IAI Accepts, published online ahead of print on 2 September 2014 Infect. Immun. doi:10.1128/IAI.02131-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

1

The type III secretion effector NleF of enteropathogenic Escherichia coli activates NF-

2

κB early during infection

3 4 5 6

Running title: The EPEC effector NleF activates NF-ĸB

7

Mitchell A. Pallett,a Cedric Berger,a Jaclyn S. Pearson,b Elizabeth L. Hartlandb and Gad

8

Frankela#

9 10

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences,

11

Imperial College London, UKa, Department of Microbiology and Immunology, University of

12

Melbourne at the Peter Doherty Institute for Infection and Immunity, Victoria 3010,

13

Australiab

14 15 16 17

#Corresponding author

18

Gad Frankel

19

CMBI, Flowers Building

20

Imperial College

21

London, SW7 2AZ

22

[email protected]

23

Tel: +44 20 7594 5253

1

24

Abstract

25

The enteric pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli

26

employ a T3SS to manipulate the host inflammatory response during infection. Previously, it

27

has been reported that EPEC, in a T3SS-dependent manner, induces an early pro-

28

inflammatory response through activation of NF-κB via ERK1/2 and PKCζ. However, no

29

effector has yet been attributed to the activation of NF-κB during infection. At later time

30

points post infection NF-κB signalling is inhibited through the translocation of multiple

31

effectors including NleE and NleC. Here we report that the highly conserved non-LEE

32

encoded effector F (NleF) shows both diffuse and mitochondrial localization during ectopic

33

expression. Moreover, NleF induces nuclear translocation of NF-κB p65 and the expression

34

of IL-8 following ectopic expression and during EPEC infection. Furthermore, the pro-

35

inflammatory activity and localization of NleF was dependent on the C-terminal amino acids

36

LQCG. Whilst the C-terminal domain of NleF was previously shown to be essential for

37

interaction with caspase-4, -8 and -9; the pro-inflammatory activity of NleF was independent

38

of caspase-4, -8 or -9 interaction. In conclusion EPEC, through the T3SS-dependent

39

translocation of NleF, induces a pro-inflammatory response in an NF-κB dependent manner

40

in the early stages of infection.

2

41

Introduction

42

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are

43

important enteric human pathogens. EPEC is a leading aetiological agent of paediatric

44

diarrhoeal disease (1); EHEC infection can lead to haemorrhagic colitis and in more severe

45

cases to kidney damage and the progression to haemolytic-uremic syndrome (HUS) (2).

46

Although the clinical outcomes differ, EHEC and EPEC share a common strategy as they

47

colonize the intestinal gut mucosa via attaching and effacing (A/E) lesions. A/E lesions are

48

characterized by local effacement and destruction of the enterocyte brush-border microvilli,

49

intimate attachment to gut enterocytes and formation of pedestal-like structures beneath

50

attached bacteria through host cytoskeletal rearrangement (3). The ability to cause A/E

51

lesions is associated with the Locus of Enterocyte Effacement (LEE) pathogenicity island

52

which encodes a type 3 secretion system (T3SS), gene regulators, the adhesin intimin,

53

chaperons and 7 effector proteins (4-6). Through T3SS translocation of the effectors, EPEC

54

and EHEC subvert and manipulate the host cell signalling during infection, including

55

intracellular trafficking, apoptosis and inflammation (7).

56

A key regulator of inflammation is the transcriptional regulator nuclear factor-kappa B (NF-

57

ĸB) (8, 9). The major subunits of NF-κB are p65 and p50; in the inactive state p65/p50 are

58

bound to IĸB (inhibitor of κ light chain gene enhancer in B cells) and are restricted to the

59

cytoplasmic compartment (9, 9). The NF-κB signalling pathway is activated in response to a

60

number of stimuli including stress signals, pro-inflammatory cytokines interleukin-1 beta (IL-

61

1β) and tumor necrosis factor (TNF) and pathogen associated recognition patterns (PAMPs)

62

(9). Activation of the NF-ĸB signalling pathways converges on IκB Kinase (IκK) and

63

phosphorylation of IĸBα, which leads to its proteasomal degradation. This results in nuclear

64

translocation of NF-κB (p65/p50) (9) and expression of multiple pro-inflammatory cytokines

65

including interleukin-8 (IL-8) (9).

3

66

The effector EspT, present in the atypical EPEC strain E110019, induces an NF-κB

67

dependent pro-inflammatory response through the activation of the GTPase Rac1 (10).

68

However the pro-inflammatory activity of EspT is restricted to infection of immune cells and

69

furthermore EspT is only present in 1% of clinical EPEC isolates (10). More commonly,

70

multiple recent publications have shown that a number of conserved EPEC and EHEC

71

effectors, particularly NleC and NleE, inhibit TNF and IL-1β-induced NF-κB activation (7).

72

NleC, a zinc metalloprotease, directly cleaves the p65 subunit of NF-κB (11, 12); NleE,

73

through its methyltransferase activity, inhibits transforming growth factor-beta-activated

74

kinase 1 (TAK1) and downstream degradation of IκBα (13-16). In addition, NleH1 targets the

75

human ribosomal protein S3 (RPS3), a co-factor of NF-κB, repressing nuclear translocation

76

of NF-κB (17). Despite inhibition by these effectors some evidence suggests the NF-κB

77

pathway is also activated during EPEC infection in a T3SS-dependent manner. For example

78

infection with the EPEC E2348/69 ΔescN mutant (T3SS deficient) does not promote p65

79

translocation to the nucleus (15). In contrast, the EPEC E2348/69 ΔPP4/IE6 double-island

80

mutant, which lacks the genes encoding the effectors NleE, NleB1, EspL, NleB2, NleC, NleD

81

and NleG, is able to significantly activate NF-κB (12, 15). This suggests that the NF-κB

82

pathway is activated and inhibited in a T3SS-dependent manner; however, no effector has yet

83

been attributed to the activation of this pathway by EPEC during infection of epithelial cells.

84

The non-LEE encoded effector F (NleF) (18, 19) is highly conserved among EPEC and

85

EHEC strains (20). NleF can inhibit the proteolytic activity of the inflammatory caspases;

86

caspase-4 and -8, as well as the activity of caspase-9 (21). The C-terminal 4 amino acids of

87

NleF, with the motif LQCG, are essential for this interaction (21). Furthermore Olsen and

88

colleagues identified TMP21, a type-1 transmembrane protein involved in vesicular transport,

89

as a binding partner of NleF (22). The aim of this study was to further characterize the

90

biological activity of NleF.

4

91

Materials and Methods

92 93

Bacterial strains, growth conditions and reagents

94

The E. coli strains used in this study are listed in Table 1. E. coli were cultured in Luria Broth

95

(LB) at 37⁰C, at 200 rpm with the appropriate antibiotics: 100 µg/ml ampicillin and 50 µg/ml

96

kanamycin. All reagents were purchased from Sigma unless otherwise stated.

97 98

Tissue Culture

99

HeLa cells were grown in Dulbecco's Modified Eagle's Medium - low glucose (1000 mg/L;

100

DMEM) supplemented with 10% (v/v) heat inactivated foetal calf serum (FCS; Gibco), 2mM

101

Glutamax (Invitrogen) and 0.1 mM non-essential amino acids in a 5% CO2 atmosphere.

102 103

Plasmid construction

104

The plasmids and primers used in this study can be found in Table 1 and Table 2,

105

respectively. nleF was amplified from E. coli O127:H6 E2348/69 genomic DNA by PCR

106

using KOD Hot Start Polymerase (Novagen). nleF and nleF1-185 were cloned into the

107

eukaryotic expression vector pRK5::myc following amplification with the primer pair 1F/1R

108

and 1F/2R respectively. PCR products were cloned into the BamHI/PstI sites of pRK5::myc

109

to generate plasmids pICC1661 and pICC1667, respectively. Alternatively nleF was cloned

110

into pEGFP-C2 following amplification with primer pair 7F/7R using the restriction sites

111

EcoRI/BamHI to generate pICC1675. nleF was cloned into the EcoRI/HindIII sites of

112

pBAD22 using the primer pair 6F/6R to construct the plasmid pICC1662. Plasmids were

113

codon optimized to remove a poly-thymine rich region using the primer pair InvF/InvR. All

114

constructs were verified by DNA sequencing.

5

115

Mutant construction

116

nleF was deleted from EPEC E2348/69 using the lambda red system (23), generating strain

117

ICC1082. The primer pair pKD4F/R was used to amplify the Kanamycin cassette from

118

pKD4. Primer pair 3F/R were used to amplify nleF from Escherichia coli O127:H6 E2348/69

119

genomic DNA with a 500 bp 5’ and 400 bp 3’ flanking sequences. The PCR product was

120

cloned into TOPO Blunt II (Invitrogen) by blunt ligation. nleF was removed by inverse PCR

121

with the primer pair 4F/R and replaced with the Kanamycin cassette using blunt end ligation

122

to construct the plasmid pICC1674. The linear product for recombination was amplified from

123

pICC1674 using primer pair 3F/R and mutagenesis was confirmed by sequencing using the

124

primer pair 5F/R.

125 126

Transfection and siRNA

127

HeLa cells were grown until ~40% confluent in 24 well plates containing 13 mm glass cover-

128

slips (VWR International). 16 h post seeding monolayers were transfected with the

129

eukaryotic expression vectors listed in Table 1 using the Gene Juice kit (Novagen/Merck), to

130

the manufacturer’s specifications. Transfected cells were left for 24 h and where indicated

131

stimulated with TNF at 20 ng/ml for 30 min for immunofluorescence or 16 h for RT-qPCR

132

before washing 3 times in PBS. Plates were stored at -80⁰C for RT-qPCR or fixed for

133

immunofluoresence as described below. For knockdown ON-TARGETplus SMARTpool

134

scrambled (D-001810-10-05), caspase-4 (L-004404-00-0005), -8 (L-003466-00-0005) and -9

135

siRNA (L-003309-00-0005; Dharmacon) were transfected at 20 nM using Hi-perfect

136

(Qiagen) following the manufacturer’s instructions. Knockdown efficiency was analysed by

137

RT-qPCR and Western blotting as described below.

138 139

Isolation of mRNA and RT-qPCR

6

140

Cells were washed in PBS and mRNA was isolated following manufacturer’s instructions

141

(Qiagen; RNeasy mini kit). Samples were treated with DNase (Promega) at 37⁰C for 30 min

142

followed by 15 min at 72⁰C. Reverse transcription PCR was carried out following the

143

manufacturer’s instructions (Promega) with the following adaptations: RNasin (Promega)

144

was added to the reaction buffer at 2-4 U/µl. IL-8, caspase-4, caspase-8, caspase-9 and

145

GAPDH cDNA was amplified with primer pairs IL-8F/R, CASP4F/R, CASP8F/R,

146

CASP9F/R and GAPDHF/R, respectively, by RT-qPCR using the 7300 Real Time PCR

147

System (Applied Biosystems) under standard cycle conditions. Changes in gene expression

148

levels were analysed relative to the control (see figure legend for individual experiments)

149

using GADPH as a standard and using the ΔΔCT method.

150 151

Infection assay

152

HeLa cells were washed three times with PBS 3 h prior to infection before replacing the

153

media with DMEM with no additives. Bacterial strains grown overnight for 17 h in LB were

154

diluted 1:100 and primed in DMEM with no additives for 3 h at 37⁰C, in a 5% CO2

155

atmosphere without agitation. Arabinose or IPTG were added at 1 mM or 0.5 mM to ∆nleF

156

pnleF and ∆PP4/IE6 pnleE bacterial cultures 0.5 h before infection, respectively. After 3 h of

157

priming the media was removed from the HeLa cells and replaced with 0.5 ml of a 1:3

158

dilution of primed bacterial culture in DMEM. The infection was carried out for 1.5 h and 4

159

h, and where indicated treated with 20 ng/ml of TNF for 30 mins after 3.5 h of infection. For

160

the 1.5 h or 4 h time point cells were washed 3 times with PBS 1 h post infection and

161

incubated for a further 0.5 h or 3 h. Cells were washed 3 times in PBS and either fixed for

162

immunofluorescence or lysed for Western blot analysis as described below.

163 164

Immunofluorescence staining and antibodies

7

165

For immunofluorescence staining cells were fixed with 3% paraformaldehyde and washed a

166

further 3 times in PBS before quenching in 50 mM NH4Cl, permeabilizing with 0.2% Trition-

167

X100 and washing 3 times in PBS. Coverslips were blocked in 1% Bovine serum albumin

168

(BSA)/PBS. All antibodies, unless stated otherwise, were diluted directly in 1% BSA-PBS.

169

Primary antibodies were incubated for 45 min followed by incubation of the secondary

170

antibodies and reagents for 30 min. Mouse monoclonal antibodies anti-Myc clone 4A6 (05-

171

724; Millipore), anti-caspase-4 clone 4B9 (sc-56056; Santa Cruz), Myc-FITC Clone 9E10

172

(F2047-100), anti-Alpha-Tubulin clone DM1A (T6199), anti-TOMM22 (ab57523; Abcam),

173

rabbit polyclonal antibodies anti-N-terminal NF-κB p65 clone A (sc-109; Santa Cruz), and

174

anti-IκBα (Cell signalling; 2942) were used as primary antibodies. Donkey anti-mouse IgG

175

(H+L)

176

immunofluorescence and Goat anti-mouse-HRP (Jackson Immunoresearch; 115-035-008)

177

and Goat anti-rabbit-HRP for western blot analysis (Jackson Immunoresearch; 111-035-008)

178

were used as secondary antibodies. Phalloidin-TRITC (P1951-.1MG; Sigma) and DAPI were

179

used to visualize actin and the nucleus respectively. Cover-slips were then mounted using

180

Prolong Gold anti-fade reagent (Invitrogen) before visualising on the Zeiss Axioimager

181

immunofluorescence microscope using the Zeiss Axiovision Rel 4.5 software.

conjugated

to

DyLight-488

(715-485-150;

Jackson

Immunoresearch)

for

182 183

Western blotting

184

90 minutes post infection cells were lysed in loading dye (LDS; 250 mM Tris pH 6.8, 40%

185

glycerol, 6 % SDS, 1 % Bromophenol blue and 0.8% β-mecaptoethanol) and boiled for 10

186

mins, run on SDS page and then transferred to a PVDF membrane. The membranes were

187

blocked in Tris buffer saline pH 7.4 (10 mM Tris, 150 mM NaCl)/0.1% Tween (TBS-T)

188

containing 5% semi-skimmed milk (blocking buffer) for 1 h before incubation with primary

189

antibody in blocking buffer for 16 h at 4⁰C. The membrane was washed in TBS-T and the

8

190

secondary antibody was incubated for 2 h in blocking buffer. Western blots were visualized

191

by the addition of ECL solution (GE Healthcare) and developed by the LAS-3000 imager

192

(Fuji). Densitometry was analysed using ImageJ.

193 194

Luciferase assay

195

An NF-κB dual luciferase reporter system (Promega) was employed as described previously

196

(15), with the following adaptations. HeLa cells were seeded as desribed above and co-

197

transfected with pEGFP-C2 or pEGFP-nleF together with pNF-κB-Luc (Clontech) and pRL-

198

TK (Promega). 24 h post transfection, for each condition, the expression of firefly luciferase

199

was measured and normalized for Renilla luciferase measurements and NF-κB activity was

200

expressed relative to HeLa-pEGFP.

201 202

Statistics

203

All data was analysed using GraphPad Prism software, using one-way Annova and

204

Bonferroni’s Multiple comparison test when required. A P

The type III secretion effector NleF of enteropathogenic Escherichia coli activates NF-κB early during infection.

The enteric pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli employ a type 3 secretion system (T3SS) to manipulate the...
2MB Sizes 5 Downloads 12 Views