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Article

In Silico Study of Cell Surface Structures of Parabacteroides distasonis Involved in Its Maintenance within the Gut Microbiota

by
Jordan Chamarande
1,
Lisiane Cunat
1,
Corentine Alauzet
1,2 and
Catherine Cailliez-Grimal
1,*
1
Stress Imunnity Pathogens (SIMPA), Université de Lorraine, F-54000 Nancy, France
2
CHRU de Nancy, Service de Microbiologie, F-54000 Nancy, France
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2022, 23(16), 9411; https://doi.org/10.3390/ijms23169411
Submission received: 11 July 2022 / Revised: 11 August 2022 / Accepted: 12 August 2022 / Published: 20 August 2022
(This article belongs to the Special Issue Gut Microbiota–Host Interactions: From Symbiosis to Dysbiosis 2.0)
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Abstract

The health-promoting Parabacteroides distasonis, which is part of the core microbiome, has recently received a lot of attention, showing beneficial properties for its host and potential as a new biotherapeutic product. However, no study has yet investigated the cell surface molecules and structures of P. distasonis that allow its maintenance within the gut microbiota. Moreover, although P. distasonis is strongly recognized as an intestinal commensal species with benefits for its host, several works displayed controversial results, showing it as an opportunistic pathogen. In this study, we reported gene clusters potentially involved in the synthesis of capsule, fimbriae-like and pili-like cell surface structures in 26 P. distasonis genomes and applied the new RfbA-typing classification in order to better understand and characterize the beneficial/pathogenic behavior related to P. distasonis strains. Two different types of fimbriae, three different types of pilus and up to fourteen capsular polysaccharide loci were identified over the 26 genomes studied. Moreover, the addition of data to the rfbA-type classification modified the outcome by rearranging rfbA genes and adding a fifth group to the classification. In conclusion, the strain variability in terms of external proteinaceous structure could explain the inter-strain differences previously observed of P. distasonis adhesion capacities and its potential pathogenicity, but no specific structure related to P. distasonis beneficial or detrimental activity was identified.

1. Introduction

Gut microbiota (GM) is now considered as a new organ system mainly due to the microorganisms’ specific biochemical interaction with their hosts and their systemic integration into the host biology [1,2]. Bacteria that are predominant in the GM are mainly defined by anaerobic bacteria part of the Firmicutes and Bacteroidetes phyla [3]. Advances in sequencing methods have facilitated the characterization and understanding of the contribution of the GM to the host well-being, which is now indisputable. In fact, it is now well-defined that the cooperation between the GM and its host is essential to regulate the development and function of the immune, metabolic and nervous system. In turn, one of the major roles of the immune system is to control and maintain its relationships with the GM. The intestinal microbiota, in addition to contributing to the development of the immune system and to intervene into host metabolic and nervous function, also creates a protective barrier against external pathogens and participate in maintaining the structure and integrity of the gastrointestinal tract [4,5,6]. In the long run, the GM can modulate host behavior and nervous system function through dynamic and bidirectional communication along the gut–brain axis [7].
Although mechanisms underlying host–microbiota interactions are not fully described, it is now well-established that cell surface molecules and structures of the GM play a key role in such relationships via conserved microbe-associated molecular patterns (MAMPs) that will be recognized by pattern recognition receptors (PRRs) of immune system cells, including Toll-like receptors (TLRs). An interaction between MAMP and TLR will then initiate the immune response if the MAMP is identified as pathogenic [8,9]. The study of secreted and surface molecules of microbiota members is also fundamental for their involvement in the establishment of species in the versatile and competitive environment of the gut and their key role as a potential virulence factor [10]. Among cell surface markers are capsular polysaccharide (CPS), fimbriae and pili, all well-described for their crucial role in microorganism colonization of the host epithelium.
In Gram-negative anaerobic bacteria, various systems have been described for each of these cell surface markers, including the CPS of Bacteroides fragilis, the fimbriae system (Fim) of Porphyromonas gingivalis, the type V pilus system (Mfa) of P. gingivalis and Bacteroides thetaiotaomicron and the immunogenic component of lipopolysaccharide (LPS); O-antigen (Rfb) is well-described in the facultative anaerobic Escherichia coli [11,12,13,14,15].
Among the gut microbiota members is Parabacteroides distasonis, a Gram-negative bacterium strictly anaerobe belonging to the Tannerellaceae family within the Bacteroidetes phylum. This bacterial species, part of the core microbiome, has recently received a lot of attention, showing beneficial properties for its host. In fact, although strain-dependent, P. distasonis display anti-inflammatory/cancer properties and activities on decreasing weight gain, hyperglycemia and hepatic steatosis in ob/ob and high-fat diet-fed mice [16,17,18]. The importance of P. distasonis membrane in these disease treatments has been pointed out in numerous studies. Notably, it has been shown to largely suppress production of pro-inflammatory cytokines in obese animal models [19] and induce apoptosis in colon cancer cell lines, suggesting anti-inflammatory and anti-cancer effects [20]. The membrane components of P. distasonis have also been reported to decrease the severity of gut inflammation in the non-immunocompromised mouse models that had induced acute and chronic colitis [21]. Many studies have highlighted these abilities to promote P. distasonis as a new potential biotherapeutic product [22,23,24]. In our previous work, we explored P. distasonis capacities related to its maintenance within the digestive tract and the electrokinetic properties of its cell peripheral regions to provide a first qualitative picture of its surface structure [25]. This work evidenced a strain-dependent ability to adhere and to form a biofilm related to the putative presence of cell surface structures such as CPS, fimbriae, pili or capsule.
Although numerous studies described the beneficial aspects of P. distasonis or its ability to colonize the intestine, few explore mechanisms behind these aptitudes. Moreover, while P. distasonis is strongly recognized as intestinal commensal specie with benefits for its host, several studies displayed controversial results, showing P. distasonis as an opportunistic pathogen [26,27,28,29]. In this study, we investigated the cell surface structures of P. distasonis that may influence host–P. distasonis crosstalk and play an essential role in its maintenance and stability within the GM. We reported gene clusters potentially involved in the synthesis of capsule, fimbriae-like and pili-like outer membrane structure and applied the new rfbA-typing classification on 26 genomes of P. distasonis including 13 new clinical strains (CS) in order to investigate its maintenance within the digestive tract and its potential pathogenicity [30]. In this study, the designation “pilus” is used to describe the external cell surface structure originating from the “minor fimbriae” Mfa system [31,32]. “Fimbriae” refer to structures arising from the Fim system. However, the use of this designation does not mean that Mfa structures are minor and short in comparison with Fim fimbriae [33]; rather, it serves to better clarify the origin of the external appendages described.

2. Results

2.1. P. distasonis Genomes Characterization

Thirteen nonredundant P. distasonis CS were isolated (Table 1) by the Clinical Microbiology Laboratory of the University Hospital of Nancy, France, and sequenced using Illumina technology. All genomes were then integrated in the Microbial Genome Annotation and Analysis Platform (MaGe) in addition to 13 other public P. distasonis genomes (Figure 1).
The length of P. distasonis CS genomes range from ~4.8 to 5.6 Mb with an average GC content of 45.00% and a percentage of protein coding density of approximately 91.00%. The pan-genome analysis revealed 2479 functional genes presented in all strains (core-genome), between 1680 and 2479 genes presented in at least two strains function (dispensable genomes) and an average of 253 genes specific to one strain (specific genomes).
The evolutionary relationships among these strains were then investigated by constructing a phylogenetic tree based on the pairwise distances using a neighbor joining algorithm (MaGe).
The tree revealed a partial evolution of P. distasonis strains and some similarities notably with FDAARGOS_1234 and ATCC 8503T genomes that appear to be relatively closed. This genome similarity is notably highlighted by the poor specific genomes of both strains.

2.2. Identification of P. distasonis Genes Potentially Involved in Capsule, Fimbriae-like and Pilus-like Synthesis

In order to determine the potential presence of capsule, fimbriae or pili at the surface of P. distasonis, reference genes involved in their synthesis were selected from B. fragilis (gut), B. thetaiotaomicron (gut) and P. gingivalis (oral cavity), as three strictly anaerobe Gram-negative bacteria part of the Bacteroidetes phyla, and referenced as opportunistic pathogens [39,40,41]. Indeed, B. fragilis is well-known for its numerous divergent polysaccharides loci all starting by genes designated as UpxY and UpxZ families, where x goes from a to h depending on the locus. upxY genes are transcriptional antitermination factors essential to the CS synthesis, while upxZ genes inhibit their secretions [11]. P. gingivalis, for its part, is well described for its proteinaceous, filamentous appendages at its surface including fimbriae and pili, synthesized through the Fim (fimA-E) and Mfa (mfa1-5) systems, respectively [15]. A similar Mfa system including only mfa1 and mfa2 has also been described in B. thetaiotaomicron [13].
Synteny analysis of reference genes on P. distasonis genomes revealed a set of genes whose function possibly approaches that of the reference genes. To refine the search, only genes with an automatic functional assignation linked to the synthesis of the sought structures were listed (Table 2).
No result was found for up(a-g)Y and up(a-h)Z, while 15 genes from 15 distinct strains were referred to as potentially uphY-like with homologies ranging from 33.10% to 36.90%. Concerning the fim gene cluster, although homologies are relatively low (from 22.50% to 27.10%), all reference genes possess a synteny in at least one genome of P. distasonis with an auto-assigned function related to the fimbriae synthesis. The synteny analysis between the mfa cluster of B. thetaiotaomicron VPI-5482T and P. distasonis genomes revealed only one positive result for B. thetaiotaomicron mfa2, while the mfa gene cluster of P. gingivalis ATCC 33277T permitted the listing of multiple genes for P. gingivalis mfa1, mfa2 and mfa4. No result was found for P. gingivalis mfa3 and mfa5 genes.
Multiple sequence alignments of the listed genes (Figure S1) revealed either the conservation of one sequence (fimB-like, fimC-like, fimD-like, Bt mfa2-like, Pg mfa1-like and Pg mfa4-like) or the presence of two distinct sequences (uphY-like 1/2, fimA-like 1/2, fimE-like 1/2 and Pg mfa2-like 1/2).

2.2.1. P. distasonis Gene Cluster Potentially Involved in Capsule Synthesis

BLAST of the consensus sequences uphY-like 1 against P. distasonis genomes revealed genes with high similarity (from 99% to 100%) in 21 of the 26 studied genomes (Figure 2A). Among these genes, 15 are from the syntenic analysis while 6 are from BLAST. These last six sequences were not found during the syntenic analysis probably due to variations in their genomic organizations. On the contrary, the uphY-like 2 was identified in only three genomes with still an important sequence conservation (from 86% to 100%). Each uphY-like genomic region was then analyzed to allow the discovery of very conserved regions with a high gene homology (Figure 2B). Among them are genes linked to the CPS synthesis including glycosyltransferase, polysaccharide export, polysaccharide biosynthesis and CPS biosynthesis genes. Each CPS cluster is also composed of downstream gene encoding an integrase. The three genes similar to uphY-like 2 were analyzed and integrated at the syntenic analysis. The genomic environment of uphY-like 2 appear to be relatively close to the first loci identified with uphY-like 1, including an integrase, a glycosyltransferase, a polysaccharide export, a polysaccharide biosynthesis and a CPS biosynthesis gene, too.
Specific research on P. distasonis ATCC 8503T CPS loci genomes allowed us to find a 14th CPS loci, in addition to the 13 already identified [42]. All CPS loci were then explored on other P. distasonis genomes (Table 3, gene details in Table S1). Among the 26 P. distasonis genomes, only ATCC 8503T and FDAARGOS_1234 possess the 14 CPS loci identified. Loci 3, 6, 9, 13 and 14 are shared between all P. distasonis, while only few strains possess loci 10, 11 and 12. Loci 7 and 8 are also conserved over genomes, but important intra-variations have been identified within these loci. Moreover, not all gene loci are different: 2 and 8 show high gene sequence conservation with a similar upxY-like gene. Locus 5 appear to be relatively close to 2 and 8 too, but with more variations. In the same way, the locus 4 shows some similarities with 2, 5 and 8 but has a different upxY. On the contrary, locus 13 possesses a similar upxY to 2, 5 and 8 but a different locus. Loci 3, 6, 7 and 1, 11, 12 also display similarities, especially between 6, 7 and 11, 12. Locus 1, although close to 11 and 12, presents a distinct upxY. Moreover, the conserved part of locus 9 does not always seem to be the one involved in the CPS synthesis.
In addition to upxY genes, several of these CPS loci contain a phage insertion (N-acetylmuramoyl-L-alanine amidase, homolog of phage T7 lysozyme) that may modulate its expression (Table 3). Among them, CPS loci 1 and 13 of the 26 P. distasonis genomes all harbor these insertions. For CPS loci 1, this inserted segment (light blue arrows in Figure 2) is oriented in the opposite direction to upxY-like gene downstream of the CPS biosynthesis genes (red arrows in Figure 2).

2.2.2. P. distasonis Gene Cluster Potentially Involved in Fimbriae-like Synthesis

Almost all the fim-like genes investigated have been identified in the 26 P. distasonis genomes (Figure 3). The few genes not found by BLAST have been highlighted in the syntenic analysis showing fimA-E on every P. distasonis genome. Notably, BLAST of fimC-like allowed the identification of another fimC-like gene possessed by 24 of the 26 studied genomes. The identified fim-like gene cluster is composed of a various gene blocks, including one main block of four genes (fimA-like, fimB-like and fimC-like); a second block of two genes (fimD-like and fimE-like) that are always together but not located in the same region as fimA-C; and several genes showing a slight homology but a synteny with fimA-like 2, which are located sometimes in and sometimes out of the main block of genes. One nonsense mutation was found on the fimE-like gene of the CL03T12C09 that probably avoid its synthesis. Compared to the P. gingivalis fim cluster, whose genes all follow each other, P. distasonis fim-like cluster appeared to be relatively close in terms of organization, with only fimD-like and fimE-like displaying a different location.

2.2.3. P. distasonis Gene Cluster Potentially Involved in Pili-like Synthesis

Homologue sequences of Bt mfa2-like genes were found in only two P. distasonis genomes, while Pg mfa1-like/Pg mfa2-like 2 and Pg mfa2-like 1/Pg mfa4-like genes were found on five and eight genomes, respectively (Figure 4A). Interestingly, the five genomes containing Pg mfa1-like gene correspond to the five genomes holding Pg mfa2-like 2. In the same way, the eight genomes positive to the BLAST are the same for Pg mfa2-like 1 and Pg mfa4-like genes. The syntenic analysis of Bt mfa2-like gene (Figure 4B) revealed a conserved gene downstream of Bt mfa2-like gene showing similarities with Bt mfa1, identified as putative Bt mfa1-like gene. Some strains harbor several mfa-like clusters, such as putative Bt mfa1-like/Bt mfa2-like + Pg mfa2-like 1/Pg mfa4-like genes or Pg mfa1-like/Pg mfa2-like 2 + Pg mfa2-like 1/Pg mfa4-like genes.

2.3. rfbA Classification and Investigation

In order to determine the potential pathogenicity of P. distasonis, the new rfbA-type classification was applied to the 26 studied genomes (Figure 5A, gene details in Table S2). The addition of new data modified the classification. A fifth group was identified and the previous gene repartition changed, notably with the presence of a rfbA-type 1 gene in all the 26 P. distasonis strains. In order to better understand the variation between each rfbA-type gene, the multiple sequence alignment of all the rfbA genes was explored (Figure 5B). The analysis revealed the presence of three gaps, two in 5′ and one in 3′. The rfbA-type 1 seems to be characterized by the presence of gaps 1 and 2, leading to a shorter rfbA sequence (876 nucleotides) with some point mutation observable. The rfbA-type 2, in addition to being characterized by the gaps 1 and 2, shows specific variations compared to the rfbA-type 1. Interestingly, a start codon ATG is observable in position 73 of every rfbA-type 2 gene that could lead to the suppression of the gap 1. The rfbA-type 3 is also identified by the gap 1 and variations from rfbA-type 1 that are relatively closed to rfbA-type 2. The gap 1 is also present for the types 4 and 5 which, however, display very unique sequences.

2.4. Implication of P. distasonis Cell Surface Structures in Its Potential Pathogenicity

All the data generated in this study were compiled in order to determine the implication of P. distasonis cell surface markers in its potential pathogenicity (Table 4). Strains were classified as commensal (ATCC 8503T and NBRC 113806) or potential pathogens (CavFT-hAR46 and CS1-20 except CS6) on the basis of the health status of their original host (based on the isolation source of each strain, Table 1), and as beneficial or detrimental based on the literature [16,20,26,27,36]. The comparison of outer membrane structure from both categories does not bring to the fore any specific structure. Indeed, all the external structures harbored by the potential pathogen strains are identified in at least one of the commensal strains. In the same way, all structures absent from the surface of commensal strains are not systematically carried by the potential pathogens.

3. Discussion

The human GM and its trillion of bacteria are now well-known for their commensal and symbiotic relationships with the host. One of the GM members is P. distasonis, a Gram-negative anaerobe part of the core microbiome. While a large number of studies promotes this species as a new potential biotherapeutic product due to its multiple benefits provided to its host, controversial results have identified it as an opportunistic pathogen [22,23,24,26,27,28,29]. Although there is still a lot to understand about the mechanisms involved in the GM–host interaction, the implication of cell surface structures of GM members is now well-defined [9,43,44]. In the present study, we investigated cell surface structures of 26 P. distasonis genomes in order to better understand its maintenance within the digestive tract and its potential virulence. Among the 26 genomes, 13 new clinical strain genomes of the member of the distal gut microbiome P. distasonis were sequenced and computed on the MaGe platform. The general features of new genomes were very similar to other P. distasonis genomes already available, with an average size of 5.2 Mb and a core-genome of 2479 CDS. The phylogenetic analysis did not highlight any special difference between CS genome from this study and other P. distasonis genome with a homogenous distribution of CS genomes over the tree.
A previous investigation of CPS loci revealed the presence of the UpxY regulator on P. distasonis ATCC 8503T genome, leading to the identification of 13 putative CPS loci over its genome [42]. In our study, a 14th putative locus was identified on the ATCC 8503T genome. Although well conserved, not all 14 CPS loci are conserved over the 26 P. distasonis strains investigated in this study. Surprisingly, any of the upxY genes identified seem to be coupled with a upxZ regulator genes. However, if UpxY positively regulates B. fragilis CPS synthesis by preventing premature transcription termination in the untranslated region, UpxZ is indispensable to limit production of multiple CPSs, as described in B. fragilis and B. thetaiotaomicron [45,46]. Consequently, P. distasonis surface polysaccharide seems to result in the combination of multiple CPS loci whose expression is potentially controlled by inversions of the promoter region, leading to phase variable synthesis [47]. Th presence of phage insertions within several of these CPS loci may also modulate its expression. In addition, P. distasonis strains do not all display the same number of CPS loci and can also have sequence variations over the loci, emphasizing the strain-dependent nature of P. distasonis CPS.
In addition to external polysaccharides, another proteinaceous surface structure involved in host–microbiota interaction is the fimbriae. One of the most described fimbriae organization is the P. gingivalis Fim system strongly identified as a virulence factor [15]. A previous study identified an analogous typical pilin encoding operon on the P. distasonis ATCC 8503T genome [48]. In our work, almost all the fim-like genes investigated were identified in the 26 P. distasonis genomes, revealing the important conservation of a gene cluster involved in the fimbriae-like synthesis. Among these clusters, two distinct type of fimbriae were identified. The first one is present on 24 of the 26 studied genomes and seems to be conserved, while the second one is only harbored by two genomes. Both clusters are composed of fimA-B-C-D-E-like genes with some variations, including the putative presence of other fimA-like genes through the Fim clusters or different gene sequences such as the fimD-E of CBBP-1 and CS20 strains that display low similarity with others fimD-E. Fimbriae do not necessarily mean pathogenicity by contributing to host epithelium colonization, thus forming a protective barrier against external pathogens and stimulating the host immune system, as recently demonstrated by the recombinant pLA-K88/Lactobacillus casei strain [49].
Pili, as well as capsular polysaccharides and fimbriae, are external proteinaceous structures involved in host–GM interaction. P. gingivalis that display fimbriae also harbor pili, also called “minor fimbriae” [33]. The Mfa system of P. gingivalis involved in the pilus synthesis has also been identified in the gut commensal B. thetaiotaomicron [13]. Although partially found, no complete mfa-like gene cluster has been identified in the studied P. distasonis strains. However, 11 of the 26 genomes possess a pair of genes composed of either Bt mfa1/mfa2, Pg mfa1/mfa2 or Pg mfa4/mfa2 with mfa1/mfa4 encoding for an external polymer and mfa2 involved in the anchoring of the pilus and length regulation of Mfa1. The absence of pilus gene cluster on other genomes could be explained either by a greater diversity of pilus with pili showing important differences from the investigated ones or by the absence of pili on more than half of the studied strains. As for fimbriae, the presence of pili does not necessarily imply pathogenicity. The well-studied probiotic Lactobacillus rhamnosus GG and its spaCBA-encoded pili confirmed this by showing multiple benefits for its host despite its proteinaceous heteropolymeric extracellular appendages [50].
The identification of fimbriae-like and pili-like gene clusters allowed the representation of cell surface markers potentially present at the surface of P. distasonis (Figure 6). Two distinct fimbriae-like and three pili-like gene clusters have been represented depending on the gene clusters found. The first type of fimbriae (left) is harbored by 24 P. distasonis while the second one (right) is harbored by the last two studied strains (CL11T00C22 and CS12). Concerning the pili, four strains (ATCC 8503T, FDAARGOS_1234, 82G9 and CS17) harbor only the first type (left), three (CavFT-har46, CS12 and CS18) harbor only the second type (middle) and none harbor only the third type (right). Some strains also presented combination of several pilus: two (CL06T03C10 and CL11T00C22) harbor the first and second pili type and two (CL03T12C09 and FDAARGOS_759) harbor the first and third type.
In order to discriminate strains regarding their LPS, all the rfbA genes of the 26 P. distasonis genomes have here been referenced, and RfbA-type classification has been applied. As described in Bank et al., 2022 [30], most of the listed rfbA genes belong to type I, highlighting the conservation of this LPS type with the ATCC 8503T rfbA belonging to the type I, and that was isolated more than 80 years ago. However, the addition of new data modified the classification with the identification of a fifth type and a new repartition of the rfbA genes within the five types. The analysis of rfbA-type variation revealed the presence of three main gaps and multiple sequence variations that shape the rfbA-type organization, including one major gap potentially non-existent due to the presence of a start codon within some rfbA gene sequences. Unlike the previous classification, the new rbfA gene repartition shows CS possessing type 1 rfbA and no specific rfbA-type allowing the distinction of CS from other P. distasonis strains. Thus, this typing does not seem to be adequate to differentiate pathogenic from non-pathogenic strains.
The comparison of outer membrane structures from commensal to potential pathogenic strains does not allow the identification of specific surface markers responsible for the putative pathogenicity of P. distasonis. The inter-strain variability observed for P. distasonis properties and potential pathogenicity could be explained by the association of all differences observed in this study, including the presence/absence of cell surface markers, loci/clusters organization and gene sequences. These variations are correlated with the phylogenetical analysis where, for example, P. distasonis ATCC 8503T and FDAARGOS_1234 strains, which are genetically close, display the exact same external structures. In the same way, FDAARGOS_759 and CL09T03C24 that are the most genetically different strains appear to differ from each other in the presence/absence of seven outer structures. Moreover, the synthesis of these external structures seems to depend on numerous factors, including genetic regulators themselves potentially contingent on environment conditions in which bacteria are evolving [47,51,52]. Thus, one plausible response to the pathogenic effects of P. distasonis is the involvement of other mechanisms than its CPS, pili, fimbriae or LPS/O-antigen membrane fractions and to the dissemination of this species from the GM to sterile sites, as an opportunistic pathogen.
Concerning P. distasonis maintenance within the GM, the presence of such external proteinaceous structures could explain its ability to adhere and persist in this complex and competitive environment. These results are consistent with our previous study that illustrates the adhesion and biofilm formation capacity of the 13 P. distasonis [25]. Although all the strains were able to adhere, an inter-strain variation was observable. These differences could be explained by a different shape of the external surface of the strains. Interestingly, the CS12 that displays the lowest adhesion capacity is also the only CS strain that does not harbor the type 1 fimbriae gene cluster identified in this study. This result could highlight the potential involvement of the type 1 fimbriae in the maintenance of P. distasonis within the GM. However, it does not seem that there is a link between the presence of a special cell surface marker for the higher adhesion or biofilm abilities. In fact, the CS1 that displays the most important adhesion capacity does not show a specific cell surface appendage that could explain this adhesion capacity. In the same way, the CS8 that has the strongest biofilm formation capacity does not show a particular cell surface structure explaining its greater capacity.
In conclusion, this work permitted the identification of several gene clusters involved in the capsule, fimbriae and pili synthesis. The presence or absence of these cell surface structures coupled with variations in gene sequences could explain P. distasonis maintenance within the GM and the inter-strain variability observed for its beneficial capacities and potential pathogenicity. However, no specific external cell surface structure that could explain P. distasonis behavior was identified. This study provides a better comprehension of the preservation of P. distasonis through the human gut and tools to better understand and characterize the beneficial/pathogenic behavior related to P. distasonis strains.

4. Materials and Methods

4.1. Whole-Genome Sequencing

The genomic DNA of the 13 CS of P. distasonis was extracted by the QiaAmpDNA MiniKit (Qiagen, Courtaboeuf, France). De Novo Microbial Genome Sequencing using Illumina technology was used to sequence the 13 CS of P. distasonis (Eurofins Genomics, Ebersberg, Germany). All genomes were integrated in the MaGe platform [53] (v3.15.3; The LABGeM, CEA/Genoscope and CNRS UMR8030).

4.2. Genome Data Used

In silico analyses of cell surface structures were performed on the 13 CS of P. distasonis and 13 public genomes available on the MaGe platform.

4.3. Pan and Core-Genome Analysis

The pan and core-genome of P. distasonis were calculated with the Pan/Core-Genome tool of MaGe, based on MicroScope gene families (MICFAM) which are computed using an algorithm implemented in the SiLiX software. The following were used as stringent parameters: 80% amino acid identity and 80% alignment coverage.

4.4. Phylogenetic Analysis

P. distasonis whole-genome sequences were used to determine the phylogenetic relationship among the isolates and public databases. Reference genomes (P. gingivalis ATCC 33277T, B. thetaiotaomicron VPI-5482T and B. fragilis ATCC 25285T) used in this study were added to the tree to demonstrate their closeness with P. distasonis. The phylogenetic tree was computed on MaGe using the Genome Clustering tool and reworked on the Interactive Tree Of Life online tool [54] (iTOL).

4.5. Comparative Genome Analysis

In order to determine the potential presence of fimbriae, pili and/or capsular polysaccharides at the surface of P. distasonis, reference genes involved in their synthesis were selected from species related to P. distasonis (Table 5).
Synteny enabled us to identify the conservation of homologous genes and gene order between genomes of different strains or species. Synteny blocks between references and P. distasonis genomes were investigated using the Genome Browser/Syntonome tools of MaGe and allowed the selection of a pool of genes potentially involved in the synthesis of the targeted structures. To reduce the number of P. distasonis genes and refine the search, only genes with an auto-assignation function related to the synthesis of the sought element were preserved and listed. The automatic functional assignation of MaGe follows an algorithm based on homologous relations with model organisms and completion of gene editor (gene name, product, EC numbers, roles…) using various programs or databases (RefGen, SwissProt, UniFIRE, TrEMBL…).
Multiple sequence alignments of each pool of genes related to each reference gene were then performed using CLC Viewer 8.0 to obtain one or several consensus sequences related to each reference gene.
Consensus sequences were then used for BLAST investigation against the 26 P. distasonis genomes using the Blast and Pattern Searches tool of MaGe.
Matching genes were then used to generate a syntenic block analysis between P. distasonis genome for each cell surface structure studied.

4.6. rfbA-Type Determination and Analysis

In order to determine the rfbA-type genes of the latest sequenced P. distasonis genomes, the classification method recently described by Bank et al. was used [30].
rfbA genes of P. distasonis were first referenced and aligned using CLC Viewer 8.0. The multiple sequence alignment was then used to generate a phylogenetic tree, allowing the classification of new rfbA genes.
As the rfbA-type genes obtained in this study were different from the previous classification, analyses of nucleotide sequences and gaps of distinct rfbA-type genes were performed in order to determine and better understand the variation between each type.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23169411/s1.

Author Contributions

Conceptualization, J.C., C.C.-G. and L.C.; methodology, J.C. and C.C.-G.; software, J.C. and C.C.-G.; formal analysis, J.C.; investigation, J.C. and C.C.-G.; writing—original draft preparation, J.C., C.C.-G. and L.C.; writing—review and editing, J.C., C.C.-G., L.C. and C.A.; supervision, C.C.-G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the French PIA project Lorraine Université d’Excellence, reference ANR-15-IDEX-04-LUE.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The genome sequencing data generated are indexed under the BioProject accession number PRJNA838851.

Conflicts of Interest

No potential conflict of interest was reported by the author(s).

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Figure 1. P. distasonis genomes characterization. (A) General features of P. distasonis genomes used in this study. (B) Graphical representations of the pangenome characteristics. From the center outward: core, dispensable and specific genome. (C) Phylogenetic analysis of 26 strains of P. distasonis. B. thetaiotaomicron VPI-5482T [39], P. gingivalis ATCC 33277T [40] and B. fragilis ATCC 25285T [41] were added as reference genomes used in this study. Bifidobacterium animalis subsp. lactis DSM 10140T was used as outgroup genome.
Figure 1. P. distasonis genomes characterization. (A) General features of P. distasonis genomes used in this study. (B) Graphical representations of the pangenome characteristics. From the center outward: core, dispensable and specific genome. (C) Phylogenetic analysis of 26 strains of P. distasonis. B. thetaiotaomicron VPI-5482T [39], P. gingivalis ATCC 33277T [40] and B. fragilis ATCC 25285T [41] were added as reference genomes used in this study. Bifidobacterium animalis subsp. lactis DSM 10140T was used as outgroup genome.
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Figure 2. Identification of upxY-like gene clusters on P. distasonis genomes. (A) BLAST of upxY-like consensus sequences against P. distasonis genomes. (B) Syntenic analysis of P. distasonis capsular polysaccharide gene clusters centered on uphY-like 1 gene. Vertical and diagonal striped arrows refer to genes having a high homology and sharing synteny with reference gene. (*) indicate genes with low homology but sharing synteny over P. distasonis genomes. CDS: coding sequences are represented by gray arrows.
Figure 2. Identification of upxY-like gene clusters on P. distasonis genomes. (A) BLAST of upxY-like consensus sequences against P. distasonis genomes. (B) Syntenic analysis of P. distasonis capsular polysaccharide gene clusters centered on uphY-like 1 gene. Vertical and diagonal striped arrows refer to genes having a high homology and sharing synteny with reference gene. (*) indicate genes with low homology but sharing synteny over P. distasonis genomes. CDS: coding sequences are represented by gray arrows.
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Figure 3. Identification of fim-like gene clusters on P. distasonis genomes compared to P. gingivalis cluster. (A) BLAST of fim-like consensus sequences against P. distasonis genomes. (B) Syntenic analysis of P. distasonis fimbriae gene clusters centered on fimA-like 1 gene. Vertical striped arrows refer to genes having a high homology and sharing synteny with reference gene. (*) indicate genes with low homology but sharing synteny over P. distasonis genomes. CDS: coding sequences are represented by gray and black arrows. Horizontal striped arrows are used for the Fim cluster of the reference genome: P. gingivalis ATCC 33277T.
Figure 3. Identification of fim-like gene clusters on P. distasonis genomes compared to P. gingivalis cluster. (A) BLAST of fim-like consensus sequences against P. distasonis genomes. (B) Syntenic analysis of P. distasonis fimbriae gene clusters centered on fimA-like 1 gene. Vertical striped arrows refer to genes having a high homology and sharing synteny with reference gene. (*) indicate genes with low homology but sharing synteny over P. distasonis genomes. CDS: coding sequences are represented by gray and black arrows. Horizontal striped arrows are used for the Fim cluster of the reference genome: P. gingivalis ATCC 33277T.
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Figure 4. Identification of pili-like gene clusters on P. distasonis genomes compared to P. gingivalis and B. thetaiotaomicron cluster. (A) BLAST of mfa-like consensus sequences against P. distasonis genomes. (B) Syntenic analysis of P. distasonis pilus gene clusters centered on fimA-like 1 gene. CDS: coding sequences are represented by gray arrows. Horizontal striped arrows are used for the Mfa cluster of the reference genomes: B. thetaiotaomicron VPI-5482T and P. gingivalis ATCC 33277T.
Figure 4. Identification of pili-like gene clusters on P. distasonis genomes compared to P. gingivalis and B. thetaiotaomicron cluster. (A) BLAST of mfa-like consensus sequences against P. distasonis genomes. (B) Syntenic analysis of P. distasonis pilus gene clusters centered on fimA-like 1 gene. CDS: coding sequences are represented by gray arrows. Horizontal striped arrows are used for the Mfa cluster of the reference genomes: B. thetaiotaomicron VPI-5482T and P. gingivalis ATCC 33277T.
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Figure 5. Classification and characterization of rfbA genes of P. distasonis. (A) rfbA copy number and classification of each P. distasonis strain. Color code: presence (green) or absence (red) of the rfbA gene of the indicated type. (B) rfbA-type nucleotide sequences and gaps analysis. Gaps are framed and numerated from 1 to 3. rfbA-type 1 single-nucleotide polymorphism are surrounded in white. Variation of rfbA-type 2 and 3 from rfbA-type 1 are highlighted by black circles. rfbA-type 4 and 5 are framed in black due to the important number of variations compared to the rfbA-type 1.
Figure 5. Classification and characterization of rfbA genes of P. distasonis. (A) rfbA copy number and classification of each P. distasonis strain. Color code: presence (green) or absence (red) of the rfbA gene of the indicated type. (B) rfbA-type nucleotide sequences and gaps analysis. Gaps are framed and numerated from 1 to 3. rfbA-type 1 single-nucleotide polymorphism are surrounded in white. Variation of rfbA-type 2 and 3 from rfbA-type 1 are highlighted by black circles. rfbA-type 4 and 5 are framed in black due to the important number of variations compared to the rfbA-type 1.
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Figure 6. Hypothetical schematic representation of P. distasonis (A) fimbriae and (B) pili from the Fim and Mfa system, respectively. The different type of fimbriae and pili have been identified from 1 to 2 and from 1 to 3, respectively. The table below each structure represents the number of strains harboring the gene cluster encoding the hypothetical structure. The color code corresponds to the syntenic analysis. “Or” indicates that one P. distasonis strain can harbor only one of the proteins encoding genes concerned. For example, type 1 Fim cluster of P. distasonis contains either fimE-like or striped fimE-like genes but never both in the 24 identified clusters. “And Or” indicates that one P. distasonis strain can harbor one or several of the protein encoding genes. For example, various fimA-like 2 genes combinations can be found within type 1 Fim cluster of P. distasonis.
Figure 6. Hypothetical schematic representation of P. distasonis (A) fimbriae and (B) pili from the Fim and Mfa system, respectively. The different type of fimbriae and pili have been identified from 1 to 2 and from 1 to 3, respectively. The table below each structure represents the number of strains harboring the gene cluster encoding the hypothetical structure. The color code corresponds to the syntenic analysis. “Or” indicates that one P. distasonis strain can harbor only one of the proteins encoding genes concerned. For example, type 1 Fim cluster of P. distasonis contains either fimE-like or striped fimE-like genes but never both in the 24 identified clusters. “And Or” indicates that one P. distasonis strain can harbor one or several of the protein encoding genes. For example, various fimA-like 2 genes combinations can be found within type 1 Fim cluster of P. distasonis.
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Table 1. P. distasonis strain isolation.
Table 1. P. distasonis strain isolation.
StrainType of SampleHost StatusIsolation DateIsolation CountryReferences
Parabacteroides distasonisATCC 8503THuman fecesApparently normal1933USA[34]
APCS2/PDHuman fecesUnknown2017IrelandNCBI
CavFT-hAR46Human intramural gut wallSevere Crohn’s disease2019USA[35]
CBBP-1FecesUnknownUnknownUnknown[36]
CL03T12C09UnknownUnknownUnknownUnknownNCBI
CL06T03C10Human fecesUnknown2009USA[37]
CL09T03C24UnknownUnknownUnknownUnknownNCBI
CL11T00C22Human fecesUnknown2009USA[37]
FDAAROS_1234UnknownUnknownUnknownUnknownNCBI
FDAARGOS_615Human fecesUnknownUnknownUnknownNot Published
FDAARGOS_759Human fecesUnknownUnknownUSA[38]
NRBC 113806Human fecesNormalUnknownUnknownNCBI
82G9Human fecesUnknownUnknownJapanNCBI
CS1Peritoneal fluidPeritonitis2016France[25]
CS2Peritoneal fluidPeritonitis2016France[25]
CS4VulvectomyVulvar infection2016France[25]
CS5Peritoneal fluidPeritonitis2016France[25]
CS6Sterility control of mesenchymal stem cellsUnknown2016France[25]
CS7Peritoneal fluidPeritonitis2016France[25]
CS8Blood cultureBacteremia2016France[25]
CS12Bone, sacrumOsteo-articular infection2016France[25]
CS13Peritoneal fluidPeritonitis2016France[25]
CS15Peritoneal fluidPeritonitis2016France[25]
CS17Small intestine collectionAbdominal abscess2017France[25]
CS18Abdominal collectionAbdominal abscess2017France[25]
CS20Peritoneal fluidPeritonitis2017France[25]
T—type strain in microbiology.
Table 2. P. distasonis genes sharing synteny with reference genes and auto-assigned as part of CPS, fimbriae or pili synthesis.
Table 2. P. distasonis genes sharing synteny with reference genes and auto-assigned as part of CPS, fimbriae or pili synthesis.
StructureReference GenePdist StrainLabelLength (aa)Automatic Assignation of Biological Function% Homology
Capsuleup(a-g)YNo match
up(a-g)ZNo match
uphYCS12PDI_v1_160022185Transcription antitermination protein UpdY36.90
CL09T03C24AGZN01_v1_510002192Transcription antitermination protein UpdY36.00
CS4PDI_v1_220060192Transcription antitermination protein UpdY36.00
FDAARGOS_615FOB23_12755179UpxY family transcription antiterminator33.72
APCS2/PDFQN59_13885179UpxY family transcription antiterminator33.70
CS2PDI_v1_140109179Transcription antitermination protein UpdY33.70
CS5PDI_v1_140028179Transcription antitermination protein UpdY33.70
CS6PDI_v1_170031179Transcription antitermination protein UpdY33.70
CS8PDI_v1_150106179Transcription antitermination protein UpdY33.70
CS15PDI_v1_340019179Transcription antitermination protein UpdY33.70
CL03T12C09AGZM01_v1_20031179Transcription antitermination protein UpdY33.14
FDAARGOS_759FIU22_01625179UpxY family transcription antiterminator33.14
82G9E0E49_RS00075179UpxY family transcription antiterminator33.14
CS1PDI_v1_140105179Transcription antitermination protein UpdY33.10
CS7PDI_v1_130113179Transcription antitermination protein UpdY33.10
upgZNo match
FimbriaefimA82G9E0E49_RS19850444fimbrial protein26.21
ATCC 8503TBDI_3514444putative fimbrial protein precursor25.99
CavFT-hAR46FE931_00755444fimbrial protein25.99
FDAARGOS_759FIU22_19490444fimbrial protein25.99
CS6PDI_v1_70115432Fimbrial protein25.60
CS13PDI_v1_70087432Fimbrial protein25.60
CL11T00C22INE94_02450431Major fimbrium subunit FimA type-225.30
CS12PDI_v1_10340431Major fimbrial subunit protein (FimA)25.10
CS1PDI_v1_20076434Major fimbrial subunit protein type II24.90
CS2PDI_v1_300040419Fimbrial protein24.20
CS15PDI_v1_330008419Fimbrial protein24.20
CS20PDI_v1_10539419Fimbrial protein24.20
APCS2/PDFQN59_10875419fimbrial protein24.10
CS4PDI_v1_10167419Fimbrial protein24.10
CL06T03C10INE86_01122420Major fimbrium subunit FimA type-224.00
CS18PDI_v1_50210420Fimbrial protein24.00
CS8PDI_v1_30239421P_gingi_FimA domain-containing protein23.70
CS5PDI_v1_240063421P_gingi_FimA domain-containing protein23.70
CS17PDI_v1_20464421P_gingi_FimA domain-containing protein23.70
FDAARGOS_1234I6J64_10580421fimbrial protein23.50
CS7PDI_v1_30250437Fimbrial protein23.20
fimB82G9E0E49_RS19870303FimB/Mfa2 family fimbrial subunit29.90
CBBP-1HHO38_19050303FimB/Mfa2 family fimbrial subunit29.90
CL06T03C10INE86_01123303Fimbrillin-A associated anchor proteins Mfa1 and Mfa229.90
FDAARGOS_1234I6J64_10575303FimB/Mfa2 family fimbrial subunit29.90
FDAARGOS_759FIU22_19510303FimB/Mfa2 family fimbrial subunit29.90
CS1PDI_v1_20075303Fimbrillin-A associated anchor proteins Mfa1 and Mfa229.90
CS6PDI_v1_70114303FimB/Mfa2 family fimbrial subunit29.90
CS12PDI_v1_10341305Fimbrillin-A associated anchor proteins Mfa1 and Mfa229.90
CS13PDI_v1_70088303FimB/Mfa2 family fimbrial subunit29.90
CL11T00C22INE94_02449305Fimbrillin-A associated anchor proteins Mfa1 and Mfa229.00
fimCCL11T00C22INE94_02448375Putative fimbrium tip subunit Fim1C22.50
fimDCS2PDI_v1_10054684P_gingi_FimA domain-containing protein26.40
CS15PDI_v1_140036684P_gingi_FimA domain-containing protein26.40
CS12PDI_v1_60229685P_gingi_FimA domain-containing protein26.10
CS17PDI_v1_40059685P_gingi_FimA domain-containing protein26.10
CS18PDI_v1_40032685P_gingi_FimA domain-containing protein26.10
CL03T12C09AGZM01_v1_210059684P_gingi_FimA domain-containing protein26.02
CS5PDI_v1_120056675P_gingi_FimA domain-containing protein25.70
CS8PDI_v1_160055675P_gingi_FimA domain-containing protein25.70
CS4PDI_v1_100056677P_gingi_FimA domain-containing protein25.10
CL09T03C24AGZN01_v1_280002678P_gingi_FimA domain-containing protein24.76
fimECL11T00C22INE94_03253632Major fimbrium tip subunit FimE27.10
CL06T03C10INE86_00220632Major fimbrium tip subunit FimE25.30
CBBP-1HHO38_14390688FimB/Mfa2 family fimbrial subunit23.01
PilusBt mfa1No match
Bt mfa2FDAARGOS_759FIU22_05440350FimB/Mfa2 family fimbrial subunit28.98
Pg mfa1CS12PDI_v1_130034509Fimbrillin_C domain-containing protein26.50
CS18PDI_v1_30088509Fimbrillin_C domain-containing protein26.50
CL06T03C10INE86_02000392Minor fimbrium subunit Mfa125.40
CL11T00C22INE94_00002509Major fimbrial subunit protein type IV25.40
Pg mfa2CL06T03C10INE86_02001329Minor fimbrium anchoring subunit Mfa231.20
CL11T00C22INE94_00003329Minor fimbrium anchoring subunit Mfa231.20
CS12PDI_v1_130033329FimB/Mfa2 family fimbrial subunit30.90
CS18PDI_v1_30089329putative Minor fimbrium anchoring subunit Mfa230.40
FDAARGOS_759FIU22_15640300FimB/Mfa2 family fimbrial subunit24.32
82G9E0E49_RS15860300FimB/Mfa2 family fimbrial subunit24.32
Pg mfa3No match
Pg mfa4FDAARGOS_759FIU22_15635463Mfa1 fimbrilin C-terminal domain-containing protein20.83
ATCC 8503TBDI_2708463putative outer membrane protein20.51
CL03T12C09AGZM01_v1_210028463Fimbrillin_C domain-containing protein20.51
82G9E0E49_RS15855463Mfa1 fimbrilin C-terminal domain-containing protein20.20
Pg mfa5No match
aa: amino acid; Bt: B. thetaiotaomicron; Pg: P. gingivalis..
Table 3. Identification of CPS loci in 26 P. distasonis genomes and phage insertion within clusters. Color code: presence (green), partial presence (orange) or absence (red) of the CPS locus by comparison with ATCC 8503T CPS loci. Partial clusters include loci either possessing similar genes compared to ATCC 8503T loci but no upxY-like gene or an identical upxY-like gene to ATCC 8503T but a different gene locus. ● indicate loci containing phage gene insertions.
Table 3. Identification of CPS loci in 26 P. distasonis genomes and phage insertion within clusters. Color code: presence (green), partial presence (orange) or absence (red) of the CPS locus by comparison with ATCC 8503T CPS loci. Partial clusters include loci either possessing similar genes compared to ATCC 8503T loci but no upxY-like gene or an identical upxY-like gene to ATCC 8503T but a different gene locus. ● indicate loci containing phage gene insertions.
P. distasonis
ATTC 8503TAPCS2/PDCavFT-hAR46CBBP-1CL03T12C09CL06T03C10CL09T03C24CL11T00C22FDAARGOS_1234FDAARGOS_615FDAARGOS_759NBRC 11380682G9CS1CS2CS4CS5CS6CS7CS8CS12CS13CS15CS17CS18CS20
Capsular polysaccharide loci1●● ● ●●●●●●●●●●●●●● ●●● ●
2 ● ● ●
3●
4
5●●●● ●●●●●● ● ●
6
7
8●● ●●● ●●●● ● ● ●
9 ●● ● ●
10
11 ●
12
13●●●●●●●●●●●●●●●●●●●●●●●●●●
14
Table 4. Identification of cell surface structures present on 26 P. distasonis strains based on host status. The beneficial or detrimental activity of strains (based on the literature) was added in order to compare potential pathogen from probiotic strains. Color code: beneficial properties (blue), detrimental properties (black), presence (green), partial presence (orange), absence (red). ATCC 8503T is represented as blue/black for its beneficial/detrimental activities due to various results found in the literature. Dashes (-) have been added for unknown status.
Table 4. Identification of cell surface structures present on 26 P. distasonis strains based on host status. The beneficial or detrimental activity of strains (based on the literature) was added in order to compare potential pathogen from probiotic strains. Color code: beneficial properties (blue), detrimental properties (black), presence (green), partial presence (orange), absence (red). ATCC 8503T is represented as blue/black for its beneficial/detrimental activities due to various results found in the literature. Dashes (-) have been added for unknown status.
Ijms 23 09411 i001
Ijms 23 09411 i002
Table 5. Reference genes used to determine external structures of P. distasonis.
Table 5. Reference genes used to determine external structures of P. distasonis.
StructureReference GenomeGeneLabelLength (Aa)Reference
CapsuleBacteroides fragilis ATCC 25285TupaYBF1367172[11,42,55]
upaZBF1368157
upbYBF1893174
upbZBF1894161
upcYBF1009172
upcZBF1010130
updYBF3699179
updZBF3698161
upeYBF2606172
upeZBF2605160
upfYBF1549199
upfZBF1550160
upgYBF0731178
upgZBF0732162
uphYBF3466179
uphZBF3465161
FimbriaePorphyromonas gingivalis ATCC 33277TfimAPGN_0180383[12,15]
fimBPGN_0181118
fimCPGN_0183462
fimDPGN_0184670
fimEPGN_0185550
PilusBacteroides thetaiotaomicron VPI-5482Tmfa1BT_3147388[13]
mfa2BT_3148430
Porphyromonas gingivalis ATCC 33277Tmfa1PGN_0287563[14,33,48]
mfa2PGN_0288324
mfa3PGN_0289446
mfa4PGN_0290333
mfa5PGN_02911228
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Chamarande, J.; Cunat, L.; Alauzet, C.; Cailliez-Grimal, C. In Silico Study of Cell Surface Structures of Parabacteroides distasonis Involved in Its Maintenance within the Gut Microbiota. Int. J. Mol. Sci. 2022, 23, 9411. https://doi.org/10.3390/ijms23169411

AMA Style

Chamarande J, Cunat L, Alauzet C, Cailliez-Grimal C. In Silico Study of Cell Surface Structures of Parabacteroides distasonis Involved in Its Maintenance within the Gut Microbiota. International Journal of Molecular Sciences. 2022; 23(16):9411. https://doi.org/10.3390/ijms23169411

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Chamarande, Jordan, Lisiane Cunat, Corentine Alauzet, and Catherine Cailliez-Grimal. 2022. "In Silico Study of Cell Surface Structures of Parabacteroides distasonis Involved in Its Maintenance within the Gut Microbiota" International Journal of Molecular Sciences 23, no. 16: 9411. https://doi.org/10.3390/ijms23169411

APA Style

Chamarande, J., Cunat, L., Alauzet, C., & Cailliez-Grimal, C. (2022). In Silico Study of Cell Surface Structures of Parabacteroides distasonis Involved in Its Maintenance within the Gut Microbiota. International Journal of Molecular Sciences, 23(16), 9411. https://doi.org/10.3390/ijms23169411

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