Could plasmids and conjugation mechanisms be used against antibiotic-resistant bacteria? [closed]

Question

I'm surprised no one has mentioned something like this.

Plasmids are often exchanged between bacteria, sometimes through conjugation. In particular, conjugation could be considered an "open-port" vulnerability.

Intuitively, there are several schemes that exploit plasmids and bacterial conjugation that could form alternatives to antibiotics:

1: Pwnage by Conjugation

A specially engineered nanoscale capsule or specially rigged bacterial cell is programmed to call out to target bacteria for conjugation. When the conjugation tunnel is opened, the capsule or rigged bacterial cell floods the target bacterium with toxins or malicious RNA designed to sabotage it.

2: Fake Conjugation

A specially designed novel antibiotic contains a conjugation-signaling structure. When a bacterium docks with the antibiotic molecule, the antibiotic irreversibly latches on and tears a massive hole in the bacterium from the inside.

3: Backdoor Plasmids

Plasmids that confer significant advantages are isolated from naturally-occuring bacteria and a special "crash gene" is added to the plasmids. The modified plasmids are implanted back into bacteria that are known to spread them around.

The crash gene's promoter listens for a small set of chemical cues. If it detects a certain threshold of 1 of these cues, it becomes active and sabotages the bacterium. The gene could code for a toxin that specifically damages bacteria. Alternately the promoter could be specifically designed so it never releases the chemical cue, forcing the bacterium to repeatedly produce a garbage produce and waste its resources.

4: Bad Neighbor Plasmid

This might be more difficult to build. A synthetic plasmid encodes 2 products: The first confers a significant benefit to prevent bacteria from discontinuing it through natural selection. The other product is periodically ("randomly") transcribed and vandalizes other plasmids, causing large numbers of nonsense mutations. A set of special markers on the synthetic plasmid protects it from vandalism by its own product. This could silently remove antibiotic resistance from bacterial populations and the bacteria have no way of "knowing" they've been sabotaged until it's too late.

Answer 1

If we assume all of these things could be practically done at least at some point in the future, here are the problems I see with the suggestions, and why they might not work (others may spot additional ones).

1 Translocation of a sabotaging 'agent' via the conjugation apparatus.

This basically already exists, look up the various secretion systems of bacteria. In particular, the Type 6 Secretion System does exactly this (though with a somewhat limited target range).

The first paragraph on wiki summarises:

The type VI secretion system (T6SS) is molecular machine used by a wide range of Gram-negative bacterial species to transport proteins from the interior (cytoplasm or cytosol) of a bacterial cell across the cellular envelope into an adjacent target cell.

The reason this wouldn't work is that bacteria have already evolved to deal with such mechanisms, and one we engineer is unlikely to fare much differently.

You can take it from me (as my PhD concerns exactly this!) that changing what the secretion systems translocate, while possible, is very difficult.

2 Phony conjugation

Antibiotics tend to be small molecules. They bind and dock with enzymes or substrates in order to inhibit some kind of activity typically, e.g. cell wall synthesis in the case of Beta-lactam antibiotics such as penicillin. I'm not sure how you imagine it would "tear a hole in the bacterium" really?

Related to the T6SS, are R-type Pyocins, which are co-opted bacteriophages that sort of do this. It requires no conjugation, and the currently proposed mechanism is that they either deliver a payload of toxins or molecules in to the cell by puncturing the exterior, or they simple create a pore in the membrane causing cellular depolarisation and death. As with phage and antibiotics however, these mechanisms can have resistance evolved against them.

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As an additional point, unless you could somehow guarantee that the conjugations were sabotaged or ineffectual in some way, promoting conjugation artificially is almost certainly going to aid the spread of resistance and virulence determinants, actually worsening the situation.

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3 A suicide plasmid

It is not trivial to 'confer significant advantages' on a cell. Most plasmids are maintained by amelorating a significant disadvantage that is often supplied artificially, e.g. auxotrophy or antibiotic selection. Given the 'choice' bacteria tend to kick out the plasmids unless they simply cannot afford to live without it. Maintaining the replicon is an expensive process in terms of cellular metabolism and resource utilisation.

'In the wild' the weaponised plasmid would likely not persist long enough to be translocated in to any target of interest in a significant enough proportion to have a real effect. The key is that and of these ideas mentioned cannot simply kill a single target cell, as they will not have an appreciable effect on the population. The bacteria will recombine or straight up remove the plasmid if it's in any way deleterious or even neutral to them, until they've 'corrected' the problem.

Addiction modules and toxin-antitoxin systems are common to see in natural plasmids, as with the other ideas mentioned. Bacteria can recombine out the 'poison' though, and eventually over time lose the plasmids. If the toxin is encoded on the chromosome, it's a little more robust.

4 Bad plasmids

See addiction modules and toxin-antitoxin systems above.

I'm afraid in general I think you're underestimating evolution!

Answer 2

Could plasmids and conjugation mechanisms be used against antibiotic-resistant bacteria?

Apparently yes. At least, it's plausible. The study is very new and, even though it was conducted in vivo, it was still under artificial conditions. We'll have to see what comes out of it.

Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing (‘addiction’) systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative ‘interference plasmids’ were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored.

The idea is that a natural, addictive plasmid conferring antibiotic resistance is displaced from a bacterial community by an incompatible plasmid expressing the appropriate antitoxin for the addiction system, and tetracycline resistance.

Replicon (solid circle), antitoxin and toxin genes (arrowhead, arrow) and antibiotic resistance genes (CTXR, orange and TETR, black solid blocks). Interference plasmid not excluded by entry exclusion system (EES) is incompatible (INC) with resident CTXR plasmid and is selected by TET.

In the top pathway, conjugation has not occurred and the bacterium retains the original plasmid. Tetracycline kills this population. In the bottom pathway, conjugation has occurred and, because of the incompatibility between the natural plasmid and the interfering plasmid, binary fission results in asymmetric segregation of them. Again, the natural addiction system is bypassed because the interfering plasmid expresses the antitoxin. The population containing the original plasmid is killed by tetracycline but that containing the interfering plasmid is resistant. However, because the this plasmid doesn't have the complete addiction system, it is lost over time in the absence of selection. Thus the resulting bacterial population is antibiotic susceptible.

You may ask why not just use tetracycline to kill all the cells in the first place. That's not the point! This is a proof of concept.

Purifying selection using antibiotics is not ideal, both because of the effects of the drug on other cells and because effective antibiotics for this purpose may be increasingly hard to identify in future. TETR bacteria are not uncommon in the human gut but TETR (or FOSR) populations that arise by plasmid acquisition will not only lose their TETR or FOSR plasmids spontaneously but are made immediately vulnerable to other commonly used antibiotics (e.g. GEN, CTX) in the process. A target plasmid that acquires FOSR by recombination with the interference plasmid will likely acquire the immediately adjacent antitoxin. An important future challenge is to develop interference plasmids with non-antibiotic selection but the inclusion of fosA3 also means that plasmid eradication is compatible with existing use of fosfomycin as a ‘rescue’ therapy of last resort.

Answer 3

Your ideas are not bad, but in all cases there are a few reasons that make them pretty much unusable in a real world (medical) application. Also none of your approaches solve the problem of resistance: bacteria can just become resistant to this new type of treatment (the bacteria using slightly different conjugation methods are immune and will have an evolutionary advantage).

1) This could work - but not today. You either need some sort of nanobots or very advanced genetic engineering to pull it off. While the technology to make such advanced 'killer machines' should be available within the next few centuries, the ethical question of 'letting them loose' into a human body or the environment will always remain very tricky.

2) I think this is too complicated. Antibiotic drugs are very small chemical molecules whereas the bacterial conjugation machinery is very large biological complex. Trying to make something bind the machinery and then make it able to kill the bacteria is superfluous - you could just make antibodies against the bacteria in the first place (they can also be coupled with effectors). Also it does in now solve the problem of evolving resistance

3/4) The added level of complexity does not help. Trying to trick bacteria with 'trojan horse' plasmids won't work. First of all evolution will work against the whole plasmid and just because it has one favourable gene does not been bacteria can't just get rid of it, if it would kill them otherwise. Secondary, mutations can act on the genes on the plasmid indecently so the bacteria can just shut down the part that's supposed to kill them and keep the rest.