Dr Clare Kirkpatrick’s research has led to the discovery of a bacterial toxin-antitoxin system with unique features. This information can be used in future studies to identify novel molecular pathways in bacteria which can be targeted by new antibiotics.

Antibiotic resistance is a growing concern for microbiologists and poses an increasingly serious health problem globally. Following the discovery of antibiotics, starting with the isolation of penicillin in 1928 by Alexander Fleming, their use became increasingly widespread around the end of the second world war. The number of highly antibiotic resistant strains of bacteria has increased concomitantly ever since, leading to the emergence of MRSA, to give one example. Before doctors and scientists understood the potentially catastrophic consequences of their overuse, the use of antibiotics was largely uncontrolled until recently. In many parts of the world it remains unregulated, and antibiotics can be bought freely over the counter. The number of new antibiotic classes being discovered has also slowed down dramatically, with almost all of the antibiotics currently in use having been discovered prior to the 1990s. Without new classes of antibiotics, the risk of the emergence of pathogenic strains of bacteria which show resistance to all known antibiotics, becomes increasingly likely over time.

The issues surrounding antibiotic resistance are highly complex and require a sustained effort worldwide from politicians, the pharmaceutical industry and scientists in order to be resolved. Failure to solve this problem, in the worst case scenario, could lead to a situation where bacterial infections which would be considered trivial by today’s standards, could become incurable and lethal in the near future. Even now, some hospital-acquired infections by the most resistant strains can fall into this category.

Scientists such as Dr Clare Kirkpatrick, who conduct research into the molecular pathways within bacteria, are at the forefront of this research effort, by defining potential new targets and modes of activity for antibiotics. ‘Development of new antibiotics is unlikely to ever be a source of significant revenue for drug companies, very little industry resources have been focused on it and the rate at which new antibiotics come to the clinic is very low’, Dr Kirkpatrick tells Scientia. She believes therefore, that academia is a more likely setting for generating knowledge in this area, rather than industry, which by its nature remains profit-oriented. She also believes that since the number of known antibiotic targets within bacteria is so low, new potential targets and pathways may need to be discovered before novel antibiotics can be found. New molecular targets may also help to teach us how existing antibiotics can be used more effectively in different combinations.

The HigBA toxin-antitoxin system

Working under the supervision of Prof Patrick Viollier at the University of Geneva, Dr Kirkpatrick helped to discover a novel regulatory pathway within Caulobacter crescentus, which was published in the prestigious journal, Nature Microbiology. The pathway involved a set of proteins constituting a ‘toxin-antitoxin system’ (TAS), in which a set of connected genes code for proteins which have distinct functions, one as a poison and the other as its antidote. The antitoxin part of the system generally works by binding to the toxin protein and inhibiting its function. Sometimes this might be solely to ensure that the two genes are passed on to the next generation, where a cell divides in two, with one new cell retaining genes for both proteins and the other losing them. In this situation, the toxin is generally more stable than the antitoxin in the new cell, so when the antitoxin eventually degrades, all that remains is the toxin and the cell is killed. Over the whole bacterial population, this ensures the proliferation of the two genes. Another purpose for TASs, is to cause bacterial cells to enter a dormant state under stressful conditions. This means that the only remaining cells will be the ‘persister cells’ which tolerate the stress conditions or antibiotic in question without needing to acquire genetic resistance mutations. Scientists have been considering using TASs for some time, in the fight against antibiotic resistance, but depending on the specific TAS, the toxin may either kill the cell or place it into a dormant state, during which it is insensitive to antibiotics, even at high doses. When these cells awaken from dormancy, for example when a patient finishes a course of antibiotics, they are just as infective as before.

One such system is called HigBA, where HigA is the antitoxin, working by inhibiting the activity of the toxin and also by preventing the ‘reading’ of the gene which codes for it (known as transcriptional repression). The toxin, HigB, is a type of enzyme which targets and breaks down RNA, a molecule similar to DNA which is required for genes to be read by the cell and coded into protein.

To give an analogy for this process, if the genome (made up of DNA) is a library, then RNA could be thought of as photocopies of particular pages, giving instructions for building different things, which would be proteins. Each gene will produce specific RNA molecules which can be read by the cell’s protein-producing machinery. If the RNA is degraded, the protein cannot be produced. One of the key features of this particular system, which makes it such a novel discovery is that unlike previously described TASs, the HigBA system targets particular RNA molecules, representing specific genes, rather than just degrading every bit of RNA available. The system is also unique in that it appears to regulate the way the cell grows and divides (the cell cycle), and responds only to a particular and highly specific kind of stress: DNA damage.

HigBA and the DNA damage response

Before Dr Kirkpatrick and her colleagues learned how HigBA responds to the presence of DNA damage, they were looking in a different direction, at a mutated Caulobacter containing a defective gene for TipN. TipN is a protein involved in defining cell polarity (determining which end of the cell is the front and which is the back) by driving the placement of proteins essential for assembly of the flagellum (which could be described as the cell’s propeller, required for motility). These polarity mutants displayed the unexpected property of increased sensitivity to the antibiotic nalidixic acid. This antibiotic normally slows the rate of bacterial growth by inhibiting the cells from replicating their genomes, which is required prior to cell division. This seemed to happen in the TipNdefective mutant even though the known target of nalidixic acid in Caulobacter has a natural mutation making it resistant to it. The team discovered that this curious situation was caused by nalidixic acid dislodging a transcriptional repressor, allowing the expression of a particular efflux pump protein complex (the function of these proteins being to selectively pump specific molecules, such as antibiotics, out of the cell). The mutant lacking TipN did not tolerate increased expression of this efflux pump, for reasons that are still unknown. HigBA was found to be involved in this process, as the toxin, HigB, decreases the expression of the efflux pump, by specifically targeting its RNA and degrading it. If the antitoxin, HigA, is mutated to become nonfunctional, the toxin is set free and the cell’s resistance to nalidixic acid is partially restored. The toxin, therefore, provides increased tolerance to this antibiotic by reducing expression of the efflux pump, and actually promotes bacterial cell growth, rather than inhibiting it in this situation. Again, this is unusual as efflux pumps would usually confer resistance, by pumping antibiotics out of the cell, rather than increasing antibiotic sensitivity.

Interestingly, the opposite is true during the DNA damage response, where bacteria without a functional HigB toxin gene show increased resistance to DNA damage-inducing agents such as mitomycin C and ciprofloxacin. This was discovered by testing the TipN-defective mutant’s growth in the presence of a chemical library containing a wide range of antibiotics, to search for others which, like nalidixic acid, were specifically inhibitory for this mutant. Two other antibiotics with these properties were found, but surprisingly the HigB toxin did not protect against them. Because they both belonged to the family of quinolone antibiotics, that block DNA replication, these findings were also tested in cells which are mutated to constantly behave as they do in the presence of DNA damage, by mutation of a gene called LexA. This experiment showed that HigB was only protective if the antibiotic did not cause DNA damage; otherwise it contributed to cell death. Indeed, this is what makes the HigBA system unique. Other TASs seem to work in concert, responding to general stress to the cell, whereas the HigBA system seems to respond specifically to stress induced by DNA damage. The HigBA system is involved further still in the DNA damage response pathway. The LexA gene, it turned out, explained why it is even possible to produce mutants which lack a functional HigA gene. Usually, in a TAS such as this one, disrupting the function of the antitoxin element of the system leads to inevitable cell death resulting from unregulated activity of the toxin, which is pretty much the point of these systems. In the HigBA system, LexA binds to the HigBA gene in the same way that HigA does, by performing transcriptional repression. Only in the presence of DNA damage, or when both LexA and HigA are deleted, is the toxin fully derepressed and allowed to kill the cell.

The influence of HigBA on the cell cycle

Although HigBA certainly is involved in the DNA damage response, that does not appear to be its sole function, with the system also having a role in regulating the cell cycle. The cell cycle is the highly complex ‘program’, involving a large number of molecular pathways, which cells use in order to carry out the processes of growth, DNA replication and cell division in the appropriate order and at the appropriate time. While looking at the RNA being targeted by the HigB toxin, Dr Kirkpatrick and her colleagues found that in addition to the efflux pump RNA, HigB also targeted CtrA. CtrA is a regulatory protein involved in controlling the cell’s transition into the DNA replication stage of the cell cycle, necessary for subsequent cell division. Its function is to maintain the cells in the swimming stage of the cell cycle (in which they do not replicate their DNA) and clearance of CtrA out of the cell allows the DNA replication stage of the cell cycle to start. They found that when the antitoxin was defective, there were fewer CtrA-dependent swimming cells in the population. So when the HigB toxin levels are higher, its action against CtrA allows the cells to proceed more quickly to the DNA replication and division stages of the cell cycle. Again, this makes the HigBA system unique among TASs in its ability to fine-tune the cell cycle, and it may further contribute to the DNA damage response since actively replicating cells are more sensitive to DNA damaging agents. ‘HigBA is highly specific both in its activation conditions and its response,’ explains Clare. ‘It is dedicated exclusively to the DNA damage response in these bacteria and attacks a small set of essential targets in the cell, leading to inescapable cell death.’

‘Discovery of new pathways within bacteria that could provide a source of new drug targets, as well as new molecules that interfere with them, is a challenge that can more easily be met in an academic setting without the requirement to generate profits for shareholders.’

The future

Dr Kirkpatrick’s new lab at the University of Southern Denmark will be focussed on chemical screening for compounds which inhibit the growth of Pseudomonas bacteria. Pseudomonas species can be disease-causing and are the second most widespread cause of hospital acquired infections. Antibiotic resistance is also known to be on the increase in this genus. This screening will be designed specifically to identify compounds which affect the pathways involved in determining cell polarity. Initial experiments performed in this screen, such as the one which identified additional TipN-specific inhibitor compounds, tested normal Pseudomonas cells against those which were defective (mutated) in genes involved in regulating polarity, expecting that the compounds of interest would inhibit cell growth in the mutants but have no effect (or less of an effect) in the normal cells. ‘I was very surprised to find that a few had the opposite effect,’ says Dr Kirkpatrick, ‘the mutation of the gene appeared to confer a protective effect on the cells against the antibiotic, even in cases where the gene in question was not known to be involved in the usual mechanism of the antibiotic.’ She plans to prioritise the study of these compounds as they may provide information on previously unknown pathways and drug targets. She also plans to use techniques which mutate large numbers of bacterial genes in order to find candidate genes which either protect or inhibit the bacteria in their resistance to potential antibiotics. This screen will be focused on finding genes which are involved in defining cell polarity, as her work on Caulobacter demonstrated this to be a promising avenue.

Dr Clare Kirkpatrick’s interest in antibiotic resistance began while studying vancomycin-resistant enterococci as an undergraduate at the University of Cambridge, UK. After completing her PhD in bacterial genetics at the University of Cambridge, she then carried out her research on Caulobacter crescentus as a post-doc in Patrick Viollier’s lab at the University of Geneva and subsequently acquired a Swiss National Science Foundation/Marie Heim-Vögtlin career development grant to initiate an independent research project on Pseudomonas aeruginosa chemical genetics. She is now starting her own laboratory group as an assistant professor at the University of Southern Denmark, Department of Biochemistry and Molecular Biology in March 2017.

CONTACT

P: (+41) 22 379 551
E: Clare.Kirkpatrick@unige.ch
W: researchgate.net/profile/Clare_Kirkpatrick

FUNDING

Fondation Pierre Mercier pour la Science

Swiss National Science Foundation (Marie Heim-Vögtlin PMPDP3_158295)

KEY COLLABORATORS

Patrick H. Viollier, University of Geneva

Peter Redder, University of Geneva

Gerardo Turcatti, NCCR Chemical Biology, Ecole Polytechnique Fédérale de Lausanne

Marc Chambon, NCCR Chemical Biology, Ecole Polytechnique Fédérale de Lausanne

Julien Bortoli Chapalay, NCCR Chemical Biology, Ecole Polytechnique Fédérale de Lausanne

REFERENCES

CL Kirkpatrick, D Martins, P Redder, A Frandi, J Mignolet, J Chapalay, M Chambon, G Turcatti and PH Viollier, Growth control switch by a DNAdamage inducible toxin–antitoxin system in Caulobacter crescentus, Nature Microbiology, 2016, 16008.

CL Kirkpatrick and PH Viollier, Synthetic Interaction between the TipN Polarity Factor and an AcrAB-Family Efflux Pump Implicates Cell Polarity in Bacterial Drug Resistance, Chemistry & Biology, 2014, 21, 657–665.

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