Functional characterization of SsaE, a novel chaperone protein of the type III secretion system

1

1

2

3

4

Functional characterization of SsaE, a novel chaperone protein of the 5

type III secretion system encoded by Salmonella pathogenicity island 2 6

7

Tsuyoshi Miki,1 Yoshio Shibagaki,2 Hirofumi Danbara,1 and Nobuhiko Okada1* 8

9

Department of Microbiology,1 and Department of Biochemistry,2 School of Pharmacy, 10

Kitasato University, Minato-ku, Tokyo 108-8641, Japan 11

12

Running title: Role of SsaE in SPI-2 secretion 13

14

*Corresponding author. Mailing address: Department of Microbiology, School of 15

Pharmacy, Kitasato University, Minato-ku, Tokyo 108-8641, Japan. Phone: 16

+81-3-5791-6256; Fax: +81-3-3444-4831; E-mail: [email protected] 17

18

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00863-09 JB Accepts, published online ahead of print on 18 September 2009

on January 12, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

http://jb.asm.org/

2

ABSTRACT 19

20

The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 21

(SPI-2) is involved in systemic infection and intracellular replication of Salmonella 22

enterica serovar Typhimurium. In this study, we investigated the function of SsaE, a 23

small cytoplasmic protein encoded within the SPI-2 locus, which shows structural 24

similarity to the T3SS class V chaperones. An S. enterica serovar Typhimurium ssaE 25

mutant failed to secrete SPI-2 translocator SseB and SPI-2 dependent effector PipB 26

proteins. Co-immunoprecipitation and mass spectrometry analyses using an 27

SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative 28

T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG 29

fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for 30

protein-protein interactions. Although SseA was reported to be a chaperone for SseB 31

and to be required for its secretion and stability in the bacterial cytoplasm, an sseA 32

deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was 33

overexpressed. In contrast, ssaE mutant strains could not transport SseB extracellularly 34

under the same assay conditions. In addition, an ssaEI55G point-mutated strain that 35

expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil 36

structure showed attenuated virulence comparable to that of a SPI-2 T3SS null mutant, 37

suggesting that the coiled-coil interaction of SsaE is absolutely essential for the 38

functional SPI-2 T3SS and Salmonella virulence. Based on these findings, we propose 39

that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III 40

secretion machinery. 41

42


.org/D

ownloaded from

http://jb.asm.org/

3

INTRODUCTION 43

44

A number of Gram-negative pathogenic bacteria use a type III secretion system (T3SS) 45

to interact with eukaryotic host cells. T3SS delivers bacterial effectors through the 46

needle-like structure extending across the inner and outer membranes of the bacterium 47

and into the cytosol of eukaryotic cells (28). Salmonella enterica serovar Typhimurium 48

is an enteropathogenic bacterium that causes gastroenteritis in humans and typhoid-like 49

fever in mice. Salmonella possesses two different T3SSs encoded by Salmonella 50

pathogenicity island 1 (SPI-1) and Salmonella pathogenicity island 2 (SPI-2). Upon 51

entry into host cells, the S. enterica serovar Typhimurium resides in a special 52

membrane-bound compartment termed the Salmonella-containing vacuole (23). 53

Expression of SPI-2 is induced within the vacuole (8) and is essential for intracellular 54

replication and virulence of S. enterica serovar Typhimurium (26, 44). 55

56

Functional SPI-2 genes are clustered within six large transcriptional units. Thirty-one 57

potential open reading frames on the SPI-2 region encode proteins that are directly 58

involved in the assembly and regulation of the T3SS (50). The Ssa proteins are involved 59

in the assembly of the syringe-like type III secretion injectisome, the so-called 60

nanomachine (41). A set of nine Ssa proteins conserved among T3SSs forms the 61

injectisome core. Transport of some effectors through the injectisome is facilitated by 62

formation of a complex between an effector and a chaperone encoded in SPI-2. For 63

example, SscB, a protein encoded immediately upstream of sseF, acts as a chaperone 64

for SseF (14). In addition to effectors, the translocators SseB (a component of the 65

oligomeric filament structure of the type III secretion apparatus), SseC, and SseD (the 66


.org/D

ownloaded from

http://jb.asm.org/

4

pore-forming translocator complex) are also secreted through the SPI-2 encoded type III 67

secretion machinery. Efficient secretion of SseC and SseD is required for the presence 68

of SseB, but SseB secretion is independent of these two translocators (31, 42). 69

Furthermore, stable expression of these translocators requires the chaperone SseA for 70

SseB and SseD and a putative chaperone SscA for SseC (49, 62, 63). 71

72

Several different types of chaperones have been classified on the basis of structural and 73

functional analyses (57). Class I chaperones are small, acidic (pI 4~5), usually dimeric 74

proteins that interact with a single T3SS effector or with two or three effectors and 75

mediate targeting of their cognate binding partners to the type III secretion apparatus 76

(35). Class II chaperones bind to translocator proteins of the T3SS. They possess in the 77

part of tetratricopeptide repeats, which form a curved layer of α-helices. These 78

chaperones play a regulatory role in type III secretion (6 , 20). Class III is represented 79

by flagellar chaperones (such as FliS), which have an entirely different structure. FliS 80

prevents polymerization of flagellin protein FliC in the cytoplasm prior to secretion (19). 81

The CesA of enteropathogenic Escherichia coli (EPEC), which acts as a chaperone for 82

EspA, is structurally different from other classes of chaperones. Therefore, it has been 83

designated as a class IV chaperone. The aggregation of EspA in the bacterial cytosol is 84

prevented through binding with CesA (59). Class V chaperones are a heterogeneous 85

group of proteins that interact with the needle subunit of the type III secretion apparatus. 86

These include Yersinia YscE and YscG, and Pseudomonas aeruginosa PscE and PscG. 87

Polymerization of needle components Yersinia YscF and P. aeruginosa PscF in the 88

bacterial cytoplasm is prevented by binding to these chaperones, YscE/YscG and 89

PscE/PscG, respectively (47). 90


.org/D

ownloaded from

http://jb.asm.org/

5

91

To date, some proteins encoded within SPI-2 remain uncharacterized. One of these is 92

SsaE. The ssaE open reading frame of 243-bp is predicted to encode a protein of 80 93

amino acids containing three predicted α-helical regions. SsaE has homology to EscE 94

(Orf2) of EPEC and Citrobacter rodentium, YscE of Yersinia pestis, PscE of 95

Pseudomonas aeruginosa, and AscE of Aeromonas hydrophila. Yersinia yscE mutants 96

are unable to export their effectors (16). In P. aeruginosa, PscE is required for 97

functional T3SS and cytotoxicity by controlling the needle complex biogenesis of T3SS 98

(47). While the core type III components are generally conserved, allowing the function 99

and/or cellular locations of some of the proteins to be inferred (4, 28), these small 100

proteins are not broadly conserved among other T3SSs or flagellar export systems. For 101

example, no homologue of PscG is present in S. enterica serovar Typhimurium. 102

103

In this study, we have characterized the role of SsaE in the secretion and translocation 104

via SPI-2 T3SS. A Salmonella mutant that lacks SsaE failed to secrete SseB (a 105

translocator) and PipB (a SPI-2 effector). Using pull-down assays, we showed that SsaE 106

directly interacts with SseB and a putative ATPase, SsaN. Furthermore, deletion and 107

site-directed mutagenesis of SsaE identified a C-terminal coiled-coil domain involved in 108

protein-protein interactions of SseB. We finally found that Salmonella expressing the 109

point-mutated SsaEI55G, which is unable to form a C-terminal coiled-coil domain, has 110

dramatic defects in virulence comparable to those of the SPI-2 null mutant. These data 111

suggest that the chaperone-like small molecule SsaE plays a crucial role in the SPI-2 112

secretion by interacting with the SseB via the C-terminal coiled-coil domain. 113

114


.org/D

ownloaded from

http://jb.asm.org/

6

MATERIALS AND METHODS 115

116

Bacterial strains, plasmids, and growth conditions. The Salmonella strains and 117

plasmids used in this study are listed in Table 1. In this study, S. enterica serovar 118

Typhimurium strain SL1344 was used as wild-type strain, and strains harboring ssaV 119

mutation and spiAC133S point-mutation were used as general SPI-2 T3SS component 120

mutants. Escherichia coli strains DH5α (Gibco BRL) and MC1061 (7) were used for 121

molecular cloning and expression of recombinant proteins. E. coli strain S17.1λpir (38) 122

was used for propagation of π-dependent plasmids and for conjugation. Bacteria were 123

routinely grown in LB broth (Sigma) at 37˚C overnight with aeration. Ampicillin (100 124

µg/ml), chloramphenicol (25 µg/ml), kanamycin (25 µg/ml), and streptomycin (25 125

µg/ml) were used when required. 126

127

Strain construction. Deletion mutants of strain SL1344 were constructed using the 128

λ Red disruption system (15). S. enterica serovar Typhimurium wild-type strain SL1344 129

derivatives with chromosomally encoded FLAG and tandem HA fusion proteins were 130

constructed using the integrational plasmid pLDΩKm2 by conjugation (37) and the 131

λ Red disruption system (15), respectively. Double mutant strains were created by 132

phage P22-mediated transduction. To consider the effect of dwonsteam gene expression, 133

a Salmonella strain containing an SsaE initiation codon mutation (ssaEmut) and a strain 134

expressing a point mutated SsaEI55G were constructed by allele exchange using the 135

temperature- and sucrose-sensitive suicide vector pCACTUS containing ssaEmut or 136

ssaEI55G as described previously (37). All constructs were verified by PCR and DNA 137

sequencing. 138


.org/D

ownloaded from

http://jb.asm.org/

7

139

Plasmid construction. For construction of the complementing plasmids pSsaE and 140

pACYC-SseB, ssaE and sseB genes were amplified from the genomic DNA of strain 141

SL1344 with the primers ssaE-SacI-FW and ssaE-SphI-RV, and sseB-FW and sseB-RV, 142

and were ligated into pMW118 and pACYC184, respectively. 143

144

To construct the plasmid encoding the N-terminal GST-tagged SseB fusion protein, sseB 145

genes were amplified from the genomic DNA of strain SL1344 with the primers 146

sseB-FW-BamHI and sseB-XhoI-RV and cloned into pGEX-6P-1 (GE Healthcare). 147

148

To construct the plasmids encoding the C-terminal FLAG-tagged fusion proteins, the 149

DNA fragments containing the respective genes were amplified by PCR with specific 150

primers, and cloned into pFLAG-CTC (Sigma). To obtain the plasmids encoding 151

truncated SsaE proteins fused with FLAG, inverse PCRs were performed with the 152

primers ssaE-StuI-R3 and ssaE-StuI-R4 (for pSsaE∆1-FLAG), ssaE-StuI-R1 and 153

ssaE-StuI-R2 (for pSsaE∆2-FLAG), or ssaE-StuI-R7 and ssaE-StuI-R8 (for 154

pSsaE∆3-FLAG). The point mutation on pSsaE-FLAG was created by a QuickChange 155

Site-directed mutagenesis kit (Stratagene) using the primers sense-ssaEI55G and 156

antisense-ssaEI55G to replace isoleucine with glycine at position 55 in the SsaE. The 157

mutation was confirmed by DNA sequencing. 158

159

To construct the plasmids encoding the C-terminal 2HA-tagged fusion protein, plasmids 160

p2HA-CTC, pACHS-2HA and pACPJ-2HA were used. For construction of p2HA-CTC, 161

inverse PCR was performed using pFLAG-CTC circular DNA as the template with 162


.org/D

ownloaded from

http://jb.asm.org/

8

primers 2HA-StuI-1 and 2HA-StuI-2. For construction of pACHS-2HA, the DNA 163

fragment containing a 2HA-tag sequence amplified from p2HA-CTC with primers 164

FLAG-SphI-FW and FLAG-BamHI-RV was cloned into pACYC184. To construct 165

pACPJ-2HA that contains the gene encoding the 2HA-tagged protein under control of 166

the sseJ promoter, DNA fragments containing the sseJ promoter region amplified from 167

the genomic DNA of the SL1344 strain with primers ProsseJ-SalI-FW and 168

ProsseJ-SphI-RV and a 2HA-tag sequence amplified from p2HA-CTC with primers 169

FLAG-SphI+ATG-FW and FLAG-BamHI-RV were cloned into pACYC184. To 170

generate the plasmids encoding the C-terminal CyaA-2HA fusion protein, a DNA 171

fragment encoding the catalytic domain of CyaA was amplified from pMS109 with 172

primers cyaA-BglII-FW and cyaA-XhoI-RV and cloned into pACPJ-2HA. The primers 173

used for the construction of all constructs are listed in Table 2. 174

175

Antibodies. Rabbit anti-Salmonella O4 polyclonal antibody (Denka Seiken) was used 176

at a dilution of 1:5000. Mouse polyclonal anti-SseB and rabbit polyclonal anti-PagC 177

antibodies were used as described previously (37, 43). The mouse monoclonal 178

antibodies, anti-FLAG (1:20000) (Sigma), anti-GST (1:2000) (Upstate, Lake Placid, 179

NY), and anti-DnaK (1:2000) (Calbiochem), were used as primary antibodies for 180

immunoblotting. The mouse monoclonal anti-HA epitope tag HA.11 was used at a 181

dilution of 1:2000 (Covance) for immunofluorescence microscopy and immunoblot 182

analysis. Alexa 488-conjugated goat anti-mouse IgG and Alexa 594-conjugated goat 183

anti-rabbit IgG secondary antibodies (dilution of 1:500) were obtained from Molecular 184

Probes. Alkaline phosphatase-conjugated goat anti-mouse and anti-rabbit IgG antibodies 185

were purchased from Sigma and were used at a dilution of 1:10000. 186


.org/D

ownloaded from

http://jb.asm.org/

9

187

Cell culture, bacterial infection and immunofluorescence microscopy. HeLa cells 188

were grown in MEM (Sigma) supplemented with 10% FBS, and were cultured in the 189

presence of gentamicin (100 µg/ml) and kanamycin (60 µg/ml) at 37°C in a 5% CO2 190

atmosphere. Bacterial infections of HeLa cells were performed as described previously 191

(37). For immunofluorescence labelling, cells were fixed, permeabilized, and probed 192

with various antibodies as described previously (37). Labeled cells were analyzed by a 193

Zeiss confocal laser scanning microscope (LSM510 META). 194

195

Protein translocation assay. Translocation of a CyaA fusion protein by SPI-2 T3SS 196

into infected host cells was measured using a cAMP enzyme immunoassay system 197

(Amersham Biosciences) to quantify intracellular levels of cAMP. HeLa cells were 198

infected with Salmonella strains carrying the plasmids encoding CyaA fusion proteins 199

for 22 h. After infection, cells were lysed and processed according to the manufacturer’s 200

instructions. The data represent the mean ± S.D. of triplicate determinants. 201

202

Subcellular fractionation. Salmonella strains grown in LPM medium (pH 5.8) were 203

fractionated. The culture supernatant fraction was obtained as described previously (10). 204

Bacterial fractionation was based on the method of Gauthier et al. (24). Bacteria grown 205

in 100 ml of LPM medium (pH 5.8) for 16 h at 37°C were used for the isolation of 206

cytoplasmic, periplasmic and membrane fractions. Samples were run on 12% 207

SDS-PAGE and transferred to PVDF membranes (Imobilon; Millipore). The gels were 208

hybridized with various antibodies and developed using a Sigma Fast™ BCIP/NBT 209

detection system as described previously (37). 210


.org/D

ownloaded from

http://jb.asm.org/

10

211

Immunoprecipitation. Immunoprecipitation of the FLAG-tagged protein complexes 212

from Salmonella and E. coli lysates was performed by using FLAG beads conjugated 213

with anti-FLAG M2 antibody (Sigma) as previously described (55). Lysates were 214

clarified by centrifugation and incubated with FLAG beads for 2 h at 4°C. Beads were 215

washed seven times with ice-cold PBS containing 1 mM PMSF (PBS-P), and bound 216

proteins were competitively eluted from the beads using FLAG peptide (Sigma) at a 217

final concentration of 90 µg/ml. The eluted protein fraction was then analyzed by 218

SDS-PAGE and Western blotting. 219

220

Pull-down assay. GST and GST-SseB fusion proteins were purified by affinity 221

chromatography on glutathione-Sepharose 4B beads according to the manufacturer’s 222

instructions (GE Healthcare). For GST pull-down assays, glutathione-Sepharose 4B 223

beads immobilized with GST or GST-SseB were incubated with cleared extracts from 224

the Salmonella ssaEmut mutant or E. coli DH5α strains expressing SsaE-FLAG for 2 h at 225

4°C. Beads were washed five times with ice-cold PBS-P and the bound proteins were 226

eluted from the beads using 10 mM glutathione. The eluted protein fraction was then 227

analyzed by SDS-PAGE and Western blotting. 228

229

Mass spectrometry analyses. The S. enterica serovar Typhimurium strain TM312 that 230

expresses SsaE-FLAG was resuspended in PBS containing lysozyme and protease 231

inhibitor cocktails (Roche), disrupted by sonication, and cleared by centrifugation. The 232

sample co-precipitated with SsaE-FLAG using FLAG beads was subjected to trypsin 233

digestion. The digested peptides were analyzed by nano-LC-ESI-MS/MS using a DiNa 234


.org/D

ownloaded from

http://jb.asm.org/

11

nano-LC system (KYA Technologies) with a L-column 2 ODS 0.05 mm × 100 mm, 3 235

µm (CERI, Japan) coupled to a QSTAR Elite hybrid liquid chromatography tandem 236

mass spectrometry (LC/MS/MS) system (Applied Biosystems). Peptide and Protein 237

identification were performed using Protein Pilot version 2.0 software (Applied 238

Biosystems) with default parameters. Each MS/MS spectrum was searched for species 239

of S. enterica serovar Typhimurium against the NCBI-database. 240

241

Mouse mixed infections. Female BALB/c mice (5- to 6-week-old) were used for the 242

mixed infection assay and were housed at Kitasato University according to the standard 243

Laboratory Animal Care Advisory Committee guidelines. At least three mice were 244

inoculated intraperitoneally with a mixture of two strains comprising 5 x 104 cfu of each 245

strain in physiological saline, and the number of viable bacteria from infected spleens 246

was determined at 48 h after infection as described previously (37). The competitive 247

index (CI) was calculated by the formula CI = output (mutant strain/wild-type 248

strain)/inoculum (mutant strain/wild-type strain). In the case of tested two strains that 249

have same virulence, the CI indicates 1.0. In contrast, in the case of the mutated gene 250

that contribute to the virulence, the CI indicates < 1.0. Each CI value is the mean of at 251

least three independent infections ± SD. CI data were analyzed by Mann-Whitney U 252

test for statistical significance. p values of 0.05 or less were considered as significant. 253

254

RESULTS 255

256

SsaE has several features of a T3SS chaperone. In the SPI-2 region of S. enterica 257

serovar Typhimurium, ssaE is flanked by spiB (also referred to as ssaD) and sseA (Fig. 258


.org/D

ownloaded from

http://jb.asm.org/

12

1A). SsaE shares some common characteristics with other type III chaperone proteins 259

(reviewed in (2, 11, 56)). SsaE has a predicted molecular mass of 9.4 kDa and a 260

predicted pI of 4.9, both of which characteristics are common in T3SS chaperones. In 261

addition, SsaE was predicted to have three α-helices by PSI-PRED (30). Among them, 262

α-3 could be predicted as a perfect amphipathic helix in the C-terminus. We searched 263

for similarities between SsaE and other T3SS chaperones using SKE-CHIMERA (54) 264

and the fold recognition algorithm SPARKS2 (61) (Fig. 1B). The model of the 265

three-dimensional structure of the SsaE was built with the FAMS program (45) on the 266

basis of this alignment using a Yersinia pestis T3SS-specific chaperone, YscE (PDB ID: 267

1ZW0), as template (Fig. 1C and D). These observations indicated that SsaE encodes a 268

protein as the T3SS-specific chaperone. 269

270

SsaE is a cytoplasmic protein, but not an effector. The subcellular localization of 271

SsaE was determined by fractionation of a Salmonella strain, followed by SDS-PAGE 272

and immunoblotting. An S. enterica serovar Typhimurium strain encoded 273

chromosomally by an ssaE-FLAG fusion gene was grown in LPM (pH 5.8), and the cell 274

components were separated into cytoplasm, periplasm, and membrane fractions. SsaE 275

(SsaE-FLAG) was not secreted and was localized to the cytoplasmic fraction (Fig. 2A 276

and B). We further attempted to detect translocation of SsaE into the host cell cytosol by 277

confocal immunofluorescence microscopy. As an HA epitope-tagged derivative of 278

effector proteins has been used to detect SPI-2-dependent translocation in host cells (29, 279

32), a plasmid (pSsaE-2HA) expressing SsaE-2HA was generated and transformed into 280

Salmonella wild-type SL1344. In HeLa cells infected with the wild-type strain 281

expressing the SsaE-2HA, SsaE was not detected in the host cell cytosol (data not 282


.org/D

ownloaded from

http://jb.asm.org/

13

shown). Furthermore, a lack of SsaE translocation into host cells was confirmed 283

quantitatively by a cAMP assay using the wild-type strain expressing the 284

SsaE-CyaA-2HA fusion protein. While SsaE-CyaA-2HA was determined to be stable in 285

Salmonella (data not shown), background levels of cAMP were detected in cells 286

infected with the wild-type strain harboring either pACPJ-CyaA-2HA (15.6 ± 0.5 fmol 287

cAMP/µg protein) or harboring pSsaE-CyaA-2HA (15.5 ± 0.6 fmol cAMP/µg protein). 288

In the same assay, the SPI-2 effector SseF-CyaA-2HA fusion protein was used as a 289

positive control for SPI-2 T3SS translocation, and intracellular cAMP was clearly 290

elevated by SseF-CyaA-2HA of wild-type strain (335.8 ± 107.6 fmol cAMP/µg protein) 291

relative to that of SPI-2 T3SS component mutant (∆ssaV) harboring pSseF-CyaA-2HA 292

(10.9 ± 3.0 fmol cAMP/µg protein). These results suggest that SsaE is a cytoplasmic 293

protein, but not an effector translocated by SPI-2 T3SS. 294

295

A Salmonella ssaE mutant strain is deficient in secretion of the SPI-2 T3SS 296

translocator SseB and effector PipB. To investigate the role of SsaE in SPI-2 T3SS, 297

we constructed a site-directed mutation in the initiation codon of ssaE (ssaEmut; ATG to 298

CTT) and then examined the ability of the ssaEmut mutant strain to secrete the 299

translocator SseB into the culture supernatant. As shown in Fig. 3 A, SseB in the 300

Salmonella mutant strain carrying an ssaEmut mutation accumulated within bacterial 301

cells, but was not secreted into the supernatant, when bacteria were grown under 302

conditions that induced SseB expression and secretion. Complementation of the ssaEmut 303

mutant with a wild-type ssaE allele restored the secretion of SseB to a level comparable 304

to that in the wild-type strain. The mutation could be also complemented by ssaE-FALG 305

or ssaE-2HA introduced on a plasmid (data not shown). These results strongly suggest 306


.org/D

ownloaded from

http://jb.asm.org/

14

that SsaE is required for secretion of SseB. In addition, Salmonella strains containing 307

sseAB::lacZ transcriptional fusion on the chromosome both in an ssaE+ and ssaE

- 308

(ssaEmut) background were grown under SPI-2-inducing conditions, and β-galactosidase 309

activity was measured. Consistent with the above results, transcription of sseAB was not 310

affected by loss of SsaE (data not shown). 311

312

Recently, it has been demonstrated that the SPI-2-encoded regulatory molecules SsaL, 313

SsaM, and SpiC, the latter two of which occur in complex, are required for the 314

translocator protein secretion (10, 60). However, mutations of ssaL and ssaM resulted in 315

enhanced secretion of SPI-2 effectors encoded outside of SPI-2 (10, 60). Therefore, to 316

further determine the role of SsaE in the secretion of SPI-2 effectors, we constructed 317

ssaL mutant with an ssaE+ and ssaE

- background, and tested the secretion of PipB. As 318

expected, no detectable SseB was secreted by any of the Salmonella mutants tested. In 319

contrast, the PipB-2HA fusion protein was detected in secreted protein fractions from 320

the ∆ssaL single mutant, but not the double mutants carrying mutations in ∆ssaL and 321

ssaEmut or ∆ssaL and spiAC133S, a point mutation in the outer membrane secretin SpiA 322

(37) (Fig. 3B), suggesting that SsaE is reqired for secretion of PipB in wild-type cells 323

and in cells lacking SsaL. Similar results were obtained from other SPI-2 effectors, 324

SopD2 (SopD2-2HA), and SspH2 (SspH2-2HA) or when the Salmonella ∆ssaM mutant 325

was used (data not shown). Thus, phenotypically, a mutant lacking SsaE is different 326

from a mutant lacking SsaL or SsaM. 327

328

Identification of SsaE-binding partners. Several physical features and predicted 329

structural characteristics of SsaE indicated that SsaE functions as a chaperone. 330


.org/D

ownloaded from

http://jb.asm.org/

15

Therefore, to identify the SsaE partner proteins encoded by SPI-2 in Salmonella, we 331

next performed co-immunoprecipitation assays using lysates prepared from the 332

wild-type strain expressing SsaE-FLAG grown under SPI-2-inducing conditions. The 333

bound proteins were eluted and subjected to mass spectrometry analyses. Seven SPI-2 334

proteins, i.e., SsaH, SsaK, SsaN, SsaQ, SseA, SseB, and SseC, were identified as 335

SsaE-interacting proteins. In general, translocator proteins of T3SSs are known to 336

require chaperones before secretion by the T3SS pathway. Thus, we further 337

characterized the SsaE interaction with SPI-2 T3SS translocator proteins SseB and 338

SseC. 339

340

To examine the binding specificity of SsaE with SseB, we performed FLAG pull-down 341

assays using lysates prepared from the wild-type strain SL1344 carrying a plasmid 342

(pSsaE-FLAG) expressing SsaE-FLAG. The bound protein was analyzed by SDS-PAGE 343

and immunoblotting with mouse anti-SseB antibody. Controls for binding specificity 344

included binding to bacterial alkaline phosphatase (BAP)-FLAG and blotting with 345

anti-PagC antibody, as PagC is a SPI-2-unrelated outer membrane protein. The 346

wild-type strain harboring a plasmid (pSsaE) expressing untagged SsaE was also used 347

as a control for non-specific binding to anti-FLAG beads. The results showed that SseB 348

was co-precipitated by SsaE-FLAG but not BAP-FLAG or untagged SsaE (Fig. 4A, B 349

and data not shown). In contrast, PagC was co-precipitated by neither SsaE-FLAG, 350

untagged SsaE, or BAP-FLAG, suggesting that SsaE is capable of interacting with SseB 351

(Fig. 4A, B and data not shown). This interaction was also confirmed by reverse 352

experiments using Salmonella extracts from the wild-type strain expressing 353

SseB-FLAG and SsaE-2HA grown under the same conditions (data not shown). 354


.org/D

ownloaded from

http://jb.asm.org/

16

355

Next, to clarify whether the interaction between SsaE and SseB is direct or requires 356

other SPI-2 proteins bridging two proteins, we performed GST pull-down assays using 357

SsaE-FLAG expressed in E. coli DH5α that does not encode the Salmonella SPI-2 358

proteins. Cell lysate was incubated with glutathione-Sepharose beads conjugated with 359

GST-SseB or GST, and the bound proteins were analyzed by SDS-PAGE and 360

immunoblotting with anti-FLAG antibody. SsaE-FLAG was co-eluted with GST-SseB, 361

whereas GST alone did not bind to the SsaE-FLAG (Fig. 4D). These results strongly 362

suggest that SsaE interacts with SseB and none of the additional proteins encoded by 363

SPI-2 are required for this interaction. 364

365

Under the same assay conditions, when SsaE-FLAG and SseC-2HA were co-expressed 366

from plasmids (pSsaE-FLAG and p2HA-SseC) in the S. enterica serovar Typhimurium, 367

SseC-2HA co-immunoprecipitated with SsaE-FLAG onto FLAG beads (data not 368

shown). However, SseC-2HA did not co-elute with SsaE-FLAG-containing beads when 369

SseC-2HA was expressed in E. coli (data not shown), indicating that the interaction 370

between SsaE and SseC is indirect binding. 371

372

The coiled-coil domain in the C-terminal region of SsaE is critical for interaction 373

with SseB. SsaE is predicted to have three α-helical regions (the amino acids spanning 374

regions 5 to 11, 24 to 32, and 52 to 68; Fig. 1B and C). Thus, to identify the SsaE 375

domain involved in interaction with partner proteins, we constructed a series of 376

SsaE-FLAG derivatives and subjected the resulting proteins, pSsaE∆1-FLAG 377

(SsaE21-80-FLAG), pSsaE∆2-FLAG (SsaE∆21-41-FLAG), and pSsaE∆3-FLAG 378


.org/D

ownloaded from

http://jb.asm.org/

17

(SsaE∆52-68-FLAG), to FLAG pull-down assays using bacterial lysates prepared from the 379

Salmonella wild-type strain (Fig. 5 A). Similar to the full-length SsaE-FLAG, SseB was 380

precipitated with SsaE∆1-FLAG and SsaE∆2-FLAG, but not with SsaE∆3-FLAG (Fig. 381

5 A), showing that SseB interacts with the C-terminal portion of SsaE encompassing 382

amino acid residues 52 to 68. 383

384

Coiled-coil sequences in proteins consist of seven residues, termed the heptad repeat 385

(a-b-c-d-e-f-g)n, which include the non-polar hydrophobic residues at positions a and d 386

(25). Residues in positions a and d typically form a core in the center of the α-helical 387

bundle, providing the driving force for protein-protein interaction (25, 36). The Ile-55 of 388

the SsaE protein within the C-terminal coiled-coil sequence was determined to be a 389

non-polar hydrophobic residue at position a by the Coils program 390

(http://www.ch.embnet.org/software/COILS_form.html), which is a tool for predicting 391

coiled-coil domains (Fig. 5B). Thus, to further confirm the importance of the C-terminal 392

coiled-coil domain of SsaE for protein-protein interaction, site-directed mutagenesis 393

was performed by using the plasmid pSsaE-FLAG as a template and replacing the 394

Ile-55 with glycine. While the resulting SsaEI55G-FLAG protein lacking the C-terminal 395

coiled-coil structure was stable in Salmonella, a substitution of Ile-55 to glycine in SsaE 396

resulted in a loss of the ability to interact with SseB (Fig. 5A). 397

398

Binding of SseB to SsaE is required for secretion of SseB. Next, to investigate the 399

functional importance of the C-terminal coiled-coil domain of SsaE, we constructed a 400

Salmonella mutant strain expressing SsaEI55G (chromosome mutant) and examined its 401

SseB secretion. This mutant strain was unable to secrete SseB into the supernatant in 402


.org/D

ownloaded from

http://jb.asm.org/

18

LPM medium at pH 5.8, and the SseB secretion could be restored by introduction of the 403

wild-type ssaE gene on the plasmid into the SsaEI55G-expressing Salmonella strain (Fig. 404

6A). These results suggest that the interaction between SseB and SsaE is essential for 405

SseB secretion. In addition, we further examined the secretion of SPI-2 effector PipB in 406

an ssaEI55G mutant strain. Similar to SseB, less PipB-2HA fusion protein was secreted 407

into the supernatant from ssaEI55G mutant strain (Fig. 6B), suggesting that the 408

C-terminal coiled-coil domain of SsaE is important for secretion of the SPI-2 effectors. 409

410

It has been reported that SseA functions as a chaperone for SseB and is required for 411

stabilization of SseB in the bacterial cytosol and for SseB export to the bacterial surface 412

(9, 49, 62, 63). Therefore, to demonstrate the presence of the SseA-SseB-SsaE protein 413

complex in the Salmonella cytoplasm, the immunoprecipitation by FLAG pull-down 414

assays using lysates from the Salmonella strain expressing SseA-2HA (chromosomal 415

mutation) harboring the plasmids either pSsaE-FLAG or pBAP-FLAG were carried out. 416

The pulled-down proteins were detected by immunoblotting with anti-FLAG (for 417

SsaE-FLAG and BAP-FLAG), anti-HA (for SseA-2HA) and anti-SseB antibodies. As 418

shown in Fig. 6C, SseA-2HA and SseB were co-precipitated by SsaE-FLAG, but not 419

BAP-FLAG (data not shown). Thus, both SseA and SsaE were able to associate with 420

SseB in the Salmonella cytoplasm. 421

422

We next confirmed that SseA plays a role in SseB secretion using a Salmonella sseA 423

mutant strain. As previously reported, the sseA mutant strain exhibited a dramatic 424

decrease in the intracellular accumulation of SseB compared to that of the wild-type 425

strain, and secreted SseB was not detectable in the supernatant fraction by Western blot 426


.org/D

ownloaded from

http://jb.asm.org/

19

analysis (data not shown). To rule out the possibility that reduced intracellular 427

production of SseB responsible for the defective secretion in the sseA mutant, we 428

constructed a plasmid (pACYC-SseB) expressing SseB constitutively and introduced it 429

into the wild-type strain and mutant strains deficient in sseA and ssaE genes. In addition, 430

Salmonella strains that lack SseB and SsaV harboring pACYC-SseB were used as 431

positive and negative controls for the SseB secretion. SseB was found in the 432

cytoplasmic fraction at levels similar to those for the wild-type strain when mutant 433

strains harbored a plasmid expressing SseB constitutively (Fig. 6D). However, no 434

detectable SseB was found by Western blot analysis of the supernatant fraction in the 435

ssaEmut and ssaEI55G mutant strains harboring the plasmid pACYC-SseB (Fig. 6D and 436

data not shown). In contrast, unlike in a previously reported study (49), SseB was easily 437

detected in the supernatant of the sseA mutant strain harboring pACYC-SseB (Fig. 6D). 438

The same results were obtained when detached fractions containing bacterial surface 439

proteins from these strains were analyzed by Western blotting using anti-SseB antibody 440

(data not shown). Our results suggest that, in addition to SseA, SsaE plays an important 441

role in SseB secretion. 442

443

SsaE, but not SseA, interacts with the putative SPI-2 T3SS ATPase SsaN. The type 444

III secretion apparatus is associated with an ATPase that presumably provides the 445

energy for the secretion process. Interestingly, mass spectrometer analysis revealed that 446

SsaE co-eluted the predicted SPI-2 T3SS ATPase SsaN. We thus examined whether the 447

binding of SsaE to SsaN could be mediated by direct SsaE-SsaN interaction or bridging 448

interactions with other SPI-2 proteins. To confirm this, SsaE was expressed in E. coli as 449

a FLAG-tagged fusion protein, and SsaN was expressed as a 2HA-tagged fusion protein. 450


.org/D

ownloaded from

http://jb.asm.org/

20

SsaE-FLAG-fusion protein prepared from E. coli lysate was immobilized on FLAG 451

beads and then the SsaN-2HA fusion protein prepared from E. coli lysate was incubated 452

with FLAG-fusion protein immobilized beads. While the BAP-FLAG control protein 453

did not interact with SsaN-2HA (data not shown), SsaE-FLAG precipitated the 454

SsaN-2HA (Fig. 7A). This binding required the C-terminal coiled-coil domain of SsaE, 455

since SsaEI55G-FLAG and SsaE∆3 did not pull-down SsaN-2HA (Fig. 7B and data not 456

shown). 457

458

Furthermore, we investigated whether the SPI-2 T3SS chaperone SseA interacts with 459

SsaN. To do this, we performed FLAG pull-down assays using the E. coli lysates 460

expressing SseA-FLAG and SsaN-2HA fusion proteins. As shown in Fig. 7C, there was 461

no detectable interaction between SsaN-2HA and SseA-FLAG. These results suggest 462

that SsaE, but not SseA, interacts directly with SsaN, and this interaction requires the 463

C-terminal coiled-coil domain of SsaE. 464

465

SsaE is required for virulence. Finally, to examine the virulence function of ssaE in 466

vivo, we performed mixed infections in mice. Control experiments with the wild-type 467

strain and the wild-type strain harboring pMW118 showed a competitive index (CI) of 468

1.13 ± 0.19. The CI of the wild-type strain versus the ssaEI55G mutant strain harboring 469

pMW118 was significantly decresed to 0.047 ± 0.013 (p<0.01), compared to the SPI-2 470

mutant strain (the CI of the wild-type strain versus the ∆ssaV mutant harboring 471

pMW118 was 0.017 ± 0.026). In addition, the CI of the wild-type strain versus the 472

ssaEI55G mutant strain expressing intact SsaE from a plasmid was 1.16 ± 0.21 (no 473

significant difference from the CI of the wild-type strain), showing that the replication 474


.org/D

ownloaded from

http://jb.asm.org/

21

defect of the ssaE point-mutant strain was due to the loss of SsaE function. These 475

results clearly demonstrate that ssaE contributes to Salmonella virulence in the mouse 476

model of systemic infection. 477

478

DISCUSSION 479

480

Bacterial type III secretion machines are involved in the transport of virulence effectors 481

directly into the cytoplasm of target cells (22). In the initial stages of the assembly of 482

the type III secretion apparatus, the T3SS is involved in protein secretion into the 483

extracellular space, while efficient translocation of effectors to the host cells takes place 484

when the T3SS is completely developed. In this study, we have characterized SsaE, 485

which is encoded by an operon for components of the type III secretion apparatus 486

within the SPI-2 locus. We show that SsaE is localized in the bacterial cytoplasm, 487

indicating that SsaE functions as neither a SPI-2 T3SS core component nor an effector. 488

The structural model of SsaE indicates that it is a protein similar to class V chaperones 489

of YscE family. Similar to generalized SPI-2 core component mutants, the S. enterica 490

serovar Typhimurium strain lacking SsaE failed to secrete translocator and effector 491

proteins, suggesting that ssaE is required for the complete SPI-2 T3SS function. In 492

addition, we demonstrated that SsaE was involved in Salmonella pathogenesis in a 493

mouse model of infection. 494

495

Generally, five different classes of T3SS proteins - i.e., proteins for apparatus, 496

translocators, effectors, chaperones, and regulators - are encoded by pathogenicity 497

islands. In addition to these proteins, a new class of type III secretion proteins has 498


.org/D

ownloaded from

http://jb.asm.org/

22

recently been identified. This class of proteins is intracellularly localized and is 499

necessary for the ordered secretion of translocator and effector proteins through the 500

T3SS pathway. In Salmonella, InvE of SPI-1 (34) and SsaL and SsaM/SpiC of SPI-2 501

(10, 60) are suggested to be involved in the recognition of translocator complexes and to 502

establish the ordered secretion. Since some studies suggest that chaperones play a role 503

in setting the secretion hierarchy (3, 5), it is possible to speculate that SsaE is a protein 504

that can distinguish translocator protein SseB from other secreted proteins, and enable 505

the export of SseB at the appropriate time. In this study, however, we showed that while 506

Salmonella mutant strains carrying either SsaE, SsaL or SsaM mutation could not 507

secrete SseB into the culture supernatant, SsaL and SsaM mutants, but not the SsaE 508

mutant, were able to transport the SPI-2-dependent effectors including PipB, SopD2 and 509

SspH2, extracellularly. Thus, SsaE is functionally different from SsaL and SsaM/SpiC. 510

From these results, although we could not provide evidence concerning the effect of 511

ssaE mutation on the SPI-2 type III secretion apparatus, it is possible to note that, like 512

other T3SS class V chaperones, the inability of ssaE mutant strains to secrete SPI-2 513

effectors is due to failure to assemble the needle complex. Further analyses of secretion 514

and/or translocation of SPI-2 effectors by ssaE-deficient mutant strains are needed to 515

clarify SsaE function on SPI-2 type III secretion apparatus assembly. 516

517

The homologues of SsaE are conserved in several bacterial pathogens, including PscE 518

of P. aeruginosa (16%), YscE of Y. pestis (25%), EscE of EPEC (27%), and AscE of A. 519

hydrophila (33%), which function as a T3SS chaperone (class V). While Salmonella 520

SsaE shares some common characteristics with these T3SS class V chaperones, the role 521

of SsaE as a chaperone for SseB appears to be unique. In P. aeruginosa, PscE forms a 522


.org/D

ownloaded from

http://jb.asm.org/

23

1:1:1 ternary heteromolecular complex composed of a putative chaperone PscG and a 523

needle component protein PscF, which is necessary to maintain a monomeric state of 524

PscF (47). Recently, the crystal structures of the PscE/PscF55-85/PscG complex (48), the 525

YscE/YscF/YscG complex (53), and the AscE/AscF/AscG complex (39) have resolved 526

the detailed protein-protein interactions between the chaperone-needle subunit complex 527

of T3SS. However, Salmonella harbors none of the PscG/YscG/AscG homologues. In 528

addition, SsaE is not associated with the stability of binding partner SseB, since the 529

ssaE mutant strain could express SseB at the same level as the wild-type strain, and 530

SseA has been shown to be required for stable expression of SseB as a chaperone (49, 531

62). These findings provide evidence that Salmonella SsaE should be categorized 532

structurally as a class V chaperone, although it has a distinct function from other class V 533

chaperones. 534

535

Although SseA has previously been found to be essential for export of SseB to the 536

bacterial surface in addition its principal role in stabilization of SseB within the 537

bacterial cytoplasm (49), our results showed that SseA is dispensable for SseB secretion 538

into culture supernatant. This apparent inconsistency is probably due to the difference in 539

sample-preparation procedures between the present study and the previous ones. We 540

adopted the in vitro secretion assays established by Coombes et al. (2004) (10) to detect 541

SPI-2-dependent secreted proteins using minimal medium previously reported to induce 542

the expression of SPI-2 genes (42) and an antibody specific for SseB (37). This 543

procedure is optimized for isolation of SPI-2 translocator proteins without the need to 544

extract the bacterial surface with n-hexadecane (49) or mechanical shearing (42), which 545

had previously been required to detect sufficient levels of SPI-2 translocator proteins. 546


.org/D

ownloaded from

http://jb.asm.org/

24

Under the same assay conditions, the ssaE mutant strain did not secrete SseB into the 547

supernatant, suggesting that SsaE, rather than SseA, is essential for export of SseB. 548

549

Multiple chaperones have been reported to act with T3SS translocators for their 550

complete secretion. For example, EspA of EPEC is one of the T3SS substrates that 551

forms a needle structure on the bacterial membrane surface, and efficient secretion of 552

EspA requires two distinct chaperones, CesAB and CesA2. These chaperones increase 553

the stability of EspA and show direct EspA-binding activity (12, 13, 52). Recently, in 554

addition to CesAB and CesA2, it has been reported that EscL interacts directly with 555

EspA and enhances the stability of intracellular EspA, and is also essential for the 556

complete secretion of EspA (33). Similarly, we found that two small T3SS molecules, 557

SsaE and SseA, function as chaperones for the Salmonella SPI-2 translocator SseB, an 558

EspA homologue. Both chaperones have some common chaperone properties, which 559

include a low molecular mass (>15 kDa), an acid pI, and a C-terminal amphipathic helix 560

(2, 11, 56). However, these chaperones are functionally different, since SsaE associates 561

with the putative T3SS-associated ATPase SsaN, while SseA does not. Recent evidence 562

strongly suggests that the T3SS-associated ATPase promotes the initial docking of T3SS 563

substrates to the secretion apparatus, and subsequent translocation of proteins via T3SS 564

dependents on the protein motive force (1, 39, 46, 58). Thus, it might be expected that 565

SseB is efficiently escorted by SsaE to the type III secretion apparatus via specific 566

binding to SsaN. 567

568

In conclusion, the SPI-2-encoded small protein SsaE was shown to interact with the 569

T3SS translocator SseB and the putative ATPase SsaN. The C-terminal coiled-coil 570


.org/D

ownloaded from

http://jb.asm.org/

25

domain of SsaE is critical for these protein-protein interactions. Furthermore, a point 571

mutation of Ile55 to glycine in SsaE completely abolishes the function of SsaE, and a 572

Salmonella strain expressing the point-mutated SsaE exhibits attenuated virulence in 573

mice, much like the SPI-2 component null mutant, suggesting that the coiled-coil 574

mediated protein-protein interactions of SsaE are absolutely essential for the functional 575

expression of SPI-2 T3SS. Since no T3SS component has been found to have a function 576

identical to that of SsaE, further studies will be needed to identify T3SS proteins that 577

play a role equivalent to that of SsaE. 578

579

ACKNOWLEDGMENTS 580

581

We are grateful to Kazuhiko Kanou and Hideaki Umeyama for the protein modeling and 582

for their helpful input. We also thank Seisuke Hattori for advice on mass spectrometry 583

experiments. 584

585

This work was supported in part by a Grant-in-Aid for Young Scientists (B) (17790292) 586

from the Japanese Ministry of Education, Culture, Sports, Science, and Technology and 587

by a Grant-in-Aid for Scientific Research C (21590490) from the Japan Society for the 588

Promotion of Science. Support (to T. M.) was also received in the form of a Kitasato 589

University Research Grant for Young Researchers (2009). 590

591

REFERENCES 592

593

1. Akeda, Y., and J. E. Galan. 2005. Chaperone release and unfolding of 594 substrates in type III secretion. Nature 437:911-5. 595

2. Bennett, J. C., and C. Hughes. 2000. From flagellum assembly to virulence: 596 the extended family of type III export chaperones. Trends Microbiol 8:202-4. 597


.org/D

ownloaded from

http://jb.asm.org/

26

3. Birtalan, S. C., R. M. Phillips, and P. Ghosh. 2002. Three-dimensional 598 secretion signals in chaperone-effector complexes of bacterial pathogens. Mol 599 Cell 9:971-80. 600

4. Blocker, A., K. Komoriya, and S. Aizawa. 2003. Type III secretion systems 601 and bacterial flagella: insights into their function from structural similarities. 602 Proc Natl Acad Sci U S A 100:3027-30. 603

5. Boyd, A. P., I. Lambermont, and G. R. Cornelis. 2000. Competition between 604 the Yops of Yersinia enterocolitica for delivery into eukaryotic cells: role of the 605 SycE chaperone binding domain of YopE. J Bacteriol 182:4811-21. 606

6. Broms, J. E., P. J. Edqvist, K. E. Carlsson, A. Forsberg, and M. S. Francis. 607 2005. Mapping of a YscY binding domain within the LcrH chaperone that is 608 required for regulation of Yersinia type III secretion. J Bacteriol 187:7738-52. 609

7. Casadaban, M. J., and S. N. Cohen. 1980. Analysis of gene control signals by 610 DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179-207. 611

8. Cirillo, D. M., R. H. Valdivia, D. M. Monack, and S. Falkow. 1998. 612 Macrophage-dependent induction of the Salmonella pathogenicity island 2 type 613 III secretion system and its role in intracellular survival. Mol Microbiol 614 30:175-88. 615

9. Coombes, B. K., N. F. Brown, S. Kujat-Choy, B. A. Vallance, and B. B. 616 Finlay. 2003. SseA is required for translocation of Salmonella pathogenicity 617 island-2 effectors into host cells. Microbes Infect 5:561-70. 618

10. Coombes, B. K., N. F. Brown, Y. Valdez, J. H. Brumell, and B. B. Finlay. 619 2004. Expression and secretion of Salmonella pathogenicity island-2 virulence 620 genes in response to acidification exhibit differential requirements of a 621 functional type III secretion apparatus and SsaL. J Biol Chem 279:49804-15. 622

11. Cornelis, G. R., and F. Van Gijsegem. 2000. Assembly and function of type III 623 secretory systems. Annu Rev Microbiol 54:735-74. 624

12. Creasey, E. A., R. M. Delahay, S. J. Daniell, and G. Frankel. 2003. Yeast 625 two-hybrid system survey of interactions between LEE-encoded proteins of 626 enteropathogenic Escherichia coli. Microbiology 149:2093-106. 627

13. Creasey, E. A., D. Friedberg, R. K. Shaw, T. Umanski, S. Knutton, I. 628 Rosenshine, and G. Frankel. 2003. CesAB is an enteropathogenic Escherichia 629 coli chaperone for the type-III translocator proteins EspA and EspB. 630 Microbiology 149:3639-47. 631

14. Dai, S., and D. Zhou. 2004. Secretion and function of Salmonella SPI-2 632 effector SseF require its chaperone, SscB. J Bacteriol 186:5078-86. 633

15. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of 634 chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl 635 Acad Sci U S A 97:6640-5. 636

16. Day, J. B., I. Guller, and G. V. Plano. 2000. Yersinia pestis YscG protein is a 637 Syc-like chaperone that directly binds YscE. Infect Immun 68:6466-71. 638

17. DeLano, W. L. 2002. The PyMoL molecular graphics system. DeLAno 639 Scientific, Palo Alto, CA, USA. 640

18. DeLano, W. L. 2002. Unraveling hot spots in binding interfaces: progress and 641 challenges. Curr Opin Struct Biol 12:14-20. 642

19. Evdokimov, A. G., J. Phan, J. E. Tropea, K. M. Routzahn, H. K. Peters, M. 643 Pokross, and D. S. Waugh. 2003. Similar modes of polypeptide recognition by 644 export chaperones in flagellar biosynthesis and type III secretion. Nat Struct 645


.org/D

ownloaded from

http://jb.asm.org/

27

Biol 10:789-93. 646 20. Francis, M. S., S. A. Lloyd, and H. Wolf-Watz. 2001. The type III secretion 647

chaperone LcrH co-operates with YopD to establish a negative, regulatory loop 648 for control of Yop synthesis in Yersinia pseudotuberculosis. Mol Microbiol 649 42:1075-93. 650

21. Frishman, D., and P. Argos. 1995. Knowledge-based protein secondary 651 structure assignment. Proteins 23:566-79. 652

22. Galan, J. E., and A. Collmer. 1999. Type III secretion machines: bacterial 653 devices for protein delivery into host cells. Science 284:1322-8. 654

23. Garcia-del Portillo, F., M. B. Zwick, K. Y. Leung, and B. B. Finlay. 1993. 655 Salmonella induces the formation of filamentous structures containing 656 lysosomal membrane glycoproteins in epithelial cells. Proc Natl Acad Sci U S A 657 90:10544-8. 658

24. Gauthier, A., J. L. Puente, and B. B. Finlay. 2003. Secretin of the 659 enteropathogenic Escherichia coli type III secretion system requires components 660 of the type III apparatus for assembly and localization. Infect Immun 71:3310-9. 661

25. Harbury, P. B., T. Zhang, P. S. Kim, and T. Alber. 1993. A switch between 662 two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. 663 Science 262:1401-7. 664

26. Hensel, M., J. E. Shea, S. R. Waterman, R. Mundy, T. Nikolaus, G. Banks, A. 665 Vazquez-Torres, C. Gleeson, F. C. Fang, and D. W. Holden. 1998. Genes 666 encoding putative effector proteins of the type III secretion system of 667 Salmonella pathogenicity island 2 are required for bacterial virulence and 668 proliferation in macrophages. Mol Microbiol 30:163-74. 669

27. Hoiseth, S. K., and B. A. Stocker. 1981. Aromatic-dependent Salmonella 670 typhimurium are non-virulent and effective as live vaccines. Nature 671 291:238-239. 672

28. Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of 673 animals and plants. Microbiol Mol Biol Rev 62:379-433. 674

29. Jantsch, J., C. Cheminay, D. Chakravortty, T. Lindig, J. Hein, and M. 675 Hensel. 2003. Intracellular activities of Salmonella enterica in murine dendritic 676 cells. Cell Microbiol 5:933-45. 677

30. Jones, D. T. 1999. Protein secondary structure prediction based on 678 position-specific scoring matrices. J Mol Biol 292:195-202. 679

31. Klein, J. R., and B. D. Jones. 2001. Salmonella pathogenicity island 2-encoded 680 proteins SseC and SseD are essential for virulence and are substrates of the type 681 III secretion system. Infect Immun 69:737-43. 682

32. Knodler, L. A., J. Celli, W. D. Hardt, B. A. Vallance, C. Yip, and B. B. Finlay. 683 2002. Salmonella effectors within a single pathogenicity island are differentially 684 expressed and translocated by separate type III secretion systems. Mol Microbiol 685 43:1089-103. 686

33. Ku, C. P., J. C. Lio, S. H. Wang, C. N. Lin, and W. J. Syu. 2009. 687 Identification of a third EspA-binding protein that forms part of the type III 688 secretion system of enterohemorrhagic Escherichia coli. J Biol Chem 689 284:1686-93. 690

34. Kubori, T., and J. E. Galan. 2002. Salmonella type III secretion-associated 691 protein InvE controls translocation of effector proteins into host cells. J 692 Bacteriol 184:4699-708. 693


.org/D

ownloaded from

http://jb.asm.org/

28

35. Lee, S. H., and J. E. Galan. 2004. Salmonella type III secretion-associated 694 chaperones confer secretion-pathway specificity. Mol Microbiol 51:483-95. 695

36. Lupas, A. 1996. Coiled coils: new structures and new functions. Trends 696 Biochem Sci 21:375-82. 697

37. Miki, T., N. Okada, and H. Danbara. 2004. Two periplasmic disulfide 698 oxidoreductases, DsbA and SrgA, target outer membrane protein SpiA, a 699 component of the Salmonella pathogenicity island 2 type III secretion system. J 700 Biol Chem 279:34631-42. 701

38. Miller, V. L., and J. J. Mekalanos. 1988. A novel suicide vector and its use in 702 construction of insertion mutations: osmoregulation of outer membrane proteins 703 and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 704 170:2575-83. 705

39. Minamino, T., and K. Namba. 2008. Distinct roles of the FliI ATPase and 706 proton motive force in bacterial flagellar protein export. Nature 451:485-8. 707

40. Morona, R., L. van den Bosch, and P. A. Manning. 1995. Molecular, genetic, 708 and topological characterization of O-antigen chain length regulation in Shigella 709 flexneri. J Bacteriol 177:1059-68. 710

41. Mota, L. J., L. Journet, I. Sorg, C. Agrain, and G. R. Cornelis. 2005. 711 Bacterial injectisomes: needle length does matter. Science 307:1278. 712

42. Nikolaus, T., J. Deiwick, C. Rappl, J. A. Freeman, W. Schroder, S. I. Miller, 713 and M. Hensel. 2001. SseBCD proteins are secreted by the type III secretion 714 system of Salmonella pathogenicity island 2 and function as a translocon. J 715 Bacteriol 183:6036-45. 716

43. Nishio, M., N. Okada, T. Miki, T. Haneda, and H. Danbara. 2005. 717 Identification of the outer-membrane protein PagC required for the serum 718 resistance phenotype in Salmonella enterica serovar Choleraesuis. Microbiology 719 151:863-73. 720

44. Ochman, H., F. C. Soncini, F. Solomon, and E. A. Groisman. 1996. 721 Identification of a pathogenicity island required for Salmonella survival in host 722 cells. Proc Natl Acad Sci U S A 93:7800-4. 723

45. Ogata, K., and H. Umeyama. 2000. An automatic homology modeling method 724 consisting of database searches and simulated annealing. J Mol Graph Model 725 18:258-72, 305-6. 726

46. Paul, K., M. Erhardt, T. Hirano, D. F. Blair, and K. T. Hughes. 2008. Energy 727 source of flagellar type III secretion. Nature 451:489-92. 728

47. Quinaud, M., J. Chabert, E. Faudry, E. Neumann, D. Lemaire, A. Pastor, S. 729 Elsen, A. Dessen, and I. Attree. 2005. The PscE-PscF-PscG complex controls 730 type III secretion needle biogenesis in Pseudomonas aeruginosa. J Biol Chem 731 280:36293-300. 732

48. Quinaud, M., S. Ple, V. Job, C. Contreras-Martel, J. P. Simorre, I. Attree, 733 and A. Dessen. 2007. Structure of the heterotrimeric complex that regulates type 734 III secretion needle formation. Proc Natl Acad Sci U S A 104:7803-8. 735

49. Ruiz-Albert, J., R. Mundy, X. J. Yu, C. R. Beuzon, and D. W. Holden. 2003. 736 SseA is a chaperone for the SseB and SseD translocon components of the 737 Salmonella pathogenicity-island-2-encoded type III secretion system. 738 Microbiology 149:1103-11. 739

50. Shea, J. E., M. Hensel, C. Gleeson, and D. W. Holden. 1996. Identification of 740 a virulence locus encoding a second type III secretion system in Salmonella 741


.org/D

ownloaded from

http://jb.asm.org/

29

typhimurium. Proc Natl Acad Sci U S A 93:2593-7. 742 51. Sory, M. P., and G. R. Cornelis. 1994. Translocation of a hybrid 743

YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol 744 Microbiol 14:583-94. 745

52. Su, M. S., H. C. Kao, C. N. Lin, and W. J. Syu. 2008. Gene l0017 encodes a 746 second chaperone for EspA of enterohaemorrhagic Escherichia coli O157 : H7. 747 Microbiology 154:1094-103. 748

53. Sun, P., J. E. Tropea, B. P. Austin, S. Cherry, and D. S. Waugh. 2008. 749 Structural characterization of the Yersinia pestis type III secretion system needle 750 protein YscF in complex with its heterodimeric chaperone YscE/YscG. J Mol 751 Biol 377:819-30. 752

54. Takeda-Shitaka, M., G. Terashi, D. Takaya, K. Kanou, M. Iwadate, and H. 753 Umeyama. 2005. Protein structure prediction in CASP6 using CHIMERA and 754 FAMS. Proteins 61 Suppl 7:122-7. 755

55. Thomas, N. A., W. Deng, J. L. Puente, E. A. Frey, C. K. Yip, N. C. 756 Strynadka, and B. B. Finlay. 2005. CesT is a multi-effector chaperone and 757 recruitment factor required for the efficient type III secretion of both LEE- and 758 non-LEE-encoded effectors of enteropathogenic Escherichia coli. Mol 759 Microbiol 57:1762-79. 760

56. Wattiau, P., B. Bernier, P. Deslee, T. Michiels, and G. R. Cornelis. 1994. 761 Individual chaperones required for Yop secretion by Yersinia. Proc Natl Acad 762 Sci U S A 91:10493-7. 763

57. Wilharm, G., S. Dittmann, A. Schmid, and J. Heesemann. 2007. On the role 764 of specific chaperones, the specific ATPase, and the proton motive force in type 765 III secretion. Int J Med Microbiol 297:27-36. 766

58. Wilharm, G., V. Lehmann, K. Krauss, B. Lehnert, S. Richter, K. 767 Ruckdeschel, J. Heesemann, and K. Trulzsch. 2004. Yersinia enterocolitica 768 type III secretion depends on the proton motive force but not on the flagellar 769 motor components MotA and MotB. Infect Immun 72:4004-9. 770

59. Yip, C. K., B. B. Finlay, and N. C. Strynadka. 2005. Structural 771 characterization of a type III secretion system filament protein in complex with 772 its chaperone. Nat Struct Mol Biol 12:75-81. 773

60. Yu, X. J., M. Liu, and D. W. Holden. 2004. SsaM and SpiC interact and 774 regulate secretion of Salmonella pathogenicity island 2 type III secretion system 775 effectors and translocators. Mol Microbiol 54:604-19. 776

61. Zhou, H., and Y. Zhou. 2004. Single-body residue-level knowledge-based 777 energy score combined with sequence-profile and secondary structure 778 information for fold recognition. Proteins 55:1005-13. 779

62. Zurawski, D. V., and M. A. Stein. 2003. SseA acts as the chaperone for the 780 SseB component of the Salmonella Pathogenicity Island 2 translocon. Mol 781 Microbiol 47:1341-51. 782

63. Zurawski, D. V., and M. A. Stein. 2004. The SPI2-encoded SseA chaperone has 783 discrete domains required for SseB stabilization and export, and binds within the 784 C-terminus of SseB and SseD. Microbiology 150:2055-68. 785

786 787


.org/D

ownloaded from

http://jb.asm.org/

30

788 FIGURE LEGENDS 789

790

Figure 1. SsaE encodes a protein that represents structurally the characteristics of 791

class V chaperones. (A) Genetic organization of SPI-2 genes involved in the virulence 792

of S. enterica serovar Typhimurium. Six representative promoters (P) within SPI-2 are 793

indicated. Genes encoding proteins for apparatus (yellow), effectors and translocators 794

(red), chaperones (orange), and regulators (green) are colored. The ssaE gene is 795

indicated by blue. The genes spiBAC and spiR are also referred to as ssaDCB and ssrA, 796

respectively. (B) Sequence alignment between SsaE and its reference structure of Y. 797

pestis YscE (PDB ID: 1ZW0). The numbering of the SsaE primary sequence is 798

indicated above the sequences. The secondary structure elements are indicated under the 799

sequences. The regions of SsaE that were predicted to be α-helices by PSI-PRED (30) 800

are highlighted in red. The regions of YscE that were assigned as α-helices by STRIDE 801

(21) are highlighted in red. (C) Ribbon representation of SsaE predicted by the FAMS 802

program (45) in stereo view at two different angles. Images were prepared using the 803

program PyMol (17, 18). Three predicted α-helical regions, α1, α2 and α3, are 804

indicated. (D) Superposotion of the model of SsaE (green) and the structure of YscE 805

(yellow). The root mean square deviation of these structures is 0.26Å. 806

807

Figure 2. SsaE is a cytoplasmic protein. (A) The S. enterica serovar Typhimurium 808

wild-type strain expressing the SsaE-FLAG from the chromosome was grown in LPM 809

medium (pH 5.8). Bacterial cells were fractionated into periplasm, cytoplasm and 810

membrane fractions. The fractions were analyzed by Western blotting using anti-FLAG 811

antibody to detect SsaE-FLAG. DnaK and PagC were used as markers of the 812

cytoplasmic and membrane protein fractions, respectively. (B) Western blot analysis of 813

secreted proteins (Supernatant) and whole cell lysates (Cell lysate) prepared from the 814

wild-type, the spiAC133S mutant and the wild-type strain expressing SsaE-FLAG. Protein 815

fractions were blotted with anti-SseB (left) and anti-FLAG antibodies (right). 816

817

Figure 3. SsaE is required for Salmonella SPI-2 T3SS-dependent secretion. (A) S. 818

enterica serovar Typhimurium strains were grown in LPM medium (pH 5.8), and the 819


.org/D

ownloaded from

http://jb.asm.org/

31

culture supernatants were collected by centrifugation. Secreted proteins were 820

precipitated by the addition of TCA. The protein samples from equal numbers of 821

bacteria were analyzed by Western blotting with anti-SseB antibody in the secreted 822

protein fraction (Supernatant) and the whole cell lysates (Cell lysate). (B) S. enterica 823

serovar Typhimurium wild-type and isogenic mutants harboring the plasmid 824

pPipB-2HA were grown in LPM medium (pH 5.8), and the protein samples from equal 825

numbers of bacteria were subjected to Western blot analysis to detect PipB-2HA and 826

SseB in secreted protein fractions (Supernatant) and the whole cell lysates (Cell lysate). 827

828

Figure 4. SsaE interacts with the SPI-2 T3SS translocator protein SseB. Interaction 829

of SsaE with SseB was analyzed by pull-down assay using bacterial lysates of 830

Salmonella wild-type strains harboring the plasmid pBAP-FLAG as a negative control 831

(A) or pSsaE-FLAG (B). Samples were taken before the binding of beads (Lysate), after 832

the final washing with beads (Final wash), and after the elution of fraction with FLAG 833

peptide (Elute). Equal amounts of samples were analyzed by SDS-PAGE and Western 834

blotting with anti-FLAG, anti-SseB, and anti-PagC antibodies. (C) GST pull-down assay 835

using GST or GST-SseB and probed for SsaE-FLAG. Bacterial lysate of E. coli strain 836

DH5α harboring pSsaE-FLAG was incubated with GST or GST-SseB immobilized 837

glutathione Sepharose beads. Samples from lysates before the addition of beads (Lysate), 838

and proteins retained on beads (Elute) were analyzed by SDS-PAGE and Western 839

blotting with anti-FLAG and anti-GST antibodies. A Silver-stained SDS-PAGE gell of 840

the elution fractions from pull-down experiments shows the proteins in each sample 841

(bottom). 842

843

Figure 5. The C-terminal coiled-coil domain of SsaE is required for protein-protein 844

interaction. (A) Predicted α-helical regions, α1, α2 and α3, in SsaE (grey) are 845

indicated (top). The full-length SsaE-FLAG fusion protein and a series of mutant 846

derivatives are represented by a solid bar with the corresponding results of the FLAG 847

pull-down assays (right). The region of the internal in-frame deletions in SsaE∆2-FLAG 848

and SsaE∆3-FLAG are indicated by a thin line. The signs + and – indicate binding and 849

no binding, respectively. Interactions of SsaE and its derivatives with SseB in the 850

pull-down assay were visualized by immunoblotting with anti-SseB antibody. (B) The 851


.org/D

ownloaded from

http://jb.asm.org/

32

Helix Wheel model of the C-terminal coiled-coil motif of the SsaE α3 region spanning 852

amino acids 52 to 68. The hydrophobic amino acids within the amphipathic helix are 853

denoted by red. 854

855

Figure 6. SsaE, but not SseA, is required for SseB secretion in vitro when SseB is 856

overexpressed. (A) The expression and secretion of SseB in the whole cell lysate (Cell 857

lysate) and culture supernatant (Supernatant) from the S. enterica serovar Typhimurium 858

wild-type, ∆ssaV, ssaEI55G, and ssaEI55G harboring pSsaE were analyzed by Western 859

blotting with anti-SseB antibody. (B) The expression and secretion of PipB-2HA in the 860

whole cell lysate (Cell lysate) and culture supernatant (Supernatant) from the S. 861

enterica serovar Typhimurium wild-type, ssaEI55G, ∆ssaL, and ∆ssaLssaEI55G harboring 862

pPipB-2HA were analyzed by Western blotting with anti-HA antibody. (C) The 863

formation of SsaE-SseB-SseA ternary complex in the Salmonella cytoplasm was 864

analyzed by FLAG pull-down assays using bacterial lysates from Salmonella wild-type 865

strain expressing the SseA-2HA from the chromosome and harboring pSsaE-FLAG. 866

Equal amounts of samples (Lysate, Final wash, and Elute) were analyzed by 867

SDS-PAGE and Western blotting with anti-HA (for SseA-2HA), anti-FLAG (for 868

SsaE-FLAG), and anti-SseB antibodies. (D) The S. enterica serovar Typhimurium 869

wild-type strain and isogenic mutants harboring the plasmid pACYC-SseB were grown 870

in LPM medium (pH 5.8), and secreted proteins were precipitated by the addition of 871

TCA. Samples from secreted protein fraction (Supernatant) and whole cell lysates (Cell 872

lysate) were analyzed by Western blotting with anti-SseB antibody. 873

874

Figure 7. SsaE interacts with a putative SPI-2 T3SS ATPase SsaN by C-terminal 875

coiled-coil binding. E. coli strain MC1061 harboring the plasmids expressing 876

SsaE-FLAG (A), SsaEI55G-FLAG (B), and SseA-FLAG (C) was grown in LB medium. 877

FLAG-fusion proteins from lysates were immobilized on the anti-FLAG M2 affinity 878

beads. Bacterial lysate prepared from E. coli strain MC1061 harboring the plasmid 879

expressing SsaN-2HA was then incubated with FLAG-fusion protein-immobilized 880

beads. After the final washing with beads (Final wash), FLAG fusion proteins binding 881

to the affinity beads were eluted by adding FLAG peptide (Elute). Samples from 882

bacterial lysates before the addition of beads, and coprecipitated proteins were analyzed 883


.org/D

ownloaded from

http://jb.asm.org/

33

by Western blotting with anti-FLAG and anti-HA antibodies. 884


.org/D

ownloaded from

http://jb.asm.org/

34

885

TABLE 1. Salmonella strains and plasmids used in this study Strain or plasmid Description Reference Salmonella strains SL1344 serovar Typhimurium, rpsL, hisG (27) TM312 SL1344 derivative expresses SsaE-FLAG This study TM133 SL1344 derivative containing spiAC133S (37) TM131 Non-polar deletion of ssaV (∆ssaV) This study

TM323 SL1344 derivative containing ssaEmut This study TM198 Non-polar ssaL::kan mutant This study

TM203 Double mutant, ∆ssaL::kan, spiAC133S This study

TM204 Double mutant, ∆ssaL::kan, ssaEmut This study

TM205 Double mutant, ∆ssaL::kan, ssaEI55G This study

TM325 SL1344 derivative expresses SsaEI55G This study TM192 Non-polar deletion of sseA (∆sseA) This study TM194 Non-polar deletion of sseB (∆sseB) This study TM254 SL1344 derivative expesses SseA-2HA This study Plasmids pMW118 Low-copy-number expression vector Nippon Gene pFLAG-CTC FLAG fusion vector Sigma pBAP-FLAG pFLAG-CTC expressing BAP-FLAG Sigma pGEX-6P-1 GST fusion vector GE Healthcare pACYC184 Middle-copy-number expression vector New England Biolabs

pCACTUS Suicide vector, sucrose-sensitive, repts (40) pMS109 Plasmid containing cyaA (51) pLDΩKm2 pLD54 derived suicide plasmid This study pACPJ-2HA pACYC184 derivative contains 2HA with sseJ promoter This study pACPJ-CyaA-2HA pACPJ-2HA contains cyaA This study pACHS-2HA pACYC184 contains 2HA This study p2HA-CTC pFLAG-CTC derivative contains 2HA instead of FLAG This study pLD-SsaE-FLAG ssaE-FLAG in pLDΩKm2 This study pSsaE-2HA pACPJ-2HA expressing SsaE-2HA This study pSseF-2HA pACPJ-2HA expressing SseF-2HA This study pSsaE-CyaA-2HA pACPJ-CyaA-2HA expressing SsaE-CyaA-2HA This study pSseF-CyaA-2HA pACPJ-CyaA-2HA expressing SseF-CyaA-2HA This study

pCACTUS-ssaEmut ssaEmut in pCACTUS This study

pCACTUS-ssaEI55G ssaEI55G in pCACTUS This study pSsaE ssaE in pMW118 This study pPipB-2HA pACHS-2HA expressing PipB-2HA from pipB promoter This study pSsaE-FLAG pFLAG-CTC expressing SsaE-FLAG This study

pSsaE∆1-FLAG pFLAG-CTC expressing SsaE21-80-FLAG This study

pSsaE∆2-FLAG pFLAG-CTC expressing SsaE∆21-41-FLAG This study

pSsaE∆3-FLAG pFLAG-CTC expressing SsaE∆52-68-FLAG This study

pSsaEI55G-FLAG pFLAG-CTC expressing SsaEI55G-FLAG This study pSseA-FLAG pFLAG-CTC expressing SseA-FLAG This study pGEX-SseB pGEX-6P-1 expressing GST-SseB This study


.org/D

ownloaded from

http://jb.asm.org/

35

pACYC-SseB sseB in pACYC184 This study pSsaN-2HA p2HA-CTC expressing SsaN-2HA This study


.org/D

ownloaded from

http://jb.asm.org/

36

886 TABLE 2. Primers used in this study Primer Sequence (restriction site) ssaV-red-FW 5'-ATTATATCGTTTGTCACTCACAATCAGCACATCACGG CTGGTGTAGGCTGGAGCTGCTTC-3' ssaV-red-RV 5'-TCTGCGTCTTATGACGAGACGACAGCGCAGTATAGG TCCCCATATGAATATCCTCCTTAG-3' ssaL-red-FW 5'-GAAGATAAAAGAGGTAGCGATGAATATTAAAATTAA TGAGGTGTAGGCTGGAGCTGCTTC-3' ssaL-red-RV 5'-AAAGCCAATTTACCTAAATATTGAAAGCCAGGTATC AGAACATATGAATATCCTCCTTAG-3' sseB-red-FW 5'-GGGAAGTCAAAACCCTATTGTGTTTAAAAATAGCTT CGGCGTGTAGGCTGGAGCTGCTTC-3' sseB-red-RV 5'-TCTGCGCTATCATACTGGAAATTTCCCCCCACTTACT GATCATATGAATATCCTCCTTAG-3' sseA-red-FW 5'-TAGTTAGCACGTTAATTATCTATCGTGTATATGGAGG GGAGTGTAGGCTGGAGCTGCTTC-3' sseA-red-RV 5'-TTCCCCATAAGATGTTTCCTGAAGACATTATGCTTTA CCTCATATGAATATCCTCCTTAG-3' sseA-2HA-red-FW 5'-TCAAGCCCCGGTGGAGATACCGTCAGGAAAAACAAA AAGGTATCCGTATGATGTGCCGGA-3’ sseA-2HA-red-RV 5'-CAATAGGGTTTTGACTTCCCCATAAGATGTTTCCTGA AGACATATGAATATCCTCCTTAG-3’ ssaE-SacI-FW 5'-CCAGAGCTCCCGATGTGGTAACGATTAAAC-3' (SacI) ssaE-SphI-RV 5'-AAAGCATGCAAGAGCCTATCCCATTAGGGC-3' (SphI) sseB-FW 5'-ACGACTGAAACAACTTAATGCTCAAGCCCC-3' sseB-RV 5'-AACCGCATCGTGTCATGTGCCTGTTGTAGG-3' sseB-FW-BamHI 5'-CCCGGATCCTCTTCAGGAAACATCTTATGG-3' (BamHI) sseB-XhoI-RV 5'-AAGCTCGAGTCATGAGTACGTTTTCTGCGC-3' (XhoI) ssaE-XhoI-FW 5'-GGGCTCGAGACTTTGACCCGGTTAGAAGAT-3' (XhoI) ssaE-BglII-RV 5'-GGGAGATCTCTCTTGCTCACTCACTACAAG-3' (BglII) sseF-FW-XhoI 5'-AAACTCGAGATTCATATTCCGTCAGCGGCA-3' (XhoI) sseF-RV-BamHI 5'-AAAGGATCCTGGTTCTCCCCGAGATGTATG-3' (BamHI) sseA-XhoI-FW 5'-GGGCTCGAGATAAAGAAAAAGGCTGCGTTT-3' (XhoI) sseA-BamHI-RV 5'-CCCGGATCCCCTTTTTGTTTTTCCTGACGG-3' (BamHI) PropipB-XhoI-FW 5'-AAACTCGAGTACGTCTGAACTACACCAGCG-3' (XhoI) pipB-BglII-RV 5'-CCCAGATCTAAATATCGGATGGGGGAAAAG-3' (BglII) ssaE-StuI-R3 5'-CCCAGGCCTCTCGAGAAGCTTCATATGATA-3' (StuI) ssaE-StuI-R4 5'-CCCAGGCCTATAATTTTACAATTAAGGGCT-3' (StuI) ssaE-StuI-R1 5'-TTTAGGCCTGCCTTTGGCCTCTTCACGCGA-3' (StuI) ssaE-StuI-R2 5'-TTTAGGCCTCCGCAGCAATATCAGCAAAAC-3' (StuI) ssaE-StuI-R7 5'-CCCAGGCCTCAATAAGGTGTTTTGCTGATA-3' (StuI) ssaE-StuI-R8 5'-ACCAGGCCTTACCATACCAGCGCACTTGTA-3' (StuI) sense-ssaEATGmut 5'-TAGATTTTAAGTGAGTGGAAGCTTACAACTTTGACCCGGTTAGAA-3' (HindIII) antisense-ssaEATGmut 5'-TTCTAACCGGGTCAAAGTTGTAAGCTTCCACTCACTTAAAATCTA-3' (HindIII) sense-ssaEI55G 5'-AACACCTTATTGCTTGAAGCCGGCGAGCAGGCCGAAAATATC-3' (NaeI) antisense-ssaEI55G 5'-GATATTTTCGGCCTGCTCGCCGGCTTCAAGCAATAAGGTGTT-3' (NaeI) 2HA-StuI-1 5'-GGGAGGCCTTATCCGTATGATGTGCCGGATTATGCGTATCCGTATGATGTGCC GGATTATGCGTAGGACTACAAGGACGACGATGACAAG-3' (StuI) 2HA-StuI-2 5'-TTTAGGCCTGTCGACAGATCTGGTACCCGGGAATTC-3' (StuI) FLAG-SphI-FW 5'-CCTGCATGCTCACACAGGAGATATCATCTG-3' (SphI)


.org/D

ownloaded from

http://jb.asm.org/

37

FLAG-BamHI-RV 5'-CCCGGATCCTATTGTCTCATGAGCGGATAC-3' (BamHI) ProsseJ-SalI-FW 5'-GGGGTCGACTCACATAAAACACTAGCACTT-3' (SalI) ProsseJ-SphI-RV 5'-CCCGCATGCAGTGTCCTCCTTACTTTATTA-3' (SphI) FLAG-SphI+ATG-FW 5'-CCTGCATGCTCACACAGGAGATATCATATG-3' (SphI) cyaA-BglII-FW 5'-AAAAGATCTCAGCAATCGCATCAGGCTGGT-3' (BglII) cyaA-XhoI-RV 5'-AAACTCGAGGTCATAGCCGGAATCCTGGCG-3' (XhoI) ssaN-XhoI-FW 5'-GGGCTCGAGAATGAATTGATGCAACGTCTG-3' (XhoI) ssaN-BamHI-RV 5'-CCCGGATCCCTCGGTGAGTATTTGGTGTAA-3' (BamHI)

887 888


.org/D

ownloaded from

http://jb.asm.org/

a3

Na1

C

a2

PssaM

ssaU T S R Q P O N V M L K J IHG sscB sscA ssaE D C B ssrA B

sseG F E D C BA spiB A C R

PssaG PsseA PspiC PssrA PssrB

Figure 1

1 10 20 30 40 MTTLTRLEDLLLHSREEAKGIILQLRAARKQLEENNGKLQ

---MTQLEEQLHN-VETVRSITMQLEMALTKLKKDMMRGG

a1

a3

a2

41 50 60 70 80

DPQQYQQNTLLLEAIEQAENIINIIYYRYHNSALVVSEQE

DAKQYQVWQRESKALESAIAIIH-----YVAGDL

SsaE

YscE

SsaE

YscE

A

B

C

a3

N

a1

C

a2

D

N C

a1a2

a3


.org/D

ownloaded from

http://jb.asm.org/

Figure 2

DnaK

PagC

SsaE-FLAG

Perip

lasm

Cyt

opla

sm

Mem

bran

e

A

Supernatant

Cell lysate

blot: anti-SseB

wt sp

iA C13

3S

wt (

SsaE-F

LAG

)

blot: anti-FLAG

B

wt sp

iA C13

3S

wt (

SsaE-F

LAG

)


.org/D

ownloaded from

http://jb.asm.org/

Figure 3

Cell lysate

Supernatant

wt ss

aV

ssaE

mut

ssaE

mut

/pM

W11

8

ssaE

mut

/pSsa

E

blot: anti-SseB

A

ssaL

ssaL

ssaE

mut

Supernatant

Cell lysate

ssaL

spiA

C13

3S

Supernatant

Cell lysate

PipB-2HA

SseB

B

wt/p

ACH

S-2H

A

wt

spiA

C13

3S

ssaE

mut

+ pPipB-2HA

blot: anti-SseB

blot: anti-HA

SseB


.org/D

ownloaded from

http://jb.asm.org/

A

Lysat

e

Final

was

h

Elute

SL1344/

pBAP-FLAG

IP: FLAG

BAP-FLAG

SseB

PagC

Lysat

e

Final

was

h

Elute

SL1344/

pSsaE-FLAG

IP: FLAG

SseB

PagC

SsaE-FLAG

B

Figure 4

SsaE-FLAG

GST-SseB

GST

SsaE-FLAG

GST

GST-SseB

Lysat

e

a-FLAG

a-GST

PD: GST

- + -

- - +

Elute

Silver stain

28

19

16

10

37435570

(kDa)

GST-SseB

GST

SsaE-FLAG

C


.org/D

ownloaded from

http://jb.asm.org/

a1

SsaE-FLAG

SsaE

SsaE

SsaE

SsaEI55G-FLAG

801

1 20 42 80

21 80

1 80

SseB

binding

51 80691

SsaE N C

a2 a3

Lysat

e (S

seB)

Final

was

h

Elute

blot:anti-SseB

Lysat

e (F

LAG

)

+

+

+ - -

I55G

IP: FLAG

blot:anti-FLAG

SseB

Figure 5

B

e

f

g

a

b

d

c

N48

I55

I62

T49

E56

N63

L50

Q57

I64

L51

A58

I65

L52

E59

Y66

E53

N60

Y67

A54

I61

R68

SsaE a3

A


.org/D

ownloaded from

http://jb.asm.org/

A

wt ss

aV

ssaE

I55G

ssaE

I55G /p

SsaE

Supernatant

Cell lysate

blot: anti-SseB

SseB

Figure 6

Supernatant

wt ss

aV

ssaE

mut

wt ss

eBss

eA

blot: anti-SseB

Cell lysate

+pACYC-SseB

SseB

B

Supernatant

Cell lysate

wt

ssaE

I55G

ssaL

ssaLss

aE

I55G

blot: anti-HA

+pPipB-2HA

PipB-2HA

C

a-SseB

Elute

Lysat

e

Final

was

h

a-FLAG

a-HA

pSsaE-FLAG

SL1344 sseA-2HA/

IP: FLAG

D


.org/D

ownloaded from

http://jb.asm.org/

Figure 7

SsaE-F

LAG

SsaN

-2H

A

Final

was

h

SsaE-FLAG

SsaN-2HA

Elute

SsaN-2HA

SseA-FLAG

SseA

-FLA

G

SsaN

-2H

A

Final

was

h

IP: FLAG

Elute

A B

C

SsaEI55G-FLAG

SsaN-2HA

SsaEI5

5G-F

LAG

SsaN

-2H

A

Final

was

h

IP: FLAG

Elute

IP: FLAG


.org/D

ownloaded from

http://jb.asm.org/

Functional characterization of SsaE, a novel chaperone protein of the type III secretion system

Documents

Transcript of Functional characterization of SsaE, a novel chaperone protein of the type III secretion system