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Microbiology and Molecular Biology Reviews, December 2004, p. 692-744, Vol. 68, No. 4
1092-2172/04/$08.00+0     DOI: 10.1128/MMBR.68.4.692-744.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Type V Protein Secretion Pathway: the Autotransporter Story

Division of Immunity and Infection, University of Birmingham, Birmingham,¹ Division of Microbiology & Infectious Diseases, University Hospital, Nottingham, United Kingom,⁴ Department of Cell Biology, CINVESTAV, Mexico City, Mexico,² Department of Microbiology & Immunology, University of British Columbia, Vancouver, British Columbia, Canada³

SUMMARY

INTRODUCTION

SECRETION BY NUMBERS: PROTEIN SECRETION MECHANISMS IN GRAM-NEGATIVE BACTERIA

Type I Protein Secretion

Two-Step Type II Protein Secretion

The Injectisome: Type III Protein Secretion

Type IV Protein and Nucleoprotein Secretion

TYPE V PROTEIN SECRETION

Autotransporter Secretion Pathway (Type Va)

Inner membrane transport.

Periplasmic transit.

ß-Domain.

Translocation of the passenger domain.

Processing of autotransporters at the surface.

Energy of transport.

Evolution and distribution of autotransporter proteins.

Two-Partner Secretion Pathway (Type Vb)

Type Vc

CLUSTER 1: SUBTILASE FAMILY OF AUTOTRANSPORTERS

Ssp

PspA and PspB

Ssa1

SphB1

AspA/NalP

CLUSTERS 2 AND 11: HELICOBACTER PYLORI AUTOTRANSPORTERS

VacA, the Vacuolating Cytotoxin

Molecular and structural features.

Vacuolating activity.

Other functions.

Epidemiology of H. pylori infection.

BabA, SabA, and AlpA

CLUSTERS 3 AND 4: AIDA AUTOTRANSPORTER FAMILY

AIDA-I

Antigen 43

IcsA

TibA, ShdA, and MisL

AutA and AutB

CLUSTERS 5 AND 8: SERINE PROTEASE AUTOTRANSPORTERS

IgA1 Proteases

H. influenzae Adhesion and Penetration Protein (Hap)

N. meningitidis App and MspA Autotransporter Proteins

Serine Protease Autotransporters of the Enterobacteriaceae

Tsh.

SepA.

EspC.

EspP.

Pet.

Pic.

SigA.

Sat.

Vat.

Others.

CLUSTER 6: BORDETELLA AUTOTRANSPORTERS

Identification of Autotransporters in the Bordetella Genomes

Pertactin

BrkA, TcfA, and Vag8

CLUSTER 7: CHLAMYDIAL AUTOTRANSPORTERS

CLUSTER 9: RICKETTSIAL AUTOTRANSPORTERS

CLUSTER 10: LIPASES AND ESTERASES

EstA

ApeE

Lip-1

McaP

OTHER AUTOTRANSPORTER PROTEINS

Adhesins

Sialidases

FUTURE DIRECTIONS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Until the 1960s, protein secretion through biological membranes was thought to be a relatively rare phenomenon. Furthermore, this view was governed by the assumption that where a protein was secreted it crossed the membrane via its own dedicated and specific mechanism. In the intervening years this view has radically changed, with the growing realization that both prokaryotic and eukaryotic cells secrete a prodigious array of proteins and possess protein translocation pathways which are surprisingly conserved throughout the phylogenetic kingdoms.

A surprising amount of information regarding bacterial protein secretion has been derived from the study of bacterial pathogenesis. Indeed, molecular and genetic investigations of bacteria have shown that pathogenic organisms may be differentiated from their nonpathogenic counterparts by the presence of genes encoding specific virulence determinants and that those proteinaceous virulence determinants, in the form of adhesins, toxins, enzymes, and mediators of motility, are usually secreted to or beyond the bacterial cell surface (139). Thus, by decorating their cell surfaces with such proteins, pathogens can directly or indirectly increase their ability to survive and multiply in the host. However, a facet often ignored by those studying bacterial pathogenesis and protein secretion is that many nonpathogenic organisms also secrete proteins which are adaptive to their life-styles; e.g., saprophytic bacteria may secrete cellulases or other degradative enzymes. However, in both cases, secretion of these factors is governed in large part by the structure of the bacterial cell envelope.

Gram-positive bacteria produce a single plasma membrane, the cytoplasmic membrane, followed by a thick cell wall layer. In contrast, gram-negative bacteria produce a double-membrane system consisting of a cytoplasmic membrane, also called the inner membrane, and an outer membrane which sandwich the peptidoglycan and periplasmic space between them. Protein secretion across the inner membrane of both gram-positive and gram-negative organisms generally follows the same routes, normally involving the Sec-dependent pathway (often referred to as the general secretory pathway [GSP]), although other routes have recently been identified, e.g., the Tat and signal recognition particle (SRP) pathways (96, 109, 116, 119, 208, 338, 419, 434). However, once across the inner membrane, the fate of the translocated proteins diverge. In gram-positive organisms, the proteins are either released into the extracellular environment or incorporated into the cell wall layer by virtue of one of several cell wall peptide-anchoring mechanisms (78, 319). In contrast, translocation across the gram-negative inner membrane results in release of the protein into the periplasmic space. Thus, proteins that are targeted for the cell surface or extracellular milieu must also cross the additional barrier to secretion formed by the outer membrane. To achieve translocation of these proteins to the cell surface and beyond, gram-negative bacteria have evolved several dedicated secretion systems, some of which bypass the Sec-dependent system and integrate both inner and outer membrane transport in a temporally linked fashion (see below).

In this review, we comprehensively describe the autotransporter secretion pathway, which is present in gram-negative organisms including animal and plant pathogens. We examine the roles of these proteins in the context of bacterial pathogenesis and highlight areas for future research. Readers are referred to more concise reviews of the autotransporter secretion pathway (101, 102, 203) and the roles of these proteins in pathogenesis (201).

SECRETION BY NUMBERS: PROTEIN SECRETION MECHANISMS IN GRAM-NEGATIVE BACTERIA

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Recent progress in the molecular analysis of the protein secretion pathways of gram-negative bacteria has revealed the existence of at least five major mechanisms of protein secretion. These pathways are highly conserved throughout the gram-negative bacterial species and are functionally independent mechanisms with respect to outer membrane translocation; commonalities exist in the inner membrane transport steps of some systems. For better or worse, these pathways have been numbered type I, II, III, IV, and V. Several more extensive reviews of the gram-negative protein secretion systems have recently been published (4, 32, 64, 66, 147, 156, 264, 274, 294, 377, 395, 428, 429). Figure 1 gives an overview of the Type I, II, III and IV secretion pathways.

View larger version (43K): [in this window] [in a new window]   FIG. 1. Schematic representation of the type I, II, III, and IV protein secretion systems. The type I pathway is exemplified by hemolysin A (HlyA) secretion in E. coli, the type III system is exemplified by Yop secretion in Yersinia, the type II system is exemplified by pullulanase secretion in Klebsiella oxytoca, and the type IV system is exemplified by the VirB system in A. tumefaciens. ATP hydrolysis by HlyB, YscN, SecA, and VirB11 is indicated. Secreted effector molecules are depicted as grey ovals. The type II and in some cases the type IV secretion systems utilize the cytoplasmic chaperone SecB, although the Tat export pathway has recently been implicated in the secretion of molecules via the type II pathway. Type III secretion also involves cytoplasmic chaperones (SycE); however, they do not interact with the Sec inner membrane translocon. The major structural proteins of each system are depicted in relation to their known or deduced position in the cell envelope. EM, extracellular milieu; OM, outer membrane; Peri, periplasm; IM, inner membrane; Cyto, cytoplasm.

Secretion of HlyA, and similar effector molecules, occurs in a Sec-independent manner and in a continuous process across both the inner and outer membranes. Furthermore, proteins secreted by TOSSs are not processed during secretion and do not form distinct periplasmic intermediates. Rather, the TOSS consists of three proteins: a pore-forming outer membrane protein, a membrane fusion protein (MFP), and an inner membrane ATP-binding cassette (ABC) protein (32). These proteins are represented in E. coli by TolC, HlyD and HlyB, respectively. Evidence suggests that the MFP (HlyD) and the ABC transporter (HlyB) in E. coli interact before binding of the effector (156). The secretion process begins when a secretion signal located within the C-terminal end of the secreted effector molecule interacts with the ABC transporter protein (224). In general, this signal sequence is specific and is recognized only by the dedicated ABC transporter. Once binding of the effector molecule occurs, interaction of HlyD with the outer membrane protein TolC is triggered, allowing secretion of the effector molecule to he external milieu (450). However, this model is somewhat controversial, since investigation of the E. chrysanthemi TOSS indicates that the ABC transporter and MFP interact only after binding of the effector molecule and subsequently MFP interacts with the pore-forming outer membrane protein (282). Nevertheless, it is apparent that in both cases the bridging formed by MFP and the outer membrane protein collapses after export of the effector molecule (485). ATP hydrolysis by the ABC transporter provides the impetus to drive secretion of the effector molecule across the cell envelope to the external milieu once bridging of all molecules has occurred. Hydrolysis of ATP is not required for interaction of the effector with the ABC protein or assembly of the TOSS complex (261, 262).

Recent data have indicated a possible structure for the TOSS complex. Resolution of the crystal structure demonstrated that TolC exists as a trimer and that the portion spanning the outer membrane formed a ß-barrel structure (Fig. 2) (263). However, unlike the typical porins, each monomer of TolC contributes four ß-strands to form a 12-strand antiparallel ß-barrel. In addition to forming the ß-barrel structure, TolC possesses a novel protein structure: the -helical barrel. This extends from the integral outer membrane ß-barrel structure into the periplasm (263). HlyD is also known to form a trimeric structure which projects from the inner membrane into the periplasm. Thus, it is easy to envisage a scenario where the monomers of HlyD and TolC, while forming their trimeric complexes, can extend to form a continuous channel across the inner and outer membranes at the appropriate time. Nevertheless, further investigations are required to fully appreciate the roles of each protein in the TOSS.

View larger version (45K): [in this window] [in a new window]   FIG. 2. Structure of TolC. The structure of TolC was solved to 2.1-Å resolution. The functional TolC unit consists of three TolC monomers oligomerized into a regular structure embedded in the outer membrane and extending into the periplasm such that it forms a continuous solvent-accessible conduit. The portion of the trimeric molecule that is embedded in the outer membrane adopts a 12-strand ß-barrel conformation consisting of four ß-strands per monomer. The molecule is 140 Å long, the ß-barrel is 40 Å long (spanning the outer membrane), and the remaining portion of the molecule adopts a 100 Å -helical conformation spanning the periplasmic space. This -helical domain consists of 12 -helices, such that the -helices extend from or connect to the periplasmic side of each ß-strand. It is the -helical domain that is predicted to interact with the MFP. Each monomer is highlighted in a separate color. Adapted from reference 263 and the Protein Data Bank (30).

Secretion via the MTB is exemplified by pullulanase (PulA) secretion from Klebsiella oxytoca (478, 57). PulA is a starch-hydrolyzing lipoprotein which forms micelles once it has been secreted into the external environment (478). Since the discovery of the MTB in K. oxytoca, the pathway has been identified in a variety of other bacterial species exporting a range of proteins with diverse functions including the cholera toxin of Vibrio cholerae, exotoxin A of P. aeruginosa, and several cell wall-degrading enzymes (428, 429). Furthermore, analyses of the MTBs and type IV fimbrial systems at the genetic and structural levels have shown sufficient similarity between the two systems to suggest that the fimbrial biogenesis pathway be classed as a type II secretion pathway (34).

Secretion of substrate proteins via the MTB occurs in a two-step Sec-dependent fashion. Thus, all proteins secreted via a type II secretion pathway are synthesized with an N-terminal signal sequence which targets proteins for secretion to the Sec inner membrane secretion pathway (for in-depth reviews, see references 115; 245; and 509). The Sec machinery is composed of an ATPase, SecA, several integral inner membrane proteins (SecD, SecE, SecF, SecG, and SecY, and a signal peptidase (116, 338). SecB, a cytoplasmic chaperone, does not recognize the signal sequence but recognizes the mature part of the protein targeted for secretion and subsequently directs the protein to the Sec translocon (109). SecA provides the energy for transport through the translocon, and the signal peptidase cleaves the signal sequence, releasing the remainder of the protein into the periplasm. Evidence suggests that, once in the periplasm the remaining protein adopts a quasi-native state which is facilitated by certain chaperones such as disulfide bond isomerase, DsbA (486). These folding steps are necessary for translocation across the outer membrane.

Transport across the outer membrane requires an additional 12 to 16 accessory proteins, which are collectively referred to as the secreton (428, 429, 486). A consensus nomenclature has been defined by the letters A to O and S for homologous genes and their products (the Xcp proteins of P. aeruginosa are labelled P to Z). The organization of the loci encoding the MTB components is relatively conserved, and most of the genes are transcribed from a single operon in which several of the genes are overlapping (428, 429). Evidence from both the Klebsiella Pul system and the Erwinia Out system for pectinase secretion suggests that protein C contributes to a species-specific interaction defining the transport of specific substrate proteins (42, 290, 404, 405). Protein D belongs to a family of proteins termed secretins. These proteins are integral outer membrane proteins predicted to consist largely of transmembrane ß-strands and form a ß-barrel structure in the outer membrane, with 12 to 14 subunits forming a complex (34, 188, 257). Several possible configurations for the secretin complex have been proposed. The most popular of these include a complex where monomers of protein D oligomerize to form a ring with a central channel and a model reminiscent of the TolC structure, where each monomer contributes ß-strands to form a single central channel (486). The C-terminal region is conserved among the secretins and is thought to be embedded in the outer membrane. It is interesting that homologues of the protein D secretin proteins have been found in the type III secretion pathways (see below) (486). Evidence suggests that protein E may act as a kinase regulating the secretion process by supplying energy to promote translocation and assembly of the pilin-like subunits, proteins G to K (430). No protein-protein interactions have been demonstrated for protein F (488). Proteins G to K possess homology to the type IV pili and are thought to form a pseudopilus (112, 264). Furthermore, these proteins are N-terminally processed and methylated by a prepilin peptidase, protein O (36, 390). A lipoprotein, protein S, has been demonstrated to play a role in stabilization of the outer membrane component protein D (428). Several other accessory proteins, which are not present in all MTB pathways, have a demonstrated requirement for substrate secretion in certain MTB systems. Evidence suggests that this may be related to the type of proteins being secreted or to substrate recognition by the MTB. In summary, the available evidence indicates that most if not all of the components of an MTB interact to form a multiprotein complex spanning both the inner and outer membranes (Fig. 1). Many more comprehensive reviews are available which describe the structure and function of the components of the MTB (428, 429).

Genes encoding components required for TTSS are generally located on a single plasmid or chromosomal locus. Comparison of the sequences demonstrates that the genes at these loci are conserved among different species, suggesting that these loci are inherited as a distinct genetic unit and that a common mechanism exists for the recognition and secretion of effector molecules (4, 64, 294, 395).

Like the TOSS, the TTSS translocate their effector molecules across the inner and outer membranes in a Sec-independent fashion. However, it is important to note that the Sec translocon, as in the case of TOSS, is required for secretion of structural components across the inner membrane. Controversy exists about the mechanism of effector molecule recognition and targeting to the TTSS. One hypothesis suggests that the signal resides in the 5' mRNA, which may target the ribosome-RNA complex to the TTSS, permitting temporal coupling of translation and secretion (11). A second proposal suggests that the N-terminal 20 amino acids serve as a binding site for cytoplasmic chaperones which specifically target the effector molecules to the TTSS (295). Notwithstanding the differences in these hypotheses, it is apparent that the region encoding the first 20 amino acids (either the untranslated mRNA or the first 20 amino acids of the polypeptide) is essential for secretion and the process is highly regulated (214).

Once targeted to the cytoplasmic side of the TTSS, secretion of the effector molecule may occur without the formation of periplasmic intermediates, with secretion proceeding through a needle-like structure composed of approximately 20 different proteins (214, 269). Unfortunately, a description of the voluminous evidence to support the roles of specific proteins in the formation of the TTSS complex is beyond the scope of this review. Based on the current evidence, the placement of specific proteins in the TTSS complex is indicated in Fig. 1. However, it is worth reiterating that the TTSS possesses a secretin-like component homologous to those found in the MTB systems (486). This secretin-like protein has been demonstrated to form ring-shaped structures with large central pores, which facilitates secretion of effector molecules and stabilizes the needle-like complex formed by the other components of the TTSS (153). This complex spans both the inner and outer membranes, resembling a hypodermic needle. The formation of a needle-like structure is supported by the visualization of the S. enterica and S. flexneri TTSS by electron microscopy (269). These studies demonstrated a striking resemblance between the TTSS and the flagellar basal body, and since the TTSS were shown to form long pilus-like structures, it was assumed, through analogy to the flagellar system, that the pilus structure allows protein transport across the bacterial outer membrane to the external milieu (214, 307). This was supported by investigations showing structural and sequence similarities between the EspA pilus protein of the E. coli TTSS and the flagellar filament (89, 255).

Perhaps the most striking feature of TTSS is their ability to target effector proteins directly into eukaryotic cells. This phenomenon is triggered in some cases when the bacterium comes in contact with a eukaryotic cell; hence, secretion via the type III pathway has been termed "contact dependent" (545). The analogy between the TTSS and the hypodermic syringe, coupled with the ability of the systems to inject proteins directly into the host cytosol, has allowed the phrase "injectisome" to be coined for this protein secretion system. Nevertheless, investigators should be reminded that not all TTSS are contact dependent and some effector molecules secreted by TTSS are released into the external environment (75).

As in other secretion pathways, the effector molecules secreted by the TFSS have a wide variety of functions. Arguably, the best-characterized function is that of pertussis toxin, which belongs to the A-B₅ toxin family (130, 481). Unlike the other type IV systems, the pertussis toxin is secreted to the extracellular milieu rather than directly into a host cell (481). The B domain (subunits S2 to S5) interacts with the host cell glycoprotein receptors and mediates translocation of the A domain (subunit S1) into the host cytosol. Once in the cytosol, the S1 subunit ribosylates the subunits of G-proteins, interfering with signaling pathways (310). In contrast, the A. tumefaciens system secretes several effector molecules, VirD2, VirE2, and VirF, into the host cell cytosol (67, 212). VirD2 is secreted as a nucleoprotein complex; the protein remains covalently associated with a single-stranded copy of T-DNA (266). Once in the host cytosol, VirE2 interacts with the VirD2/T-DNA complex, mediating delivery of the T-DNA to the host cell nucleus and resulting in crown gall tumor formation. A role for the VirF protein has yet to be defined conclusively. Unlike the pertussis toxin, the Vir effector proteins are secreted separately through the TFSS.

Controversy exists over how the substrate molecules for TFSS pass through the inner membrane. For the A. tumefaciens protein-DNA complex, export is postulated to take place in a single continuous step from cytoplasm to the interior of the cell. In contrast, export of pertussis toxin, like the type II secretion pathway effector molecules, occurs in two steps; the toxin subunits are first translocated across the inner membrane via the Sec machinery and are subsequently targeted for export across the outer membrane (51, 52). Thus, one evolutionary branch of the TFSS appears to be a branch of the GSP whereas the division encompassing T-DNA transfer is not.

The process governing transfer across the outer membrane is best understood for the A. tumefaciens TFSS. The quantity of information to support the individual roles of separate proteins is too great to review here; however, it is worth mentioning the roles of a few major proteins. Transfer of T-DNA and assembly of the pilus requires VirA, VirB1 to VirB11, VirD1 to VirD4, VirE2, and VirG. VirB2 is the major pilus subunit, although it is not yet clear whether this pilus emanates from the inner or outer membrane. VirB4 and VirB11 are inner membrane proteins, and VirD4 is a cytoplasmic protein, all of which possess ATPase activity. It has been suggested that these proteins provide the energy for translocation of the effector molecules through the pilus structure in a fashion analogous to the TOSS and TTSS. The remaining proteins are variously associated with the inner and outer membranes and with each other; the proposed placement of each protein is indicated in Fig. 1. TFSS has been divided into two subclasses: type IVa corresponds to protein machinery containing VirB homologues of A. tumefaciens, and type IVb corresponds to functional secretion systems assembled from Tra homologues of the Incl Collb-P9 plasmid of Shigella flexneri (66). Several excellent reviews have covered the functions of each subunit in more detail (66, 111, 143, 274, 277, 448).

TYPE V PROTEIN SECRETION

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Perhaps the simplest protein secretion mechanisms are those which are included under the umbrella of type V secretion. This family of secreted proteins includes those secreted via the autotransporter system (type Va or AT-1), the two-partner secretion pathway (type Vb), and the recently described type Vc system (also temed AT-2) (99, 102). Proteins secreted via these pathways have similarities in their primary structures as well as striking similarities in their modes of biogenesis. Figure 3 provides a schematic overview of the type V secretion pathways.

View larger version (30K): [in this window] [in a new window]   FIG. 3. Schematic overview of the type V secretion systems. The secretion pathway of the autotransporter proteins (type Va) is depicted at the bottom left of the diagram, the two-partner system (type Vb) is depicted in the center of the diagram, and the type Vc or AT-2 family is depicted on the right. The four functional domains of the proteins are shown: the signal sequence, the passenger domain, the linker region, and the ß-domain. The autotransporter polyproteins are synthesized and generally exported through the cytoplasmic membrane via the Sec machinery. Interestingly, effector proteins with an unusual extended signal sequence, which purportedly mediates Srp-dependent export, are found in all three categories of type V secretion. Once through the inner membrane, the signal sequence is cleaved and the ß-domain inserts into the outer membrane in a biophysically favored ß-barrel structure that forms a pore in the outer membrane. After formation of the ß-barrel, the passenger domain inserts into the pore and is translocated to the bacterial cell surface, where it may or may not undergo further processing.

Since this initial description, many more proteins which follow this pathway of secretion have been identified among the gram-negative bacteria (201). In all cases, the primary structure of the autotransporter protein is reminiscent of the IgA1 protease, consisting of a modular structure composed of three domains (231). These domains have been given different names throughout the literature. In this review, we use a consistent nomenclature throughout; thus, the domains are termed the signal sequence, the passenger domain, and the translocation unit. The signal sequence (also called the signal peptide or leader sequence) is present at the N-terminal end of the protein and allows targeting of the protein to the inner membrane for its further export into the periplasm (203). The next domain is the passenger domain (also called the -domain, N-passenger domain, or N-domain), which confers the diverse effector functions of the various autotransporters. The last main domain, located at the C-terminal end of the protein, is the translocation unit (also called the ß-domain, helper domain, C-domain, transporter domain, or autotransporter domain), consisting of a short linker region with an -helical secondary structure and a ß-core that adopts a ß-barrel tertiary structure when embedded in the outer membrane (317, 364, 365, 475), facilitating translocation of the passenger domain through the outer membrane.

Inner membrane transport. Bioinformatic analysis of the gonococcal IgA1 protease N terminus using SignalP (352) predicts that this domain functions as a signal sequence, allowing targeting of the protein to the inner membrane. Similarly, the N-terminal regions of all other autotransporter proteins display characteristics of the prototypical Sec-dependent signal sequence, for which SecB is presumed to act as the molecular chaperone (45, 203). These signal sequences possess (i) an n-domain with positively charged amino acids, (ii) an h-domain containing hydrophobic amino acids, and (iii) a c-domain with a consensus signal peptidase recognition site which often contains helix-breaking proline and glycine residues as well as uncharged and short lateral-chain residues at positions –3 and –1 that determine the site of signal sequence cleavage (315). However, some autotransporters, like AIDA-1, Pet, or Hbp, exhibit an unusually long signal sequence consisting of a C-terminus resembling a normal signal sequence with a hydrophobic core and a consensus signal peptidase cleavage site but presenting a conserved extension of the n-domain which contributes most to the variation in overall length (203). In silico analyses of completed bacterial genomes has revealed the presence of at least 80 proteins possessing these extended signal sequences across the breadth of the Proteobacteria (Fig. 4) (M. Desvaux and I. R. Henderson, unpublished observations). Interestingly, these extended signal sequences appear to be associated exclusively with proteins larger than 100 kDa. All of these atypical signal sequences, consisting of at least 42 amino acids, appear to consist of two charged domains (n1 and n2) and two hydrophobic domains (h1 and h2), in addition to the C-domain signal peptidase I recognition site (Fig. 4). While the n1 and h1 domains of these autotransporters are well conserved, the sequence of the n2 domain is variable and that of the following h2 domain even more so (203). The variability within the n2 and h2 domains is expected and consistent with Sec-dependent signal sequences, although, notably, the n2 domain contains an unusual number of charged amino acids (49). In contrast, the conservation within the n1 and h1 domains is highly unusual and is characterized by the presence of conserved aromatic amino acids in the n1 domain and a glutamate residue in the h1 domain (Fig. 4). Such sequence conservation is frequently indicative of the existence of a specialized biological function, suggesting that the extended n1-h1 portion of the signal sequence directs inner membrane export through a pathway different from the Sec system or that it recruits accessory proteins to work in tandem with the Sec pathway.

View larger version (93K): [in this window] [in a new window]   FIG. 4. Structure and alignment of the extended signal sequences. The extended signal sequences belonging to autotransporter proteins from a wide range of gram-negative bacteria are depicted. Blue shading indicates the positions of the positively charged n1 and n2 domains. The positions of the hydrophobic h1 and h2 domains are denoted by yellow shading. The signal peptidase recognition sites are indicated by green shading. The n2, h2 and C-domains are characteristic of a typical signal sequence secreted via the Sec-dependent translocon in conjunction with the SecB chaperone. The in silico-predicted and/or empirically determined cleavage site between the signal sequence and the passenger domain is indicated. Conserved residues are highlighted.

Interestingly, several recent reports have described autotransporter proteins, namely, the H. pylori AlpA, B. pertussis SphB1, and N. meningitidis AspA/NalP proteins (82, 358, 508), with typical lipoprotein signal sequences which are indicative of processing by signal peptidase II. While investigations of AlpA demonstrated that it was processed as a lipoprotein when expressed in E. coli DH5, no such processing could be demonstrated in wild-type H. pylori (358). An alternative hypothesis is that AlpA is processed further downstream by signal peptidase I. Indeed, the protein may be acylated and processed by signal peptidase II and may subsequently undergo processing by signal peptidase I prior to insertion into the outer membrane. Using a chimeric SphB1-ß-lactamase fusion, Coutte et al. (83) demonstrated that SphB1, like AlpA, was processed as a lipoprotein in E. coli. However, such processing could not be demonstrated directly in the natural B. pertussis host. Nevertheless, growth in the presence of globomycin, a specific inhibitor of signal peptidase II, prevented localization of SphB1 in the outer membrane (83). Furthermore, the localization of AspA/NalP (also termed NalP) was also inhibited by treatment with globomycin (508). Interestingly, the presence of the lipoprotein signal sequence was found to be necessary for the functional localisation of SphB1 in the outer membrane, suggesting that it is required for anchoring SphB1 on the bacterial cell surface (83).

Periplasmic transit. After export through the inner membrane, the autotransporter proprotein exists as a periplasmic intermediate. The original model of outer membrane secretion predicts that the first of the ß-strands passes from the periplasmic space to the external surface, thus leaving the passenger domain temporarily extending into the periplasm (203). Using a reporter single-chain antibody as a reporter passenger domain, the secretion process has been investigated (510). It was concluded that the folding of the passenger domain takes place in the periplasm before, or at least simultaneously with its own translocation through the outer membrane. However, a recent investigation by Oliver et al. (365) revealed the presence of an intramolecular chaperone in BrkA, spanned by residues 606 to 702. This region corresponds to PD002475 in the ProDom database (76) and is found in many autotransporters but not in other proteins in the database. In the model derived from this study, the complete folding of the protein occurs on the surface of the bacteria (see the discussion of outer membrane transport below). This suggests that previous findings by Veiga et al. (510) may have been influenced by the study of a passenger domain unrelated to the autotransporters. Furthermore, it is unlikely that large molecules, such as the passenger domains, can pass through a pore of 2 nm while fully folded, and therefore the molecules must be maintained in a translocation-competent unfolded state in the periplasm.

Thus, the status of the autotransporter proteins in the periplasm remains controversial. Of particular interest is how such large proteins can maintain an unfolded, or partially folded, state and resist degradation by periplasmic proteases. For IcsA, the soluble periplasmic form of the autotransporter appears transient (46); hence, it was suggested that the ß-barrel inserts rapidly into the outer membrane or may interact with a general or autotransporter-specific periplasmic chaperone (46). It was proposed that the chaperone activity of DegP was involved (410). It is worth mentioning here that the formation of disulfide bonds in the passenger domain in the presence of the periplasmic disulfide bond-forming enzyme DsbA decreases the efficiency of secretion of the passenger domain (46, 232, 499, 510). This suggests that the proprotein is accessible to periplasmic enzymes and that at least partial folding of the autotransporter may arise in the periplasm. However, it remains possible that the decrease in secretion efficiency is a result of the translocation of a nonnative passenger domain rather than the formation of disulfide bonds per se. Indeed, the IcsA protein forms disulfide bonds in the periplasm and is efficiently secreted via its native translocating unit (46). Nevertheless, the paucity of cysteine residues which contribute to disulfide bond formation is also a unifying characteristic of autotransporter passenger domains; thus, the secretion of proteins with secondary structure appears to be an exception rather than a rule (231).

ß-Domain. ß-Barrels can have different topologies; the simplest of those theoretical topologies is the all-next-neighbor connection between adjacent strands, in which each ß-strand aligns in an antiparallel fashion to form a ß-sheet. The ß-barrel architecture has been found in all integral outer membrane proteins whose structures have been solved (97); -helical bundles have been found only in the cytoplasmic membrane (257, 444). Generally, it is assumed that this differentiation originates from the biogenesis of the outer membrane proteins, whose polypeptide chains have to cross the cytoplasmic membrane, where, if they contained hydrophobic -helix-rich regions, they would be stuck. As with all hitherto known integral outer membrane proteins, the C-terminal domain of autotransporter proteins is predicted to consist of ß-pleated sheets in the form of a ß-barrel (303).

Generally, the C-terminal domains of autotransporters consist of 250 to 300 amino acid residues. The ß-domain of autotransporters are all homologous but extremely diverse in sequence. Bioinformatic analyses of the C-terminal sequences of highly diverse autotransporters have permitted a consensus signature sequence to be proposed for this domain, which has been proven useful for the identification of new autotransporters from databases (203). While the exact shape of the ß-barrels of various autotransporters is still subject to speculation, some general features can be determined from bioinformatic analysis. Using the AMPHI algorithm (223) or the WHAT and AveHAS programs in combination (543, 544), it was predicted that the ß-domains of most autotransporters exhibit 14 antiparallel amphipathic strands consisting of 9 to 12 residues (303, 540).

In addition to the overall ß-sheet structure, the ß-domains share a consensus amino acid motif at the extreme C terminus, which represents the final spanning segment (231, 303). The terminal amino acid is always phenylalanine or tryptophan, preceded by alternating hydrophilic (charged or polar) and hydrophobic residues, i.e., (Y/V/I/F/W)-X-(F/W). This motif is also found in other gram-negative outer membrane proteins; in the case of PhoE, an E. coli porin, substitution or deletion of the C-terminal phenylalanine has a drastic effect on protein folding and stabilization of the monomer, resulting in an ineffective trimerization and outer membrane localization of the protein (95, 470). This observation has also been extended to the autotransporter proteins, since deletion of the final three amino acid residues of the H. influenzae Hap autotransporter, i.e., YSF, abolishes outer membrane localization of the protein (206). However, mutation of individual amino acids in the terminal three residues does not seem to affect its localization significantly.

By analogy to the mechanism of porin biogenesis (480), the current model proposes that the autotransporter proprotein spontaneously inserts into the outer membrane in a biophysically favoured ß-barrel conformation as it interacts with the local nonpolar environment of the membrane (Fig. 3) (203). The first and last ß-strands spontaneously form hydrogen bonds in an antiparallel fashion to close the ring conformation, permitting the establishment of a molecular pore. The alternating hydrophobic side chains of amino acids are embedded within the hydrophobic lipid bilayer, while the hydrophilic side chains project into an aqueous environment in the center of the barrel (507). However, investigations with other outer membrane proteins, like PhoE and OmpA, suggest that lipopolysaccharide (LPS) and the periplasmic chaperone Skp are required for efficient assembly into the outer membrane (95, 148). Recently, Bulieris et al. (50) proposed the first assisted-folding pathway of an integral membrane protein by using the example of OmpA. In this model, once OmpA is translocated into the periplasm, it binds to three molecules of Skp, which maintains OmpA in an unfolded state. This OmpA-Skp complex interacts with two to seven LPS molecules to form a folding-competent intermediate that facilitates insertion and folding of OmpA into the outer membrane lipid bilayer. It was suggested that the cochaperone function of Skp and LPS could be involved in the folding of a large number of outer membrane proteins (50). However, it also appeared that deletion of the skp gene does not entirely eliminate the presence of outer membrane proteins in the outer membrane, indicating this Skp-LPS assisted-folding pathway was certainly not the only mechanism by which outer membrane proteins insert and fold into the outer membrane.

Recently, the involvement the surface antigen Omp85 challenged the idea of a spontaneous assembly of outer membrane proteins into the outer membrane (517). Omp85 is a highly conserved protein since homologues are present in all the complete genome sequences of gram-negative bacteria. Interestingly, the gene is located in close proximity to the skp gene and to the lpxA and lpxB genes involved in LPS biogenesis. This protein is essential for cell viability and for outer membrane protein assembly in mitochondria and bacteria and demonstrates sequence similarity to Toc75 of the chloroplast protein import machinery, suggesting a common evolutionary origin for all systems (155, 518). It has been shown that, following Omp85 depletion, unassembled forms of various outer membrane proteins, including autotransporters, accumulated. Thus, Omp85 could possess a general function in the assembly of outer membrane proteins, although the molecular mechanisms underlying this function remain to be elucidated. However, Genevrois et al. challenged this view by demonstrating that Omp85 was in fact involved in lipid export from the inner to the outer membrane (154). In this study, the localization of some outer membrane proteins was not impaired upon Omp85 depletion but degradation products were observed. Interestingly, the isolation of an omp85 knockout mutant in an LPS-deficient mutant, which would have definitively demonstrated that Omp85 was involved only in the LPS transport, was impossible, suggesting additional function(s) for Omp85. Thus, a concerted mechanism where Omp85 permits the phospholipid and LPS transport and assembly of the outer membrane as well as the assembly of outer membrane proteins cannot be completely ruled out and necessitates further investigations (518).

Translocation of the passenger domain. Originally, it was proposed that the secretion of the unfolded passenger domain occurs through the hydrophilic channel formed by the monomeric ß-barrel and that a globular conformation was not compatible with this mode of translocation (252-254). In support of this hypothesis, a recent study has demonstrated the existence of an ion channel in the ß-domain of BrkA with an average conductance of 3.0 nS in 1 M KCl (449). However, recent investigations have challenged this long-held hypothesis. Several investigations suggested that the presence or absence of DsbA, the major periplasmic disulfide bond-forming catalyst in E. coli, did not make any difference to the stability and targeting of the passenger domain to the outer membrane (46, 499, 510). These investigations questioned whether a structured polypeptide could be translocated through the somewhat narrow hydrophilic channel of a ß-barrel (355). Indeed, an alternative model of secretion was proposed after in vitro investigations of the secretion of gonococcal IgA1 protease using the ß-domain embedded in multilamellar liposomes. This model suggests that the passenger domain is instead secreted through an oligomeric ring-shaped structure consisting of a minimum of six ß-barrels and forming a central hydrophilic 2-nm-diameter pore (512). Nevertheless, these researchers also suggested that the oligomeric ring could consist of as many as 10 monomers and that the exact structure of the oligomeric ring remained speculative. Such a structure would be analogous to the outer membrane complexes found in other secretion systems, e.g., secretin or fimbrial ushers (459, 487, 540). This study has allowed the proposal of an alternative mechanism for autotransporter secretion through a 2-nm pore depicted as large enough to tolerate the passage of certain protein domains in a folded state (Fig. 5) (512). The use of different immunoglobulin domains, with distinct and defined folding properties, fused to the IgA1 protease ß-domain tends to demonstrate that type Va secretion is compatible with folded passenger domains containing disulfide bonds following the action of periplasmic or intramolecular chaperones prior to the outer membrane translocation (511). However, based on the available evidence from a variety of different systems, which each offer only a partial view of the translocation process, several other hypotheses have been proffered for the outer membrane translocation step. These include scenarios where the export of the passenger domain occurs through a common channel shared by the different subunits assembled into a single complex and also where the passenger domain is not secreted through any pore but, rather, extends directly from the translocating unit into the extracellular milieu, in a manner analogous to the YadA-like proteins (Fig. 5) (422, 474).

View larger version (44K): [in this window] [in a new window]   FIG. 5. Alternative mechanisms of autotransporter protein secretion. (A) Several different hypotheses have been proffered for the mechanism of autotransporter protein biogenesis. From left to right are depicted the traditional view of secretion originally proposed by Pohlner et al. (401), in which the passenger domain passes through the pore of the ß-barrel to the outside; a recently proposed structure where the passenger domain extends directly from the ß-barrel into the extracellular milieu; the secretion pathway proposed by Veiga et al. (512), in which a central channel is formed through which the passenger domains are secreted to the outside, and the pathway proposed by Hoiczyk et al. (210) for the type Vc autotransporter family, in which several molecules contribute ß-strands to make a large ß-barrel pore through which the proteins are secreted to the external side of the outer membrane. (B) Crystal structure of the AspA/NalP translocating unit to 2.6 Å. A side view and a stereo-top view are depicted. The protein forms a 12-strand ß-barrel structure characterized by short periplasmic turns and longer external loops. The barrel interior is highly hydrophilic due to the presence of charged amino acids. Within the barrel is embedded an -helical region, which is attached to the first transmembrane ß-strand such that the extreme N terminus of the protein, and to which it is presumed a native passenger domain would be attached, is located on the extracellular surface. Adapted from reference 366 and the Protein Data Bank (30).

Oliver et al. (364, 365) recently discovered an autochaperone domain in BrkA. This well-conserved domain, found upstream of the translocation unit, is present within the passenger domains of several autotransporters, like AIDA-I, IgA1 protease, Ag43, Hap, and IcsA, but was not detected in some autotransporters such as TcfA and Hia (365). It was demonstrated that this domain is important for the folding of the BrkA passenger domain, probably by triggering or initiating correct folding of the passenger domain. In BrkA, this domain could act as an intramolecular building block (305). It was speculated that this domain might function as a general chaperone to scaffold the folding of any protein linked to it. This autochaperone domain is cleaved from the passenger domain of PrtS but not from BrkA, suggesting that the folding mechanism(s) may differ depending on the autotransporter (365).

Assuming that the model described by Pohlner et al. (401) is correct, the passenger domain forms a hairpin structure and appears to remain unfolded, or partially folded, as it travels through the channel. Indeed, the structure demonstrated for the ß-barrel of AspA/NalP revealed a pore sufficiently wide to accommodate two extended polypeptide chains passing through the pore simultaneously (366). Then, as the passenger domain emerges from the ß-domain channel, folding is triggered beginning vectorially from a C-terminal direction on the bacterial cell surface. Evidence that passenger folding can occur on the bacterial surface has been provided by studies of PrtS autotransporter (362). Interestingly, the passenger domains of the autotransporters demonstrate a high degree of ß-helix structure, as predicted from the BetaWrap program (44), suggesting a structure analogous to pertactin (123) (see "Cluster 6: Bordetella autotransporters" below); thus, translocation of the passenger domains may actually be assisted by C-terminal winding of the ß-helix. However, it was shown recently that while autotransporters lacking the autochaperone domain could be translocated and displayed on the cell surface, they did not fold into their native conformation (365). Unfortunately, the influence of the autochaperone domain deletion on the efficiency and kinetics of the passenger domain secretion is unknown.

Processing of autotransporters at the surface. Once at the bacterial surface, several alternative processing steps have been demonstrated for autotransporters. First, the passenger domain may be processed and released into the extracellular milieu, e.g., Pet and EspP (49, 124). In contrast, for some autotransporters like Ag43 (373), AIDA-I (24) and the pertactins (279, 287), once the passenger domain is cleaved, it remains in contact with the bacterial surface via a noncovalent interaction with the ß-domain. However, the passenger domain is not necessarily cleaved but may also remain intact as a large protein with a membrane-bound C-terminal domain and an N-terminal domain extending into the external milieu, e.g., Hia or Hsr (367, 465). Nevertheless, the cleavage between the passenger domain and the translocation unit can occur either well upstream of the linker region or within the predicted -helical region, like BrkA (364). In this latter case, BrkA remains steadfastly associated with the bacterial outer membrane, raising the possibility that the linker region could also act as an anchor for the passenger domain.

Controversy still exists over how cleavage of the passenger domain from the translocation unit occurs, especially whether cleavage is a result of a membrane-bound protease or an autoproteolytic event. For IgA1, Hap, and, more recently, App, it has been demonstrated that the cleavage was the result of an autoprocessing event involving the integral serine protease active sites of the autotransporter, present in the passenger domain (206, 401, 445). AIDA-I is also thought to be autoprocessed, although no serine protease motif has been found (472). Passenger domains may also be processed at several sites and possibly by a variety of protease as observed with Hap, where the primary autocleavage site was mutated but the protein was still cleaved at alternative sites (206). Some autotransporters have been shown to undergo processing even after deletion of their serine protease motif, suggesting the action of alternative proteases (22, 409). It has been demonstrated that in S. flexneri, IcsA (also called VirG) is cleaved by another membrane protease, IcsP (also called SopA) (117, 451).

Interestingly, the IcsA autotransporter has the unusual characteristic of being localized to a single pole of the bacillus (166). The current model suggests that, on translocation into the outer membrane at one pole, IcsA, which is anchored in the membrane by its ß-domain, diffuses laterally within the outer membrane such that some of it drifts down the sides of the bacillus towards the septum (463). IcsP, which is localized in the outer membrane, slowly cleaves IcsA at all sites on the bacterial surface, but since the insertion of IcsA is occurring exclusively at one pole, it results in a unipolar distribution of IcsA (463). This study raises the question of the fate of the ß-barrel once cleavage of the passenger domain has occurred. By analogy to other outer membrane proteins, the ß-barrel of autotransporters might be a rather stable structure (444). Results from cellular localization studies seem to corroborate the persistence of the ß-barrel in the outer membrane (60). However, this question has not yet been satisfactorily addressed. One might speculate that accumulation of pore-forming structures in the outer membrane would be lethal for the bacteria and that some kind of degradation or regulation process must therefore occur. However, the degradation mechanism(s) of periplasmic and outer membrane proteins remains elusive and requires further investigation, although it might involve periplasmic enzymes such as DegP and DegQ or outer membrane proteases such as OmpT.

Energy of transport. Comprehensive understanding of the energy requirements used to translocate proteins across the bacterial outer membrane has lagged behind the wide-ranging studies investigating inner membrane transport (378). Since neither ATP nor GTP occurs in the periplasm, hydrolysis of these molecules cannot be the driving force for the translocation of the passenger domain through the outer membrane. Moreover, there is still no evidence of the existence of a proton motive force (p) across the outer membrane. The presence of P-loop nucleotide motifs in several members of the autotransporter family has been reported (203). Proteins bearing a P-loop motif are not always ATP- or GTP-binding proteins, and even proteins binding ATP or GTP do not always possess this motif (433, 522). Subsequently, it was suggested that since autotransporters can be correctly targeted and exported in nonnative gram-negative bacterial backgrounds, e.g., gonococcal IgA1 protease in E. coli, no additional secretion function is required and thus the energy source driving translocation across the outer membrane is derived from the correct folding of the passenger domain on the bacterial cell surface (254). From the study by Veiga et al. (510), where a nonnative single-chain antibody reporter passenger domain was used, it appeared unlikely that the folding of the passenger domain on the cell surface would provide the energy required for the translocation through the outer membrane. Unfortunately, this set of experiments used a nonnative passenger domain lacking the intramolecular chaperone domain. Thus, a common sorting mechanism assisting the assembly and export of autotransporter proteins and involving the autochaperone domain may exist. However, as mentioned above, BrkA lacking the autochaperone domain is still secreted, suggesting that other factors provide the necessary driving force for secretion. In conclusion, the energy dependence of the autotransporter secretion is still controversial and speculative and further investigation is required to clarify this issue.

Evolution and distribution of autotransporter proteins. While the translocation units of autotransporters are highly homologous, the passenger domains are very diverse. To date, all characterized passenger protein domains have been implicated in virulence by displaying enzymatic activity (protease, peptidase, lipase, and esterase); mediating actin-promoted bacterial motility; acting as adhesins, immunomodulatory proteins, toxins or cytotoxins, or, as more recently discovered, permitting the maturation of other virulence proteins (82, 201, 203, 540). The autotransporters are present only in the Bacteria kingdom and are most prevalent in the phylum Proteobacteria, including the -, ß-, -, and -Proteobacteria classes (540). Like the TTSS, the only other phylum in which autotransporters have also been identified is the phylum Chlamydiae, genera Chlamydia and Chlamydophila (198, 213). In most cases, multiple genes encoding autotransporters are found throughout the genome sequence of the microorganism, e.g., E. coli (530), Bordetella spp. (382), Yersinia pestis (98), Helicobacter pylori (313), Pseudomonas aeruginosa (306) and Neisseria (91). It has been suggested through genome sequence analysis that the passenger domain is not always covalently linked to the ß-domain, since some open reading frames (ORFs) code either only for a passenger domain or a ß-domain. However, these could simply be pseudogenes, since there is no experimental evidence that these proteins are expressed (540).

It has been proposed that autotransporter proteins could have evolved by domain shuffling (91, 303). Functionally novel autotransporters could arise by linking a new active passenger domain to a generic ß-barrel domain. Phylogenetic analysis showed that horizontal transfer between distant organisms of the portions of the gene encoding the autotransporter ß-domain was a rare evolutionary event; instead, close homologues appear to arise almost exclusively by speciation and late gene duplication events within a single organism. Such analyses have allowed Yen et al. to classify the autotransporters into 10 distinct phylogenetic clusters (540). However, reinvestigation of the phylogenetic relationship of the autotransporters reveals that the proteins can be grouped into at least 11 clusters (Fig. 6) (I. R. Henderson, unpublished data). However, it is clear that the functional passenger domains have spread by horizontal transfer (91, 201). For example, while the ß-domains of the IgA1 proteases and the serine protease autotransporters of the Enterobacteriaceae (SPATE) have different evolutionary lineages (cluster 8 and 5, respectively), the functional passenger domains are evolutionarily related and belong to the same clan of serine proteases (Table 1, Fig. 6). These data suggest that most autotransporters have arisen through fusion events between passenger domains and ß-domains.

View larger version (37K): [in this window] [in a new window]   FIG. 6. Phylogenetic tree of the autotransporter proteins. The CLUSTALX and TREE programs were used for multiple alignments and construction of a phylogenetic tree for the functionally characterized autotransporter proteins. The tree depicted in this figure is derived from analyses of the C-terminal translocating domains of the autotransporters. Proteins are clustered according to the pattern proposed by Yen et al. (540). A single additional cluster (cluster 11) is indicated.