Team:Paris Bettencourt/Project/Sabotage

   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. We demonstrated the validity of this trojan horse strategy by converting back to a sensitive state populations of bacteria initially resistant to antibiotics like chloramphenicol or kanamycin.

   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 them with tailormade small RNAs. In order to design our silencing device (small RNA) we used the protocol described in Na et al 2013 and inserted a 24bp sequence complementary to the RBS area of the target genes inside the scaffold sequence that allow the stabilization of the hybridization through recruitment of the protein Hfq. We designed three sRNA respectively targeting Kanamycine resistance gene located on a pCOLA Duet Vector, Chloramphenicol resistance on pACYC Duet Vector and lac Z on E. coli's chromosome.

   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 immediately 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.

  



Design of a sequential killing strategy
To perform our proof of concept experiments, our targeted genes were different antibiotic resistance genes on commercial plasmids that were transformed in MG1655 cells. This model of plasmid bearing resistance was chosen because it was more close to real life situation. Indeed resitance genes are mostly located on plasmids. The experimental set up consisted of a sequential strategy ; a phage infection followed by a treatment with antibiotics. The phage infection leads to the silencing of the antibiotic-resistance genes. After the silencing is complete, antibiotics are used to kill the cells to prove the efficiency of the silencing and the possibility of using this strategy as a therapy.

   We received the phagemid Helper system used in this project from Monica Ortiz. it consists of a helper plasmid, M13K07 bearing Kanamycine resistance and a phagemid, Litmus-28 bearing GFP and a Ampiciline resistance. Using those different markers, we characterized the system by infecting the cells with a ratio of phage of 1 to 100 and plating appropriate dilutions on different medias (LB,Kan, Amp, Kan/Amp). We show that the phagemid is spread a thousand fold more than the helper plasmid. There are as many cells infected with the helper as cells infected by both helper and phagemid, that is to say, phages producing cells. THose results show the validity of using this system to spread a genetic element in a bacterial population. Most of the cells bear the desired genetic element and the spread is carried on by a minor quantity of cells producing the vector.

   We used the a phagemid bearing the sRNA anti-Cm we designed to infect a population of chloramphenicol resistant cells. By applying the sequential killing strategy described in Figure 3, we efficiently killed 99,1% of the bacterial population (chloramphenicol concentration 500ug/ul). We tested this strategy with different concentrations of chloramphenicol. We show that the rise of chloramphenicol concentration directly lead to drastic reduction of the survival of the infected cells.

   In order to show the general application of our system, we then applied the same strategy on MG1665 transformed with pCOLA Duet. Those kanamycine resistant cells were infected with phages bearing our sRNA anti Kan. 99,87% of the population was killed (standard deviation 0,23 %) at 500mg/mL of kanamycine.

   One of the main limit of our system could be the development of resistances. Indeed, there are at least two ways to become resistant to our system: by avoiding infection or by developing a resistance to the silencing. In order to get a better idea of what is the main cause of resistance we screened the survivor colonies for GFP expression. GFP positive colonies correspond to infected cells with malfunctioning silencing. We show that 70% of resistance is caused by a resistance towards the sRNA. This resistance could be due to a mismatch between the sRNA sequence and the target sequence resulting from a single point mutation.

   Several sRNA on the same phagemid : Although our silencing small RNAs were assembled in phagemids with Gibson assembly, the Biobricks cutting sites included in our BBG adapter method provide full modularity. In our case a direct application is the assembly of multiple silencing modules (promotor+sRNA+terminator) on a single phagemid vector. As the main clinical problem with bacteria is multi or even pan-resistance this modularity provide an interesting strategy to explore as it allows simultaneous downregulation of multiple target genes.

   Building a library of sRNA : As our silencing strategy rely on base pair complementarity, a main concern from the clinical point of view would be the fast emergence of resistances to silencing through single point mutations. Indeed even if the majority of mutations that would decrease the efficiency of the silencing would also lead to an impaired or non functionnal protective protein, some neutral mutations will still be made possible by the redundancy of the genetic code. To tackle this issue an interesting solution would be to build a library of small RNA containing single point mutation on those particular neutral positions and load it on our phagemids. By being one move ahead of evolution we expect that such an anticipating strategy would considerably slow down the emergence of resistance to silencing.