Overview

As resistance against antibiotics are growing and pharmaceutical pipelines are drying up, we decided to investigate a new strategy based on specific silencing of the genes responsible for resistance through bioengineered stealth bacteriophages. The silencing of the genes is obtained with a simple and modular system of tailormade small RNAs and the spreading of this construct in a bacterial population is based on an autonomous phagemid/helper phage system.

Objective

Our objective is to make an antibiotic-resistant bacterial population sensitive again to those same antibiotics.

Design

Design a silencing device

Most resistance against antibiotics come from specific proteins that bacteria can produce such as efflux pump that extract the antibiotics from the cell or enzymes that quickly metabolize the antibiotic molecules to make them inactive. We choose a straight forward strategy to force bacteria to stop producing those proteins by targeting the mRNA coding for those proteins with tailormade small RNAs.

This simple silencing system is driven by the complementarity between a small RNA and the ribosome binding site area of a given mRNA. As the small RNA will hybridize with this key area of the mRNA it will prevent the binding of the ribosome and therefore prevent translation into protein. Moreover the small RNA we are using have a consensus sequence for recruiting Hfq protein which result in the stabilization of the hybridation between our small RNA and its target mRNA thus increasing the silencing efficiency.

Design a genetic element that spread in a population

In nature, non-lytic filamentous bacteriophages are quite good at spreading genetic elements in bacterial populations and we thus imediately thought about them as vectors of choice. However being infected by a phage represent a huge burden for an individual bacteria which we thought would be detrimental for our construct to be able to maintain itself long enough in a population for this system to be clinically relevant. Therefore we choose to use a phagemid/helper system which is composed of two mobile interacting elements. The « heavy » elements of this system is an M13 phage which genome contains an altered packaging signal thus reducing the probability of packaging in a protein capside and escaping from the cell. Its role is to produce capside proteins which will in fact be used by a « light » element called phagemid which is a normal plasmid harboring a packaging signal. As a result a cell containing both elements will produce and secrete a lot of phagemid encapsulated plus some helper phages from time to time. We expect such a system to infect with light elements the majority of cells in a population and to be able to spread and maintain itself thanks to a small number of coinfected cells harboring both light (phagemid) and heavy (helper phage) elements transforming them into phage-producing cells.

Our silencing device will be loaded on the light element in order to spread it efficiently. As the post-transcriptional regulation we are using only rely on RNA and doesn’t require synthesis of any protein, its cost for the cell will be very low. Moreover producing protective proteins against antibiotics is costly for the cell and lower its fitness in an antibiotic free environment, we thus expect our silencing device to be a temporary relief for the infected cell which should avoid early counterselection dynamics.

A non counter selected system Phagemid system

Sequential method to kill

Results

Characterization of the phagemid system

Sequential killing

Generalization: results for Kan

Origin of resistance

Conclusion and perspectives

Several sRNA on the same plasmid :

Building a library of sRNA :

Possible Applications :