Active role of the stringent response in antibiotic tolerance

From time to time we try killing bacteria with antibiotics. Most of the bugs die, but not all. These survivors fall into two categories: resistant bugs and tolerant bugs. Resistant bugs have specific mechanisms counteracting the drug: mutations in the target site, enzymes destroying the antibiotic, etc. Tolerant bugs are not getting killed using some more general approach, such as forming a biofilm efficiently shielding them from contact with the drug or shutting down its biosynthetic activity and waiting for the better days to come.

The stringent response is a mechanism rewiring the bacterial physiology under stress. It changes many things simultaneously, and, not surprisingly, functionality of the stringent response is linked to antibiotic tolerance. However, the big question here is the nature of this link: do bugs need functional stringent response in order to tolerate the drug just because relaxed bugs do not shut down their growth when needed and die, or does the stringent response induce production of certain specific enzymes protecting from the drug?

Recent report by Nguyen and colleagues seems to settle this question. Using series of in vivo experiments with E. coli knock-out strains deficient either in stringent response per se (knock-outs of RelA and SpoT) or in down-stream stringent response-regulated targets they show that the main source of antibiotic tolerance is not a general biosynthetic shut-down. Specifically, they identify two genes induced during the stringent response - superoxide dismutase (SOD) and catalase - to be crucial for bacterial survival in the presence of antibacterials.  What these do, they protect the bug from the hydroxyl radical. And build-up the latter was recently identified as a common mechanism causing the cell death during treatment by different unrelated antibacterials. 

References:

Nguyen at al., Science (2011) 334, pp. 982-986 PIMD 22096200

Kohanski et al. Cell (2007) 130, pp. 797-810 PIMD 17803904

The peculiar story of PTC124

Several years ago a very, very cool drug was discovered - PTC124. This one was inhibiting NMD (nonsense mediated mRNA decay), and since NMD is implicated in several deseases, PTC124 was of great interest. I have discussed this story here.

Well, it seems that there is a twist in this story. PTC124 was discovered using firefly luciferase (FLuc) as a reporter. And now is seems that it rather than acting on the NMD level, PTC124 interacts with the FLuc itself and modulates its activity!

What's especially interesting, is that work on PTC124 continues, and many more papers are getting published, not necessarily using FLuc... So what was it? Just a fluke?

...this story is somehow similar to that one.

References:

Auld, D. S., Lovell, S., Thorne, N., Lea, W. A., Maloney, D. J., Shen, M., Rai, G., et al. (2010). Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124 Proceedings of the National Academy of Sciences of the United States of America, 107(11), 4878–4883. PIMD 20194791

Auld, D. S., Thorne, N., Maguire, W. F., & Inglese, J. (2009). Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression Proceedings of the National Academy of Sciences of the United States of America, 106(9), 3585–3590. PIMD 19208811

Methicillin-resistant S. aureus (MRSA) JKD6229, freaking out and deadly

Stringent response, as I wrote here, here, and here, and here, is a central regulator of bacterial physiology, which decides whether to grow happily churning out new proteins without a care or to shut down all of the unnecessary systems, relocate all  resources to amino acid production and put up a fight. So what happens if a mutation hyper-activates it in Staphylococcus aureus? Wonder no more - the pathogen goes berserk!

The strain in question is called JKD6229 and it was reported in the resent paper by Gao at al. in PloS Pathogens. It was discovered among the clinical isolates, and rigorous analysis showed that it has continuously activated stringent response with elevated levels of ppGpp alarmone. Previous studies implicated stringent response in bacterial virulence, demonstrating that inhibition of this mechanism renders bacteria inapt for any sort of shenanigans (for review see Dalebroux et al. 2010).

Digging deeper, authors discovered that JKD6229 had a whole set of mutations making it nasty.

First was a set of mutations rendering it resistant to several antibiotics. Modified topoisomerase IV gave it resistance to cyproflaxacin, RNA polymerase was altered to give it resistance to rifampin and ribosomal large subunit methyltransferase RlmN had an insertion making it insensitive to linezolid. It short - this bug was really, really hard to kill. 

Second, authors report that the bug had mutated RelA, which had low activity in ppGpp hydrolysis, leading to accumulation of high ppGpp levels. High ppGpp level inhibits ribosome synthesis and results in slow growth, so the strain was dubbed as "Small Colony Variant". At the very same time, all the defense mechanisms were on high alert, and JKD6229 was not expecting anything good from the world around it. It was not growing fast, but it was hard to kill and it was fighting back. 

One very, very pissed off bacterium. 

And there are several ways to deal with it.

First, you can kill it (see my earlier account on the development of antibacterials). Surely, JKD6229 will be trying hard to survive, accumulating even more resistance markers.

Second, you can try calm it down, make it non-virulent. This approach is an emerging strategy in development of antibacterials - inhibiting virulence, but not killing the bug. There are two potential benefits. First, lower selective pressure for resistance mutants since in most of the tissues virulence is not needed for survival. Second, potentially higher selectivity - only the bad bugs perish, and the good ones live. However, there are problems with this approach. Virulence mechanisms are very diverse, and therefore drugs targeting them will have a very narrow spectrum. And usually you do not know exactly what sort of bug is causing the problem. Development of rapid diagnostic methods can fix this, and then 'narrow spectrum' becomes 'selectivity', and this is a good thing.

PS: Wait! Mutations in Staphylococcus aureus RelA? Well, surely not. Staphylococcus aureus does not have RelA. The protein authors refere to is Rel, a bi-functional protein capable of both producing and hydrolyzing ppGpp, as opposed of RelA, which is able only of ppGpp syntheses. For more information on phylogenetic relationships between RelA, Rel and SpoT see Mittenhuber 2001.

References:

Gao W, Chua K, Davies JK, Newton HJ, Seemann T, Harrison PF, Holmes NE, Rhee HW, Hong JI, Hartland EL, Stinear TP, & Howden BP (2010). Two novel point mutations in clinical Staphylococcus aureus reduce linezolid susceptibility and switch on the stringent response to promote persistent infection. PLoS pathogens, 6 (6) PMID: 20548948

Dalebroux ZD, Svensson SL, Gaynor EC, & Swanson MS (2010). ppGpp conjures bacterial virulence. Microbiology and molecular biology reviews : MMBR, 74 (2), 171-99 PMID: 20508246

Mittenhuber G (2001). Comparative genomics and evolution of genes encoding bacterial (p)ppGpp synthetases/hydrolases (the Rel, RelA and SpoT proteins). Journal of molecular microbiology and biotechnology, 3 (4), 585-600 PMID: 11545276

Escaich S (2010). Novel agents to inhibit microbial virulence and pathogenicity. Expert opinion on therapeutic patents, 20 (10), 1401-18 PMID: 20718591

Mendeley group on stringent response

specific inhibition of nonsense-mediated mRNA decay by small molecule

The last step of protein synthesis is called translation termination. During this step the stop codon is recognized by the protein factor called "release factor" and finished protein is cleaved off the tRNA. Mutations which cause premature termination (nonsense mutations) lead to shortened protein which is usually defective, and in order to avoid accumulation of these proteins such mRNA are recognized by nonsense mediated mRNA decay (NMD) machinery and degraded.

This is usually a good thing, but imagine that the gene which has this nonsense mutation is very important for survival of the cell, and imagine that the truncated version is still somewhat functional. In this case it would be beneficial to produce it even if it is somewhat compromised: something is better than nothing.

Therefore in some cases NMD is actually a cause of a desease, such as Duchenne muscular dystrophy and cystic fibrosis - over-zealous NMD removes all the damaged mRNAs and no protein product is produced. In this case suppression of NMD is needed.

Treatment with antibiotic gentamicin wich causes error-prone translation is a solution, but unfortunately gentamicin is not specific for NMD and causes errors on all the steps of translation. Specific inhibition of NMD was sought after for many years, and finally a smal molecule which inhibits it was discovered.

The molecule is called PTC124, and we know that it does the trick (inhibits NMD) and works well against the DMD. It was discovered in 2007, and by now we still don't know to what it binds (ribosome? NMD factors?) and what is the mechanism. The reason for that is that it is extremely complicated to construct an in vitro system for studying PTC124 (you will need purified translational and NMD components, and that is a lot of components). So for now we have no idea how it works, but it does!

Update: ...or may be it doesn't!

References:

Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P, Paushkin S, Patel M, Trotta CR, Hwang S, Wilde RG, Karp G, Takasugi J, Chen G, Jones S, Ren H, Moon YC, Corson D, Turpoff AA, Campbell JA, Conn MM, Khan A, Almstead NG, Hedrick J, Mollin A, Risher N, Weetall M, Yeh S, Branstrom AA, Colacino JM, Babiak J, Ju WD, Hirawat S, Northcutt VJ, Miller LL, Spatrick P, He F, Kawana M, Feng H, Jacobson A, Peltz SW, & Sweeney HL (2007). PTC124 targets genetic disorders caused by nonsense mutations. Nature, 447 (7140), 87-91 PMID: 17450125

Manuvakhova M, Keeling K, & Bedwell DM (2000). Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system. RNA (New York, N.Y.), 6 (7), 1044-55 PMID: 10917599

Searching for antibacterials under the lamppost

We all know that we need more antibiotics and the ones we have are no good. We all also know that bugs become more and more skilled in avoiding being killed, and this is no good either.

The question here is why don't we have new antibiotics coming. And it turns out to be real hard to make one. Initially we had a discovered a whole bunch of these from natural components. All these were developed by bacteria and fungi to kill other bacteria and fungi, and these chaps had billions of years to develop these components. We just hijacked their research, really.

When all these started to age because of the pathogens rapidly acquiring resistance, big pharma decided to have a go at making new antibacterials. Hey, we are pretty good at fighting cancer, killing some bugs should be a walkover.

Here is a great example how we fail. By "we" I mean GlaxoSmithKline, who are no amateurs. They constructed a library of constructs, which would produce genes of interest at different expression level - and that would be all the genes in the genome they could clone that way!,  and tested all these against all the compounds they had on their shelves. ...WOW!

Many, many monies later: 10 hits, no leads, no drugs. Game over.

But let us imagine that there would be drugs developed. What next? These would be the last line of defense, and will be proscribed very, very rarely. This creates a problem of profit in the antibiotics R and D: even if you develop a new antibacterial, there will be no money coming you way.

Well then, pharma can not help us... How about academia?

There is some hope there. A couple of new approaches has emerged.  One is grafting one antibiotic scaffold on another, creating a hybrid werwolf antibiotic, which kills like hell. However, majority of the academia peoples just figured out that anything is an antibiotic target, and that anything is an antibiotic, and they started killing their favorite proteins or RNA in vitro. And yes, if you inhibit something important, your bug will die.

RelA is no exception. Several ppGpp analogues were developed as lovely antibacterials: exhibit 1 and  2. (A quick reminder - RelA makes ppGpp from one ATP and one GDP molecule, and ppGpp in turn acts as a molecular messenger remodeling the whole bacterial physiology, thus RelA is very important for bacterial virulence - a great target for an antibacterial indeed!).

Does it look promising? Not at all. It is not enough to inhibit something important. You also need to be specific and you also need to get into the cell. The latter is a very, very big problem - you need to follow the Lipinski's rule of five, and ppGpp is a very, very bad scaffold to start with in this case: loads of hydrogen bond donors, loads of acceptors, too big and not hydrophobic enough.  All bad. Well, the only things that are good is grants acquired and papers published.

So here we are. Big pharma bails out, academia has no idea as to what they are doing. Any chances of our survival?

Yes. Small start-up pharma packed with academia-derived know-how. Rib-X, Tetraphase. Pray for them, please.

References:

Payne DJ, Gwynn MN, Holmes DJ, & Pompliano DL (2007). Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nature reviews. Drug discovery, 6 (1), 29-40 PMID: 17159923

Charest MG, Lerner CD, Brubaker JD, Siegel DR, & Myers AG (2005). A convergent enantioselective route to structurally diverse 6-deoxytetracycline antibiotics. Science (New York, N.Y.), 308 (5720), 395-8 PMID: 15831754

Mendeley group on stringent response