Specific targeting of intestinal Prevotella copri by a ... · the intestinal phase of infection, we...
Transcript of Specific targeting of intestinal Prevotella copri by a ... · the intestinal phase of infection, we...
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Title: Specific targeting of intestinal Prevotella copri by a Listeria monocytogenes bacteriocin 1
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Short title: Targeting of intestinal Prevotella by a bacteriocin 3
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Authors : Nathalie Rolhion1,2,3*, Benoit Chassaing4,5, Marie-Anne Nahori1,2,3, Jana de Bodt6, 5
Alexandra Moura7,8, Marc Lecuit7,8,9, Olivier Dussurget1,2,3,10, Marion Bérard11, Massimo 6
Marzorati6, Hannah Fehlner-Peach12 , Dan R. Littman12,13, Andrew T. Gewirtz5**, Tom Van de 7
Wiele6**, Pascale Cossart1,2,3*** 8
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1Institut Pasteur, Unité des Interactions Bactéries-Cellules, Département de Biologie Cellulaire 10
et Infection, F-75015 Paris, France 11
2Inserm, U604, F-75015 Paris, France 12
3INRA, Unité sous-contrat 2020, F-75015 Paris, France 13
4Neurosciences Institute, Georgia State University, Atlanta, Georgia, USA 14
5Center for Inflammation, Immunity and Infection, Institute for Biomedical Sciences, Georgia 15
State University, Atlanta, Georgia, USA 16
6Center of Microbial Ecology and Technology (CMET), Faculty of Bioscience Engineering, 17
Ghent University, Ghent, Belgium 18
7Institut Pasteur, Unité Biologie des Infections, F-75015 Paris, France 19
8Inserm, U1117, F-75015 Paris, France 20
9Paris Descartes University, Sorbonne Paris Cité, Division of Infectious Diseases and Tropical 21
Medicine, Necker-Enfants Malades University Hospital, Institut Imagine, F-75743 Paris, 22
France 23
10Université de Paris, F-75013 Paris, France 24
11Animalerie Centrale, Department of Technology and Scientific Programmes (DTPS), Institut 25
Pasteur, F-75015 Paris, France 26
12The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University 27
School of Medicine, New York, New York, 10016, USA 28
13Howard Hughes Medical Institute, New York, New York 10016, USA 29
* current address: Centre de Recherche Saint-Antoine, équipe « Microbiote, Intestin et 30
Inflammation », Inserm UMRS 938, F-75012 Paris, France 31
** these authors contributed equally to the work 32
*** correspondence to: [email protected] 33
Key words: Listeria, bacteriocin, microbiota, Lmo2776, M-SHIME® 34
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Authors contributions: P.C. and N.R. designed the project. P.C. supervised the project. N.R 35
initiated the project and performed all the experiments, otherwise stated. B.C. performed 16S 36
samples preparation, 16S sequencing data analysis and FISH experiments. M.A.N. was 37
involved in all the in vivo work. J.B. helped with the M-SHIME® experiments. A.M. and M.L. 38
performed the Listeria genomic analyses. O.D. helped with analysis of data. M.B. helped with 39
in vivo experiments. H.F-P. and D.R.L. provided human isolates of P. copri. A.T.G. provided 40
advice for microbiota-related experiments. T.V.W. and M.M. provided support and advices for 41
the M-SHIME® experiments. N.R. and P.C. wrote the paper, with editing and discussions from 42
B.C., O.D., A.M., M.L., H.F-P., D.R.L., M.M., A.T.G. and T.V.W. 43
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Abstract 44
Deciphering the specific function of every microorganism in microbial gut communities is a 45
key issue to interrogate their role during infection. Here, we report the discovery of a Listeria 46
bacteriocin, Lmo2776, that specifically targets the abundant gut commensal Prevotella copri 47
and affects Listeria infection. Oral infection of conventional mice with almo2776 mutant 48
leads to a thinner intestinal mucus layer and higher Listeria loads both in the intestinal content 49
and deeper tissues compared to WT Listeria, while no difference is observed in germ-free mice. 50
This microbiota-dependent effect is phenocopied by precolonization of germ-free mice before 51
Listeria infection, with P. copri, but not with other commensals,. Together, these data unveil a 52
role for Prevotella in controlling intestinal infection, highlighting that pathogens may 53
selectively deplete microbiota to avoid excessive inflammation. 54
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Introduction 55
Prevotella is classically considered a common commensal bacterium due to its presence in 56
several locations of the healthy human body, including the oral cavity, gastrointestinal tract, 57
urogenital tract and skin (1). The Prevotella genus encompasses more than 40 different 58
culturable species of which three, P. copri, P salivae and P. stercorea, can be isolated from the 59
gut. Prevotella has been reported to be associated with opportunistic infections, e.g. 60
periodontitis or bacterial vaginosis (1). Moreover, Prevotella is the major genus of one of the 61
three reported human enterotypes (2), but how Prevotella behaves in different gut ecosystems 62
and how it interacts with other bacteria of the microbiota and/or with its host is not well defined. 63
In addition, high levels of genomic diversity within Prevotella strains of the same species have 64
been observed (3), which adds another layer of complexity for predicting the effects of 65
Prevotella strains. Recent studies have linked higher intestinal abundance of P. copri to 66
rheumatoid arthritis (4-6), metabolic syndrome (7), low-grade systemic inflammation (7) and 67
inflammation in the context of human immunodeficiency virus (HIV) infection (8-10), 68
suggesting that some Prevotella strains may trigger and/or worsen inflammatory diseases (1, 69
11, 12) 70
The microbiota plays a central role in protecting the host from pathogens, in part through a 71
process referred to as colonization resistance (13). In the case of Listeria monocytogenes, the 72
foodborne pathogen responsible for listeriosis, the intestinal microbiota provides protection, as 73
germfree mice are more susceptible to infection than conventional mice (14, 15). Treatment 74
with probiotics such as Lactobacillus paracasei CNCM I-3689 or Lactobacillus casei BL23 75
was shown to decrease L. monocytogenes systemic dissemination in orally inoculated mice (16). 76
Unravelling the interactions between the host, the microbiota and pathogenic bacteria is critical 77
for the design of new therapeutic strategies via manipulation of the microbiota. However, 78
identifying the specific molecules and mechanisms used by the commensals to elicit their 79
beneficial action is challenging due to the high complexity of the microbiome, together with 80
technical issues in culturing many commensal species. In addition, cooperative interactions 81
between commensal species are likely to be central to the functioning of the gut microbiota 82
(17). So far, the mechanism or the molecules underlying the impact of commensals on the host 83
have been elucidated only for a few species. Segmented filamentous bacteria (SFB) were shown 84
to coordinate maturation of T cell responses towards Th17 cell induction (18, 19). 85
Glycosphingolipids produced by the common intestinal symbiont Bacteroides fragilis have 86
been found to regulate homeostasis of host intestinal natural killer T cells (20). A 87
polysaccharide A (PSA) also produced by B. fragilis induces and expands Il-10 producing 88
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CD4+ T cells (21-23). Finally, the microbial anti-inflammatory molecule (MAM) secreted by 89
Faecalibacterium prausnitzii impairs the nuclear-factor (NF)-B pathway (24). 90
Conversely, enteric pathogens have evolved various means to outcompete other species in the 91
intestine and access nutritional and spatial niches, leading to successful infection and 92
transmission (25, 26). In this regard, the contribution of bacteriocins and type VI secretion 93
system effectors during pathogen colonization of the gut is an emerging field of investigation. 94
Here, by studying the impact of a novel L. monocytogenes bacteriocin (Lmo2776) on infection, 95
we discovered P. copri, an abundant gut commensal, as the primary target of Lmo2776 in both 96
the mouse and human microbiota and as a modulator of infection. 97
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Results 99
Lmo2776 limits Listeria intestinal colonization and virulence in a microbiota-dependent 100
manner 101
A recent reannotation of the genome of the Listeria monocytogenes strain EGD-e revealed that 102
the lmo2776 gene, absent in the non-pathogenic Listeria innocua species (Figure S1A), 103
potentially encodes a secreted bacteriocin of 107 amino acids (27, 28), homologous to the 104
lactococcin 972 (Lcn972) secreted by Lactococcus lactis (29) and to putative bacteriocins of 105
pathogenic bacteria Streptococcus iniae (30), Streptococcus pneumoniae and Staphylococcus 106
aureus (Figure S1B). This gene belongs to a locus containing two other genes lmo2774 and 107
lmo2775 genes, encoding potential immunity and transport systems (28). This locus is present 108
in Lineage I strains responsible for the majority of Listeria clinical cases (31) and in some 109
Lineage II strains, such as EGD-e (Figure S1C). Little is known about this bacteriocin family 110
and most studies have focused on Lcn972. Lmo2776 shares between 38 to 47% overall amino 111
acid sequence similarity with members of the lactococcin 972 family. Because expression of 112
lmo2774, lmo2775 and lmo2776 genes is significantly higher in stationary phase compared to 113
exponential phase of EGD-e at 37 °C in BHI (Figure S2A), all experiments described below 114
were conducted with Listeria grown up to stationary phase. 115
We first examined the effect of Lmo2776 on infection. We inoculated conventional BALB/c 116
mice with either the WT, the lmo2776 or the Lmo2776 complemented strains and compared 117
Listeria loads in the intestinal lumen and deeper organs, the spleen and liver. We had verified 118
that the deletion of lmo2776 was not affecting the expression of surrounding genes, lmo2774, 119
lmo2775 and lmo2777 (Figure S2B) or bacterial growth in vitro (Figure S2C). Inoculation of 120
mice with lmo2776 strain resulted in significantly higher bacterial loads in the small intestinal 121
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lumen 24h post-inoculation compared to the WT strain (Figure 1A). These differences 122
persisted at 48 and 72h post-inoculation (Figure S2D). Bacterial loads of lmo2776 were also 123
significantly higher in the spleen and liver at 72h post-inoculation compared with both WT and 124
Lmo2776-complemented strains (Figure 1B and C). Similar results were observed in 125
C57BL/6J mice (data not shown). Together, these results indicate a key role for Lmo2776 in 126
bacterial colonization of the intestine and deeper organs. Following intravenous inoculation of 127
BALB/c mice with 5.103 WT or lmo2776 bacteria, bacterial loads at 72h post-inoculation 128
were similar in the spleen and liver (Figure S2E), revealing that Lmo2776 exerts its primary 129
role during the intestinal phase of infection and not later. 130
Considering that lmo2776 is predicted to encode a bacteriocin and that it significantly affects 131
the intestinal phase of infection, we hypothesized that Lmo2776 might target intestinal bacteria, 132
thereby impacting Listeria infection. To address the role of intestinal microbiota in infection, 133
we orally inoculated germ-free mice with WT or lmo2776 strains and compared bacterial 134
counts 72h post-inoculation. Strikingly, no significant difference was observed between WT 135
and lmo2776 strains in the small intestinal content (Figure 1D), nor in spleen and liver 136
(Figure 1E and F). These results showed that the Lmo2776 bacteriocin limits the virulence of 137
wild-type Listeria in a microbiota-dependent manner. 138
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Lmo2776 specifically targets Prevotella in mouse and human microbiota 140
In order to identify which intestinal bacteria were targeted by Lmo2776, we compared 141
microbiota compositions of conventional mice orally infected with WT or lmo2776 strains by 142
16S rRNA gene sequencing. We first verified that the fecal microbiota composition of all mice 143
was indistinguishable at day 0 (Figure 2A). As expected, the microbiota composition at day 1 144
post-infection was dramatically altered by infection with Listeria WT (Figure S3A). These 145
alterations in microbiota composition included reduced levels of Bacteroidetes phylum (relative 146
abundance before infection: 65.4% and at day 1 post-infection: 42.4%) and increased levels of 147
Firmicutes (relative abundance before infection: 29.9% and at day 1 post infection: 54.0%) 148
(Figure S3B to E). The increased levels in the Firmicutes were mainly due to an increase of 149
the Clostridia class (relative abundance before infection: 27.4% and at day 1 post-infection: 150
50.7%). Of note, the relative abundance of Listeria was around 0.1% and cannot therefore 151
explain by itself the increased levels of Firmicutes observed between day 0 and day 1. 152
Importantly, at 24h and 48h post-infection, intestinal microbial community compositions 153
differed in mice orally inoculated with the lmo2776 strain compared to the WT strain (Figure 154
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2A). We focused on operational taxonomic units (OTUs) for which the relative abundance was 155
identical before the infection with the Listeria strains (day -3 to day 0) and was subsequently 156
altered by at least a 2-fold difference at day 1 post-infection in mice infected with lmo2776 157
compared to mice infected with WT strain. In independent experiments, the relative abundance 158
of 12 OTUs was lower in mice infected with the WT strain compared to the lmo2776 mutant 159
(Figure 2B and C) (OTU 355746, 216524, 421792, 258849, 331772, 346870, 430194, 447141, 160
465433, 208409, 353012 and 364179) at day 1 and also at day 2 post-infection. Phylogenetic 161
analyses revealed that all these 12 OTUs belong to the Prevotella cluster (Figure 2D). A 162
decrease of Prevotella in mice infected with WT strain at day 1 and day 2 post-inoculation 163
compared to mice infected with lmo2776 strain was also observed by qPCR analysis, using 164
primers specific for Prevotella, confirming that Lmo2776 targets Prevotella in the intestinal 165
microbiota (Figure 2E). 166
Important differences exist between mouse and human gut microbiota composition. Indeed, 167
Prevotella abundance is known to be low in the mouse intestinal content (less than 1%) while 168
it can reach up to 80 % in the human gut microbiota (32, 33). As Listeria is a human pathogen, 169
we searched to investigate the impact of Lmo2776 on human intestinal microbiota. For this 170
purpose, we used a dynamic in vitro gut model (mucosal-simulator of human intestinal 171
microbial ecosystem (M-SHIME®)), which allows stable maintenance of human microbiota in 172
vitro, in the absence of host cells but in presence of mucin-covered beads (34-37) and therefore 173
studies on human microbiota independently of the host responses (such as inflammation). The 174
microbiota of a healthy human volunteer was inoculated to the system which was then infected 175
with WT or lmo2776 Listeria. Application of 16S sequencing to luminal and mucosal M-176
SHIME® samples indicated that before Listeria inoculation, the bacterial composition in all 177
vessels was similar (Figure 3A and data not shown). In contrast, following Listeria addition, 178
luminal microbial community compositions were different in vessels containing WT bacteria 179
compared to both non-infected vessels and vessels infected with the lmo2776 isogenic mutant 180
(Figure 3A). No difference was observed in mucosal microbial community composition. The 181
relative abundance of 7 OTUs (313121, 518820, 346938, 588929, New.0.ReferenceOTU20, 182
173565 and 89083) was lower in the case of the WT strain compared to the non-inoculated 183
condition or upon addition of the lmo2776 strain (Figure 3B). These 7 OTUs all belonged to 184
Prevotella copri species (Figure 3C), revealing that Lmo2776 targets P. copri in the human 185
microbiota in a host-independent manner. 186
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As short-chain fatty acid (SCFA) levels serve as a classical read-out for gut microbiota 187
metabolism and as Prevotellae are known to produce propionate (38), we quantified SCFAs 188
production in the luminal M-SHIME® samples. A specific decrease in propionate production 189
upon infection with WT bacteria was observed as early as 6h post-infection (Figure 3D) 190
compared to non-infected and lmo2776-infected vessels. This difference was continuously 191
observed up to 3 days post-infection, while no significant difference was observed for butyrate, 192
isobutyrate, acetate and isovalerate (Figure S4). Although propionate is produced by many 193
bacterial species, the decrease in propionate production observed upon inoculation of M-194
SHIME® with WT Listeria is in agreement with the decrease in Prevotella population. 195
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Lmo2776 targets P. copri in vitro 197
We first addressed the direct inhibitory activity of Lmo2776 on P. copri by growing P. copri at 198
37 °C in anaerobic conditions in the presence of culture supernatants of Listeria strains and 199
counting the viable CFUs on agar plates. Growth of P. copri dramatically decreased (up to 3 200
Log) in the presence of the WT Listeria supernatant compared to the lmo2776 supernatant or 201
medium alone (Figure 3E), suggesting that Lmo2776 is secreted and targets directly P. copri. 202
To definitively assess the function of Lmo2776, a peptide of 63 aa (Gly69 to Lys131) 203
corresponding to the putative mature form of Lmo2776 was synthesized. Its activity was first 204
analyzed on P. copri and B. thetaiotaomicron, another prominent commensal bacterium 205
(Figure 3F). A dose-dependent effect of Lmo2776 peptide was observed on the growth of P. 206
copri while no effect was observed on the growth of B. thetaiotaomicron, demonstrating that 207
Lmo2776 targets P. copri and not B. thetaiotaomicron. We then tested the effect of the peptide 208
on several other intestinal bacteria, either aerobic (Enterococcus faecalis, Escherichia coli) or 209
anaerobic (Akkermansia muciniphila) bacteria. No effect was observed on any of these bacteria 210
(Figure 3G). Moreover, Lmo2776 peptide did not inhibit the growth of seven other Prevotella 211
species (P. salivae, P. oris, P. nigrescens, P. pallens, P. corporis, P. melaninogenica and P. 212
bivia). We next tested the peptide activity on 7 P. copri isolated from healthy humans and 213
patients. Strikingly, 6 out of the 7 strains were sensitive to the bacteriocin (Figure 3G). 214
We also tested the effect of the Lmo2776 peptide on known targets of the bacteriocins of the 215
lactococcin-972 family (B. subtilis, L. lactis MG1614). Growth of B. subtilis decreased 216
significantly in presence of the peptide (Figure 3G), while no effect was observed on L. lactis 217
MG1614. Growth of Bacillus subtilis was also specifically and significantly reduced in the 218
presence of WT Listeria and of Lmo2776 complemented strains compared to the lmo2776 219
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strain (Figure S5A-B). Addition of the culture supernatant of WT Listeria to B. subtilis 220
significantly decreased the number of B. subtilis compared to the addition of lmo2776 culture 221
supernatant (Figure S5C). (30). These results indicate that Lmo2776 is a bona fide bacteriocin 222
that targets both P. copri and B. subtilis in vitro. 223
To evaluate the effect of the Lmo2776 peptide in vivo in animals, we used an approach 224
previously described to bypass degradation by enzymes of the upper digestive tract. 225
Conventional BALB/c mice were inoculated intra-rectally with Lmo2776 peptide or water, 226
taken as a control. Levels of total bacteria, Prevotella and Akkermansia muciniphila were 227
determined by quantitative PCR on feces collected between 1 and 4h post-administration. While 228
no effect was observed on the levels of total bacteria or A. muciniphila, fecal levels of P. copri 229
decreased following administration of Lmo2776 peptide, demonstrating that similar to bacteria, 230
Lmo2776 alone was effective in reducing P. copri in vivo (Figure 3H). 231
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Colonization of germ-free mice by P. copri phenocopies the effect of the microbiota on 233
Listeria intestinal growth in conventional mice 234
To decipher the role of P. copri during Listeria infection in vivo, germ-free C57BL/6J mice 235
were orally inoculated with either P. copri, B. thetaiotaomicron or P. salivae, another 236
Prevotella present in the gut, or stably colonized with a consortium of 12 bacterial species 237
(termed Oligo-Mouse-Microbiota (Oligo-MM12), representing members of the major bacterial 238
phyla in the murine gut: Bacteroidetes (Bacteroides caecimuris and Muribaculum intestinale), 239
Proteobacteria (Turicimonas muris), Verrucomicrobia (Akkermansia muciniphila), 240
Actinobacteria (Bifidobacterium longum subsp. Animalis) and Firmicutes (Enterococcus 241
faecalis, Lactobacillus reuteri, Blautia coccoides, Flavonifractor plautii, Clostridium 242
clostridioforme, Acutalibacter muris and Clostridium innocuum) (39)). Two weeks after 243
colonisation, these mice were orally inoculated with WT Listeria or lmo2776 strains and 244
Listeria loads in the intestinal lumen and target organs were compared 72h post-infection. 245
Compared to the WT strain, the lmo2776 mutant strain displayed significantly higher loads in 246
the intestinal lumen (Figure 4A), the spleen (Figure 4B) and liver (Figure S5D) in mice 247
colonized with P. copri, while no difference between the two strains was observed in mice 248
precolonized with B. thetaiotaomicron, P. salivae or the OligoMM12 consortium. In addition, 249
the number of P. copri significantly decreased in WT inoculated P. copri-colonized animals 250
compared to lmo2776-inoculated animals (Figure 4C). Altogether, these results indicate that 251
the greater ability of the lmo2776 mutant to grow in the intestine and reach deeper tissues 252
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compared to the WT strain is dependent on the presence of Prevotella in the microbiota, as it is 253
observed in either conventional mice or mice colonized with P. copri. 254
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P. copri modifies the mucus layer and its permeability 256
The intestinal mucus layer of conventional animals forms a physical barrier of about 30µm that 257
is able to keep bacteria at a distance from the epithelium (40). A mucus-eroding microbiota 258
promotes greater epithelial access (41). Prevotella, through production of sulfatases that induce 259
mucus degradation (42), might impair the mucosal barrier function and therefore contribute to 260
better accessibility to intestinal epithelial cells and to local inflammation. We thus compared 261
the mucus layer thickness of conventional mice infected with WT Listeria or lmo2776 by 262
confocal microscopy, using mucus-preserving Carnoy fixation and FISH (43). The average 263
distance of bacteria from colonic epithelial cells was significantly smaller in mice infected with 264
lmo2776 compared to mice infected with WT Listeria at 24 and 48h (Figure 4D and E), 265
suggesting that Prevotella present in the microbiota of mice infected with lmo2776 decreases 266
the mucus layer thickness and consequently increases its permeability. Of note, these distances 267
were also smaller than in uninfected mice, indicating that Listeria infection by itself can affect 268
the mucus layer thickness. To confirm the effect of P. copri on mucus layer in the context of 269
listerial infection, germ-free C57BL/6J mice were precolonized with P. copri, B. 270
thetaiotaomicron or P. salivae, then orally inoculated with WT Listeria or lmo2776 strains 271
and mucus layer thickness was analysed by FISH. In mice precolonized with P. copri, the 272
average distance of bacteria from colonic epithelial cells was significantly smaller in lmo2776-273
infected mice compared to WT Listeria-infected mice (Figure 4F and G). Strikingly, such 274
difference was not observed in germ-free mice or in mice precolonized with P. salivae or B. 275
thetaiotaomicron, revealing that mucus erosion is dependent on P. copri. 276
Since disruption of the mucosal barrier by Prevotella could favour invasion of the host by 277
bacteria and contribute to intestinal inflammation, we quantified faecal lipocalin-2 (LCN-2) as 278
a marker of intestinal inflammation (44). LCN-2 is a small secreted innate immune protein 279
which is critical for iron homeostasis and whose levels increase during inflammation. Faecal 280
LCN-2 levels were thus analysed after colonization of germ-free mice with P. copri compared 281
to non-colonized mice or mice colonized with B. thetaiotaomicron. A significant increase of 282
faecal LCN-2 was observed in germ-free mice monocolonized with P. copri compared to non-283
colonized animals or to animals monocolonized with B. thetaiotaomicron (Figure 4H), 284
revealing that P. copri induces intestinal inflammation. Altogether, these results showed that 285
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presence of Prevotella in the intestine is associated with a thinner mucus layer and increased 286
levels of faecal LCN-2. They are consistent with previous reports describing Prevotella as a 287
bacterium promoting a pro-inflammatory phenotype (1, 6, 45). 288
289
Discussion 290
Outcompeting intestinal microbiota stands among the first challenging steps for 291
enteropathogens. Pathogens may secrete diffusible molecules such as bacteriocins or T6SS 292
effectors to target commensals and consequently promote colonization and virulence. In most 293
cases, the molecular mechanisms underlying the interplay between pathogenic and commensal 294
bacteria in the intestine remain elusive. We previously reported that most strains responsible 295
for human infections, such as the F2365 strain, secrete a bacteriocin that promotes intestinal 296
colonization by Listeria (46). When overexpressed in mouse gut, this bacteriocin, named 297
Listeriolysin S (LLS), decreases Allobaculum and Alloprevotella genera known to produce 298
butyrate or acetate, two SCFAs reported to inhibit transcription of virulence factors or growth 299
of Listeria (47, 48). However, whether physiological concentrations of LLS have a direct or an 300
indirect role on these genera is still under investigation and is a question particularly difficult 301
to address as LLS is highly post-translationally modified and therefore difficult to purify or to 302
synthetize. In the case of Salmonella enterica serovar Typhimurium infection, killing of 303
intestinal Klebsiella oxytoca via the its T6SS is essential for Salmonella enterica gut 304
colonization of gnotobiotic mice colonized by K. oxytoca (49), but whether K. oxytoca and 305
other members of the gut microbiota are targeted by the Salmonella T6SS in conventional mice 306
is unknown. Finally, Shigella sonnei uses a T6SS to outcompete E. coli in vivo but the effectors 307
responsible for this effect are unknown (50). 308
Here, we demonstrated that the Lmo2776 Listeria bacteriocin targets Prevotella in mouse and 309
in in vitro reconstituted human microbiota. This effect is direct and specific to P. copri as (i) P. 310
copri are killed by Listeria culture supernatant and by the purified Lmo2776 in vitro and (ii) 311
despite the complexity of the microbiota and its well-controlled equilibrium, no other genus of 312
the intestinal microbiota was found to be impacted by Lmo2776. By studying Lmo2776, we 313
have unveiled a so far unknown role for intestinal Prevotella copri in controlling bacterial 314
infection. The intestinal microbiota, in some cases, has already been reported to promote 315
bacterial virulence by producing metabolites that enhance pathogens virulence gene expression 316
and colonization in the gut (25, 26). For example, B. thetaiotaomicron enhances Clostridium 317
rodentium colonization by producing succinate (51, 52) and Akkermansia muciniphila 318
exacerbates S. Typhimurium-induced intestinal inflammation by disturbing host mucus 319
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homeostasis (53). P. copri increases the mucus layer permeability and increases propionate 320
concentration and levels of fecal LCN-2, in agreement with previous studies reporting that P. 321
copri exacerbates inflammation (6, 45). In addition, Prevotella enrichment within the lung 322
microbiome of HIV-infected patients has been observed and is associated with Th17 323
inflammation (54). Prevotella sp. have also been associated with bacterial vaginosis and their 324
role in its pathogenesis has been linked to the production of sialidase, an enzyme involved in 325
mucin degradation and increased levels of pro-inflammatory cytokines (55, 56). Our data 326
strongly indicate that P. copri, by modifying the mucus layer permeability and changing the gut 327
inflammatory condition, promotes greater epithelial access and therefore infection by Listeria 328
(Figure 4I). We can speculate that individuals with high abundance of intestinal Prevotella 329
might be more susceptible to enteric infections. Interestingly, it was recently shown that 330
subjects with higher relative abundance of P. copri could be at higher risk to traveler's diarrhea 331
and to the carriage of multidrug-resistant Enterobacteriaceae (57). On the other hand, Lmo2776 332
Listeria bacteriocin allows a selective depletion of P. copri in intestinal microbiota. This could 333
therefore prevent excessive inflammation and allow Listeria persistence and long lasting 334
infection, eventually leading to menngitis. Further work is required to determine why Listeria 335
strains would gain an advantage by keeping the lmo2776 gene. We showed here that the 336
Lmo2776 bacteriocin also targets B. subtilis, a Gram-positive bacterium found in the soil, 337
suggesting that Lmo2776 could give an advantage to Listeria in that environment. It is possible 338
that Lmo2776 is critical for species survival and replication in a so far unknown niche, 339
consequently favoring transmission or dissemination. B. subtilis is also found in the human 340
gastrointestinal tract (58) and could be targeted by Lmo2776 in the intestine as well. B. subtilis 341
is also targeted by Sil, another member of the lactococcin 972 family (30). The role of the 342
homologs of lactococcin 972 in other human pathogenic bacteria such as S. pneumoniae and S. 343
aureus remains to be determined, but the conservation of the bacteriocin in different pathogenic 344
bacteria associated with mucosa strongly suggests an important role. 345
Taken together, our data indicate that P. copri can modulate human disease and using Lmo2776 346
might represent an effective therapeutic tool to specifically reduce P. copri abundance in the 347
gut without affecting the remaining commensal microbiota. 348
349
Figure legends 350
Figure 1. Lmo2776 limits Listeria virulence in a microbiota-dependent manner. (A-C) 351
BALB/c mice were inoculated orally with 5×109 Listeria monocytogenes WT (EGDe), 352
Δlmo2776 or Lmo2776 complemented (p2776) bacteria. CFUs in the intestinal luminal content 353
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13
(A), the spleen (B) and the liver (C) were assessed at 72h post-infection. (D-F) Germ-free 354
C57BL/6J were inoculated with 5×109 Listeria WT or Δlmo2776 for 72h and CFUs in the 355
intestinal luminal content (D), the spleen (E) and the liver (F) were assessed. Each dot 356
represents the value for one mouse. Statistically significant differences were evaluated by the 357
Mann–Whitney test. (*p< 0.05, NS, not significant). 358
359
Figure 2. Lmo2776 targets Prevotella in mouse microbiota. (A) Principal coordinates 360
analysis of the weighted Unifrac distance matrix of mice infected with WT strain (blue) or 361
lmo2776 (red) at day 0 (left), day 1 (center) and day 2 (right). Permanova: at day 0, P=0.383; 362
at day 1, P=0.05864; and at day 2, P=0.360. (B) Relative abundance of 12 OTUs in gut 363
microbiota of mice inoculated with WT or lmo2776 strains at day 0, day 1 and day 2. Each 364
dot represents the value for one mouse. (C) Heat-map analysis of the relative abundance of 12 365
OTUs in gut microbiota of mice inoculated with WT or lmo2776 strains at day 0, day 1 and 366
day 2. Each raw represents one mouse. The red and blue shades indicate high and low 367
abundance. (D) Phylogenetic tree of 16S rRNA gene alignment of 5 representative bacteria for 368
each phylum of the bacteria domain, together with OTUs showing significantly different 369
relative abundances in gut microbiota of mice infected with WT or lmo2776 strains at day 1. 370
The 12 OTUs with an increased abundance in lmo2776-infected mice compare to Listeria 371
WT-infected mice are shown in red and Prevotella genus is indicated in blue. (E) PCR 372
quantification of Prevotella in feces of mice inoculated with WT strain or lmo2776 at day 0 373
and day 1. 374
375
Figure 3. Lmo2776 targets P. copri in human microbiota and in vitro. (A) Relative 376
abundance of genera in SHIME® vessels non-infected or infected with WT or lmo2776 strains 377
at day 0, day 1 and day 2. The four more abundant genera are indicated. (B) Relative abundance 378
of 7 different OTUs in SHIME® vessels infected with WT or lmo2776 strains or non-infected 379
at day 0 and day 1. Each dot represents the value for one vessel. (C) Phylogenetic tree of 16S 380
rRNA gene alignment of several Prevotella species, together with OTUs showing significantly 381
different relative abundances in vessels inoculated with WT or lmo2776 strains at day 1. Six 382
OTUs with an increased abundance in lmo2776-inoculated vessels compare to Listeria WT-383
inoculated vessels are shown in red and Prevotella copri is indicated in blue. (D) Levels of 384
propionate in SHIME® vessels infected with WT (orange) or lmo2776 (red) strains or non-385
infected (blue) overtime. Results are expressed as mean ± SEM for 2 to 3 individual vessels. 386
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14
(E) Numbers of P. copri after incubation with supernatant of WT (Lm) or lmo2776 strains. 387
(F) Relative abundance of P. copri (white) and B. thetaiotamicron (black) after 24h incubation 388
with increasing dose of Lmo2776 peptide (3 (+), 6 (++) and 9(+++) µg/ml) relative to their 389
abundance without Lmo2776 peptide. (G) Relative abundance of different bacteria after 24h 390
incubation with Lmo2776 peptide (3µg/ml) relative to their abundance without Lmo2776 391
peptide. Results are expressed as mean ± SEM of a least 3 independent experiments and P-392
values were obtained using two-tailed unpaired Student’s t-test (*p<0.05, ***p<0.005). (H) 393
PCR quantification of Prevotella and A. muciniphila in the feces of mice treated with Lmo2776 394
peptide (1 mg) or with water relative to their levels before treatment. Each dot represents the 395
value for one mouse. Statistically significant differences were evaluated by Student’s t-test 396
(**p< 0.01). 397
398
Figure 4. P. copri controls Listeria infection by modifying mucus layer and promoting 399
inflammation. (A-B) Assessment of listerial CFUs in the intestinal luminal content (A) and in 400
the spleen (B) of germ-free (GF) C57BL/6J mice colonized or not with P. copri, P. salivae or 401
B. thetaiotamicron or stably colonized with 12 bacterial species (Oligo-MM12) for 2 weeks and 402
then inoculated with L. monocytogenes WT or Δlmo2776 for 72h. (C) Numbers of P. copri 403
CFUs in the intestinal luminal content of GF C57BL/6J mice colonized with P. copri and then 404
inoculated with Listeria WT or Δlmo2776 for 72h. (D) Confocal microscopy analysis of 405
microbiota localization in colon of BALB/c mice infected with 5×109 Listeria WT or Δlmo2776 406
bacteria for 24 or 48h. Muc2 (green), actin (purple), bacteria (red), and DNA (Blue). Bar = 407
20μm. White arrows highlight the 3 closest bacteria. Pictures are representatives of 5 biological 408
replicates. (E) Distances of closest bacteria to intestinal epithelial cells per condition over 5 409
high-powered fields per mouse, with each dot representing a measurement. (F) Confocal 410
microscopy analysis of microbiota localization in colon of GF C57BL/6J mice colonized with 411
P. copri, B. thetaiotamicron or P. salivae for 2 weeks and inoculated with 5×109 Listeria WT 412
or Δlmo2776 bacteria. Muc2 (green), actin (purple), bacteria (red), and DNA (Blue). Bar = 413
20μm. (G) Distances of closest bacteria to intestinal epithelial cells per condition over 5 high-414
powered fields per mouse, with each dot representing a measurement. (H) Levels of the 415
inflammatory marker Lcn-2 in faeces of mice 2 weeks post-inoculation with P. copri or B. 416
thetaiotamicron. (I) Model depicting the effect of Prevotella on Listeria infection. In A, B and 417
E, each dot represents one mouse. Statistically significant differences were evaluated by the 418
Mann–Whitney test (A, B), one way-ANOVA test (E, G) or two-tailed unpaired Student’s t-419
test (C and H) (*p< 0.05, ***p<0.005). 420
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was notthis version posted July 2, 2019. . https://doi.org/10.1101/680801doi: bioRxiv preprint
15
421
Acknowledgments 422
We thank the CDTA (Cryopréservation, Distribution, Typage et Archivage animal, CNRS, 423
Orléans) and the Institut Pasteur Animalerie Centrale staff, especially the technicians of the 424
Centre for Gnotobiology Platform of their help with animal work (Karim Sébastien, Thierry 425
Angélique, Marisa Gabriela Lopez Dieguez, Martine Jacob and Eddie Maranghi). We thank 426
Grégory Jouvion and Magali Tichit from the Unité d'Histopathologie humaine et modèles 427
animaux de l’Institut Pasteur for technical help and the Centre Ressources Biologiques de 428
l’Institut Pasteur for providing strains. We thank Laurence Maranghi and Juienne Blondou for 429
essential technical support and Christophe Bécavin, Andrew Hryckowian, Beatriz Martinez, 430
Gérard Eberl and Gunnar Hansson for helpful discussion. 431
N.R. was supported by an EMBO short-term fellowship (ASTF 399-2015). B.C. is supported 432
by a Career Development Award from the Crohn’s and Colitis Foundation, an Innovator Award 433
from the Kenneth Rainin Foundation and a Seed Grant from the GSU’s Brain & Behaviour 434
program. O.D. was supported by Idex UP2019. H. F-P. was supported by an NYU-HCC CTSI 435
grant (1TL1TR001447). D.R. L. was supported by the Howard Hughes Medical Institute and 436
the Colton Center for Autoimmunity. This work was supported by grants to P.C. (European 437
Research Council (ERC) Advanced Grant BacCellEpi (670823), ANR BACNET (BACNET 438
10-BINF-02-01), ANR Investissement d'Avenir Programme (10-LABX-62-IBEID), Human 439
Frontier Science Program (HFSP; RGP001/2013), ERANET Infect-ERA PROANTILIS 440
(ANR-13-IFEC-0004-02) and the Fondation le Roch les Mousquetaires). P.C. is a Senior 441
International Research Scholar of the Howard Hughes Medical Insitute. 442
443
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575
List of supplementary materials 576
Materials and Methods 577
Fig S1-S5 578
References (59-72) 579
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Figure 1
* * NS
A
WT D Cplt100101102103104105106107
intestinal content
CFU/
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ntent
WT D p2776
* *
B
WT D Cplt100101102103104105106107108109
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WT D Cplt100101102103104105106107108109
101 0liver
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Figure 2
A
B
C
day 0PC2 (19.19%)
PC3 (14.73%)PC1 (47.24%)
WTD
D
OTUs targetedby Lmo2776
Prevotella genus
0.07
day 1PC2 (24.28%)
PC3 (4.96%) PC1 (60.11%)
WTD
day 2
wtD227
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6 wtD227
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2
4
6
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DWT DWT DWT day 0 day 1 day 2 wt
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D2276 wt
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8
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6 wtD227
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6 wtD227
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6 wtD227
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2
3
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DWT DWT DWT day 0 day 1 day 2
DWT DWT DWT day 0 day 1 day 2
DWT DWT DWT day 0 day 1 day 2
DWT DWT DWT day 0 day 1 day 2
DWT DWT DWT day 0 day 1 day 2
DWT DWT DWT day 0 day 1 day 2
DWT DWT DWT day 0 day 1 day 2
DWT DWT DWT day 0 day 1 day 2
DWT DWT DWT day 0 day 1 day 2
relative
abund
ance
relative
abund
ance
relative
abund
ance
relative
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ance
relative
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ance
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ance
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ance
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ance
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ance
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ance
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ance
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DWT DWT DWT day 0 day 1 day 2
relative
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ance
WTD
PC3 (11.81%)
PC2 (13.55%)
PC1 (61.45%)
day0
day1
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Figure 3
B
D
1,00E+00
1,00E+01
1,00E+02
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Prevotella
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WTNI D NI WT D
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200250300350400450500550600
0 6h 24h 48h 72h
propionate
NI WT D NI WT D0.00
0.05
0.10
0.15
relati
ve ab
unda
nce
313121 518820 346938 588929
NI WT D NI WT D0.00
0.05
0.10
0.15
0.20
relati
ve ab
unda
nce
NI WT D NI WT D0.00
0.05
0.10
0.15
relati
ve ab
unda
nce
NI WT D NI WT D0
20
40
60
relati
ve ab
unda
nce
day 0NI WT D
day 1NI WT D
day 0NI WT D
day 1NI WT D
day 0NI WT D
day 1NI WT D
day 0NI WT D
day 1NI WT D
020406080
100120140160 Lm Prevotella
P. copri
******
**
******
****
control copripeptide copricontrol ak peptide ak10- 510- 410- 310- 210- 1100101102
no + +noLmo2776 peptide :
relative
abund
ance of
bacte
ria Prevotella A. muciniphila
NI WT D NI WT D0
2
4
6
8
10
relati
ve ab
unda
nce
NI WT D NI WT D0.0
0.2
0.4
0.6
0.8
1.0
relati
ve ab
unda
nce
day 0NI WT D
day 1NI WT D
day 0NI WT D
day 1NI WT D
New Ref OTU20
NI WT D NI WT D0.0
0.5
1.0
1.5
2.0
relati
ve ab
unda
nce
day 0NI WT D
day 1NI WT D
173565 189083
relative
abund
ance of
bacte
ria **
C
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was notthis version posted July 2, 2019. . https://doi.org/10.1101/680801doi: bioRxiv preprint
GF WTGF D
copri WTcopri D
B theta WTB theta
D
salivae
WTsali
vae D
MM12 WTMM12 D
103
104
105
106
107
Lister
ia (CF
U/mg)
I
E
WT 24
Dlmo2776 24 WT 48
Dlmo2776 48
0
10
20
30
40
distan
ce of
bacte
ria
from
IEC (µ
m)
DWT24 h
DWT48 h
* *C
WT DGF + P. copri
*
Prevo
tella/
mg co
ntent WT D
24 h48 h
WT(Lmo2776 secretion) D2776
PrevotellaListeria
F G
D
B
WT
D
GF GF + P. copri GF + B. thethaiotamicron
WT D WT D WT DGF GF + P. copri GF + P. salivae.
WT DGF + B.theta.
WT DOligoMM12
CFU/
splee
n
GF + P. salivae
Figure 4
H
GF
GF + Prevotella GF + Bt
0
50
100
150200250
lipocal
in-2 (
ng/ml
)Lcn
-2 (n
g/g f
eces
)
germ-free + P. copri + B. thethaiotamicron
*GF WT
GF DPc W
TPc D Bt WT Bt D
Ps WT
Ps D0
1020304050
distan
ce of
bacte
ria
from
IEC (µ
m)
WT D WT D WT DGF GF + P. copri GF + P. salivae.
WT DGF + B.theta.
**
GF WTGF D
copri WTcopri D
B theta WTB theta
D
salivae
WTsali
vae D
MM12 WTMM12 D
10 3
10 4
10 5
10 6
10 7
10 8A *** NSNS
CFU/
mg co
ntent
WT D WT D WT DGF GF + P. copri GF + P. salivae
WT DGF + B.theta
WT DOligoMM12
NS NS ** NSNS NS NS
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was notthis version posted July 2, 2019. . https://doi.org/10.1101/680801doi: bioRxiv preprint