We started our Microbiology Journal Club of the new year, technically a new decade. We started off with a bang, well a bang from a physics perspective, more of a whimper from a microbiology perspective.The paper under discussion was:
Helicobacter pylori moves through mucus by reducing mucin viscoelasticity. by Celli JP et al. Proc Natl Acad Sci U S A. 2009 Aug 25;106(34):14321-6.
The basic premise is that H. pylori, a spiral shaped bacteria is thought to burrow its way through the gastric mucus, like a cork screw to get to the underlying epithelial cells lining your stomach. However, this work suggests another mechanism. But let's back up a little.
How do the epithelial cells lining your stomach survive? Great question, Im glad you asked. The epithelial cells secrete several things to protect themselves one of which is mucus. Gastric mucus has an amazing property of being able to change its viscosity. Kind of like molasses is watery when heated and almost solid when cooled. There is no decision making going on by the mucus, this is strictly a property of physics and chemistry. Now gastric mucus is interesting because it is more 'solid' the lower the pH, like 2, and more 'liquid' at higher pH, like 7. What this means is that where the mucus is exposed to acid it forms a protective shell, but closer to the epithelial cells, it is less viscous.
What does this have to do with H. pylori? Another great question. I should point out that H. pylori does not grow at pH 2, its killed by this environment much like most everything else.
 H. pylori - Yutaka Tsutsumi, MD |
How do the epithelial cells lining your stomach survive? Great question, Im glad you asked. The epithelial cells secrete several things to protect themselves one of which is mucus. Gastric mucus has an amazing property of being able to change its viscosity. Kind of like molasses is watery when heated and almost solid when cooled. There is no decision making going on by the mucus, this is strictly a property of physics and chemistry. Now gastric mucus is interesting because it is more 'solid' the lower the pH, like 2, and more 'liquid' at higher pH, like 7. What this means is that where the mucus is exposed to acid it forms a protective shell, but closer to the epithelial cells, it is less viscous.
What does this have to do with H. pylori? Another great question. I should point out that H. pylori does not grow at pH 2, its killed by this environment much like most everything else.
H. pylori actually modifies the local pH through an enzyme called urease that generates ammonia, which has a pH of around 12. Pretty good way to deal with stomach acid. Ill also point out that H. pylori mutants that cannot make urease are absolutely killed in the stomach. So the idea has been that H. pylori makes urease until it can burrow through the mucus to get to the epithelial cells where the pH is much more conducive to life.
But how does H. pylori burrow through the mucus when it is like a shell? That is the $64,000 question and the one considered by the authors of the paper. Remember that the viscosity of the mucus is affected by pH and that H. pylori can make the local environment less acidic. This led to the hypothesis that H. pylori increases the pH (making it less acidic), which also reduces the viscosity of the mucus (its easier to swim through) and that's how H. pylori gets through the mucus to the epithelial cells. The authors then did a number of studies to support their hypothesis, although did little to rule it out. In fact one the disappointments is that important controls were essentially non-existant and the authors could have had a much tighter more informative study if they had done them. (I should point out the paper was published in the "Biophysics and computational biology" section of PNAS not the "Microbiology" section.)
One of the first experiments done is to compare gastric mucus at pH 4 (control) with gastric mucus + H. pylori (which quickly becomes pH 7). There are two variables here, does everyone see them? The first, the one the authors care about is the presence/absence of bacteria. The second is pH. So when I see data like in Figure 1 (1B is shown), I have thoughts.
This panel is showing the nonlinear viscoelastic response of the samples, don't worry I don't know what that means either. Regardless, the white lines, no bacteria pH 4 samples, are constant (flat) until the applied stress reaches ~10 Pa, and then they drop like a rock. The red lines, + bacteria pH 7 samples, show a different response. So the authors conclusion is that the bacteria have an effect, which I agree with. However, I want to see the data for the no bacteria pH 7 experiment (not done) or the + bacteria pH 4 experiment (not done. This latter experiment could easily be done by simply using the urease mutant, which does not increase the pH). These experiments would demonstrate whether the difference in viscoelasticity was due ONLY to the pH effect caused by the bacteria or due to the pH effect and additional effects. These bacteria also secrete proteases that could degrade the mucus, which would also reduce the viscosity.
The authors include some movies and pictures of cells and also measure some things in these datasets. However, it is really impossible to determine what is being measured or how cells were chosen. For example, one movie uses mucus at pH 4 (without urea, so the bacteria cannot change the pH) and the one bacterium shown tries real hard to swim, but doesn't go far (were there any others?). The other movie uses mucus at pH 6 and a number of bacteria, three of which move around quite well, the others do not move much at all. So when the authors are mapping the movement of bacteria (Figure 2) did they simply ignore the cells not moving? do a different experiment? what? I can't tell.
Regardless, I think the authors are on to something. I do not believe they have thrown out the burrowing model described previously. But I do believe they have refined it and made it more biologically meaningful. By looking at the true nature of gastric mucus and thinking about the bacteria in this context, I think the authors have gained some biological insights into what these bacteria need to do to survive and how the gastric mucus protects us (from acid, already known, and maybe from other bacteria, not previously known at least not via this mechanism).
Finally, I thought this work suggested something else. The model being used is that the bacteria get into your stomach, don't ask how you don't really want to know how your GI tract is colonized. The stomach is a hostile environment and the bacteria have a short time to get to safety by the epithelial cells. The bacteria secrete a bucketload of urease, increase the pH to survive a bit longer and also to reduce the viscosity of the mucus so they can swim through it to safety. Once there, the bacteria can cause chronic inflammation, which damages the epithelial cells, and can eventually lead to gastric cancer.
I suggest another possibility. The bacteria usually get in to us as infants when our stomach pH is not a acidic as it is when we are older. The bacteria survive long enough to colonize our stomachs and things go on there merry way. If the bacteria grow too much, there is sufficient reduction in the local viscosity of the mucus that acid can leak through and damage the epithelial cells (remember they don't like it either). Damage leads to inflammation and chronic damage can cause ulcers to form and eventually cancer.
These two ideas are not mutually exclusive and there is still the question why do some people get gastric illness from H. pylori and others do not.
Celli, J., Turner, B., Afdhal, N., Keates, S., Ghiran, I., Kelly, C., Ewoldt, R., McKinley, G., So, P., Erramilli, S., & Bansil, R. (2009). Helicobacter pylori moves through mucus by reducing mucin viscoelasticity Proceedings of the National Academy of Sciences, 106 (34), 14321-14326 DOI: 10.1073/pnas.0903438106
But how does H. pylori burrow through the mucus when it is like a shell? That is the $64,000 question and the one considered by the authors of the paper. Remember that the viscosity of the mucus is affected by pH and that H. pylori can make the local environment less acidic. This led to the hypothesis that H. pylori increases the pH (making it less acidic), which also reduces the viscosity of the mucus (its easier to swim through) and that's how H. pylori gets through the mucus to the epithelial cells. The authors then did a number of studies to support their hypothesis, although did little to rule it out. In fact one the disappointments is that important controls were essentially non-existant and the authors could have had a much tighter more informative study if they had done them. (I should point out the paper was published in the "Biophysics and computational biology" section of PNAS not the "Microbiology" section.)
One of the first experiments done is to compare gastric mucus at pH 4 (control) with gastric mucus + H. pylori (which quickly becomes pH 7). There are two variables here, does everyone see them? The first, the one the authors care about is the presence/absence of bacteria. The second is pH. So when I see data like in Figure 1 (1B is shown), I have thoughts.
This panel is showing the nonlinear viscoelastic response of the samples, don't worry I don't know what that means either. Regardless, the white lines, no bacteria pH 4 samples, are constant (flat) until the applied stress reaches ~10 Pa, and then they drop like a rock. The red lines, + bacteria pH 7 samples, show a different response. So the authors conclusion is that the bacteria have an effect, which I agree with. However, I want to see the data for the no bacteria pH 7 experiment (not done) or the + bacteria pH 4 experiment (not done. This latter experiment could easily be done by simply using the urease mutant, which does not increase the pH). These experiments would demonstrate whether the difference in viscoelasticity was due ONLY to the pH effect caused by the bacteria or due to the pH effect and additional effects. These bacteria also secrete proteases that could degrade the mucus, which would also reduce the viscosity.
The authors include some movies and pictures of cells and also measure some things in these datasets. However, it is really impossible to determine what is being measured or how cells were chosen. For example, one movie uses mucus at pH 4 (without urea, so the bacteria cannot change the pH) and the one bacterium shown tries real hard to swim, but doesn't go far (were there any others?). The other movie uses mucus at pH 6 and a number of bacteria, three of which move around quite well, the others do not move much at all. So when the authors are mapping the movement of bacteria (Figure 2) did they simply ignore the cells not moving? do a different experiment? what? I can't tell.
Regardless, I think the authors are on to something. I do not believe they have thrown out the burrowing model described previously. But I do believe they have refined it and made it more biologically meaningful. By looking at the true nature of gastric mucus and thinking about the bacteria in this context, I think the authors have gained some biological insights into what these bacteria need to do to survive and how the gastric mucus protects us (from acid, already known, and maybe from other bacteria, not previously known at least not via this mechanism).
Finally, I thought this work suggested something else. The model being used is that the bacteria get into your stomach, don't ask how you don't really want to know how your GI tract is colonized. The stomach is a hostile environment and the bacteria have a short time to get to safety by the epithelial cells. The bacteria secrete a bucketload of urease, increase the pH to survive a bit longer and also to reduce the viscosity of the mucus so they can swim through it to safety. Once there, the bacteria can cause chronic inflammation, which damages the epithelial cells, and can eventually lead to gastric cancer.
I suggest another possibility. The bacteria usually get in to us as infants when our stomach pH is not a acidic as it is when we are older. The bacteria survive long enough to colonize our stomachs and things go on there merry way. If the bacteria grow too much, there is sufficient reduction in the local viscosity of the mucus that acid can leak through and damage the epithelial cells (remember they don't like it either). Damage leads to inflammation and chronic damage can cause ulcers to form and eventually cancer.
These two ideas are not mutually exclusive and there is still the question why do some people get gastric illness from H. pylori and others do not.
Celli, J., Turner, B., Afdhal, N., Keates, S., Ghiran, I., Kelly, C., Ewoldt, R., McKinley, G., So, P., Erramilli, S., & Bansil, R. (2009). Helicobacter pylori moves through mucus by reducing mucin viscoelasticity Proceedings of the National Academy of Sciences, 106 (34), 14321-14326 DOI: 10.1073/pnas.0903438106
2 comments:
You need some basic grammar and spelling help -- commas ≠ periods, effect ≠ affect.
Thanks for adding to the discussion.
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