Pathogens and Disease Advance Access published May 17, 2015
1
OmpA family proteins and Pmp-like autotransporter: new adhesins
2
of Waddlia chondrophila
3 4
Carole Kebbi-Beghdadi1, Andreas Domröse2, Elisabeth Becker2, Ousmane H. Cisse1,
5
Johannes H. Hegemann2, Gilbert Greub1.
6 7
1
8
University Hospital Center and University of Lausanne, Lausanne, Switzerland, 2
9
2
10
Center for Research on Intracellular Bacteria (CRIB), Institute of Microbiology,
Institut für Funktionelle Genomforschung der Mikroorganismen, Heinrich-Heine-
Universität, Düsseldorf, Germany.
11 12
Corresponding author:
13
Prof. Gilbert Greub
14
Institute of Microbiology
15
Rue du Bugnon 48
16
1011 Lausanne
17
Switzerland
18
Tel: 0041 21 314 4979
19
Fax 0041 21 314 4060
20
e-mail: [email protected]
21
1
21
Abstract
22
Waddlia chondrophila is an obligate intracellular bacteria belonging to the
23
Chlamydiales order, a clade that also includes the well-known classical Chlamydia
24
responsible for a number of severe human and animal diseases. Waddlia is an
25
emerging pathogen associated with adverse pregnancy outcomes in humans and
26
abortion in ruminants. Adhesion to the host cell is an essential prerequisite for
27
survival of every strict intracellular bacteria and, in classical Chlamydia, this step is
28
partially mediated by polymorphic outer membrane proteins (Pmps), a family of
29
highly diverse autotransporters that represent about 15% of the bacterial coding
30
capacity. Waddlia chondrophila genome however only encodes one putative Pmp-
31
like protein. Using a proteomic approach, we identified several bacterial proteins
32
potentially implicated in the adhesion process and we characterized their expression
33
during the replication cycle of the bacteria. In addition, we demonstrated that the
34
Waddlia Pmp-like autotransporter as well as OmpA2 and OmpA3, two members of
35
the extended Waddlia OmpA protein family, exhibit adhesive properties on epithelial
36
cells. We hypothesize that the large diversity of the OmpA protein family is linked to
37
the wide host range of these bacteria that are able to enter and multiply in various
38
host cells ranging from protozoa to mamalian and fish cells.
39 40
2
40
Introduction
41
Waddlia chondrophila are strict intracellular bacteria belonging to the Chlamydiales
42
order. This clade is currently divided in 9 family-level lineages (Stride, Polkinghorne
43
et al. 2013) which, according to a recent report by Lagkouvardos et al., largely
44
underestimates the huge diversity of these bacteria, especially in the marine
45
environment (Lagkouvardos, Weinmaier et al. 2014). Bacteria belonging to the
46
Chlamydiales order are strictly intracellular; some represent harmless environmental
47
species multiplying in amoebae whereas others are important human and animal
48
pathogens. Chlamydia trachomatis, for example, causes urogenital infections
49
associated with infertility (Baud, Regan et al. 2008) and Chlamydia pneumoniae is a
50
recognized agent of pneumonia (Hahn, Azenabor et al. 2002). Other members of the
51
Chlamydiaceae family, such as Chlamydia abortus or Chlamydia psittaci, are well-
52
known animal pathogens associated with important economic repercussions and
53
having a significant zoonotic potential (Everett 2000).
54
Waddlia chondrophila was isolated twice, in two different locations, from aborted
55
bovine foetuses. Abortion was also observed after inoculation of these bacteria to
56
bovines (Dilbeck, Evermann et al. 1990, Henning, Schares et al. 2002, Dilbeck-
57
Robertson, McAllister et al. 2003). Moreover, a strong correlation was observed
58
between anti-Waddlia antibodies and abortion in ruminants (Dilbeck-Robertson,
59
McAllister et al. 2003) and, more recently, Waddlia DNA was detected in vaginal
60
swabs of 8% of 150 cows suffering from abortion (Barkallah, Gharbi et al. 2014). In
61
humans, Waddlia chondrophila seropositivity is associated with adverse pregnancy
62
outcomes such as miscarriage or ectopic pregnancy (Baud, Thomas et al. 2007,
63
Baud, Goy et al. 2014, Hornung, Thuong et al. 2015). Moreover, Waddlia has been
64
detected by qPCR and immunohistochemistry in vaginal swabs and placentas of
65
women suffering from miscarriage (Baud, Goy et al. 2011, Baud, Goy et al. 2014). In
66
addition, these bacteria were also detected in clinical samples of patients with
67
community-acquired pneumonia or bronchiolitis, which suggest their implication in
68
lower respiratory tract infections (Haider, Collingro et al. 2008, Goy, Croxatto et al.
69
2009).
70
Waddlia, like all other members of the Chlamydiales order studied so far, undergo a
71
biphasic replication cycle that involves an infectious form able to adhere to and enter
72
into the host cell, the elementary body (EB) and a replicative form, the reticulate body
73
(RB), which is strictly intracellular and divides by binary fission. When bacteria 3
74
encounter stress conditions such as antibiotic treatment or nutrient starvation, a third
75
form, called an aberrant body (AB) can also be observed. Thus, aberrant bodies
76
have been observed when Waddlia was grown in endometrial cells in a low nutrient
77
medium (Kebbi-Beghdadi, Cisse et al. 2011) and when it was exposed to penicillin
78
derivatives and to phosphomycin (Jacquier, Frandi et al. 2014). Aberrant bodies
79
correspond to a dormant form of the bacteria that can revert to infectious forms when
80
conditions improve and that are associated with long term persistence in the host
81
organism (Beatty, Morrison et al. 1994, Hogan, Mathews et al. 2004).
82
For obligate intracellular bacteria such as member of the Chlamydiales order,
83
attachment and entry in the host cell are essential requirements for survival and
84
replication. In Chlamydia, several bacterial proteins are implicated in adhesion.
85
These include two important constituents of the chlamydial membrane, the major
86
outer membrane protein (MOMP) (Su, Watkins et al. 1990, Swanson and Kuo 1994,
87
Su, Raymond et al. 1996) and OmcB, a cysteine-rich protein thought to play a role in
88
the stabilization of the cell envelope by disulfide bonds (Hatch 1996, Stephens,
89
Koshiyama et al. 2001). OmcB binds to heparan sulfate-like glycosaminoglycans
90
(GAGs) on the surface of host cells via basic amino acids located in its N-terminal
91
region (Zhang and Stephens 1992, Fadel and Eley 2007, Moelleken and Hegemann
92
2008, Fechtner, Stallmann et al. 2013), this interaction being probably the first step in
93
the attachment process. GroEL1 and Heat shock proteins (HSP) 70 are also
94
localized on the surface of infectious EBs and play a role in the establishment of
95
infection (Raulston, Davis et al. 2002, Wuppermann, Molleken et al. 2008).
96
In addition, chlamydial adhesion also relies on a specific family of highly diverse
97
autotransporters, the polymorphic outer membrane proteins (Pmps) (Longbottom,
98
Russell et al. 1996, Longbottom, Russell et al. 1998, Grimwood, Olinger et al. 2001).
99
As many as 21 different Pmps, classified in 6 subtypes, have been identified in
100
Chlamydia pneumoniae. They exhibit little similarity in amino acid sequences but all
101
contain multiple repeats of the tetrapeptide motifs GGA(ILV) and FxxN. Pmp6,
102
Pmp20 and Pmp21, representing 3 of the 6 Pmp subtypes, were shown to be
103
implicated in C. pneumoniae adhesion to human epithelial cells precisely via their
104
repeated motifs (Molleken, Schmidt et al. 2010). The epidermal growth factor
105
receptor (EGFR) on the host cell was recently identified as the receptor for the C.
106
pneumoniae adhesin and invasin Pmp21 (Molleken, Becker et al. 2013). The
107
Chlamydia trachomatis Pmp family comprises nine members (pmpA to pmpI), also 4
108
classified in the same 6 subtypes, that all mediate adhesion to human epithelial and
109
endothelial cells (Becker and Hegemann 2014).
110
We have previously demonstrated that Waddlia chondrophila is able to enter and
111
multiply in various host cells ranging from mammalian epithelial cells and
112
macrophages to fish and insect cells and to protists (Goy and Greub 2009, Croxatto
113
and Greub 2010, Kebbi-Beghdadi, Batista et al. 2011, Kebbi-Beghdadi, Cisse et al.
114
2011, Kebbi-Beghdadi et al. unpublished). Nothing is known about the molecules
115
implicated in the adhesion of Waddlia chondrophila to its host cell but OmcB and
116
HSP60 (GroEL) are present in the Waddlia genome (Bertelli, Collyn et al. 2010).
117
However, Waddlia OmcB lacks the XBBXBX sequence present in the N-terminal
118
region of all Chlamydia OmcB and that was shown to be crucial for adhesion
119
(Moelleken and Hegemann 2008, Fechtner, Stallmann et al. 2013). A more detailed
120
analysis of this genome also revealed the presence of a family of 11 genes coding for
121
putative porins with a barrel structure. These OmpA proteins could represent the
122
functional homologs of MOMP, a major chlamydial protein that is notably absent in
123
the Waddlia genome. Furthermore, one putative autotransporter protein has also
124
been described (gene wcw_0271) that displays a predicted overall structure
125
somehow similar to the chlamydial Pmps with an N-terminal leader sequence
126
followed by a passenger domain and a barrel C-terminal domain. In addition, this
127
Waddlia Pmp-like protein also harbours 6 tetrapeptide repeats identical to those
128
implicated in adhesion of the chlamydial Pmps.
129
In the present study, we identified, using a proteomic approach, the Waddlia
130
molecules implicated in adhesion and we characterized their transcriptional and
131
protein expression profiles. We also evaluated in vivo on epithelial cells the adhesion
132
potential of these molecules and of the putative Pmp-like protein, knowing that
133
understanding this crucial step of host-pathogen interactions is a major stone on the
134
way towards efficient diagnostic and treatments of this emerging pathogen.
135 136
Material and methods
137
Cell culture and bacterial strains
138
Human lung carcinoma cell line A549 (ATCC CCL-185), Vero cells derived from
139
kidney epithelial cells of the African Green Monkey (ATCC CCL-81) and the human
140
endometrial adenocarcinoma Ishikawa cell line were cultivated as described in 5
141
Kebbi-Beghdadi et al. (Kebbi-Beghdadi, Cisse et al. 2011). HEp-2 cells (ATCC CCL-
142
23 ) were routinely maintained in high glucose Dulbecco’s modified minimal essential
143
medium (DMEM; Gibco, Life Technologies, Zug, Switzerland) supplemented with
144
10% foetal calf serum (Biochrom AG, Berlin, Germany), 1% non essential amino
145
acids
146
W. chondrophila strain ATCC VR-1470 was grown at 32°C within Acanthamoeba
147
castellanii strain ATCC 30010 in 25 or 75 cm2 cell culture flasks (Corning, New York,
148
USA) with peptone-yeast extract-glucose broth (Jacquier, Aeby et al. 2013). EBs
149
were purified as described elsewhere (Greub and Raoult 2002b). For infection of
150
epithelial cells, bacteria were recovered from a 4 days-old amoebal co-culture and
151
filtered through a 5 µm filter (Millipore, Carrigtwohill, Ireland) to eliminate trophozoites
152
and cysts.
and
1%
vitamins
(Gibco,
Life
Technologies).
153 154
Infection procedure
155
Epithelial cells were harvested from Corning culture flasks with 0.25% trypsin
156
(Sigma-Aldrich, Buchs, Switzerland), washed with fresh medium and seeded, one
157
day before infection, at 2-5 x105 cells per well in 24-wells microplates (Corning) or at
158
4 x 106 cells per 25 cm2 flasks for RNA extraction and total protein preparation. Cells
159
were infected with a 1/2000 dilution of bacteria (MOI 0.1-1). Plates were then
160
centrifuged at 1790g for 10 min at room temperature. After 15 min of incubation at
161
37°C in the presence of 5% CO2, cells were washed with fresh medium to remove
162
non-internalized bacteria and were then incubated for different periods of time at
163
37°C in a 5% CO2 atmosphere.
164 165
Outer membrane proteins extraction and 2D gel electrophoresis
166
Purified EBs were incubated 1 hour at 37°C in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA
167
and 2% sodium lauroyl sarcosinate (sarkosyl) (Fluka BioChemika, Buchs,
168
Switzerland). Detergent-insoluble complexes were pelleted by ultracentrifugation at
169
100’000g for 1 hour. Outer membrane proteins were solubilized by a 10 minutes
170
incubation at 56°C in 0.125M Tris-HCl (pH6.8), 0.33M urea and 50 mM DTT. 100 g
171
of sarkosyl-insoluble proteins were resuspended in 37.5 mM Tris, 7M urea, 2M
172
Thiourea, 50 mM DTT, 4% CHAPS, 0.5% Triton X-100 and 1% ASB and separated
173
by 2D gel electrophoresis as described by Centeno et al. (Centeno, Repici et al.
6
174
2007). Proteins were visualized by Coomassie Blue staining or transferred to
175
nitrocellulose for overlay assays.
176 177
Biotinylation of epithelial cell membrane proteins
178
Confluent monolayer of A549, Vero and Ishikawa cells in 75 cm2 flasks were washed
179
3 times with ice-cold PBS then scraped with a rubber policeman. 2.5 x 107 cells were
180
incubated 1 hour at 37°C with 10 mg/ml EZ-link sulfo-NHS-LC-biotin (Pierce, Thermo
181
Fisher Scientific, Lausanne, Switzerland) in PBS, washed 3 times with PBS, 100 mM
182
glycin (Biosolve Chimie, Dieuze, France) and lysed by sonication in PBS. Cellular
183
debris were removed by 15 minutes centrifugation at 13'000 rpm, 4°C and
184
biotinylated proteins were kept at -80°C.
185 186
Overlay assays
187
Nitrocellulose membranes were blocked over night in PBS, 0.05% Tween 20, 1%
188
BSA and washed 3 times 15 minutes in PBS, 0.05% Tween 20, 0.1% BSA.
189
Membranes were incubated 1.5 hours at 4°C with biotinylated epithelial cells diluted
190
1/1000 in PBS, 0.05% Tween 20, 0.1% BSA. After three washing steps of 15 minutes
191
with PBS, 0.05% Tween 20, 0.1% BSA, membranes were incubated during 1.5 hours
192
with horseradish peroxidase-labelled streptavidin (Amersham, GE Healthcare,
193
Glattbrugg, Switzerland) diluted 1/10’000 in PBS, 0.05% Tween 20, 5% BSA. The
194
membranes were then washed four times 15 minutes with PBS, 0.05% Tween 20
195
and reactive proteins were detected with a chemiluminescence-based kit (Lite-
196
AblotTM, Euroclone, Milano, Italy).
197 198
Mass spectrometry
199
For identification of proteins, Coomassie Blue stained spots were excised from 2D
200
gels and transferred to special 96-well plates (Perkin Elmer Life Sciences,
201
Schwerzenbach, Switzerland). MALDI-MS-MS analysis was performed on a 4700
202
Proteomics Analyser (Applied Biosystems, Framingham, MA, USA) as described in
203
Kebbi-Beghdadi et al. (Kebbi-Beghdadi, Lienard et al. 2012). Briefly, in-gel proteolytic
204
cleavage with trypsin was performed in an automated workstation and digests were
205
resuspended in alpha-cyano-hydroxycinnamic acid matrix before being deposited in
206
duplicate
207
After MALDI-TOF MS analysis, the 10 most intense ion signals were selected for
on
a
target
plate.
7
208
MS/MS analysis. Non-interpreted peptide tandem mass spectra were used for direct
209
interrogation of W. chondrophila open reading frames. With the parameters used, the
210
threshold for statistical significance (p
1
OmpA family proteins and Pmp-like autotransporter: new adhesins
2
of Waddlia chondrophila
3 4
Carole Kebbi-Beghdadi1, Andreas Domröse2, Elisabeth Becker2, Ousmane H. Cisse1,
5
Johannes H. Hegemann2, Gilbert Greub1.
6 7
1
8
University Hospital Center and University of Lausanne, Lausanne, Switzerland, 2
9
2
10
Center for Research on Intracellular Bacteria (CRIB), Institute of Microbiology,
Institut für Funktionelle Genomforschung der Mikroorganismen, Heinrich-Heine-
Universität, Düsseldorf, Germany.
11 12
Corresponding author:
13
Prof. Gilbert Greub
14
Institute of Microbiology
15
Rue du Bugnon 48
16
1011 Lausanne
17
Switzerland
18
Tel: 0041 21 314 4979
19
Fax 0041 21 314 4060
20
e-mail: [email protected]
21
1
21
Abstract
22
Waddlia chondrophila is an obligate intracellular bacteria belonging to the
23
Chlamydiales order, a clade that also includes the well-known classical Chlamydia
24
responsible for a number of severe human and animal diseases. Waddlia is an
25
emerging pathogen associated with adverse pregnancy outcomes in humans and
26
abortion in ruminants. Adhesion to the host cell is an essential prerequisite for
27
survival of every strict intracellular bacteria and, in classical Chlamydia, this step is
28
partially mediated by polymorphic outer membrane proteins (Pmps), a family of
29
highly diverse autotransporters that represent about 15% of the bacterial coding
30
capacity. Waddlia chondrophila genome however only encodes one putative Pmp-
31
like protein. Using a proteomic approach, we identified several bacterial proteins
32
potentially implicated in the adhesion process and we characterized their expression
33
during the replication cycle of the bacteria. In addition, we demonstrated that the
34
Waddlia Pmp-like autotransporter as well as OmpA2 and OmpA3, two members of
35
the extended Waddlia OmpA protein family, exhibit adhesive properties on epithelial
36
cells. We hypothesize that the large diversity of the OmpA protein family is linked to
37
the wide host range of these bacteria that are able to enter and multiply in various
38
host cells ranging from protozoa to mamalian and fish cells.
39 40
2
40
Introduction
41
Waddlia chondrophila are strict intracellular bacteria belonging to the Chlamydiales
42
order. This clade is currently divided in 9 family-level lineages (Stride, Polkinghorne
43
et al. 2013) which, according to a recent report by Lagkouvardos et al., largely
44
underestimates the huge diversity of these bacteria, especially in the marine
45
environment (Lagkouvardos, Weinmaier et al. 2014). Bacteria belonging to the
46
Chlamydiales order are strictly intracellular; some represent harmless environmental
47
species multiplying in amoebae whereas others are important human and animal
48
pathogens. Chlamydia trachomatis, for example, causes urogenital infections
49
associated with infertility (Baud, Regan et al. 2008) and Chlamydia pneumoniae is a
50
recognized agent of pneumonia (Hahn, Azenabor et al. 2002). Other members of the
51
Chlamydiaceae family, such as Chlamydia abortus or Chlamydia psittaci, are well-
52
known animal pathogens associated with important economic repercussions and
53
having a significant zoonotic potential (Everett 2000).
54
Waddlia chondrophila was isolated twice, in two different locations, from aborted
55
bovine foetuses. Abortion was also observed after inoculation of these bacteria to
56
bovines (Dilbeck, Evermann et al. 1990, Henning, Schares et al. 2002, Dilbeck-
57
Robertson, McAllister et al. 2003). Moreover, a strong correlation was observed
58
between anti-Waddlia antibodies and abortion in ruminants (Dilbeck-Robertson,
59
McAllister et al. 2003) and, more recently, Waddlia DNA was detected in vaginal
60
swabs of 8% of 150 cows suffering from abortion (Barkallah, Gharbi et al. 2014). In
61
humans, Waddlia chondrophila seropositivity is associated with adverse pregnancy
62
outcomes such as miscarriage or ectopic pregnancy (Baud, Thomas et al. 2007,
63
Baud, Goy et al. 2014, Hornung, Thuong et al. 2015). Moreover, Waddlia has been
64
detected by qPCR and immunohistochemistry in vaginal swabs and placentas of
65
women suffering from miscarriage (Baud, Goy et al. 2011, Baud, Goy et al. 2014). In
66
addition, these bacteria were also detected in clinical samples of patients with
67
community-acquired pneumonia or bronchiolitis, which suggest their implication in
68
lower respiratory tract infections (Haider, Collingro et al. 2008, Goy, Croxatto et al.
69
2009).
70
Waddlia, like all other members of the Chlamydiales order studied so far, undergo a
71
biphasic replication cycle that involves an infectious form able to adhere to and enter
72
into the host cell, the elementary body (EB) and a replicative form, the reticulate body
73
(RB), which is strictly intracellular and divides by binary fission. When bacteria 3
74
encounter stress conditions such as antibiotic treatment or nutrient starvation, a third
75
form, called an aberrant body (AB) can also be observed. Thus, aberrant bodies
76
have been observed when Waddlia was grown in endometrial cells in a low nutrient
77
medium (Kebbi-Beghdadi, Cisse et al. 2011) and when it was exposed to penicillin
78
derivatives and to phosphomycin (Jacquier, Frandi et al. 2014). Aberrant bodies
79
correspond to a dormant form of the bacteria that can revert to infectious forms when
80
conditions improve and that are associated with long term persistence in the host
81
organism (Beatty, Morrison et al. 1994, Hogan, Mathews et al. 2004).
82
For obligate intracellular bacteria such as member of the Chlamydiales order,
83
attachment and entry in the host cell are essential requirements for survival and
84
replication. In Chlamydia, several bacterial proteins are implicated in adhesion.
85
These include two important constituents of the chlamydial membrane, the major
86
outer membrane protein (MOMP) (Su, Watkins et al. 1990, Swanson and Kuo 1994,
87
Su, Raymond et al. 1996) and OmcB, a cysteine-rich protein thought to play a role in
88
the stabilization of the cell envelope by disulfide bonds (Hatch 1996, Stephens,
89
Koshiyama et al. 2001). OmcB binds to heparan sulfate-like glycosaminoglycans
90
(GAGs) on the surface of host cells via basic amino acids located in its N-terminal
91
region (Zhang and Stephens 1992, Fadel and Eley 2007, Moelleken and Hegemann
92
2008, Fechtner, Stallmann et al. 2013), this interaction being probably the first step in
93
the attachment process. GroEL1 and Heat shock proteins (HSP) 70 are also
94
localized on the surface of infectious EBs and play a role in the establishment of
95
infection (Raulston, Davis et al. 2002, Wuppermann, Molleken et al. 2008).
96
In addition, chlamydial adhesion also relies on a specific family of highly diverse
97
autotransporters, the polymorphic outer membrane proteins (Pmps) (Longbottom,
98
Russell et al. 1996, Longbottom, Russell et al. 1998, Grimwood, Olinger et al. 2001).
99
As many as 21 different Pmps, classified in 6 subtypes, have been identified in
100
Chlamydia pneumoniae. They exhibit little similarity in amino acid sequences but all
101
contain multiple repeats of the tetrapeptide motifs GGA(ILV) and FxxN. Pmp6,
102
Pmp20 and Pmp21, representing 3 of the 6 Pmp subtypes, were shown to be
103
implicated in C. pneumoniae adhesion to human epithelial cells precisely via their
104
repeated motifs (Molleken, Schmidt et al. 2010). The epidermal growth factor
105
receptor (EGFR) on the host cell was recently identified as the receptor for the C.
106
pneumoniae adhesin and invasin Pmp21 (Molleken, Becker et al. 2013). The
107
Chlamydia trachomatis Pmp family comprises nine members (pmpA to pmpI), also 4
108
classified in the same 6 subtypes, that all mediate adhesion to human epithelial and
109
endothelial cells (Becker and Hegemann 2014).
110
We have previously demonstrated that Waddlia chondrophila is able to enter and
111
multiply in various host cells ranging from mammalian epithelial cells and
112
macrophages to fish and insect cells and to protists (Goy and Greub 2009, Croxatto
113
and Greub 2010, Kebbi-Beghdadi, Batista et al. 2011, Kebbi-Beghdadi, Cisse et al.
114
2011, Kebbi-Beghdadi et al. unpublished). Nothing is known about the molecules
115
implicated in the adhesion of Waddlia chondrophila to its host cell but OmcB and
116
HSP60 (GroEL) are present in the Waddlia genome (Bertelli, Collyn et al. 2010).
117
However, Waddlia OmcB lacks the XBBXBX sequence present in the N-terminal
118
region of all Chlamydia OmcB and that was shown to be crucial for adhesion
119
(Moelleken and Hegemann 2008, Fechtner, Stallmann et al. 2013). A more detailed
120
analysis of this genome also revealed the presence of a family of 11 genes coding for
121
putative porins with a barrel structure. These OmpA proteins could represent the
122
functional homologs of MOMP, a major chlamydial protein that is notably absent in
123
the Waddlia genome. Furthermore, one putative autotransporter protein has also
124
been described (gene wcw_0271) that displays a predicted overall structure
125
somehow similar to the chlamydial Pmps with an N-terminal leader sequence
126
followed by a passenger domain and a barrel C-terminal domain. In addition, this
127
Waddlia Pmp-like protein also harbours 6 tetrapeptide repeats identical to those
128
implicated in adhesion of the chlamydial Pmps.
129
In the present study, we identified, using a proteomic approach, the Waddlia
130
molecules implicated in adhesion and we characterized their transcriptional and
131
protein expression profiles. We also evaluated in vivo on epithelial cells the adhesion
132
potential of these molecules and of the putative Pmp-like protein, knowing that
133
understanding this crucial step of host-pathogen interactions is a major stone on the
134
way towards efficient diagnostic and treatments of this emerging pathogen.
135 136
Material and methods
137
Cell culture and bacterial strains
138
Human lung carcinoma cell line A549 (ATCC CCL-185), Vero cells derived from
139
kidney epithelial cells of the African Green Monkey (ATCC CCL-81) and the human
140
endometrial adenocarcinoma Ishikawa cell line were cultivated as described in 5
141
Kebbi-Beghdadi et al. (Kebbi-Beghdadi, Cisse et al. 2011). HEp-2 cells (ATCC CCL-
142
23 ) were routinely maintained in high glucose Dulbecco’s modified minimal essential
143
medium (DMEM; Gibco, Life Technologies, Zug, Switzerland) supplemented with
144
10% foetal calf serum (Biochrom AG, Berlin, Germany), 1% non essential amino
145
acids
146
W. chondrophila strain ATCC VR-1470 was grown at 32°C within Acanthamoeba
147
castellanii strain ATCC 30010 in 25 or 75 cm2 cell culture flasks (Corning, New York,
148
USA) with peptone-yeast extract-glucose broth (Jacquier, Aeby et al. 2013). EBs
149
were purified as described elsewhere (Greub and Raoult 2002b). For infection of
150
epithelial cells, bacteria were recovered from a 4 days-old amoebal co-culture and
151
filtered through a 5 µm filter (Millipore, Carrigtwohill, Ireland) to eliminate trophozoites
152
and cysts.
and
1%
vitamins
(Gibco,
Life
Technologies).
153 154
Infection procedure
155
Epithelial cells were harvested from Corning culture flasks with 0.25% trypsin
156
(Sigma-Aldrich, Buchs, Switzerland), washed with fresh medium and seeded, one
157
day before infection, at 2-5 x105 cells per well in 24-wells microplates (Corning) or at
158
4 x 106 cells per 25 cm2 flasks for RNA extraction and total protein preparation. Cells
159
were infected with a 1/2000 dilution of bacteria (MOI 0.1-1). Plates were then
160
centrifuged at 1790g for 10 min at room temperature. After 15 min of incubation at
161
37°C in the presence of 5% CO2, cells were washed with fresh medium to remove
162
non-internalized bacteria and were then incubated for different periods of time at
163
37°C in a 5% CO2 atmosphere.
164 165
Outer membrane proteins extraction and 2D gel electrophoresis
166
Purified EBs were incubated 1 hour at 37°C in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA
167
and 2% sodium lauroyl sarcosinate (sarkosyl) (Fluka BioChemika, Buchs,
168
Switzerland). Detergent-insoluble complexes were pelleted by ultracentrifugation at
169
100’000g for 1 hour. Outer membrane proteins were solubilized by a 10 minutes
170
incubation at 56°C in 0.125M Tris-HCl (pH6.8), 0.33M urea and 50 mM DTT. 100 g
171
of sarkosyl-insoluble proteins were resuspended in 37.5 mM Tris, 7M urea, 2M
172
Thiourea, 50 mM DTT, 4% CHAPS, 0.5% Triton X-100 and 1% ASB and separated
173
by 2D gel electrophoresis as described by Centeno et al. (Centeno, Repici et al.
6
174
2007). Proteins were visualized by Coomassie Blue staining or transferred to
175
nitrocellulose for overlay assays.
176 177
Biotinylation of epithelial cell membrane proteins
178
Confluent monolayer of A549, Vero and Ishikawa cells in 75 cm2 flasks were washed
179
3 times with ice-cold PBS then scraped with a rubber policeman. 2.5 x 107 cells were
180
incubated 1 hour at 37°C with 10 mg/ml EZ-link sulfo-NHS-LC-biotin (Pierce, Thermo
181
Fisher Scientific, Lausanne, Switzerland) in PBS, washed 3 times with PBS, 100 mM
182
glycin (Biosolve Chimie, Dieuze, France) and lysed by sonication in PBS. Cellular
183
debris were removed by 15 minutes centrifugation at 13'000 rpm, 4°C and
184
biotinylated proteins were kept at -80°C.
185 186
Overlay assays
187
Nitrocellulose membranes were blocked over night in PBS, 0.05% Tween 20, 1%
188
BSA and washed 3 times 15 minutes in PBS, 0.05% Tween 20, 0.1% BSA.
189
Membranes were incubated 1.5 hours at 4°C with biotinylated epithelial cells diluted
190
1/1000 in PBS, 0.05% Tween 20, 0.1% BSA. After three washing steps of 15 minutes
191
with PBS, 0.05% Tween 20, 0.1% BSA, membranes were incubated during 1.5 hours
192
with horseradish peroxidase-labelled streptavidin (Amersham, GE Healthcare,
193
Glattbrugg, Switzerland) diluted 1/10’000 in PBS, 0.05% Tween 20, 5% BSA. The
194
membranes were then washed four times 15 minutes with PBS, 0.05% Tween 20
195
and reactive proteins were detected with a chemiluminescence-based kit (Lite-
196
AblotTM, Euroclone, Milano, Italy).
197 198
Mass spectrometry
199
For identification of proteins, Coomassie Blue stained spots were excised from 2D
200
gels and transferred to special 96-well plates (Perkin Elmer Life Sciences,
201
Schwerzenbach, Switzerland). MALDI-MS-MS analysis was performed on a 4700
202
Proteomics Analyser (Applied Biosystems, Framingham, MA, USA) as described in
203
Kebbi-Beghdadi et al. (Kebbi-Beghdadi, Lienard et al. 2012). Briefly, in-gel proteolytic
204
cleavage with trypsin was performed in an automated workstation and digests were
205
resuspended in alpha-cyano-hydroxycinnamic acid matrix before being deposited in
206
duplicate
207
After MALDI-TOF MS analysis, the 10 most intense ion signals were selected for
on
a
target
plate.
7
208
MS/MS analysis. Non-interpreted peptide tandem mass spectra were used for direct
209
interrogation of W. chondrophila open reading frames. With the parameters used, the
210
threshold for statistical significance (p
