the BfpD ATPase to recruit BfpD to the cytoplasmic membrane (53). Interaction with BfpC and BpfE greatly enhances the ATPase activity of the hexameric BfpD complex (54). Crowther et al. proposed a model wherein ATP hydrolysis allows BfpD to transmit mechanical force to the BFP assembly machinery to drive the extraction of bundlin subunits from the cytoplasmic membrane and their assembly into the pilus fiber (54). BfpI, BfpJ, and BfpK are prepilin-like proteins (pseudopilins) that are processed by the BfpP prepilin peptidase, similar to the BfpA bundlin subunit (222). The function of these proteins is not clear, but they may form a pilus-like structure in the periplasm that facilitates the secretion of the nascent pilus fiber across the outer membrane (102). BfpB is an outer membrane lipoprotein belonging to the secretin family that forms the channel through which the growing pilus is secreted (221). The secretin is the only integral outer membrane component of the secretion machinery. Secretins form highly stable, oligomeric, ring-shaped complexes in the outer membrane and contain large central channels, 5 to 10 nm in diameter (21, 45, 192). Channels of this size are capable of accommodating an assembled pilus fiber.

Structures of Component Proteins and Physical Properties.

The structural subunit proteins (pilins) of type IV fimbriae contain a characteristic and highly conserved N-terminal prepilin leader sequence. Type IVa fimbriae contain a short, positively charged leader sequence, 5 to 6 residues long. The cleavage of the leader sequence occurs following a conserved glycine, and the first amino acid of the mature protein is phenylalanine (102, 266). In addition, a conserved glutamate is present at position +5 of the mature protein, followed by a conserved hydrophobic region of approximately 25 residues (51, 266). The conserved glutamate is required for the processing and assembly of pilin subunits (157, 211, 264). The remainder of the pilin sequence exhibits variability, except for the presence of two C-terminal cysteines that define a disulfide-bonded antigenic loop, or D region (266). Type IVb pilins have longer leader sequences, generally 15 to 30 residues; the first mature amino acid is not phenylalanine; and the mature pilins are larger.

The structures of type IVa pilin subunits from N. gonorrhoeae (207) and P. aeruginosa (99, 130) and type IVb pilin subunits from V. cholerae, S. enterica serovar Typhi, and EPEC (51, 220, 306) have been solved previously. These structures revealed that pilins adopt a ladle-like shape, with the N terminus forming an extended, hydrophobic α-helix and the C terminus folding into a globular domain comprising mixed alpha and beta structures. The globular domains of the different type IV pilin subunits adopt different folding patterns but appear to pack similarly into the pilus fiber, producing structures essentially identical in form and function (50, 220). In assembly models for type IV fimbriae, the hydrophobic N-terminal helices pack into the center of the pilus as a coiled coil, forming a hydrophobic core that stabilizes the assembled fiber, while the C-terminal domains are located towards the surface, providing specific subunit-subunit contacts and functionally variable exposed regions of the pilus (Fig. 5E) (50). Prior to assembly into the pilus, the hydrophobic N-terminal helix is thought to anchor subunits in the cytoplasmic membrane for processing by the prepilin peptidase, providing a membrane-localized pool of subunits for pilus biogenesis. In addition, during pilus retraction, the subunits are thought to be disassembled from the base of the fiber, with the liberated subunits entering back into the cytoplasmic membrane via their N termini. The globular C-terminal domains of type IVa pilins from N. gonorrhoeae and P. aeruginosa fold into an α-ß roll conformation, whereas the type IVb pilins have a larger globular head domain and exhibit topology that differs from that of their type IVa counterparts. Nuclear magnetic resonance analysis of the structure of BfpA revealed an α-ß fold consisting of four α-helices that surround a platform formed by a seven-stranded ß-sandwich (220). Two distinct regions of the C-terminal head domain of pilins form exposed surfaces in the pilus fiber that are important for subunit-subunit interactions as well as interactions with the environment: an α-ß loop region and the D region that contains the conserved disulfide bond (Fig. 5E) (50). The D region in type IVb fimbriae is longer than that in type IVa fimbriae (~50 versus ~20 residues) and exhibits the highest degree of sequence variation (50, 220). The disulfide loop region of the C-terminal domain is required for the assembly of the pilus fiber, is an antigenically important domain, and may also mediate the binding activity of the fimbriae. In P. aeruginosa, the receptor-binding site is located in the D region (147). The restriction of the P. aeruginosa binding activity to the pilus tip is explained because the D region appears to be at least partly buried by subunit-subunit interactions along the length of the fiber but exposed at the pilus tip to allow binding. Residues in both the D region and the α-ß loop appear to contribute to the binding specificity of S. enterica serovar Typhi type IV fimbriae (306). For some type IV fimbriae, such as those of Neisseria, a separate, tip-located minor pilin subunit may serve as the pilus adhesin (232).

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