Small Things Considered

Fine Reading: Endogenous Retroviruses

by Elio

I can't think of anything more startling, more promising, and more intractable in biology than the business of endogenous retroviruses. They seem to be ubiquitous in both invertebrates and vertebrates, and even in plants. In humans, they make up at least 8% of the genome, plus some bits and pieces. They are ancient; in our lineage they go back perhaps 100 million years. Let's face it, if we hadn't been told about them, we could never have conjured up such a strange story. It’s like hearing that you possess a limb or an organ that you didn't know about.

The very existence of endogenous retroviruses (ERVs) touches us in a number of ways. How is evolution shaped by the acquisition of such viral packets of genes? How often has it happened? Did it arise by infection, as seems likely? Is this an ongoing process that leads to further evolution? What does this have to do with the evolution of viruses? What does it reveal about the regulation of gene expression? On balance, do they do more good or more harm? All these questions have arisen elsewhere, but they are brought to the fore by these strange entities.

There is much to be learned, but certain facts stand out. A striking example of the good ERVs have done is seen in the case of the syncytin gene, whose product is involved in some essential way in the development of the placenta. It is thought that ERVs may also play a role in the immunosuppression needed to avoid fetal rejection by the mother. On the other hand, can they cause cancer? With some laboratory-induced exceptions, ERVs have not been found in tumors. On the other hand, ERVs in humans and great apes control the formation of a protein (GTAp63) that protects the germ line from cancers. In addition, ERVs may contribute to resistance to exogenous retroviruses. So much for viruses being “bad.”

It follows that knowing about endogenous retroviruses should be part of the education of all biologists, not just retrovirologists. I recommend a review by Jonathan Stoye entitled Studies of Endogenous Retroviruses Reveal a Continuing Evolutionary Saga. Here you will find a clearly written account of ERV genomic architecture, reproductive cycle, and phylogeny. Also discussed are some of the consequences of their presence, including the expression of viral proteins, the control of host processes by ERVs, and their cost-benefit balance. The complex subject of controlling retroviral replication is explained with clarity, as is the evolutionary interplay between the ERVs and their host. You might as well start somewhere, and this is a good place for it.

Where Mathematicians & Biologists Meet

by Joe Mahaffy

Source.

Mathematics and Biology have a long history together. It goes back to early studies on epidemiology (such as John Snow‘s on cholera and the Broad Street pump), and includes Ross’s quantitative studies that show how malaria can be controlled by careful analysis of data. And, of course, there are many others. In the early 20^th century, population models with differential equations were developed to describe the dynamics of populations, such as the studies of Alfred Lotka, who felt that natural selection could be quantified by physical laws, and Vito Volterra, who created a model to explain the predator-prey ratios in the Italian fish markets. These early models provide excellent tools because in their simplicity they show biologists how mathematics can help explain noteworthy biological phenomena. Mathematicians enjoy such models because the examples themselves make it easier to explain what the equations are describing.

The Broad Street Pump (modern
replica). Source.

        Dr. Snow. Source.

Key to the development of the collaboration of molecular biologists and mathematicians was the seminal work of A. L. Hodgkin and A. Huxley on the action potentials in nerve cells. They modeled the nerve cell membrane as if it were an electric circuit, using paradigms developed from physics, and they ran extensive experiments to find the best fitting functions and parameters for their model. This work is unique in that the mathematical model contains such admirable detail that even 60 years later it continues to reveal molecular insights, such as the properties of ion channels.

Vito Volterra (1860-1940).
Source.

Alfred J. Lotka (1880-1949).
Source.

Mathematicians who work with biological problems generally fall into three categories. The first group takes existing models from mathematical biology, then adds a variety of terms to explore the robustness of the mathematics. By concentrating on mathematical properties that expose weaknesses in the models, they don’t contribute much to biological understanding, generally speaking. The second group considers biological phenomenon, looks for mathematical patterns that match experimental observations, and tries to connect the equations to the biology. This technique was made famous by René Thom and his 1970 catastrophe theory, but it turned off many biologists because of the ad hoc nature of this kind of modeling. The last group consists of mathematicians who work closely with biologists to obtain detailed models that help disclose key underlying biological principles.

Brian Goodwin, a key founder of theoretical biology.
Source.

A classic example of collaboration: Jacob and Monod developed the biochemical basis of genetic repression and induction—the operon model. Then, in 1963, B. C. Goodwin transformed it into systems of differential equations. This was followed by an explosion of research in the mathematical modeling of biochemical control loops. These control systems can exhibit complex dynamics, which makes them interesting for mathematicians to study, while biologists appreciate how their precise descriptions can differentiate between several possible outcomes. An example from my own experience is my collaboration with Judith Zyskind when she was studying the cell cycle of E. coli. We designed a model based on the then known biology that supported the now commonly accepted key role of DnaA protein in controlling the initiation of replication. The mathematical model helped elucidate key steps in the complex control loop, while the biological features were key to constructing the mathematical model.

The Lotka-Volterra model produces regular oscillatory
cycles in predator and prey populations. Source.

Such models based on biochemical kinetics have provided enticing studies for mathematicians in various fields. Biological examples of interesting dynamical systems run the gamut from robust stable systems to regular oscillatory conduct to chaotic behavior. Mathematically, these topics belong to the area of dynamical systems and bifurcation theory. Mathematicians took notice of interesting complex biological problems when in 1976, May showed that the discrete logistic growth of organisms exhibits chaotic behavior. This chaos theory has exploded due  both to the increasing computational power available and the challenges that it poses mathematically. It has been proposed for explaining many biological phenomena, including observed patterns in organizational biology, using fractal geometry (made most famous through the work of Mandelbrot).

Mathematical models of stochastic processes can help elucidate the behavior of biological systems because many biological events in the cell are governed by small numbers of molecules. McAdams and Arkin, for example, used stochastic models to show how phage l could choose either a lytic or a lysogenic pathway under similar conditions simply due to random fluctuations in the underlying molecular events. Given enough computing power, these stochastic simulations can be used to study bifurcating paths of evolution or co-evolution of paired species.

The repressilator genetic regulatory network. Source.

The emerging field of synthetic biology has a tight connection to mathematics. This merger really started in 2000 with Collins’ genetic toggle switch and Elowitz and Leibler’s “repressilator,” which showed how theoretical models could be used to reproduce certain biological control systems. A wealth of new designs have followed that help explain observed biological behaviors, and new tools have been created to explore biochemical pathways. These endeavors require that mathematician and biologist work closely together in the design of particular models. The biochemical pathway in question must be carefully engineered due to the limited range of the model’s parameters that follow from mathematical bifurcation studies of the governing differential equations. The actual experiments often show differences from the desired behavior, which in turn lead to further mathematical studies to design a more robust control loop.

The above discussion highlights only the bridges between mathematics and molecular kinetics in biology via the use of dynamical systems. Many other connections exist between biology and mathematics, for example between cell motility and fluid dynamics or between bacterial growth and pattern theory. Simply put, there exists a huge array of complex biological problems where the valuable tools of mathematics have enabled better understanding of biological processes, and where the biology challenges mathematicians with difficult analytic problems. These are problems that evolution has solved but that remain a mystery to us.

Courtesy of Jim Crowley, Executive Director, SIAM.

Mathematical biology is still a relatively young field of study, and it is growing rapidly. Many of the best attended and most exciting talks at major conferences are coming from this collaborative field. The Society of Mathematical Biology was one of the first professional groups formed about 35 years ago, while the largest applied mathematics group, the Society for Industrial and Applied Mathematics (SIAM), formed a special group for mathematical biology about 10 years ago. I present a graph showing the growth of the Life Science Activity Group of SIAM. Even the largely pure mathematics organization, American Mathematics Society, has created sessions in mathematical biology in many of their regional meetings in the last 10-20 years.

Yet there are hurdles remaining. A large gap in understanding and communication still exists between biologists and mathematicians. Many biological problems appear to be too complex for detailed mathematical analysis. For example, complex biochemical pathways with their many parameters can rarely be completely modeled. Mathematicians can point out key elements in a pathway, but they may miss important small variations critical to biological adaptation. On the positive side, the advancement of mathematical biology has been accelerated by the increase in computer power, by better numerical tools, and by more students of mathematics accepting the challenge of biological applications. Paralleling these developments on the biological side is the more rigorous quantitative training of young biologists. The groups are meeting in collaborations and interdisciplinary work, to the clear benefit of both fields. This is an incremental process, as each field learns what can and cannot be done in the specific areas. Collaborations between biologists and mathematicians will continue to be a major growth area in the future as the disciplines become increasingly intertwined.

Joe Mahaffy is Professor of Applied Mathematics in the Department of Mathematical Sciences, San Diego State University.

The Bacterial Resistome is Both Ancient and Surprising

by S. Marvin Friedman

The Soda Straw Room in the Lechuguilla Cave. These pencil-thin
stalactites are called soda straws from their size and the fact
they have a central canal through which mineral-laden water flows.
Although it is not uncommon for soda straws to grow three to five
feet long, the ones in this room are as long as 15 feet. Credit: Norm
Thompson. Source.

One of the many interesting controversies that microbiologists can ponder today is whether the alarming proliferation of antibiotic-resistant strains is primarily a consequence of the widespread use of antibiotics in humans and in animal husbandry. An examination of bacteria isolated from terrestrial animals in the Galapagos, a remote location with limited exposure to humans, revealed  an absence of antibiotic resistance genes. Likewise, plasmids from bacterial collections that predate the antibiotic era were mostly devoid of resistance elements. Furthermore, a study of Dutch soil samples showed an increase in antibiotic resistance genes in contemporary samplings as compared to those from the pre-antibiotic era. On the other hand, antibiotic resistance genes were both abundant and diverse in ancient DNA recovered from Pleistocene deposits (30,000 years ago). Likewise, a survey of present-day actinomycetes revealed that multidrug resistance was prevalent even though human sources of antibiotics were presumed absent in this environment. It should be noted, however, that the actinomycetes are prolific antibiotic producers and thus it is not surprising to find resistance elements enriched in this group.

In order to further explore the evolution of the resistome, Bhullar and co-workers cultured bacteria isolated from the Lechuguilla Cave located within Carlsbad National Park in New Mexico. The deep recesses of this cave have been isolated from surface input for the past 4-7 million years and therefore constitute an ideal locale for carrying out this study. The Lechuguilla bacterial collection, consisting of 31 Gram-positive and 62 Gram-negative strains, was screened against 26 antimicrobial agents. Similar to their surface cousins, most of the cave strains were multidrug resistant; a few were resistant to 14 of the antimicrobials tested. Unlike surface bacteria, however, few or none of the surveyed cave bacteria exhibited resistance to the synthetic antibiotics ciprofloxacin and linezolid or to the natural product antibiotics tetracycline, vancomycin, and rifampicin. A previous study found that all of 480 soil-derived bacterial isolates were resistant to daptomycin, compared to only about 1/6 of these from the cave.

Resistance levels of Lechuguilla cave bacteria at 20 μg/ml against various antibiotics: (top) Gram-positive strains (bottom) Gram-negative strains. Antibiotics are grouped according to their mode of action/target, where each color represents a different target. Source.

These researchers identified several antibiotic inactivation mechanisms in cave bacteria that had not been previously observed in those particular species, two of which are of special interest. Four strains of Paenibacillus lautus inactivated daptomycin probably via a metalloesterase that hydrolytically opens the ring structure. Daptomycin inactivation had not previously been found in the Firmicutes. Macrolide inactivation in the cave bacterium Brachybacterium paraconglomeratum resulted from phosphorylation by a kinase of the MPH class. Previously identified mph genes are encoded on plasmids found in resistant isolates of pathogenic strains of Escherichia coli, Staphylococcus aureus, Pasteurella multocida, and Pseudomonas aeruginosa. Were those mph genes housed in the pathogens acquired by horizontal gene transfer from environmental saprophytes such as this cave dweller?

The take-home message from this paper is that the bacterial antibiotic resistome is both ancient and genetically diverse. The warning here for the clinical community is that there are still plenty of new genes out there in the environment that can be appropriated by disease-producing bacteria in order to escape death by the action of our antimicrobial agents. On the other hand, the good news coming from these findings is that there are many compounds with antibiotic activity just waiting to be discovered.

Marvin is Professor Emeritus in the Department of Biological Sciences at Hunter College of CUNY in New York City, and an Associate Blogger for Small Things Considered.

Bhullar K, Waglechner N, Pawlowski A, Koteva K, Banks ED, Johnston MD, Barton HA, & Wright GD (2012). Antibiotic resistance is prevalent in an isolated cave microbiome. PloS One, 7 (4) PMID: 22509370

If It Walks Like DNA, and Talks Like DNA…

by Merry

The Hawk Moth Caterpillar inflates its thorax as a
defense mechanism, resulting in a snakelike appearance,
complete with false eyes. Source.

Conjugative plasmids and transposons have been found guilty of spreading antibiotic resistance genes from pathogen to pathogen.  But how do they get past the bacterial defenses against incoming foreign DNA? Most bacteria have some sort of restriction-modification system to take care of just such molecular invaders. With such a system, they modify their own chromosomal DNA by adding methyl groups to specific residues within a short recognition sequence which is different for different enzymes. Incoming DNA that doesn’t have those particular sites methylated is “restricted,” i.e., cleaved, by the restriction endonuclease. This is end of game for an incoming phage or other mobile element that had replicated in a different strain or species.

So, how come horizontal gene transfer (HGT) in general, and in particular HGT by conjugative plasmids ferrying antibiotic resistance factors, occurs as frequently as it does? One reason is that plasmids (like phages) use a number of tricks to avoid restriction. A particularly interesting example is provided by a plasmid-encoded endonuclease inhibitor that protects the incoming plasmid DNA, thereby opening the door for plasmid transfer between unrelated bacterial cells.

Conjugative plasmids are self-starters. They provide all the mechanisms they need to transfer without any help. First they direct the establishment of cell-cell contact with a recipient cell. Then the plasmid in the donor cell replicates by a rolling circle mechanism, and transfers one strand of its DNA genome, the T-strand, to the recipient cell. Since restriction endonucleases act on double-stranded DNA, the transferred single-stranded genome has temporary immunity. It becomes vulnerable only when the complementary strand has been synthesized. This buys the plasmid some time to establish its anti-restriction defense, perhaps as much as an hour.

Some plasmids, including the colicin 1B plasmid (Col1b), encode a protein that provides protection against restriction endonucleases. There is a large family of such proteins called Ard for Alleviation of Restriction of DNA; the particular one encoded by this plasmid is a member of the ArdA subgroup. These proteins get around. They are encoded by plasmids in enterobacteria including E. coli and Yersinia pestis, by conjugative transposons, and by chromosomes in Actinobacteria, Cyanobacteria, Proteobacteria, and Firmicutes.

ArdA inhibits the restriction activity of Type I restriction endonucleases in vivo. (Type I restriction-modification systems are a diverse lot [classified in four families], are widely distributed, and have been identified in more than half of the sequenced bacterial genomes.) Just carrying a gene for an endonuclease inhibitor isn’t enough to protect an incoming plasmid. The plasmid has to ensure that enough of the inhibitor protein is synthesized in the recipient cell by the time its genome becomes the vulnerable duplex DNA. The colicin 1B plasmid handles this quite elegantly. Its ardA gene is located near the leading end of the T-strand, the region of the plasmid DNA that enters the cell first. This gene is rapidly transcribed from the single-stranded genome by the E. coli RNA polymerase due to an unusual promoter—a region of secondary structure formed by imperfect intramolecular base pairing. Synthesis of the complementary DNA strand ends this burst of ardA transcription by disrupting the base-paired promoter and ushers in the phase of normal transcription from duplex plasmid DNA.

The dimeric structure of ArdA and its distribution of negative charge clearly mimic a DNA double helix. (a) The ArdA dimer showing the linear arrangement of domain 1 (red and fuchsia), domain 2 (orange and yellow), and domain 3 (blue and cyan). (c) The surface of ArdA with acidic residues coloured red and basic residues coloured light blue. The negatively charged residues form a helical pattern across the surface. (d) An overlay of a selection of the acidic residues in the dimer, viewed from the same angle as in (c), onto the enzyme-bound DNA duplex. Only the phosphate backbone of DNA is shown (red and white) with the acidic residues from ArdA matching one strand shown in yellow and those matching the other strand shown in green. Source.

 

How does ArdA inhibit the host’s restriction endonuclease? This makes an interesting story. You might expect that such a protein inhibitor would compete with the enzyme by binding to the specific DNA sequence recognized by the endonuclease. Good guess, but that doesn’t appear to be the case because the same ArdA protein not only inhibits endonucleases from all four families, but inhibits ones with different recognition site specificities. Instead, ArdA mimics the shape and charge of a ~42 bp section of B-form DNA. It mimics it so well that the enzyme is tricked into binding to it, instead of to its usual DNA substrate. We’re all familiar with biological mimicry, but this is quite a stunning example at the molecular level.

All the members of the ArdA protein family encode small (166-169 amino acids), highly acidic proteins that carry a net charge of -22 to -29. Structural studies of the ArdA protein monomer from conjugative transposon Tn916 showed a very unusual shape. Its three domains are arrayed in almost a straight line, thereby producing a very elongated molecule (70 Ǻ × 20 Ǻ). This protein is virtually all surface, with almost none of the usual hydrophobic core. In fact, only eight amino acid residues are shielded from the solvent. The biologically active form is a head-to-head dimer, thus longer still. The location of the many negatively-charged amino acids produces two ribbons of negative charge on a raised surface that entwine around the entire length of the molecule—strikingly similar to the distribution of negative charges of a helical DNA phosphate backbone. The overlay of the dimer on a segment of B-form DNA (see figure) makes the mimicry obvious. In light of this, it is not surprising that while the amino acid sequences of the various ArdA proteins show considerable variation, the location  of the many charged amino acids is highly conserved. Furthermore, there is even a surface groove that runs the length of the dimer akin to the major groove of the DNA double helix. Lastly, to complete the deception, the dimer is curved, mimicking the shape of a DNA helix when bound to the active site of the endonuclease. No wonder the enzyme is fooled.

A similar elongated acidic protein has been found in another place—the restriction endonuclease inhibitor encoded by coliphages T3 and T7. This one, dubbed Ocr (Overcoming Classical Restriction), is unrelated to ArdA but it plays a similar role, actually doing it a little better—because it has to. The incoming phage genome is dsDNA, thus is immediately vulnerable to the endonuclease. There isn’t time to synthesize much Ocr. Protection must be rapid and effective. Ocr binds the endonucleases more strongly that does ArdA, so much so that about 1700 time as much ArdA is needed for the same level of inhibition. Ocr binds the enzyme 50 times more strongly than does DNA itself. Quite a good mimic, wouldn’t you say?

The wings of a South African speckled emperor moth.
Source.

We are reminded that to be a successful mimic does not require exact duplication. You only need to possess the few key attributes that signal loudly to the receiver. Thus, here it matters little that you build your mimic of amino acids rather than nucleotides so long as you end up with something of the right size, shape, and surface distribution of negative charges. These examples of molecular mimicry also convey a message to us, telling us something about “perception” at the level of molecules.

McMahon SA, Roberts GA, Johnson KA, Cooper LP, Liu H, White JH, Carter LG, Sanghvi B, Oke M, Walkinshaw MD, Blakely GW, Naismith JH, & Dryden DT (2009). Extensive DNA mimicry by the ArdA anti-restriction protein and its role in the spread of antibiotic resistance. Nucleic Acids Research, 37 (15), 4887-97 PMID: 19506028

Walkinshaw MD, Taylor P, Sturrock SS, Atanasiu C, Berge T, Henderson RM, Edwardson JM, & Dryden DT (2002). Structure of Ocr from bacteriophage T7, a protein that mimics B-form DNA. Molecular Cell, 9 (1), 187-94 PMID: 11804597

Talmudic Question #87

By John Ingraham

Can you think of a species that has been wiped out by an infectious agent?

Polar Enchantment

by Elio

NASA's Polar spacecraft captured the first-ever movie of
auroras dancing simultaneously around both of Earth's
polar regions. Source.

All bacteria, even those that are spherical, display geometric asymmetries during at least part of their life cycle, and it appears that cells take advantage of these asymmetries to localize cellular components to specific sites. So begins a paper by Li and Young that describes a novel approach for detecting E. coli proteins that preferentially localize to the cell poles. There is already a considerable catalog of “polar” proteins in various bacteria, some involved in chemotaxis, signaling, regulation of septation, flagella synthesis, phage attachment, and a raft of other phenomena. Pretty soon you may be tempted to ask which proteins do not localize to the poles. This may sound facetious, but it illustrates the recent changes in mindset regarding the structural complexities of the prokaryotic cell. But many questions remain. We readily subscribe to the notion that bacteria are not bags of enzymes but this begs the broad question “what are they?”

As the authors point out, findings of pole-localized proteins have been largely accidental, when researchers weren’t specifically looking for them. They contend that the time has come to be more systematic about this matter. Granted, some workers had earlier looked at minicells as proxies for the poles because each had started life as a pole of their mother cell. Others had fused specific cellular proteins to fluorescent proteins such as GFP and then simply screened the cells under the microscope looking for those where fluorescence accumulated at the poles. These researchers, instead, set out to develop a novel technique to intentionally and specifically pull out “polar” proteins. Their technique uses affinity chromatography of an inner membrane protein known to be localized in the polar regions. The idea was to tag the protein with an epitope and retrieve membrane vesicles that contain it by using beads coated with an antibody against that epitope. The underlying rationale is that neighboring membrane proteins in the same vesicles can be assumed to come along for the ride. Sure enough, this approach worked: they identified this way some new “polar proteins” as well as some previously known to be pole-located .

Protein profiles in polar and non-polar vesicles. Pole-derived
vesicles labelled with Tar-P-FLAG or Tar-C-FLAG and non-
pole-derived vesicles labelled with Tar-P-FLAGD were isolated
by affinity capture by using anti-FLAG beads. Triton X-100-
and SDS-eluted samples were separated on a 12% SDS-PAGE
gel and stained with Sypro Ruby. Total inner membrane (IM)
proteins from E. coli MG1655 cell envelopes were solubilized
with Triton X-100 and loaded for comparison (lane 1). Protein
bands to the left of each capital letter were removed and
identified by mass spectrometry. (A) Tar, Tsr, Tap and Trg;
(B) MukB; (C and D) Tar multimers. Tar-FLAG bands are
indicated by arrows. M, gel lanes subjected to full-lane
GeLC-MS/MS analysis. Source.

The authors chose as the affinity lure a pole-associated protein, the chemoreceptor Tar. This is a well-studied inner membrane protein that is relatively abundant at the poles. They tagged it with a short epitope, a widely used octapeptide called FLAG, which they engineered into a periplasmic loop of Tar. The methods and controls used in the paper are worthwhile reading. In brief, they used a plasmid carrying the modified Tar gene to express the Tar-FLAG protein in E. coli cells. Next they ran the cells through a high shear fluid processor to generate membrane vesicles. Because the FLAG tag extends into the periplasm, it would be exposed on the outside of any right-side out inner membrane vesicles. They now fished out the vesicles containing Tar-FLAG by mixing them with beads coated with anti-FLAG antibody. SDS-agarose electrophoresis showed that they had enriched for specific protein bands that were identified by mass spec. By using a modified Tar protein that has lost its polar specificity, they also showed that this technique could retrieve non-polar membrane proteins.

The authors identified an impressive number of proteins (22) found only at the poles. Some were known, some were new. Another set consisted of proteins were detected at both polar and non-polar locations, with some of them being enriched at the poles. Among the most abundant proteins at the poles were, as expected, the chemotaxis proteins (Tar, Tsr, Tap, Trg, and CheA). To establish that their other candidate “polar” proteins are, in fact, polar, they selected 15 of them for construction of GFP fusions. Four of the fusion proteins (Aer, YqjD, TnaA, and GroES) exhibited detectable GFP fluorescence by microscopy.

The plague bacillus, Yersinia pestis, in a bloody
exudate. Note the characteristic safety pin
appearance of the bacteria, which are the small
blue rods in the midst of red and white blood
cells. Source: CDC.

There are pertinent caveats when interpreting experimental data as evidence of polar localization, one being that denatured or misfolded proteins tend to aggregate into inclusion bodies that find their way to the poles. This leads me to a small digression: In my early days, I learned that stained cells of the plague bacillus (Yersinia pestis), the agent of melioidosis (Burkholderia pseudomallei), and some others, look like a safety pin, that is, the dye used for staining them accumulates at the poles. What is enriched are polyphosphate storage granules or, in other species, stored glycogen, sulfur, or polyhydroxyalkanoates. Back to this paper, the authors were able to show that their polar proteins were not in inclusion bodies. They used an mCherry-tagged chaperone protein (IbpA) that is involved in processing protein aggregates and which labels them in vivo. They detected no fluorescence in the cells. Another nice control: the polar location of the GFP-fused proteins was not somehow determined by Tar because the same fluorescence results were obtained in the absence of Tar.

Origin of TnaA and GroES polar foci. Percen-
tages of nascent (smaller) TnaA foci observed
at the old pole, at the new pole or at midcell.
Note that nascent polar foci might have
originated at midcell in a previous generation.
Nascent GroES foci were located most often
at midcell. Source.

Two of the three fusion proteins found to be polar, TnaA–GFP and GroES–GFP, formed only a single focus per cell. Since the proteins were localized at the poles at different times during growth of the cultures, they concluded that TnaA and GroES get there independently and are each unaffected by the other, even when expressed in excess. Thus, there are different ways to get to the poles. Of interest was also the observation that polar localization of three protein, TnaA, GroES, and YqjD, was disrupted in cells lacking the MinCDE proteins, which is known to operate in cell division by helping to place the septum at mid-cell. This suggests that this system may also help localize proteins that are not involved in cell division.

Where are we now regarding polar location? The authors posit that our knowledge is seriously incomplete. There is no comprehensive inventory of polar constituents, we know little about how they travel to the poles. Are they attached to something? Is the location transient or continuous? The list of possible explanations for polar predilection is rather short but a few examples stand out. For instance, in a rod-shaped cell with a single flagellum, such as Caulobacter or Vibrio, it would make little sense to sprout the structure in the middle of the cell. Ditto for bacteria such as Listeria or Shigella which, when inside host cells, recruit cytoplasmic actin to a pole-located protein and use that for movement. And doesn't it seem sensible that chemotactic proteins should be associated with one pole, which functions as a “nose” to “smell” attractants or repellents? The list goes on a bit further to include the role of the poles in chromosome replication and segregation, phage receptors, and DNA uptake.

So, this is a mixed bag. In some cases, the polar location of proteins and cell structures makes good sense and helps us understand their function. Surely, as implied in this paper, these salient examples are but the tip of the polar iceberg. Much more awaits discovery. You can say that also for the two poles of our earthly concern.

Li G, & Young KD (2012). Isolation and identification of new inner membrane-associated proteins that localize to cell poles in Escherichia coli. Molecular Microbiology, 84 (2), 276-95 PMID: 22380631

What Happened to Our Friendly Enterococci?

by Merry

Enterococci had been generally regarded as benign commensals, a part of our healthy intestinal microbiota. They were even invited in, being used as probiotics. But then, in the late 1970s, the first multiple drug-resistant strains appeared, and vancomycin-resistant strains followed in 1981. In recent decades, they have taken center stage as one of the pathogens most commonly associated with nosocomial infections. Enterococcal infections are particularly difficult to manage now because these bugs have accumulated numerous virulence genes and antibiotic resistance factors. What happened?

First, a few facts about these organisms. Enterococcus is a genus of Gram-positive lactic acid bacteria in the phylum Firmicutes. They used to be called streptococci because their typically paired cells look so similar. The two species found in our intestines, E. faecalis (90–95%) and E. faecium (5–10%), are facultative anaerobes.

Now here’s your first clue as to their change into pathogens. Hospital-adapted strains have more DNA than their benign relations—more than 600 kb of additional coding potential, enough for hundreds of genes—mostly in the form of acquired mobile elements. The first enterococcus to have its genome sequenced was one such hospital-adapted E. faecalis strain. I’ll have more to say about this strain (V583) later. One quarter of its genome is composed of mobile elements, including three independently replicating plasmids, three chromosomally integrated plasmid remnants, seven prophages, and a pathogenicity island. Here reside several virulence genes as well as antibiotic resistance factors.

Functional CRISPR loci and acquired antibiotic resistance in a
historical collection of E. faecalis strains. (Click figure for larger
version.) E. faecalis strains are listed by date of isolation, from
oldest to most recent. Acquired antibiotic resistance is shown
in red, and the presence of a functional CRISPR defense is
shown in green. Antibiotic resistance genes: tetracycline (tetL
and tetM), erythromycin (ermB), gentamicin (aac6’–aph2’’),
chloramphenicol (cat), ampicillin (blaZ), and vancomycin (vanA
and vanB). ** = strains for which draft or complete genome
sequences are available. The specific commensal oral strain
and multiple drug-resistant hospital-adapted strain discussed
in the text are noted. Source.

The second clue: That same sequenced hospital-adapted strain does not have a functional CRISPR defense against incoming mobile elements and it carries multiple drug resistance factors (and virulence genes). In contrast, an antibiotic-sensitive oral E. faecalis isolate (OG1RF) has a CRISPR defense and contains few mobile elements. Hmmmm… makes one wonder. To see if this correlation applies to other strains, researchers analyzed 45 E. faecalis isolates, some of which predated the antibiotic era. Strains without CRISPRs were associated with more antibiotic resistance traits (see figure). They also surveyed eight E. faecium genomes and found the same pattern. This seems to make sense. One might expect that strains that had lost their CRISPR defense would be more apt to acquire mobile elements carrying antibiotic resistance factors. Such strains would fare well, i.e., would be under strong positive selection, in the antibiotic-rich hospital environment.

Correlations, even statistically significant correlations such as this, do not prove causation. However, here’s a piece of supporting experimental evidence. These strains carry pheromone-responsive plasmids that are experts at HGT between closely-related bacterial strains. These plasmids shut down the synthesis of specific pheromones by their host. When in the vicinity of plasmid-free cells, the plasmids respond to the pheromones by inducing their own host to initiate mating, thus greatly increasing the frequency of plasmid transfer to suitable host cells. Earlier these researchers showed that plasmids in the hospital-adapted strain can transfer large segments of chromosomal DNA, including the vancomycin resistance factor, into the chromosome of the oral commensal strain. In a more recent paper, they demonstrate that a single conjugative transfer event can produce a new strain that is both antibiotic resistant and lacking a functional CRISPR locus. Under some circumstances, such a strain would be at a disadvantage because it would be less able to defend its genome against invasion by foreign elements. However, the ability to acquire mobile elements carrying useful antibiotic resistance factors and virulence genes would provide the new strain with a ticket for success in a hospital setting.

So perhaps this is what happened to our commensal enterococci. If so, then we played a part in their replacement. Our antibiotics can be effective in ways we don’t even realize.

Palmer KL, & Gilmore MS (2010). Multidrug-resistant enterococci lack CRISPR-cas. mBio, 1 (4) PMID: 21060735

Are Phages the Answer?

by S. Marvin Friedman

The emergence of multiple drug-resistant bacterial strains, the prevalence of recalcitrant biofilm configurations, and the reluctance of the pharmaceutical industry to initiate new antibiotic discovery programs have led to the development of a formidable population of bacterial pathogens that is increasingly difficult to control. After a long but successful era of research that had all but eliminated serious threats from bacterial infections, we are now facing this dire problem once again. In response, researchers have recently been exploring alternative approaches to antibiotic therapy including identifying chemical agents that antagonize quorum sensing and thus prevent population-wide expression of virulence genes, as well as employing either intact bacteriophages or their isolated lysins to directly kill their pathogenic bacterial hosts. Lysins kill Gram-positive bacteria by hydrolyzing the peptidoglycan in the cell wall, thereby causing cell lysis. Gram-negative bacteria are immune to their action because their outer membrane does not allow the lysins access to their peptidoglycan. I will now summarize two recent papers that use intact phages to combat two important bacterial pathogens, both in vitro and in vivo.

Pseudomonas. aeruginosa colonies showing the
characteristic green color. Source: Gloria Delisle,
Microbe Library, ASM.

One of the important applications for phage therapy is for treating cystic fibrosis (CF). CF is an inherited genetic disorder where a defective enzyme results in the production of unusually viscous, sticky mucus and chloride-containing secretions in ducts and body cavities. The lungs, in particular, are seriously compromised and are readily infected, typically by Pseudomonas aeruginosa. Initial colonization usually occurs during early childhood. The ensuing chronic infection eventually causes death due to respiratory failure in 80–95% of CF patients. Treatment of these patients is impeded by the multiple mechanisms of antibiotic resistance harbored by these strains of P. aeruginosa and by their ability to form biofilms in the lung.

A research team has recently investigated the efficacy of phage therapy in the treatment of CF. They isolated two phages (φMR299-2 and φNH-4) from sewage and showed that both were virulent for P. aeruginosa. The virions of one have the isometric capsids and short tails characteristic of members of the family Podoviridae, whereas the other possesses an isometric capsid and long contractile tail and therefore is classified in the Myoviridae. The authors used equal numbers of the two phages in all experiments.

(A) Growth of lux-tagged Pseudomonas biofilms
on the surface of a CF-bronchial epithelia cell
monolayer. Light was measured 1, 5, and 24 h.
(B) Readings from 6 wells. Source.

The researchers employed bioluminescence imaging to assess the ability of this phage mix to kill Pseudomonas cells in biofilms on the surface of a monolayer of a cystic fibrosis bronchial epithelial cell line. They inoculated confluent monolayers with two lux-tagged Pseudomonas strains, one mucoid, the other non-mucoid. They also provided arginine, which is required for good biofilm formation. After 24 hours, bioluminescence indicative of the growing bacterial biofilm increased one hundred-fold. Most of the lux-tagged Pseudomonas cells resisted washing, indicating that they were anchored to the epithelial monolayer. Numerous Pseudomonas cells were packed within the biofilm matrix and attached to the epithelial cells, as shown by staining with Calcofluor white, a fluorescence dye that binds to polysaccharides in biofilm matrices.

Fluorescent image of 24-h-old culture of P. aeruginosa cells grown on a CF-epithelial cell monolayer after Calcofluor white staining. Staining confirms that P. aeruginosa strain NH57388A (A) and strain MR299 (B) are embedded in an exopolysaccharide structure prior to phage exposure. After 24-h incubation with mixed phages, matrices become open and, with reduced numbers of cells for both NH57388A (C) and MR299 (D). (E) The phage titers over the 24-h incubation. Source.

Is the phage mixture effective against the Pseudomonas biofilms? When the Pseudomonas cells were cultured for 24 hours in the presence of the phages, Calcofluor white staining showed only weak and open matrices, indicating considerable disruption of the biofilm architecture. Phage titers increased about one hundred-fold during the 24 hour period, confirming that substantial phage replication had occurred. In addition, direct plating confirmed that this reduced luminescence resulted from the killing of Pseudomonas cells by the phages. The conclusion is that the phage mixture was effective in killing Pseudomonas cells embedded in biofilms growing on a bronchial epithelial cell line.

To test effectiveness of the phages in vivo, they infected 8 week-old female BALB/c mice intranasally with lux-tagged Pseudomonas and confirmed the presence of the labeled bacteria in the lungs 2 hours later. Notice the simple elegance of this model. Not only do the Pseudomonas cells localize almost eclusively in the respiratory tract, but their whereabouts can be seen by simply taking the mice to a dark room. At the 2 hour point, the researchers divided the mice into control and test groups and inoculated the test group intranasally with the phage mixture. At 6 hours, the luminescence recorded in lungs of the control group had reached its maximum level, a three-fold increase. The luminescence recorded from the phage-treated mice decreased significantly during the same period. It sounds promising.

Mice were infected with nonmucoid P. aeruginosa MR299 (A) and mucoid NH57388A (B). Test mice (+) were treated with the phage mix. Phage was given 2 h after infection with Pseudomonas. Control mice (−) did not receive the phage mix. Source.

The story of phages as therapeutic tools goes back to their earliest history. In 1919, D’Herelle, one of their co-discoverers, successfully treated a patient with dysentery. Over the course of time, phage therapy has had its ups and downs. The allure of using phages when other measures fail has continued and current day research may well restore them to a deserved place in the antimicrobial armamentarium.

Currently, phage therapy is successfully practiced in several European countries such as Georgia, Poland, and Russia. In the United States, the Food and Drug Administration has recently approved the use of phage on food to prevent contamination of meat and poultry. Reluctance to extend the use of phage therapy to control bacterial infections in humans stems from concerns over immunogenic reactions due to the presence of large numbers of phage in the circulation. In addition, the release of endotoxins from Gram-negative bacteria attacked poses another potential problem. Lastly, defined dosages cannot be formulated since successful treatment releases more progeny phage, thus potentially increasing the “dosage” available to reinfect host cells.

Paul et al. have overcome these objections by engineering a lysin-deficient phage. For this purpose, they modified a temperate phage (P954) that infects Staphylococcus aureus. Because methicillin-resistant strains of S. aureus (MRSA) infect about 94,000 people in the U.S. each year, resulting in almost 19,000 deaths, new therapies are urgently needed. The researchers disrupted the native lysin gene carried on the prophage by introducing the chloramphenicol acetyl transferase (cat) gene via homologous recombination using a plasmid construct. A lysin-deficient phage does not degrade the bacterial cell wall and thus cannot lyse the cell, but the phage still produces a holin protein that creates gaping holes in the inner membrane at the close of the phage replication cycle. As a result, the cell can no longer carry out respiration and death rapidly follows. To test the efficacy of lysin-deficient phage P954, they injected immunocompromised mice IP with the MRSA isolate B911 at a dose that causes 80% mortality. IP administration of lysin-deficient phage P954 immediately and 2 hours after challenge fully protected the mice from the lethal effect of MRSA. Importantly, the lysiin-deficient phage alone was neither toxic nor lethal for the mice. Thus, lysin-deficient phage P954 appears to be an excellent candidate for treating difficult MRSA infections in humans.

Phage therapy has had a checkered and intense history. The final word may not be in, but studies such as these make it appear that it has a substantive future. That would be a good thing, not only for cystic fibrosis patients, but perhaps for all who suffer from recalcitrant infections.

Marvin is Professor Emeritus in the Department of Biological Sciences at Hunter College of CUNY in New York City, and an Associate Blogger for Small Things Considered.

Alemayehu D, Casey PG, McAuliffe O, Guinane CM, Martin JG, Shanahan F, Coffey A, Ross RP, & Hill C (2012). Bacteriophages φMR299-2 and φNH-4 can eliminate Pseudomonas aeruginosa in the murine lung and on cystic fibrosis lung airway cells. mBio, 3 (2) PMID: 22396480

Paul VD, Sundarrajan S, Rajagopalan SS, Hariharan S, Kempashanaiah N, Padmanabhan S, Sriram B, & Ramachandran J (2011). Lysis-deficient phages as novel therapeutic agents for controlling bacterial infection. BMC microbiology, 11 PMID: 21880144

What Is This Link to Mushrooms in Works of Art?

by Elio

Pseudo Fardella, Italian, active in Tuscany second half, 17th
century. A Basket of Cherries, Apples, Plums, Chestnuts,
Asparagus and Porcini on a Ledge. Private collection.

On the left side of this blog, in amongst the Blogroll links, is a somewhat strange entry, “Mushrooms in Works of Art.” I’ll save you the trouble of clicking on it. This is the website of a registry that lists works of art, mainly Western, that display mushrooms. Now, why would anyone care about this? The project started about 10 years ago when mycologist Hanns Kreisel from Greifswald University in Germany and chemist Tjakko Stijve from Switzerland and I came together, impelled by the same thought, which was that depictions of mushrooms in art would give us some insight into their relationship to people of various times and cultures.

Otto Marseus van Schrieck, Dutch, 1614/1620-1678. Still Life
with Insects and Amphibians. Herzog Anton Ulrich Museum,
Braunschweig, Germany.

We exchanged lists of such works of art and found that the couple of hundred items each of us had didn’t overlap much. This suggested that the “population” of such works was quite large and that further efforts were called for. We became ever more alerted to “finds” in museums, in art books, and, of course, on the internet. The records of old sales by auction houses proved to be particularly fruitful. In time, new people came on board. The project currently is in the hands of a Belgian mycologist, Daniel Thoen, an artist and University of Wisconsin art professor, Nancy Mladenoff, and myself. The registry now consists of over 1200 items, mainly paintings. The single best represented genre is Baroque still lifes, mainly Italian, Flemish, and Dutch.

Giovanni Francesco Barbieri, known as “Il
Guercino,” Italian, 1591-1666. The Greengrocer.
Private collection.

What have we learned? Basically, what we already suspected, namely that some countries, especially Italy, are especially mycophilic. Mushrooms can be found in perhaps 10% of all Italian Baroque depictions of fruits and vegetables. Most commonly, the species portrayed are the highly prized Caesar’s mushroom (Amanita caesarea) and the King bolete or porcino (Boletus edulis), which have been consumed avidly from Roman antiquity to this day. Some artists were particularly fond of mushrooms and painted them in a large number of their surviving works. Among them is an Italian only known by the unflattering name of Pseudo Fardella (meaning in the style of Fardella) and the Dutch artist of the forest understory, Otto Marseus van Schrieck.

Walter Jenks Morgan, British, 1847-1924. A Fairy Ring.

A number of paintings show mushrooms being offered for sale by vegetable and fruit vendors. This is a good indication of what species were consumed at that time. Few poisonous mushrooms are seen in paintings from before the 18^th century. This is surprising because the archetype of mushrooms, the red one with white dots (Amanita muscaria or the fly agaric) is cosmopolitan, abundant, and showy. One can speculate that this species was omitted because it is toxic enough that nowadays the trip of people who take it for “recreational” purposes” is often to the emergency room. One can imagine that displaying poisonous mushrooms above one’s mantle was not exactly popular. This has changed and the fly agaric may well be the species that is most often depicted in recent times. Of interest is that in otherwise non-mushroom loving Great Britain, mushrooms had a great ascendancy in the 19^th century. A particular genre, known as the Victorian Fairy Paintings, deals with the underworld of fairies and gremlins, often looking like Barbie dolls with wings. These imaginary figures are seen cavorting on the forest floor, often around or over mushrooms. Mystical mushrooms?

The registry is classified by period and region of origin. It provides a brief description of the mushrooms and, we hope, will entice scholars to probe more deeply into the historical questions that mushrooms elicit.

Why do I bring this up now? The reason is that the registry has recently been moved to the website of the North American Mycological Association and, thanks to its webmaster David Rust and with the help of Marjorie Young, has been expanded considerably. You are welcome to visit it and taste the delights of “artistic” mushroom hunting in cyberspace.

On Retrons

We reprint this article from Habib Maroon’s blog Biobabel, with his kind permission.

by Habib Maroon

The secondary structure of msDNA Ec73. The
76 nt RNA (in box), is joined to a 73nt ssDNA.
Note the 2'-5 phosphodiester bond connecting
the two molecules at the branching guanosine.

Retrons are an understudied type of prokaryotic retroelement responsible for the synthesis of an enigmatic species of small extra-chromosomal satellite DNA termed multicopy single-stranded DNA (msDNA). msDNAs are actually composed of both a single-stranded (ss) DNA and a ssRNA. The 5' end of the msDNA is covalently bonded to an internal guanosine residue of the msRNA by a unique 2'-5' phosphodiester bond, whilst the 3' ends of the molecules are joined by a small stretch of base-pairing. msDNAs are therefore a sort of looped hybrid molecule, but extensive internal base pairing creates various stem-loop/hairpin secondary structures (see figure). The retron, (i. e., the genetic loci encoding the msRNA and msDNA molecules (msr and msd) and the gene encoding the reverse transcriptase (ret) responsible for the synthesis of msDNA) is transcribed as an operon.

Retrons are present in a wide variety of eubacterial, and some archaeal, genomes. A recent study identified 97 different retron-like reverse transcriptase genes within bacteria, however their distribution is sporadic. For instance, seven distinct retron elements have been found amongst E. coli strains, but only 15% of natural E. coli isolates produce msDNAs. Based on their sporadic occurrence and analysis of codon usage, retrons have been suggested to be a recent addition to the E. coli genome.

A major exception to the sporadic distribution found in most bacteria is within the myxobacteria, where all ten genera include msDNA-producing species. Myxobacterial retrons form a phylogenetically related group. These features, as well as sequence divergence, suggest that the common ancestor of the extant myxobacteria contained a retron as much as 150 million years ago, which has been vertically transmitted.

Retrons have not been shown to be mobile genetic elements, although the presence of reverse transcriptase does suggest this possibility. A clue to their propagation is the association of many of them with prophage sequences, suggesting their spread could be associated with bacteriophage. However, as with many observations about retrons, there are plenty of exceptions.

Organisation of a retron operon. Note the inverse orientations and short
overlap of msr and msd.

msDNA is essentially a cDNA produced from a short region of an mRNA template. During msDNA synthesis, an RNA template derived from the operon mRNA and composed of msr and msd, is folded into a specific secondary structure due to flanking inverted repeat sequences. The msd sequence is then reverse transcribed by the retron reverse transcriptase, using the 2'OH group of the 'branching' guanosine residue as a primer. The lagging RNA template strand is then degraded by RNaseH activity (probably host cell derived), leaving the msDNA covalently bonded at it's 5' end and base paired to the msRNA at their 3' ends.

No function has been unequivocally attributed to msDNA. Mutating retron ret genes to prevent synthesis of E. coli or myxococcal msDNAs produces no detectable effects. Overexpression of certain E. coli msDNAs has been shown to increase mutation rate. msDNAs generally form hairpin structures by complementary base pairing of inverted repeat sequences (see figure). However, in many msDNA hairpins the base pairing is imperfect. It appears that the overexpression associated mutation rate phenotype is due to mismatch-containing msDNAs sequestering the mismatch repair enzyme MutS. Overexpression of msDNAs without mismatch-containing hairpins does not cause similar effects. It is possible that msDNA could be regulating MutS availability by this titration mechanism in normal conditions or as part of a stress response. However, the overexpression experiments lead to msDNA concentrations far beyond normal physiological levels, so can yield no more than hints of normal function.

Studies on retrons from Vibrio cholerae suggest potentially important roles for msDNAs. Epidemic cholera is caused by V. cholerae serotypes O1 and O139, both of which contain the retron Vc95. Non-O1, non-O139 strains rarely contain Vc95. This retron is not associated with the CTXphi prophage that encodes cholera toxin, however it's presence correlates with pathogenicity. Interestingly, the genomic location of Vc95 is occupied by other species of retron or by insertions of non-coding sequences in other V. cholerae strains. This implies that retrons are indeed mobile elements, however analysis of this site has not yielded many clues about potential mechanisms of integration or excision.

In conclusion, the lacunae in our understanding of retrons and msDNA are far more striking than the known facts. Are retrons parasitic elements? Or do msDNAs have physiological roles in their host cells? Are retrons mobile elements? Just what does msDNA do? Judging from the literature, interest in retrons peaked around 1990, and recent years have been very fallow. I do hope that funding agencies and researchers keep pursuing the answers to these questions and don't let them remain as an interesting oddity in the literature.

Lampson, B., Inouye, M., & Inouye, S. (2005). Retrons, msDNA, and the bacterial genome. Cytogenetic and Genome Research, 110 (1-4), 491-499 DOI: 10.1159/000084982

Simon, D., & Zimmerly, S. (2008). A diversity of uncharacterized reverse transcriptases in bacteria. Nucleic Acids Research, 36 (22), 7219-7229 DOI: 10.1093/nar/gkn867

Inouye, K., Tanimoto, S., Kamimoto, M., Shimamoto, T., & Shimamoto, T. (2011) Two novel retron elements are replaced with retron-Vc95 in Vibrio cholerae. Microbiology and Immunology, 55(7), 510-513. DOI: 10.1111/j.1348-0421.2011.00342.x

The Winner of the 2012 Peter Wildy Prize: Vincent Racaniello

by Elio

The winner of the 2012 Peter Wildy Prize For Microbiology Education is Vincent Racaniello of Columbia University, a fellow blogger (Virology Blog) and the podcast host of TWiV (This Week in Virology), TWiP (This Week in Parasitism), and TWiM (This Week in Microbiology).

This prize is awarded annually by the Society for General Microbiology (UK) for an outstanding contribution to microbiology education, without restriction on the area of microbiology in which the award is made. This may include university teaching or education of the general public, school pupils or professional groups. The winner receives £1000 and gives a lecture on his/her work at a Society meeting. Vincent’s talk to the SGM was entitled Educating the World about Microbes and can be heard by clicking here.

Frankly, I can't think of anyone more deserving of this award. Vincent, with whom I have had the pleasure of working on TWiM, is a consummate master of communication. Seldom in my experience have I met a scientist who can deliver the pleasures of science in such an articulate way. To listen to him is to become enthralled with whatever it is he is talking about. I can bear witness to his fervor, his gentleness, his humor. The French have an expression for it: un homme engagé (roughly translated, a committed man). Vincent is indeed engagé!

The Immunological Synapse Goes Viral

by Merry

Structural proteins in the HTLV-I virion include Gag (a
capsid protein) and Env (the surface glycoprotein required
for infectivity). Fluorescently-labeled antibodies showed
that these proteins were not polarized in isolated infected
T cells. In cell-cell conjugates, the proteins accumulated at
the cell-cell junction within 40 min. This particular confocal
image shows polarization of HTLV-I Gag p19 (red) to the
cell-cell junction. Source.

Here’s yet another tale of how a cunning virus has converted one of our antiviral defenses into a tool for its own purposes. The co-opted mechanism is one used by cytotoxic T-cells to kill virus-infected cells: the immunological synapse. More on this in a moment. The virus is Human T-Lymphotropic Virus Type I (HTLV-1). The appropriated tactic enables the virus to spread efficiently to new host cells.

When discovered in 1977, HTLV-1 was the first known human retrovirus (HIV not being identified until six years later). While not as devastating as HIV, it currently infects 10–20 million people, 2–3% of whom will develop adult T-cell leukemia/lymphoma while another 2-3% develop a chronic inflammatory condition (HAM/TSP). Like HIV, it infects primarily CD4+ T cells. And like HIV, HTLV-1 also transmits from one person to the next in blood, milk, or semen. But just how it does this was puzzling because, unlike HIV, few free virions are found in the blood and, of those, only one virion in a million is infectious. More clues: Only enveloped HTLV-1 virions are infectious and they acquire their envelope from the lymphocyte plasma membrane as they bud from their host cell. Efficient transfer of the virus between cells requires cell-cell contact, both in vitro and in vivo.

Combined these observations suggest that perhaps the virions bud from one cell and immediately enter their next host cell without ever wandering free in the liquid milieu. What would such a strategy require? First, an infected CD4+ T cell must dock with an uninfected CD4+ T cell, something it normally does not do. The mature virions in the infected cell must be transported to the zone of surface contact and be released there, acquiring their envelope as they exit. The now-infectious virions must then enter their new host cell directly.

Models of the immunological synapse and viral synapse. (A) When an immunological synapse forms between a cytotoxic T lymphocyte and a target cell, the microtubule-organizing center is polarized towards the synapse (orange ellipses = centrioles). Lytic granules containing cytotoxins (red discs) are transported along microtubules (black lines) to the synaptic cleft. The cytotoxins (red points) are released into the synaptic cleft and taken up by the target cell. (B–C) Possible modes of virus transmission through the HTLV-1 viral synapse. (B) Enveloped HTLV-1 particles (purple-blue discs) bud into the synaptic cleft and are transmitted to the target cell. (C) Enveloped virus particles bud at the periphery of the synapse and are transmitted through the extracellular space (observed in some infected cell lines but not in naturally-infected CD4+ T cells). Source.

A viral synapse. Conjugates were allowed to form for
40 min between infected and uninfected CD4⁺ T cells.
A cell adhesion protein (talin, green) accumulates at
the surface of the infected cell in a pattern that
represents the outer ring of a bullseye; the HTLV-I
capsid protein (Gag, red) is localized at the surface in
the center of the talin ring. Bar = 5 μm. Source.

This is exactly what HTLV-1 achieves by manipulating the components of our immunological synapse. The immunological synapse is a specialized cellular structure used by T-killer cells to destroy virus-infected cells and tumorigenic cells. When a T-killer cell meets a suspect cell, it forms an immunological synapse at its own cell periphery in the region of contact. Picture a bullseye on the T-killer cell surface. Cell-cell adhesion molecules localized in the outer ring are used to adhere to the other cell. In the center is the recognition site used by the T-killer cell to determine if the contacted cell should be destroyed. If the verdict is “guilty,” the microtubule organizing center of the T-killer cell polarizes towards the synapse. Secretory lysosomes move along the microtubules to the center of the bullseye where their contents are secreted. The noxious secretion contains both perforin (to make holes in the target cell’s membrane) and granzymes (serine protease granules that induce apoptosis). This door-to-door delivery is deadly.

Conjugates were allowed to form for 40 min between
infected and uninfected CD4⁺ T cells. The microtubule-
organizing center reorients to lie adjacent to the
polarized HTLV-I capsid protein (Gag) at the cell-cell
junction. Tubulin-alpha (green), HTLV-I Gag p19 (red).
Bar = 5 μm. Source.

Turning the tables, HTLV-1 uses an analogous cell-cell junction called a viral synapse to deliver its progeny virions to uninfected lymphocytes. Lymphocytes don’t normally form stable cell-cell junctions with each other. However, CD4⁺ T cells infected with HTLV-1 do, at least with uninfected CD4⁺ T cells. When mixed together, they form such contacts within 40 minutes. By using immunofluorescence and fluorescent in-situ hybridization (FISH), researchers showed that both HTLV-1 proteins and viral RNA genomes accumulate at the surface in the region of cell-cell contact and then transfer into the uninfected cell. The microtubule-organizing center of the infected cell polarizes toward the synapse, as was seen at the immunological synapse, only here the virus is orchestrating the process and the transported cargo is viral. The addition of 33 nM nocodazole, which blocks microtubule polymerization, also prevents transfer of the viral proteins to the uninfected cell.

What exactly takes place at a viral synapse? One group of researchers used electron tomography to explore the contact zone formed where the surface of an infected cell interacts with an uninfected cell. Within that region they distinguished a synaptic cleft bounded by a region of closely opposed cell membranes. Enveloped HTLV-1 virions were seen budding from the infected cell into the synaptic cleft. Typically these virions were not free, but appeared to be in contact with the infected cell, the target cell, or both, suggesting that often the exiting virions make contact with the new host cell before letting go of the old. This is further evidence that cell-cell contact is necessary for efficient infection.

Tomogram slices and surface representations of the virological synapse between a target cell and an HTLV-1 infected cell from a chronically HTLV-1-infected cell line. (A) The viral synapse is characterized by a tight membrane-membrane contact with an inter-membrane spacing of about 20 nm. Viral budding sites (black arrowheads) and virus particles (white arrowhead) can be detected within a synaptic pocket and at the periphery of the synapse. Virus budding at the periphery is not seen in naturally-infected CD4⁺ T cells). (B and D) Slices through the synaptic cleft and the periphery, respectively, as indicated by the white rectangles in (A), (C and E) The corresponding surface representations (yellow = cell membranes; blue = virus envelope; magenta = virus core; red = viral core protein at budding site). Bars = 500 nm (A) and 200 nm (B-E). Source.

However, this may not be the only trick used by this virus, as other researchers have reported another mechanism that could also support efficient cell-to-cell transfer. They found clumps of HTLV-1 virions attached to the cell surface encased in a virally-induced, carbohydrate-rich, extracellular matrix (a “viral biofilm”). In their model, these sticky assemblies rapidly adhere to a target cell upon contact—another way virions could transfer without ever losing their cellular connection. Either way, HTLV-1 transfers from cell to cell without exposing itself to our immune defenses. Furthermore, the budded virions are treated like the valuable progeny they are. Instead of relying on random collisions with a potential host cell in the bloodstream, they are efficiently delivered to an appropriate doorstep.

I recently blogged about the tactics used by poxviruses, and now here’s another example where a virus not only escapes destruction but does so by borrowing a trick from our immune system. Do these stories of subversion —and there are others—leave you a tad disappointed in our immune system? Could be. Or perhaps, like me, you are even more in awe of its capabilities, given the challenges presented by these ingenious viruses.

Igakura, T. (2003). Spread of HTLV-I Between Lymphocytes by Virus-Induced Polarization of the Cytoskeleton Science, 299 (5613), 1713-1716 DOI: 10.1126/science.1080115
Majorovits E, Nejmeddine M, Tanaka Y, Taylor GP, Fuller SD, & Bangham CR (2008). Human T-Lymphotropic Virus-1 Visualized at the Virological Synapse by Electron Tomography. PloS One, 3 (5) PMID: 18509526