specific targeting of intestinal prevotella copri by a ... · the intestinal phase of infection, we...

1

Title: Specific targeting of intestinal Prevotella copri by a Listeria monocytogenes bacteriocin 1

2

Short title: Targeting of intestinal Prevotella by a bacteriocin 3

4

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

9

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

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

https://doi.org/10.1101/680801

2

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


https://doi.org/10.1101/680801

3

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


https://doi.org/10.1101/680801

4

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


https://doi.org/10.1101/680801

5

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

98

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


https://doi.org/10.1101/680801

6

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

139

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


https://doi.org/10.1101/680801

7

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


https://doi.org/10.1101/680801

8

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

196

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


https://doi.org/10.1101/680801

9

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

232

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


https://doi.org/10.1101/680801

10

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

255

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


https://doi.org/10.1101/680801

11

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


https://doi.org/10.1101/680801

12

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


https://doi.org/10.1101/680801

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


https://doi.org/10.1101/680801

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


https://doi.org/10.1101/680801

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

References 444

1. J. M. Larsen, The immune response to Prevotella bacteria in chronic inflammatory disease. 445 Immunology 151, 363-374 (2017). 446

2. M. Arumugam et al., Enterotypes of the human gut microbiome. Nature 473, 174-180 (2011). 447 3. V. K. Gupta, N. M. Chaudhari, S. Iskepalli, C. Dutta, Divergences in gene repertoire among the 448

reference Prevotella genomes derived from distinct body sites of human. BMC Genomics 16, 449 153 (2015). 450

4. D. Alpizar-Rodriguez et al., Prevotella copri in individuals at risk for rheumatoid arthritis. Ann 451 Rheum Dis, (2019). 452

5. Y. Maeda et al., Dysbiosis Contributes to Arthritis Development via Activation of Autoreactive 453 T Cells in the Intestine. Arthritis Rheumatol 68, 2646-2661 (2016). 454

6. J. U. Scher et al., Expansion of intestinal Prevotella copri correlates with enhanced 455 susceptibility to arthritis. Elife 2, e01202 (2013). 456

7. H. K. Pedersen et al., Human gut microbes impact host serum metabolome and insulin 457 sensitivity. Nature 535, 376-381 (2016). 458


https://doi.org/10.1101/680801

16

8. S. M. Dillon et al., Gut dendritic cell activation links an altered colonic microbiome to mucosal 459 and systemic T-cell activation in untreated HIV-1 infection. Mucosal Immunol 9, 24-37 (2016). 460

9. C. A. Lozupone et al., HIV-induced alteration in gut microbiota: driving factors, consequences, 461 and effects of antiretroviral therapy. Gut Microbes 5, 562-570 (2014). 462

10. U. S. Kaur et al., High Abundance of genus Prevotella in the gut of perinatally HIV-infected 463 children is associated with IP-10 levels despite therapy. Sci Rep 8, 17679 (2018). 464

11. R. E. Ley, Gut microbiota in 2015: Prevotella in the gut: choose carefully. Nat Rev Gastroenterol 465 Hepatol 13, 69-70 (2016). 466

12. G. Precup, D. C. Vodnar, Gut Prevotella as a possible biomarker of diet and its eubiotic versus 467 dysbiotic roles-A comprehensive literature review. Br J Nutr, 1-24 (2019). 468

13. C. G. Buffie, E. G. Pamer, Microbiota-mediated colonization resistance against intestinal 469 pathogens. Nat Rev Immunol 13, 790-801 (2013). 470

14. C. Archambaud et al., The intestinal microbiota interferes with the microRNA response upon 471 oral Listeria infection. MBio 4, e00707-00713 (2013). 472

15. S. Becattini et al., Commensal microbes provide first line defense against Listeria 473 monocytogenes infection. J Exp Med 214, 1973-1989 (2017). 474

16. C. Archambaud et al., Impact of lactobacilli on orally acquired listeriosis. Proc Natl Acad Sci U 475 S A 109, 16684-16689 (2012). 476

17. S. Rakoff-Nahoum, K. R. Foster, L. E. Comstock, The evolution of cooperation within the gut 477 microbiota. Nature 533, 255-259 (2016). 478

18. V. Gaboriau-Routhiau et al., The key role of segmented filamentous bacteria in the coordinated 479 maturation of gut helper T cell responses. Immunity 31, 677-689 (2009). 480

19. Ivanov, II et al., Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139, 481 485-498 (2009). 482

20. D. An et al., Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal 483 natural killer T cells. Cell 156, 123-133 (2014). 484

21. S. K. Mazmanian, C. H. Liu, A. O. Tzianabos, D. L. Kasper, An immunomodulatory molecule of 485 symbiotic bacteria directs maturation of the host immune system. Cell 122, 107-118 (2005). 486

22. S. K. Mazmanian, J. L. Round, D. L. Kasper, A microbial symbiosis factor prevents intestinal 487 inflammatory disease. Nature 453, 620-625 (2008). 488

23. J. L. Round, S. K. Mazmanian, Inducible Foxp3+ regulatory T-cell development by a commensal 489 bacterium of the intestinal microbiota. Proc Natl Acad Sci U S A 107, 12204-12209 (2010). 490

24. E. Quevrain et al., Identification of an anti-inflammatory protein from Faecalibacterium 491 prausnitzii, a commensal bacterium deficient in Crohn's disease. Gut 65, 415-425 (2016). 492

25. A. J. Baumler, V. Sperandio, Interactions between the microbiota and pathogenic bacteria in 493 the gut. Nature 535, 85-93 (2016). 494

26. N. Rolhion, B. Chassaing, When pathogenic bacteria meet the intestinal microbiota. Philos 495 Trans R Soc Lond B Biol Sci 371, (2016). 496

27. M. Desvaux, E. Dumas, I. Chafsey, C. Chambon, M. Hebraud, Comprehensive appraisal of the 497 extracellular proteins from a monoderm bacterium: theoretical and empirical exoproteomes 498 of Listeria monocytogenes EGD-e by secretomics. J Proteome Res 9, 5076-5092 (2010). 499

28. P. Glaser et al., Comparative genomics of Listeria species. Science 294, 849-852 (2001). 500 29. B. Martinez, J. E. Suarez, A. Rodriguez, Lactococcin 972 : a homodimeric lactococcal bacteriocin 501

whose primary target is not the plasma membrane. Microbiology 142 ( Pt 9), 2393-2398 502 (1996). 503

30. M. F. Li, B. C. Zhang, J. Li, L. Sun, Sil: a Streptococcus iniae bacteriocin with dual role as an 504 antimicrobial and an immunomodulator that inhibits innate immune response and promotes 505 S. iniae infection. PLoS One 9, e96222 (2014). 506

31. M. M. Maury et al., Uncovering Listeria monocytogenes hypervirulence by harnessing its 507 biodiversity. Nat Genet 48, 308-313 (2016). 508


https://doi.org/10.1101/680801

17

32. F. Hildebrand et al., Inflammation-associated enterotypes, host genotype, cage and inter-509 individual effects drive gut microbiota variation in common laboratory mice. Genome Biol 14, 510 R4 (2013). 511

33. T. L. Nguyen, S. Vieira-Silva, A. Liston, J. Raes, How informative is the mouse for human gut 512 microbiota research? Dis Model Mech 8, 1-16 (2015). 513

34. B. Chassaing, T. Van de Wiele, J. De Bodt, M. Marzorati, A. T. Gewirtz, Dietary emulsifiers 514 directly alter human microbiota composition and gene expression ex vivo potentiating 515 intestinal inflammation. Gut 66, 1414-1427 (2017). 516

35. A. Geirnaert et al., Interindividual differences in response to treatment with butyrate-517 producing Butyricicoccus pullicaecorum 25-3T studied in an in vitro gut model. FEMS Microbiol 518 Ecol 91, (2015). 519

36. P. Van den Abbeele et al., Butyrate-producing Clostridium cluster XIVa species specifically 520 colonize mucins in an in vitro gut model. ISME J 7, 949-961 (2013). 521

37. P. Van den Abbeele et al., Incorporating a mucosal environment in a dynamic gut model results 522 in a more representative colonization by lactobacilli. Microb Biotechnol 5, 106-115 (2012). 523

38. D. Flint H.J., S.H. , in Encyclopedia Food Microbiology, C. A. Batt, Ed. (Elsevier, 2014), pp. 203-524 208. 525

39. S. Brugiroux et al., Genome-guided design of a defined mouse microbiota that confers 526 colonization resistance against Salmonella enterica serovar Typhimurium. Nat Microbiol 2, 527 16215 (2016). 528

40. M. E. Johansson et al., The inner of the two Muc2 mucin-dependent mucus layers in colon is 529 devoid of bacteria. Proc Natl Acad Sci U S A 105, 15064-15069 (2008). 530

41. M. S. Desai et al., A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus Barrier 531 and Enhances Pathogen Susceptibility. Cell 167, 1339-1353 e1321 (2016). 532

42. D. P. Wright, D. I. Rosendale, A. M. Robertson, Prevotella enzymes involved in mucin 533 oligosaccharide degradation and evidence for a small operon of genes expressed during 534 growth on mucin. FEMS Microbiol Lett 190, 73-79 (2000). 535

43. M. E. Johansson, G. C. Hansson, Preservation of mucus in histological sections, immunostaining 536 of mucins in fixed tissue, and localization of bacteria with FISH. Methods Mol Biol 842, 229-235 537 (2012). 538

44. B. Chassaing et al., Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker 539 for intestinal inflammation. PLoS One 7, e44328 (2012). 540

45. E. Elinav et al., NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. 541 Cell 145, 745-757 (2011). 542

46. J. J. Quereda et al., Bacteriocin from epidemic Listeria strains alters the host intestinal 543 microbiota to favor infection. Proc Natl Acad Sci U S A 113, 5706-5711 (2016). 544

47. C. E. Ostling, S. E. Lindgren, Inhibition of enterobacteria and Listeria growth by lactic, acetic 545 and formic acids. J Appl Bacteriol 75, 18-24 (1993). 546

48. Y. Sun, B. J. Wilkinson, T. J. Standiford, H. T. Akinbi, M. X. O'Riordan, Fatty acids regulate stress 547 resistance and virulence factor production for Listeria monocytogenes. J Bacteriol 194, 5274-548 5284 (2012). 549

49. T. G. Sana et al., Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to 550 establish in the host gut. Proc Natl Acad Sci U S A 113, E5044-5051 (2016). 551

50. M. C. Anderson, P. Vonaesch, A. Saffarian, B. S. Marteyn, P. J. Sansonetti, Shigella sonnei 552 Encodes a Functional T6SS Used for Interbacterial Competition and Niche Occupancy. Cell Host 553 Microbe 21, 769-776 e763 (2017). 554

51. M. M. Curtis et al., The gut commensal Bacteroides thetaiotaomicron exacerbates enteric 555 infection through modification of the metabolic landscape. Cell Host Microbe 16, 759-769 556 (2014). 557

52. J. A. Ferreyra et al., Gut microbiota-produced succinate promotes C. difficile infection after 558 antibiotic treatment or motility disturbance. Cell Host Microbe 16, 770-777 (2014). 559


https://doi.org/10.1101/680801

18

53. B. P. Ganesh, R. Klopfleisch, G. Loh, M. Blaut, Commensal Akkermansia muciniphila 560 exacerbates gut inflammation in Salmonella Typhimurium-infected gnotobiotic mice. PLoS One 561 8, e74963 (2013). 562

54. M. K. Shenoy, S. V. Lynch, Role of the lung microbiome in HIV pathogenesis. Curr Opin HIV 563 AIDS, (2017). 564

55. A. M. Briselden, B. J. Moncla, C. E. Stevens, S. L. Hillier, Sialidases (neuraminidases) in bacterial 565 vaginosis and bacterial vaginosis-associated microflora. J Clin Microbiol 30, 663-666 (1992). 566

56. J. Si, H. J. You, J. Yu, J. Sung, G. Ko, Prevotella as a Hub for Vaginal Microbiota under the 567 Influence of Host Genetics and Their Association with Obesity. Cell Host Microbe 21, 97-105 568 (2017). 569

57. S. Leo et al., The intestinal microbiota predisposes to traveler's diarrhea and to the carriage of 570 multidrug-resistant Enterobacteriaceae after traveling to tropical regions. Gut Microbes, 1-11 571 (2019). 572

58. H. A. Hong et al., Bacillus subtilis isolated from the human gastrointestinal tract. Res Microbiol 573 160, 134-143 (2009). 574

575

List of supplementary materials 576

Materials and Methods 577

Fig S1-S5 578

References (59-72) 579


https://doi.org/10.1101/680801

Figure 1

* * NS

A

WT D Cplt100101102103104105106107

intestinal content

CFU/

mg co

ntent

WT D p2776

* *

B

WT D Cplt100101102103104105106107108109

spleen

CFU/

splee

n * *

WT D p2776

C

WT D Cplt100101102103104105106107108109

101 0liver

CFU/

liver

WT D p2776

D

WT D100101102103104105106107108

intestinal content

CFU/

mg co

ntent

WT D

NS

E

CFU/

splee

n

WT D100101102103104105106107108109

spleenNS

WT D

F

CFU/

liver

WT D100101102103104105106107108109

liver

WT Dgerm freemiceconventionalmice


https://doi.org/10.1101/680801

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

6 wtD227

6 wtD227

60

2

4

6

relati

ve ab

unda

nce

258849

346870 430197 447141

355746 216524 421792

208409 353012 364179

wtD227

6 wtD227

6 wtD227

60.0

0.5

1.0

1.5

2.0

relati

ve ab

unda

nce

DWT DWT DWT day 0 day 1 day 2 wt

D2276 wt

D2276 wt

D2276

0

2

4

6

8

relati

ve ab

unda

nce

wtD227

6 wtD227

6 wtD227

60

1

2

3

relati

ve ab

unda

nce

wtD2276 wt

D2276 wtD2276

0

2

4

6

relati

ve ab

unda

nce

wtD227

6 wtD227

6 wtD227

60.0

0.5

1.0

1.5

relativ

e abu

ndan

ce

wtD227

6 wtD227

6 wtD227

60.0

0.5

1.0

1.5

2.0

relati

ve ab

unda

nce

wtD227

6 wtD227

6 wtD227

60.0

0.5

1.0

1.5

relati

ve ab

unda

nce

wtD227

6 wtD227

6 wtD227

60

2

4

6

relati

ve ab

unda

nce

wtD227

6 wtD227

6 wtD227

60

1

2

3

relati

ve ab

unda

nce

DWT DWT DWT day 0 day 1 day 2









relative

abund

ance

relative

abund

ance

relative

abund

ance

relative

abund

ance

relative

abund

ance

relative

abund

ance

relative

abund

ance

relative

abund

ance

relative

abund

ance

relative

abund

ance

331772

wtD227

6 wtD227

6 wtD227

60

1

2

3

4

relati

ve ab

unda

nce


relative

abund

ance

465433

wtD227

6 wtD227

6 wtD227

60.0

0.5

1.0

1.5

relati

ve ab

unda

nce


relative

abund

ance

WTD

PC3 (11.81%)

PC2 (13.55%)

PC1 (61.45%)

day0

day1

+ WT

+ D

OTUs

355746

216524

421792

258849

331772

346870

430197

447141

465433

208409

353012

364179

+ WT

+ D

+ WT

+ Dday2

E

wtD227

6 wtD227

610

100

1000

10000

100000

pgof Pr

evotella

/mg

DWT DWT day 0 day 1


https://doi.org/10.1101/680801

Figure 3

B

D

1,00E+00

1,00E+01

1,00E+02

1,00E+03

1,00E+04

1,00E+05

1,00E+06

1,00E+07

1 2WT D

10 710 610 5

10 4

10 3

0

10 2

10 1Prevot

ella CFU

/ml

Lm SN :

E ***

HG

F

*****

***

relative

abund

ance

of bact

eria

020406080

100120

no peptide + ++ +++no[Lmo2776 peptide]

day 0 day 1

Prevotella

Lachnospiraceae

BacteroidesParabacteroides

WTNI D NI WT D

A day 2

NI WT D

WT

DNI

mmol/L

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


https://doi.org/10.1101/680801

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


https://doi.org/10.1101/680801

specific targeting of intestinal prevotella copri by a ... · the intestinal phase of infection, we...

Documents