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Arctic genes make new vaccines!

August 6, 2010

I’m back from a few weeks doing research at sea.  I read a paper today that I would like to tell you about.  Essential genes from Arctic bacteria used to construct stable, temperature-sensitive bacterial vaccines was published in PNAS this month.  It describes how genes from arctic bacteria were used to transform pathogenic bacteria into temperature sensitive versions of themselves that are killed at core mammalian body temperature.  Basically, the take home message is that Arctic bacteria may allow scientists to create important new vaccines!

Cold loving (psychrophilic) bacteria have evolved over geologic time (potentially 2 billion years) to have slightly different versions of many key genes that all bacteria share.  These changes might be due in part to the randomness of evolution, or it may be a tradeoff for being functional at low temperatures, but many of these genes do not function properly at warm temperatures where typical bacteria thrive.

In this study, scientists replaced some of the genes in pathogenic bacteria with their cold loving cousin’s version of the same gene.  In many cases, the bacteria with the new temperature sensitive version of the gene died at 33, 35, or 37°C.  The human body is about 37°C, so this means that these pathogens would not survive in our core, however other parts of our body are not quite that warm, so the bacteria would be able to survive in those locations.

In this study they used mice and rats to show that temperature sensitive versions of pathogens could trigger immunity… very cool!  Basically the bacteria survived in the cooler region of the mice, not able to reproduce enough to be harmful to the animals.  At the same time, the bacteria’s presence allowed the mice to develop immunity to the original version of the bacteria.  After the live, temperature sensitive immunization, the mice were injected with the original version of the pathogen, and did not show signs of infection.

They started off with one specific pathogen named Francisella tularensis that infects mice but not humans, and subsequently tried the technique on other pathogens including a strain of Salmonella enterica and Mycobacterium smegmatis (a close relative of the tuberculosis causing Mycobacterium tuberculosis).  Their success with these other bacteria indicates the potential for safe and effective vaccines to some important human diseases.  There are potential non-medical applications as well.  If temperature strains of harmful bacteria can be engineered that die at 37°C, research on those organisms could be safer, easier, and cheaper because they would be simple and easy to kill.

One potential problem with this approach is that the engineered strains could mutate and revert to the original version that is not temperature sensitive.  This would be problematic in many ways.  However, because these particular genes have evolved over such a long time, some of them are different enough that there is no quick and easy mutation that turns them back to the heat tolerant version.  If scientists are smart about which genes they pick, the risk of this type of mutational reversion would be very low.

Oral vaccines – awesome, but no panacea

July 28, 2010

ERV has a great post about a new paper in PNAS that details the mechanism of immunity granted by muco-Rice, a great solution for cholera protection in the developing world:

So scientists have been working really hard to create vaccines to cholera and ETEC, and a group of folks have figured out a really cool strategy– genetically modify rice to express part of the cholera toxin. When someone eats the vaccine rice, their immune system gets a cheat-sheet for what the real toxin will look like. Their body starts secreting IgA, antibodies that recognize the toxin, in their mouth/tears/digestive tract/breast milk. If that person is exposed to real cholera, they already have antibodies around to neutralize the toxin, thus are able to prevent or limit the extent of that infection!

BONUS: If the person who gets the vaccine is a breast-feeding mom, these beneficial anti-cholera-toxin IgA antibodies are passed down to their babies via breast milk, thus also protect their babies from cholera!

BONUS BONUS: This plant-based vaccine strategy is also beneficial because it requires no sterile needles, no freezing/refrigeration, and the vaccine would have a ridiculous shelf-life of at least three years.

Sounds awesome! Let’s make all our vaccines into food products – no needles, long shelf-life, cheap! Right?

Wrong. Unfortunately, this method will probably have very limited use. Most of the things you eat generate immune tolerance, not protective immunity. In fact, when so-called oral tolerance breaks down, you get food allergies. There’s so much foreign crap (pardon the pun) in your digestive tract that the immune system has evolved to actively ignore and suppress the immune response to the things you eat. But wait – what about the oral polio vaccine? That works because it’s a live-attenuated (weakened) virus. The virus actually causes a local infection, which pisses off the immune system and circumvents oral tolerance.

But Cholera-toxin is a protein, not a virus, so at first glance, it seems like it should induce oral tolerance. After all, you don’t have immune responses every time you eat a steak (unless you’re unfortunate enough to have a steak allergy – would life even be worth living?). But cholera toxin is special – it can invade cells on its own. In fact, that’s why it causes so much damage in the first place. When you’re infected with Vibrio cholera, it secretes this toxin which is composed of a bunch of different subunits (parts). The B-subunit is able to latch on to cells and get pulled inside, then the A-subunit punches a hole in the vesicle containing it and runs amok. This rice vaccine only uses the B-subunit, so it can invade cells and piss off the immune system without actually causing much damage (it does cause a little bit of damage, which is what alerts the immune system in the first place). This is great news for treating cholera (and a few other bacterial toxin-induced diseases) as ERV mentioned, but unfortunately any dreams of ditching needles for good will have to be put on hold.

Biofilms and the bacterial subversion of the immune system

July 23, 2010

I was just talking about how our mucosal surfaces have anti-microbial peptides to keep bacteria at bay. I also mentioned how whatever defenses we have, pathogens find a way to get around them. Case in point:

Our results suggest that curli and cellulose exhibit differential and complementary functions. Both of these biofilm components were expressed by a high proportion of clinical E. coli isolates. Curli promoted adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, but also increased the induction of the proinflammatory cytokine IL-8. Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys.

Uropathogenic E. coli (UPEC) cause most urinary tract infections, and they form specialized structures called biofilms. As a group of these bacteria divide, they secrete a matrix of proteins and other components that allow them to aggregate and adhere to their environment. And bacteria in biofilms are often behave differently from their free-living counterparts. In fact, it’s been shown that Vibrio cholerae (which causes cholera), is only pathogenic in its bio-film form.

This paper is showing that the biofilm made by uropathogenic E. coli has components called “curli fimbriae” that inhibit the anti-microbial peptide LL-37. They took bacteria samples from patients with UTIs and cultured them, checking for the expression of biofilm components. By far the most infectious strains expressed curli, and if the curli were removed, these trains were no longer infectious. Interestingly, in a classic example of Red Queen evolution (I hope to write more on that later), the immune system has learned to recognize curli fimbriae as signs of infection and mount stronger immune responses. Of course, the bacteria are smarter than we are, and have a counter-counter-measure, cellulose, which prevents the immune system from detecting the curli. Almost all of the strains these guys cultured made cellulose, and when they removed the ability to make cellulose, the UPEC induced a strong immune response and were unable to establish persistant infections.

It would be great to have a way to simply disrupt these biofilms as a treatment for UTIs, since using non-specific antibiotics can have all kinds of other side-effects. This research is probably a long way off, but it’s an important first step.

Single celled farmer

July 23, 2010
by

There’s a great post on Skeptic Wonder about a single-celled protist that secretes a nutritious substance as it crawls along seaweed. The secretion attracts colonies of bacteria to grow, and the protist comes back and “harvests” them. Pretty neat:

Thus, a ‘mere’ single celled organism can produce organised tracks of nutritious material, wait for their bacterial crop to grow, and subsequently harvest it. We like to think we invented agriculture. The more biologically-oriented among us point out leafcutter ant fungus gardens and aphid farming. Yet, agriculture has also evolved on the unicellular scale in a small humble foraminiferan living among blades of seagrass. Humbling, isn’t it?

The microbes’ motto: “Anything you can do, I can do better.”

Immune response from start to finish: Part 1

July 23, 2010

[I’ve been hooked on the immune system since I was a kid and my dad showed me electron micrographs of macrophages eating bacteria in Scientific American. Now that I’m in graduate school studying immunology, and macrophages in particular, my dad asked if I could give a play-by-play of an immune response. Here you go Dad:]

Part 1: Invasion and detection, the innate immune system

Most immunology classes I’ve taken have begun with a simple, but profound truth: the best immune response is one that prevents pathogens from ever gaining entry (pathogen = disease-causing organism). Hence, we are covered in barriers. Skin is the most obvious example of a barrier – it’s water-tight, protected by layers of dead cells and covered in things called anti-microbial peptides which are basically tiny protein antibiotics.But other bits of our body can’t be sealed off so completely – the mucosal tissues lining our oral, nasal, genital and gastrointestinal tracts all have to be permeable to carry out their functions – our lungs for instance, which are exposed to the microbial world every time we breathe, would be useless if they were as impenetrable as skin! But that doesn’t mean these tissues are defenseless – they are composed of specialized epithelial cells, which form “tight-junctions,” and secrete mucous and anti-microbial peptides in an effort to be inhospitable.

But these barriers are far from perfect. Evolution has forced compromise – skin is elastic to allow for ease of movement, but that means it’s susceptible to getting cut; the gut epithelium is permeable to nutrients, but also to microbes. In addition, pathogens are masters at subverting even our best defenses (in fact, this is sort of a theme in immunology – we know something is important if we find a pathogen that has learned to get around it). So, once a bug gets past past the initial barriers, what’s next? The immune system needs to know that something is wrong, and that’s where pattern recognition comes in.

I’ve described pattern recognition before, because it’s what I study, but I’ll mention a few things briefly here. Every cell in the body has specialized receptors to detect invading pathogens. These receptors are called pattern-recognition receptors (PRRs) because they recognize parts of pathogens called “PAMPs” – pathogen-associated molecular patterns. All organisms are made of the same basic building blocks (proteins, nucleic acid, lipids and carbohydrates), but bacteria and viruses have some features that are unique, and can therefore be recognized as foreign. Double-stranded RNA, for instance, is never present in the absence of a viral infection. Lipopolysaccharide (LPS) is a sugar that is found in bacterial cell walls, but not in mammals. Not all cells express every PRR, but most cells can at least recognize internally if they get infected.

There are other specialized cells, like macrophages, that are professional pathogen seekers. Macrophages express pattern recognition receptors on their cell-surface called Toll-like receptors (TLRs) that can recognize external bacteria and viruses. The macrophages can then eat the intruders as well as release signals called cytokines that cause inflammation and alert nearby cells of the danger. Inflammation also triggers the influx of neutrophils from the bloodstream – these cells are like kamikazes, eating and destroying everything in their path. Another cell type, natural killer (NK) cells, can recognize signs of infection and stress and force those cells to commit suicide.

These events are enough to clear the vast majority of potential infections, and you would never notice any symptoms. These responses are called the innate immune system, because it’s more or less present in the same form at birth. And the response very general, most viruses and most bacteria will be dealt with in essentially the same way. But real pathogens are sneaky, and they know how to get around these defenses. In Part 2, I’ll talk about the adaptive immune system and the generation of highly specific, coordinated responses to clear prolonged infections.

Immune response from start to finish, the series
Part 1: Invasion and detection: Innate immunity (current)
Part 2: T-cells, B-cells and adaptive immunity
Part 3: Immune Memory

Your viruses are your own… in your poop

July 19, 2010

More fun news out of your intestines last week – a paper published in Nature shows that the viruses living in the bacteria living in your gut are unique to you:

Here we report sequencing of the viromes (metagenomes) of virus-like particles isolated from faecal samples collected from healthy adult female monozygotic twins and their mothers at three time points over a one-year period. We compared these data sets with data sets of sequenced bacterial 16S ribosomal RNA genes and total-faecal-community DNA. Co-twins and their mothers share a significantly greater degree of similarity in their faecal bacterial communities than do unrelated individuals. In contrast, viromes are unique to individuals regardless of their degree of genetic relatedness.

This is a pretty small study – they only looked at 4 families – so you should take these conclusions with a grain of salt. Still, the results are pretty striking. These researchers took stool samples from 4 mothers and their monozygotic (identical) twin daughters. It’s been known for a long time that mothers and their offspring share a great deal of their commensal bacteria (especially if they were born naturally as opposed to via c-section), but here they decided to look at the viruses. What they found was pretty remarkable: the variation in viruses was as large between mothers and daughters (and between twins) as it was between unrelated individuals.

As far as I can tell, there’s nothing wrong with the science here, but there are a few caveats. If you read the piece by Carl Zimmer I linked to earlier (and if you haven’t, go do it now), you’ll know that we can’t actually grow these microbes outside the human body. The scientists here extracted all the viruses (or virus-like particles), then threw them together, amplified the DNA with PCR (which can introduce biases), and then sequenced it. Finally, they used mathematical algorithms (which I don’t fully understand) to group the DNA they found with databases of known virus genomes. However, over 80% of the reads could not be matched to any known viruses, so they have to use other algorithms to predict what sorts of viruses they are and what kinds of bacteria they infect. There’s a lot of room here for biases to be introduced.

Also, as of yet there’s not functional information gained from this sort of analysis. I think the main point of the paper is to make a case that we need to look at the virus content in order to get a complete understanding of the influence that the microbes in our bodies can have on our physiology. And considering the millions of dollars that the NIH is planning to dish out for the human microbiome project in the coming years, this report couldn’t have come at a more opportune time.

The new(ish) science of microbiomics

July 15, 2010

I can’t do any better than Carl Zimmer:

In 2008, Dr. Khoruts, a gastroenterologist at the University of Minnesota, took on a patient suffering from a vicious gut infection of Clostridium difficile. She was crippled by constant diarrhea, which had left her in a wheelchair wearing diapers. Dr. Khoruts treated her with an assortment of antibiotics, but nothing could stop the bacteria. His patient was wasting away, losing 60 pounds over the course of eight months. “She was just dwindling down the drain, and she probably would have died,” Dr. Khoruts said.

Dr. Khoruts decided his patient needed a transplant. But he didn’t give her a piece of someone else’s intestines, or a stomach, or any other organ. Instead, he gave her some of her husband’s bacteria.

The transplant of bacteria (via stool) from here husband cured the pathogenic infection. Zimmer uses this story as a launching point to discuss the new science of microbiomics that I’ve been talking a bit about recently. Read the whole thing, it’s fascinating.

My favorite part:

Only 13 percent of the species on two people’s hands are the same. Only 17 percent of the species living on one person’s left hand also live on the right one.

What the hell?

Rare minerals and the fight against pathogenic bacteria

July 10, 2010

I’ve kinda been on a microbe love-fest recently, but don’t get me wrong – there are some seriously nasty bugs out there. We have antibiotics, which are great, but a lot of pathogens are developing resistance, and antibiotics can do all kinds of crazy stuff that we don’t understand to the natural commensals in our gut and elsewhere. Vaccines would be great, but it’s really hard to make vaccines against bacteria.

Most vaccines against viruses work through neutralizing antibodies, which can bind to incoming viruses and prevent them from fusing with cells. But compared to viruses, bacteria are huge, and anyway they don’t infect cells in the same way (if they even infect cells at all). Some bacteria make toxins that can be neutralized (that’s how the tetanus vaccine works), but toxins are usually small molecules, so neutralizing antibodies can be effective. So what other options do we have?

An new paper in PLoS Pathogens has an idea:

In a search for putative vaccine components, we have characterized here a new receptor of Neisseria meningitidis, a resident of the nasopharynx that occasionally causes sepsis and meningitis. We show that expression of this receptor is induced under zinc limitation and that the protein is involved in the uptake of zinc[…] the protein appears an excellent candidate for the development of a vaccine against N. meningitidis, for which no universal vaccine is available yet.

Bacteria can generally make all the biological components they need to survive from relatively simple precursors. But they also need a few trace metals as co-factors, and these metals in very short supply. To get what they need, bacteria have special pumps in their outer membrane to pull those metals in, and these authors reasoned that blocking these pumps with antibodies might work as a vaccine target. It looks like they were right.

Building on work done on an iron pump, these guys found a pump that is necessary for zinc uptake in the bacterium Neisseria meningitidis. Crucially, this protein is highly conserved, meaning it’s almost identical across many different strains (making a vaccine is way less useful if it only targets a small subset of the bacteria you’re after). Then they tested to see whether they could immunize mice with this protein and generate an antibody response that was bacteriocidal.

Unfortunately, they didn’t show any data to say that these immunized mice were actually protected against subsequent infection (they just took antibodies from their blood and showed that they could kill the bugs ex vivo), but this is a good first step, and a potentially cool new way to make vaccines against some of the more dangerous bacteria that infect us.

Fly STD’s, and the help of Bacteria

July 9, 2010

Sweet:

Here we demonstrate that a maternally transmitted bacterium, Spiroplasma, protects Drosophila neotestacea against the sterilizing effects of a parasitic nematode, both in the laboratory and the field. This nematode parasitizes D.neotestacea at high frequencies in natural populations, and, until recently, almost all infections resulted in complete sterility. Several lines of evidence suggest that Spiroplasmais spreading in North American populations of D. neotestacea and that a major adaptive change to a symbiont-based mode of defense is under way.

Here’s the deal – flies have bacteria, and flies have worms. There’s a parasitic worm infection that causes sterility in female fruit-flies (the worm hijacks the reproductive tract, causing worm eggs to incubate in the fly rather than fly eggs). But one of the fly’s commensals (Spiroplasma) learned how to block this sterilizing effect, which is vital since the bacterium is transfered from mother fly to off-spring. In other words, no fly babies, no more Spiroplasma.

Even cooler, this “protective symbiosis” seems to have evolved in the relatively recent past. In the 80’s this particular worm wasn’t a big problem, but over the last 30 years, it’s come to infect a large portion of the fly population. The sterilizing effect puts a strong selective pressure on both the fly and the Sprioplasma (which can only be transmitted if the fly has babies), but the bacteria can evolve faster and so they’ve born the brunt of the protection. It’s natural selection in action!

Update: There’s a pretty good write-up of this paper in the Guardian if you want more info.

Everything’s “Contaminated”

July 8, 2010

A while back I heard an NPR story about bacteria growing in reusable grocery bags, and now there’s a piece from WaPo’s health blog about bacteria fround on 3-D glasses you get at movie theaters:

In its July issue, Good Housekeeping magazine tested seven pairs of 3-D glasses, three that were wrapped and four unwrapped, and found that none of them were bacteria-free.

Well, duh. Bacteria are everywhere! When working with cell culture in lab, everything is done in a special “hood,” a box with fans and filters specifically designed to prevent anything outside from getting in, I wear gloves, and spray everything with ethanol, and I even have high doses of antibiotics in the media. Stuff still gets contaminated every once in a while.

What matters is what kinds of bacteria are present, and if they are actually pathogenic. The NPR story goes to great lengths to explain that the bacteria found in the grocery bags are basically benign, but the WaPo piece…

While most of the bacteria (collected via swabs that were sent to an independent lab) was deemed harmless, one set of glasses bore Staphylococcus aureus bacteria, which can cause pinkeye and other infections.

And some strains of E. coli can cause severe food poisoning, but there’s billions of non-pathogenic E. coli in your gut right now. They then say you should wipe the glasses with alcohol swabs, but in the very next sentence is

Even if you don’t think to take any such measures, though, your risk of getting sick from movie glasses is pretty low[…]

Ugh. So, there are bacteria in your grocery bags and on your 3-D glasses, which are harmless, but you should wash them anyway, but if you don’t it doesn’t matter. My take: after you wipe the glasses with alcohol swabs, you’ll probably just re-contaminate them with the S. aureus that’s already on your nose.