From soil to human: evolution of bacterial immune evasion mechanisms

by Brooke Napier, dedicated to Christian Raetz

Most, if not all, pathogenic bacteria did not get their start infecting humans, no, most of them started off as meager soil or water bacteria, just hoping to catch a rock to colonize. A lot of biologists have dedicated their lives to understanding the genetics or mechanisms behind harmless soil/water bacteria evolving into lean-mean human killing machines.

This month in PNAS, Li et al. took a closer look into one of the mechanisms pathogenic and antibiotic-resistant bacteria use to evade the immune system and antibiotics, LPS remodeling, and how this mechanism has evolved from non-pathogenic soil bacteria.

First off, WTF is LPS remodeling?

Gram-negative bacteria (depicted below in yellow) have an inner and outer lipid membrane (in blue) separated by the periplasm (this is distinct from Gram-positive bacteria that only have one membrane and a thick periplasm on the outer surface). The outer-leaflet of the outer membrane (in orange below) is the LPS, or lipopolysacchride (lipids and sugars, yall). The LPS it the first thing the host immune system can see, since it's the outer most surface of the bacteria (unless there is a capsule, which is an entirely different post).

Not surprisingly the mammalian host has developed a quick response to LPS by the cellular receptor TLR4. Stimulation of TLR4 leads to inflammation in the host, which can lead to destruction of the invading bacteria; therefore, it is intuitive that the bacteria have evolved mechanisms to escape detection of the host immune system by remodeling the LPS, or the outer surface of the bacteria.

 

Remodeling of the LPS can occur in many forms, you can modify the sugars or the lipids of the LPS Lipid A (pictured bottom left, bottom yellow structure), the biologically active portion of the LPS, to evade host detection.

viaMany books and hundreds of papers have been devoted to understanding modification of Lipid A sugars and the adjoining phosphates, however Li et al. describe a specific modification of the lipids in the LPS Lipid A that can be turned on when the bacteria senses that is within a human host.

How are these bacteria sensing the environment? 

It's briliant really, the temperature. The temperature of the environment (ie, water or soil) is generally 15°C, whereas the human body is 37°C, and bacteria can sense this temperature difference and regulate Lipid A modifications accordingly. Yersinia pestis, the causative agent of the plague, can colonize rodents and is transmitted by fleas. In response to these different environments Y. pestis can synthesis alternative Lipid A structures in different growth temperatures - one for the temperature of a flea (21°C) and another for the temperature of a mammalian host (37°C). 

The flea-specific LPS has a hexaacylated Lipid A, or contains 6 fatty-acid chains (as pictured in the Lipid A to the left), to protect the bacteria from conditions in the flea digestive tract or external environment, whereas the tetraacylated Lipid A, or contains 4 fatty-acid chains, found within the mammalian host allows bacteria to evade detection by TLR4.

What about bacteria that are soil bacterium and now pathogenic?

To answer this question Li. et al looked at Francisella tularensis, the causative agent of tularemia, that is been identified in a wide array of environments and hosts (soil, water, fresh water protozoans, arthropods, and mammals), indicating its need for adaptation among various conditions. Since Lipid A remodeling has been identified as temperature-sensitive, in this study they looked at Francisella Lipid A modifications in response to temperature changes. 

What is the role of temperature in modulating Francisella membrane remodeling?

The Francisella tetraacylated Lipid A is depicted to the right, with a characteristic galactosamine (GalN - green) addition on the 1 phosphate and 4 acyl (or fatty acid) chains (black). As noted at the bottom the acyl chains are 14-18 carbons in length, this is important because these are the carbons affected by temperature.

As shown below, when subjected to different temperatures Francisella Lipid A acyl chains vary in length. In the figure below, A) shows at 37°C, mammalian host temperature, the acyl chains are 16, 18, 18, and 18 carbons in length, B) at 25°C, insect temperature, the acyl chains are 16, 18, 18, and 16 carbons in length, and C) at 15°C, environmental or soil/water temperature, the acyl chains are 16, 16, 18, 16 carbons in length. These data indicate that Francisella Lipid A is modified by number of carbons of the 4 acyl chains in response to environmental vs. host temperatures.

Additionally, they identified two different acyltransferases, LpxD1 and LpxD2, that are responsible for Lipid A acyl chain modifications. LpxD1 adds longer acyl chains upon transmission to mammalian host temperatures and LpxD2 adds shorter acyl chains to Lipid A, allowing growth and adaptability in cold-blooded hosts or soil/water. Both genes encoding LpxD1 and LpxD2 are temperature regulated, allowing for "exquiste control over this process and suggesting the process is critical for survival". 

Ok, so what does this mean for the bacteria?

As I mentioned before, Lipid A modifications are important for bacteria to survive in the host in order to evade TLR4 recognition, subsequent inflammation and bacterial destruction. However, Francisella is not detected by TLR4 - so why is this modification important? 

Their last figure shows that a lpxD1 deletion mutant cannot survive in a mouse, which means the gene responsible for acyl chain modifications in mammalian host temperatures is required for survival in the host. Therefore, this acyl chain modification is an adaptation required for Francisella to causes disease in humans.

In this paper they did not take this idea any further, but it would be interesting to understand why there is protection of Francisella in the host with this acyl chain modification. If TLR4 does not recognize Francisella, does TLR4 recognize Francisella without modified acyl chains (in the lpxD1 deletion mutant)? Or is it another host receptor?

Anyone want to do this as a post-doc project in my lab? I should have one going by the 2090 ;)

Li Y, Powell DA, Shaffer SA, Rasko DA, Pelletier MR, Leszyk JD, Scott AJ, Masoudi A, Goodlett DR, Wang X, Raetz CR, & Ernst RK (2012). LPS remodeling is an evolved survival strategy for bacteria. Proceedings of the National Academy of Sciences of the United States of America PMID: 22586119

Quick Hit: Water ball

by Sarah Scoles

You may have seen this, as it's a few days old, but it's a hit-home visualization, so I thought  I'd share it anyway.

Below, you will see the Earth and a gigantic blue War-of-the-Battleship-Alien-Worlds marble hovering above it. Do not be fooled, though. This is no ordinary War-of-the-Battleship-Alien-Worlds marble. It's the sphere you could make if you gathered up all water on the Earth and, you know, made it into a sphere.

The USGS made us this graphic in order to make us go, at once, "Gee whiz, that's a lot of water!" "Gee whiz, Earth is big!" "Gee whiz, that's not a lot of water compared to how much Earth there is!"

As Gizmodo says, "The sphere includes all the water in the oceans, seas, ice caps, lakes and rivers as well as groundwater, atmospheric water, and even the water in you, your dog, and your tomato plant." Especially your tomato plant.

This sphere has a diameter of 860 miles, or approximately the distance from Brooklyn to Atlanta (this globe shows it spanning some distance in the West and Midwest, but as an Easterner, I have a hard time knowing what "from Salt Lake City to Topeka" really means).

The sphere's volume is approximately 332,500,000 cubic miles, more than Pluto's main moon Charon.

If you took all this water and slopped it across the US (and stopped it from slipping off the coasts and into Canada), the continental states would be covered 90 miles deep in water.

Other cool facts you can glean from the USGS data include

• 97.54% of Earth's water is salty
• 0.0001% of it is in you, me, your mom, that tree over there, and chinchillas.
• 68.6% of the fresh water is in ice caps and glaciers. So it's a good thing they're melting!
• Psych.
• Of fresh water that is liquid, 99% is groundwater and thus largely inaccessible.
• If that liquid fresh water were made into a sphere, it would have a diameter of about 170 miles (20% of the all-water sphere's diameter). However, that diameter means that the sphere would contain only 0.7% of the original sphere's volume.

Mostly, this, to me, makes the point that while we think of Earth as "Earth's surface" because that's the part we walk on and interact with, it's got that whole center going on. We're used to hearing "The Earth's surface is 70% water! Land is in the minority!" and so we (or at least I) in my everyday life go about thinking about how much water there is. But when you compare it to how much stuff is underneath all our feet, and how many miles it takes you to get from East Toyko to West Toyko if you start by going East, there is hardly any water at all.

Check out the USGS's webpage on this topic, which includes a table detailing the water content. You can mess around with the numbers and come up with your own mind-boggling percentages, such as 0.000000000000000000000000000000000000000000000000000000000000000
00000000000000000000000000000000000000000000000000000000000000001% of the water in the world is frozen in wooly mammoths that are frozen in glaciers.

What do scientists think of science outreach?

by Sarah Scoles

The Public Library of Science recently published a paper entitled "How Academic Biologists and Physicists View Science Outreach." While there are many peer-reviewed, quantitatively substantial papers about outreach and "the public," few such papers exist on the topic of outreach and "the scientists." This May 2012 paper reports on biologists' and physicists' participation in and attitudes toward outreach.

The paper is very readable, is freely available online, and was published just three months after it was received, because PLoS agrees that science is for everybody. So I encourage you to read the paper itself, but here are some interesting statistics:

Of those scientists surveyed:

• 80% of female scientists with children do outreach; 66% of female scientists without children do outreach.
• 72% of female scientists participate in outreach, while only 43 of male scientists do.
• 50% of male scientists with children do outreach; 37% of male scientists without children do outreach.
• 42% of scientists engage in no public outreach.
• 29% of scientists say that scientists are bad at communicating, or are peceived as bad communicators.
• 25% of scientists think that the public is the main barrier to effective outreach.70% of these scientists perceive the public as scientifically ignorant, and 30% perceive the public as disinterested.
• 19% of scientists say that they would pursue more outreach experiences if they received more recognition and respect for their efforts.
• 15% of scientists think that non-scientists should run outreach programs.
• 5% of scientists are not interested in outreach because they don't see it as part of their role as scientists..
• 5% of scientists do 50% of the outreach.

Others spoke of the "Sagan Effect," or the perception that if you are a popularizer of science, your research is not as extreme/hard-core, and you probably have subpar scientific abilities...if you can communicate well, energize the public, and inspire the scientists of the future (Jensen, et al., 2008).

This image could show either how scientists sometimes perceive "communicators" OR how "the public" sometimes perceives scientists' communication. Scientists may fear that journalists and outreachers will not grasp their work or will simplify it, messing up the message. When scientists present their work publicly in the same way they present it to their collaborators, it can sound like gibberish to the uninitiated (Image credit: Business Week).

Further Questions

1. Is communicating research one of scientists' responsibilities, or is adding their results to the human knowledge base enough?
2. Some scientists believe that non-scientists are unable to comprehend the complexities of their research and thus cannot communicate it well, but they do not want to do the communication themselves or are not good at it, but they do want their results to reach a wide audience. Who is to do the communicating, then? Other scientists who do value outreach?
3. Is it best to separate the scientists and the science communicators?
4. Should scientists be encouraged to do more outreach? Or should they just be encouraged to respect outreach and leave it to those with writing/speaking skills, charisma, and well-tailored pants?
5. How much scientific background is necessary to do outreach/communication well?
6. What is different about those 5% of scientists who do so much outreach? Do they place less value on their research and the time/energy they have for it? Are their priorities those of "true scientists" if that is the case?
7. How much is outreach based on altruism and/or a desire to "give back"? On a sense of responsibility? On a desire for recognition and fame, since few scientists will be on Jersey Shore? Does having been inspired by a science superhero as a kid affect the likelihood that you'll try to become a science superhero for someone else?

I'll post some of these as discussion topics on our nascent forum, so if you have ideas, please share!

Ecklund, E., James, S., & Lincoln, A. (2012). How Academic Biologists and Physicists View Science Outreach PLoS ONE, 7 (5) DOI: 10.1371/journal.pone.0036240  

Jensen, P., Rouquier, J., Kreimer, P., & Croissant, Y. (2008). Scientists who engage with society perform better academically Science and Public Policy, 35 (7), 527-541 DOI: 10.3152/030234208X329130

Further Reading Material:

What factors predict scientists' intentions to participate in public engagement of science activities? (Poliakoffl & Webb, 2007)

Scientists and public outreach: participation, motivations, and impediments. (Andrews, et. al, 2004)

Out of the loop: why research rarely reaches policy makers and the public and what can be done. (Shanley & Lopez, 2008)

Constructing Communcation: talking to scientists about talking to the public. (Davies, 2008) [sorry, I couldn't find a free version of this one]

It's feeding time for more supermassive black holes than once thought

by Sarah Scoles

Just like some people are active and some are not, some galaxies have active nuclei, and some do not. But while for people inactivity means parking in the space closest to the grocery store's automatic doors, inactivity for a galaxy's nucleus means that it is not actively accreting, or eating, matter. Active galactic nuclei are eating things all the time, and all that feasting presumably affects the way the galaxy grows up. But how? That's the question researchers like Lisa Winter, a Hubble Fellow at the University of Colorado, Boulder, are asking.  

What is a galactic nucleus?

Like the nucleus of an atom, the term nucleus, in this context, means the part at the center that is considerably massive. For an atom, this is a combo of protons and neutrons. For a galaxy, this is a supermassive black hole. An active galactic nuclei, or AGN, has matter around it, and that matter is falling into the black hole.

But even though light doesn't come from the black hole, we can see light from the matter that hasn't fallen in--the material that is swirling around the black hole, and the material that, while swirling, gets directed outward in jets.

For more information on seeing evidence of black holes, see this post.

This is what activity looks like. Artist concept credit: ESA/AOES Medialab. 

Are there different types of AGN, or are all active galaxies active in the same way?

While all people who are active do not throw javelins on the weekends, all AGN might be active in the same way. While we observe AGN with lots of different properties, unified models of AGN state that different subclasses could be united by considering the angle at which we see the galaxy. This viewing angle could cause us both to see or to miss, say, soft X-ray emission.

So many arrows. Credit: Pierre Auger Observatory.

The universe doesn't care at all whether we look an AGN head-on or see it from the side, so we see AGN from all different sides, and, depending on the side, we can see different properties.  

Does this mean all AGN are the same? No, they are all special snowflakes, and they do have differing characteristics. But mechanisms and structures are likely similar across time and space.

What don't we know about AGN?

Oh, tons.

1. Why are some galaxies active and some not?

2. Why were there more AGN in the early universe than there are now?

3. Were galaxies that are now couch-potatoes once marathon runners?

4. If so, what processes, exactly, led them from one way of being to the other?

5. If an AGN is eating all this gas, does that mean it will have a lower star formation rate, since it will have less star-birthing material?

6. Can we build a wormhole to one that comes out right at the event horizon and just see what is going on? And then die? For science?

Astronomers are also still figuring out how, exactly, to find AGN. If a galaxy is active, that's because there's a bunch of gas around its black hole. This gas blocks and absorbs light. Light is all our telescopes get. If the light isn't reaching us, we can't learn anything about its source.

However, different kinds of light waves are blocked and absorbed more than others. Optical light and soft (lower energy) X-rays have a hard time getting from the AGN to us. Infrared light looks the same as light from newly forming stars, so we can't know for sure whether it's coming from a black hole or from baby stars. 

Higher energy X-rays to the rescue!

These "hard" X-rays are unambiguous indicators of nucleic activity.

There's a telescope for that.

It's called SWIFT. It has an instrument called the BAT that can detect these kinds of X-rays. And the results from the BAT suggest that by looking at infrared, optical, and wussy soft X-rays, we have missed a significant population of AGN. According to the Lisa Winter's, and her collaborators', results, 24% of the galaxies BAT detected don't show up in anything but hard X-rays. There's a whole demographic of "hidden" AGN that no one knew and few cared about.

Sad.

Hidden AGN are important to general AGN study: if you're missing a whole subset of an object whose physics you're trying to untangle, you can't really untangle the physics very well.

For a NASA article on this topic, click here.

Why do AGN matter to me?

Well, maybe our galaxy used to be active (it current eats some stuff, but not enough for anyone to use the acronym). Maybe that affected the way the galaxy evolved, which means that it was a key factor in determining how the galaxy (and you) look now. Or maybe it wasn't active. Maybe some galaxies are never active. But, then, that would have affected the way the galaxy evolved, too. If having an active nucleus is a factor in how galaxies grow up, then our galaxy became the stand-up fellow it is today by either having an active nucleus, or not.

Now here's a different question to consider before you go to bed tonight: How did galaxies get supermassive black holes at their centers? How did they form?

Or, alternatively, as a student I was helping with a science fair project said, "What are galaxies for?"

Lisa M. Winter (2011). Uncovering Local Absorbed Active Galactic Nuclei with Swift and Suzaku 2011 arXiv: 1112.0545v1

Prenatal exposure to a common pesticide linked with brain abnormalities

via

Virginia A. Rauh, et al. published in PNAS (Early Edition Articles) this week that prenatal exposure to chlorpyrifos (CPF), a widely-used organophosphate insecticide, is associated with neurobehavioral deficits in humans. 

WTF are CPF?

CPF have a shady history, to say the least, starting in 1965 (Dow Chemical Company) when CPF was first registered as a home and garden insecticide. In 2001, the Enivornmental Protection Agency (EPA) set regulatory action to phase out residential use; however, despite reports of chronic exposure to CPF being linked with neurological effects, developmental disorders, and autoimmune disorders – this product is still widely used in agriculture. 

For more history: go here. 

In high-doses CPF works by inhibiting acetylcholinesterase (AChE), an enzyme required to hydrolyze the neurotransmitter acetylcholine. AChE is important mainly to terminate synaptic transmission and inhibitors of AChE lead to muscular paralysis, convulsions, bronchial constriction, and death by asphyxiation. CPF, a class of organophosphates, are a class of irreversible AChE inhibitors. 

AChE mechanism of action. (via)

CPF are the same type of AChE inhibitors used in nerve gases during chemical warfare (ex: Sarin and Soman). High exposure (such as an insect on an apple) to AChE-inhibiting CPF will result in death, however humans receive only low doses. 

That begs the question:

What happens to animals and humans when exposed to "real" doses of CPF?

Me? (via)

Previous animal models (most likely carried out to assess exposure and safety) have shown that low-level exposure to CPF during developmental stages lead to developmental and neurobehavioral abnormalities. Developmentally-challenged neonatal rats anyone?

Interestingly, a very large component of the toxicity found in these studies is likely unrelated to the inhibition of AChE (yes, it does other horrible, nasty things in low-doses as well).

Instead, CPF additionally disrupt cellular machinery that control neuronal replication, differentiation, apoptosis, axon formation, synaptogenesis, and neural circuit formation – all of which sound pretty important for healthy development of a baby if you ask me. With this horrifying information, researchers have recently looked to study if low-levels of CPF exposure had similar consequences in children.

Unfortunately it is known that CPF is detectible in amniotic fluid and readily crosses the placental barrier, and prenatal exposures have been linked to smaller head sizes, lower birth weights, attention problems, and results in lower IQs (references all included in the primary article, let me know if you’d like it).

So what does this study show?

Despite all of this evidence for neurodevelopmental toxicity of CPF, specific effects on brain structure that could lead to behavorial or cognitive differences in humans, have not been studied.

Virginia A. Rauh, et al. was studying a cohort of children from 5.9-11.2 years of age that were prenatally exposed to low-doses of CPF. She found that this low-exposure to CPF caused damage in the brain regions that deal with mood, behavior and cognition (septal nucleus, striatum, somatosensory cortex, and hippocampus). 

This damage came in many forms including: general glial cell numbers (glial cells are non-neuronal cells that provide support and protection for neurons in the brain), changes in number and types of neurons, and delayed alterations in hippocampal cell number along with swelling and early stages of glial scarring (that occurs in response to cellular injury).

What does this mean for us?

In her discussion at the end of the article Virginia Rauh discusses the public health implications of her findings. She mentions that the neurotoxic effects are not only harmful in utero, but have long-term effects extending (at least) into the early school years. Additionally, these studies mimick previous animal model data, suggesting that the neural damage is irreversible.

The big and scary end of the story is that current safety limits are set according to levels needed to achieve inhibition of cholinesterase (mentioned above), because it has been long-assumed that only high-levels of CPF can cause toxicity. However, this study and previous animal studies have shown that low-levels of CPF result in neuronal damage (independent of inhibition of cholinesterase) that result in these horrifying brain abnormalities.

Therefore it’s high time we adjust “toxicity” levels of CPF based on something other than inhibition of cholinesterase. Either that or eat organic foods (that’s my only PSA for the year, I swear).

 -Brooke N.

Rauh, V., Perera, F., Horton, M., Whyatt, R., Bansal, R., Hao, X., Liu, J., Barr, D., Slotkin, T., & Peterson, B. (2012). Brain anomalies in children exposed prenatally to a common organophosphate pesticide Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1203396109