Extraction of Peptidoglycan from L. paracasei subp. Paracasei X12 and Its Preliminary Mechanisms of Inducing Immunogenic Cell Death in HT-29 Cells

Abstract

L. paracasei subp. paracasei X12 was previously isolated from a Chinese traditional fermented cheese with anticancer activities and probiotic potential. Herein, the integral peptidoglycan (X12-PG) was extracted by a modified trichloroacetic acid (TCA) method. X12-PG contained the four representative amino acids Asp, Glu, Ala and Lys, and displayed the similar lysozyme sensitivity, UV-visible scanning spectrum and molecular weight as the peptidoglycan standard. X12-PG could induce the production of apoptotic bodies observed by transmission electron microscopy (TEM). X12-PG could significantly induced the translocation of calreticulin (CRT) and the release of high mobility group box 1 protein (HMGB1), the two notable hallmarks of immunogenic cell death (ICD), with the endoplastic reticulum (ER) damaged and subsequently intracellular [Ca(2+)] elevated. Our findings implied that X12-PG could induce the ICD of HT-29 cells through targeting at the ER. The present results may enlighten the prospect of probiotics in the prevention of colon cancer.

Keywords: calcium; calreticulin; immunogenic cell death; peptidoglycan.

Figure 1

Structural features of the integral peptidoglycan isolated from L. paracasei subp. paracasei X12 (X12-PG) and the L. paracasei subp. paracasei X12 bacteria (X12) observed by transmission electron microscope (TEM). (A,B) The structure of X12 (×20,000, ×40,000); (C,D) The cylindrical structure of internal X12-PG (×20,000, ×40,000).

Figure 2

Quantitative and qualitative analysis of X12-PG. (A) The degradation models of X12-PG and X12 when exposed to lysozyme. A_{450 nm} of X12-PG declined faster than that of X12; (B) The UV-visible scanning spectrum of X12-PG and peptidoglycan standard. The standards and samples were scanned between 190 and 700 nm in a UV/VIS spectrophotometer; and (C) The molecular weight (kDa) of X12-PG quantified by SDS-PAGE. The gels were stained with Coomassie Brilliant Blue. The molecular weight was 14 kDa, corresponding to the peptidoglycan standard. Each graph represents the average of more than three replications.

Figure 3

Apoptosis of HT-29 cells after treatment of X12-PG. (A–D) were respectively treated with 0, 400, 800, 1600 μg/mL X12-PG for 48 h. HT-29 cells were observed by TEM (×6000). The arrows indicate the apoptotic bodies.

Figure 4

Structural damages of endoplasmic reticulum (ER) after treatment of X12-PG. (A–D) were respectively treated with 0, 400, 800, 1600 μg/mL X12-PG for 48 h. Cells were observed by TEM (×20,000). The encircled regions show the location of ER.

Figure 5

Calreticulin (CRT) exposure detected by immunofluoroscence assay after treatment of X12-PG. (A–D) were respectively treated with 0, 400, 800, 1600 μg/mL X12-PG for 48 h, then incubated with primary antibody (Anti-Calreticulin, diluted 1:200 in blocking buffer) and detected with the FITC-labeled secondary antibody. Fluorescence intensity was visualized by immunofluorescence microscopy (×200).

Figure 6

CRT exposure detected by flow cytometry after treatment of X12-PG. Prior to analysis, cells were respectively treated with 0, 400, 800, 1600 μg/mL (A–D) X12-PG for 48 h, then harvested and labeled with FITC. The concentration of CRT on cell membrane was expressed as mean fluorescence intensity (MFI) which has excluded background (Isotype-matched IgG was used as a control: Data not shown). (Error bars are mean ± SD, ** p < 0.01 versus control)

Figure 7

Intracellular Ca²⁺ concentration ([Ca²⁺]) after treatment of X12-PG. (A–D) were respectively treated with 0, 400, 800, 1600 μg/mL X12-PG for 48 h. Cytosolic [Ca²⁺] was increased in a dose-dependent manner after exposure to X12-PG. HT-29 cells were loaded with Fluo-3/AM and analyzed by flow cytometry. Fluo-3/AM was excited at the 488 nm line of an argon laser and the fluorescence intensity was measured at an emission wavelength 530 nm. [Ca²⁺] was expressed as mean fluorescence intensity (MFI). (Error bars are mean ± SD, * p < 0.05 versus control, ** p < 0.01 versus control).

Figure 8

Properties of immunogenic cell death (ICD) induced by X12-PG in HT-29 cells. As a result of endoplasmic reticulum stress (stimulated by X12-PG), cancer cells expose CRT on their plasma membrane at a pre-apoptotic stage. This facilitates the recruitment of dendritic cells (DCs) into the tumor bed, the engulfment of tumor antigens by DCs (stimulated by CRT), and optimal antigen presentation to T cells (stimulated by HMGB1). CRT served as an “eat-me” signal, and CRT exposure may be regulated by cytosolic [Ca²⁺].