Fimbriae

TOC

Fimbriae

Fimbriae

Chapter 11

DAVID LOW, BRUCE BRAATEN, and MARJAN van der WOUDE

INTRODUCTION

Definitions

Fimbriae (plural, from Latin for thread or fiber) are proteinaceous, hairlike appendages, ranging from 2 to 8 nm in diameter, on the surface of bacteria (46). Escherichia coli and Salmonella spp. express a wide variety of different fimbriae with different binding specificities. In most cases a single bacterial isolate can express multiple fimbrial types. The term "fibrillae" has been used to designate those fimbriae with diameters of 2 to 3 nm, such as K88 and K99 fibrillae (88). Some fimbriae such as Pap and type 1 are composites, consisting of a long rigid shaft of 6 to 8 nm in diameter and a short, thin fibrillar tip of 2 to 3 nm in diameter.

Fimbriae are thinner and generally shorter and more numerous than flagella (between 100 and 1,000 fimbriae per cell). Fimbriae are usually not involved in cellular motility, with the exception of type IV fimbriae, which have the property of "twitching motility" (162). The term "pili" (plural, from Latin for hairs or fur) is currently used interchangeably with fimbriae, although it was suggested previously that "pili" be reserved for appendages such as F pili (see chapter 126) that play a role in the transfer of DNA between bacterial cells during conjugation (138).

Fimbriae are composed of protein subunits termed fimbrins that range in size from 14 to 30 kDa (Table 1). In the pyelonephritis-associated pili (pap) operon, the PapA fimbrin forms most of the fimbrial shaft, and the fibrillar tip is composed of the PapE and PapF fimbrins. The PapA fimbrin is present in about a 100-fold molar excess over other pap-encoded fimbrins and therefore is designated as the major fimbrial subunit, whereas the PapE and PapF fimbrins are minor fimbrial components. The PapG fimbrin, located at the fibrillar tip, confers upon bacteria expressing Pap fimbriae the ability to bind to specific receptors on host target cells (99, 110) and has therefore been denoted an adhesin subunit. In contrast to Pap, in some fimbriae the major fimbrial subunit confers adhesive specificity as well. For example, the FaeG protein of K88 fibrillae forms the bulk of the fibrillar structure and confers binding specificity towards Galα(1–3)Gal. For more in-depth coverage of the role of fimbriae in adhesion, we refer the reader to chapter 150.

Table 1Chemical and biological properties of E. coli and Salmonella fimbriae

Overview of Classification Schemes

Historically, E. coli fimbriae have been classified based on their phenotypic traits. Orskov and Orskov (136) have used antigenic properties to group fimbriae into "F-types" (see Table 1). The adhesive properties of fimbriae have also been used for classification. In many cases, E. coli that express fimbriae will agglutinate red blood cells of various species. Initial studies indicated that type 1 fimbriae bind to mannose-containing host cell receptors, since type 1 fimbrial hemagglutination is inhibited by addition of d-mannose (mannose-sensitive or "MS" adhesin). In contrast, most other fimbrial types agglutinate red blood cells in the presence of mannose (mannose-resistant or "MR" adhesin). The MR class of fimbrial adhesins is very diverse, and a number of different binding specificities have already been identified (Table 1). Because most E. coli and Salmonella strains express multiple fimbrial types, each one needs to be isolated to determine its binding specificity.

The usefulness of adhesive properties for the classification of fimbriae is limited by a number of factors. In some cases, as noted above, the fimbrial protein containing adhesin activity is only a minor fimbrial component, distinct from the major fimbrial subunit (78). Later studies showed that in both type 1 (2, 71, 113) and S (119) fimbriae, the adhesin protein is distinct from the major fimbrial subunit. In contrast, for the K88, K99, and F1845 fimbriae, among others, the major structural subunit appears to confer adhesin activity (Table 1). Therefore, identification of adhesin binding specificities does not necessarily address the nature of the major fimbrial subunits themselves, which can be determined by DNA and amino acid sequence analyses. On the basis of primary amino acid sequence data of major fimbrial subunits, we have classified E. coli and Salmonella fimbriae into seven major groups (Table 1).

Fimbrial genes are organized in operons that encode major and minor fimbrial subunits as well as chaperone and assembly proteins required for their assembly on the bacterial cell surface. In addition, each fimbrial gene cluster codes for one or more proteins that regulate gene expression. Structure-function analyses have shown that certain groups of operons encode regulatory proteins sharing amino acid similarities, providing a basis for the classification scheme shown in Table 2. For example, the PapI and PapB regulatory proteins coded for by the pap operon share amino acid similarities with regulatory proteins encoded by the sfa, daa, afa, fae, and pef operons (see Table 2).

Table 2Genetic and regulatory classification of E. coli and Salmonella fimbrial operons

A comparative analysis of the classification schemes shown in Tables 1 and 2 shows that although two fimbrial operons may be closely related on the basis of operon regulatory properties, they may be in separate classes based on the primary amino acid sequences of their major fimbrial subunits. For example, the major fimbrial subunits of the pap and fap operons share amino acid similarities, but their regulatory proteins and regulatory DNA sequences are distinct from each other. Thus, analysis of any single fimbrial gene product provides only a limited amount of information regarding the relatedness of fimbrial operons.

In this chapter we present what is currently known about fimbrial biochemistry, assembly, and genetics. Because the pap operon has been extensively studied, we use it as a paradigm to which other fimbrial operons are compared. The classification schemes described above (Tables 1 and 2) are discussed in the context of the biochemistry and genetics of fimbriae.

BIOCHEMISTRY OF FIMBRIAE

Primary Amino Acid Sequence

The primary amino acid sequences of a number of major fimbrial subunit proteins have been reported, providing a basis for the classification scheme shown in Table 1 (denoted by numerical superscripts indicating the major subunits). As discussed above, many fimbrial operons also code for minor fimbrial subunits, which may or may not share similarities with the major subunit. In the pap operon, the PapE minor fimbrin resembles the PapA major subunit, whereas in the fae operon, the FaeC minor subunit is in a different class from the FaeG major subunit. In Table 1, we restrict our analysis to the major fimbrial subunits.

Major subunits of class 1 fimbriae, including Pap and type 1, have two cysteine residues spaced 38 to 43 amino acids apart and may form a cystine bridge. Other amino acids conserved are a Phe residue, located 8 amino acids from the N-terminal cysteine, and the penultimate Tyr at the COOH terminus (106). This Tyr appears essential for K99 fimbrial expression and may be important for interactions with periplasmic chaperones (157). The 987P major fimbrial subunit shares many features of class 1 fimbrins but lacks the penultimate Tyr at the carboxyl terminus (38).

Class 2 fimbriae include F1845 as well as adhesins that do not form fimbriae, the afimbrial adhesins AFA-I, AFA-III, and Dr (O75X) (15) (A. Labigne, personal communication). Similar to class 1 fimbriae, there are two conserved cysteine residues in class 2 fimbriae, but they are spaced 31 amino acids apart. Also, the conserved Phe residue is 16 amino acids from the N-terminal cysteine and a Tyr residue is located 4 amino acids from the COOH terminus, with the exception of AFA-I which contains a Phe residue at this position. Notably, AfaE3 (AFA-III) and DaaE (F1845) share 56.9% sequence identity, and both gene clusters appear to be highly conserved, raising the question of why AFA-III has an amorphous structure whereas DaaE subunits are assembled into fimbriae. Le Bouguenec et al. hypothesized that the absence of an AFA-III fimbrial structure may be due either to a lack of expression of an accessory protein or to a low rate of expression of the afaE3 gene product (103).

Class 3 fimbriae, which include K88, CS31A and F41, are distinct from fimbrial classes 2 and 3 due to the lack of cysteine residues although they share a penultimate tyrosine in the carboxyl terminus like class 1. K88 and CS31A share 46% sequence similarity, and F41 is more distantly related (62). All three fimbriae share 15 amino acids in their leader sequences as well as four conserved proline residues in the mature fimbrin sequence. Other members of this group are NFA-4 (76), isolated from uropathogenic E. coli, and PCF-09 (73) from enteropathogenic E. coli. CS5 fimbriae share many features of class 3 fimbriae but lack the penultimate tyrosine as well as two conserved prolines (29).

The bundle-forming fimbrin BfpA shares sequence similarities with type IV fimbriae isolated from Pseudomonas, Neisseria, Moraxella, and Vibrio spp., especially at the amino terminus (162). BfpA shares the highest amino acid sequence identity with the toxin-coregulated pilus (TCP) from Vibrio cholerae and is the sole member of class 4.

The CFA/I and CS1 fimbriae share 55% sequence identity and are distinct from the other fimbrial classes. These fimbriae lack cysteines and COOH-terminal Tyr residues and are designated as class 5 (141).

The CsgA fimbrin from E. coli and AgfA (SEF17) fimbrin from Salmonella enteritidis are members of the GVVPQ family (class 6) and share significant sequence similarity at the amino terminus (7, 34). Finally, the SEF14 (sefA) fimbrin has no homology to any other known fimbrins, forming a separate class (32), as does the sefD gene of SEF18, which is widely distributed among Salmonella and Serratia spp. but not other members of the Enterobacteriaceae (31).

Structure of Fimbriae

The major fimbrial subunits discussed above polymerize into fibers with characteristic diameters and shapes. Although polymerization can occur in vitro (see Physical Properties of Fimbriae, below), assembly of fimbrin subunits into the fimbrial structure in vivo requires a number of additional fimbrial gene products (see Assembly of Fimbriae, below). Here we describe what is known about the structure of Pap fimbriae and use it as a paradigm to analyze structures of other fimbriae.

Pap fimbriae are composite structures consisting of a long, rigid base section and a short, flexible, open helical structure (78). The rigid section is composed mostly of the PapA fimbrin subunit arranged in a tightly packed right-handed helix (Fig. 1). X-ray fiber diffraction analysis indicates that Pap fimbriae contain 3.2 fimbrin subunits per turn of the helix and have a helical pitch of 2.4 nm (25, 66). These fimbriae are about 7 nm in diameter with a central axial hole of 1.5 nm in diameter. Since these fimbriae usually extend about 1 to 2 μm away from the bacterial cell surface, a single rigid fiber is composed of about 1,200 to 2,400 fimbrin monomers. In contrast, the short, flexible tip, composed of PapE monomers, is 2 nm in diameter with a 15-nm pitch. Neither the PapA nor the PapE fimbrins confer binding specificity. Instead, specific binding to the Gal(α1→ 4)βGal receptor is carried out by the PapG monomer located at the end of the flexible tip.

Fig. 1Pap fimbriae of E. coli. (A) Electron micrograph of a uropathogenic E. coli strain (DL855) stained with colloidal gold-labeled antibodies against Pap fimbriae. The open arrow shows a flagellum, and the closed arrow shows a single Pap fimbria. (B) Right-handed helical structure of Pap fimbriae as determined by X-ray diffraction (66).

Source: (Reprinted with permission from Academic Press.)

The structure of type 1 fimbriae appears to be very similar to that of Pap (25). In contrast, K88, K99, F41, and CS3 fimbriae consist only of thin (ca. 2-nm), extended flexible fibers (fibrillae) without an axial hole (59) (Table 1). These structures resemble the flexible tip of Pap fimbriae. Notably, the FaeG (K88) and FanC (K99) major fimbrins have adhesin activity, unlike the major fimbrins PapA (P fimbriae) and FimA (type 1 fimbriae) which are not adhesive.

Structure-Function Analyses of Major Fimbrial Subunits.

Assembly of the Pap fimbrial-adhesin complex requires specific interactions between the PapA major fimbrial subunit and the PapC, PapD, and PapK assembly components. In addition, polymerization requires PapA-PapA monomer interactions. The main approach that has been taken to identify amino acids that are important for the various protein-protein interactions has been to alter the fimbrin primary sequence and determine the effect on fimbrial expression and structure.

Analysis of the FsoA (F7₁) and FelA (F11) major subunits of Pap fimbriae showed that spacing between blocks of amino acids that are conserved among Pap fimbrial variants is critical for fimbrial expression (174). Replacement of "hypervariable" regions (defined as regions that are highly variable between different PapA subunits) with other amino acid sequences, including an epitope of foot-and-mouth disease virus, did not block formation of fimbriae, although fimbrial expression was reduced and the fimbriae were shorter than normal. A similar strategy for amino acid replacement was carried out using the FaeG major subunit of K88 fimbriae (11). Replacement of FaeG amino acids 163 to 173, which are variable among K88ab, K88ac, and K88ad fimbriae, with other amino acids reduced the stability of K88 fimbriae although fimbriae were still expressed. Together, these results indicate that certain variable regions of fimbrins are important as spacers but their structures are not critical for fimbrin export and polymerization into fimbriae.

Mutational analysis of the FanC major subunit of K99 fimbriae indicated that Trp-67 and the penultimate tyrosine residue are critical for fimbrial stability (85, 157). Notably, a penultimate tyrosine residue is characteristic for fimbrins of class 1 including Pap (see above and Table 1), suggesting a role for this conserved amino acid in class 1 fimbrial stability. Although specific amino acids have been identified which appear to be essential for fimbrial stability, it is not yet clear which protein-protein interactions are mediated by these amino acids or how they contribute to fimbrial stability.

Physical Properties of Fimbriae

Fimbriae vary in their susceptibility to dissociation into monomer subunits. Pap fimbriae can be dissociated in a low-ionic-strength buffer lacking divalent cations. In contrast, type 1 fimbriae are more stable and are not readily dissociated even by detergents such as sodium dodecyl sulfate. Incubation of type 1 fimbriae in saturated guanidine hydrochloride at 37°C results in complete dissociation. Renaturation of the dissociated subunits into a fimbrial fiber occurs upon dialysis against Tris buffer containing Mg²⁺ ion, with partial reacquisition of mannose binding (51, 79). In contrast, addition of glycerol (50% [vol/vol]) causes unraveling of the tight helical coil of type 1 fimbriae into flexible 2-nm fibers which show a higher binding affinity for mannose and are sensitive to trypsin digestion, unlike native fimbriae. Removal of glycerol by dialysis results in reassembly into fimbrial structures that appear identical to native fimbriae (4).

Depolymerization of type 1 fimbriae causes loss of certain quaternary structure-specific epitopes as well as exposure of new epitopes that are normally hidden in the native fimbrial structure (3). Type 1 fimbriae that have been disassociated and reconstituted by polymerization are morphologically identical to native fimbriae, on the basis of analysis with monoclonal antibodies recognizing native fimbrial epitopes (3).

ASSEMBLY OF FIMBRIAE

In this section, we briefly outline the processes involved in the expression of fimbriae, focusing once again on Pap fimbriae since they have been most intensively studied. Proteins with functions similar to the Pap assembly machinery have been identified for other fimbrial types (96, 146, 158). There are nine known gene products required for Pap fimbrial expression, including the PapA major fimbrial subunit (78, 79). All nine proteins have leader sequences that enable them to be transferred across the cytoplasmic membrane by the protein secretion machinery. The SecA protein was shown to be essential for expression of type 1 fimbriae (41). The PapD protein (28.5 kDa) is a chaperone that localizes to the periplasmic region, binds to the PapA, PapH, PapK, PapE, PapF, and PapG proteins, and carries them to PapC. This outer membrane protein has been designated as an "usher" due to its ability to allow the ordered passage of different subunits (42). PapD appears to stabilize fimbrial precursor proteins, and by analogy with the GroEL and Hsp70 chaperones, PapD might maintain them in the unfolded state (100).

The PapC protein escorts the PapD-subunit complexes into a fimbrial-adhesin structure composed of a tightly coiled, rigid base of PapA monomers and a flexible tip fibrillar structure consisting of PapE, PapF, and PapG. The PapF protein acts as an adaptor which attaches the PapG adhesin at the fimbrial tip to PapE, the major subunit of the tip fibrillae (84). The PapK protein appears to regulate the length of the tip fibrillum (84). PapC binds to PapD in a complex with the three tip fibrillar components PapE, PapF, and PapG, but not with PapD-PapA complexes, providing a mechanism by which tip fibrillae are assembled before the fimbrial rod (42). The PapH subunit is located at the base of the fimbrial rod and may be the last fimbrial subunit incorporated (10). Capping of the fimbrial rod with PapH might prevent further polymerization, thus controlling fimbrial length. The FimG protein of type 1 fimbriae appears to have a similar function since mutations in fimG result in abnormally long fimbriae (114, 146).

Type 1 fimbrial subunits are incorporated into the fimbrial structure at the base, in contrast to flagella which are assembled at the tips. Unlike fimbrins, flagellin proteins do not have signal sequences and are transported through the wide core of the flagellum to the tip, where they are assembled (109). The small diameter of the fimbrial core (1.5 to 2 nm) is too narrow for transport of fimbrial subunits.

ORGANIZATION OF FIMBRIAL OPERONS

Fimbrial operons have either a plasmid or a chromosomal location (Table 2). In many cases the genes involved in fimbrial biosynthesis and regulation are contiguous, whereas in other cases, such as coo (CS1) and cfa (CFA/I), they are physically separated, comprising separate operons. Even when fimbrial genes are contiguous, they may be organized within separate operons. In the pap, sfa, afa, daa, and fae operons, the papI, sfaC, afaF, daaF, and faeA genes, respectively, are divergently transcribed from the other fimbrial genes (Table 2). In the fan gene cluster encoding K99 fimbriae, at least three different operons with the same transcriptional orientation appear to be present. The fanG(H) genes, involved in fimbrial biogenesis, appear to be under separate regulatory control from the fanC gene encoding the major fimbrial subunit (82).

The transcriptional organization of fimbrial genes is also variable. In the pap and sfa gene clusters, transcription initiation occurs upstream of the papB and sfaB regulatory genes, respectively, and transcription proceeds through the gene coding for the major fimbrial subunit followed by genes coding for proteins involved in fimbrial assembly and expression. In contrast, the daaE fimbrial subunit gene is located downstream of the daaB-daaD genes involved in F1845 fimbrial biogenesis (14). The fae operon, coding for K88 fimbriae, has a similar genetic organization (139).

TRANSCRIPTIONAL REGULATION

Analysis of Fimbrial Operons Based on Regulatory Properties

E. coli and Salmonella spp. are exposed to a number of different environmental conditions both within and outside of their hosts. These bacteria have developed mechanisms that enable them to respond to environmental cues, including regulation of fimbrial expression. All fimbrial operons examined code for regulatory proteins that either activate or repress fimbrial gene expression. In addition, most fimbrial operons are controlled by global regulatory networks such as the CAP and Lrp regulons, coordinating fimbrial expression with other cellular gene expression in response to environmental stimuli.

In Table 2, a number of fimbrial operons are grouped on the basis of similarities in their regulatory properties. The pap, prs, sfa, daa, afa, and fae operons all contain conserved DNA sequences designated as GATC box I and GATC box II (see Methylation-dependent phase variation, below), which share 11 and 8 base pairs of sequence identity, respectively (14, 77, 103, 120, 168, 169). Also, each operon contains genes with significant identity to the papI and papB regulatory genes in the pap operon (Table 3). Moreover, leucine-responsive regulatory protein (Lrp) acts as a positive regulator of pap, sfa, and daa, but as a negative regulator of fae (77, 168). In the latter operon, an additional GATC box I is present near the RNA polymerase binding site, which may account for the negative effect of Lrp on transcriptional activation (77). Notably, although fimbrial expression from pap, daa, and sfa is under OFF-ON phase variation control (see below), K88 fimbrial expression from fae is not (168) (M. W. van der Woude and D. Low, unpublished data). The pef operon, located on the 60-mDa plasmid in Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) also is a member of the pap group based on conserved GATC box sequences and the presence of regulatory proteins with amino acid sequence similarities to the PapI and PapB regulatory proteins (58). Pef fimbrial synthesis is regulated by Lrp, although it is not known whether expression is under phase variation control (R. Kadner, personal communication).

Table 3Three families of fimbrial regulatory proteins of E. coli and S. typhimurium

The fan operon is regulated by Lrp, similar to the pap-like operons described above, but is not under phase variation control and lacks both a PapI-like regulatory gene as well as GATC box regions. Moreover, leucine and alanine repress fan transcription via the Lrp protein, whereas pap, daa, and sfa are not responsive to these aliphatic amino acids (24, 39, 145, 168).

The E. coli fim operon, encoding type 1 fimbriae, is also regulated by Lrp and is subject to phase variation, but by a DNA inversion mechanism distinct from the other fimbrial operons. The rate of type 1 fimbrial switching in both directions is increased by aliphatic amino acids via the Lrp protein (17, 61). In some E. coli strains, designated as agar phase-locked, expression of type 1 fimbriae and mannose adhesion is abolished after transfer from broth to agar media (80, 153). This effect may be the result of regulation of transcription of fimB and fimE by an unidentified DNA binding protein not present in agar-grown cells (J. Duncan, personal communication).

The fim gene cluster in S. typhimurium, which is very similar to SEF21 (type 1) in S. enteritidis (30, 123, 142, 164; W. W. Kay, personal communication), is under a regulatory control mechanism distinct from fim (encoding type 1 fimbriae) of E. coli even though both operons code for a fimbria-adhesin complex with specificity for mannose and both fimbriae are under phase variation control (164).

Another group of fimbrial operons includes the coo, cfa, fap, and agg fimbrial gene clusters, which all code for regulatory proteins sharing sequence identity with the DNA binding domain of AraC, the transcriptional regulator of the arabinose operon (27, 28, 60, 93, 124). The Rns, CfaD, FapR, and AggR regulatory proteins share additional sequence similarities, especially at the amino termini (Table 3). These regulatory proteins are designated Rns-like, based on the first published sequence of this class of regulatory protein identified in the coo gene cluster coding for CS1 fimbriae (27). Currently, it is not known whether the Rns class of regulatory proteins bind DNA or whether there are additional factors in the regulatory network.

Fimbriae composed of CsgA monomers have been designated "curli" for their thin, coiled, fibrillar structure (133). The expression of curli fimbriae is activated by the Crl regulatory protein at 26°C but not at 37°C (7). In addition, the RpoS sigma factor, involved in the control of many genes activated in the stationary phase of growth, is required for curli fimbrial expression, whereas the histonelike protein H-NS acts to repress curli expression (132).

Fimbrial operons that share common regulatory properties (Table 2) are not necessarily in the same class, based on the primary amino acid sequence of their major subunits (Table 1). For example, Pap, F1845, and K88 fimbriae are structurally distinct from each other, but the operons coding for their expression share common regulatory properties such as conserved GATC box sites and regulatory proteins. These results suggest the possibility that exchange of DNA sequences between different fimbrial operons has occurred. In some operons such as fap, afa, and fae, insertion sequences are located within regulatory regions, which may promote the exchange and transfer of flanking DNA sequences (77, 94) (C. Le Bouguenec and A. Labigne, personal communication).

Environmental Control of Fimbrial Expression

Fimbrial expression is regulated by a number of environmental factors including carbon source (via the catabolite activator protein [CAP]), aliphatic amino acids (via Lrp), iron (via the ferric uptake regulator Fur), electron acceptors other than oxygen (via Fnr), and temperature (possibly via H-NS and RimJ) (Table 2). In general, fimbrial expression is activated by poor growth conditions (low glucose and amino acid levels) and is repressed by temperatures less than 26 to 28°C. An exception is curli fimbriae, in which temperatures greater than 26°C repress expression (7). Notably, several strains of S. enteritidis appear to constitutively express GVVPQ fimbriae, which are closely related to curli (see Table 1) (35). Currently, very little is known about the physiologic significance of these signals, except for the temperature response which provides a mechanism to shut off fimbrial synthesis outside of homeothermic hosts.

TCP (the toxin-coregulated pilus) in V. cholerae and the type IV fimbriae of Pseudomonas aeruginosa are controlled by the ToxR and PilR/PilS two-component regulatory systems, respectively (74, 118). In these systems, an environmental stimulus alters the conformation of the sensor component, which then transmits the signal via phosphate transfer to a regulatory component such as a DNA binding protein. Two-component regulatory control of fimbrial expression in E. coli and Salmonella spp. has not been described. However, a possible candidate is bfp, encoding bundle-forming pili (fimbriae). The major structural subunit, BfpA, is a member of the type IV fimbriae and is most closely related to TCP fimbriae (43, 64, 162).

Transcriptional Regulatory Mechanisms

Expression of fimbriae is either uniform or subject to phase variation. Under the latter control mechanism, fimbrial expression reversibly switches between ON (fimbriae-positive) and OFF (fimbriae-negative) states, in contrast to uniform regulatory control in which distinct fimbrial expression states are not observed. Under both uniform and phase variation control mechanisms, fimbrial transcription can be environmentally responsive through alterations in transcript levels or switch frequency rates, respectively (Table 2). In addition, it is also possible that the level of transcription in phase ON cells can be controlled by environmental signals.

Methylation-Dependent Phase Variation.

Phase variation in the pap operon is controlled by the methylation states of two GATC sites, GATC-I and GATC-II. These GATC sites are located within GATC boxes I and II, respectively, in the pap regulatory region and are located within pap DNA binding domains for the Lrp global regulatory protein. Methylation of these sites by deoxyadenosine methylase (Dam) inhibits Lrp binding (22, 24, 169). Binding of Lrp, in turn, regulates the methylation states of these GATC sites, forming methylation patterns characteristic of phase OFF and phase ON cells (Fig. 2) (19). PapI, an 8.8-kDa regulatory protein, is required to switch from the OFF state to the ON state. PapI binds to Lrp, resulting in a shift in Lrp binding from the GATC-II site to the GATC-I site (128). Binding of Lrp at GATC-I stimulates pap transcription and forms the phase ON DNA methylation pattern (GATC-I site nonmethylated, GATC-II site methylated). The binding of Lrp-PapI to the GATC-I site is inhibited if this site is fully methylated, which is the methylation state of OFF cells (GATC-I site methylated, GATC-II site nonmethylated). These results indicate that the switch between OFF and ON expression states requires DNA replication to alter the methylation states of the pap GATC sites. Following DNA replication, the GATC-I site is transiently hemimethylated, providing the opportunity for binding of Lrp-PapI since binding of Lrp-PapI to DNA containing a hemimethylated GATC-I site occurs with low affinity (128).

Fig. 2Pap phase variation model. Transcriptionally active (phase ON) and inactive (phase OFF) states of pap are shown. The transition between these transcription states is thought to require DNA replication (19, 23). The GATC-I and GATC-II sites are represented by a square and circle, respectively. Methylation of these GATC sites in the ON and OFF states is indicated by "?CH3." Proteins involved in pap transcriptional regulation are shown, as well as the papI and papB genes. Dam, deoxyadenosine methylase; RNA pol, RNA polymerase holoenzyme; Lrp-PapI, complex between the Lrp and PapI regulatory proteins. Hatched areas indicate putative interactions occurring between regulatory proteins and RNA polymerase. The papBA and papI transcription start sites are shown by wavy lines.

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Unlike many Lrp-regulated operons, the expression of Pap fimbriae is not controlled by leucine or alanine (24). However, Pap fimbrial expression is regulated by carbon source via the catabolite activator protein (CAP). CAP stimulates transcription of papI and thus regulates the phase switch frequency by controlling PapI levels (56, 67). CAP also may directly affect the switch between OFF and ON states (67). The PapB regulatory protein, like CAP, activates papI transcription, in addition to acting as a feedback regulator of papBA transcription (55).

Dam levels also regulate Pap phase variation. Under conditions of only a fourfold increase in Dam, phase variation is blocked as a result of increased methylation of the GATC-I site, which inhibits binding of Lrp-PapI and formation of the ON state (19, 23). In the absence of Dam, cells are also locked in the OFF state because methylation of the GATC-II site is required for pap transcription. Possibly, methylation of GATC-II blocks binding of Lrp to this site which, in turn, opens the adjacent RNA polymerase binding site of the papBA promoter (23). Finally, H-NS appears to act as a repressor of pap transcription, although our preliminary results indicate that it is not essential for Pap phase variation (56, 68; M. W. van der Woude, L. S. Kaltenbach, and D. A. Low, Mol. Microbiol., in press).

Inversion-Dependent Phase Variation.

In contrast to Pap, expression of type 1 fimbriae coded for by the fim operon in E. coli is regulated by a methylation-independent mechanism (47, 57). Phase variation is regulated by inversion of a 314-base-pair DNA sequence containing the promoter of the fimA gene coding for the major fimbrial subunit by a RecA-independent mechanism (Fig. 3) (1). Two genes adjacent to fimA, fimB and fimE (also designated hyp) (95, 135), code for proteins with sequence similarities to site-specific recombinases such as phage λ integrase. The fimE gene product appears to promote inversion of the promoter-containing DNA fragment primarily in the ON to OFF orientation, whereas the fimB gene product promotes recombination in both directions (115, 116). In rich medium at 37°C, FimE-promoted inversion is so rapid that colonies formed from phase ON cells are mostly in the phase OFF orientation (61). Notably, many previous studies were carried out using E. coli K-12 isolates that lack FimE activity, which display aberrant fimbrial expression phenotypes due to a reduction in the ON to OFF phase switch frequency (18). Moreover, inversion-independent phase variation has also been recently reported, although the mechanism is not known (115).

Fig. 3Type 1 fimbrial phase variation model. Transcriptionally active (phase ON) and inactive (phase OFF) states of fim are shown. The inverted repeat region (IR) consists of the 9-base-pair sequence "TTGGGGCCA," which flanks a 314-base-pair DNA fragment containing the transcription start site for fimA designated as pA. The fimA gene codes for the major fimbrial subunit, whereas fimB and fimE are regulatory genes required for inversion-dependent phase variation. The effects of Lrp, IHF, and histonelike protein H-NS on fim inversion are shown.

Source:

Expression of type 1 fimbriae is controlled by at least three global regulatory factors, integration host factor (IHF) (45, 49), H-NS (PilG) (92), and leucine-responsive regulatory protein (Lrp) (17). IHF is necessary for fim switch inversion. IHF could play a direct role in site-specific recombination, as it does in phage λ integration and excision, and/or it could modulate transcription of fimB or fimE or both (45, 49). The histonelike protein H-NS appears to repress Fim switching since switch rates in both directions increase in a pilG mutant (92). In contrast to H-NS, the global regulatory protein Lrp stimulates fim phase switching in both directions (Fig. 3). Lrp has only a minimal effect on transcription of fimB and fimE and appears to stimulate recombination by binding within the switch region (17) (I. Blomfield, personal communication).

POSTTRANSCRIPTIONAL REGULATION

A critical factor in fimbrial biogenesis is the expression of appropriate levels of each of the components of the assembly machinery as well as the fimbrial subunits. Because the major fimbrial subunit constitutes up to 99% of fimbriae, more of these fimbrin molecules are required than other assembly and minor fimbrin components. One way in which this is achieved is through RNA processing and differential stabilization of the cleaved RNAs. In the pap operon, transcription initiated at the papBA promoter proceeds through the papB regulatory gene and the papA major fimbrin subunit gene. Although most transcription is stopped by a terminator located between papA and papH, some transcriptional read-through occurs which is believed to allow expression of the remaining genes of the pap operon (9). Posttranscriptional processing of the papBA polycistronic mRNA occurs by RNase E cleavage at the site UUUGU↓AUUGAUC, located between papB and papA (9, 125). The papB-containing mRNA is highly unstable, with a half-life of only 2.5 min, whereas the papA mRNA has a half-life of about 27 min (9). A stem-loop structure 2 base pairs from the 5' end of the papA mRNA as well as the transcription terminator at the 3' end of papA (between papA and papH) may be involved in stabilization against exonucleolytic attack (50).

Processing and differential stabilization of mRNAs also occur in the cfa operon, coding for CFA/I fimbriae (90), and in the daa operon, coding for F1845 fimbriae (13). In cfa, transcription starts upstream of the cfaA gene, which probably codes for an assembly protein based on its homology with cooB (155), and continues through the cfaB gene coding for the major fimbrin subunit. Cleavage of this polycistronic mRNA occurs between the cfaA and cfaB genes, followed by rapid degradation of cfaA-containing mRNA and stabilization of the cfaB gene coding for the major fimbrin subunit. In the daa operon, transcription initiates upstream of daaA and proceeds through daaBCDE. This transcript is processed between daaD and the daaE gene, coding for the major fimbrin subunit, followed by rapid degradation of the daaBCD portion of the mRNA (13). In contrast to pap, processing of both the cfa and daa mRNAs is not carried out by RNase E, but instead by unidentified RNases. For both the daa and cfa operons, stem-loop structures which act as transcription terminators have been proposed to also block the two known 3'→ 5' exonucleases, polynucleotide phosphorylase and RNase II, from degrading these mRNAs (13, 90).

CONCLUDING REMARKS

Most E. coli and Salmonella spp. isolates contain the genetic information for expressing multiple types of fimbriae. In some cases, cross-talk between fimbrial operons such as sfa and pap occurs, in which expression of one operon influences the other (121). Moreover, most fimbrial operons belong to modulons, which are sets of operons controlled by a single global regulator such as Lrp and CAP (105) (Table 2). This allows fimbrial expression to be coordinated and integrated with the metabolic state of cells. This ability to coordinate the expression of multiple fimbrial types may be important for host colonization since, under certain conditions, expression of fimbriae can be deleterious. For example, although type 1 fimbriae of E. coli are important for oropharyngeal colonization (16), expression of type 1 fimbriae can also lead to enhanced phagocytosis and activation of polymorphonuclear neutrophils (65, 156).

ACKNOWLEDGMENTS

We are very grateful to the following colleagues for providing preprints and unpublished results: I. Blomfield, S. Clegg, F. de Graaf, J. Duncan, B. Eisenstein, D. Evans, S. Falkow, J. Hacker, R. Hull, S. Hull, R. Isaacson, W. Kay, A. Labigne, C. Le Bouguenec, L. Makowski, P. Manning, S. Moseley, S. Normark, P. Orndorff, F. Orskov, I. Orskov, M. Rhen, J. Scott, B. E. Uhlin, and I. van Die. We also thank L. Kaltenbach for help in preparing Table 3. This work was supported by grants from the National Institutes of Health (grant 2 RO1 AI23348) and the National Science Foundation (grant MCB-93055166).

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