Established in 1979, Shenzhen transformed
dramatically from a tiny town into a modern metropolis within 41 years.
Rapid urbanization brought plenty of challenges including water
pollution and public health risk. To deal with this uncertain future,
Shenzhen has established a program called the “Shenzhen Water Strategy”,
which includes the protection of water source, the recovery of water,
the guarantee of water safety, and the enhancement of visible water.
Sewage treatment has been regarded as one of
the most essential parts of Shenzhen water governing strategy, and
biofilm, which is composed of numerous microbes and multiple types of
organic matters, plays an important role in the bioremediation of
sewage. However, in this process, a common problem often arises that the
pipelines of sewage treatment plants are easily blocked as a result of
the overgrowth of biofilm. One proposed solution is to constantly
replace the blocked pipeline. However, this method is not only too
costly for large-scale applications but will also completely destroy the
structure of biofilm, which can hardly be regenerated in a short period
of time. Among all microbes existing in the biofilm, Pseudomonas
putida contribute vastly to waste processing due to its complex
metabolism. Therefore, we are inspired to design a system through which
we can utilize phage infection to reduce the density of overgrown
biofilm and then stop the excessive damage of phage infection using
drugs that can protect bacteria from phage infection.
The conserved Aca protein in
Pseudomonas phage acts on the CRISPR-Cas system
in Pseudomonas. The CRISPR-Cas system is an
acquired immune system in bacteria and archaea that
helps them resist the invasion of foreign genetic
material, such as bacteriophage, by integrating the
foreign DNA sequence into its own genome1.
Phages express anti-CRISPR (Acr) proteins to inhibit the
CRISPR-Cas system, preventing their own genomes from
being destroyed. acr genes encode proteins that
bind to acr-promoter and regulate adjacent
anti-CRISPR-associated (aca) genes1.
Through binding to acr-promoter, Aca proteins
could act as crucial inhibitors of the transcription of
anti-CRISPR genes1. After the DNA of phage is
injected into host cells, immediate high-level
transcription of acr gene helps phage to
survive under the pressure of the CRISPR-Cas system in
host cells, but such high transcription level of
acr gene induces dysregulation of other
critical genes, leading to the death of
phage1. With the presence of Aca proteins,
transcription of acr gene is downregulated and
phage can express essential genes for
survival1. In all, if the function of Aca
proteins can be artificially inhibited via small
molecule compounds, we will be able to increase the
defense system of the bacterium against phages.
Figure 1. The working mechanism of the
anti-CRISPR system
With the conserved aca genes in
Pseudomonas phage, we are inspired to find an effective drug to
protect Pseudomonas, an important bacterium in the sewage
treatment system, from excessive phage infection in our expected system.
Project Description
As the binding between Aca1 protein and acr-promoter
plays an essential role in the survival of Pseudomonas phage,
we aimed to find a drug that could disrupt the binding between Aca1
protein and acr-promoter. In this way, we can combine phages
that are able to reduce overgrown Pseudomonas and that drug
together to regulate the state of Pseudomonas biofilm.
Based on the structure of Aca1 binding with acr-promoter,
we obtained some inhibitor candidates using the Target-Directed
Screening (TDS) technique. Then, we designed a two-plasmid system in
E. coli to simulated the anti-CRISPR operon with the
aca1 gene with egfp as a reporter. With such a system,
we were able to screen potential inhibitors among those candidates.
Next, we modified potential inhibitors to further improve its
efficiency. Finally, we designed experiments to test the efficiency of
the optimized inhibitor in protecting Pseudomonas from phage infection,
but such an experiment remained unaccomplished because COVID-19 forced
us to delay our laboratory work.
Reference
Stanley, S. Y., et al. Anti-CRISPR-Associated Proteins Are
Crucial Repressors of Anti-CRISPR Transcription. Cell178, 1452-1464.e1413,
doi:https://doi.org/10.1016/j.cell.2019.07.046 (2019).
