IAI Accepts, published online ahead of print on 5 November 2007 Infect. Immun. doi:10.1128/IAI.00684-07 Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 NleE/OspZ family of effector proteins is required for PMN 2 transepithelial migration, a characteristic shared by 3 enteropathogenic Escherichia coli and Shigella flexneri 4 infections D E 5 T P 6 Daniel V. Zurawski1, Karen L. Mumy2, Luminita Badea3, Julia A. Prentice3, Elizabeth L. 7 Hartland3,4, Beth A. McCormick2, and Anthony T. Maurelli1* 8 9 E C 1 Department of Microbiology and Immunology, Uniformed Services University of the 10 Health Sciences, Bethesda, Maryland 11 2 Mucosal Immunology Laboratory, Massachusetts General Hospital, Charlestown; and 12 Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, 13 Massachusetts 14 3 Department of Microbiology, Monash University, Victoria, Australia. 15 4 Department of Microbiology and Immunology, University of Melbourne, Victoria, 16 Australia. C A 17 18 * Corresponding author. Address: Department of Microbiology and Immunology, 19 4301 Jones Bridge Rd., Bethesda, MD 20814-4799 Email: amaurelli@usuhs.mil 20 Telephone: 301-295-3415, Fax: 301-295-1545 21 22 Running Title: OspZ and NleE are required for PMN migration 23 Abstract 24 Enteropathogenic Escherichia coli (EPEC) and Shigella flexneri are human host-specific 25 pathogens that infect intestinal epithelial cells. However, each bacterial species employs 26 a different infection strategy within this environmental niche. EPEC attach to the apical 27 surface of small intestine enterocytes, causing microvillus effacement and rearrangement 28 of the host cell cytoskeleton beneath adherent bacteria. In contrast, S. flexneri invades 29 large intestine epithelium at the basolateral membrane, replicates, and spreads cell-to- 30 cell. Both EPEC and S. flexneri rely on type III secretion systems (T3SS) to secrete 31 effectors into host cells, and both pathogens recruit polymorphonuclear leukocytes 32 (PMNs) from the submucosa to the lumen of the intestine. In this report, we compared 33 the virulence functions of the EPEC T3SS effector NleE and the homologous Shigella 34 protein, Orf212. 35 renamed this protein OspZ. Infection of polarized T84 intestinal epithelial cells with an 36 ospZ deletion mutant of S. flexneri resulted in reduced PMN transepithelial migration 37 when compared to infection by wild-type. An nleE deletion mutant of EPEC showed a 38 similar reduction of PMN migration. The ability to induce PMN migration was restored 39 in both mutants when either ospZ or nleE was expressed from a plasmid. An infection of 40 T84 cells with the ospZ mutant resulted in reduced ERK phosphorylation and NF-ΚB 41 activation compared to infection by wild-type. Therefore, we conclude that OspZ and 42 NleE share a similar role in the upstream induction of host signaling pathways required 43 for PMN transepithelial migration in Shigella and EPEC infections. D E T P E C We discovered Orf212 was secreted by the S. flexneri T3SS and C A 2 44 Introduction 45 Shigella species and enteropathogenic E. coli (EPEC) are Gram-negative bacterial 46 pathogens that mediate infections in the human intestine often causing life-threatening 47 diarrhea in immunocompromised individuals (1, 3, 12, 20, 22, 25). Those most at risk 48 are children in under-developed nations where contamination of food and water is 49 common (1, 25). Remarkable progress has been made to identify virulence determinants 50 required to mediate the pathogenesis of both species of bacteria. However, there are still 51 aspects of virulence that are not fully understood, efficacious vaccines have not been 52 produced, and antibiotic resistance is increasing across all diarrhoeagenic E. coli and 53 Shigella species (41). D E T P E C 54 While infections caused by these enteric pathogens cause similar symptoms, they 55 exhibit different pathogenesis strategies. Shigella flexneri only invades colonic epithelial 56 cells at the basolateral membrane, which the bacteria may reach by transcytosis through 57 M cells and tight junction modification (44, 45). Alternatively, S. flexneri may reach the 58 basolateral membrane through the activation of polymorphonuclear leukocyte (PMN) 59 migration. Secreted proteins and lipopolysaccharide (LPS) activate host cell signaling 60 pathways, including the extracellular-related kinase (ERK1/2) pathway, leading to the 61 production of molecules that attract PMNs from the bloodstream to the lumen of the large 62 intestine (24, 31, 39, 40, 55). Then, Shigella pass through the gaps created between 63 epithelial cells by the migrating PMNs to gain access to the submucosa (39, 45). C A 64 In contrast to S. flexneri, EPEC attaches intimately to the apical surface of the 65 intestinal epithelium where the bacteria induce characteristic attaching and effacing (A/E) 66 lesions (20, 22). EPEC remains extracellular and recruits focal adhesion proteins and 3 67 other proteins required for the actin polymerization which forms a pedestal on the surface 68 of the cell (20, 22). Despite these differences, both S. flexneri and EPEC recruit PMN to 69 the lumen from the submucosa, a process which requires attachment and IL-8 secretion in 70 EPEC infections (49). 71 infections in vivo requires the activation of the ERK1/2 pathway and IL-8 secretion (24, 72 33). EPEC infections also activate the ERK1/2 pathway and IL-8 secretion (49), but this 73 activity has not been linked to PMN transepithelial migration yet. 74 transmigration of PMNs across the intestinal epithelium is a pathogenesis trait shared by 75 both S. flexneri and EPEC infections (31, 39, 49). PMN transepithelial migration associated with S. flexneri D E Regardless, the T P E C 76 Both S. flexneri and EPEC also use the type three secretion system (T3SS) to 77 secrete effector proteins into the host cell to manipulate the environment to one that 78 fosters replication and spread of the bacteria (5, 11, 20, 32, 46). NleE is a T3SS effector 79 discovered in Citrobacter rodentium that is encoded in a two gene operon downstream of 80 nleB (8, 21) and has homologues in Shigella and A/E E. coli species (53). Recently, it 81 was shown that infection of mice with an nleE mutant of C. rodentium results in a 82 reduction of both bacterial load and colonic hyperplasia suggesting that nleE plays a role 83 in colonization and disease (53). In this study, we characterized the NleE homologue, 84 Orf212, from S. flexneri. orf212 is encoded on the virulence plasmid of S. flexneri and 85 other Shigella species, and is found between two sets of IS elements. We discovered that 86 Orf212 was secreted by S. flexneri in a T3SS-dependent manner. A deletion mutation of 87 orf212 indicated that the gene was dispensable for invasion and cell-to-cell spread, but 88 was required for effective transepithelial migration of PMNs in an in vitro assay. 89 Interestingly, we discovered that nleE was required for EPEC-induced PMN migration C A 4 90 and that either nleE or orf212 could complement the EPEC and S. flexneri deletion 91 mutations across species. Therefore, we renamed orf212, ospZ because it is the last open 92 reading frame (orf) (#212 of 212 orfs) that was discovered on the virulence plasmid (5) 93 and because it shares a function similar to other Osp proteins (55). D E T P E C C A 5 94 Materials and Methods 95 Bacterial strains and growth conditions. All E. coli and S. flexneri strains used in this 96 study are listed in Table 1. E. coli and S. flexneri were cultured in Luria broth (LB) or in 97 tryptic soy broth (TSB), both at 37°C with aeration. Antibiotics were used in growth 98 media and plates when required for selection at the following concentrations: kanamycin, 99 50 µg/ml; chloramphenicol, 25 µg/ml; spectinomycin, 100 µg/ml; streptomycin, 25 100 D E µg/ml; and ampicillin, 200 µg/ml. T P 101 102 Tissue culture. HeLa cells and L2 mouse fibroblast cells were cultured in Dulbecco's 103 modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). 104 The human epithelial colon cancer-derived cell line T84 (passages 46 to 66) was 105 maintained in DMEM/F-12 supplemented with 15 mM HEPES (pH 7.5) and 10% FBS. 106 To obtain polarized monolayers, T84 cells were grown on 0.33 cm2 (PMN migration) or 107 4.7 cm2 (harvesting, immunoblotting) collagen-coated permeable polycarbonate filters 108 (Costar) as previously described (24, 31), and were utilized after they reached a confluent 109 and differentiated state. All tissue culture media were purchased from Invitrogen, and all 110 cell lines were maintained in the presence of 5% CO2 at 37°C. E C C A 111 112 Plasmid construction. All plasmids used in this study are described in Table 1. ospZ 113 from S. flexneri 2457T was amplified by PCR using Taq (Qiagen) polymerase with 114 upstream (5’-GGTACCCACATATAAAACGTCTTTATTG-3’) and downstream (5’- 115 GGATCCATAGACTTTAATCTCTGGCGA-3’) 6 primers, cloned into pGEM-T 116 (Promega), and sequenced. Subsequently, ospZ was subcloned into pDZ2 and EGFP-C1 117 using KpnI and BamHI restriction sites. 118 constructs for cross-species complementation, both nleE and ospZ were cloned into 119 pBAD24. 120 CCATGGATGATTAGTCCCATCAAG 121 AAGCTTATAGACTTTAATCTC-3’). nleE was amplified from EPEC with primers 122 upstream (5’-CCATGGATGATTAATCCTGTTACTAAT–3’) and downstream (5’- 123 AAGCTTCGAATTCTCCTCAATTTTAGAAAGTTT– 3’). Both genes were cut with 124 NcoI/HindIII restriction enzymes and ligated into pBAD24 cut with the same enzymes. 125 pTrcNleE-2HA plasmid was made by amplifying nleE using upstream (5’- 126 CATGCCATGGTTAATCCTGTTAC-3’) 127 CGGAATTCCTACGCATAATCCGGCACATCATACGGATACGCATAATCCGGCA 128 CATCATACGGATACTCAATTTTAGAAAG-3’) cloned into the pTrc99A vector. The 129 pTrcOspZ-2HA was generated by cutting an intermeadiate OspZ-2HA from pBAD24 130 using NcoI and HindIII and ligating into pTrc99A cut with the same restriction enzymes. 131 Lastly, 132 TTAATCCTGTTACTA-3’) and downstream primers (5’-TCCCCGCGGCTCAATTTT 133 AGAAAG-3’), and cloned into pEGFP-C1 to generate an N-terminal GFP-fusion. was ospZ In order to generate arabinose-inducible amplified with -3’) and C A nleE was amplified using upstream downstream upstream (5’- D E downstream T P E C and primers primers (5’- (5’- (5'-AAGAATTCATGA 134 135 Mutant construction. 136 allelic exchange using a modification of the method of Datsenko and Wanner (7, 35) 137 previously described (55). Primers were used to amplify the chloramphenicol resistance 138 cassette (cat) with sequences at the 5' and 3' ends identical to sequences 20 bp internal to The ∆ospZ deletion mutant of S. flexneri was generated by 7 139 and 30 bp upstream (5’ - TGTTTATATTTGAGTATAGAGATTAAAAATG 140 ATTAGTCCCATCAAGAATTGTGTAGGCTGGAGCTGCTTC – 3’) and downstream 141 (5’ - CATTTCACGAGCAATAATAATCTCAGATTTAATAGACTTTAATCTCTGG 142 CATATGAATACCTCCTTAGTTCC – 3’) of ospZ. After transformation of BS766 and 143 recombination, bacteria were plated on TSB plates containing Congo red and 144 chloramphenicol at a concentration of 5 µg/ml. 145 purified and screened by PCR using three different primer sets to identify the ∆ospZ 146 deletion mutant. To remove cat from BS819, this strain was transformed with pCP20 and 147 incubated at 42°C (7) to generate BS821. The EPEC ∆nleE mutant was also generated 148 by the method of Datsenko and Wanner (7, 35) using pKD3 as a template and the primer 149 pairs 5’ - GCGTGTCCCCTATAAATACTAAATATGCTGAACATGTGGTGAAAA 150 ATATTTACCTGTGTAGGCTGGAGCTGCTTC - 3’ and 5’ - CAATTTTAGAAAGTT 151 TATTATTTATGTATTTCATATAACTGTCTATTTCCCCAGGCCATATGAATATCC 152 TCCTTA - 3’. Lastly, we verified that all mutations created in S. flexneri or EPEC did 153 not have an effect on growth in LB. D E Cmr colonies on these plates were T P E C 154 C A 155 Secretion assays. Secretion of T3SS effector proteins from S. flexneri was analyzed as 156 previously described (55). Briefly, bacterial cultures were subcultured in LB and grown 157 at 37°C. Once late log phase was reached, bacterial samples were normalized to the same 158 OD600, and Congo red (7 µg/ml) was added. After 1 h, whole-cell lysates and supernatant 159 fractions were prepared. For whole cell preparations 1 ml of culture was centrifuged and 160 resuspended in 100 l of sodium dodecyl sulfate-polyacrylamide gel electrophoresis 8 161 (SDS-PAGE) sample buffer. Supernatant fractions were prepared by trichloroacetic acid 162 (TCA) precipitation of 25 ml of culture passed through a 0.2 micron filter to ensure the 163 removal of bacteria. Secretion of T3SS effectors from EPEC was analyzed as previously 164 described (54). Briefly, overnight cultures were washed, subcultured in DMEM, grown 165 at 37°C for 5 hr, and whole cell and supernatant fractions were prepared as above. D E 166 T P 167 Polyacrylamide gel electrophoresis and immunoblot analysis of proteins. 168 protein analysis, samples were resolved on 10% or 13% Tris-glycine SDS-PAGE gels 169 (26). For immunoblotting, proteins were transferred to pure nitrocellulose membranes 170 (Biorad), and two hemagglutinin (2HA)-tagged proteins were detected by addition of 171 mouse anti-HA monoclonal antibody HA.11 (Covance). To analyze ERK and NF-ΚB 172 signaling, T84 cells were seeded on 6-well plates, infected, and cells were scraped at 173 different time points. Infected cells were pelleted, resuspended in sample buffer, and 174 frozen until they were boiled and run on SDS-PAGE. Samples were immunoblotted 175 using mouse monoclonal antibody against phospho-ERK1/2 (Thr202/Tyr204) and rabbit 176 monoclonal antibody against phospho-IKKalpha/beta (Ser176/180) (Cell Signaling, Inc.). 177 Total protein amounts were evaluated using rabbit polyclonal antibody against ERK1/2 178 (Cell Signaling, Inc.). Bands were visualized using sheep anti-mouse and sheep anti- 179 rabbit secondary antibodies conjugated to horseradish peroxidase (Amersham). Primary 180 and secondary antibodies were used at a 1:1000 dilution. Blots were developed using 181 Visualizer (Upstate), and images were captured with a charge-coupled device camera For E C C A 9 182 from the LAS-3000 CH imaging system (Fuji). Densitometry of visualized bands was 183 determined using Image Gauge V4.22. 184 D E 185 Shigella virulence assays. S. flexneri invasion assays were carried out as previously 186 described (15), and colony forming units (CFU) were counted and compared to the 187 amount of input bacteria to calculate the invasion efficiency. 188 performed as previously described (37). The Serény test (50) was used to assess invasion 189 and the in vivo inflammatory response in guinea pigs. Three guinea pigs were used to 190 evaluate each strain used for each experiment, and symptoms were monitored for four 191 days. 192 Plaque assays were T P E C C A 193 Immunofluorescence and transfection analysis. Immunofluorescence was carried out 194 as before with S. flexneri using mouse anti-HA monoclonal antibody HA.11 (Covance) 195 and goat anti-mouse antibody conjugated to AlexaFluor 488 (Invitrogen) (54), except 196 ImageiT-FX blocker (Invitrogen) was used to further eliminate background staining. 197 EPEC infected cells were fixed with 3% paraformaldehyde and stained with anti-HA 198 antibody as above. Transfections were carried out as before (55) except Lipofectimine 199 2000 or Lipofectimine LTX (Invitrogen) was used as the transfection reagent according 200 to the manufacturer’s instructions. Cells were sometimes stained with 4',6'-diamidino-2- 201 phenylindole (DAPI) (0.5 µg/ml) for 20 min. Shigella-infected cell images and EPEC- 202 infected cell images were acquired with an Olympus BX51 microscope using an 10 203 Olympus DP-70 digital camera and merged using DP controller software version 204 1.1.1.71. Transfected cell images were acquired with an Olympus 1X81 microscope 205 using a SensiCam charge-coupled device camera (Cooke) and merged with IPLabs 206 software version 3.1 or the Olympus BX51 microscope and software above. D E 207 208 PMN transepithelial migration assay. The PMN migration assay for S. flexneri and 209 EPEC was performed as previously described (31, 49). Briefly, S. flexneri and EPEC 210 were subcultured in LB, washed in Hank's buffered salt solution (HBSS), and added to 211 the basolateral or apical compartment, respectively, of Transwells at a multiplicity of 212 infection (MOI) of ~100 for 90 min. After 90 min, bacteria were thoroughly washed 213 away. 214 technique (31) and added to the basolateral compartment. Transmigration to the apical 215 compartment was quantified by assaying for the PMN azurophilic granule marker 216 myeloperoxidase. For inducible complementation of deletion mutations, arabinose was 217 added at a final concentration of 0.6% to the HBSS during the PMN migration assay. 218 Unpaired Student t tests were used to analyze statistical significance in comparisons 219 between wild-type EPEC and S. flexneri and experimental strains. T P E C Human PMNs were purified from whole blood by a gelatin sedimintaton C A 11 220 Results 221 Orf212 is a homologue of the EPEC T3SS effector NleE 222 It was shown previously that NleE has homologues in Citrobacter, EHEC, and 223 Shigella species (53). In A/E effacing pathogens, the two T3SS effectors nleB and nleE 224 are encoded together in the same operon (21). Interestingly, Shigella species evolved to 225 incorporate only an nleE homologue, orf212, on the virulence plasmid. In contrast, 226 Salmonella enterica serovar Typhimurium species evolved to only incorporate an nleB 227 homologue, STM4157, on its chromosome (BLAST analysis). It should also be noted 228 that Salmonella enterica serovar Typhi and Paratyphi do not have a copy of nleB or nleE 229 (BLAST analysis). D E T P E C 230 Further study of orf212, the S. flexneri nleE homologue, found that this gene 231 harbored a frame-shift mutation that creates a stop codon, which truncates the protein 232 product at amino acid 188 in the sequenced strains of S. flexneri 2a (301 and 2457T) and 233 S. flexneri 5 M90T. We re-sequenced orf212 from S. flexneri 2a 2457T, the wild-type 234 strain used in this study, to confirm the mutation. We also sequenced orf212 from 235 clinical isolates of the same related cluster of Shigella (27), which included S. flexneri 236 types 1a and 3a, and S. boydii type 12 to see if the frame-shift mutation was conserved in 237 these isolates. Lastly, we sequenced orf212 from enteroinvasive E. coli (EIEC), a close 238 relative of Shigella species. C A 239 We found that S. flexneri 1a does indeed have the same frame-shift mutation 240 which leads to a stop codon at amino acid 188 (Fig. 1) whereas, S. flexneri 3a has an IS 241 element (IS3) inserted after just 34 base-pairs of DNA sequence leading to a gene 12 242 deletion (data not shown). The EIEC orf212 also had a frame-shift mutation that leads to 243 a stop codon, but the truncation was at amino acid 152 (Fig. 1). S. boydii 12 had the 244 same mutation that would lead to a protein product truncated at amino acid 152 (Fig. 1). 245 Therefore, it appears that there is some plasticity within this region of the virulence 246 plasmid, but the majority of related S. flexneri serotypes (1, 2, and 5) have a frame-shift 247 mutation leading to a truncated protein product at amino acid 188. 249 D E T P 248 Orf212 is secreted by the S. flexneri T3SS E C 250 NleE is a T3SS effector protein that is secreted and translocated into host 251 eukaryotic cells by EPEC (8, 21). Since orf212 on the S. flexneri virulence plasmid has 252 such a high degree of homology to nleE (53), we wanted to determine if Orf212 was 253 secreted by the S. flexneri T3SS. Using a strategy implemented previously (55), we 254 generated a two-hemagglutinin (2HA) tagged version of Orf212. The pDZ9 (Orf212- 255 2HA) plasmid was transformed into an ∆orf212 deletion mutant (BS821) that was 256 generated by allelic exchange using the lambda red recombination system. The resulting 257 strain was designated BS822. This plasmid was also transformed into a spa47 mutant 258 (BS652) to generate BS824. Spa47 is the ATPase required for a functional T3SS of S. 259 flexneri (19), and we previously used this background strain to test the T3SS-dependent 260 secretion of putative effectors (55). After one hour of exposure to Congo red to induce 261 the S. flexneri T3SS, Orf212-2HA (~23 kDa) was found in the whole cell and supernatant 262 fractions of BS822 (Fig. 2). 263 untransformed 2457T or in the supernatant of the T3SS mutant BS824 (Fig. 2). C A In contrast, no signal was detected in either the 13 264 Therefore, Orf212 is secreted by the S. flexneri T3SS, and we renamed this protein OspZ 265 because it is the last annotated orf on the virulence plasmid (5), and the potential exists 266 for more T3SS effectors to be discovered (orf9-orf186). 267 268 D E The intracellular localization of OspZ and NleE 269 Since OspZ and NleE are homologues, we hypothesized that these proteins may 270 share a similar localization inside the host cell. Therefore, a semi-confluent monolayer of 271 HeLa cells was infected with 2457T or ospZ/pTrc-OspZ-2HA (BS841) for four hours, 272 and the OspZ-2HA fusion protein was detected using indirect immunofluorescence with 273 an anti-HA antibody and an Alexa Fluor 488 secondary antibody. We counter-stained 274 with DAPI, which stains the DNA of the bacteria and nucleus of the host cell. We found 275 that the anti-HA signal was localized primarily with the bacteria or in the vicinity of the 276 bacteria in the cytoplasm (Fig. 3A). To a lesser degree some signal was also found inside 277 the nucleus (Fig. 3A). As a negative control, cells infected with wild-type 2457T (no 278 pTrc-OspZ-2HA) displayed no anti-HA signal above background levels (Fig. 3A) T P E C 279 C A 280 cloned nleE in frame with a 2HA tag (pTrc-NleE-2HA), and transformed this plasmid 281 into EPEC E2348/69. HeLa cell monolayers were infected with wild-type EPEC (no 282 pTrc-NleE-2HA) or carrying the pTrc-NleE-2HA plasmid for five hours, and the secreted 283 NleE-2HA fusion protein was detected using immunofluorescence with anti-HA 284 antibody. We observed that the immunofluorescence signal localized with the bacteria, 285 but also more definitively in the nucleus of the host cells when compared to OspZ (Fig. 286 3B). No HA signal was found in the nucleus infected with the negative control (Fig. 3B). To determine if NleE exhibited a similar localization in EPEC infected cells, we 14 287 To confirm the localization of NleE and OspZ, we ectopically expressed these 288 proteins fused to green fluorescent protein (GFP) at their N-terminus. OspZ and NleE 289 were cloned in frame with EGFP to generate the N-terminal fusion proteins. A semi- 290 confluent monolayer of HeLa cells was transiently transfected with plasmids expressing 291 GFP-OspZ or GFP-NleE, and cells were fixed after 12 hours. 292 observed two distinct localization phenotypes in the host cell. GFP-OspZ was found in 293 punctate staining in the cytoplasm of transfected cells (Fig. 4A), and occasionally, GFP- 294 OspZ localized in the nucleus (~25-30% of transfected cells) (Fig. 4A). To confirm the 295 nuclear localization of GFP-OspZ, the GFP-OspZ plasmid was co-transfected with RFP- 296 OspF, which has been shown previously to localize in the nucleus of transfected cells 297 (55). 298 together (yellow merged signal) (Fig. 4B). We also confirmed this observation with 299 DAPI staining (data not shown). In contrast, GFP-NleE was found primarily in the 300 nucleus of the transfected HeLa cells (Fig. 4C) and no punctate staining was observed. 301 The nuclear staining with the ectopically expressed proteins was consistent with the 302 immunofluorescence results observed with infected cells. 303 304 D E Upon analysis, we T P E C In co-transfected cells, GFP-OspZ and RFP-OspF were found in the nucleus C A Virulence phenotypes of a ospZ mutant 305 Since OspZ from S. flexneri was secreted in a T3SS-dependent manner, we 306 wanted to determine whether the ∆ospZ deletion mutant (BS821) was attenuated in 307 various tests of S. flexneri virulence. First, we compared ∆ospZ to wild-type 2457T in an 308 invasion assay using HeLa cells and polarized T84 cells and found no discernible 309 difference in invasion efficiency (data not shown). 15 310 Next, we compared ∆ospZ and 2457T in a plaque assay to determine the ability of 311 each strain to invade, replicate intracellularly, and spread cell-to-cell. 312 significant difference was observed between ∆ospZ and 2457T with the efficiency of 313 plaque formation (2457T = 1.1 ± 0.13%, ∆ospZ = 1.3 ± 0.15%) and size of plaques being 314 equivalent (~1.5 mm). Lastly, we compared ∆ospZ and 2457T infections using the 315 Serény test, one of a limited number of animal infection models for S. flexneri (50). 316 Three guinea pigs were infected with ∆ospZ or 2457T at a dose of 2.5 x 108 CFU in one 317 eye of each animal. Over the course of four days guinea pigs infected with either ∆ospZ 318 or 2457T developed conjunctivitis and a strong inflammatory response with no 319 discernible difference between wild-type and ∆ospZ. D E 322 T P E C 320 321 Again, no PMN transepithelial migration assays C A PMN transepithelial migration is a hallmark of S. flexneri infection and requires 323 the T3SS (31). We identified two T3SS effectors, OspF and OspC1, that contribute to 324 Shigella-induced PMN migration (55). The PMN migration assay allowed us to detect 325 subtle differences between strains which other S. flexneri virulence assays do not (24, 31, 326 55). We postulated PMN migration requires multiple, upstream factors, and wanted to 327 see if OspZ contributed to this aspect of S. flexneri pathogenesis. 328 Mutant and wild-type strains of S. flexneri were used to infect polarized T84 329 monolayers. After 90 min of infection, isolated human PMNs were added to the 330 basolateral side of the Transwell and transepithelial migration was evaluated. When 331 ospZ (BS821) was compared to 2457T, we observed a statistically significant decrease 16 332 in the amount of neutrophils that migrated from the basolateral to the apical 333 compartment, the physiologic direction of PMN recruitment/infiltration (Fig. 5A). 334 EPEC is also capable of inducing PMN transepithelial migration (49). Therefore, 335 we wanted to see if infection with a nleE deletion mutant resulted in a similar 336 deficiency as was observed with the deletion of ospZ of S. flexneri. PMN transmigration 337 induced by the EPEC nleE mutant was significantly reduced in comparison to wild-type 338 EPEC (Fig. 6A), and this reduction mirrored the reduction observed with the 339 deletion mutant of S. flexneri. D E T P ospZ 340 Since OspZ and NleE have nearly identical primary amino acid sequences (Fig. 341 1), and the deletion mutants showed a comparable phenotype in the PMN transmigration 342 assay, we hypothesized that nleE should complement the Shigella ∆ospZ deletion mutant, 343 and conversely, ospZ should complement the EPEC ∆nleE mutant in the PMN migration 344 assay. To address each hypothesis, we generated two arabinose-inducible, untagged 345 constructs by cloning ospZ and nleE into pBAD24, and each plasmid was transformed 346 cross-species into the corresponding deletion mutant. 347 E C C A ∆ospZ/pBAD24::nleE (BS834) was used to infect polarized T84 cells in 348 Transwells in the presence of 0.6% 349 ∆ospZ/pBAD24::ospZ (BS833) served as a positive control. When normalized to 2457T, 350 T84 cells infected with BS834 induced nearly equivalent levels of PMN migration to the 351 apical compartment of the Transwell when compared to 2457T (Fig. 5A). Similarly, the 352 reduction could be restored by an OspZ expressing plasmid, pBAD24::ospZ (BS833) 353 (Fig. 5A). These results implied that nleE was secreted by the S. flexneri T3SS and 354 complemented the function of ospZ in the ∆ospZ deletion mutant. We verified that NleE 17 arabinose and compared to 2457T. 355 was secreted by 2457T by analyzing supernatants of 2457T transformed with pTrc-NleE- 356 2HA (BS840) (Figure 5B). 357 Since NleE expression could complement an ospZ deletion mutant of S. flexneri, 358 we hypothesized that OspZ expression should complement the PMN transepithelial 359 migration defect for for the 360 was used to infect polarized T84 cells, and the level of PMN migration was compared to 361 wild-type EPEC infection. ∆nleE/pBAD24::nleE (ATM898) was used as a positive 362 control. 363 complemented the 364 migration compared to wild-type EPEC and ∆nleE/pBAD24::nleE (Fig. 6A). Therefore, 365 ospZ can substitute for nleE in the PMN transepithelial assay. 366 D E nleE mutant of EPEC. ∆nleE/pBAD24::ospZ (ATM897) T P When normalized to wild-type EPEC infections, ∆nleE/pBAD24::ospZ nleE mutant, and actually, induced significantly more PMN E C Lastly, to verify that OspZ was secreted by the heterologous EPEC T3SS, we C A 367 transformed pTrc-OspZ-2HA into wild-type EPEC (ATM928). ATM928 and wild-type 368 EPEC were grown overnight, subcultured into DMEM, and secretion was evaluated after 369 five hours. As expected from the PMN migration results, OspZ was secreted by the 370 EPEC T3SS (Fig. 6B). 371 372 OspZ plays a role in the induction of host signaling pathways 373 Since we have previously shown that S. flexneri infections with other osp deletion 374 mutants have a deficiency in PMN transepithelial migration and display a deficiency in 375 ERK1/2 pathway activation (55), we evaluated ERK1/2 phosphorylation in BS821, the 376 ∆ospZ deletion mutant during infection. We infected T84 cells with 2457T or BS821 and 377 evaluated ERK1/2 phosphorylation by Western blot over the first hour of infection using 18 378 a phospho-specific antibody that recognizes the activation of ERK1/2. As a loading 379 control, we blotted for total ERK2 protein. 380 phosphorylation after 30 minutes of infection in T84 cells infected with BS821 compared 381 to 2457T (Fig. 7). We also evaluated NF-KB activation since it has been shown to be 382 activated the first hour of S. flexneri infection (40). As shown in Figure 7, we used an 383 antibody against phospho-specific residues indicating NF-KB activation to show that 384 cells infected with BS821 also displayed reduced NF-KB activation with respect to wild- 385 type infection (Fig. 7). We used densitometry on blots from three different experiments 386 at the 45 min time point and found that cells infected with the ∆ospZ had 63.53 ± 3.92% 387 of the amount of ERK1/2 phosphorylation and 52.82 ± 11.95% of NF-KB activation 388 when compared to wild-type infection (normalized to 100%). Therefore, we conclude 389 that OspZ plays a role in activating host inflammatory pathways required for PMN 390 transepithelial migration. 391 We saw a marked reduction in ERK1/2 D E T P E C C A 19 392 Discussion 393 The large virulence plasmid of S. flexneri encodes a T3SS and at least twenty 394 effector proteins that are secreted by this apparatus (5, 23, 32, 45, 55). Many of these 395 proteins were identified in a previous study that used N-terminal sequencing to identify 396 the secreted proteins from a constitutive secretion mutant ( ipaB) of S. flexneri serotype 397 5a (5). OspZ was not among the proteins identified. However, the amount of OspZ 398 secreted by this strain may not have been sufficient for N-terminal sequencing. In this 399 study, OspZ piqued our interest because of its similarity to the EPEC T3SS effector, 400 NleE, and we confirmed that OspZ is indeed a secreted S. flexneri T3SS effector. OspZ 401 can therefore be added to a growing list of Shigella T3SS effectors that have homologues 402 in other Gram-negative pathogens. For example, OspF of Shigella has homologues in 403 Salmonella species, Pseudomonas syringae, and Chromobacterium violaceum (4). It has 404 also been shown that a family of effectors, IpgB1/B2 (Shigella), SifA/B (Salmonella), 405 and Map (EPEC/EHEC) mimic the function of RhoGTPases in the host cell (2). In 406 addition, Shigella and EPEC share other homologous effectors such as VirA (Shigella) 407 and EspG/G2 (EPEC), which disrupt host microtubules (10, 52). 408 D E T P E C C A While some of these shared T3SS effectors have highly homologous amino acid 409 sequences, it is nevertheless important to confirm their related functions since even small 410 changes can affect protein function. For example, the Shigella OspF localizes in the 411 nucleus of host cells while the homologous SpvC (>70% homology) from Salmonella 412 remains in the cytoplasm (55). At the same time, OspF and SpvC both share a function 413 as a lyase to remove phosphate groups from mitogen-activated protein kinases (MAPK) 414 (29). In the current study we encountered a similar localization difference between OspZ 20 415 and NleE. While OspZ-2HA and GFP-OspZ were found in the nucleus in some 416 instances, the tagged NleE was found in the nucleus 100% of the time. A classic or non- 417 classical bipartite or monopartite nuclear localization signal (NLS) was not detected in 418 either NleE or OspZ using NLS scanning software. However, it is possible that each 419 protein interacts directly with a host protein that has an NLS. Another possibility exists 420 where OspZ/NleE could enter the nucleus via an interaction with another secreted 421 bacterial protein which also localizes in the host nucleus. 422 expression of GFP-OspZ and GFP-NleE showed that both proteins can enter the nucleus 423 in the absence of bacterial infection. The localization difference observed between NleE 424 and OspZ (including the punctuate staining of GFP-OspZ) could be attributed to the 425 additional 36 amino acids found at the C-terminus of NleE (Fig. 1). While the additional 426 C-terminal domain does not contain an NLS, it is possible this domain serves to more 427 effectively target NleE to the nucleus. We are currently investigating this possibility and 428 have preliminary data which suggests the C-terminus is required for NleE nuclear 429 localization (Badea and Hartland, unpublished). 430 localization could be a product of diffusion since it was not found consistently in the 431 nucleus, and both tagged proteins are smaller than the nuclear pore (~60 kDa). The 432 importance of nuclear localization is unclear since both ospZ and nleE can complement 433 the PMN migration deficiency during infection. Therefore, the nuclear localization is 434 unlinked to the PMN migration phenotype. D E However, the ectopic T P E C C A In contrast, the OspZ nuclear 435 Since OspZ and NleE both played a role in the induction of PMN migration 436 across a polarized T84 monolayer during S. flexneri and EPEC infections, we speculate 437 that the function these proteins share is linked to influencing host signaling pathways. 21 438 Interestingly, the expression of OspZ in the 439 migration when compared to wild-type EPEC. Therefore, the S. flexneri version of this 440 T3SS effector may be more active than its EPEC counterpart. This hyper-migration is 441 not unexpected given that S. flexneri infections cause more acute inflammation than 442 EPEC infections (20), and it could be attributed to the missing C-terminal 36 amino 443 acids. 444 nleE mutant led to increased PMN D E It is clear from previous work that the activation of the MEK/ERK pathway is T P 445 required for Shigella-induced PMN migration (24, 55). Our data suggest OspZ is 446 required for the full activation of the ERK1/2 and NF-KB pathways by an unknown, 447 upstream mechanism (Fig. 7). EPEC stimulates ERK1/2 phosphorylation to induce an 448 inflammatory response which includes NF-KB activation and IL-8 production (47, 48). 449 We are currently investigating if NleE plays a role in ERK1/2 and NF-KB activation and 450 if it can be linked to PMN migration phenotype, but it would not be a surprising 451 connection given the shared homology/function to OspZ and the reduced hyperplasia 452 observed in infections with the ∆nleE mutant of Citrobacter (53). It should be noted that 453 IL-8 (a product of NF-KB activation) is secreted basolaterally in vivo, and a separate 454 factor, hepoxilin A3 (HXA3) has been identified to be essential in recruiting PMN across 455 the epithelial layer to the apical side in Pseudomonas and Salmonella infections (18, 33, 456 34). Therefore, it is likely that the same host signaling pathways required for PMN 457 migration are required for HXA3 production and apical secretion, and we are 458 investigating this possibility. 459 directly, but it is more likely that receptors or kinases upstream of ERK1/2 and NF-KB 460 are being activated, and OspZ and NleE play a role in this activation. Therefore, ERK1/2 E C C A EPEC and Shigella may target ERK1/2 and NF-KB 22 461 and NF-KB may only be a fraction of the host signaling pathways whose activation is 462 required to mediate the PMN migration phenotype in EPEC and Shigella-infected 463 epithelial cells (9, 34). 464 phosphatidylinositol (PI) 3-kinase pathway. S. flexneri and EPEC activate PI3-kinase 465 (38, 42), but EPEC can also inhibit PI3-kinase at a different time point during infection 466 and in macrophages (6) so it would be interesting if these responses were altered during 467 infection with ospZ/ nleE mutants. One upstream pathway we are investigating is the D E T P 468 The initial activation of the inflammatory response is just one aspect of Shigella 469 and EPEC interactions with the innate immune system. At later time points both Shigella 470 and EPEC down-regulate inflammation in a T3SS-dependent fashion. In the case of 471 Shigella, OspG binds to UbcH5 to promote the ubiquitination of phosphorylated inhibitor 472 of NF-ΚB type alpha (phospho-IΚBα) (23). OspF removes the phosphate groups of 473 members of the MAPK family in the nucleus and plays a role in chromatin remodeling to 474 down-regulate AP-1 regulated genes (4, 29). EPEC also down-regulates MAPK family 475 members in a T3SS-dependent manner, but the identity of the effector(s) has not been 476 elucidated (43). In addition, MOI plays a role in whether EPEC induces a pro- or anti- 477 inflammatory response in the host (51). When a lower MOI of EPEC is used to infect 478 HT-29 intestinal cells, IL-8 production is increased with contributions from an 479 unidentified T3SS effector and flagellin (51). A higher MOI of EPEC leads to the down- 480 regulation of IL-8 in an T3SS-dependent manner (51). Lastly, EPEC rearranges host 481 proteins from the basolateral to apical membrane, which again may impact the kinetics of 482 inflammatory signaling (36, 42). Interestingly, Shigella lack flagella, which could be 483 why the bacteria evolved to use multiple T3SS effectors (Osp proteins and IpgD) to E C C A 23 484 activate the signaling pathways required to induce inflammation and PMN transepithelial 485 migration (9, 38, 55). Therefore, in both Shigella and EPEC infections, the regulation of 486 inflammatory signaling is a complex and dynamic balance. While the activation of PMN 487 migration and other inflammatory processes contributes to pathogenesis, too much 488 inflammation may activate the host immune system to clear the infection (9, 39, 40, 45). 489 The challenge in understanding this paradox will be to identify the T3SS effectors 490 involved (in this case, OspZ and NleE) and their function at different times during 491 infection. Shigella and EPEC appear to utilize T3SS effectors with different kinetics and 492 in different host compartments (e.g. cytoplasm vs. nucleus) with most likely different 493 host targets to balance the innate immune response. D E T P E C 494 In conclusion, this study identifies for the first time that OspZ is a secreted 495 Shigella T3SS effector and is required for virulence. OspZ has a role, along with OspF 496 and OspC1 (55) and most likely other secreted proteins in mediating the PMN migration 497 phenotype. NleE is the first T3SS effector of EPEC shown to play a role in PMN 498 transepithelial migration, but given the shared virulence strategies of related bacterial 499 pathogens, we anticipate that other EPEC T3SS effectors also contribute to this 500 phenotype. C A 24 501 Acknowledgements 502 We would like to thank Nancy Adams and Reinaldo Fernandez for technical assistance, 503 and Stephanie Perry for bioinformatics help. This work was supported by National 504 Institutes of Allergy and Infectious Diseases Grant AI24656 (to A.T.M.). B.A.M. is 505 supported by National Institutes of Health Grants DK56754 and DK33506. K.L.M. is 506 supported by a T32 Training Grant sponsored by Harvard Medical School and the 507 Department of Surgery at Massachusetts General Hospital. J.A.P. was supported by an 508 Australian Postgraduate Scholarship, and E.L.H. is supported by grants from the 509 Australian National Health and Medical Research Council and the Australian Research 510 Council. 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Immun. 749 74:5964-5976. T P E C 750 751 C A 36 752 Table 1 – Strains and plasmids used in this study. Strain Genotype/Description Referencea Shigella 2457T Wild-type Shigella flexneri 2a 13 BS103 Virulence plasmid-cured derivative of 2457T 30 BS508 Shigella flexneri 1a BS510 Shigella flexneri 3a BS652 2457T/ spa47 (spa47::aadA); SpecR BS685 Shigella boydii 12 BS766 2457T transformed with pKM208; AmpR D E CDC CDC T P E C 55 CDC 55 2457T/ ospZ (ospZ::cat); CmR This study 2457T/ ospZ – unmarked This study C A BS821 transformed with pDZ9; CmR This study BS652 transformed with pDZ9; CmR This study BS833 BS821 transformed with pBAD24::ospZ; CmR This study BS834 BS821 transformed with pBAD24::nleE; CmR This study BS840 2457T transformed with pTrc-NleE-2HA; AmpR This study BS841 BS821 transformed with pTrc-OspZ-2HA; AmpR This study endA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 16 BS819 BS821 BS822 BS824 Escherichia coli DH5α ( lacIZYA-argF)U169 deoR (Φ80 dlac [lacZ] M15) EIEC Enteroinvasive E. coli serotype O124:NM 17 EPEC Enteropathogenic E. coli E2348/69 wild-type O127:H6 28 37 EPEC E2348/69/ nleE; CmR This study EPEC transformed with pTrc-NleE-2HA; AmpR This study ATM897 nleE transformed with pBAD24::ospZ; CmR This study ATM900 nleE transformed with pTrc-NleE-2HA; AmpR This study ATM928 nleE transformed with pTrc-OspZ-2HA; AmpR nleE EPEC/pTrc-NleE-2HA Plasmids D E T P pKD3 bla cat oriR6K; AmpR CmR pKM208 Temperature-sensitive red-, gam-, and lacI expressing This study 7 35 Plasmid expressing FRT recombinase; AmpR Cloning intermediate; CmR 55 OspZ-2HA fusion. Expression driven by POspZ; CmR This study pTrc-NleE-2HA E C 7 NleE-2HA fusion. Expression driven by PTrc; AmpR This study pTrc-OspZ-2HA OspZ-2HA fusion. Expression driven by PTrc; AmpR This study pBAD24 Arabinose inducible vector; pBR322ori; AmpR 14 pBAD24::nleE nleE cloned into NcoI/HindIII sites of pBAD24; AmpR This study pBAD24::ospZ ospZ cloned into NcoI/HindIII sites of pBAD24; AmpR This study pDsRed2-C1 Red fluorescent protein vector (RFP); KanR Clontech pEGFP-C1 Green fluorescent protein vector (GFP); KanR Clontech EGFP::NleE nleE cloned into pEGFP-C1; KanR This study EGFP::OspZ ospZ cloned into BglII/HindIII sites of pEGFP-C1; KanR This study plasmid driven by PTac promoter; AmpR pCP20 pDZ2 C A pDZ9 753 a Centers for Disease Control and Prevention, Nancy Strockbine. 38 754 Figure Legends 755 Fig. 1 –NleE and OspZ are homologues found in Shigella species and attaching and 756 effacing strains of E. coli. Accession numbers in parentheses. Sequence from S. flexneri 757 1a, S. flexneri 2a 2457T, S. flexneri 5a M90T (AF348706), S. dysenteriae (YP406222), S. 758 sonnei (YP313493), 759 (epath100c11_p1cb_ORF_3157 coliBase), and EIEC were aligned using ClustalW 760 (http://www.ebi.ac.uk/clustalw/) 761 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Shaded regions are homologous 762 residues where * represents identical amino acids,: are conservative substitutions, and . 763 are semi-conservative substitutions all based on the GONNET250 matrix. 766 and generated Fig. 2 – T3SS-dependent secretion of OspZ. C A by T P E C 764 765 D E S. boydii (YP406371), S. boydii 12, EPEC E2348/69 2457T, ospZ/pDZ9 (BS822), and spa47/pDZ9 (BS824) whole cell fractions and supernatant were prepared after one hour 767 of induction with Congo red. 768 immunoblotted with anti-HA antibody to visualize OspZ-2HA (~22 kD band). 769 770 BioEdit Samples were run on a 13% SDS-PAGE gel and Fig. 3 – Localization of OspZ-2HA and NleE-2HA in infected host cells. (A) 771 ospZ/pTrc-OspZ-2HA (BS841) was used to infect a semi-confluent monolayer of HeLa 772 cells. After 4 h, cells were fixed and evaluated by immunofluorescence. Images are 773 representative of DAPI staining, Anti-HA, and the merged fields. The negative control is 774 HeLa cells infected with wild-type 2457T (minus pTrc-OspZ-2HA). (B) EPEC/pTrc- 775 NleE-2HA and EPEC (pTrc99A, empty plasmid) were used to infect a semi-confluent 776 monolayer of HeLa cells. After 5 h, cells were fixed and evaluated by 39 777 immunofluorescence. Images in the middle column are representative of the 778 immunofluorescence anti-HA signal observed with each strain, the left-hand column 779 shows nuclei and bacteria stained with DAPI, and the right-hand column is the merged 780 fields. D E 781 782 Fig. 4 – Ectopic expression of GFP-OspZ and GFP-NleE in HeLa cells. Semi-confluent 783 monolayers of HeLa cells were transiently transfected for 12 h with mammalian 784 expression vectors encoding: (A) GFP-OspZ, (B) GFP-OspZ and RFP-OspF (co- 785 transfection), or (C) GFP-NleE. Panel (A) has two different representative fields of GFP- 786 OspZ (left and middle column), nuclear and cytoplasmic staining. The far right column 787 is the middle column enlarged (dotted white box) to highlight the punctate staining in the 788 cytoplasm. In panel (C) nuclei were stained with DAPI. 789 T P E C C A 790 Fig. 5 - NleE can complement the S. flexneri 791 assays. (A) T84 polarized monolayers were infected basolaterally with 2457T, 792 (BS821), 793 90 min were evaluated for PMN migration. HBSS represents uninfected monolayers 794 (buffer only). 795 performed three times in triplicate. The data are means ± standard deviations (error bars) 796 of triplicate samples and represent one of the three experiments performed in which 797 similar results were obtained. Test samples were individually compared to 2457T using 798 an unpaired Student t test, and statistically significant differences (P values ≤ 0.01) are 799 indicated by an asterisk. (B) 2457T transformed with pTrc-NleE-2HA (BS840) was ospZ/pBAD24::ospZ (BS833), and ospZ in PMN transepithelial migration ospZ/pBAD24::nleE (BS834) and after All strains were normalized to wild-type 2457T. 40 ospZ Experiments were 800 grown in secretion conditions and compared to the negative control, untransformed wild- 801 type 2457T. Whole cell fractions and supernatant were prepared. Samples were boiled 802 and run on a 13% SDS-PAGE gel, and immunoblotted with anti-HA antibody to visualize 803 Nle-2HA (~25 kD band). D E 804 805 Fig. 6 – OspZ can complement EPEC 806 (A) T84 polarized monolayers were infected apically with EPEC E2348/69, 807 nleE/pBAD24::ospZ (ATM897), and nleE in PMN transepithelial migration assays. T P nleE, nleE/pBAD24::nleE (ATM898), and after 90 808 min were evaluated for PMN migration. HBSS represents uninfected monolayers (buffer 809 only). All strains were normalized to wild-type EPEC E2348/69. Experiments were 810 performed three times in triplicate. The data are means ± standard deviations (error bars) 811 of triplicate samples and represent one of the three experiments performed in which 812 similar results were obtained. Test samples were individually compared to 2457T using 813 an unpaired Student t test, and statistically significant differences (P values ≤ 0.01) are 814 indicated by an asterisk. (B) 815 conditions and compared to the negative control EPEC E2348/69 with no plasmid and the 816 positive control, ospZ/pTrc-OspZ-2HA (BS841). Whole cell fractions and supernatant 817 were prepared. Samples were boiled and run on a 13% SDS-PAGE gel, and 818 immunoblotted with anti-HA antibody to visualize OspZ-2HA (~22 kD band). E C C A nleE/pTrc-OspZ-2HA was grown in EPEC secretion 819 820 Fig. 7 – ERK1/2 and NF-KB have reduced activation in cells infected with the S. flexneri 821 ospZ mutant. T84 cells were infected with wild-type 2457T or ospZ (BS821). Cells 822 were scraped at each time point, boiled, and run on a 10% SDS-PAGE gel, and 41 823 immunoblotted with anti-phospho-ERK1/2 and anti-phospho NF-KB antibody to 824 visualize signaling pathway activation. Samples were blotted with anti-ERK1/2 antibody 825 to monitor equal loading. The blot is representative of an experiment repeated three 826 times. D E T P E C C A 42 827 Fig. 1 D E T P E C 828 C A 43 829 Fig. 2 D E T P E C C A 44 830 Fig. 3 D E T P E C C A 831 45 832 Fig. 4 D E T P E C C A 833 46 834 Fig. 5 835 A D E 836 T P 837 E C 838 839 840 841 C A B 842 843 47 844 Fig. 6 845 A D E 846 847 T P 848 849 E C 850 851 852 853 854 C A B 855 856 857 858 859 48 860 Fig. 7 D E T P 861 862 E C C A 49