A Movable Defense

In the evolutionary arms race between pathogens and hosts, genetic elements known as transposons are regularly recruited as assault weapons for cellular defense.

By | January 1, 2015

JUMPERS: Transposable elements, which make up as much as 90 percent of the corn genome and are responsible for the variation in kernel color, may also be at the root of diverse immune defenses.COMPOSITE IMAGE. © ARINA P. HABICH/SHUTTERSTOCK.COM; © ISTOCKPHOTO.COM/JAUHARI1

Researchers now recognize that genetic material, once simplified into neat organismal packages, is not limited to individuals or even species. Viruses that pack genetic material into stable infectious particles can incorporate some or all of their genes into their hosts’ genomes, allowing remnants of infection to remain even after the viruses themselves have moved on. On a smaller scale, naked genetic elements such as bacterial plasmids and transposons, or jumping genes, often shuttle around and between genomes. It seems that the entire history of life is an incessant game of tug-of-war between such mobile genetic elements (MGEs) and their cellular hosts.

MGEs pervade the biosphere. In all studied habitats, from the oceans to soil to the human intestine, the number of detectable virus particles, primarily bacteriophages, exceeds the number of cells at least tenfold, and maybe much more. Furthermore, MGEs and their remnants constitute large portions of many organisms’ genomes—as much as two-thirds of the human genome and up to 90 percent in plants such as corn.

MOBILE DNA: A false-color transmission electron micrograph of a transposon, a segment of DNA that can move around chromosomes and genomes© PROFESSOR STANLEY N. COHEN/SCIENCE SOURCEDespite their ubiquity and prevalence in diverse genomes, MGEs have traditionally been considered nonfunctional junk DNA. Starting in the middle of the 20th century, through the pioneering work of Barbara McClintock in plants, and over the following decades in a widening range of organisms, researchers began to uncover clues that MGE sequences are recruited for a variety of cellular functions, in particular for the regulation of gene expression. More-recent work reveals that many organisms also use MGEs for a more specialized and sophisticated function, one that capitalizes on the ability of these elements to move around genomes, modifying the DNA sequence in the process. Transposons seem to have been pivotal contributors to the evolution of adaptive immunity both in vertebrates and in microbes, which were only recently discovered to actually have a form of adaptive immunity—namely, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated genes) system that has triggered the development of a new generation of genome-manipulation tools.

Multiple defense systems have evolved in nearly all cellular organisms, from bacteria to mammals. Taking a closer look at these systems, we find that the evolution of these defense mechanisms depended, in large part, on MGEs—those same elements that are themselves targets of host immune defense.

Layers of defense

As cheaters in the game of life, stealing resources from their hosts, parasites have the potential to cause the collapse of entire communities, killing their hosts before moving on or dying themselves. But hosts are far from defenseless. The diversity and sophistication of immune systems are striking: their functions range from immediate and nonspecific innate responses to exquisitely choreographed adaptive responses that result in lifelong immune memory after an initial pathogen attack.1

 

Transposons seem to have been piv­otal contributors to the evolution of adap­tive immunity both in vertebrates and in microbes.

Over the last two decades or so, it has become clear that nearly all organisms possess multiple mechanisms of innate immunity.2 Toll-like receptors (TLRs), common to most animals, recognize conserved molecules from microbial pathogens and activate the appropriate components of the immune system upon invasion. Even more widespread and ancient is RNA interference (RNAi), a powerful defense system that employs RNA guides, known as small interfering RNAs (siRNAs), to destroy invading nucleic acids, primarily those of RNA viruses. Conceptually, the biological function of siRNAs is analogous to that of TLRs: an innate immune response to a broad class of pathogens.

 

Prokaryotes possess their own suite of innate immune mechanisms, including endonucleases that cleave invader DNA at specific sites and enzymes called methylases that modify those same sites in the prokaryotes’ own genetic material to shield it from cleavage, a strategy known as restriction modification (RM).3 If overwhelmed by pathogens, many prokaryotic cells will undergo programmed cell death or go into dormancy, thereby preventing the spread of the pathogen within the organism or population. In particular, infected bacterial or archaeal cells can activate toxin-antitoxin (TA) systems to induce dormancy or cell death. Normally, the toxin protein is complexed with the antitoxin and thus inactivated. However, under stress, the antitoxin is degraded, unleashing the toxin to harm the cell.

Many viruses that infect microbes also encode RM and TA modules.4 These viruses are, in effect, a distinct variety of MGEs that sometimes have highly complex genomes. Viruses use RM systems for the very same purpose as their prokaryotic hosts: the methylase modifies the viral genome, whereas the endonucleases degrade any unmodified genomes in the host cell, thereby providing nucleotides for the synthesis of new copies of the viral genome. And the TA system can ensure retention of a plasmid or virus within the cell. The toxin and antitoxin proteins dramatically differ in their vulnerability to proteolytic enzymes that are always present in the cell: the toxin is stable whereas the antitoxin is labile. This does not matter as long as both proteins are continuously produced. However, if both genes are lost (for example, during cell division), the antitoxin rapidly degrades, and the remaining amount of the toxin is sufficient to halt the biosynthetic activity of the cell and hence kill it or at least render it dormant. A plasmid or virus that carries a TA module within its genome thus implants a self-destructing mechanism in its host that is activated if the MGE is lost. (See illustration.)

When an MGE inserts into the host genome, it inevitably modifies that genome, typically using an MGE-encoded recombinase (also known as integrase or transposase) as a breaking-and-entering tool. Speaking in deliberately anthropomorphic terms, the MGEs do so for their own selfish purposes, to ensure their propagation within the host genome. However, given the ubiquity of MGEs across cellular life forms, it seems extremely unlikely that host organisms would not recruit at least some of these naturally evolved genome manipulation tools in order to exploit their remarkable capacities for their own purposes. Immune memory that involves genome manipulation is arguably the most obvious utility of these tools, and in retrospect, it is not surprising that unrelated transposons and their recombinases appear to have made key contributions to the origin of both animal and prokaryotic forms of adaptive immunity.

Guns for hire

DISRUPTING COLOR: The variations in color seen in this dahlia “flower” (actually a cluster of small individual flowers, or florets) can be caused by transposon-induced mutations. © ISTOCKPHOTO.COM/BEDOUntil recently, prokaryotes had been thought to entirely lack the sort of adaptive immunity that dominates defense against parasites in vertebrates. This view has been overturned in the most dramatic fashion by the discovery of the CRISPR-Cas, RNAi-based defense systems found to be present in most archaea and many bacteria studied to date.5 In 2005, Francisco Mójica of the University of Alicante in Spain and colleagues,6 and independently, Dusko Ehrlich of the Pasteur Institute in Paris,7 discovered that some of the unique sequences inserted between CRISPR, known as spacers, were identical to pieces of bacteriophage or plasmid genomes. Combined with a detailed analysis of the predicted functions of Cas proteins, this discovery led one of us (Koonin) and his team to propose in 2006 that CRISPR-Cas functioned as a form of prokaryotic adaptive immunity, with memory of past infections stored in the genome within the CRISPR “cassettes”—clusters of short direct repeats, interspersed with similar-size nonrepetitive spacers, derived from various MGEs—and to develop a detailed hypothesis about the mechanism of such immunity.8

Subsequent experiments from Philippe Horvath’s and Rodolphe Barrangou’s groups at Danisco Corporation,9 along with several other studies that followed in rapid succession, supported this hypothesis. (See “There’s CRISPR in Your Yogurt,” here.) It has been shown that CRISPR-Cas indeed functions by incorporating fragments of foreign bacteriophage or plasmid DNA into CRISPR cassettes, then using the transcripts of these unique spacers as guide RNAs to recognize and cleave the genomes of repeat invaders. (See illustration.) A key feature of CRISPR-Cas systems is their ability to transmit extremely efficient, specific immunity across many thousands of generations. Thus, CRISPR-Cas is not only a bona fide adaptive immunity system, but also a genuine machine of Lamarckian evolution, whereby an environmental challenge—a virus or plasmid, in this case—directly causes a specific change in the genome that results in an adaptation that is passed on to subsequent generations.10

When a mobile genetic element (MGE) inserts into the host genome, it inevitably modifies that genome, typically using an MGE-encoded recombinase as a breaking-and-entering tool.

A torrent of comparative genomic, structural, and experimental studies has characterized the extremely diverse CRISPR-Cas systems according to the suites of Cas proteins involved in CRISPR transcript processing and target recognition.5,11 While Type I and Type III systems employ elaborate protein complexes that consist of multiple Cas proteins, Type II systems perform all the necessary reactions with a single large protein known as Cas9. These findings opened the door for straightforward development of a new generation of genome editing. Cas9-based tools are already used by numerous laboratories all over the world for genome engineering that is much faster, more flexible, and more versatile than any methodology that was available in the pre-CRISPR era.12

And it seems that humans are not the only species to have stolen a page from the CRISPR book: viruses have done the same. For example, a bacteriophage that infects pathogenic Vibrio cholera carries its own adaptable CRISPR-Cas system and deploys it against another MGE that resides within the host genome.13 Upon phage infection, that rival MGE, called a phage inducible chromosomal island-like element (PLE), excises itself from the cellular genome and inhibits phage production. But at the same time, the bacteriophage-encoded CRISPR-Cas system targets PLE for destruction, ensuring successful phage propagation.

Consequently, in prokaryotes, all defense systems appear to be guns for hire that work for the highest bidder. Sometimes it is impossible to know with any certainty in which context, cellular or MGE, different defense mechanisms first emerged.

Transposon origins of adaptive immunity

THE ULTIMATE MOBILE ELEMENT: Bacteriophages converging on an E. coli cell can be seen injecting their genetic material (blue-green threads) into the bacterium. Often, viral DNA will be taken up by the host genome, where it can be passed through bacterial generations.© EYE OF SCIENCE/SCIENCE SOURCE. COLORIZATION BY MARY MADSENRecent evidence from our groups supports an MGE origin of the CRISPR-Cas systems. The function of Cas1—the key enzyme of CRISPR-Cas that is responsible for the acquisition of foreign DNA and its insertion into spacers within CRISPR cassettes—bears an uncanny resemblance to the recombinase activity of diverse MGEs, even though Cas1 does not belong to any of the known recombinase families. As a virtually ubiquitous component of CRISPR-Cas systems, Cas1 was likely central to the emergence of CRISPR-Cas immunity.

During a recent exploration of archaeal DNA dark matter—clusters of uncharacterized genes in sequenced genomes—we unexpectedly discovered a novel superfamily of transposon-like MGEs that could hold the key to the origin of Cas1.14 These previously unnoticed transposons contain inverted repeats at both ends, just like many other transposons, but their gene content is unusual. The new transposon superfamily is present in both archaeal and bacterial genomes and is highly polymorphic (different members contain from 6 to about 20 genes), with only two genes shared by all identified representatives. One of these conserved genes encodes a DNA polymerase, indicating that these transposons supply the key protein for their own replication. While diverse eukaryotes harbor self-synthesizing transposons of the Polinton or Maverick families, this is the first example in prokaryotes. But it was the second conserved protein that held the biggest surprise: it was none other than a homolog of Cas1, the key protein of the CRISPR-Cas systems.

We dubbed this new transposon family Casposons and naturally proposed that, in this context, Cas1 functions as a recombinase. In the phylogenetic tree of Cas1, the casposons occupy a basal position, suggesting that they played a key role in the origin of prokaryotic adaptive immunity.

In vertebrates, adaptive immunity acts in a completely different manner than in prokaryotes and is based on the acquisition of pathogen-specific T- and B-lymphocyte antigen receptors during the lifetime of the organism. The vast repertoire of immunoglobulin receptors is generated from a small number of genes via dedicated diversification processes known as V (variable), D (diversity), and J (joining) segment (V(D)J) recombination and hypermutation. (See illustration.) In a striking analogy to CRISPR-Cas, vertebrate adaptive immunity also seems to have a transposon at its origin. V(D)J recombination is mediated by the RAG1-RAG2 recombinase complex. The recombinase domain of RAG1 derives from the recombinases of a distinct group of animal transposons known as Transibs.15 The recombination signal sequences of the immunoglobulin genes, which are recognized by the RAG1-RAG2 recombinase and are necessary for bringing together the V, D, and J gene segments, also appear to have evolved via Transib insertion.

The two independent origins of adaptive immune systems in prokaryotes and eukaryotes involving unrelated MGEs show that, in the battle for survival, organisms welcome all useful molecular inventions irrespective of who the original inventor was. Indeed, the origin of CRISPR-Cas systems from prokaryotic casposons and vertebrate V(D)J recombination from Transib transposons might appear paradoxical given that MGEs are primary targets of immune systems. However, considering the omnipresence and diversity of MGEs, it seems likely that even more Lamarckian-type mechanisms have, throughout the history of life, directed genomic changes in the name of host defense.16

Moreover, the genome-engineering capacity of immune systems provides almost unlimited potential for the development of experimental tools for genome manipulation and other applications. The utility of antibodies as tools for protein detection and of RM enzymes for specific fragmentation of DNA molecules has been central to the progress of biology for decades. Recently, CRISPR-Cas systems have been added to that toolkit as, arguably, the most promising of the new generation of molecular biological methods. It is difficult to predict what opportunities for genome engineering could be hidden within still unknown or poorly characterized defense systems. 

© KIMBERLY BATTISTA

Eugene V. Koonin is a group leader at the National Library of Medicine’s National Center for Biotechnology Information in Bethesda, Maryland. Mart Krupovic is a research scientist at the Institut Pasteur in Paris, France.

References

  1. T. Boehm, “Design principles of adaptive immune systems,” Nat Rev Immunol, 11:307-17, 2011.
  2. R. Medzhitov, “Approaching the asymptote: 20 years later,” Immunity, 30:766-75, 2009.
  3. K.S. Makarova et al., “Comparative genomics of defense systems in archaea and bacteria,” Nucleic Acids Res, 41:4360-77, 2013.
  4. J.E. Samson et al., “Revenge of the phages: defeating bacterial defences,” Nat Rev Microbiol, 11:675-87, 2013.
  5. R. Barrangou, L.A. Marraffini, “CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity,” Mol Cell, 54:234-44, 2014.
  6. F.J. Mójica et al., “Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements,” J Mol Evol, 60:174-82, 2005.
  7. A. Bolotin et al., “Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin,” Microbiology, 151:2551-61, 2005.
  8. K.S. Makarova et al., “A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action,” Biol Direct, 1:7, 2006.
  9. R. Barrangou et al., “CRISPR provides acquired resistance against viruses in prokaryotes,” Science, 315:1709-12, 2007.
  10. E.V. Koonin, Y.I. Wolf, “Is evolution Darwinian or/and Lamarckian?” Biol Direct, 4:42, 2009.
  11. K.S. Makarova et al., “Evolution and classification of the CRISPR-Cas systems,” Nat Rev Microbiol, 9:467-77, 2011.
  12. H. Kim, J.S. Kim, “A guide to genome engineering with programmable nucleases,” Nat Rev Genet, 15:321-34, 2014.
  13. K.D. Seed et al., “A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity,” Nature, 494:489-91, 2013.
  14. M. Krupovic et al., “Casposons: a new superfamily of self-synthesizing DNA transposons at the origin of prokaryotic CRISPR-Cas immunity,” BMC Biology, 12:36, 2014.
  15. V.V. Kapitonov, J. Jurka, “RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons,” PLOS Biol, 3:e181, 2005.
  16. E.V. Koonin, M. Krupovic, “Evolution of adaptive immunity from transposable elements combined with innate immune systems,” Nature Rev Genet, doi:10.1038/nrg3859, December 9, 2014.
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Avatar of: mlerman

mlerman

Posts: 53

January 6, 2015

The quetion is why the immune system fails in case of cancer?

Cancer genes fall into two main categories: cancer-causing genes, CCG, that drive malignant transformation and maintain tumor growth, and CAN genes that orchestrate local invasion and further spread of metastatic cells. CCG show high mutation rates (~100%) while the CAN genes show low mutation rates (5-10%). The number of genes involved in causation and cancer spread may be estimated from death frequencies as function of age (6-8 genetic steps to death from cancer to be in the range of 3-4 assuming both alleles affected; allowing haploinsufficiency for some of these genes the number may be higher but not to exceed 4-6. This small number is therefore responsible for malignant transformation, initial local growth and finally cancer spread by invasion and metastasis culminating in the death of the patients. The obvious discrepancy between this estimate and the much larger number of cancer genes as reflected in various “gene signatures” (http://cgap.nci.nih.gov/) suggests that most cancer genes associated with a particular cancer are not mutated. The over-/under-expression of these genes results from altered function of the original small set of mutated genes and their downstream targets; among these earliest targets there might be genes (“relay genes”) that multiply/diversify the genetic pathways (like HIF 1 and 2 alpha and BHLHB2). The total number of cancer genes assuming there are about 200 different human cancers will not much exceed 6,000-12,000 assuming 10%-5% mutation rates for CAN genes respectively. The number of CCG may not exceed 200. It is important to emphasize the fundamental role of VHL in tumor progression and creation of cancer stem cells (CSC). Michael Lerman, Ph.D., M.D.

Avatar of: James V. Kohl

James V. Kohl

Posts: 349

January 6, 2015

Thank you for  this comprehensive review.

I think the article supports this claim: Evolution in all its main steps occurs by competent agent-driven natural genome editing. (or genome dynamics events)

I think it refutes this claim: "...genomic conservation and constraint-breaking mutation is the ultimate source of all biological innovations and the enormous amount of biodiversity in this world." (p. 199)

I would like clarification from anyone who thinks that evolution occurs outside the agent-driven context of genome dynamics events linked to nutrient-dependent RNA-directed DNA methylation and RNA-mediated amino acid substitutions in animals.

I think biological energy from the sun must link light-induced changes in energy to amino acid substitutions that differentiate cell types in micobes, plants, and animals. Unless the evolution of biodiversity occurs via mutaitons, I think ecological variation can be linked from light-induced amino acid substitutions to nutrient-dependent cell type differentiation and ecological adaptations at the level of self vs non-self recognition, which may arise with energy level changes in 2 or 4 atoms.

The energy level changes may be stabilized by nutrient-dependent amino acid substitutions fixed in organized genomes of species from microbes to man by RNA-mediated protein folding and the physiology of nutrient-dependent reproduction.

The atoms to ecosystems model of cell type differentiation I just briefly detailed appears to be exemplified in unicellular and multicellur organisms with nutrient-dependent pheromone-controlled physiologies of reproduction. The physiologies arise in the context of biological energy from the sun that links plant life to animal life via the exchange of information carried by chemical signals in food odors and pheromones. 

The chemical signals regulate protein biosynthesis and degradation that is fine-tuned to fit with ecological variation that probably lead to ecological adaptions via immune system responses, which are negatively impacted by nutrient stress or by social stress.

For example, excess radiation energy  or chemical energy stress can be expected to begin with energy changes in atoms and changes in pre-mRNA, which are manifested in cell type-specific cancers. The cancers would be indirectly associated with genetic predispositions and life history transitions transgenerationally linked to nutrient stress or to social stress but directly linked from hormone-organized and hormone-activated morphological and behavioral development via RNA-mediated sex differences in the cell types of yeasts to breast cancer in human females

Avatar of: James V. Kohl

James V. Kohl

Posts: 349

Replied to a comment from mlerman made on January 6, 2015

January 6, 2015

I think the immune system fails due to accumulation of nutrient-stress and social stress induced DNA instability that goes outside the boundaries of what would typically occur only in the context of nutrient-dependent RNA-directed DNA methylation and DNA repair linked from RNA-mediated amino acid substitutions to DNA stability in organized genomes via protein folding.

I think that's what is now indicated in the context of nutrigenomics and pharmacogenomics that link metabolic networks to genetic networks via the bio-physically constrained chemistry of proteing folding manifested in differences in morphological and behavioral phenotypes but also in amino acid substitutions linked to genomic stability or instability in species from microbes to man via their nutrient-dependent pheromone-controlled physiology of reproduction.

January 7, 2015

There are lots and lots of detail here, along with as many acronyms. I try to see a big picture, aligned with Paul Ewald's idea that threatening an agent pushes it toward resisting, while providing a safe place but contolling growth pushes it toward commensalism. If our target is friendly adaptation rather than resistance then we ought to be looking at the latter instead of more deadly antibiotics.

It sounds like Crisper responds to threats in ways that benefit its posterity. That is helpful down the road. Does it do anything for diversity--a much better measure of the system's health?

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