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Applied and Environmental Microbiology, October 2007, p. 6351-6359, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.00920-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity{triangledown}

Jens Brockmeyer,1* Martina Bielaszewska,1 Angelika Fruth,2 Marie Luise Bonn,1 Alexander Mellmann,1 Hans-Ulrich Humpf,3 and Helge Karch1

Institute for Hygiene and the National Consulting Laboratory on Hemolytic Uremic Syndrome, University of Münster, Robert Koch Strasse 41, 48149 Münster, Germany,1 National Reference Center for Salmonella and Other Enteric Pathogens, Robert Koch Institute, Branch Wernigerode, Burgstrasse 37, 38855 Wernigerode, Germany,2 Institute for Food Chemistry, University of Münster, Correnstrasse 45, 48149 Münster, Germany3

Received 24 April 2007/ Accepted 5 August 2007


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ABSTRACT
 
We investigated the prevalence, distribution, and structure of espP in Shiga toxin-producing Escherichia coli (STEC) and assessed the secretion and proteolytic activity of the encoded autotransporter protein EspP (extracellular serine protease, plasmid encoded). espP was identified in 56 of 107 different STEC serotypes. Sequencing of a 3,747-bp region of the 3,900-bp espP gene distinguished four alleles (espP{alpha}, espPß, espP{gamma}, and espP{delta}), with 99.9%, 99.2%, 95.3%, and 95.1% homology, respectively, to espP of E. coli O157:H7 strain EDL933. The espPß, espP{gamma}, and espP{delta} genes contained unique insertions and/or clustered point mutations that enabled allele-specific PCRs; these demonstrated the presence of espP{alpha}, espPß, espP{gamma}, and espP{delta} in STEC isolates belonging to 17, 16, 15, and 8 serotypes, respectively. Among four subtypes of EspP encoded by these alleles, EspP{alpha} (produced by enterohemorrhagic E. coli [EHEC] O157:H7 and the major non-O157 EHEC serotypes) and EspP{gamma} cleaved pepsin A, human coagulation factor V, and an oligopeptide alanine-alanine-proline-leucine-para-nitroaniline, whereas EspPß and EspP{delta} either were not secreted or were proteolytically inactive. The lack of proteolysis correlated with point mutations near the active serine protease site. We conclude that espP is widely distributed among STEC strains and displays genetic heterogeneity, which can be used for subtyping and which affects EspP activity. The presence of proteolytically active EspP in EHEC serogroups O157, O26, O111, and O145, which are bona fide human pathogens, suggests that EspP might play a role as an EHEC virulence factor.


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INTRODUCTION
 
Infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) are associated with a broad spectrum of outcomes ranging from asymptomatic shedding, to uncomplicated diarrhea, hemorrhagic colitis (HC), and hemolytic-uremic syndrome (HUS) (30, 52), the leading cause of acute renal failure in children (49). A subset of highly pathogenic STEC strains which can cause severe diseases, including HC and HUS, and usually possess, besides Stx, additional virulence factors, such as intimin encoded by eae, has been classified as enterohemorrhagic E. coli (EHEC) (29, 30, 34, 40) to differentiate the strains from less-pathogenic STEC (40) The most common EHEC serotype implicated in human diseases is E. coli O157:H7 (30, 31, 52), but infections caused by other serotypes, especially O26:H11/NM (where NM is nonmotile), O91:H21, O103:H2, O111:H8/NM, O113:H21, O145:H25/H28/NM, and O157:NM, have also been identified (3, 7, 17, 20, 27, 28, 30, 41). Stxs are considered the principal virulence factors of STEC (31, 45, 52), but severe clinical outcomes have also been associated with E. coli strains that do not possess stx genes (38, 47), indicating that additional virulence factors might contribute to the pathogenesis of HUS and HC. For example, the plasmid-encoded serine protease EspP from EHEC O157:H7 was shown to cleave coagulation factor V in human plasma (8), and this effect has been proposed to contribute to the mucosal hemorrhages observed in patients with HC (8).

EspP is one of the serine protease autotransporters of Enterobacteriaceae (SPATEs) (8, 23, 24). Autotransporters are a family of proteins (24, 25) that, by definition, contain within one single molecule all components necessary for translocation through the periplasm and the outer membrane of gram-negative bacteria (12, 23, 25, 43). The extracellular transport of the biologically active passenger domain is accomplished by the type V or autotransporter secretion pathway (12, 23, 24, 25). The N-terminal signal sequence leads the autotransporter through the inner membrane via the Sec mechanism. Secretion through the outer membrane is performed by the C-terminal ß domain which forms a ß-barrel-shaped pore in the outer membrane that facilitates the transport of the passenger domain to the extracellular compartment. After transport, the passenger domains of some autotransporters including EspP are cleaved and released from the bacterial cell into the external milieu. Molecular mechanisms involved in different stages of the autotransporter process of EspP, some of which are unique for this autotransporter, have been extensively characterized (10, 42, 50, 51, 53). Whereas the domains responsible for the translocation are conserved within the SPATE family (23, 24, 35), the enzymatically active passenger domains are more heterogeneous in their amino acid sequences and putative biological functions (15, 24). A certain degree of genetic diversity has also been reported for espP genes from a limited spectrum of STEC serotypes (8, 13), but the relationship between the structure of EspP and its function has not been examined.

In this study we investigated a large collection of EHEC and STEC clinical isolates to assess the frequency and sequence of the espP gene. Furthermore, we determined if the structural differences in espP genes result in functional disorders of EspP such as altered secretion or lack of the proteolytic activity. espP structural polymorphisms also enabled us to develop allele-specific PCRs to subtype espP for diagnostic and epidemiological purposes.


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MATERIALS AND METHODS
 
Bacterial strains.
A total of 575 EHEC and STEC strains were isolated between 1990 and 2004 from epidemiologically unrelated patients with HUS (n = 198) or diarrhea (n = 377) in Germany (n = 526), Austria (n = 25), the Czech Republic (n = 16), Denmark (n = 2), France (n = 1), Italy (n = 2), and the United States (n = 3). The strains belonged to 107 different serotypes as determined by a microtiter serotyping method with antisera against E. coli O antigens 1 to 181 and H antigens 1 to 56 (44). A subset of these strains was characterized for the presence of other putative virulence and fitness genes in previous studies (4, 6, 38).

PCR.
The strains were screened for the presence of espP using PCR with primers esp-A and esp-B (8) which amplify a 1,830-bp internal fragment of the gene. For restriction fragment length polymorphism (RFLP) and nucleotide sequence analyses, a 3,760-bp region of espP, which covers 96% of the 3,900-bp espP gene of E. coli O157:H7 strain EDL933 (EMBL-GenBank accession number X97542), was amplified using the primer pair espPlong-1 and espPlong-2 (Table 1). To distinguish espP{alpha}, espPß, espP{gamma}, and espP{delta}, we developed PCRs specific for each of these alleles. PCR primers and conditions are listed in Table 1. PCRs were performed in a Biometra TGradient 96 cycler (Biometra GmbH, Göttingen, Germany) using reagents from PEQLAB Biotechnologie (Erlangen, Germany) and primers from Sigma-Genosys (Haverhill, United Kingdom). The 50-µl PCR mixture consisted of 5 µl of bacterial DNA purified with InstaGene Matrix (Bio-Rad, München, Germany), 30 pmol of each primer, a 250 µM concentration of each deoxynucleoside triphosphate, 5 µl of 10x reaction buffer Y, 10 µl of 5x Enhancer, 1.5 µl of 50 mM MgCl2, and 2 units AmpliTaq DNA polymerase. The PCR products were analyzed using agarose gel electrophoresis and ethidium bromide staining.


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  		TABLE 1. Primers and conditions used in PCRs developed in this study

espP RFLP.
Five hundred nanograms of the PCR product created with primers espPlong-1 and espPlong-2 (Table 1) was digested with 5 units of AccI, DraI, RsaI, or SspI (New England Biolabs, Frankfurt, Germany) according to the manufacturer's instructions. Restriction fragments were separated on a 1.2% (wt/vol) agarose gel and visualized by staining with ethidium bromide.

Nucleotide sequencing.
Plasmid DNA purified with NucleoBond BAC 100 cartridges (Macherey-Nagel, Düren, Germany) or the 3,760-bp espP PCR product (Table 1) purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (GE Healthcare, München, Germany) was used as a template for bidirectional sequencing with an automated ABI Prism 3130 Avant Genetic Analyzer (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany), ABI Prism BigDye Terminator Ready Reaction Cycle Sequencing kit (Perkin-Elmer Applied Biosystems), and customized primers. Sequences were analyzed using MEGA software, version 3.1 (32), and aligned with ClustalW (32), and homology was determined using the EMBL-GenBank database (http://www.ncbi.nlm.nih.gov/BLAST).

Phylogenetic analysis.
The phylogenetic structure of espP genes was analyzed with the Splits Tree program, version 4.6, using the split decomposition algorithm (1, 26). The espP sequence from E. coli O157:H7 strain EDL933 (EMBL-GenBank accession number X97542) was included for comparison.

Purification of EspP.
Strains were grown overnight in 50 ml of Luria-Bertani broth at 37°C with vigorous shaking. The cultures were centrifuged (6,000 x g for 30 min at 4°C), supernatants were passed through a 0.20-µm-pore-size filter (Corning Inc., Corning, NY), and proteins were precipitated (1 h at 4°C) by adding ammonium sulfate (Merck, Darmstadt, Germany) to 55% saturation. The precipitate was collected by centrifugation (6,000 x g for 30 min at 4°C), and the pellet was dissolved in 500 µl of 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer containing 150 mM NaCl (pH 7.4). EspP was purified using HiTrap Benzamidine FF columns (GE Healthcare) according to the manufacturer's instructions. The fractions enriched for EspP were collected and concentrated using a 10-kDa Vivaspin spin-down filter (Vivascience-Sartorius, Göttingen, Germany).

SDS-PAGE and immunoblotting.
EspP preparations were separated using a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel in a Mini-Protean II dual slab cell (Bio-Rad) (33) and either stained with Coomassie blue or transferred to a nitrocellulose membrane (Protran; Schleicher and Schuell, Dassel, Germany) using a Mini-Trans-Blot cell (Bio-Rad). EspP was identified by immunoblot analysis using rabbit anti-EspP antibody (8) (diluted 1:1,000) and a horseradish peroxidase-conjugated goat anti-rabbit antibody (diluted 1:40,000; Dianova, Hamburg, Germany). Bound antibodies were visualized by incubation (5 min) with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL), followed by the detection of the chemiluminescence signal using ChemiDoc XRS imaging system (Bio-Rad). The detection limit of the assay was ≥50 ng of protein.

Cleavage of pepsin and coagulation factor V.
To determine the proteolytic activity of EspP, 20 µg of porcine pepsin A (Roche Diagnostics, Mannheim, Germany) or 8 µg of human coagulation factor V (Merck Biosciences, Schwalbach, Germany) was incubated (15 h at 37°C) with 1 µg of purified EspP in 25 µl of 10 mM HEPES buffer containing 150 mM NaCl. Samples were separated on a 12% SDS-PAGE gel, and reaction products were stained with Coomassie blue. The EspP-producing clone DH5{alpha}(pB9-5), which contains espP from E. coli O157:H7 EDL933 (8), and the vector control strain DH5{alpha}(pK18) without the espP insert (8) were used as the positive and negative control, respectively.

Cleavage of an oligopeptide substrate.
One microgram of purified EspP was incubated (15 h at 37°C) with a 2 mM solution of the para-nitroaniline-conjugated oligopeptide substrate alanine-alanine-proline-leucine (Ala-Ala-Pro-Leu-pNA) (Bachem, Weil am Rhein, Germany) in 10 mM HEPES buffer with 150 mM NaCl. Reactions were performed in 96-well microtiter plates in total volumes of 100 µl. The absorbance was determined at 0 min and 15 h at 405 nm using a microplate reader (Dynex Technologies, Chantilly, VA). Samples were defined as proteolytically active when the difference of the absorbance readings was ≥0.03 units. Positive and negative controls were as described above.

Nucleotide sequence accession numbers.
Nucleotide sequences of espP alleles have been deposited in the EMBL-GenBank database under accession numbers AM691835 to AM691848 (Table 2).


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  		TABLE 2. Characteristics of espP alleles, encoded EspP subtypes, and accession numbers of espP sequences determined in this study


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RESULTS
 
Frequency and distribution of espP among STEC clinical isolates.
A total of 575 STEC clinical isolates belonging to 107 serotypes were screened by PCR for the presence of espP using primers esp-A and esp-B (8). espP was found in 360 (62.6%) strains (298 eae-positive and 62 eae-negative strains), which belonged to 56 serotypes (Table 3). Only strains of serotype O157:H7 uniformly (81 of 81) contained espP. In serotypes O26:H11/NM, O111:H8/NM, and O145:H25/H28/NM, where at least 20 isolates were examined, 62.2%, 4.7%, and 88.3% of strains, respectively, were espP positive. For 51 serotypes, only espP-negative strains (n = 117) were detected; 58 of these strains contained eae and 59 were eae negative. The espP-negative, eae-positive strains belonged to serotypes O8:H2, O11:H2, O55:H6, O55:H7 (2 strains), O103:H2 (25 strains), O103:H18 (3 strains), O103:H21, O103:NM (2 strains), O119:H2, O121:H10, and O157:NM (20 strains, all sorbitol-fermenting). The strains that were negative for both espP and eae were of serotypes O3:H2, O3:H10, O6:H10, O8:H10 (2 strains), O8:HNT (2 strains; HNT is H antigen nontypeable), O31:NM, O40:H8 (4 strains), O62:NM (2 strains), O70:H35, O73:H18, O75:H8 (2 strains), O76:H19 (2 strains), O91:H4, O92:HNT, O100:NM, O112:H8, O112:NM (2 strains), O113:H4 (4 strains), O117:H7, O125:H4, O128:H2, O136:HNT, O138:H8, O146:H20, O146:H21 (7 strains), O146:H28 (2 strains), O146:NM, O152:H4, O158:H18, O165:NM, O174:H8, O174:H21, ONT:H2 (ONT is O antigen nontypeable), ONT:H8, ONT:H12, ONT:H19, ONT:H32, Orough:H2 (where Orough indicates autoagglutinable), Orough:H21, and Orough:HNT.


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  		TABLE 3. espP alleles and biological activities of the encoded EspP subtypes in STEC strains isolated from humans

RFLP analysis of espP.
To gain insight into structural differences of espP in STEC strains of different serotypes, 3,760 bp of the 3,900-bp espP gene were amplified in 98 representative strains of 56 espP-positive serotypes (Table 3) and digested with AccI, DraI, RsaI, or SspI. All 98 strains produced an amplicon of the expected size, indicating that none of them contained espP with large insertions or deletions. Digestion with AccI or DraI resulted in no differences in the espP restriction patterns. In contrast, restriction with RsaI and SspI demonstrated two distinct RFLP patterns, R1 and R2 with RsaI and S1 and S2 with SspI (Fig. 1, lanes 1 to 4). These patterns occurred in three different combinations among the strains investigated. Seventy-one strains (33 serotypes), 19 strains (15 serotypes), and 8 strains (8 serotypes) displayed the combinations R1-S1, R2-S2, and R1-S2, respectively (Table 3), suggesting the presence of at least three different espP alleles.


Figure 1
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  		FIG. 1. RFLP analysis of espP. PCR products of 3,760 bp obtained with primers espPlong-1 and espPlong-2 were digested with RsaI or SspI and separated using agarose gel electrophoresis. Lanes 1 and 2, RsaI restriction patterns R1 and R2, respectively; lanes 3 and 4, SspI restriction patterns S1 and S2, respectively; M, molecular weight marker (100-bp ladder; PEQLAB Biotechnologie).

Biological activity of EspP.
To determine if STEC strains displaying different RFLP patterns produce active EspP, we sought immunoreactive EspP in supernatants of overnight cultures using immunoblotting with an EspP-specific antibody and tested the proteolytic activity of the secreted protein based on its ability to cleave porcine pepsin A and the oligopeptide substrate Ala-Ala-Pro-Leu-pNA. In all 19 strains with espP RFLP pattern R2-S2, EspP was both secreted into the supernatant and proteolytic (Table 3). In contrast, EspP in the eight strains with the espP RFLP pattern R1-S2 was either not secreted (n = 2) or secreted but inactive against the substrates tested (n = 6) (Table 3). We distinguished two groups among 71 strains with the R1-S1 espP RFLP pattern. In 52 strains of 17 serotypes, EspP was transported into the supernatant and cleaved pepsin A and Ala-Ala-Pro-Leu-pNA; in the remaining 19 strains of 16 serotypes, EspP was either not secreted (5 strains) or was not proteolytic (14 strains) (Table 3). Samples displaying negative results in either proteolytic activity or secretion were repeated at least twice to confirm the findings.

espP alleles identified by nucleotide sequencing.
To gain insight into the functional differences of EspP proteins, we sequenced the 3,760-bp espP amplicons from 11 strains that displayed different RFLP patterns and/or varied in EspP autotransporter or proteolytic activity, and we identified four alleles (espP{alpha}, espPß, espP{gamma}, and espP{delta}) (Table 2 and Fig. 2).


Figure 2
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  		FIG. 2. Nucleotide sequence differences in espP{alpha}, espPß, espP{gamma}, and espP{delta} alleles compared to espP from E. coli O157:H7 strain EDL 933 (EMBL-GenBank accession number X97542). The gray bars represent the analyzed 3,747-bp sequences of espP. Synonymous point mutations are depicted by white pins, and nonsynonymous point mutations are indicated by black pins. The espI fragment introduced into espP by a putative recombination is depicted by a black bar. The black vertical arrows indicate the two nonsynonymous point mutations which may be involved in the lack of proteolytic activity. The black arrowhead indicates the unique point mutation in the linker domain of strain 89/04. Positions of PCR primers to identify the respective espP alleles are indicated by horizontal arrows, and sizes of the corresponding amplicons are indicated below the amplicon lines. The size scale and relevant functional regions of espP are indicated at the bottom of the graph.

(i) espP{alpha}.
The espP{alpha} allele in serotypes O157:H7, O26:H11, O111:NM, and O145:NM produced uniformly proteolytic EspP (Table 2) and was highly (99.9%) homologous to espP of E. coli O157:H7 strain EDL933 (EMBL-GenBank accession number X97542), with five or fewer point mutations (Fig. 2). Two point mutations in the espP gene of EHEC O157:H7 strain 4805/00 resulted in two amino acid exchanges in the encoded protein, whereas all the other mutations were synonymous.

(ii) espPß.
The espPß allele in strains of serotypes O7:H18, O163:H19, and O174:H2 in which EspP either was not secreted or was proteolytically inactive (Table 2) contained up to 30 point mutations compared to espP of EDL933 (Fig. 2) (99.2% nucleotide sequence homology). The nonsynonymous point mutations in nucleotide positions 938 and 948 which led to the exchange of tyrosine at position 313 to phenylalanine (Y313F) and of asparagine at position 316 to lysine (N316K), respectively, are located in the proximity of the serine protease active site (Fig. 2) and may be thus responsible for the lack of proteolytic activity. Strain 89/04 in which no extracellular secretion of EspP was observed (Table 2) differed from the other two strains that did secrete EspP (Table 2, strains 36/03 and 107/04) by a unique nonsynonymous point mutation in the putative linker domain (Fig. 2), which leads to the amino acid exchange from arginine to glutamine at position 1005 (R1005Q); this point mutation might hinder EspP autotransport.

(iii) espP{gamma}.
The espP{gamma} allele in strains of serotypes O77:H18 and O127:HNT, which secrete proteolytically active EspP (Table 2), demonstrates 95.3% nucleotide sequence homology to espP of EDL933. This allele is characterized by a 200-bp DNA fragment between positions 2722 and 2921 which is 95.5% homologous to the espI gene which encodes another E. coli serine protease, EspI, and has been recently identified within the pathogenicity island "locus for proteolysis and adherence" of an eae-negative STEC strains (48). A 172-bp core region of this fragment (position 2736 to 2907) shows homology only to espI, whereas the flanks display at least 93% homology to both espP and espI, suggesting that this fragment was introduced into the gene by recombination. The integrated DNA fragment of espI is three nucleotides shorter than the replaced espP fragment, resulting in a 3-bp shorter espP. In addition, espP{gamma} has up to 27 point mutations compared to espP of strain EDL933 (Fig. 2), and two of these are unique for espP{gamma} whereas the others are also present in espPß and/or espP{delta}.

(iv) espP{delta}.
The espP{delta} allele in strains of serotypes O84:H4 and O156:NM (Table 2) is least homologous to espP of EDL933 (95.1%). This allele combines features of espP{gamma} and espPß by containing the 200-bp insert of espI and the two nonsynonymous point mutations in the proximity of the serine protease active site (positions 938 and 948) (Fig. 2). Accordingly, similar to espPß, espP{delta} encodes EspP that is not proteolytic (Table 2). Furthermore, the espP{delta} allele displays six unique point mutations (Fig. 2).

Development of espP allele-specific PCRs.
We exploited the espP sequence polymorphism to develop a set of PCRs that distinguish the four espP alleles (Table 1 and Fig. 2). The specificity of these PCRs was evaluated by applying them to the 11 sequenced strains (Table 2). As expected, strains harboring espP{delta}, which shares with espP{gamma} the espI insert (Fig. 2), were positive in PCRs targeting espP{gamma} as well as in the espP{delta}-specific PCR.

Identification of espP alleles in STEC clinical isolates.
We further applied the espP allele-specific PCRs to determine the distribution of the four espP alleles among the 98 representative espP-positive STEC isolates of 56 serotypes (Table 3). espP{alpha}, espPß, espP{gamma}, and espP{delta} alleles were found in strains of 17, 16, 15, and 8 serotypes, respectively (Table 3). In all 46 strains determined by PCR to contain espPß, espP{gamma}, or espP{delta}, the specificity of the PCRs was confirmed by sequencing the fragments of the espP genes typical for each allele (Fig. 2). All 19 strains that were positive in the espPß-specific PCR displayed the two nonsynonymous mutations associated with the absence of proteolytic activity but not the espI insert. All 19 strains positive in the espP{gamma}-specific PCR contained the espI insert; and all 8 strains positive in the espP{delta}-specific PCR harbored both the two nonsynonymous point mutations present in espPß and the espI insert (data not shown). Similar to the sequenced strains harboring the respective espP alleles (Table 2), the presence of espP{alpha} and espP{gamma} in these 98 clinical isolates completely correlated with the production of extracellularly secreted and proteolytically active EspP{alpha} and EspP{gamma}, respectively, whereas espPß and espP{delta} regularly encoded EspP subtypes (EspPß and EspP{delta}, respectively) which lacked the capacity for autotransporter or proteolytic activity (Table 3).

Cleavage of human coagulation factor V.
Representative preparations of EspP{alpha} (from strain 18/03, serotype O145:NM), EspPß (strain 107/04, serotype O174:H2), EspP{gamma} (strain 100/04, serotype O127:HNT), and EspP{delta} (strain 4795/97, serotype O84:H4) (Table 2) were tested for their proteolytic activity against human coagulation factor V, which may be relevant to the pathogenesis of human disease. Similar to pepsin A and the oligopeptide Ala-Ala-Pro-Leu-pNA, factor V was cleaved by EspP{alpha} and EspP{gamma} but not by EspPß and EspP{delta} (data not shown).

Phylogenetic analysis.
To confirm the finding of different espP alleles, we investigated the phylogenetic distribution of the espP sequences (Table 2). The sequence comparison using split decomposition yielded an unrooted dendrogram, suggesting four distinct espP alleles, as indicated by the main edges of the phylogenetic network (Fig. 3). The role of recombination in the emergence of the different espP alleles is supported by boxes in the phylogram.


Figure 3
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  		FIG. 3. Splits Tree dendrogram of the espP{alpha}, espPß, espP{gamma}, and espP{delta} sequences and the corresponding sequence of espP from E. coli O157:H7 strain EDL933 (EMBL-GenBank accession number X97542). The four distinct edges of the dendrogram indicate the different espP alleles. The boxes in the dendrogram indicate recombination events between the analyzed sequences.


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DISCUSSION
 
Structural and functional properties of autotransporters have been intensively studied (15, 23, 25) for several reasons. First, because the autotransporter secretion pathway has been discovered only recently, new members of this family have been emerging (25, 35, 37). Second, the members of the SPATE subfamily have been, with some exceptions (22), detected only in pathogenic organisms (24, 25), suggesting that they might be virulence factors (24). This is supported by the fact that the SPATE proteins are the most abundant proteins secreted by their producers (15, 24, 25). Third, despite their homology, SPATE proteins demonstrate distinct substrate specificities, suggesting diverse pathogenetic functions (15). Indeed, it has been shown that these proteins can act as adhesins, proteases, enterotoxins, or cytotoxins (8, 13, 15, 23, 24, 35, 37, 39). However, the functional studies of the SPATE proteins have usually been performed with limited numbers of prototype strains or with clones harboring the SPATE genes (8, 9, 15, 25, 37, 39). Therefore, we used a large collection of wild-type STEC strains from patients to investigate to what extent genetic diversity occurs in espP genes and how the structural variations affect the functional properties of the encoded proteins.

Using nucleotide sequencing, we identified four different espP alleles that encode four EspP subtypes. EspP{alpha} and EspP{gamma} are secreted and proteolytic, while EspPß and EspP{delta} are impaired in their autotransporter or proteolytic activity. Our findings demonstrate that mutations within espP can affect the properties of the encoded protein. We are currently performing mutagenesis experiments to determine the role of the two nonsynonymous point mutations which result in the two amino acid exchanges near the serine protease motifs in EspPß and EspP{delta} in the absence of proteolytic activity of these EspP subtypes. The same approach is being applied to determine if there is an association between the unique point mutation within the linker domain of strain 89/04 and the lack of the extracellular transport of Espß from this strain (Table 2). This possibility is supported by the observation by Velarde et al. (53), who demonstrated that particular mutations in the EspP linker domain led to a significant reduction of the extracellular secretion of the protein. Interestingly, EspP{gamma} encoded by the espP{gamma} allele in which 200 bp of the linker domain has been replaced by a fragment of the espI gene is active in both autotransporter and proteolytic activity. This suggests that the fragment of espI introduced into the espP linker domain, probably by recombination, can complement the function of the linker domain of EspP. This observation is in accordance with the concept of domain shuffling, which has been implicated in the evolution of other autotransporters (11, 36). A recombination event is also likely to have been involved in the evolution of espP{delta} which is, schematically, in the 5' half of its sequence highly homologous to espPß, whereas the 3' half of the sequence is highly homologous to espP{gamma} (Fig. 2). These observations support the importance of recombinations as one of the leading mechanisms in the evolution of the E. coli genome (14) and suggest that additional espP alleles in STEC strains can emerge in future studies.

Polymorphism within espP enabled us to develop PCRs to distinguish espP{alpha}, espPß, espP{gamma}, and espP{delta}. Within serotypes where multiple strains were analyzed, all contained the same espP allele, suggesting that the observed espP recombinations are not frequent enough to affect the overall clonal evolution of this gene within serotype lineages. The scheme developed in this study for espP subtyping using allele-specific PCRs might be useful for several purposes. First, identification of the espP allele harbored by an STEC strain allows the prediction of activity of the encoded EspP protein and forms the basis for investigations of EspP as a virulence factor. The determination of the mere presence of espP (5), without further subtyping of the gene, does not allow a reliable assessment of the potential contribution of EspP to human disease because a subset of espP genes present in STEC strains encode EspP, which is biologically inactive, at least in vitro. Second, subtyping of espP genes identifies EspP subtypes that are secreted but are not proteolytic; such EspP proteins might be useful as vaccines to elicit the development of specific anti-EspP antibodies which would protect against biological effects of EspP during infection. Third, the espP subtyping scheme may be useful to investigate the presence and distribution of espP alleles, which we identified in human STEC isolates, in STEC strains from different populations of animals. This would allow the determination of species-specific distributions of espP alleles and would help to identify those species that are reservoirs of STEC encoding the biologically active EspP subtypes.

The secretion of proteolytically active EspP{alpha} by EHEC of serotypes O157:H7, O26:H11/NM, O111:H8/NM, and O145:H25/H28/NM, which cause severe human diseases (7, 17, 20, 30, 52), suggests that EspP might contribute to pathogenesis. Specifically, three biological effects of EspP demonstrated in vitro (8, 13, 19) might be relevant. First, the ability of EspP{alpha} to cleave (and thereby inactivate) human coagulation factor V (8; also this study), which is involved in coagulation, might be implicated in the pathogenesis of bloody diarrhea. This syndrome, which occurs either as the sole manifestation of EHEC infection (21) or as a prodromal symptom of HUS (21, 30, 52), is histopathologically characterized by mucosal hemorrhages in the large intestine (21); the interference of EspP with the coagulation cascade might contribute to this extensive mucosal bleeding. Second, the EspP homologue PssA which was identified in an EHEC O26:NM strain (13) is cytotoxic to Vero cells, opens cell junctions, and leads to cell detachment and death (13). If EspP exerts a similar effect on intestinal epithelial cells during infection, it might contribute to the mucosal erosions observed in patients with HC (21) or could be involved in the diarrhea by reducing the absorption capacity of the intestinal epithelium. Third, recent studies provide evidence that EspP mediates intestinal colonization of E. coli O157:H7 in a calf model and adherence to primary bovine intestinal epithelial cells (16). Mutation of espP impaired the adherence significantly but adherence could be restored after addition of exogenous EspP, supporting the role of EspP in the adherence. Similarly, the presence of espP in a bovine isolate of serogroup O78 has been associated with a strong adhesive capacity of this strain to the bovine epithelial MDBK cell line (19). The putative involvement of EspP in intestinal adherence is supported by numerous reports that other members of the autotransporter family also mediate or contribute to bacterial colonization of mucosal surfaces (2, 18, 24). The expression of EspP during human infection is demonstrated by the presence of antibodies against EspP in sera of patients who recovered from HUS (8, 13). However, it should be noted that the proteolytic cleavage of human coagulation factor V which has been shown in vitro is only one putative effect of EspP that may contribute to the pathogenesis of EHEC infections. In fact, the full spectrum of biological functions of EspP during infection is not known and might be broader than proteolysis, analogously to the functional diversity observed in other autotransporters (24). As with many STEC loci, it is not possible to assign with certainty a pathogenic role to the encoded molecules. It is likely that there are a variety of pathways of host injury and multifaceted proteins with diverse functions; animal models have been limited in their ability to recapitulate human infection. Prospective studies, including subtyping of espP genes in clinical STEC isolates, are necessary to evaluate, on an allele-by-allele basis, the associations of EspP with human disease and to gain insight into the roles of different EspP subtypes in the virulence of STEC.

The role of EspP in the pathogenicity of STEC strains is also indirectly supported by a recent study of a panel of autotransporter-encoding genes, including espP, in a large collection of diarrheagenic, extraintestinal, and commensal E. coli isolates from humans and avian pathogenic E. coli. espP was significantly associated with diarrheagenic E. coli and was unique to STEC strains, in contrast to enteropathogenic and enterotoxigenic E. coli (46).

In conclusion, espP is widely distributed among STEC strains and occurs in at least four alleles that encode functionally diverse EspP proteins. Subtyping of espP in STEC isolates from patients and the environment provides a tool for further investigation of the role of EspP as a putative virulence factor of STEC strains.


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ACKNOWLEDGMENTS
 
This study was supported by Deutsche Forschungsgemeinschaft Sonderforschungsbereich grant SFB293 (project A18).

We thank Phillip I. Tarr (Washington University School of Medicine, St. Louis, MO) for fruitful and extensive discussions of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Hygiene, Universität Münster, Robert Koch Str. 41, 48149 Münster, Germany. Phone: 49 251 980 2822. Fax: 49 251 980 2868. E-mail: jens.brockmeyer{at}ukmuenster.de Back

{triangledown} Published ahead of print on 17 August 2007. Back


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Applied and Environmental Microbiology, October 2007, p. 6351-6359, Vol. 73, No. 20
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