Parvimonas micra, an oral pathobiont associated with colorectal cancer, epigenetically reprograms human primary intestinal epithelial cells

Recently, an intestinal dysbiotic microbiota with enrichment in oral cavity bacteria has been described in colorectal cancer (CRC) patients. Here we characterized and investigated one of these oral pathobionts, the Gram-positive anaerobic coccus Parvimonas micra. We identified two phylotypes (A and B) exhibiting different phenotypes and adhesion capabilities. We observed a strong association of phylotype A with CRC, with its higher abundance in feces and in tumoral tissue compared with the normal homologous colonic mucosa, which was associated with a distinct methylation status of patients. By developing an in vitro hypoxic co-culture system of human primary colonic cells with anaerobic bacteria, we showed that P. micra phylotype A alters the DNA methylation profile promoters of key tumor-suppressor genes, oncogenes, and genes involved in epithelial-mesenchymal transition. In colonic mucosa of CRC patients carrying P. micra phylotype A, we found similar DNA methylations alterations, together with significant enrichment of differentially expressed genes in pathways involved in inflammation, cell adhesion, and regulation of actin cytoskeleton, providing evidence of P. micra possible role in the carcinogenic process.


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The adherent P. micra phylotype A is associated with CRC 123 Differences in the in vitro adhesion capacity of the two phylotypes might reflect a different colonization 124 ability in vivo. To assess this hypothesis, we first improved the resolution of our previous whole genome 125 sequencing (WGS) metagenomic analysis 26 , focusing on the taxonomic assignation of raw data, in order 126 to precisely investigate the occurrence and abundance of P. micra of CRC patients' feces. We observed 127 an enrichment of P. micra occurrence in the feces of CRC patients as compared to control healthy 128 individuals (27% versus 1.1%). Three taxa of P. micra were detected: 'Parvimonas micra' from phylotype

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We then analyzed Parvimonas carriage in feces of CRC patients and control individuals, as well as from 133 paired tumor and normal homologous tissues (adjacent to the tumor), using 16S rDNA sequencing of the 134 V3-V4 region. As expected, Parvimonas prevalence in feces was enriched in CRC patients (81%), with 135 no differences within the stages (international TNM (tumor-node-metastasis) staging system), compared 136 with control individuals (60%) (Figure 2A). In 78% of CRC patients, Parvimonas sequences were detected 137 in both tumoral and normal homologous samples with a significant abundance enrichment in the tumoral 138 tissues (2.20% versus 0.53% of bacterial reads, respectively; p<0.01) ( Figure 2B). Prevalence of 139 Parvimonas in tissues showed no difference according to the TNM staging, with 74% in stages I-II versus 140 84% in stages III-IV and a respective abundance of 1.34% and 1.41% of bacterial reads ( Figure 2B).

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The V3-V4 regions of the 16S rDNA was then analyzed to discriminate between Parvimonas phylotypes 142 A and B. Forty-four percent of control individuals carried the phylotype A in their feces as compared to 143 61% of the CRC patients (p<0.01), whereas no difference in the prevalence of the phylotype B was 144 observed (22% and 27%, respectively) ( Figure 2C). An increase in the abundance of the phylotype A, 6 of 36 sequencing data from feces showed that phylotype A was enriched in CMI positive patients (p<0.01) but 152 this was not the case for phylotype B ( Figure 2E). Parvimonas carriage in tissues was also associated 153 with a positive CMI (p<0.05), although the number of samples was not sufficient to discriminate between 154 the two phylotypes ( Figure 2F).

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These results indicate that Parvimonas micra phylotype A sub-species is associated with CRC and a 156 positive CMI.  (Figures 3C and D). Under these culture conditions, monolayers were 170 fully confluent three days after organoids fragments were seeded on the inserts ( Figure 3D).

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P. micra has been described as an anaerobic microorganism 28 and little is known about its oxygen 172 tolerance. To determine compatible oxygenation conditions with eukaryotic cell growth requirements, P. 173 micra was cultivated at various oxygen concentrations (0, 2, or 21% O2) and viability was assessed by 7 of 36 and showed all characteristics of a differentiated epithelium as assessed by the presence of tight 180 junctions, differentiated epithelial cells, colonocytes, goblet cells, and enteroendocrine cells ( Figure 3E).

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To investigate the possible involvement of P. micra in early oncogenic processes, we co-cultivated 182 primary cell monolayers with the two reference isolates, PmA and PmB, as well as F. magna as a control, 183 which is the closest phylogenetic taxon to P. micra. One day prior to co-culture, cells were placed in 184 hypoxic conditions (2% O2) to allow pre-adaptation. On the fourth day of the culture, cells were co-cultured

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These results suggested that P. micra might contribute to host epithelial cell transformation through a 199 NF-κB pathway-mediated effect.

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As we previously showed a correlation between Parvimonas carriage in feces and a positive CMI in 201 patients 13 , global DNA methylation was measured using a 5-methyl-cytosine dosage assay in vitro. Co-202 cultures of both P. micra phylotypes showed a significant increase in global DNA methylation of primary 203 cells as compared to NS condition (p<0.05) or to F. magna ( Figure 3I). To identify affected genes, a 204 genome-wide DNA methylation analysis was performed on the human colonic primary cells exposed or  only one in-between the two phylotypes of P. micra, and two genes for PmA and F. magna ( Figure 3L).

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Strikingly, most of the differentially-methylated promotors in PmA co-culture cells were either found in 223 oncogenes, TSGs, or genes involved in epithelial-mesenchymal transition (EMT) when compared to PmB 224 or F. magna co-cultures ( Figure 3M, 3N, 3O). Cells co-cultivated with PmA presented hyper-methylation 225 of several TSGs promoters such as SCIN, HACE1, TSPAN13, FBXO32, IGFBP7, SIX1 or CXXC5. Except 226 for the KIAA0494 gene that codes for an uncharacterized protein, all DMGs induced by PmA were 227 involved in carcinogenesis, particularly in EMT processes or cytoskeleton remodeling (Table S3). These 228 results suggest that P. micra phylotype A might altered expression of a set of genes, including well-229 characterized carcinogenesis regulators, through epigenetic promoter gene methylation. To confirm that the differences in phylotypes might influence colon cancer progression, we put our efforts 234 in isolating Parvimonas from colonic tumoral biopsies. We eventually successfully isolated one clone that 9 of 36 isolate, named PmG5, belongs to phylotype A. Indeed, PmG5 was hemolytic, "non-compact" on blood 238 agar plates, and able to adhere to Matrigel® ( Figure 4B) and to the colonic cell line TC7 in vitro ( Figure   239 4C). No spiky structures were observed on PmG5 surface by TEM ( Figure 4D). Thus, PmG5 showed all 240 the features of the PmA isolate we used for the in vitro assay.

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Having shown that P. micra phylotype A is more prevalent in CRC and associated to cancer tissue, we 242 wondered if its presence was also associated to DNA methylation changes in host colonic cells, as 243 observed in vitro. We thus performed reduced-representation bisulfite sequencing (RRBS) analysis on 244 colonic tumoral tissues either negative or highly colonized by P. micra from 3 patients in each group. We 245 observed 135 differentially methylated genomic regions (DMRs), mostly located in promotor regions 246 (45%) and in CpG island context (68%) ( Figures 4E, 4F). Albeit we did not identify the same genes as in 247 our in vitro study, differentially methylated regions were found in numerous TSGs, oncogenes, and genes 248 involved in EMT ( Figure 4G, Tables S4).

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To identify altered gene expression patterns linked with P. micra colonization of the mucosa, RNA-seq 250 was performed on colonic tumoral tissues, and transcriptome comparisons of either Parvimonas-positives 251 (n=30) or P. micra phylotype A-positive patients (n=20) versus negative patients (n=12) were analyzed.

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We found 483 and 336 differentially expressed genes (DEGs) for Parvimonas and P. micra phylotype A, 253 respectively (Log2FC>1, p<0.05) ( Figure 4H, Tables S4). To uncover potential signaling pathways that 254 are impacted by P. micra colonization, we performed Kyoto Encyclopedia of Genes and Genomes 255 (KEGG) pathway analysis ( Figure 4I) on over-expressed genes in P. micra positive patients. Over-256 expressed genes in P. micra phylotype A -positive patients revealed significant enrichment in several 257 pathways related to inflammation, such as phagosome, Toll-like receptor signaling pathway, cytokine-258 cytokine receptor interaction, chemokine signaling pathway or intestinal immune network for IgA 259 production, the latter demonstrating a localized inflammatory response to mucosal colonization ( Figure   260 4I). Affected pathways also included transcriptional misregulation in cancer, hematopoietic cell lineage, 261 and PI3K-Akt signaling pathway, a pathway well-known to be involved in cell growth, survival, cell-cycle were involved in cell adhesion and regulation of actin cytoskeleton ( Figure 4I, Tables S4).

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These findings revealed that tissues colonized with P. micra phylotype A display altered DNA methylation 265 patterns and expression in keys genes involved in carcinogenic events.

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Recently, we and others observed an epidemiological association between CRC and several oral bacteria 269 detected in feces using 16S rDNA sequencing 24,31 or metagenome analysis 12,13,32 . They mainly belong to 270 species such as Fusobacterium nucleatum, Porphyromonas gingivalis, Solobacterium moorei,

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Peptostreptococcus stomatis, Gemella morbillorum, and Parvimonas micra. Some of these oral bacteria 272 were also associated with colonic tissues of CRC patients 24,33,34 , being more abundant in tumors than 273 in normal adjacent tissues 25,34 . Interestingly, we found that these bacteria were often found in co-274 occurrence in the mucosa of patients (data not shown), suggesting that these oral microorganisms could 275 live in close community in association with the colonic mucosa, partially reproducing an ecosystem similar 276 to the oral cavity. Indeed, P. micra, F. nucleatum, P. stomatis, and G. morbilorum have been observed in 277 biofilm-like structures at the surface of the colonic mucosa of CRC patients and healthy subjects 11,[35][36][37][38][39] .

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In the present study, we particularly focused on P. micra, at the subspecies level, and quantitatively 279 measured its carriage in both feces and colonic tissues in a large cohort from Henri Mondor Hospital -

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Assistance Publique des Hôpitaux de Paris (Directed by Prof. I. Sobhani) that includes normal 281 colonoscopy, adenomatous and CRC patients. We observed, as others 40 , that P. micra is present but not 282 significantly enriched in patients with adenomas, these benign tumors being considered as precursor of 283 CRC 41 , indicating that P. micra is more likely to be involved in accelerating and/or exacerbating the 284 carcinogenic processes rather than being a primary driver bacterium, according to the "driver-passenger" 285 model 42 . And indeed, genetically predisposed murine model of intestinal adenoma formation (e.g.,

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Apc Min/+ mice) orally gavaged with P. micra exhibited a significantly higher tumor burden and were 287 associated with altered immune responses and enhanced inflammation 43,44 . In human, P. micra, like other 288 oral pathobionts, have been shown to be associated with the CMS1 subtype of CRC, which makes up 289 14% of all CRC cases 45 , and with an over-activation of genes involved in immune responses. The CMS1 290 tumors, also called "immune subtype", are characterized by a strong immune cell infiltration by CD8+ Next steps require moving from association to causality to identify the mechanisms and define more 294 aggressive phylotypes driving carcinogenesis. In the present study, we have identified different 295 phylotypes of P. micra, observed a strong association of the adherent and hemolytic phylotype A with 296 CRC, and developed a physiologically relevant low-oxygen in vitro co-culture model, to assess the 297 interaction of this particular pathobiont with primary human colonic intestinal cells. After 48h of co-culture, 298 no impact of P. micra was observed on cell proliferation, differentiation, or DNA damage. In contrast, P. 299 micra induced the activation of the central transcription factor NF-κB, a master regulator of inflammation 300 and anti-apoptotic responses, involved in gene-expression reprogramming in cancers. In a recent in vitro 301 study, tumoral colonic cell lines were infected with P. micra and presented a higher proliferation rate upon 302 infection due to the activation of the Ras signaling pathway 44 . Consistent with this observation, we showed 303 that patients carrying P. micra, have altered PI3K-Akt signaling, a downstream effector of the Ras 304 pathway, demonstrating that P. micra phylotype A might induce aberrant cellular proliferation that may 305 contribute to initiation and progression of cancer cells.

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Apart from specific somatic mutations that characterize cancerous cells, epigenetic DNA modifications, 307 particularly methylation, of TSGs promotors are main contributors in colonic carcinogenesis. We recently 308 showed that CRC-associated dysbiotic feces transplanted to mice caused epigenetic changes similar to 309 those observed in human tumors and the occurrence of murine colonic crypt aberrations 13 . Furthermore, 310 the CMS1 CRC subtype, in which P. micra and other oral bacteria enrichment has been observed 45 , is 311 associated with the phenotype of CpG islands hypermethylation in TSG promoters (high CIMP) 46 .

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Consistent with this hypothesis, Xia et al. observed an association between enrichments of F. nucleatum,

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Parvimonas spp. or other bacteria, and hypermethylation of promoter in several TSGs in CRC tumoral 314 tissue. Moreover, they observed that F. nucleatum was able to up-regulate DNA methyltransferase 315 activity in vitro 48 , although they did not focus on Parvimonas subspecies. Further, we have shown in a 316 large CRC patients' cohort that P. micra was significantly associated with methylation of the WIF1 317 promoter, a very well-known TSG 13 . In the present study, we report for the first time that P. micra 318 increases global DNA methylation of target host cells using an in vitro co-culture model of human primary 319 colonic cells. Notably, by comparing different P. micra phylotypes, we established a P. micra phylotype Scinderin gene (SCIN) coding for a Ca 2+ dependent actin-severing and capping protein, is involved in the 323 regulation of actin cytoskeleton and known to be overexpressed in CRC 49 ; Tetraspanin 13 (TSPAN13) is 324 a TSG coding for a transmembrane signal transduction protein that regulates cell development, motility, 325 and invasion 50 ; DIAPH3 gene, is a major regulator of actin cytoskeleton involved in cell motility and 326 adhesion 51 ; Semaphorin 3F (SEMA3F), is a TSG coding a secreted protein involved in cytoskeletal 327 collapse and loss of migration 52 ; and SASH1 is a TSG coding for a scaffold protein involved in the TLR4 328 signaling, and known to interact with the actin cytoskeleton to maintain stable cell-cell adhesion 53 .

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Notably, the hypermethylation of the TSG HACE1 gene promoter was also observed upon co-culture with

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In conclusion, here we described the P. micra phylotype A as the most prevalent CRC-associated    The authors thank patients for having contributed to the present research program. We thank the 352 bacteriology Departments of Cochin, Pitié Salpétrière, and Mondor Hospitals from APHP for providing P.   Figure S1 and Table S1.

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and disease history were referenced. The cumulative methylation index (CMI) score was previously 520 determined 57 . This score was calculated from the methylation status of three genes (wif1, penk and npy) 521 involved in colorectal carcinogenesis and was considered negative for CMI<2 or positive for CMI≥2.

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Clinical data are summarized in Table S2.

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The raw data have already been reported in several papers 12,26,57 . For this study, one hundred and sixty-542 six fecal samples from the CCR1 and DETECT cohorts were considered. The Diamond/MEGAN6 543 bioinformatics pipeline 59 was used for metagenomic assignment. Sequences were filtered for an average 544 quality (Phred score) greater than 20 over a window of two consecutive bases and a length greater than

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Monolayers were incubated at 37°C in 5% CO2. After three days of culture, the medium was changed  study, twenty-seven clinical isolates of P. micra, including HHM BlNa17 (PmB), were collected from the 654 bacteriology departments of three Parisian hospitals: Henri Mondor, Cochin, and Pitié Salpetrière (Table   655   S1). The identification of the different isolates was confirmed by mass spectrometry and 16S rDNA 656 sequencing analysis (Eurofins Genomics) and sequences were deposited on GenBank (Table S1).

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Cell monolayers were dissociated with TrypLE™ Express Enzyme (Gibco) at 37°C for ten to twenty 745 minutes. The cells were recovered in DMEM/F12 at RT and then centrifuged for five minutes at 300g.

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After washing the pellet in PBS, cells were stained with the Live/Dead fixable cell stain kit (ThermoFisher) 747 for twenty minutes on ice, following the supplier's instructions. Cells were analyzed using a FACS Attune

Fig. S1
iii.       E3 ubiquitin ligase involved in specific tagging of target proteins, leading to their subcellular localization or proteasomal degradation.
TSG. Down-regulated by DNA methylation in CRC. Loss or knockout of HACE1 enhanced tumor growth, invasion, and metastasis; in contrast, the overexpression of HACE1 can inhibit the development of tumors.

Li 2019 Hibi 2008 Zhang 2007
TSPAN13 (Tetraspanin 13) Transmembrane signal transduction protein that plays a role in the regulation of cell development, activation, growth, motility and invasion.
TSG. Downregulation inhibits proliferation of CRC cells.

FBXO32
(F-Box Protein 32) Astogin-1 Component of a SCF E3 ubiquitin-protein ligase complex which mediates the ubiquitination and subsequent proteasomal degradation of target proteins.
TSG. Under-expressed in CRC. Induce cell differentiation. Upstream regulator of EMT. Protein coding gene involve in actin remodeling. Regulate cell movement and adhesion.
Deficiency enhances cell motility, invasion and metastasis in many cancers.

SIX1
(sine oculis homeobox 1) Transcription factor involve in regulation of cell proliferation, apoptosis, embryonic development and tumorigenesis.
Oncogene. Overexpressed in CRC, overexpression of Six1 dramatically promotes CRC tumor growth and metastasis in vivo. Voltage-gated potassium channel Kv2.1. Contributes to the pronounced pro-apoptotic potassium current surge during neuronal apoptotic cell death in response to oxidative injury.

SASH1
(SAM and SH3 domain-containing protein 1) Scaffold protein involved in the TLR4 signaling. Stimulate cytokine production and endothelial cell migration in response to binding pathogens TSG. Downregulated in CRC. Downregulation expression was correlated with the formation of metachronous distant metastasis. Loss of SASH1 induces EMT. SASH1 inhibits metastasis formation In Vivo.
Knockdown reduced migration, invasion and tumorigenesis of ESCC in vitro and reduced the tumorigenicity of ESCC xenograft in nude mouse.

Yeo 2014
SEMA3F (Semaphorin 3F) Secreted signaling protein that are involved in axon guidance during neuronal development. Act in an autocrine fashion to induce apoptosis, inhibit cell proliferation and survival. Regulator of actin cytoskeleton.
TSG. Down-regulated by DNA methylation in CRC tissues and CRC cell lines. Associated with progressive phenotypes of CRC. Overexpression reduced proliferation, adhesion, and migratory capability of colon cancer cells. SEMA3Foverexpressing cells exhibited diminished tumorigenesis when transplanted in nude mice and reduced liver metastases.