A novel genetic screening method for antibiotic-related intestinal diseases!

Difficult Clostridium difficile infection (CDI) is a major cause of antibiotic-associated intestinal disease and can lead to severe diarrhea and fatal pseudomembranous colitis
.
TcdB, one of the basic virulence factors secreted by this bacterium, induces apoptosis
in host cells through a little-known mechanism.

This article is the original of Translational Medicine Network, please indicate the source for reprinting

Author: kope

According to recent statistics from the U.
S.
Centers for Disease Control and Prevention, Clostridium difficile caused about 230,000 cases, including 12,800 deaths, in 2017, resulting in about $100 million in treatment costs
.
As a global healthcare issue, there is an urgent need to understand the mechanisms of infection and develop effective therapies for CDI
.

Antibiotic-resistant bacteria

 01 

Clostridium difficile is an antibiotic-resistant bacterium that is prone to Clostridium difficile infection (CDI)
when antibiotics are used, especially in excess.
Typically, Clostridium difficile enters the body through the mouth and remains dormant until it becomes infected in the large intestine and releases toxins, destroying tissues
.
These toxins kill cells, which can lead to diarrhea, abdominal pain, fever, and in extreme cases, fatal
.

Clostridium difficile infection is mainly mediated by two exotoxins, Clostridium difficile toxins A and B (TcdA and TcdB).

Both toxins are cytotoxic to cells, but TcdB causes more cell death and leads to severe disease
.
Focusing on TcdB, many studies have shown that TcdB uses a multi-step strategy to intoxicate host cells
.
Enters the cell by binding to receptors on the surface of the host intestine and is then internalized
by receptor-mediated endocytosis.
TcdB can use multiple receptors simultaneously or have specific receptors
for different cell types.
Cytotoxic attacks may disrupt the intestinal epithelial barrier, leading to fluid buildup in the intestinal lumen, tissue damage, and severe inflammation
.
The important role of connexant plate globin (JUP), γ-catenin in epithelial apoptosis and the potential
to inhibit high-mobility frame 1 (HMGB1) for CDI therapy were first reported.

Novel genetic screening methods

 02 

Dr.
Yingxue Li, a member of the study and a lecturer in the Biology Laboratory at Duke Kunshan University's Department of Natural and Applied Sciences, described the experiment: "We have pioneered a new genetic screening method that uses bacterial cells to create thousands of precisely genetically targeted RNA molecules, known as small interfering RNAs (siRNAs).

We then used high-throughput methods to test these siRNAs and used biochemical assays and microscopy to understand the role
of siRNAs in cell death induced by Clostridium difficile toxin.
Finally, we preliminarily screened the most likely candidate genes and verified through various laboratory techniques whether they had an effect
in Clostridium difficile toxin-induced cell damage.
"The benefit of this method is that it can reduce costs and produce efficient small interfering RNAs
simply and cheaply.

Identification of host factors
involved in TcdB-induced apoptotic cell death using a novel RNAi screening method.
Caco-2 cells were selected as a model for the colonic epithelium, which is highly sensitive
to TcdB-induced apoptosis.
A bacterial generated RNAi library was created, customized to Caco-2 cells based on pro-siRNA technology, and used caspase 3/7 and cell viability assays as readouts
for RNAi screening.
Screening revealed multiple host factors
involved in TcdB-induced apoptosis.

To determine whether HMGB1 is also involved in TcdB-induced and mitochondria-mediated apoptosis, the researchers performed the same cytochrome c release assay
in Caco-2 cells transfected with siRNA targeting HMGB1 (siHMGB1-1).
The expression of HMGB1 in cells transfected with siHMGB1-1 is almost completely inhibited
.
These data suggest that both JUP and HMGB1 are involved in TcdB-induced and mitochondria-dependent apoptosis pathways
.
HMGB1 plays a crucial role in TcdB-induced apoptosis and can be used as a potential therapeutic target for the treatment of
CDI.

Mature inhibitors

 03 

Liquirhizin (also known as glycyrrhizic acid) acts as a maturation inhibitor of HMGB1 and is used to determine whether glycyrrhizin treatment can alleviate TcdB-induced apoptosis
.
Licotropin is a plant glycoside
extracted from the root of the licorice plant.
It has two isomers: 18β-glycyrrhizin and 18α-glycyrrhizin, derived from 18β-glycyrrhizin
by isomerization.
The injection uses glycyrrhizin as the main active ingredient and glycine and cysteine hydrochloride as additional ingredients to improve the stability, solubility and safety
of the drug.
This study used purified glycyrrhizin and CGI to evaluate their protective effects
against TcdB.

Both cell lines and animal experiments have shown that glycyrrhizin pretreatment protects the colonic epithelium from TcdB-induced damage
.
Liquirhizin pretreatment significantly reduced TcdB-induced cell rounding and cell death
.
These findings support HMGB1 as a potential drug target for the treatment of CDI
with the approved drug of the HMGB1 inhibitor glycyrrhizin.

Resources:

https://doi.
org/10.
1128/mbio.
01849-22

Note: This article is intended to introduce the progress of medical research and cannot be used as a reference
for treatment options.
If you need health guidance, please go to a regular hospital
.