Surviving antibiotics…

Killing avoidance strategies

A couple of recent research papers remind me that I promised to talk a little about a phenomenon by which bacteria can avoid being killed by antibiotics, without actually being resistant in the classical sense, i.e. they can’t actually grow in elevated concentrations of the antibiotics they survive, and those cells that do survive give rise to populations that are no more, or less, likely to survive next time.

The first paper comes from the lab of Prof. Tony Coates at the Centre for Infection at St. George’s, University of London. Prof. Coates has for a long time been heavily involved in research into the treatment of latent and persistent infections, most notably T.B./tuberculosis. His research team (as indeed are mine) are trying to understand why some antibiotics that kill actively growing bacteria of a particular species have no effect on cells of the same species that aren’t actively growing; almost akin to the bit in Jurassic Park where T. rex kills the lawyer who’s running, but wouldn’t have done had the lawyer stood still §.

One of the reasons for this is that, historically, most drug discovery has been focussed on targeting actively growing cells, but what we are increasingly finding is that persistent infections can be mediated by a recalcitrant population of slow-growing or non-growing cells.

Whilst the idea of targeting non-growing bacteria is not a wholly new idea (you can find a review on the subject by Prof. Coates in my ‘Further reading’), it does seem that together with the report’s first author, Dr Yanmin Hu, their spin-out company (Helperby Therapeutics) has developed a platform and proof of principle drug that is now in trials, demonstrating the utility of such an approach. They have identified an antibiotic compound that has potent anti-Staphylococcal activity, but importantly, acts specifically against non-multiplying cells.

In a second paper, brought to my attention by Ed Yong, the Collins lab in Boston has identified that a sub-population of super-resistant bacteria act in a charitable manner to other members of the colony that are less resistant. Whilst the super-resistant cells could satisfy their own selfishness by merely allowing all their less-resistant siblings to die out, the bacteria in this case have a mutation that maintains their production of indole (a signalling molecule) when normally its production would be shut down on exposure to the antibiotic. When released by the cell, indole stimulates non-resistant cells to enter a state of phenotypic resistance or ‘antibiotic survival’, even though continued-production of indole incurs a fitness cost.

Why might they do this?

Well, for one of the reasons that is very much the subject of my blog, bacterial fitness. As I have mentioned before, antibiotic resistance can have a fitness cost, which means that cells committing themselves to this ‘path of resistance’ may find themselves at a disadvantage come the time when the antibiotic is no longer around. The subject of my research is to document the various ways in which antibiotic resistant Staph. aureus mitigate these fitness costs so that they get to remain resistant and just as competitive as they ever were in the absence of antibiotic. It seems that in the case of the Collins’ lab’s charitable bacteria, they may mitigate the fitness cost of antibiotic resistance at a population level by maintaining the presence of non-resistant cells that can come to the fore once the antibiotic is removed.

“These few drug-resistant mutants, by enhancing the survival capacity of the overall population in stressful environments, may also help to preserve the potential for the population to return to its genetic origins should the stress prove transient. Efforts to monitor and combat antibiotic resistance are complicated by these bet-hedging survival strategies and other forms of bacterial cooperation.”

So what I want to do is briefly introduce the types of ‘antibiotic survival’ strategies seen in bacteria. It goes without saying that future drug discovery that targets the molecular/physiological underpinning for these strategies (once we’ve identified what these are!) will be important for the clinical management of infection.

Resistance or ‘killing avoidance’?

I’ve discussed in a previous post what I might describe as mechanisms of antibiotic resistance, i.e. producing a enzyme that modifies or chews up the antibiotic; or changing the component of the cell so that the antibiotic targeted to that component no longer has any effect, or pumping the antibiotic out of the cell before it does any damage.

It was recognised early on, in the heyday of antibiotics, that penicillin could kill most bacteria in a culture, but could not sterilise a culture. This has been observed with numerous antibiotic compounds, thus at a practical level you cannot achieve a 100% kill with antibiotics. Now this isn’t generally a problem for a healthy individual, as it is at this point the immune system takes over and clears away the remaining cells. However, many people receiving antibiotics aren’t well, they may be immuno-compromised, or suffering from a deep-seated infection. The persistence of a bacterial infection becomes a perfect breeding ground for classical antibiotic resistance, with each resurgence of the infection from surviving cells increasing the probability that resistance may evolve; and thus is thought to play a significant role in the failure of antibacterial treatment.

1. INDIFFERENCE. Bacteria can avoid being killed by being in a stationary phase (non-growing or metabolically inactive). This is actually the default repose of bacteria in the environment, only submitting to bursts of growth in the presence of nutrients. Most current (and old) antibiotics are specific to the particular cell components and processes of actively growing cells, there is no reason to expect that such antibiotics would have any killing effect on cells not engaging in these processes.

2. TOLERANCE. Those antibiotics that do kill bacteria don’t necessarily do so directly; they initiate a series of events, a cascade of physiological responses, which ultimately result in cell death. Unlike indifference, tolerance is not linked to the growth/metabolic state of the bacteria, but instead result from genetic changes that uncouple the killing activity of the drug from its inhibitory activity. In the clinic, tolerance seems to be specific to certain bacteria, and even then only in response to particualr antibiotics targeting the bacteria cell-wall.

3. PERSISTENCE. In a bacterial population there exists a sub-population of ‘persister’, cells that regardless of the growth state of the population as a whole, continue to exist in a stationary or growth-retarded state. It may be that persisters avoid antibiotic killing in the same way that indifferent bacteria do, but whilst there are some antibiotics that can kill indifferent cells, they don’t kill persisters; this suggests that something different is going on in these cells, and there is increasing evidence to suggest that there are defined genetic differences implicated in persistence, including changes within the stress-response pathways, but what these are (and what they do exactly) remains to be seen.

4. BIOFILMS. Finally, and most stubbornly, there is the issue of biofilms. Biofilms are like a condominium (or halls of residence) of bacteria, a structured environment where the bugs are surrounded by a gelatinous matrix of sugar chains and many other macromolecules. They are involved in some 80% of human infections and represent a major cause of antibiotic treatment failure. Within the matrix the bacteria avoid antibiotic killing through indifference and persistence, thought to be brought on by the low oxygen and low nutrient environment; the matrix also provides some protection from certain classes of antibiotics, as well as the immune system. Even if a large number of matrix surface cells are killed off, the matrix structure survives and can be re-populated by the surviving cells. For some bacteria the biofilm environment stimulates them to massively increase their rate of mutation, which can increase the rate at which antibiotic resistance can evolve.

So what do we do?

Well again it comes down to idealism versus pragmatism. The current system of drug discovery is fraught and inefficient enough without an additional burden of esoteric and poorly understood mechanisms of bacterial antibiotic survival. I do think there is some merit in drug discovery targeted at non-growing indifferent bacteria, this is particularly important in the treatment of T.B. The problem is going to be that many of these killing avoidance strategies differ between pathogens and between the particular environment in which they’re found, and also that in the absence of any ongoing preventative treatment, such as potential vaccines, by the time an infection manifests itself the antibiotic survival systems are likely to already be in place.

Other than indifference, biofilms are a system worth addressing in the immediate term. We have amassed a huge amount of data on biofilms, and demonstrated that they are of great clinical importance, thus efforts should be made to increase the number of biofilm busting compounds we have available.

Many people are familiar with antibiotic resistance, but I’m interested to hear (especially from other biologists) how much people knew about such antibiotic survival strategies. Also, as ever, please feel free to ask questions at any level. This (rather long) post barely touches the surface of this subject, there’s plenty more to be said!

^§^ The theory that T. rex would only ‘see’ moving objects is probably a little outdated.

Further reading

As always I will try to find open access material where available, and will update those references that aren’t as and when they do.

_Hu et al. (2010) A New Approach for the Discovery of Antibiotics by Targeting Non-Multiplying Bacteria: A Novel Topical Antibiotic for Staphylococcal Infections. PLoS ONE 5: e11818._
Open access ]

_Coates, A. et al. (2002) The future challenge facing the development of new antimicrobial drugs. Nature Reviews Drug Discovery 1: 895-910._
Free pdf ]

_Lee, H. et al. (2010) Bacterial charity work leads to population-wide resistance. Nature 467, 82-85._
[Sorry, article behind a paywall] – You can read Ed Yong’s post on it though.

Levin, B.R. and Rozen, D.E. (2006) Non-inhertied antibiotic resistance. Nat Rev Microbiol 4: 556-562.
free pdf ]

– A very useful grounding to the subject of phenotypic resistance, as it was understood back in 2006.

Lewis, K. (2010) Persister cells. Annu. Rev. Microbiol. 64: 357-72.
[Sorry, another paywall paper ]

– Good review of bacterial persistence.

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Compensating for alien genes…

[This post was restored from a WayBackWhen archive of an older incarnation of mentalindigestions.net]

“FROM the perspective of a bacterium, higher eukaryotes are oversexed, unadventurous and reproduce in an inconvenient way.” So says Pål Johnsen and Bruce Levin in their commentary of today’s article for discussion, and nary a truer word said. Of course, one may state that inconvenient as reproduction may be, bacteria clearly have no sense of fun.

There was once an idea that we could address the problem of antibiotic resistant bacterial strains by removing the ailing antibiotic from clinical use. In the absence of selective pressure it was thought that the evolutionary traits that enable the strain to resist the antibiotic would actually put the strain at a competitive disadvantage compared with a strain that doesn’t have such antibiotic resistance. The proposed cause of this? Fitness costs – these are imposed by a resource-expensive set of mutations, or carriage of alien DNA, that make the resistant strain compete less well once its non-resistant brethren are no longer being killed off by the antibiotic.

However, some years ago now experimental evidence suggested that this is not always the case; it may in fact be often not the case. It is worth mentioning at this point that it has been shown that in some circumstances (alt) the number of infections caused by a particular antibiotic-resistant pathogenic bacterium have become fewer on reduction (or removal) of the antibiotic to which that strain is resistant, but to assume this would be the case with all strains/antibiotics is naïve.

It is true, with few exceptions, that initially both plasmid and chromosomally encoded resistances result in fitness losses. However, when resistance has a cost it is possible for compensatory mutations in a cell to ameliorate these costs, usually without the loss of resistance. The type of compensatory mutations that mitigate the fitness cost of acquiring antibiotic resistance, or any other incoming DNA that encodes potentially useful genes, will depend very much upon the environment in which the bacteria finds itself. These include the availability of resources, i.e. the growth environment of the bacteria, the environment of the genes (mobile or chromosomal), or whether the genes are being selected for by an external factor, such as the presence of antibiotics in the case of resistance genes.

So what sort of ‘nips and tucks’ might a bacterial population undergo in order to maintain a battery of costly genes, but that may provide an ongoing advantage? Well, this is the subject of much ongoing research; one example indicated that, in the absence of selective pressure, costly genes are simply silenced – a molecular mechanism often found in higher organisms that prevents a gene from being ‘switched on’. Thus a reservoir of drug resistance determinants may remain in populations that have compensated for their presence, remaining ‘inactive’ until a selective pressure removes the silencing.

A recent study by Peter Lind (et al.), a grad student working in the lab of Dan Andersson at Uppsala, Sweden, addresses a particularly pertinent question of compensatory mutations: those associated with genes acquired by horizontal gene transfer (HGT). HGT bypasses the slow and haphazard process of evolution (via random mutation, selection and recombination) by offering an opportunity for bacteria to receive fully fledged genes encoding pathogenicity factors (genes that make bacteria better at causing disease) as well as genes that encode resistances to disinfectants and/or antibiotics, amongst others. There is no doubt that such incoming DNA may pose significant fitness costs, so Lind et. al. set out to quantify the nature of compensatory mutations on such incoming DNA.

Continue reading “Compensating for alien genes…”

Heat shocking adaptive evolution…

Research bloggingIN evolutionary theory there is a phenomenon known as canalisation, a process in which the phenotype (i.e. the outward physical appearance of an organism) remains unchanged, despite genetic or environmental influences.  This suggests that a mechanism exists to buffer the physical appearance from such changes, which may explain why some species can remain mostly unchanged for millions of years.

The buffering afforded by this mechanism permits the accumulation of genetic variation, in effect storing it up like an evolutionary capacitor. Also, presumably the accumulated genetic variation may be released by an event that overcomes the evolutionary capacitor, releasing fuel (in the form of variation) that provides a substrate for natural selection and potentially accelerating evolution. But how?

The idea of capacitance was first suggested by Rutherford and Lindquist 1 following experiments on a protein called heat shock protein 90 (Hsp90) in fruitflies. Generally speaking, heat shock proteins assist in the maintenance and correct folding of cellular proteins, especially when under temperature stress; Hsp90 plays a particular role in maintaining the unstable signalling proteins that act as key regulators of growth and development.

They suggested that in nature, a stressing event such as high or low temperatures may overcome the protective buffering effect that Hsp90 has on maintaining these key regulators. As Hsp90 becomes diverted from its usual role, due to an increase of stress-damaged proteins in the cell, those cell signalling proteins it normally maintains are free to adopt a range of altered behaviours, interfering with the development of the organism. The result is morphological variants upon which natural selection can act. Rutherford and Lindquist found as much, with chemically and environmentally compromised Hsp90 resulting in flies with abnormal wings, legs or eyes, they observed a broad variety of phenotypes.

Rutherford and Lindquist went on to demonstrate that the capacity for such remarkable variation was pre-existing, i.e. it was encoded genetically prior to the stressing event, but had been silenced. Evolutionary capacitance may therefore provide a mechanism of adaptive evolution in which a population under stress may release previously silent variation, resulting in the appearance of certain individuals with more desirable traits in that changed environment. When such revealed traits are selected for they can become fixed and independently of the buffering action of Hsp90.

This week, in a letter to Nature, Valeria Specchia et al.2 report some fascinating evidence that indicates that beyond merely acting as a gate-keeper to unleash variation, mutations of Hsp90 that compromise its functionality result in new, rather than pre-exisiting, variation. They observed that mutations in Hsp90 affect the production of piRNAs. These are small RNA molecules that are involved in the silencing of genes, particularly those involved in development, i.e. sex cells like eggs and sperm, and all the cell types that give rise to these cells. These piRNAs are also responsible for repressing genetic elements called transposons.

Continue reading “Heat shocking adaptive evolution…”

Lame theses….

[ratings]

Here is an excerpt of a philosophical lecture series going on at my institution:

The Mangoletsi Lectures 2009: God, Science and Philosophy
Peter van Inwagen, John Cardinal O’Hara Professor of Philosophy, University of Notre Dame

Lecture 4: God and Science II

I return to the topic of a possible scientific disproof of the existence of God. Unlike the discussion in the first lecture, this lecture considers a particular scientific theory in detail—the Darwinian theory of evolution. I give a statement of the theory, present some reasons for being skeptical about whether it is in every respect true, and present an argument for the conclusion that, whether the theory is true or false, its truth is consistent with the thesis that the universe was created by an intelligent being. Finally, I defend a stronger position than the consistency of the Darwinian theory with the existence of an intelligent creator; I defend the thesis that, if the Darwinian theory were true and known to be true, our knowing that it was true would not provide us with any reason to believe that the universe does not have an intelligent creator.

He takes a Papal line by stating, ‘I defend the thesis that, if the Darwinian theory were true and known to be true, our knowing that it was true would not provide us with any reason to believe that the universe does not have an intelligent creator‘.

His erroneous use of the phraseology ‘if the theory were true and known to be true‘ demonstrates a fundamental disconnect in this man’s understanding of science. What we can say is ‘the theory is not false, and has been shown (countless times) to not be false’.

What he appears to be saying is, if you can’t disprove the existence of God, then ipso facto, he exists. It is a tenuous, and rather Catholic, position he hopes to defend, that demonstrating the validity of the theory of evolution, as we have, does not give us any reason to believe there isn’t still an intelligent creator. You could just as soon state the opposite. Obviously the existence of God is not open to scientific testing as no testable hypothesis could realistically be formulated; however, we can (and have) amassed enough data to obviate a need for a God in the equation.

Obviously he’s left himself some wriggle room in the form of, ‘its truth [the theory of evolution] is consistent with the thesis that the universe was created by an intelligent being‘; yes, sure, if you want to fudge it into your own creation story go ahead. It could be consistent with whatever you like, feel free to merge the rigorous science with anecdotal and fantastic origins theory, but this does not give it any more meaning, you’re merely hand-waving on the bits for which you have no explanation, i.e. the origins of life (which evolution in itself does not describe).

Meanwhile scientists will continue to remain curious and investigate the actual origins, rather than making up answers.

Hox box…

[ratings]

WHAT do hedgehog, merlin and okra have in common with jelly belly, pimples and Genghis Khan? What if I said that hedgehog is not just a cute, spiny mammal? That Merlin is not just the name of a bird and a wizard? That okra is not just a vegetable? Would you be interested to learn that “jelly belly” is crucial for gut muscle development? “Genghis Khan”, far from being a Mongol lord who conquered Asia, is involved in the stimulation of structural components in cells. Oh, and we can look to “Merlin” to restrain cell proliferation.

Confused? Don’t be. They are all names of genes – sequences of DNA that exert influence on a creature by encoding and regulating the production of a protein. These particular genes are found in Drosophila melanogaster, otherwise known as the fruit fly.

Scientists are interested in a region of the fruit fly chromosome called the “homeobox”. The homeobox (“homeo”, from the Greek for “similar”, and “box” as the sequence is in a defined package) contains “Hox genes”. First identified in fruit flies in the early 1980s, they control the different aspects of body development: head, legs, wings or other structures. Interestingly, many other creatures, including humans, possess these genes, where they carry out similar functions. We are all basically running on the same genetic software.

Research in this area is helping us to understand why our head is where it is, why we have two arms joined to our upper body and not to our hips, and why we have feet, rather than hands, at the ends of our legs. More significantly, they are helping to identify the genetic basis of certain human diseases by helping us understand the mechanisms of this genetic control; errors in embryonic development account for a large number of spontaneous abortions in humans.

Hox genes produce simple proteins that govern the activities of other “target” genes, which result in the development of a specific body parts at specific locations; it is those “target” genes that contain the specific information about how and what appendages look like,  and the hox genes control the degree to which those target genes are switched on or off. The arrangement of genes mirror the arrangement of the body parts they control, starting with the head at one end, followed by the mid-sections, and so on. It’s a logical blueprint that works because it represents economy of information.

In the above figure the hox gene clusters of the fruit fly are colour coded for the respective sections of head-bottom development they control, and below are the homologous (performing the same function) genes in a mouse. Whilst we mammals have four clusters of these gene groups, some of which have become redundant or lost due to compensation by one of the other clusters, the startling similarity with the fruit fly hox cluster is unmistakable.

So over the course of millions of years, despite the changes to body form and function (the changes to the specific genes that the above clusters control) in the course of animal evolution, the controlling elements themselves have been conserved. This says something important about the blueprint of animals on Earth; if it works, keep it.
Continue reading “Hox box…”

Superorganisms…

[ratings]

NO, don’t get excited, I said super organisms. Yesterday’s The Scientist led with an article on Super organisms, which reminds me of my invertebrate neural and endocrinology lectures of years past. I used to be fascinated by the idea of super organisms, which is simply an organism of many organisms.

Being a prokaryotic biologist, I tend to think of things at the scale of planktonic (free-living) single cells, and occasionally we enjoy the concept of cooperative living in biofilms or other more complex structured consortia like stromatolites. Ultimately, evolution has resulted in multicellular organisms, some of which were further refined into organisms consisting of many different tissues with disparate characteristics; most people are not unfamiliar with this.

An interesting idea in biology is the idea of a super organism, where parallels can be drawn between the essential components of a complex higher organism, such as a mammal, and individual organisms within the super organism:

It is a rather contentious idea as it runs into the semanto-scientific diction of what exactly constitutes an organism. Are we limited by our usual scale-interpretation as Humans, where an abstract idea of a super organism clashes with our own biological recognition of what constitutes an organism? The big question is of course, how can such a super organism evolve? This is one of those great challenges that evolutionary biologists love.

At what scale does natural selection, the active force of evolution, have its effect? Does it act at the level of the individual? Yes, probably; I am still with Dawkins on the idea of selection acting at the level of the gene. However, for natural selection to have an effect, it depends on individual differences within a population, and crucially, on the ability of the “fittest” individual to survive and reproduce. However, in Ant colonies the Ants are sterile drones; the reproductive entity of a such a super organism is the queen of an Ant colony.

Thus we have a situation where “unfit” worker Ants can result in the collapse of a colony, therefore selection feeds back to the Queen where reproductive success is dependant upon producing workers that are capable of fulfilling their roles in the provision of food, looking after eggs, defending the colony and building infrastructure; thus a very indirect form of selection. So is the superorganism being selected or not?

Good question,  it’ll be fun finding out.

Viruses in the genes

[ratings]

THERE was a recent article in NewScientist suggesting that viruses are the unsung heroes of evolution. Whilst that is somewhat of a sensationalist position, there is a great degree of truth in it. Many anti-evolutionists seem convinced that it is mathematically impossible that genetic variation and mutation can be a sufficient substrate upon which natural selection can act.

What they forget is that whilst a mathematical proof is always the truth, it is a truth that is dependent upon whether the mathematical model accurately reflects the physical problem. Mathematics is limited to the validity of the assumptions that underpin the statement of the problem, thus in the fixing of certain variables it’s important to distinguish between getting the maths right and getting the problem right.

The variation seen in a species, upon which natural selection can act given circumstances that favour one variation over another, is encoded by alleles; this is the name given to different “versions” of the same gene, thus for eye colour, different alleles may be: brown, blue, green etc. Some alleles are dominant, some are recessive; the dominant ones win and get used, the recessive ones lose  and don’t get used. The dominant and recessive alleles are both part of your genetic make up, and this is called your genotype. The dominant alleles result in a physical attributes in the organism, such as brown eyes, and these physical attributes are known as the phenotype.

It is true to say that whilst all phenotype is derived from the genotype, not all genotype results in phenotype. Dominant traits, because they are aspects of the genotype that are reflected in the phenotype, are traits that can be acted upon by natural selection; however recessive traits are effectively hidden from natural selection unless the DNA that codes for the recessive alleles is physically linked to a piece of DNA that results in some other dominant trait that can be selected for or against. This recessivity maintains a store of genetic diversity.
Continue reading “Viruses in the genes”