Safety Assessment of Lactiplantibacillus plantarum GUANKE Based on Whole-Genome Sequencing, Phenotypic, and Anti-Inflammatory Capacity Analysis

Abstract

Lactiplantibacillus plantarum GUANKE (L. plantarum GUANKE) is a Gram-positive bacterium isolated from the feces of healthy volunteers. Whole-genome sequencing analysis (WGS) revealed that the genome of L. plantarum GUANKE consists of one chromosome and two plasmids, with the chromosome harbors 2955 CDS, 66 tRNAs, and 5 rRNAs. The genome is devoid of virulence factors and Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems. It contains three intact prophage regions and bacteriocin biosynthesis genes (plantaricins K, F, and E), as well as seventeen genomic islands lacking antibiotic resistance or pathogenicity determinants. Functional prediction outcomes identified that the genome of L. plantarum GUANKE is closely related to transcription, carbohydrate transport and metabolism, and amino acid transport and metabolism. Carbohydrate-active enzymes (CAZymes) analysis and GutSMASH analysis revealed that the genome of L. plantarum GUANKE contained 100 carbohydrate-active enzyme genes and two specialized metabolic gene clusters. Safety assessments confirmed that L. plantarum GUANKE neither exhibited β-hemolytic activity nor harbored detectable transferable drug resistance genes. The strain exhibited remarkable acid tolerance and bile salt resistance. Cellular adhesion assays demonstrated moderate binding capacity to Caco-2 intestinal epithelium (4.3 ± 0.007)%. In vitro analyses using lipopolysaccharide (LPS)-stimulated macrophage models demonstrated that L. plantarum GUANKE significantly suppressed the secretion of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β), exhibiting dose-dependent anti-inflammatory activity. In vivo experiments showed that L. plantarum GUANKE was involved in the regulation of the apical junction pathway and interferon pathway in colon tissue of normal mice.

Keywords: L. plantarum; immunological regulation; whole-genome sequencing.

Figure 1

Loop map of the genome of L. plantarum GUANKE. (a) The genome of the L. plantarum GUANKE chromosome and its distribution characteristics. (b) Genome overview and distribution characteristics of plasmid (No. CP004407). (c) Genome overview and distribution characteristics of plasmid (No. CP004408).

Figure 2

General characteristics of L. plantarum GUANKE. (a) The bacteriocin gene of L. plantarum GUANKE. (b) The gene island sequence of L. plantarum GUANKE. The red color represents the gene island predicted by the three methods, the blue represents the gene islands predicted by the IslandPath-DIMOB method, yellow represents the gene islands predicted by the SIGI-HMM method, and green represents the gene islands predicted using the IslandPick method. (c) Carbenzyme synthesis gene of L. plantarum GUANKE.

Figure 3

Genome function annotation of L. plantarum GUANKE. The eggNOG 5.0 database was used to annotate KEGG (a), GO (b), and COG (c) functions of L. plantarum GUANKE’s genes.

Figure 4

Acid and bile salt tolerance of L. plantarum GUANKE. The growth curves of L. plantarum GUANKE in normal MRS medium (a), MRS medium at different pHs (b), and MRS medium with different bile salt concentrations (c). The number of viable bacteria in MRS medium of different pHs (d) and MRS medium of different bile salt concentrations (e). The activity in artificial gastric juice and the activity in intestinal fluid of L. plantarum GUANKE (f). Data are expressed as a mean ± SEM, statistical analysis was performed by two-tailed Student’s t-test (p ≤ 0.05, *, p ≤ 0.01, **, p ≤ 0.001, ***, p ≤ 0.0001, ****, ns = not significant).

Figure 5

Hemolysis and antibiotic sensitivity of L. plantarum GUANKE. (a) Hemolytic analysis of L. plantarum GUANKE. (b) Antibiotic sensitivity analysis of L. plantarum GUANKE.

Figure 6

The cytotoxicity of L. plantarum GUANKE. After co-incubation with Caco-2 cells for 24 h, the contents of LDH (a), TNF-α (b), IL-1β (c), and IL-6 (d) in the cell culture supernatant were detected using a CytoTox 96^® assay and ELISA, respectively. Data are expressed as a mean ± SEM, statistical analysis was performed by two-tailed Student’s t-test (p ≤ 0.0001, ****, ns = not significant).

Figure 7

L. plantarum GUANKE inhibits secretion of inflammatory cytokines by LPS-induced macrophages. ELISA was used to detect the secretion of TNF-α (a), IL-1β (b), and IL-6 (c) in the supernatant of THP-1 cells and the secretion of TNF-α (d) and IL-6 (e) in the supernatant of mBMDMs cells after LPS stimulation. Data are expressed as a mean ± SEM, statistical analysis was performed by two-tailed Student’s t-test (p ≤ 0.05, *, p ≤ 0.01, **, p ≤ 0.001, ***).

Figure 8

L. plantarum GUANKE enhances the apical junction pathway and the interferon response pathway in mouse colon tissue. GSEA analysis revealed that oral administration of L. plantarum GUANKE could enhance the apical junction pathway (a), apical surface pathway (b), interferon-alpha response pathway (c) and interferon-gamma response pathway (d) in colon tissue.