[Effect on Muc2 gene knockdown in Ht29 cells by CRISPR/Cas9 on probiotics-mediated inhibition of E.coli K1 adhesion and invasion].
OBJECTIVE
To investigate the effects of Lactobacillus rhamnosus GG (LGG) for inhibiting E.coli K1 (E44) adhesion and invasion of an intestinal epithelial cell model with Muc2 gene knockdown established using CRISPR-Cas9 system.


METHODS
Two 20-25 bp sgRNAs targeting Muc2 were chemically synthesized to construct CRISPR expression vectors for transfection in wild-type human colonic cancer cell line Ht29. The efficiency of Muc2 knockdown was determined using Western blotting. After assessment of the viability and proliferation of the transfected cells with MTT assay, we evaluated the effects of the probiotics against E44 adhesion and invasion of the cells through a competitive exclusion assay.


RESULTS
Transfection of the cells with Lenticrisprv2 plasmid vectors resulted in a cell line with stable Muc2 knockdown by 81%. The inhibitory effects of probiotics against E44 adhesion and invasion of the transfected cells were markedly attenuated, and the relative adhesion and invasion rates of E44 were 72.23% (P<0.05) and 81.49% (P<0.05), respectively.


CONCLUSIONS
Muc2 knockdown causes attenuation of the inhibitory effects of probiotics against E44 adhesion and invasion of the intestinal epithelial cells, suggesting that up-regulation of Muc2 may serve as an important mechanism for the probiotics to reinforce the intestinal barrier and antagonize the pathogenic bacteria, which sheds light on a new strategy for prevention and treatment of bacterial intestinal infections.
Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine1, Southern Medical University, Guangzhou 510515, China. E-mail: qiujiawen_gz@126.com.
Jia-Wen
JW
Qiu
Xiao-Long
XL
He
Bao
B
Zhang
Lei
L
DU
Qing
Q
Zeng
Sen
S
Li
Huan-Huan
HH
Xiong
Min
M
Long
Jun
J
Luo
Hong
H
Cao
D001422:Bacterial Adhesion; D064113:CRISPR-Cas Systems; D004847:Epithelial Cells / Q000166:cytology* / Q000382:microbiology; D004926:Escherichia coli / Q000472:pathogenicity*; D055785:Gene Knockdown Techniques; D019073:HT29 Cells; D006801:Humans; D007422:Intestines / Q000166:cytology; D052201:Lacticaseibacillus rhamnosus; D055262:Mucin-2 / Q000235:genetics*; D019936:Probiotics; D014162:Transfection; D015854:Up-Regulation
C524625:MUC2 protein, human; D055262:Mucin-2