Common themes and variations in serine protease autotransporters

Review

Common themes and variations in serine protease autotransporters Yihfen T. Yen1,2, Maria Kostakioti3, Ian R. Henderson4 and Christos Stathopoulos1 1

Department of Biological Sciences, California State Polytechnic University, Pomona, CA 91768, USA Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, USA 3 Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA 4 Bacterial Pathogenesis and Genomics Unit, Division of Immunity and Infection, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK 2

The serine protease autotransporters of the Enterobacteriaceae (SPATEs) represent a group of large-sized, multi-domain exoproteins found only in pathogenic enteric bacteria. These proteins contain a highly conserved channel-forming C-terminal domain, which functions together with YaeT/Omp85 to facilitate secretion of the passenger domain to the cell surface. The C-terminal domain also mediates autoproteolytic cleavage, which releases the passenger from the bacterial cell. The passenger folds into a characteristic parallel b-helical stalk-like structure with an N-terminal globular domain that performs serine proteolytic activity. Here, we review and discuss recent findings that have led to a better understanding of these unique features in this virulence protein family, including their biogenesis, structural architecture, sequence variation, sub-grouping, evolution and biochemical function. A family of proteolytic virulence proteins secreted by the autotransporter pathway Multiple pathways for protein secretion have been described in Gram-negative bacteria [1]. The serine protease autotransporters of the Enterobacteriaceae (SPATEs) constitute a group of exoproteins secreted by pathogenic enteric bacteria of the g-proteobacteria through the autotransporter (AT) or type V pathway. The majority of proteins secreted by this pathway are virulence factors implicated in bacterial pathogenesis [2,3]. The secreted component and the secretion apparatus of an AT system are packed within the same polypeptide [2,4,5], which comprises a cleavable N-terminal signal sequence, an internal passenger domain and a C-terminal translocator domain (Figure 1a). The C-terminal domain forms a bbarrel in the outer membrane (OM), enabling the passenger to be transported to the cell surface, from which some ATs release their passengers to the extracellular environment [4,6]. The virulence properties of the ATs are associated with the passenger domain [2]. In 1994, Provence and Curtiss described the first SPATE protein: the temperature-sensitive-hemagglutinin (Tsh) of an avian pathogenic Escherichia coli strain [7]. Since then, >20 other SPATEs have been identified in Escherichia, Shigella, Citrobacter and Salmonella species (Table 1). SPATEs harbor a serine protease motif that Corresponding author: Stathopoulos, C. ([email protected]).

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confers their proteolytic capability. These ATs are found only in pathogenic strains and are usually the most abundant proteins secreted into growth media in laboratory settings [8]. Like many ATs, the SPATEs usually carry out multiple functions that are linked to the disease development of their hosts [3,9]. Although recent data obtained from structural and biochemical studies have led to a better understanding of this protein family, several questions still need to be addressed. In this review, we examine the latest findings on the biogenesis, structural architecture, sequence variation, sub-grouping, evolution and biochemical function of this family of virulence proteins. The significance of these data is interpreted and future research directions are also discussed. Biogenesis of functionally active SPATE proteins The Sec-dependent inner membrane (IM) translocation of a SPATE relies on its extended N-terminal signal peptide (SP), which ranges from 48 to 59 amino acid residues (Table 1). The extension contains a conserved sequence motif (Figure 1a) that is unique to several AT and twopartner secretion (TPS) proteins originated from the b- or g-proteobacteria [10]. It remains controversial whether the extended SP mediates a co- or post-translational secretion of the SPATE proprotein [11] and whether the signalrecognition-particle (SRP) components also participate in the process [12]. In SPATEs, the extended SP seems to have functions other than IM translocation: studies with EspP and Pet from enterohemorrhagic and enteroaggregative E. coli, respectively, show that the extension slows down the secretion across the IM, thereby preventing protein misfolding in the periplasm, which could inhibit subsequent export across the OM [13,14]. Following IM translocation, the SP is cleaved from the AT polypeptide and the mature protein is released into the periplasmic compartment [2,4], where the passenger domain attains at least a partially folded conformation before its secretion across the OM [15–17]. In addition, there is recent evidence that the C-terminal domain of EspP folds in the periplasm to achieve a b-barrel conformation [18] (Figure 1c) that resembles the crystal structure of the protein [19]. It is easy to speculate the involvement of molecular chaperones in the event because the surface presentation of Shigella flexneri IcsA, a non-SPATE AT, requires the periplasmic chaperones Skp, SurA and DegP [20]; the secretion of Bordetella pertussis BrkA, also a non-SPATE AT, needs

0966-842X/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2008.05.003 Available online 1 July 2008

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Figure 1. Conserved motifs in three domains of SPATEs. (a) The signal peptide (SP) is represented by a dark box, passenger domain a dark line and translocator domain a gray box. The translocator domain further contains the N-terminal a-helical linker and the C-terminal b-barrel-forming b-domain. Residues are numbered based on the Tsh primary structure, starting from the first methionine in the signal sequence. The letter x denotes a less conserved position. Colors correspond to the regions mapped in the tertiary structures shown in (b) and (c). Motifs are defined as conserved regions consisting of more than three consecutive amino acid residues and were obtained by aligning 20 SPATEs (Table 1) using ClustalW of BioEdit, v.7.0.5.2. (b) Motifs in the passenger domain of SPATEs. Motifs were mapped in the crystal structure of Hbp (PDB ID: 1wxr) [31], which contains the entire passenger domain without the SP and part of the translocator domain up to the cleavage site, N1100–N1101. Two views of the structure are shown. (c) Motifs in the translocator domain of SPATEs. Motifs were mapped in the crystal structure of EspP (PDB ID: 2qom) [19], the residues of which span from the cleavage site to the last residue of the polypeptide. The side and bottom views of the structure are shown. Mapping of the residues in the tertiary structures was performed with MBT Protein Workshop [52].

SurA (RC Fernandez, personal communication). More notably, at least one of these chaperones (DegP) [21] recognizes the C-terminal signature motif of integral OM proteins (OMPs), which are present also in the AT translocator domain [22]. Future research is needed to demonstrate whether a molecular chaperone is involved directly in the periplasmic folding and OM targeting of a SPATE protein.

Based on the length of the translocator, ATs can be divided into two groups: the conventional and the trimeric ATs [4,23]. SPATEs represent a typical example of conventional ATs; the b-domain of each SPATE folds into a single b-barrel formed by 277 amino acids. OM insertion and translocation of conventional ATs involve the YaeT/ Omp85 OM protein-assembly factors [24,25]; however, the mechanism is currently unknown. Passenger secretion 371

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Table 1. Serine protease autotransporters of the Enterobacteriaceaea Protein

Organism

AidA_B7A ETEC

Disease Diarrhea

SP (aa) Passenger Translocator Function (aa) (aa) 59 1030 277 Unknown

Diarrhea

57

1050

277

Unknown

48 48 56 56

1010 1010 1031 1031

277 277 277 277

Unknown Unknown Cytopathic effects on intestinal cells Unknown

NCBI accession: ZP_00714135 NCBI accession: AAW66606 [48] [48] [53] [46]

53 52 55

976 1034 968

277 277 277

Enterotoxin Unknown Cytotoxin

[54,55] [56] [57]

52

1048

277

Heme-sequestering protein

[31,58]

Enterotoxin Cytopathic effects on intestinal cells; serum resistance mediator; hemagglutinin Protease Cytotoxin; portease Vacuolating cytotoxin on kidney and bladder cells Intestinal inflammation Cytotoxin Hemagglutinin; adhesin for RBCs, Hb, ECM Unknown Vacuolating cytotoxin

[59,60] [61]

EaaA EaaC EatA EpeA

Salmonella bongori Escherichia coli Escherichia coli ETEC EHEC

EspC EspI EspP

EPEC STEC EHEC

Hbp

Human septic Escherichia coli EAEC EAEC; Shigella flexneri

? ? Diarrhea Bloody diarrhea; hemorrhagic colitis Diarrhea Diarrhea; renal failure Bloody diarrhea; hemorrhagic colitis Wound infections and septicemia Diarrhea Bloody diarrhea; shigellosis

52 55

966 1040

277 277

UPEC STEC UPEC

Urinary tract infections 55 Diarrhea, renal failure 55 Urinary tract infections 49

1038 968 969

277 277 277

56 54 55

1039 954 1048

277 277 277

55 55

1044 1045

277 277

Boa

Pet Pic

PicU PssA Sat

Shigella flexneri Shigellosis SepA Shigella flexneri Shigellosis SigA Colibacillosis; Tsh_APEC APEC septicemia Urinary tract infections Tsh_UPEC UPEC APEC Colibacillosis; Vat septicemia

Refs

[62] [63] [64] [65] [2] [7,66] [44] [44,47]

a Abbreviations: aa, amino acid; ECM, extracellular matrix; APEC, avian pathogenic E. coli; EAEC, enteroaggregative E. coli; EHEC, enterohemorrhagic E. coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli; Hb, hemoglobin; RBC, red blood cell; SP, signal peptide; STEC, shiga toxin- producing E. coli; UPEC, uropathogenic E. coli.

across the OM could occur through the pore formed by either YaeT/Omp85 (‘Omp85’ model) or the AT b-barrel (‘hairpin’ model); alternatively, AT secretion across the OM could occur through direct insertion of the already assembled protein into the membrane bilayer, perhaps facilitated by the YaeT/Omp85 complex [4,24,26,27]. Once on the cell surface, the passenger of a SPATE is cleaved from the translocator and released into the extracellular environment. Cleavage occurs in a conserved site located between two consecutive asparagine residues in the linker region joining the passenger and the translocator [27,28]. An aspartate located inside the pore of the translocator performs the cleavage task [6]. Following secretion and folding, the mature SPATE passenger becomes functionally active. The secreted passengers exhibit divergent pathogenic functions (Table 1), despite considerable sequence homology among them [9]. To date, the biogenesis of SPATEs is comparable to that of conventional ATs, although differences have been noted in the processing of the passenger domain; furthermore, the role of periplasmic chaperones in the secretion process of SPATEs and the function of the extended signal sequence in non-SPATE ATs have not yet been determined. Hypotheses proposed for the mechanisms of AT biogenesis have undergone dramatic modifications during recent years, shifting from the classical ‘hairpin’ model to the most recent Omp85 model. The hairpin model, presented 20 years ago to explain the secretion of the Neisseria IgA1 protease, favors a view in which the passenger domain goes through the b-barrel to reach the cell surface 372

[29]. Supported strongly by recent experimental data, the Omp85 model likens AT secretion to the secretion of bacterial OMPs [30] and involves chaperone-assisted folding in the periplasm and YaeT/Omp85-mediated insertion into the OM. Given the similarity of the Omp85 model and the secretion model of b-barrel OMPs, we propose that the Omp85 model be named the ‘OMP-like’ model because such a name is more inclusive. Future research will determine whether the OMP-like model will eventually gain full support in the field and whether the secretion mechanism of ATs resembles that of chaperone/usher pathway, which secretes the pilus subunits [5]. Structural arrangement of the passenger domain: the code of virulence Although equipped with the ability to perform different functions, SPATE passenger domains display a similar architecture, rich in b-strands, and are predicted to fold into a parallel b-helical structure, a fold verified by the crystal structure of the SPATE protein Hbp [31]. In agreement with the two solved crystal structures of Pertactin (Prn) from B. pertussis [32] and vacuolating toxin VacA from Helicobacter pylori [33], which are both non-SPATE ATs, the Hbp passenger comprises a ‘stalk’ formed of 24 parallel right-handed b-helical turns, each consisting of three b-strands [31] (Figure 2a). This parallel b-helical architecture seems to be a common characteristic in the passengers of most ATs and proteins secreted by the TPS system [34]. The b-strands are separated by loops and higher levels of conservation are found in the strands than

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Figure 3. Variable regions in 20 SPATEs. Variable residues were identified by aligning 20 SPATEs (Table 1) using ClustalW of BioEdit, v.7.0.5.2 and mapped in the passenger domain (PDB ID: 1wxr) [31]. Domain 2, a putative chitin-binding domain [31], is in light purple and contains a variable region colored in dark purple. The putative heme-binding domain [31] is in light green and contains a variable region colored in dark green. Other colors (red, orange and yellow) were assigned arbitrarily to the remaining variable regions. Mapping of the residues in the tertiary structures was performed with MBT Protein Workshop [52].

Figure 2. Conserved residues in 20 SPATEs. Residues are mapped onto (a) the passenger domain (PDB ID: 1wxr) [31] and (b) onto the translocator domain (PDB ID: 2qom) [19]. Two views of each structure are shown. Fully conserved regions are shown in red. Moderately conserved regions are shown in orange. In (a), the protease catalytic triad is in green and the conserved region in the a-helical linker is in blue. The conserved residues were identified by aligning 20 SPATEs (Table 1) using ClustalW of BioEdit, v.7.0.5.2. Mapping of the residues in the tertiary structures was performed with MBT Protein Workshop [52].

the loops. In addition to the b-helical core, Hbp has two Nterminal globular domains. Domain 1, consisting of residues 1–256 of the mature protein, folds into a trypsin-like protease only after the removal of the N-terminal signal sequence and is associated with the proteolytic property of the protein [31]. Domain 2, comprising residues 481–556 (Figure 3), shows homology to the chitin-binding region of chitinase but the adherence property is yet to be confirmed [31]. Our bioinformatics analysis revealed that 173 of the total 1200 residues in the passenger domains of SPATEs are conserved and are expected to contribute to their structural stability and functional capability. These conserved residues can be found in mainly two places of the tertiary structure: (i) in the stalk formed by the b-helical turns, where they seem to stack on one another (Figure 2a). Of the 173 residues, 122 are hydrophobic, indicating that non-polar interaction has a role in the stabilization of the b-helical structure; and (ii) in the interface between the stalk and the globular proteolytic domain (domain 1). This interface is located at the tip of the stalk, which can extend

above the cell surface at the later stage of SPATE secretion while the protein is still OM-bound. The interface can thus act as the substrate-recognition site. Its binding to the substrate can occur before the subsequent cleavage action carried out by the nearby proteolytic site. This hypothesis is further supported by our observation that the interface contains five conserved motifs (Figure 1a). One motif is the catalytic triad formed by H125, D153 and G257DSGS, with the first serine being the residue responsible for the proteolytic activity [35,36]. The other motifs located nearby possibly influence the efficiency of the serine proteolytic ˚ away from the catalytic triad; activity: motif DFS is 11 A ˚ other motifs DxLHKxGxGxL and LKxGxGxVxL are 8 A apart from the motif RLxKxVxE (Figure 1b). Our modeling results presented here thus indicate both structural and functional relevance of this interface region. In addition to domains 1 and 2, SPATEs possess a putative autochaperone domain formed by residues 950– 1048 of the mature protein [31]. This domain, also known as the ‘junction’ region, is located at the C terminus of the passenger domain [31] and influences folding and efficient secretion of the passenger of a non-SPATE AT across the OM [37]. Recent studies on the folding behavior of an isolated Prn passenger have shown that the Prn b-helices fold at an extremely slow rate and that folding is initiated by the extra stable C-terminal domain of the polypeptide [37]. The findings indicate that folding of an AT b-helical passenger might follow a ‘vectorial’ mechanism, in which the C-terminal ‘junction’ can serve as the template (nucleator) for folding, resulting in a folding process that needs no external energy. The slow folding rate can also confer a functional advantage to an AT by enabling the polypeptide to remain in an export-competent conformation during its secretion across the bacterial membrane. Having an 373

Review extended SP that anchors the preprotein at the IM and thus delays its passage across the OM possibly also contributes to the slowing down of the process. Taken together, these data indicate that the folding of a SPATE passenger is constrained by the presence of an N-terminal signal sequence that has to be cleaved off before the serine protease domain can fold into its native and functionally active structure. The data also indicate that folding of the C-terminal autochaperone domain has to precede folding of the remaining b-helical passenger. Translocator domain: the means of delivery Biochemical studies on the translocators of two SPATEs, Tsh and EspP, indicate that each exists as a monomeric unit in vitro [15,38]. The recent crystal structure of the EspP translocator domain confirms the conventional AT status of the SPATEs [19]. Like the previously crystallized translocator domain of conventional AT NalP from Neisseria [26], the EspP translocator forms a 12-strand b-barrel that is connected to the a-helical linker at its N terminus. Unlike NalP, the linker of which plugs the entire length of the hydrophilic pore formed by the barrel [26], the struc-

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ture of EspP indicates an a-helix that is cleaved midway in the interior of the barrel (Figure 1c). SPATE translocators are the most conserved of the three domains with >60% amino acid sequence similarity (Table 1). The conserved residues show stacking patterns in the tertiary structure of the EspP translocator (Figure 2b). Nearly half of the 174 conserved residues are polar (84), whereas the others are non-polar (90). This is unlike the predominant presence of conserved hydrophobic residues in the passenger domain and indicates that different interactions might exist among the residues in the translocator than those that form non-polar interaction in the passenger. We hypothesize that these conserved translocator residues have a role in b-barrel stabilization, partially through the stacking of aliphatic residues, or that they participate in the secretion of the passenger domain by interacting with the a-helix that localizes within the barrel. The latter hypothesis was formulated based on the observation that three conserved motifs in the b-domain (Figure 1a), located in the 3rd, 7th and 12th b-strands, seem to surround the a-helix from three sides (Figure 1c). Indeed, a residue in the 7th strand of the EspP b-barrel is

Figure 4. Phylogenetic analysis of the amino acid sequence of full-length SPATE passenger domains. The tree was generated using split decomposition analyses (SplitsTree ver. 4) and was further tested for reliability using bootstrap analysis, yielding a result of 96.9%. The tree demonstrates two major subfamilies within the SPATE passenger domain and evidence of recombination among the passenger domains. Recombination is highlighted by the boxed regions of the phylogram. The SPATE proteins demonstrating an intracellular target are clustered at the top end of the phylogram and include Pet and EspP; those with a preference for extracellular substrates are clustered at the bottom of the phylogram and include EatA, Pic, Tsh and EpeA.

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Review

No xxVHKxGxGxL LRxGxGxVxL Little conservation No Abbreviations: aa, amino acid; SP, signal peptide.

GDSGS(G); No S284

Less similar 254 (971–1225)

No

No No

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a

Hap

AAN37924 Nontypeable No: 25 1104 Haemophilus influenzae P860295 No: 42 1153 AAN71716 Neisseria App meningitidis serogroup B strain MC58 and serogroup A strain Z2491 No: 21 950 Campy_ YP_001467 Campylobacter 185 concisus 13826 1225

No DxLSKxGxGxL LSxGxGxVxL No 262 Yes; H115, Yes RLxKxVxD (1196–1457) D158, S267

No

No CAA01030 Neisseria gonorrhoeae IgA1

No: 27 1244

Motifs in passenger domain H, D, DFS RLxKxVxE GDSGS(P) Residues in 3 domains (aa) SP Passenger b-domain Organism NCBI accession

Evolution of the SPATEs A study by Yen et al. [42] suggested that all members of the AT family have arisen through speciation. Broadly speaking, this is true with only limited examples of horizontal gene transfer occurring. One example of horizontal gene transfer is the existence of the gene encoding a SPATE protein (Boa) within Salmonella bongori. The absence of SPATEs from all other Salmonella sp. and the close

AT

Variations among SPATEs New SPATEs were discovered recently in Citrobacter rodentium and the E. coli strains E22, B7A and F11 (Y.T. Yen and C. Stathopoulos, unpublished). Deviations from key characteristics of SPATEs have been noted in new members. Recently, a SPATE homolog named RpeA from rabbit enteropathogenic E. coli was found to have no cleavage site in the linker region [41], probably making this putative SPATE the first member without a C-terminal processing site. Within the bona fide SPATE members, the most variable regions seem to serve functions specific to individual SPATEs. Our bioinformatics data revealed six such variable regions. In the Tsh tertiary structure, the majority of these variable regions localize in loops (Figure 3). One of these sequences corresponds to the putative heme-binding domain of Hbp [31] and forms a helical secondary structure. This sequence is unique to Tsh, Boa and Vat, implying that Boa and Vat can have similar heme- or hemoglobin-binding activity to Tsh. Another variable region localizes within the chitin-binding domain [31]. This sequence forms a loop–strand–loop secondary structure that extends away from the b-helical stalk. Our data indicate that it is more prevalent in SPATEs, although its biological function is unknown at present. The remaining four variable regions all localize in the loops of the passenger stalk. Owing to the loop localization and distinctiveness of these variable regions, it is possible that they have a functional rather than structural role; that is, they confer distinct virulence functions to individual SPATEs.

Table 2. SPATE-like serine protease ATs outside the family of Enterobacteriaceaea

partially responsible for the cleavage of its passenger [6]. It is also possible that the highly conserved C-terminal amphipathic b-strand segment of the translocator, found in all b-barrel OMPs, might be crucial for interacting with YaeT/Omp85 [39]. The role of the b-domain in the translocation of the passenger through the OM remains controversial but the participation of the linker in this process has been confirmed in EspP and Tsh [27,40]. Present in the linker of all known SPATEs is a 14 amino acid motif EVNNLNKRMGDLRD that constitutes the longest and most conserved sequence in the entire polypeptide (Figure 1a). Mutations in certain residues of this motif do not block insertion of the translocator into the OM but do severely affect or even abolish extracellular secretion of the passenger [27]. These data support a role of the linker in the early steps of SPATE secretion across the OM, which involve protein folding and targeting to the OM. We thus speculate that the linker might have a role in the assembly and stabilization of the b-barrel at the periplasm–OM interface.

Motifs in translocator domain DLFTG EPQx-ELVxG DxLHKxGxGxL LKxGxGxVxL a-linker motif/cleavage site 261 Yes; H101, Yes R(F/N)xKxVxE DxLAKxGxGxL Yes Little No No (1272–1532) D151, S278 conservation, no cleavage site 262 Yes; H98, DLS RLxKxVxD DxLSKxGxGxL LSxGxGxVxL No No No (1130–1391) D138, S242

SAFGxYNxDxxxNAxxRYxF

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Table 3. Non-SPATE-like serine protease ATsa AT

NCBI accession

Organism

Proteobacteria classification

Ssp/PrtS

CAA42236

Serratia marcescens

g1045 Yes: confirmed p Enterobacteriacae roteolytic ability of serine

Pseudomonas fluorescen

g

YP_348415

ZP_01343849 Burkholderia mallei

b

YP_674659

Mesorhizobium sp.

a

YP_363133

Xanthomonas campestris

g

ZP_00651916 Xylella fastidiosa Dixon YP_487044

SphB1

CAC44081

Ssa1

AAA80490

g

Rhodopseudomonas a palustris HAA2 Bordetella b pertussis Mannheimia haemolytica

ZP_00135639 Actinobacillus pleuropneumoniae serovar 1 str. 4074 Neisseria AspA/AusP CAC34605 meningitidis GroupC ETE37 AAN71715 Neisseria NalP meningitides st H44/76 ZP_01410861 Campylobacter fetus subsp. Fetus 82–40

g

g

b

b

e

Size Studied (aa)

Refs SP

[67] 27

Serine protease residues D76, H112, S340

AT bdomain (aa) 285 (761– 1045)

D85, H142, S363 1120 No 27 D79, H113, S318 1006 No 29 D69, H104, S359 944 No 21; D94, Lpp SP H130, S325 976 No 21;Lpp D110, SP H145, S355 1179 No 18 D222, H264, S474 1039 Yes: a self proteolytic [50] 36; D184, subtilisin needed for Lpp SP H221, FHA processing S412 [68] 26 D58, 934 Yes: cloning, H116, expression, S351 immunogenicity 932 No 27 D83, H116, S351

278 (750– 1028) 261 (859– 1120) 280 (726– 1006) 240 (704– 944) 275 (701– 976) 267 (912– 1179) 216 (778– 1039) 273 (661– 934) 259 (673– 932)

1067 Yes: confirmed a self-processing AT

281 (786– 1067) 281 (802– 1083) 248 (927– 1175)

1028 No

35

[69] 27; D137, Lpp SP H209, S410 1083 Yes: a serine protease [51] 27; D139, AT involved in other Lpp SP H211, ATs’ processing S427 1175 No 17 D45, H81, S300

Homologs

Found in S. marcescens: Ssp-h2 (BAA11383.1) Found in other species

Found in other species Found in other species Found in other species Found in other species

Found in diff strains Found in diff strains Found in other species

a

Abbreviations: aa, amino acid; Lpp, lipoprotein; SP, signal peptide.

homology of Boa to the E. coli SPATEs indicate strongly that this has been acquired through horizontal gene transfer rather than through speciation. A recent examination of the E. coli reference (ECOR) collection, strains A, B1, B2, D and E [43], revealed that SPATE proteins were clustered in the B2, D and a subgroup of the A phylogenetic branches [44]. The absence of SPATEs from the majority of the B1 strains and the remainder of the A subgroup seems to be unusual because at least one SPATE has been identified in all of the diarrheagenic and extraintestinal E. coli pathogens whose genomes have been determined. Based on these studies, it is tempting to speculate that pathogenic E. coli evolved through the acquisition of genes encoding SPATE proteins. However, the observed diverse functions and substrates of the SPATE proteins argue against a common role for these proteins in causing disease. Despite their high levels of homology, the passenger domains of the SPATE proteins demonstrate distinct sub376

strate specificities [9]. Previously, it was suggested that the SPATE family of ATs could be divided into two groups: one demonstrating cytopathic activity with the other exhibiting a preference for extracellular targets [22]. Using split decomposition analyses [45] of the complete passenger domains to explore the phylogenetic relationships of a subset of SPATE proteins, Dutta et al. confirmed the presence of the two groups of SPATE proteins [9]. However, this investigation did not establish a specific correlation between the phylogenetic groupings and biological function. Indeed, despite their common ability to cause cytopathic effects on cultured cells and to cleave spectrin, the highly similar proteins, such as Pet and Sat (53% identity), do not share identical peptide-cleavage specificities [9]. Since this initial study, several additional members of the SPATE family have been described for E. coli, including EspI, EatA, EpeA, EaaA, EaaC and Boa [46–48]. Splitdecomposition phylogenetic analyses incorporating these new members of the SPATE family of ATs revealed that

Review the original bifurcating phylogenetic pattern proposed by Henderson et al. [22] continues to be valid for the passenger domains (Figure 4), although, again, there is no precise functional correlation among the proteins. Interestingly, scrutiny of the amino acid sequences of these two groups of proteins revealed that the proteins could be distinguished by the presence and concomitant absence of specific domains within the passenger. Thus, passenger domains of the SPATE-protein subfamily that exhibit a preference for extracellular targets possess a domain (domain 2, see Figure 3), which has been implicated in the heme-binding activity of Hbp but which is absent in those proteins that demonstrate cytotoxicity [31]. By contrast, the subfamily of SPATE proteins that demonstrate toxic effects on eukaryotic cells possess within their passenger domains a region termed ‘domain 2A’, which is absent from those proteins demonstrating a predilection for extracellular targets. Previous investigations have indicated that domain 2A is responsible for the ability of the cytopathic SPATEs to bind to receptors on the cell surface [49]. Other serine protease autotransporters SPATE-like serine protease ATs outside the Enterobacteriaceae family Several ATs outside the Enterobacteriaceae family, including the well characterized IgA proteases from Neisseria gonorrhoeae [29] and Hap from nontypeable Haemophilus influenzae [36], in addition to a new member from Campylobacter concisus, also contain the serine protease motif (Table 2). Most of the motifs present in the passenger domains of SPATEs are found in the passenger domains of non-Enterobacteriaceae serine protease ATs, suggestive of functional commonality shared by these two groups. By contrast, the extended SP present in all SPATEs is not observed in any serine protease ATs outside the Enterobacteriaceae family. Moreover, the translocators of the SPATE-like non-Enterobacteriaceae ATs are usually smaller and none of the motifs in the SPATE translocators are present. The cleavage site in the a-linker is also absent (Table 2). The main differences between the two AT groups thus lie in the two regions that govern IM and OM translocation. Such differences indicate that distinct secretion mechanisms are used by the two groups and further imply the existence of species-dependent secretion variations within the common AT pathway. Non-SPATE-like serine protease autotransporters Some serine protease ATs found in both enteric and nonenteric bacteria possess a different serine protease motif: glycine–threonine–serine or GTS (Table 3). Members of this group are widespread throughout the proteobacteria and several display auto-proteolytic-processing ability [2,36]. Unlike SPATEs with a virulence role, these nonSPATE-like counterparts seem to possess the ability to process other proteins to their mature forms. Two ATs in this group, SphB1 from B. pertussis [50] and NalP from N. meningitidis [51], are needed for the maturation of a TpsA protein and an AT, respectively. Notably, SphB1 [50] and NalP [51] are confirmed lipoproteins and several other members in this non-SPATE-like group also harbor a lipoprotein SP (Table 3).

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Box 1. Salient features of SPATEs (i) They originate from only enteropathogenic or uropathogenic bacterial hosts, including E. coli, Shigella, Salmonella and Citrobacteria species [8]. (ii) These ATs possess a signal peptide of 48–59 amino acid residues that is longer than a typical Sec-dependent signal sequence. The extended region of the signal sequence is conserved in all SPATEs and has been hypothesized to retard protein folding and therefore facilitate secretion [13,14]. (iii) In the passenger domain, aliphatic stacking stabilizes the bhelical turns that form the cell-surface protruding structure [31]. The catalytic triad (GDSGS, H, D) confers proteolytic, although not autolytic, properties to SPATEs. Folding occurs in the passenger and translocator domains in the periplasm, before OM insertion [15,17,18]. (iv) The 277 amino-acid translocator domain forms a monomeric pore structure [19,38]. OM secretion requires the intrinsic components (residues in the linker and the b-barrel) but also the exogenous factors (YaeT/Omp85 complex) [25]. Release of the protein is accomplished by autocleavage, a task performed by the b-barrel, at a cleavage site located in the linker [6]. (v) SPATEs are secreted proteins localized to the extracellular environment of their bacterial hosts. All SPATEs characterized to date exhibit virulence functions and many are multifunctional. Their effects can be exerted either within or outside the host cells [3,9].

Concluding remarks and future perspectives SPATEs are multifunctional virulence-associated proteins of the AT family that have gained increasing attention owing to their involvement in numerous enteric diseases [2,9]. Currently, >20 SPATEs have been identified. A summary of what is known currently about SPATEs is provided in Box 1. Although functional, structural and secretion studies have provided insights into the properties of several SPATE proteins [2,9], many members have not yet been characterized and several questions about this family remain unanswered (Box 2). Ultimately, inforBox 2. Unanswered questions  Does the extended SPATE signal sequence interact with components of the SRP? How does it function to slow down the export of an AT protein across the IM? How do other ATs without the extension circumvent this deficiency?  Does folding (partial or full) of a SPATE polypeptide in the periplasm involve molecular chaperones, such as Skp, SurA and DegP, which are important for b-barrel OMP biogenesis? If so, how do these chaperones recognize the unfolded AT sequence? Is the C-terminal signature motif the primary recognition site? Which conserved SPATE residues have key roles in the folding of the protein?  What is the precise role of YaeT/Omp85 in OM insertion and translocation of SPATEs? How does YaeT/Omp85 interact with the periplasmic SPATE intermediate? Does the OM translocation of a SPATE require its b-barrel to be inserted first into a pore provided by the Omp85 complex or just directly into the OM bilayer? How is the passenger domain translocated across the OM?  How is SPATE autocleavage regulated? Does autocleavage also occur in the SPATE periplasmic intermediate? Once the passenger is cleaved and released from the membrane, what is the fate of the translocator domain?  Which residues and domains of the SPATE passenger determine the pathogenic function(s) of the protein? How is the functional diversity explained at the molecular level?

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Review mation pertaining to secretion and structure of the SPATEs will not only enhance our understanding of the mode of action of these virulence proteins but will also improve our knowledge of the biogenesis of other ATs. Although this family of ATs exhibits such diverse pathogenic functions, commonality in their structural architecture and mechanism of secretion has been observed. Studying the role of these shared features could enable the design of potential universal inhibitors against all SPATEs. Targeting the conserved motifs located throughout the three domains of these proteins could prove to be an effective measure in weakening bacterial virulence. Dissecting their secretion mechanism could also aid in exploring methods that inhibit the secretion process itself, permitting the disarming of these multitasking virulent components. References 1 Stathopoulos, C. et al. (2007) Protein secretion in bacterial cells. In Bacterial Physiology: A Molecular Approach (El-Sharoud, W., ed.), pp. 129–153, Springer 2 Henderson, I.R. et al. (2004) Type V protein secretion pathway: the autotransporter story. Microbiol. Mol. Biol. Rev. 68, 692–744 3 Henderson, I.R. and Nataro, J.P. (2001) Virulence functions of autotransporter proteins. Infect. Immun. 69, 1231–1243 4 Jacob-Dubuisson, F. et al. (2004) Protein secretion through autotransporter and two-partner pathways. Biochim. Biophys. Acta 1694, 235–257 5 Kostakioti, M. et al. (2005) Mechanisms of protein export across the bacterial outer membrane. J. Bacteriol. 187, 4306–4314 6 Dautin, N. et al. (2007) Cleavage of a bacterial autotransporter by an evolutionarily convergent autocatalytic mechanism. EMBO J. 26, 1942–1952 7 Provence, D.L. and Curtiss, R., 3rd (1994) Isolation and characterization of a gene involved in hemagglutination by an avian pathogenic Escherichia coli strain. Infect. Immun. 62, 1369–1380 8 Henderson, I.R. and Nataro, J.P. (2005) Autotransporter proteins. In EcoSal- Escherichia coli and Salmonella: Cellular and Molecular Biology, ASM Press online-only – see EcoSal module 8.7.3 at (http:// www.ecosal.org/ecosal/toc/index.jsp) 9 Dutta, P.R. et al. (2002) Functional comparison of serine protease autotransporters of enterobacteriaceae. Infect. Immun. 70, 7105–7113 10 Desvaux, M. et al. (2006) The unusual extended signal peptide region of the type V secretion system is phylogenetically restricted. FEMS Microbiol. Lett. 264, 22–30 11 Rutherford, N. and Mourez, M. (2006) Surface display of proteins by gram-negative bacterial autotransporters. Microb. Cell Fact. 5, 22 12 Sijbrandi, R. et al. (2003) Signal recognition particle (SRP)-mediated targeting and Sec-dependent translocation of an extracellular Escherichia coli protein. J. Biol. Chem. 278, 4654–4659 13 Szabady, R.L. et al. (2005) An unusual signal peptide facilitates late steps in the biogenesis of a bacterial autotransporter. Proc. Natl. Acad. Sci. U. S. A. 102, 221–226 14 Desvaux, M. et al. (2007) A conserved extended signal peptide region directs posttranslational protein translocation via a novel mechanism. Microbiology 153, 59–70 15 Skillman, K.M. et al. (2005) Efficient secretion of a folded protein domain by a monomeric bacterial autotransporter. Mol. Microbiol. 58, 945–958 16 Jong, W.S. et al. (2007) Limited tolerance towards folded elements during secretion of the autotransporter Hbp. Mol. Microbiol. 63, 1524–1536 17 Brandon, L.D. and Goldberg, M.B. (2001) Periplasmic transit and disulfide bond formation of the autotransported Shigella protein IcsA. J. Bacteriol. 183, 951–958 18 Ieva, R. et al. (2008) Incorporation of a polypeptide segment into the bdomain pore during the assembly of a bacterial autotransporter. Mol. Microbiol. 67, 188–201 19 Barnard, T.J. et al. (2007) Autotransporter structure reveals intrabarrel cleavage followed by conformational changes. Nat. Struct. Mol. Biol. 14, 1214–1220

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Trends in Microbiology Vol.16 No.8 20 Purdy, G.E. et al. (2007) IcsA surface presentation in Shigella flexneri requires the periplasmic chaperones DegP, Skp, and SurA. J. Bacteriol. 189, 5566–5573 21 Walsh, N.P. et al. (2003) OMP peptide signals initiate the envelopestress response by activating DegS protease via relief of inhibition mediated by its PDZ domain. Cell 113, 61–71 22 Henderson, I.R. et al. (1998) The great escape: structure and function of the autotransporter proteins. Trends Microbiol. 6, 370–378 23 Cotter, S.E. et al. (2005) Trimeric autotransporters: a distinct subfamily of autotransporter proteins. Trends Microbiol. 13, 199–205 24 Voulhoux, R. et al. (2003) Role of a highly conserved bacterial protein in outer membrane protein assembly. Science 299, 262–265 25 Jain, S. and Goldberg, M.B. (2007) Requirement for YaeT in the outer membrane assembly of autotransporter proteins. J. Bacteriol. 189, 5393–5398 26 Oomen, C.J. et al. (2004) Structure of the translocator domain of a bacterial autotransporter. EMBO J. 23, 1257–1266 27 Kostakioti, M. and Stathopoulos, C. (2006) Role of the a-helical linker of the C-terminal translocator in the biogenesis of the serine protease subfamily of autotransporters. Infect. Immun. 74, 4961–4969 28 Navarro-Garcia, F. et al. (2001) Plasmid-encoded toxin of enteroaggregative Escherichia coli is internalized by epithelial cells. Infect. Immun. 69, 1053–1060 29 Pohlner, J. et al. (1987) Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease. Nature 325, 458–462 30 Bos, M.P. et al. (2007) Biogenesis of the Gram-negative bacterial outer membrane. Annu. Rev. Microbiol. 61, 191–214 31 Otto, B.R. et al. (2005) Crystal structure of hemoglobin protease, a heme binding autotransporter protein from pathogenic Escherichia coli. J. Biol. Chem. 280, 17339–17345 32 Emsley, P. et al. (1996) Structure of Bordetella pertussis virulence factor P.69 pertactin. Nature 381, 90–92 33 Gangwer, K.A. et al. (2007) Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain. Proc. Natl. Acad. Sci. U. S. A. 104, 16293–16298 34 Kajava, A.V. and Steven, A.C. (2006) The turn of the screw: variations of the abundant b-solenoid motif in passenger domains of Type V secretory proteins. J. Struct. Biol. 155, 306–315 35 Bachovchin, W.W. et al. (1990) Inhibition of IgA1 proteinases from Neisseria gonorrhoeae and Hemophilus influenzae by peptide prolyl boronic acids. J. Biol. Chem. 265, 3738–3743 36 Fink, D.L. et al. (2001) The Hemophilus influenzae Hap autotransporter is a chymotrypsin clan serine protease and undergoes autoproteolysis via an intermolecular mechanism. J. Biol. Chem. 276, 39492–39500 37 Oliver, D.C. et al. (2003) A conserved region within the Bordetella pertussis autotransporter BrkA is necessary for folding of its passenger domain. Mol. Microbiol. 47, 1367–1383 38 Hritonenko, V. et al. (2006) Quaternary structure of a SPATE autotransporter protein. Mol. Membr. Biol. 23, 466–474 39 Robert, V. et al. (2006) Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif. PLoS Biol. 4, e377 40 Velarde, J.J. and Nataro, J.P. (2004) Hydrophobic residues of the autotransporter EspP linker domain are important for outer membrane translocation of its passenger. J. Biol. Chem. 279, 31495–31504 41 Leyton, D.L. et al. (2007) Contribution of a novel gene, rpeA, encoding a putative autotransporter adhesin to intestinal colonization by rabbitspecific enteropathogenic Escherichia coli. Infect. Immun. 75, 4664– 4669 42 Yen, M.R. et al. (2002) Protein-translocating outer membrane porins of Gram-negative bacteria. Biochim. Biophys. Acta 1562, 6–31 43 Ochman, H. and Selander, R.K. (1984) Standard reference strains of Escherichia coli from natural populations. J. Bacteriol. 157, 690– 693 44 Parham, N.J. et al. (2005) Distribution of the serine protease autotransporters of the Enterobacteriaceae among extraintestinal clinical isolates of Escherichia coli. J. Clin. Microbiol. 43, 4076–4082 45 Huson, D.H. (1998) SplitsTree: analyzing and visualizing evolutionary data. Bioinformatics 14, 68–73 46 Leyton, D.L. et al. (2003) Transfer region of pO113 from enterohemorrhagic Escherichia coli: similarity with R64 and

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identification of a novel plasmid-encoded autotransporter, EpeA. Infect. Immun. 71, 6307–6319 Parreira, V.R. and Gyles, C.L. (2003) A novel pathogenicity island integrated adjacent to the thrW tRNA gene of avian pathogenic Escherichia coli encodes a vacuolating autotransporter toxin. Infect. Immun. 71, 5087–5096 Sandt, C.H. and Hill, C.W. (2000) Four different genes responsible for nonimmune immunoglobulin-binding activities within a single strain of Escherichia coli. Infect. Immun. 68, 2205–2214 Dutta, P.R. et al. (2003) Structure–function analysis of the enteroaggregative Escherichia coli plasmid-encoded toxin autotransporter using scanning linker mutagenesis. J. Biol. Chem. 278, 39912–39920 Coutte, L. et al. (2001) Subtilisin-like autotransporter serves as maturation protease in a bacterial secretion pathway. EMBO J. 20, 5040–5048 van Ulsen, P. et al. (2003) A neisserial autotransporter NalP modulating the processing of other autotransporters. Mol. Microbiol. 50, 1017–1030 Moreland, J.L. et al. (2005) The Molecular Biology Toolkit (MBT): a modular platform for developing molecular visualization applications. BMC Bioinformatics 6, 21 Patel, S.K. et al. (2004) Identification and molecular characterization of EatA, an autotransporter protein of enterotoxigenic Escherichia coli. Infect. Immun. 72, 1786–1794 Mellies, J.L. et al. (2001) espC pathogenicity island of enteropathogenic Escherichia coli encodes an enterotoxin. Infect. Immun. 69, 315–324 Navarro-Garcia, F. et al. (2004) The serine protease motif of EspC from enteropathogenic Escherichia coli produces epithelial damage by a mechanism different from that of Pet toxin from enteroaggregative E. coli. Infect. Immun. 72, 3609–3621 Schmidt, H. et al. (2001) Identification and characterization of a novel genomic island integrated at selC in locus of enterocyte effacementnegative, Shiga toxin-producing Escherichia coli. Infect. Immun. 69, 6863–6873 Brunder, W. et al. (1997) EspP, a novel extracellular serine protease of enterohaemorrhagic Escherichia coli O157:H7 cleaves human coagulation factor V. Mol. Microbiol. 24, 767–778

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58 Otto, B.R. et al. (1998) Characterization of a hemoglobin protease secreted by the pathogenic Escherichia coli strain EB1. J. Exp. Med. 188, 1091–1103 59 Henderson, I.R. et al. (1999) Involvement of the enteroaggregative Escherichia coli plasmid-encoded toxin in causing human intestinal damage. Infect. Immun. 67, 5338–5344 60 Navarro-Garcia, F. et al. (1999) Cytoskeletal effects induced by pet, the serine protease enterotoxin of enteroaggregative Escherichia coli. Infect. Immun. 67, 2184–2192 61 Henderson, I.R. et al. (1999) Characterization of pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli. Infect. Immun. 67, 5587–5596 62 Parham, N.J. et al. (2004) PicU, a second serine protease autotransporter of uropathogenic Escherichia coli. FEMS Microbiol. Lett. 230, 73–83 63 Djafari, S. et al. (1997) Characterization of an exported protease from Shiga toxin-producing Escherichia coli. Mol. Microbiol. 25, 771–784 64 Guyer, D.M. et al. (2002) Sat, the secreted autotransporter toxin of uropathogenic Escherichia coli, is a vacuolating cytotoxin for bladder and kidney epithelial cells. Infect. Immun. 70, 4539–4546 65 Benjelloun-Touimi, Z. et al. (1995) SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion. Mol. Microbiol. 17, 123–135 66 Kostakioti, M. and Stathopoulos, C. (2004) Functional analysis of the Tsh autotransporter from an avian pathogenic Escherichia coli strain. Infect. Immun. 72, 5548–5554 67 Miyazaki, H. et al. (1989) Characterization of the precursor of Serratia marcescens serine protease and COOH-terminal processing of the precursor during its excretion through the outer membrane of Escherichia coli. J. Bacteriol. 171, 6566–6572 68 Gonzalez, C.T. et al. (1995) Pasteurella haemolytica serotype 2 contains the gene for a noncapsular serotype 1-specific antigen. Infect. Immun. 63, 1340–1348 69 Turner, D.P. et al. (2002) Autotransported serine protease A of Neisseria meningitidis: an immunogenic, surface-exposed outer membrane, and secreted protein. Infect. Immun. 70, 4447–4461

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