1 title: functional type 1 secretion system involved in legionella
TRANSCRIPT
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Title: Functional type 1 secretion system involved in Legionella pneumophila virulence 1
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Running title: T1SS involved in L. pneumophila virulence 3
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Authors: 1 - Fabien FUCHE1-5
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2 - Anne VIANNEY1-5
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3 – Claire ANDREA1-5
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4 - Patricia DOUBLET1-5
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5 - Christophe GILBERT1-5§
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Address: 11
1 CIRI, International Center for Infectiology Research, Legionella 12
pathogenesis group, Université de Lyon, Lyon, France. 13
2 Inserm, U1111, Lyon, France. 14
3 Ecole Normale Supérieure de Lyon, Lyon, France. 15
4 Université Lyon 1, Centre International de Recherche en Infectiologie, 16
Lyon, France. 17
5 CNRS, UMR5308, Lyon, France 18
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§ Corresponding author: [email protected], Phone : 33 4 73 43 13 66, 20
FAX : 33 4 72 43 26 86 21
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Keywords: Legionella pneumophila, Type I Secretion System (T1SS), Rtx toxin, virulence 23
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JB Accepts, published online ahead of print on 24 November 2014J. Bacteriol. doi:10.1128/JB.02164-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Abstract 25
Legionella pneumophila is a Gram negative pathogen mainly found in water, either in a free-26
living form or within infected protozoans where it replicates. This bacterium can also infect 27
humans by inhalation of contaminated aerosols, causing a severe form of pneumonia called 28
legionellosis or Legionnaire’s disease. The involvement of Type II and Type IV secretion 29
systems in the virulence of L. pneumophila is now well documented. Despite bioinformatics 30
studies showing that a Type I secretion system (T1SS) could be present in this pathogen, the 31
functionality of this system based on LssB, LssD and TolC proteins has never been 32
established yet. Here, we report the demonstration of the T1SS functionality, as well as its 33
role in the infectious cycle of L. pneumophila. Using deletion mutants and fusion proteins, we 34
demonstrated that the RTX protein RtxA is secreted through an LssB-LssD-TolC-dependent 35
mechanism. Moreover, fluorescence monitoring and confocal microscopy showed that this 36
T1SS is required for entry into the host cell, although it seems dispensable to intracellular 37
cycle. Together, these results underline the active participation of L. pneumophila to its 38
internalization into the host cells, via its T1SS. 39
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Introduction 42
Legionella pneumophila is a Gram negative pathogen that colonizes aquatic environments, 43
where it survives by infecting water protozoans, especially amoebae. Pathogenic strains of L. 44
pneumophila emerge from the environment after replication in such protozoans, are 45
disseminated through aerosols of contaminated water, and reach human alveolar macrophages 46
where they begin a new infectious cycle, causing a severe form of pneumonia called 47
Legionnaire’s disease or legionellosis. The development of air-conditioning systems, cooling 48
towers and other aerosol-generating systems is frequently reported as the reason for the 49
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spread of this pathogen (1). Indeed, L. pneumophila is the second etiological agent of 50
pneumonia requiring hospitalization in intensive care units, after Streptococcus pneumoniae 51
(2). Moreover, the mortality rate of Legionnaire’s disease, even under appropriate antibiotic 52
treatment, ranges from 7 % to 25 %, thus making legionellosis a public health concern (3). 53
Moreover, L. pneumophila is a scientifically relevant model to study intracellular pathogens 54
(4). 55
Among pathogenic bacteria, secretion systems play a crucial role in virulence, whether by 56
damaging the host, or by being essential for bacterial replication, for example by hijacking 57
cellular pathways or promoting the escape from the immune system (5). Bacterial protein 58
secretion systems have been extensively studied, and to date, 7 of them have been identified 59
within 2 categories: those which address their substrates to the extracellular environment, 60
such as types I, II, V and VII secretion systems, and those whose substrates are injected 61
through the host membrane into its cytoplasm, such as types III, IV and VI secretion systems 62
(6). In L. pneumophila, Types II and IV secretion systems (T2SS and T4SS, respectively) 63
have been identified several years ago (7-9) and their implication in the virulence of this 64
bacterium is extensively studied (10, 11). The T4SS Dot/Icm is particularly investigated, as it 65
is responsible for the translocation of more than 275 effector proteins into the cytoplasm of 66
infected cells (12, 13). Those effectors are required for the entire intracellular cycle of L. 67
pneumophila, as they are involved in the creation of a replicative niche inside the host cell, 68
called Legionella-containing vacuole (LCV) that is suitable for bacterial replication. A decade 69
ago, bioinformatics studies predicted a set of genes that codes for a putative Type I Secretion 70
System (T1SS) in Legionella, but the functionality of such system has never been 71
demonstrated (14). The implication of T1SSs in the virulence of pathogenic bacteria, such as 72
uropathogenic Escherichia coli (UPEC), Bordetella pertussis or even the entomopathogen 73
Photorhabdus, has been demonstrated many years ago (15-17). To date, the most studied 74
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examples of T1SS are Hly system in UPEC (18), Apr system in Pseudomonas aeruginosa 75
(19) and Prt system in Dickeya dadantii (20), For example, in the case of UPEC, the system 76
responsible for the secretion of the hemolytic toxin HlyA It is based on HlyB, an inner 77
membrane ABC-transporter involved in substrate recognition and translocation, the 78
periplasmic membrane fusion protein HlyD, and the outer membrane-spanning porin TolC 79
(21). Together these 3 components form a channel that spans both membranes to export 80
substrates into the extracellular environment. The most commonly found substrates are toxins, 81
such as lipases and proteases, adhesins and hemophores (22). Among T1SS-secreted toxins, 82
the RTX family is the most studied. Repeat-in Toxins (RTX) proteins are large, multi-domain 83
proteins that have diverse functions, depending on the embedded functional domains: HlyA 84
exhibits a pore forming activity, CyaA of Bordetella pertussis has an adenylate cyclase 85
activity, LapA and LapF of Pseudomonas putida are adhesins involved in cell-surface or cell-86
cell interactions, respectively (23). They also contain several repeats of the glycin-rich 87
nonapeptide motif GGxGxDxxx, which is the signature of RTX proteins (24). The RtxA 88
protein of L. pneumophila also contains a high number of tandem repeats, whose exact 89
number and nature vary among strains (25). In this pathogen, RtxA has been linked to the 90
ability of the bacterium to invade and replicate within amoebas and macrophages (26, 27). 91
However, the mechanism of the potential secretion of this protein has never been investigated. 92
To date, there is no available study on T1SS functionality in L. pneumophila, neither on its 93
role in virulence. In this paper, we report that LssD-LssD-TolC is a functional T1SS, and that 94
RtxA is a substrate of this T1SS. Moreover, we showed that it is involved in virulence 95
towards amoeba and macrophages, precisely in early steps of the infection, such as the entry 96
into the host cell. 97
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Materials and Methods 99
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Strains and growth conditions 100
All strains used in these study are listed in Table S1 (see Supporting Information). Legionella 101
pneumophila strains Lens and Paris were grown on buffered charcoal yeast extract (BCYE) 102
agar or in liquid BYE medium. Unless otherwise mentioned, all cultures were grown at 30°C; 103
since we observed in our lab that the virulence towards amoebae was at an optimum at this 104
temperature. Kanamycin (10 µg ml-1
), chloramphenicol (5 µg ml-1
) or gentamycin (5 µg ml-1
) 105
were added when appropriate. Escherichia coli strains were grown at 37°C in LB medium 106
supplemented with chloramphenicol (5 µg ml-1
) or ampicillin (100 µg ml-1
) when appropriate. 107
Axenic Acanthamoeba castellanii cells were grown on proteose-yeast extract-glucose (PYG) 108
medium at 30°C and split once a week. Dictyostelium discoideum Dd04 expressing calnexin-109
green fluorescent protein (GFP) (DBS0236184) were obtained from Dicty Stock Center 110
(http://dictybase.org/StockCenter/StockCenter.html; depositor A. Muller-Taubenberger). D. 111
discoideum cells were axenically grown in HL5 medium at 22°C, supplemented with G418 at 112
20 µg ml-1
when necessary. Human acute monocytic leukemia (THP1) cells and U937 cells 113
were maintained at 37°C, 5% CO2 in RPMI 1640 medium supplemented with 10% heat-114
inactivated fetal calf serum. Differentiation into macrophages was triggered by addition of 115
phorbol 12-myristate 13-acetate (PMA) at 100 ng ml-1
final concentration. 116
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Plasmids constructions 118
All DNA constructions were made using E. coli XL1 Blue or DH5alpha as hosts. L. 119
pneumophila Lens and Paris genomic DNA were used as template for PCR reactions, with 120
primers listed in Table S2 (see Supporting Information). Plasmids pXDC50 (28) and pXDC61 121
(29) were obtained from Xavier Charpentier (see Table S1 in Supporting Information). All 122
recombinant plasmids were systematically verified by enzymatic digestion before 123
electroporation in L. pneumophila (2400 V, 200 Ω, 25 µF). 124
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Gene inactivation in L. pneumophila 126
Gene-specific knock-out strains were constructed using the homologous recombination 127
strategy. Briefly, 2 kb flanking regions located upstream and downstream of the gene to 128
inactivate were amplified by PCR. Kanamycin resistance cassette was inserted between the 129
two regions by a double joint PCR. To obtain mutant strains derived from wild type Lens, the 130
final PCR fragment was cloned into the pLaw344 plasmid (30), which can be selected on 131
chloramphenicol and counter-selected on sucrose-supplemented medium. After 132
electroporation and selection, CmR clones containing the plasmid were grown 24 hours in 133
liquid medium without antibiotic, and spread on BCYE agar plates containing kanamycin (10 134
µg ml-1
) and sucrose (5% final concentration). Recombinant clones were then analyzed to 135
confirm deletion of the target gene(s) and loss of the pLaw344. To knock-out genes in strain 136
Paris, bacteria were grown overnight at 37°C to an OD600nm of 1-1.2. One to two micrograms 137
of purified PCR DNA fragment containing the resistance cassette flanked by the two 2 kb 138
homologous regions were added to the bacteria. The tubes were incubated overnight at 30°C 139
without shaking. Bacteria were then plated on BCYE agar containing 20 µg ml-1
kanamycin. 140
Recombinant clones were verified by PCR. 141
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Intracellular growth 143
Acanthamoeba castellanii cells were seeded in a 96-wells microplate at 1 x 105 cells/well and 144
allowed to adhere for at least 2 hours at 30°C. After washing with peptone-yeast extract (PY) 145
medium, amoebas were infected at a MOI of 10 with bacterial suspensions made by dilution 146
of late stationary phase cultures of L. pneumophila strains expressing mCherry (pXDC50). 147
Plates were then centrifuged 10 min at 600 g to phase bacteria-cells contact followed by 148
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incubation for 1 hour at 30°C. Amoeba were washed 3 times to remove non adherent bacteria 149
and plates were incubated at 30°C for 48 hours. 150
Dictyostelium discoideum cells were seeded at 1 x 105 cells/well, and allowed to adhere 16 151
hours at 22°C. Infections were performed in the same way using MB medium at 25°C. 152
Intracellular growth was monitored by following the intensity of fluorescence emitted by 153
mCherry (λexcitation: 580 nm, λemission: 620 nm) using a TECAN InfinitePro plate reader. The 154
plate reader was thermostated at 25°C or 30°C for D. discoideum or A. castellanii, 155
respectively. 156
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Cytotoxicity assays 158
Cell viability, hence bacterial-induced cell mortality, or cytotoxicity, was measured by using 159
the Alamar Blue viability indicator, according to the manufacturer protocol (Life 160
Technologies). Briefly, after 24 or 48 hours of incubation, infected cells were washed 3 times 161
in the appropriate medium, and 100 µl/well of 10% Alamar Blue diluted in medium was 162
added in each well. Plate were incubated at the temperature suitable for the cells considered 163
for 4 to 16 hours, and absorbance was recorded at 570 nm and 600 nm using a BioTek 164
Instruments µQuant plate reader. Cell viability was estimated by comparing 570 nm/600 nm 165
ratios with the ratio obtained with non-infected cells. 166
167
Recruitment of the ER to LCV in D. discoideum 168
D. discoideum cells producing calnexin-GFP were seeded into sterile glass coverslips in 6-169
well plates at 5 x 106 cells per well in HL5 medium and allowed to adhere overnight. 170
Monolayers were infected at an MOI of 100 with mCherry-producing L. pneumophila grown 171
for 4 days at 30°C. The plates were centrifuged at 880 g for 10 min, and incubated 1 hour at 172
25°C. The medium was then carefully removed and the monolayers were fixed with 3.7% 173
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paraformaldehyde (30 min, 4°C). Coverslips were examined with an inverted confocal 174
microscope (Axiovert 200M; Zeiss, Thornwood, NJ). 175
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Pore-forming activity assays 177
This assay was previously described by Kirby et al. (31). Briefly, A. castellanii cells were 178
seeded on coverslips placed in a 6-well plate and infected at an MOI of 500 for 1 hour at 179
30°C. Coverslips were then inverted onto a 5 µl drop of PBS containing 25 µg ml-1
ethidium 180
bromide and 5 µg ml-1
acridine orange placed on a glass side. Coverslips were immediately 181
observed using appropriate filters with a Zeiss fluorescence microscope. 182
183
ß-lactamase fusions construction and enzymatic activity assay 184
The last 162 or 731 amino acids coding sequence of L. pneumophila Paris rtxA gene were 185
PCR amplified using primers listed in Table S2 (see Supporting Information). The resulting 186
PCR fragments were digested, and cloned in the pXDC61 in frame with the 3’ terminus of 187
blaM gene (deleted for the sequence encoding the signal peptide), creating translational 188
fusions Bla-RtxA162 and Bla-RtxA731, respectively. 189
Legionella pneumophila strains carrying pblaM-rtxA162, pblaM-rtxA731, or pblaM-fabI (no 190
secretion control) were grown in BYE liquid medium to stationary phase. Cells were then 191
pelleted by centrifugation, adjusted to OD600nm 20 in BYE medium supplemented with 1 mM 192
isopropyl-thiogalactoside (IPTG) to induce the production of the hybrid proteins, and 193
incubated 3 hours at 30°C without shaking. Bacterial suspensions were then added to a sterile 194
paper disk placed onto an Escherichia coli MG1655 layer made on an ampicillin-195
supplemented LB agar plate, and growth of E. coli around the disk was monitored after a 16 196
hours incubation at 37°C. Supernatants were also collected after centrifugation, and dropped 197
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onto an Escherichia coli MG1655 layer made on an ampicillin-supplemented LB agar plate. 198
Growth of E. coli was monitored as previously mentioned. 199
200
mCherry fusions construction and secretion assay 201
The blaM-containing fragments of plasmids pblaM-rtxA162 and pblaM-rtxA731 were excised 202
(NdeI/KpnI) and the PCR-amplified mCherry-encoding gene was cloned into the same sites, 203
generating pmCherry-rtxA162 and pmCherry-rtxA731 respectively. Plasmids plssBDh, ptolCh 204
and plssBD-tolCh were constructed by cloning the lssBD, tolC or lssBD and tolC genes in the 205
p15A-derived plasmid pACYC184kan, under the control of the constitutive pKan promoter. 206
For the secretion assay, E. coli MG1655 strains carrying one fusion-expressing plasmid and 207
one T1SS gene-expressing plasmid were grown to late exponential phase, centrifuged and 208
resuspended in fresh LB medium supplemented with 1 mM IPTG. Induction of hybrid protein 209
expression was carried out at 37°C for 30 minutes, the supernatant was collected and mCherry 210
fluorescence was monitored in a 96-wells microplate for 16 hours at 30°C in a TECAN 211
InfinitePro 212
213
Results 214
Legionella pneumophila encodes two putative members of a type I secretion system 215
Jacobi and Heuner identified two genes, lssB and lssD, of Legionella pneumophila as part of a 216
putative type I secretion, based on similarity with toxin transporter systems of Vibrio 217
cholerae, Salmonella typhi and Escherichia coli (14). Moreover, we showed in a previous 218
study that a tolC gene is present in L. pneumophila genome and that it is involved in early 219
steps of host invasion, as a ∆tolC mutant exhibits an impaired virulence towards amoebas and 220
macrophages (32). A similar phenotype has also been observed with a ∆rtxA mutant strain 221
(26, 27). Carefully analyzing LssB protein sequence, and additionally to the classical ABC 222
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transporter domains (a TransMembrane Domain (TMD) and a Nucleotide Binding Domain 223
(NBD)), we noticed the presence of a C39 peptidase-like motif (from position 16 to position 224
137), which has been recently reported as a signature of T1SS that export RTX proteins (33). 225
In the case of functional C39 peptidase, the T1SS is involved in the export of bacteriocins and 226
the corresponding activity (C39 peptidase) is essential to mature the secreted bacteriocins 227
(cleavage downstream a GG pattern of a pro-domain). At the difference, the C39 peptidase-228
like motif corresponds to a degenerated catalytic site of a C39 peptidase and the exported 229
proteins all belong to the RTX family and are not matured during the export. 230
We therefore hypothesized that LssB-LssD-TolC acts as a functional type I secretion system 231
and has a role in the virulence of this bacterium, presumably by allowing L. pneumophila to 232
secrete the RtxA protein. Therefore, we inactivated lssB, lssD and rtxA genes in the epidemic 233
strain Lens as well as in the endemic strain Paris by homologous recombination, and studied 234
the role of these genes in the physiology and virulence of the bacteria. It should be noted that 235
lssB and lssD have been described as the last genes of a 6 genes operon by studying the 236
mRNA synthesis (14), therefore reducing a possible polar effect of inactivation. Within this 237
operon, the role of the other genes products has yet to be documented. rtxA gene is annotated 238
on genomes with its own transcript unit. 239
240
241
Legionella lssB and lssD genes are required for intracellular replication 242
As a preliminary test, the growth of each strain was followed by measuring absorbance at 600 243
nm. No growth difference could be observed between wild type and mutant strains, indicating 244
that lssB and lssD are not essential for optimal growth in laboratory (see Fig. S1 in Supporting 245
Information). The ability to replicate within different hosts was then investigated using the 246
strains carrying pXDC50. The intensity of mCherry fluorescence was monitored for 2 to 6 247
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days after contact with two models of environmental hosts, Acanthamoeba castellanii and 248
Dictyostelium discoideum (Fig. 1A and 1B). As expected, a ∆dotA mutant cannot replicate 249
within amoebas. This mutant is unable to assemble a functional type IV secretion system, 250
which is necessary for the translocation of many effectors in the cytosol of infected cells and 251
for intracellular growth (34). The wild-type strain Lens show detectable replication after 8 252
hours in A. castellanii and 40 hours in D. discoideum (Fig. 1A and 1B), the difference in time 253
being attributed to the difference in the temperatures used in these infection experiments: A. 254
castellanii optimal temperature is 30°C, while D. discoideum infections were carried out at 255
25°C (closer to the optimum temperature of growth for these amoebas, 22°C). A delay was 256
clearly observed in appearance of ∆lssBD mutant strain replication in both hosts (38 hours 257
and 105 hours, respectively). A ∆lssBD mutant strain carrying a plasmid with the wild-type 258
copy of native lssB and lssD genes exhibits an intracellular replication profile in A. castellanii 259
that is similar to the wild-type strain profile. This complementation is only partial in D. 260
discoideum, but the difference with the ∆lssBD profile is still significant. 261
262
lssB and lssD mediate bacterial-induced cytotoxicity 263
Cell death induction is known to be correlated with the virulence of L. pneumophila. We 264
investigated the cytotoxicity of the ∆lssBD mutant strain towards A. castellanii amoeba, and 265
found that its ability to cause cell death was dramatically reduced (below 30 %) compared to 266
the wild type strain’s ability (70 %) (Fig. 2A). This cytotoxicity was at least partially restored 267
by complementation with a wild type copy of these genes. Observations by optical 268
microscopy confirmed this lack of cytotoxic effect from the ∆lssBD mutant (see Fig. S2 in 269
Supporting Information). Moreover, this phenotype is also observed toward THP1 270
macrophages (10 % in case of ∆lssBD mutant strain versus 65 % in case of WT) and the 271
plasmid complementation totally restored Legionella cytotoxicity (Fig. 2B). Interestingly, 272
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these results suggest that the mechanism underlying the cytotoxicity may be effective against 273
these phylogenetically distant hosts. 274
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LssB and LssD are involved in early steps of the infectious cycle 276
We analyzed by confocal microscopy the ability of the ∆lssBD mutant strain to create a 277
functional replicative niche within the infected cell (Fig. 3A). The availability of numerous 278
genetic tools and mutants library of Dictyostelium discoideum made it a good model for such 279
studies. Indeed, we used an endoplasmic reticulum (ER) marker coupled to GFP (Calnexin-280
GFP) expressed in D. Discoideum to show that the average number of bacteria per infected 281
cell was around 9 for the wild type strain (Fig. 3B), which is similar to the number observed 282
using the ∆dotA mutant strain, indicating that a functional type IV secretion system is not 283
required for an efficient entry into the host cell. Interestingly, the ∆lssBD mutant strain 284
exhibited a significantly decreased internalization level, as only 5 bacteria were found inside 285
infected host cells on average. A wild type phenotype could be restored by plasmid 286
complementation (Fig. 3B). 287
A statistical analysis was performed on the observation of more than 200 host cells from 3 288
independent experiments to estimate the level of ER recruitment to LCV within the infected 289
cells. One hour after contact, approximately 65 % of internalized WT bacteria strain 290
constituted an ER-surrounded LCV (Fig. 3C). As expected, a ∆dotA mutant strain is unable to 291
form a mature LCV and no ER recruitment could be detected (Fig. 3A and 3C). The ability of 292
∆lssBD mutant to recruit ER-derived vesicles around the LCV was found to be statistically 293
comparable to the WT strain as well as to the complemented strain. These results showed that 294
lssB and lssD genes are required in an early step of the infectious circle, presumably during 295
the entry into the host cell, but do not seem essential to the constitution of the ER-surrounded 296
LCV within the first hour of infection. 297
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298
Secretion of the protein RtxA is abolished in ∆lssBD and ∆tolC mutant strains 299
RTX toxins are known substrates of type I secretions systems. Based on the characteristics 300
shared by proteins from the RTX family, Cirillo et al. identified a rtx locus in L. pneumophila 301
AA100 genome, containing the rtxA gene, that is required for virulence towards amoeba and 302
human macrophages (27). Indeed, the ability of a ∆rtxA mutant to adhere and enter into host 303
cells was reduced to 40% of the ability of the wild type strain. We obtained similar results 304
using L. pneumophila ∆rtxA mutant strain derived from wild type strain Paris (see Fig. S3 in 305
Supporting Information). Moreover, the cytotoxicity of the ∆rtxA mutant strain towards A. 306
castellanii amoeba was reduced (40 %) compared to the wild type Paris strain’s ability (60 307
%). This phenotype was similar to the ∆lssBD phenotype (see Fig. S4 in Supporting 308
Information). 309
Although the secretion signal for T1SS substrates is not conserved, it has been shown to be 310
uncleaved and located in the last 50-60 C-terminal residues of the substrate proteins (21). To 311
assess whether LssB, LssD and TolC are able to export Legionella RtxA protein, we 312
constructed two different translational fusions between the gene encoding the ß-lactamase 313
(blaM) deleted of its signal sequence and 3’ portions of rtxA gene respectively encoding 162 314
and 731 amino acids of RtxA C-terminal region. A blaM-fabI fusion was used as a negative 315
control of secretion, as FabI is a L. pneumophila cytoplasmic protein (29). The production of 316
all three hybrid proteins was assessed by Western blotting using anti-BlaM antibodies (Fig. 317
5). Comparable amounts of protein were detected with BlaM, BlaM-RtxA731 and BlaM-FabI, 318
but it seems that lower amounts were produced in case of BlaM-RtxA162. Moreover, 2 bands 319
can be seen for hybrid proteins, certainly due to the instability of the fusion products. The 320
secretion of these hybrid proteins was then studied by monitoring the growth of ampicillin-321
sensitive E. coli in presence of L. pneumophila Paris and mutant derivatives producing these 322
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hybrid proteins. This test and the time of study was designed to limit the background resulting 323
in the detection of hybrid proteins released after cell lysis, which was followed with BlaM-324
FabI fusion protein. Strain Paris, and its derivative mutant ∆lssBD were used for these 325
experiments and the results presented in Fig. 4A showed that the secretion of the two BlaM-326
RtxA162 and BlaM-RtxA731 hybrid proteins was abolished in a ∆lssBD genetic background 327
(no growth of E. coli around the central disk containing Legionella strain) compared to the 328
active secretion in a wild type genetic background. The BlaM-FabI hybrid did not allow good 329
growth of the E. coli ampicillin-sensitive strain, indicating that there was no secretion of the 330
hybrid protein and no lysis (WT) or very few lysis (∆lssBD) of the L. pneumophila strains 331
during the time of experiment. A similar experiment was performed by using cell-free culture 332
supernatant of hybrid-producing L. pneumophila and showed that the TolC protein is also 333
involved in the secretion of the BlaM-RtxA731 hybrid protein as ampicillin was degraded and 334
allowed the E. coli ampicillin-sensitive strain to grow around the supernatant drop (Fig. 4B). 335
336
RtxA is secreted in a LssB-LssD-TolC dependent manner 337
To ensure that the secretion of the hybrid proteins observed in a L. pneumophila wild type 338
background is specific to the presence of LssB, LssD and TolC, the corresponding genes were 339
expressed in an E. coli background (wild type strain MG1655), as well as the genes encoding 340
new hybrid proteins: mCherry-RtxA162 and mCherry-RtxA731. The mCherry fluorescent 341
protein was chosen because of its slow folding rate (35), as it was shown that a rapid folding 342
of the substrate prior to the secretion by T1SS could block the export (36). The secretion of 343
mCherry-RtxA hybrid proteins can be evaluated by following the fluorescence intensity at 344
620 nm in the culture supernatant (excitation: 580 nm). The results showed that the three 345
proteins LssB, LssD and TolC are required for efficient secretion of the two tested hybrid 346
proteins in this heterologous background (Fig. 5). We also noticed a higher level of secretion 347
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(more than twice) using the shortest RtxA end (162 amino acid residues). Since those two 348
hybrid proteins were produced at a similar level (data not shown), we assumed that this is due 349
to an inappropriate folding of the larger hybrid protein. The expression of LssB-LssD proteins 350
in absence of Legionella TolC protein led to a low secretion level of fusion proteins (up to a 351
factor 5 increase compared to control strain) that might result from E. coli TolC protein 352
involvement but does not appear statistically significant. It is worth noting that the expression 353
of Legionella TolC protein alone did not result in fusion proteins secretion in E. coli. 354
355
lssB, lssD and tolC genes are required in RtxA-mediated pore-forming activity of L. 356
pneumophila 357
In addition to adherence and virulence, L. pneumophila RtxA was shown to be involved in 358
pore formation in the membranes of infected cells (20). We thus compared the pore-forming 359
activity of the ∆rtxA mutant to those of ∆lssBD and ∆tolC mutants strains (Fig. 6) and 360
observed a strong decrease of this activity (10 fold decrease) using all three mutant strains 361
compared to wild type strain Paris (75% of visualized amoeba cells lost their membrane 362
integrity). It is important to note that this decrease was similar using all mutant strains 363
(∆lssBD, ∆tolC and ∆rtxA) suggesting a shared implication in this activity. Moreover, 364
complementation of ∆lssBD mutant strain restored the pore-forming activity. Interestingly, 365
the ∆dotA mutant strain exhibited a pore-forming activity similar to that of the wild-type 366
strain, which show that, although it is necessary for intracellular replication and thus for 367
global virulence towards host cell, the T4SS is not required for the pore-forming ability of L. 368
pneumophila. These results demonstrate that RtxA pore-forming activity is nearly absent in 369
both T1SS-deficient mutants ∆lssBD and ∆tolC, indicating that the secretion of hybrid 370
proteins observed in the previous experiments was not an artefact, and actually occurs in an in 371
vivo infection situation. 372
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373
Discussion 374
The role of secretion systems in bacterial pathogenicity is well established. In Legionella 375
pneumophila, two secretion systems are clearly identified (T4ASS Icm/Dot and T2SS Lsp), 376
and their role in the virulence of this bacterium has been extensively studied over the years. 377
Recently, the possibility for the T4BSS Lvh VirD4 protein to complement the defect in 378
virulence of a T4ASS-invalidated ∆dotA mutant was reported (11, 37). Genome sequencing 379
data also predicted the existence of a putative autotransporter (T5aSS) in strain Paris (38). 380
Bioinformatics predictions also suggested that a T1SS is present in all L. pneumophila strains 381
(14, 39-42). Moreover, we pointed out the presence of a C39 peptidase-like motif in L. 382
pneumophila LssB protein, which strongly suggested its classification as a T1SS inner 383
membrane partner involved in recognition and secretion of RTX proteins (33). In this paper 384
we demonstrated that the corresponding protein RtxA in Legionella is a substrate of a LssB-385
LssD-TolC based Type I Secretion System. The structure of L. pneumophila RtxA however 386
raised several interesting perspectives concerning the role of the tandem repeats that shape 387
more than half of the protein. Using both homologous and heterologous expression of fusion 388
proteins, we showed that the last 162 amino acids of RtxA protein were sufficient to promote 389
its secretion, hence eliminating the necessity of tandem repeats for the recognition and/or 390
translocation through the T1SS. We are currently investigating the localisation of RtxA 391
during infection of host cells by L. pneumophila, as it was shown in Pseudomonas fluorescens 392
that the RtxA-homolog LapA was a surface-associated adhesion (43, 44). Although the role of 393
the different regions of such large RTX proteins remains to be experimentally assessed, the 394
variability in number and nature of the tandem repeats between RtxA proteins of different 395
strains of L. pneumophila may suggest a modulation of the virulence based on these repeats. 396
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Additionally, we documented the phenotype of a L. pneumophila strain defective for this 397
T1SS. The ∆lssBD mutant strain displayed strongly attenuated virulence towards A. 398
castellanii and D. discoideum. Moreover, preliminary results showed a moderately attenuated 399
virulence towards U937 macrophages (data not shown). We were able to study the early steps 400
of the infectious cycle, demonstrating that a ∆lssBD mutant strain is defective for the entry 401
into host cell, which is consistent with the observations of Cirillo et al. using a ∆rtxA mutant 402
strain (26). The absence of T1SS did not seem to cause a defect in the creation of the 403
replicative Legionella-containing vacuole (LCV), as the recruitment of ER-derived vacuoles 404
to the surface of the phagosome is not altered after one hour of infection. This recruitment is 405
considered as a good marker of the constitution of the LCV and of successful hijacking of 406
host vesicular trafficking (28). Therefore we hypothesized that the initial entry into the host 407
cell was responsible for the general intracellular replication defect observed. However, the 408
attenuation of entry may not to be sufficient to explain the quasi-abolished virulence towards 409
amoebae, suggesting that the increased phagosome-lysosome fusion observed in ∆rtxA mutant 410
by Cirillo and coworkers might also be involved in this phenotype. 411
Altogether, these results demonstrate for the first time the functionality of a T1SS in 412
Legionella pneumophila, and underline its importance in the virulence of this pathogen. 413
Indeed, the lss locus seems to be present in the majority of 217 L. pneumophila isolates 414
analysed by Cazalet and co-workers (25). The rtxA gene encoding the T1SS substrate is also 415
present in all tested isolates, though its structure is different between several strains groups. 416
These results underline the active participation of L. pneumophila to its internalization into 417
the host cells, via its T1SS and its cognate substrate RtxA. 418
419
Acknowledgements 420
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Fabien Fuche was supported by a fellowship from the Ministère de l'Enseignement Supérieur 421
et de la Recherche (France). 422
We are grateful to Nathalie Bailo for technical assistance, especially in microscopy. We also 423
would like to thank Xavier Charpentier (University of Lyon) for kindly providing the 424
pXDC50 and pXDC61 plasmids. 425
426
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566
Figure legends 567
Figure 1: Intracellular replication of the L. pneumophila Lens and mutant derivatives in 568
eucaryotic host cells 569
The intracellular replication of the ∆lssBD mutant in Acanthamoeba castellanii (A) and 570
Dictyostelium discoideum (B) was monitored by following the fluorescence intensity with a 571
TECAN plate reader (excitation: 580 nm; emission: 620 nm). All bacteria expressed the 572
mCherry fluorescent protein under control of an IPTG-inducible promoter (plasmid 573
pXDC50). The results are representative of at least 4 infection experiments (MOI = 10), 574
performed in triplicate (errors bars are indicated). 575
576
Figure 2: Cytotoxicity of L. pneumophila Lens and mutant derivatives towards eucaryotic 577
host cells 578
Infections of A. castellanii (A) or THP1 macrophages (B) were carried out as described in the 579
experimental procedures, and host cell mortality was quantified using the Alamar blue dye, 48 580
hours after the initial contact. Standard deviations are represented as error bars; the results are 581
means of 3 independent experiments (MOI =10) performed in triplicate (n.s., non 582
significantly different; ** p<0.01; **** p<0.0001 using a Student’s t-test). 583
584
Figure 3: L. pneumophila entry into the host cells and ER recruitment to the vacuole 585
Dictyostelium discoideum cells expressing calnexin-GFP were seeded on glass coversips and 586
infected by L. pneumophila WT Lens and mutant derivatives expressing mCherry (MOI = 587
100). After 1 hour, coverslips were fixed, mounted and observed by confocal microscopy (A). 588
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Statistical analysis was performed by observing at least 200 host cells, resulting from 3 589
independent experiments, allowing to show the average number of bacteria in each cell 590
(B) as well as the proportion of internalized bacteria (C) actually recruiting ER to their 591
LCV (n.s., non-significantly different; **** p<0.0001 using Student’s t-test). ‡ No 592
statistical analysis could be performed here as the ΔdotA mutant is by definition unable 593
to recruit ER. 594
595
Figure 4: Secretion assay 596
The secretion of BlaM-RtxA hybrid proteins in L. pneumophila Paris WT, ∆lssBD or ∆tolC 597
background was assayed by monitoring the growth of an E. coli ampicillin-sensitive strain 598
plated on an ampicillin-containing medium. The growth can be restored if ampicillin in the 599
agar plate is degraded by the secreted ß-lactamase activity. L. pneumophila strains expressing 600
hybrid proteins were dropped onto a sterile paper disk, production of hybrid proteins by 601
bacteria prior to the test was verified by western blot on whole cells extracts (anti-BlaM) (A), 602
or cell-free culture supernatants were dropped directly onto the E. coli monolayer (B). 603
604
Figure 5: Heterologous mCherry-RtxA secretion assay 605
The fluorescence intensity in cell-free culture supernatants of E. coli producing mCherry-606
RtxA hybrid proteins was monitored 16 hours after bacteria removal by centrifugation 607
(folding time). The fluorescence was normalized by the amount of fluorescence in bacteria 608
(production of the protein) and by the fluorescence of the native mCherry-producing strain, 609
for each pACYC construction. Results are expressed as the ratio of fluorescence for plssBDh, 610
ptolCh and plssBD-tolCh strains, compared to the fluorescence observed with the empty vector 611
(pACYC184kan). Tests were performed for both mCherry-RtxA162 and mCherry-RtxA731 612
hybrid proteins (n.s., non-significantly different; ** p<0.01 using Student’s t-test). 613
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614
Figure 6: Pore-forming activity of L. pneumophila Paris and mutant derivatives towards 615
amoeba cells 616
Acanthamoeba castelanii cells seeded on coverslips were infected by L. pneumophila at an 617
MOI of 500 for 1 hour. Coverslips were inverted on a drop of acridine orange/ethidium 618
bromide staining solution and analysed by fluorescence microscopy. Cells stained in green 619
were considered as intact, whereas membrane of cells stained in red were considered as 620
damaged. Standard deviations are represented as error bars; the results are means of 3 621
independent experiments (n.s., non significantly different; **** p<0.001 using Student’s t-622
test). 623
624
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