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

Abstract

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.

Keywords: Lactiplantibacillus plantarum; anti-proliferative activity; comparative genomics; genomics; phylogenetic analysis; probiotics; whole-genome sequencing.

Figure 1

Circular genome map of L. plantarum L125. 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.

Figure 2

Neighbor-joining phylogenetic tree of L. plantarum L125 and 21 potential probiotic L. plantarum strains based on orthologous genes. The tree was constructed using 1000 bootstrap replicates, calculated by MEGAX (version 10.1.8). The red arrow indicates the position of L. plantarum L125 in the phylogenetic tree.

Figure 3

(A) Average Nucleotide Identity (ANI) matrix and similarity scores between the coding regions of L. plantarum L125 and the 21 L. plantarum strains. (B) L. plantarum L125 strain-specific genes, compared to the 21 L. plantarum strains, assigned to Clusters of Orthologous Groups (COGs) functional categories. “Function Unknown (S)” and “General Function Prediction only (R)” are depicted in the category termed “Poorly Characterized”.

Figure 4

Comparison of the percentage of genes assigned to the COG functional categories of L. plantarum L125 (Brown bars) and of the 21 L. plantarum strains (Yellow bars). For each one of the 21 L. plantarum strains, the percentage of genes for each COG functional category was determined, and average values were calculated (Yellow bars). The values are depicted as mean ± standard deviation.

Figure 5

KEGG profiles comparison between L. plantarum L125 (Brown bars) and 21 L. plantarum strains (Yellow bars). For each one of the 21 L. plantarum strains, the number of genes in each KEGG functional category was determined, and average values were calculated (Yellow bars). The values are depicted as mean ± standard deviation.

Figure 6

Detailed comparison of genes inside the pln locus (plnABCD, plnEFI and plnGHSTUVW operons) between L. plantarum L125 and L. plantarum WCFCS1. Black dashed lines represent pseudogenes, while black hyphens indicate gene absence. Protein identities are also indicated.

Figure 7

Time- and dose-dependent anti-proliferative, anti-clonogenic and anti-migration activity of L. plantarum L125-derived CFCS against the human adenocarcinoma cell line, HT-29. L. rhamnosus GG was used as a reference. The SRB colorimetric assay was used to evaluate the anti-proliferative activity of undiluted (A,C) or diluted at a ratio of 1:2 (B,D) L. plantarum L125 or L. rhamnosus GG-derived CFCS, after 24 (A,B) or 48 (C,D) hour treatments. (E) Representative photos of the colony formation assay results, showing the anti-clonogenic potential of undiluted L. plantarum L125 and L. rhamnosus GG CFCS after 48 h treatments. (F) Quantitative results of the colony formation assay for the reference and tested strain. (G) The anti-migration capacity of L. plantarum L125- or L. rhamnosus GG-derived CFCS, evaluated by the wound healing assay. Photos were taken at 0, 24 and 48 h post-incubation with undiluted CFCS. Scale bar, 100 μm. Data are presented as the mean ± standard deviation. * p < 0.05, ** p < 0.001 compared to control, untreated cells.