Tet(O) cryoEM structure is out!

So it is finally out: the cryoEM structure of Tet(O) on the ribosome we have collaborated on with Joachim Frank's lab is finally published on in Nature Communications. Tet(O) is a bacterial translational GTPase that clears the ribosome from tetracycline antibiotic, and structural data provided in the paper shed light on the mechanism of Tet(O)-mediated resistance. Recently cryoEM of Tet(O)'s close relative, Tet(M) was published by Beckmann and Wilson labs, so now one can compare the two. And yes, they look very similar. No surprise there.

References:

Li et al. Nature Communications (2013) PIMD: 23403578

Dönhöfer at al. PNAS (2012) PIMD:  23027944

Relacin - a novel antibacterial targeting the stringent response, maybe

The stringent response is a promising target for novel antibacterials: it is involved in virulence and antibiotic insensitivity, and inhibiting the stringent response would disarm the bug, making is both less evil and easier to kill.

A new study is came out in PLoS Pathogens describing a novel Rel inhibitor, relacin (Fig. 1). Wexselblatt and colleagues are following up their earlier work on derivatizing ppGpp into a Rel inhibitor  and are now testing the compound not only in vitro, but also in vivo.

Fig 1: the chemical structure of relacin.

They show that relacin efficiently inhibits sporulation of Bacillus subtilis. Sporulation in this organism is driven by ppGpp, and inhibitory effect of relacin is a strong indication that it actually works. However, really high concentrations are needed to achieve significant effects: 0.5 - 2 mM. At these concentrations one would expect that in addition to hitting RelA, relacin will affect all the other ppGpp targets, i.e. translational GTPases, GTP biosynthesis enzymes etc. The authors do not test these effects. It would be easy to do it in an in vitro translational lysate... but, unfortunately, this is not done.  By using a GFP-fusion reporter, they do show that relacin inhibits translation of mid-sporulation protein SpoIIQ, but they do not check that it does not inhibit translation in general. A simple test of GFP expression would do.

With this (potential) absence of specificity relacin is unlikely to be the 'magic bullet' inhibiting just the stringent response and making bacterial less pathogenic, but still viable. However, relacin is just the first step. There is a hope that the derivatives to come will work at more in vivo-relevant concentrations and will be highly Rel-specific.

References:

Wexselblatt et al. Biomed Org Chem (2010) PIMD: 20483622

Wexselblatt et al. PLoS Pathogens (2012) 

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

Antibiotics vs the ribosome

Ok, now it is official: this summer in Tartu there will be a conference on antibiotics inhibiting protein synthesis organized by Tanel Tenson.

It is not too late to register! And it has a very, very competitive registration fee - 0 USD!

Confirmed speakers:

James Williamson (Scripps Research Institute),
Alexander Mankin (University of Illinois at Chicago),
Steven Douthwaite (University of South Denmark),
Daniel Wilson (University of Munich),
Karen Shaw (Trius Therapeutics),
Ada Yonath (Weizmann Institute of Science).
Birte Vester (University of Southern Denmark)
Joyce Sutcliffe (Tetraphase Pharmaceuticals)
Mans Ehrenberg (Uppsala University)
Chaitan Khosla (Stanford University)
Markus Zeitlinger (Medical University of Vienna)

... and me and Gem Atkinson! That sold it, right?

Antibiotics affecting ribosome's protein composition

Antibiotics kill bugs; and about a half of them are doing so by messing up translation. That usually means that the ribosome is stalled at a certain step, be it initiation or elongation or ribosomal recycling.

But it is not always just that. Sometimes antibiotics also mess up the ribosome itself  and affect its composition.

Exhibit A: kasugamycin, an antibiotic that inhibits translation initiation in bacteria by interfering with binding of the the initiator tRNA. Amazingly enough, treatment with kasugamycin results in dramatic change in the ribosomal composition which is in turn changing ribosome's functional properties. Several proteins dissociate from the small ribosomal subunit (S1, S2, S6, S12, S18 and S21) which turns the 70S ribosome into a 61S kasugamycin particle. Ribosomal protein S1 is of particular interest here, because it is very important for the mRNA:ribosome interactions and is responsible for  A/U rich sequences acting as translational activators.

The S61 particle loses the ability to translate mRNAs with Shine-Dalgarno sequences, while being able to translate leaderless mRNAs, that is the ones starting directly with the initiation codon at the 5'. These leaderless mRNAs can be translated without the help of any initiation factors, by bacterial and eukaryotic ribosomes alike, so it is no surprise that S61 particles, even though compromised can still translate these messages.

What is particularly interesting in the kasugamycin story, it is that loss of the ribosomal proteins can be reconstituted in vitro by simply mixing the drug with the 70S. This means that the effect is direct rather than mediated by the assembly process (see below for an example of the latter effect).

Exhibit B: chloramphenicol and erythromycin. These antibiotics cause defects of the ribosomal assembly, and they seem to be doing so by interfering with the expresion levels of different ribosomal proteins. Here we have Liebig's barrel in action: you interfere with levels of many components you need to have and end up running out of one, the limiting one.

All of the above is highly relevant for people using antibiotics as tools, for instance in microscopy. Chloramphenicol and kasugamycin are widely used to inhibit translation (for instance here and here). It's worth remembering that they are doing much more than that while interpreting your results. Sometimes the tool you use can have much more complicated character than one would anticipate, as I discussed here.

References:

Wilson DN (2009). The A-Z of bacterial translation inhibitors. Critical reviews in biochemistry and molecular biology, 44 (6), 393-433 PMID: 19929179

Schluenzen F, Takemoto C, Wilson DN, Kaminishi T, Harms JM, Hanawa-Suetsugu K, Szaflarski W, Kawazoe M, Shirouzu M, Nierhaus KH, Yokoyama S, & Fucini P (2006). The antibiotic kasugamycin mimics mRNA nucleotides to destabilize tRNA binding and inhibit canonical translation initiation. Nature structural & molecular biology, 13 (10), 871-8 PMID: 16998488

Schuwirth BS, Day JM, Hau CW, Janssen GR, Dahlberg AE, Cate JH, & Vila-Sanjurjo A (2006). Structural analysis of kasugamycin inhibition of translation. Nature structural & molecular biology, 13 (10), 879-86 PMID: 16998486

Kaberdina AC, Szaflarski W, Nierhaus KH, & Moll I (2009). An unexpected type of ribosomes induced by kasugamycin: a look into ancestral times of protein synthesis? Molecular cell, 33 (2), 227-36 PMID: 19187763

Siibak T, Peil L, Dönhöfer A, Tats A, Remm M, Wilson DN, Tenson T, & Remme J (2011). Antibiotic-induced ribosomal assembly defects result from changes in the synthesis of ribosomal proteins. Molecular microbiology, 80 (1), 54-67 PMID: 21320180

Nevo-Dinur K, Nussbaum-Shochat A, Ben-Yehuda S, & Amster-Choder O (2011). Translation-independent localization of mRNA in E. coli. Science (New York, N.Y.), 331 (6020), 1081-4 PMID: 21350180

Tzareva NV, Makhno VI, & Boni IV (1994). Ribosome-messenger recognition in the absence of the Shine-Dalgarno interactions. FEBS letters, 337 (2), 189-94 PMID: 8287975

Andreev DE, Terenin IM, Dunaevsky YE, Dmitriev SE, & Shatsky IN (2006). A leaderless mRNA can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors. Molecular and cellular biology, 26 (8), 3164-9 PMID: 16581790

Montero Llopis P, Jackson AF, Sliusarenko O, Surovtsev I, Heinritz J, Emonet T, & Jacobs-Wagner C (2010). Spatial organization of the flow of genetic information in bacteria. Nature, 466 (7302), 77-81 PMID: 20562858

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

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