Oral Administration of Mice with Cell Extracts of Recombinant Lactococcus lactis IL1403 Expressing Mouse Receptor Activator of NF-kB Ligand (RANKL)

Abstract

Receptor activator of NF-kB ligand (RANKL) is known to play a major role in bone metabolism and the immune system, and its recombinant form has been expressed in bacterial systems for research since the last two decades. However, most of these recombinant forms are used after purification or directly using living cells. Here, there were cell extracts of recombinant Lactococcus lactis expressing mouse RANKL (mRANKL) used to evaluate its biological activity in mice. Mice were divided into three groups that were fed phosphate-buffered saline (PBS), wild-type L. lactis IL1403 (WT_CE), and recombinant L. lactis expressing mRANKL (mRANKL_CE). The small intestinal transcriptome and fecal microbiome were then profiled. The biological activity of mRANKL_CE was confirmed by studying RANK-RANKL signaling in vitro and in vivo. For small intestinal transcriptome, differentially expressed genes (DEGs) were identified in the mRANKL_CE group, and no DEGs were found in the WT_CE group. In the PBS vs. mRANKL_CE gene enrichment analysis, upregulated genes were enriched for heat shock protein binding, regulation of bone resorption, and calcium ion binding. In the gut microbiome analysis, there were no critical changes among the three groups. However, Lactobacillus and Sphingomonas were more abundant in the mRANKL_CE group than in the other two groups. Our results indicate that cell extracts of mRANKL_CE can play an effective role without a significant impact on the intestine. This strategy may be useful for the development of protein drugs.

Keywords: Lactococcus lactis; cell extracts; microbiome; receptor activator of NF-kB ligand (RANKL); transcriptome.

Fig. 1.. Schematic diagram for construction of recombinant mRANKL expression vector system and detection of target gene expression.

(A) Plasmid vector system. (B) Western blot for detecting mRANKL from cell extracts (intracellular) and cell-free culture supernatants (extracellular), lane 1: Cell extracts of Lactococcus lactis IL1403; lane 2: Cell-free culture supernatant of L. lactis IL1403; lane 3: Cell extracts of recombinant L. lactis IL1403; lane 4: Cell-free culture supernatant of recombinant L. lactis IL1403. mRANKL, mouse receptor activator of NF-kB ligand.

Fig. 2.. Venn diagram of co-up and co-downregulated genes.

(A) Upregulated genes that control (PBS) versus treatment (WT_CE and mRANKL_CE). (B) Downregulated genes that control (PBS) versus treatment (WT_CE and mRANKL_CE). mRANKL_CE, recombinant Lactococcus lactis expressing mouse receptor activator of NF-kB ligand; WT_CE, wild-type L. lactis IL1403; PBS, phosphate buffered saline.

Fig. 3.. The relative abundance (%) of genus among that PBS, WT_CE and mRANKL_CE three groups.

(A) Lactococcus. (B) Lactobacillus. (C) Sphingomonas. (D) Bacteroides. For significance tests, a one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test were used. PBS, phosphate buffered saline; WT_CE, wild-type Lactococcus lactis IL1403; mRANKL_CE, recombinant L. lactis expressing mouse receptor activator of NF-kB ligand.