Ribosome-Engineered Lacticaseibacillus rhamnosus Strain GG Exhibits Cell Surface Glyceraldehyde-3-Phosphate Dehydrogenase Accumulation and Enhanced Adhesion to Human Colonic Mucin

Abstract

Differences in individual host responses have emerged as an issue regarding the health benefits of probiotics. Here, we applied ribosome engineering (RE) technology, developed in an actinomycete study, to Lacticaseibacillus rhamnosus GG (LGG). RE can effectively enhance microbial potential by using antibiotics to induce spontaneous mutations in the ribosome and/or RNA polymerase. In this study, we identified eight types of streptomycin resistance mutations in the LGG rpsL gene, which encodes ribosomal protein S12. Notably, LGG harboring the K56N mutant (LGG-MT_K56N) expressed high levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the cell surface compared with the LGG wild type (LGG-WT). GAPDH plays a key role in colonic mucin adhesion. Indeed, LGG-MT_K56N significantly increased type A human colonic mucin adhesion compared to LGG-WT in experiments using the Biacore system. The ability to adhere to the colon is an important property of probiotics; thus, these results suggest that RE is an effective breeding strategy for probiotic lactic acid bacteria.IMPORTANCE We sought to apply ribosome engineering (RE) to probiotic lactic acid bacteria and to verify RE's impact. Here, we showed that one mutant of RE Lacticaseibacillus rhamnosus GG (LGG-MT_K56N) bore a GAPDH on the cell surface; the GAPDH was exported via an ABC transporter. Compared to the wild-type parent, LGG-MT_K56N adhered more strongly to human colonic mucin and exhibited a distinct cell size and shape. These findings demonstrate that RE in LGG-MT_K56N yielded dramatic changes in protein synthesis, protein transport, and cell morphology and affected adherence to human colonic mucin.

Keywords: GAPDH; Lacticaseibacillus rhamnosus GG; human colonic mucin; ribosome engineering.

FIG 1

(A) The secreted proteins present in culture supernatants (spent medium) were analyzed by SDS-PAGE with CBB staining. The arrow indicates the 40-kDa protein (X) that appears to accumulate in the LGG-MTs but not in the parent. A representative image from one of three independent experiments is shown. (B) Densitometric analysis of gels from three independent SDS-PAGE experiments was used to quantitate protein X production; protein levels are normalized to that in the LGG-WT parent. Letters (i.e., a, b, and c) represent significant differences (P < 0.05) by a two-tailed one-way ANOVA. Data are means and SD (n = 3). n.d., not detected. (C) SEM photographs of LGG-WT and LGG-MT_K56N cells. Images on the right are greater magnifications of the indicated areas in the middle panels. Bar = 5 μm. (D) Growth curves of LGG-WT and LGG-MT_K56N.

FIG 2

(A) Scheme of the sample preparation procedure. (B and C) GAPDH production by LGG-WT and LGG-MT_K56N was determined by Western blot analysis of bacterial cell lysates (B) or supernatants (spent culture medium) (C). (D and E) Time-dependent display of GAPDH in LGG-WT and LGG-MT_K56N was monitored at 8, 12, 24, and 48 h in supernatants (spent culture medium) (D) and in BLF (obtained by rinsing the bacterial cell pellet with PBS at pH 7.3) (E). Arrows indicate GAPDH (B, C, D, and E). (F and G) Densitometric analysis of Western blots from three independent experiments was used to quantity GAPDH. Letters (i.e., a, b, and c) represent significant differences (P < 0.05) by two-tailed one-way ANOVA. Data are means and SD (n = 3). CELL, bacterial cells; SUP, culture supernatant; BLF, bacterial lavage fluid.

FIG 3

BLFs obtained by rinsing of cell pellets with PBS (at pH 4.2 or 7.3) were analyzed by SDS-PAGE (A), GAPDH Western blotting (B and C), and an EVOS FL imaging system (E). Arrows indicate GAPDH (B and C). Densitometric analysis of Western blots from three independent experiments was used to quantify GAPDH (D). Alexa Fluor 488-GAPDH-stained LGG was analyzed under visible light and fluorescence and as a merged image with an FL imaging system (E). Bar = 100 μm. Fluorescence index values are displayed relative to those obtained from LGG-WT (F). Statistical analysis was performed by a Student's t test. Data are means and SD (n = 3). BLF, bacterial lavage fluid.

FIG 4

Test of binding of LGG-WT and LGG-MT_K56N to human colonic mucin using Biacore analysis. The adhesion values are expressed as RUs (resonance units) at 200 s after cessation of sample addition. Data are expressed as means and SD (n = 3).