Genomic and Phylogenetic Analysis of Lactiplantibacillus plantarum L125, and Evaluation of Its Anti-Proliferative and Cytotoxic Activity in Cancer Cells

Lactiplantibacillus plantarum is a diverse species that includes nomadic strains isolated from a variety of environmental niches. Several L. plantarum strains are being incorporated in fermented foodstuffs as starter cultures, while some of them have also been characterized as probiotics. In this study, we present the draft genome sequence of L. plantarum L125, a potential probiotic strain presenting biotechnological interest, originally isolated from a traditional fermented meat product. Phylogenetic and comparative genomic analysis with other potential probiotic L. plantarum strains were performed to determine its evolutionary relationships. Furthermore, we located genes involved in the probiotic phenotype by whole genome annotation. Indeed, genes coding for proteins mediating host–microbe interactions and bile salt, heat and cold stress tolerance were identified. Concerning the potential health-promoting attributes of the novel strain, we determined that L. plantarum L125 carries an incomplete plantaricin gene cluster, in agreement with previous in vitro findings, where no bacteriocin-like activity was detected. Moreover, we showed that cell-free culture supernatant (CFCS) of L. plantarum L125 exerts anti-proliferative, anti-clonogenic and anti-migration activity against the human colon adenocarcinoma cell line, HT-29. Conclusively, L. plantarum L125 presents desirable probiotic traits. Future studies will elucidate further its biological and health-related properties.


Introduction
Lactiplantibacillus plantarum is one of the 26 phylogenetic groups of the Lactobacillaceae family that consists of facultative anaerobic, Gram-positive, non-motile and non-sporeforming rods that can occur single, in pairs or short chains, presenting high genomic diversity [1]. The L. plantarum group forms a monophyletic clade with other heterofermentative Lactobacillus and Pediococcus strains and also shares major metabolic attributes with homofermentative lactobacilli [2]. Two subspecies of this species have been identified so far: L. plantarum subsp. plantarum and L. plantarum subsp. argentoratensis [2]. L. plantarum strains generally present a nomadic lifestyle, as they can be found free living in nutrientrich environments, such as vegetables or in association with vertebrate or invertebrate hosts [3].

Detection of Genetic Elements Associated with Probiotic Characteristics
BAGEL (version 4) was used to detect and visualize gene clusters that are implicated in the biosynthesis of antimicrobial peptides [37]. The Resistance Gene Identifier (RGI) (version 5.1.1) verified the presence of antibiotic resistance genes [38]. BLAST (basic local alignment search tool) was employed to search for genetic loci that are involved in stress response and host-microbe interactions.

Cell-Free Supernatant Preparation
For the preparation of CFCS, L. plantarum L125 was cultured for 20 h in MRS broth at 37 • C under anaerobic conditions. The next day, 10 8 Colony Forming Units/mL (CFU/mL) were added in Roswell Park Memorial Institute (RPMI)-1640 cell culture medium supplemented with GlutaMAX™, 10% fetal bovine serum (FBS) and 20 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) (all from Thermo Fisher Scientific, Waltham, MA, USA) and were incubated anaerobically at 37 • C for 24 h. Then, the bacterial cells were Biomedicines 2021, 9, 1718 4 of 17 pelleted by centrifugation at 2.600× g for 15 min, and the supernatants were sterile filtered using an 0.22 µm pore size filter (Corning, New York, NY, USA). The dilution of the CFCS was performed in the cell culture medium without antibiotics.

Sulforhodamine B Colorimetric Assay
The sulforhodamine B (SRB) colorimetric assay was employed to investigate the anti-proliferative potential of CFCS of L. plantarum L125, against the human colon adenocarcinoma cell line, HT-29 (ATCC, Manassas, VA, USA). HT-29 cells were maintained in RPMI-1640 medium supplemented with GlutaMAXTM, 10% FBS, 100 µg/mL streptomycin and 100 U/mL penicillin (all from Thermo Fisher Scientific) in a humidified, sterile atmosphere at 37 • C, 5% CO 2 . Cells were seeded in 96-well plates (Corning) at a density of 7000 cells per well. The next day, cells were treated with 100 µL of CFCS (undiluted or diluted to a ratio of 1:2). Untreated cells (control) were maintained in standard cell culture medium. After 24 or 48 h treatments, the SRB assay was performed as previously described [39]. For the calculation of the cellular survival, the following formula: ((sample OD570 − media blank OD570 )/(mean control OD570 − media blank OD570 )) × 100 was applied. The assay was performed four independent times in octuplicates.

Colony Formation Assay
A colony formation assay was performed to determine the anti-clonogenic effect of CFCS on HT-29 cells, as previously described, with minor modifications [40]. Briefly, HT-29 cells (1000 cells per 100 mm plate) were treated with undiluted CFCS from L. plantarum L125 or L. rhamnosus GG for 48 h. The cells were incubated for 10 days until the formation of visible colonies. The colonies were stained with 0.5% (v/v) crystal violet, following the protocol proposed by Franken et al. 2006 [41]. Results are expressed as: Number of colonies (%) = (number of colonies treated /number of colonies untreated ) × 100.

Wound Healing Assay
The anti-migration potential of L. plantarum L125 CFCS was examined using the wound healing assay. To this end, HT-29 cells were seeded in polymer coverslip inserts in 35 mm µ-Dishes at a density of 80,000 cells per silicone insert (Ibidi, Gräfelfing, Germany) and were incubated in standard conditions overnight. The next day, the inserts were removed to reveal a 500 µm cell-free gap. Then, the cells were treated with undiluted CFCS from L. plantarum L125 or L. rhamnosus GG. Untreated cells (control) were maintained in the cell culture medium, as mentioned above. Photographs were taken with a ZEISS Primovert light microscope (Zeiss, Göttingen, Germany) equipped with a digital camera (Axiocam ERc 5 s) at 0, 24 and 48 h post-treatment.

Statistical Analysis
Statistical differences in the in vitro experiments were analyzed using 2-tailed Student's t-tests. A p < 0.05 was considered statistically significant. Results were expressed as the mean ± standard deviation of measurements. All experimental procedures were repeated three independent times unless otherwise stated.

Genome Features
The genomic characteristics of L. plantarum L125 were investigated using wholegenome sequencing and comprehensive bioinformatic analysis (Table 1), leading to the construction of its genome map ( Figure 1). The complete genome of L. plantarum L125 consists of 3,354,135 bp with a GC content of 44.34%. The 3220 predicted genes include 3024 protein-coding sequences (CDSs), 126 pseudogenes, 62 tRNA genes, 4 rRNA genes as well as 4 ncRNAs. Both PGAP and CRISPRDetect (version 2.4) provided evidence that L. plantarum L125 does not carry CRISPR arrays. In addition, one intact prophage region with a length of 35 kb was identified (Table S2). Lastly, we did not identify any transferable genetic elements related to antibiotic resistance in the genome of L. plantarum L125, which agrees with previous in vitro findings [19].  (Table S2). Lastly, we did not identify any transferable genetic elements related to antibiotic resistance in the genome of L. plantarum L125, which agrees with previous in vitro findings [19]. From the outer to inner circle, the information is displayed as follows: genome size (black), forward strand CDS (orange), reverse strand CDS (blue), pseudogenes (green), tRNA genes (red), GC content, GC skew. L. plantarum L125 was classified as the species Lactobacillus plantarum, which is now known as Lactiplantibacillus plantarum [2,19]. Based on previous in vitro findings, L. plantarum L125 exhibits good probiotic potential [19,20]; therefore, in order to determine its phylogenetic position and relationship compared to other L. plantarum strains, we constructed a neighbor-joining phylogenetic tree, including 1000 bootstrap replicates ( Figure 2). The phylogenetic tree is based on orthologous gene clusters and consists of L. plantarum L125 and 21 other potential probiotic L. plantarum strains. Among them, two well-established L. plantarum probiotic strains, L. plantarum WCFCS1 and L. plantarum 299v, were also included [6,42] (Figure 2). To assure the accurate phylogenetic placement of the newly sequenced strain, Streptococcus pneumoniae Hu17 and Leuconostoc mesenteroides SRCM102733 have been used as outgroups/controls ( Figure S1). The reliability of the phylogenetic placement is also verified by ANI analysis, as L. plantarum L125 exhibited high ANI scores (>98.6%) with all L. plantarum strains ( Figure 3A). L. plantarum L125 was classified as the species Lactobacillus plantarum, which is now known as Lactiplantibacillus plantarum [2,19]. Based on previous in vitro findings, L. plantarum L125 exhibits good probiotic potential [19,20]; therefore, in order to determine its phylogenetic position and relationship compared to other L. plantarum strains, we constructed a neighbor-joining phylogenetic tree, including 1000 bootstrap replicates ( Figure 2). The phylogenetic tree is based on orthologous gene clusters and consists of L. plantarum L125 and 21 other potential probiotic L. plantarum strains. Among them, two well-established L. plantarum probiotic strains, L. plantarum WCFCS1 and L. plantarum 299v, were also included [6,42] (Figure 2). To assure the accurate phylogenetic placement of the newly sequenced strain, Streptococcus pneumoniae Hu17 and Leuconostoc mesenteroides SRCM102733 have been used as outgroups/controls ( Figure S1). The reliability of the phylogenetic placement is also verified by ANI analysis, as L. plantarum L125 exhibited high ANI scores (>98.6%) with all L. plantarum strains ( Figure 3A).    The vast majority of strains included in the tree have been isolated from fermented food products, mainly from kimchi, in countries located in East Asia, while only 2 strains were isolated from food products in European countries: L. plantarum L125 and L. plantarum Lp790. The abovementioned geographical correlation is reflected in the phylogenetic tree, as L. plantarum Lp790, which was isolated from Morlacco cheese in Italy and showed The vast majority of strains included in the tree have been isolated from fermented food products, mainly from kimchi, in countries located in East Asia, while only 2 strains were isolated from food products in European countries: L. plantarum L125 and L. plantarum Lp790. The abovementioned geographical correlation is reflected in the phylogenetic tree, as L. plantarum Lp790, which was isolated from Morlacco cheese in Italy and showed good probiotic potential in both in vitro and in vivo studies [43], is the closest evolutionary relative of L. plantarum L125 ( Figure 2).
Furthermore, genome comparison of the 22 aforementioned L. plantarum strains revealed that L. plantarum L125 carries 220 unique genes. Strain-specific proteins were classified into COG functional categories. Notably, 60% of the genes code for proteins involved in fundamental cellular functions (Metabolism, Information Storage and Processing, Cellular Processes and Signaling). The remaining 40% of the genes are poorly characterized ( Figure 3B). Overall, L. plantarum L125 appears to be part of the L. plantarum species and possesses a number of genes with important functions.

Functional Classification
We conducted a comprehensive in silico analysis to describe the genomic traits of L. plantarum L125, as well as to compare them with the 21 selected L. plantarum strains. To gain a better insight into the functional characteristics of L. plantarum L125, its CDSs were allocated to COG and KEGG functional categories. The majority of the CDSs (94.48%) were assigned to 20 COG functional categories. Similarly, for the 21 L. plantarum strains, the CDSs of each strain were distributed into COG functional categories, and the average percentage for each COG category was calculated (Table S3). A comparison of the COG profile of L. plantarum L125 with the average values of the 21 L. plantarum strains revealed similar percentages in all COG functional categories ( Figure 4). In both cases, the "Function Unknown (S)" was the most abundant category, followed by "General Function Prediction only (R)" and "Transcription (K)" (Figure 4). More precisely, L. plantarum L125 has 19.3% of its CDSs assigned to "Function Unknown (S)", 11.5% to "General Function Prediction only (R)" and 9.5% to "Transcription (K)". Concomitantly, we performed KEGG analysis to uncover the variety and functionality of proteins coded by L. plantarum L125. More specifically, 53.20% of the L. plantarum L125 CDSs were classified into 39 KEGG functional categories and 180 pathways. These pathways notably include "biosynthesis of secondary metabolites" (ko: 01110; 170 genes), "microbial metabolism in diverse environments" (ko: 01120; 88 genes) and "ABC transporters" (ko: 02010; 78 genes). Regarding the capability of L. plantarum L125 to biosynthesize amino acids, KEGG pathway reconstruction showed that this strain can fully synthesize only 8 out of 20 amino acids: threonine, cysteine, methionine, lysine, histidine, arginine, proline and tryptophan (Table S4, Figures S2-S8), while it encodes part of the essential proteins involved in the biosynthesis of the other twelve amino acids. Furthermore, we observed that the KEGG profile of L. plantarum L125 is comparable to that of the other 21 L. plantarum strains included in the study ( Figure 5, Table S5).
Moreover, we searched the genome of L. plantarum L125 for genes encoding enzymes involved in carbohydrate metabolism. We identified 76 genes that regulate the metabolism of a wide array of carbohydrates and assigned them into five CAZymes gene classes: 36 glycoside hydrolase (GH) genes, 31 glycosyltransferase (GT) genes, 5 carbohydratebinding modules (CBMs) genes, 3 carbohydrate esterase (CE) genes and 1 Auxiliary Activity (AA) gene, (Table S6). Thus, L. plantarum L125 may be able to utilize several monoand polysaccharides as energy sources and also synthesize complex molecules, such as chitin and cellulose. This finding could support the nomadic nature of the strain, common to L. plantarum strains [3].  Concomitantly, we performed KEGG analysis to uncover the variety and functionality of proteins coded by L. plantarum L125. More specifically, 53.20% of the L. plantarum L125 CDSs were classified into 39 KEGG functional categories and 180 pathways. These pathways notably include "biosynthesis of secondary metabolites" (ko: 01110; 170 genes), "microbial metabolism in diverse environments" (ko: 01120; 88 genes) and "ABC transporters" (ko: 02010; 78 genes). Regarding the capability of L. plantarum L125 to biosynthesize amino acids, KEGG pathway reconstruction showed that this strain can fully synthesize only 8 out of 20 amino acids: threonine, cysteine, methionine, lysine, histidine, arginine, proline and tryptophan (Table S4, Figures S2-S8), while it encodes part of the essential proteins involved in the biosynthesis of the other twelve amino acids. Furthermore, we observed that the KEGG profile of L. plantarum L125 is comparable to that of the other 21 L. plantarum strains included in the study ( Figure 5, Table S5).

Identification of Genes Implicated in Stress Response, Microbe-Host Interactions and Bacteriocin Biosynthesis
The genome of L. plantarum L125 was scoured for genetic loci implicated in the interaction with the host. In this context, genome annotation revealed that L. plantarum L125 possesses two genes coding for proteins mediating survival in the GI tract [44]: cation:proton antiporter and the PBP1A family penicillin-binding protein (Table 2). Furthermore, three genes involved in the acid tolerance mechanisms [45] of L. plantarum L125 were also identified: D-alanine--poly(phosphoribitol) ligase subunit (dltA), D-alanyl-lipoteichoic acid biosynthesis protein (dltD) and glutamate decarboxylase (gadB). Moreover, an F0F1-ATPase that consists of eight subunits, known for its role in acidic tolerance [46], and three bile salt hydrolases were also detected in the genome of L. plantarum L125.
Exposure to extreme temperatures, prevalent in the food industry, can be stressful for bacteria and subsequently lead to the expression of heat and cold shock proteins. L. plantarum L125 carries five proteins involved in heat shock response ( Table 2); molecular chaperone DnaJ, molecular chaperone DnaK, nucleotide exchange factor GrpE, chaperonin GroEL, co-chaperone GroES. Accordingly, survival in low temperatures can be mediated by three proteins of the cold shock protein family.
Moreover, L. plantarum L125 codes for a plethora of cell surface proteins (Table 2). More specifically, L. plantarum L125 contains six proteins carrying cell wall anchor domains (LPxTG motifs), as well as 1 gene encoding for a collagen-binding protein. Furthermore, three proteins with mucus-binding domains and two fibronectin-binding domaincontaining proteins were also identified. Finally, the elongation factor Tu, a moonlighting protein with adhesin-like activity, was detected within the L. plantarum L125 genome. Table 2. List of proteins encoded by L. plantarum L125, involved in stress response and host-microbe Moreover, we searched the genome of L. plantarum L125 for genes encoding enzymes involved in carbohydrate metabolism. We identified 76 genes that regulate the metabolism of a wide array of carbohydrates and assigned them into five CAZymes gene classes: 36 glycoside hydrolase (GH) genes, 31 glycosyltransferase (GT) genes, 5 carbohydratebinding modules (CBMs) genes, 3 carbohydrate esterase (CE) genes and 1 Auxiliary Activity (AA) gene, (Table S6). Thus, L. plantarum L125 may be able to utilize several mono-and polysaccharides as energy sources and also synthesize complex molecules, such as chitin and cellulose. This finding could support the nomadic nature of the strain, common to L. plantarum strains [3].

Identification of Genes Implicated in Stress Response, Microbe-Host Interactions and Bacteriocin Biosynthesis
The genome of L. plantarum L125 was scoured for genetic loci implicated in the interaction with the host. In this context, genome annotation revealed that L. plantarum L125 possesses two genes coding for proteins mediating survival in the GI tract [44]: cation:proton antiporter and the PBP1A family penicillin-binding protein ( Table 2). Furthermore, three genes involved in the acid tolerance mechanisms [45] of L. plantarum L125 were also identified: D-alanine-poly(phosphoribitol) ligase subunit (dltA), D-alanyl-lipoteichoic acid biosynthesis protein (dltD) and glutamate decarboxylase (gadB). Moreover, an F0F1-ATPase that consists of eight subunits, known for its role in acidic tolerance [46], and three bile salt hydrolases were also detected in the genome of L. plantarum L125. Exposure to extreme temperatures, prevalent in the food industry, can be stressful for bacteria and subsequently lead to the expression of heat and cold shock proteins. L. plantarum L125 carries five proteins involved in heat shock response ( Table 2); molecular chaperone DnaJ, molecular chaperone DnaK, nucleotide exchange factor GrpE, chaperonin GroEL, co-chaperone GroES. Accordingly, survival in low temperatures can be mediated by three proteins of the cold shock protein family.
Moreover, L. plantarum L125 codes for a plethora of cell surface proteins (Table 2). More specifically, L. plantarum L125 contains six proteins carrying cell wall anchor domains (LPxTG motifs), as well as 1 gene encoding for a collagen-binding protein. Furthermore, three proteins with mucus-binding domains and two fibronectin-binding domaincontaining proteins were also identified. Finally, the elongation factor Tu, a moonlighting protein with adhesin-like activity, was detected within the L. plantarum L125 genome.
Over the last few years, genome analysis of numerous L. plantarum strains has revealed the presence of genetic loci responsible for the production of antimicrobial peptides, also known as bacteriocins [47]. To examine the capability of L. plantarum L125 to produce such antimicrobial peptides, we found that our strain possesses three genes that are crucial for the production of the class IIb bacteriocin: plantaricin EF. The abovementioned genes are homologous and exhibit high identity values to those of the probiotic strain L. plantarum WCFCS1 [48] (Figure 6). However, L. plantarum L125 lacks several genes of the plnABCD and plnGHTUVW operons, which are essential for transcriptional regulation and bacteriocin secretion, respectively [49] (Figure 6).

Investigation of Potential Health-Promoting Effects Induced by L. plantarum L125
Potential probiotic strains can induce a variety of beneficial actions when interacting with the host. In this study, we explored the anti-proliferative activity of L. plantarum L125 CFCS against the human colon adenocarcinoma cell line, HT-29. For that reason, cells were treated with undiluted or diluted at a ratio of 1:2 CFCS and cell survival was estimated using the SRB colorimetric assay. L. rhamnosus GG was used as a reference strain due to its well-characterized cytotoxic and anti-proliferative properties [50]. CFCS treatments induced a significant time-and dose-dependent effect ( Figure 7A-D, p < 0.01). More specifically, the undiluted L. plantarum L125 CFCS decreased cell survival by 40 and 60% after 24 and 48 h treatments, respectively ( Figure 7A,C). The reference strain induced similar effects. Furthermore, we sought to determine the anti-clonogenic potential of L. plantarum L125-derived CFCS by employing the colony formation assay. Indeed, the undiluted CFCS significantly reduced the number of viable colonies compared to control, untreated cells (p < 0.01) ( Figure 7E,F). Finally, the anti-migration capacity of the undiluted CFCS was assessed by the wound healing assay. Notably, HT-29 co-incubation with CFCS limited cell migration ( Figure 7G). On the other hand, wound healing of the untreated sample was completed after 48 h ( Figure 7G).

Investigation of Potential Health-Promoting Effects Induced by L. plantarum L125
Potential probiotic strains can induce a variety of beneficial actions when interacting with the host. In this study, we explored the anti-proliferative activity of L. plantarum L125 CFCS against the human colon adenocarcinoma cell line, HT-29. For that reason, cells were treated with undiluted or diluted at a ratio of 1:2 CFCS and cell survival was estimated using the SRB colorimetric assay. L. rhamnosus GG was used as a reference strain due to its well-characterized cytotoxic and anti-proliferative properties [50]. CFCS treatments induced a significant time-and dose-dependent effect ( Figure 7A-D, p < 0.01). More specifically, the undiluted L. plantarum L125 CFCS decreased cell survival by 40 and 60% after 24 and 48 h treatments, respectively ( Figure 7A,C). The reference strain induced similar effects. Furthermore, we sought to determine the anti-clonogenic potential of L. plantarum L125-derived CFCS by employing the colony formation assay. Indeed, the undiluted CFCS significantly reduced the number of viable colonies compared to control, untreated cells (p < 0.01) ( Figure 7E,F). Finally, the anti-migration capacity of the undiluted CFCS was assessed by the wound healing assay. Notably, HT-29 co-incubation with CFCS limited cell migration ( Figure 7G). On the other hand, wound healing of the untreated sample was completed after 48 h ( Figure 7G). cines 2021, 9,

Discussion
In this study, we announce the draft genome sequence of L. plantarum L125, a strain presenting biotechnological interest, that was originally isolated from a fermented sausage [19]. The complete genome of the strain consists of 3,354,135 bp with a GC content of 44,34%, and it contains one prophage region and no CRISPR arrays. L. plantarum L125, although originally isolated from meat products, may be able to adapt to a variety of niches, as suggested by the fact that 88 of its genes are assigned to the KEGG pathway "microbial metabolism in diverse environments". Indeed, L. plantarum strains are able to colonize a wide range of habitats such as the human GI tract, meat, fish, vegetables, dairy and other fermented products [1]. The nomadic lifestyle of the species is mirrored in the vast genetic diversity that L. plantarum strains present [51]. Previous reports indicate that during the environmental adaptation process, genomic changes may occur [52,53]; however, a strong link between genome content and niche adaptation of L. plantarum has not been established yet [3]. Indeed, our phylogenetic analysis did not reveal any grouping of the studied L. plantarum strains based on their isolation source. Interestingly, we observed that the closest evolutionary relative of L. plantarum L125 is L. plantarum Lp790, the only other strain that was isolated in Europe and, more specifically, from Italian dairy products [43]. The abovementioned genome alterations during niche adaptation, including gene gain/loss events, may affect genetic clusters associated with amino acid biosynthesis [54]. In this study, we showed that L. plantarum L125 possesses complete biosynthetic pathways for eight out of the 20 amino acids (Figures S2-S8), underlining the need for amino acid supply from nutrient-dense environments. Likewise, the KEGG reconstruction pathway revealed that all studied L. plantarum strains exhibit identical capability regarding amino acid biosynthesis.
A prerequisite for microbe-host interactions to occur is the tolerance of the host niche. A previous report revealed the ability of L. plantarum L125 to survive in highly acidic and bile-rich environments [19]. In fact, this strain did manage to withstand the abovementioned stress conditions, which are similar to those prevailing in the human GI tract [19]. In this study, we found numerous proteins that support the previous in vitro findings and are associated with either acid tolerance or bile salt resistance (Table 2). Furthermore, according to the same study, L. plantarum L125 tolerance to bile salts is accompanied by bile salt hydrolase activity. Indeed, a comprehensive bioinformatical analysis revealed the presence of bile salt hydrolases within the L. plantarum L125 genome (Table 2). Moreover, probiotics intended for biotechnological application should tolerate heat or/and cold stress conditions [55], and therefore, the presence of heat and cold shock proteins within their genome is regarded as a desirable trait. In this context, L. plantarum L125 was detected in high counts in Greek traditional dry fermented sausages that were stored at 4 • C for 160 days [20]. These findings indicate that the cold-shock family proteins we identified (Table 2) are functional and correlated with their viability at low temperatures.
The ability of lactobacilli to adhere to and interact with intestinal surfaces is considered to be crucial for their probiotic action [56]. A number of cell surface molecules such as polysaccharides and proteins have been associated with this phenotype [57]. More specifically, probiotic bacteria utilize collagen-, mucin-and fibronectin-binding proteins, as well as LPXTG domain-containing proteins, to attach to the host intestinal epithelial cells or mucosa [58]. In addition, the adhesion capability of lactobacilli is also supported by several moonlighting proteins, such as EF-Tu [59], which, among other functions, can exhibit adhesin-like activity [56]. In this study, we identified numerous cell surface adhesins and moonlighting proteins in L. plantarum L125 (Table 2). Future studies will explore the adhesion capacity of the strain in vitro and will focus on the specific mechanisms mediating this effect, as well as the biological significance of this interaction for the host cell.
Concerning the antimicrobial activity of probiotic strains, they can exert inhibitory effects by utilizing a great variety of mechanisms. Indeed, direct antimicrobial activity of L. plantarum can be induced by the secretion of inhibitory compounds, such as bacteriocins [60], fatty acids, ethanol and hydrogen peroxide [61], or by competitive pathogen exclusion [62]. In this vein, the bacteriocin produced by L. plantarum ATCC 8014 limited the proliferation of Staphylococcus aureus 99308 in a mouse model of mandibular fracture postoperative infection [63]. Furthermore, some probiotic strains can stimulate the immune response of the host, leading to pathogen clearance [64]. Our results corroborate previous studies showing that L. plantarum L125 does not present bacteriocin-like activity [19], although it carries three genes encoding for plantaricin EF (Figure 6). In greater detail, it lacks essential genes for transcriptional regulation and secretion, alluding to the fact that the plantaricin cluster may not be functional. The ability of L. plantarum L125 to inhibit pathogen colonization, expansion and biofilm formation by mechanisms other than bacteriocin synthesis will be studied in the future.
Another significant health-promoting property that specific probiotic strains possess is the ability to regulate cell cycle progression and cell death [64]. Indeed, previous studies from our lab have shown that potential probiotics, such as L. pentosus B281 and L. paracasei K5, can induce cell death in species-and strain-specific fashion [40,65]. In this context, the administration of viable L. casei ATCC 393 cells to a mouse model bearing CT26 tumors led to a reduction in tumor volume via the induction of apoptotic cell death [66]. Similarly, ferrichrome, isolated from L. casei ATCC334 CFCS, exerted tumorsuppressive effects in a BALB/c xenograft model that were also attributed to the induction of apoptosis and, more specifically, to the c-Jun N-terminal kinase (JNK) signaling pathway {15]. However, potential probiotic strains can also induce cytotoxic effects by alternative mechanisms, such as immunogenic cell death [67]. For example, the oral administration of heat-killed L. plantarum BF-LP284 to a murine syngeneic model of sarcoma and resulted in the inhibition of tumor growth and the stimulation of anti-tumor immune responses [68]. In the present study, we observed that CFCS of L. plantarum L125 can effectively limit the proliferation and migration capacity of HT-29 cells. HT-29 cells were selected in this study as an in vitro model of the human colon. The observed effects were mediated in a time-and dose-dependent manner and were comparable to the activity of L. rhamnosus GG, a well-studied probiotic strain (Figure 7). Of note, the observed reduction in cell viability was not due to the acidic pH of CFCS (data not shown). On the other hand, cell surface molecules and/or excreted metabolites may mediate these anti-proliferative actions. Regarding the nature of these active compounds, exopolysaccharides (EPS), peptidoglycans and conjugated linolenic acids (CLA), as well as S-layer proteins, have been implicated in the induction of cell death [69,70]. Interestingly, we have located clusters for EPS and CLA biosynthesis in the genome of L. plantarum L125 (data not shown). The latter was almost identical to the functional CLA biosynthesis cluster found in L. plantarum ZS2058 [71]. However, further studies are needed to determine their functionality and the potential contribution of these molecules to the observed anti-proliferative effects.

Conclusions
In this study, we presented the whole genome sequence of L. plantarum L125 and performed comprehensive bioinformatic analysis to locate genes involved in the probiotic phenotype. We found the strain codes for proteins supporting survival and adaptation in the gastrointestinal niche, as well as tolerance to conditions prevalent in the food industry. Concerning the potential health benefit of the strain, we observed that the CFCS from L. plantarum L125 can induce anti-proliferative, anti-clonogenic and anti-migration effects on the colon adenocarcinoma cell line, HT-29. Additional studies are needed to validate the putative anticancer potential of the strain in animal models of tumorigenesis and in the clinical setting. Subsequently, its incorporation in the functional food industry will further be examined.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/biomedicines9111718/s1, Figure S1: Neighbor-joining phylogenetic tree based on orthologous genes, of L. plantarum L125 and 21 L. plantarum strains. Figure S2: The KEGG pathway of Glycine, Serine and Threonine Metabolism (ko: 00260). Figure S3: The KEGG pathway of Cysteine and Methionine Metabolism (ko: 00270). Figure S4: The KEGG pathway of Lysine Metabolism (ko: 00300). Figure S5: The KEGG pathway of Arginine Metabolism (ko: 00220). Figure S6: The KEGG pathway of Arginine and Proline Metabolism (ko: 00330). Figure S7: The KEGG pathway of Histidine Metabolism (ko: 00340). Figure S8: The KEGG pathway of Tryptophan Metabolism KEGG pathway (ko: 00400). Table S1: The probiotic properties of the 21 L. plantarum strains, used in the comparative genomic analysis. Table S2: Prophage sequences in genome assembly of L. plantarum L125 by PHAge Search Tool Enhanced Release (PHASTER). Table S3: Detailed presentation of the percentages of genes assigned to the COG functional categories of L. plantarum L125 and of the 21 L. plantarum strains. Table S4: List of complete KEGG modules and pathways that reflect the amino acid biosynthesis capability of L. plantarum 125. Table S5: Detailed presentation of the number of genes assigned to the KEGG functional categories of L. plantarum L125 and of the 21 L. plantarum strains. Table S6: L. plantarum L125 genes assignment into CAZymes families aided by the CAZy database.  and co-financed by Greece and the European Union (European Regional Development Fund) and "PUZL-Molecular identification and utilization of indigenous people of hop varieties for the production of high quality beers with name production" (MIS 5056124) under the Regional Operational Program "Sterea Ellada 2014-2020", co-funded by the European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation.
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: The L. plantarum strain L125 genome sequence has been deposited at DDBJ/ENA/GenBank under the accession JAIGOE000000000. The version described in this paper is version JAIGOE010000000.

Acknowledgments:
We acknowledge the support of the M.Sc. program «Translational Research in Biomedicine» of the Department of Molecular Biology and Genetics, Democritus University of Thrace.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.