H. pylori Finds its Home

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00:00:12;16 Hi.
00:00:13;13 My name is Julie Huang
00:00:14;15 and I'm a microbiologist.
00:00:16;07 And today I'd like to tell you a story
00:00:18;14 about a bacterium that has evolved
00:00:20;14 some really clever strategies
00:00:22;11 to survive in one of the harshest environments in the human body.
00:00:26;06 And this environment is something that
00:00:28;04 we're all intimately familiar with
00:00:29;24 -- it's our stomach.
00:00:31;20 So, this morning, I had a nice a nice big breakfast
00:00:33;21 and by now the food has gone down into the stomach
00:00:36;09 and it's well on its way to digestion.
00:00:38;16 Our stomach produces a lot of hydrochloric acid
00:00:41;14 and digestive enzymes
00:00:43;05 to help us break down the food
00:00:44;22 so that we can get the energy
00:00:47;03 to go on with the rest of our day.
00:00:49;06 But the stomach is also meant to do a second function:
00:00:52;13 the acid is thought to sterilize
00:00:54;23 whatever we take into our body
00:00:56;21 so that we don't end up with food poisoning
00:00:58;19 and other diseases.
00:01:00;24 So for the longest time,
00:01:02;09 because of this idea that the
00:01:04;22 acid is supposed to sterilize whatever we take in,
00:01:07;08 people didn't believe that any living organism
00:01:10;00 can actually live in this harsh, acidic environment.
00:01:13;10 But this idea was challenged in the 1980s.
00:01:16;27 Let me tell you the story of how this happened.
00:01:19;22 So, this story begins with two scientists from Australia,
00:01:22;25 Barry Marshall and Robin Warren.
00:01:25;01 And in the 1980s,
00:01:26;24 one of the most devastating diseases affecting people at the time
00:01:29;14 were stomach ulcers.
00:01:31;16 So, stomach ulcers are essentially open wounds in the stomach
00:01:34;27 and in some cases people were
00:01:37;18 literally bleeding out of these wounds
00:01:39;18 and dying from these ulcers.
00:01:41;09 And at the time people believed that
00:01:43;05 ulcers were caused by things like
00:01:45;10 eating spicy foods or having too much stomach acid
00:01:48;09 or even things like stress.
00:01:51;04 But Barry Marshall and Robin Warren really wanted to
00:01:53;25 put this to the test and identify
00:01:55;25 what was the cause of these stomach ulcers
00:01:57;21 that were killing so many people.
00:02:01;05 And so they took biopsies from patients
00:02:03;05 that were suffering from ulcers
00:02:05;07 and then looked at it underneath the microscope,
00:02:07;22 and to their surprise
00:02:10;04 they actually found that there were all these spiral-shaped bacteria found
00:02:13;13 living in the tissue,
00:02:15;06 and so they proposed the idea that
00:02:17;04 perhaps this spiral-shaped bacterium
00:02:19;26 can actually be linked to ulcers.
00:02:22;00 And when they told scientists in the community about this,
00:02:24;21 to their dismay,
00:02:26;15 they were actually rejected very, very quickly.
00:02:28;29 Scientists were really holding onto this idea that
00:02:31;19 the stomach was so hostile, so acidic
00:02:33;13 that nothing could really grow there,
00:02:35;04 and the idea that it was linked to ulcers...
00:02:38;06 that was something that they just couldn't accept.
00:02:41;00 Barry Marshall, being a very persistent scientist,
00:02:43;23 decided that he was going to prove them wrong,
00:02:46;06 so he did the ultimate experiment...
00:02:49;00 on himself.
00:02:50;20 He took a vial of live culture of this spiral-shaped bacteria
00:02:53;26 and drank it and,
00:02:57;00 definitely, days later,
00:02:59;00 after he had drunk this culture,
00:03:00;22 he started to feel the symptoms that patients with ulcers
00:03:03;23 would start to feel too.
00:03:05;13 He was cramping, he was vomiting,
00:03:07;05 just felt completely sick.
00:03:08;25 And a few days after he had drank this vial,
00:03:10;29 he took a biopsy of his own stomach
00:03:13;10 and, sure enough, underneath the microscope
00:03:15;16 he saw those spiral-shaped bacteria
00:03:18;03 that was found in the stomachs of those ulcer patients he had seen.
00:03:23;03 He didn't let the infection progress to the point
00:03:24;24 that he would develop ulcers
00:03:26;13 and, luckily, because he knew that
00:03:28;28 this was a bacterium,
00:03:30;16 he could take antibiotics to clear this infection.
00:03:32;15 And sure enough, a few days after taking antibiotics,
00:03:35;28 he was cleared of the infection
00:03:37;20 and those symptoms went away
00:03:39;11 and, most importantly, he never developed ulcers.
00:03:42;11 And so this evidence really was
00:03:45;00 important in linking the fact that there was
00:03:47;14 actually a live bacterium
00:03:49;10 living in the stomach and it was
00:03:52;06 causing ulcers in these people.
00:03:54;23 And so Barry Marshall and Robin Warren
00:03:56;15 continued on with their studies on this bacterium,
00:03:59;25 and this bacteria was eventually named Helicobacter pylori.
00:04:03;11 And the name refers to the fact that the bacterium
00:04:06;00 actually has this helical corkscrew shape
00:04:08;29 and the pylori part of its name refers to the fact that,
00:04:11;26 initially, the bacteria was isolated
00:04:14;02 from the pyloric region of the stomach,
00:04:16;11 which is down here,
00:04:18;03 because this is where the ulcers were happening.
00:04:20;01 And so much of the studies coming...
00:04:23;07 since then, that seminal discovery,
00:04:25;14 have revealed that, actually, H. pylori
00:04:28;00 is one of the most successful pathogens to infect humans.
00:04:31;25 It actually infects half of the world's population,
00:04:34;17 so, right now,
00:04:36;21 one in two people in the entire world is actually
00:04:40;19 infected with this bacterium.
00:04:42;08 And it turns out that in about
00:04:44;26 10-15% of those who are infected,
00:04:47;03 those individuals were end up developing these
00:04:49;11 duodenal and gastric ulcers,
00:04:51;25 like you see here in this image.
00:04:53;22 But actually more importantly, perhaps,
00:04:56;06 is the fact that now that we know it's a bacterium,
00:04:59;16 people are able to take antibiotics right away,
00:05:02;11 as soon as they're feeling symptoms in the stomach,
00:05:04;20 and so people are no longer dying from things like ulcers.
00:05:08;21 But there was another link that was
00:05:11;06 really surprising coming from studies
00:05:13;08 that came after this discovery of H. pylori,
00:05:16;10 and that was the link of H. pylori with stomach cancer.
00:05:20;00 So, stomach cancer is the
00:05:22;06 third leading cause of cancer deaths in the world.
00:05:25;00 Each year, a million new cases occur
00:05:28;20 and about 700,000 people will die
00:05:30;25 from this type of cancer.
00:05:32;19 And the idea that an organism also causing,
00:05:36;08 or being linked and associated to, a cancer
00:05:39;28 was completely unfathomable,
00:05:41;26 until we realized that H. pylori
00:05:44;15 was able to live in the stomach
00:05:46;27 and it actually gave more...
00:05:50;02 it actually caused people to have a higher risk
00:05:52;23 of actually developing stomach cancer.
00:05:55;08 And it's such a bad risk factor
00:05:58;13 that the World Health Organization
00:06:00;25 has actually designated it to be a type I carcinogen,
00:06:03;25 so this is as bad as smoking is to lung cancer.
00:06:07;10 And so this is really devastating
00:06:09;26 because so many people are affected
00:06:11;29 if they're infected with H. pylori,
00:06:14;18 but what we believe is,
00:06:16;29 by better understanding the biology of H. pylori in the stomach,
00:06:20;23 we can develop novel therapies to treat these infections.
00:06:24;29 So, why would we need novel therapies
00:06:26;17 when we have antibiotics?
00:06:28;22 Well, that's because antibiotic resistant strains are popping up very quickly now,
00:06:34;09 and in a few years the antibiotics that we have
00:06:37;15 are no longer going to be able to
00:06:40;03 effectively clear these infections.
00:06:42;16 So, imagine, just 30 years ago,
00:06:45;12 people had no idea what was causing
00:06:47;22 ulcers or stomach cancer,
00:06:49;16 and because of the courageous experiments
00:06:51;29 that Barry Marshall did on himself,
00:06:54;06 and the studies he did together with his colleague
00:06:56;18 Robin Warren,
00:06:58;09 we now know that there's a bacterium
00:06:59;29 that we can target.
00:07:01;13 And because of their seminal work,
00:07:02;25 Barry Marshall and Robin Warren actually won the Nobel Prize in 2005
00:07:07;11 for linking H. pylori to the peptic ulcers
00:07:11;17 and the cause for gastric cancer.
00:07:14;05 So, now I kind of want to take us back to the stomach.
00:07:17;22 So, as I referred to in the beginning,
00:07:19;20 the stomach is thought to be this
00:07:21;21 very hostile environment
00:07:23;04 because of the high abundance of the hydrochloric acid
00:07:26;03 that the cells release.
00:07:27;28 The hydrochloric acid is really strong
00:07:30;11 -- it's as strong as the acid that you find in battery acid,
00:07:33;19 which can corrode even metal.
00:07:35;18 So if you were actually to drop
00:07:38;01 a spot of hydrochloric acid from the stomach
00:07:41;19 onto a piece of wood,
00:07:43;06 it would literally eat through that piece of wood
00:07:45;01 and cause a hole to form.
00:07:46;06 So how in the world are our cells actually surviving in this,
00:07:48;27 our own, harsh acidic environment.
00:07:50;24 Well, our cells are pretty smart too.
00:07:53;07 They're not just going to produce the acid
00:07:54;27 and not have a way to protect itself,
00:07:57;29 so it actually forms also
00:08:01;00 this very thick mucus layer
00:08:03;02 that covers the cells and effectively
00:08:05;09 prevents the acid from ever reaching the cells,
00:08:08;11 creating this very well-buffered zone,
00:08:11;26 and this is how the cells of our body,
00:08:14;13 in the stomach,
00:08:15;29 actually survive the harsh, acidic environment.
00:08:17;29 But what about H. pylori?
00:08:19;23 It's not naturally in the stomach to begin with?
00:08:22;14 How does it survive?
00:08:24;17 Is it because it's an acidophile?
00:08:26;27 Well, it turns out that it's not.
00:08:29;00 It actually hates acid.
00:08:31;00 And the way that H. pylori
00:08:33;08 has figured out how to survive in the stomach
00:08:37;06 is by being able to move and swim
00:08:39;11 using these tail-like flagella at the poles
00:08:43;06 and that allows it to move away from danger
00:08:45;26 in the environment
00:08:47;04 and move towards things that are good.
00:08:49;09 And, for H. pylori, what it's figured out
00:08:52;20 is that the stomach...
00:08:54;14 in the stomach, the cells in the mucus layer
00:08:56;27 is where the pH is near neutral,
00:08:58;22 so it needs to home in to that specific location.
00:09:02;18 So, where is H. pylori specifically in the stomach?
00:09:05;23 I first want to orient you to
00:09:07;27 what the stomach topology looks like.
00:09:09;15 This is a cross-section through the gastric epithelium
00:09:12;06 or the stomach tissue,
00:09:13;22 and what you can see is that
00:09:15;22 at the surface here,
00:09:17;11 the surface epithelium,
00:09:18;20 this is where the mucus,
00:09:20;00 as well as the food that we digest,
00:09:21;14 would lie.
00:09:23;02 But you also notice that on the top here
00:09:24;21 there are these openings called gastric pits,
00:09:27;10 and it's through the gastric pits
00:09:29;12 that will lead us deeper into these folds of the tissue
00:09:32;15 that make up the gastric glands.
00:09:34;22 And the cells of the glands
00:09:36;16 are what produces the mucus, as well as the acid,
00:09:38;20 that gets released into the lumenal space
00:09:41;01 where our food would be.
00:09:42;23 So if you take a biopsy from an infected individual,
00:09:46;11 what you see under the microscope
00:09:48;11 is that a majority of the bacteria
00:09:50;07 reside in this overlying mucus layer
00:09:52;08 in a free-swimming state.
00:09:54;18 But if you were to look closer at the cells
00:09:56;16 that make up the epithelium,
00:09:58;12 you can also find that there are bacteria
00:10:00;27 directly attached to the cells,
00:10:03;05 and this particular population that's adhered
00:10:05;22 and attached to the cells
00:10:07;13 is what we believe is the important population
00:10:10;10 that can cause disease.
00:10:12;18 And that's because H. pylori is known to
00:10:15;07 have several toxins that it can inject into cells
00:10:17;22 and, upon injection,
00:10:19;27 these cells will essentially get damaged.
00:10:22;07 And a previous graduate student in the lab
00:10:24;01 actually discovered that
00:10:26;09 not only do the bacteria
00:10:28;12 attach to the cells and inject these toxins,
00:10:30;13 but they can cause the cells to release nutrients,
00:10:33;11 allowing it to grow and replicate
00:10:36;13 on the cell surface
00:10:38;10 into these really beautiful clusters that we call microcolonies.
00:10:42;03 And so all of this was observed in cell culture,
00:10:44;21 meaning that we were growing these cells in the lab
00:10:47;05 and infecting them,
00:10:48;21 and we discovered that they can grow on cells,
00:10:51;05 but we wondered,
00:10:52;25 where is this happening within the stomach?
00:10:55;07 So, unlike Barry Marshall,
00:10:57;20 most of us don't want to experiment on ourselves
00:11:00;04 to be able to study how H. pylori lives in the stomach,
00:11:03;12 but luckily for us we were able to
00:11:05;26 develop an animal model using the mouse.
00:11:08;19 So, the mouse has allowed us
00:11:11;23 to be able to obtain entire stomachs
00:11:14;12 infected with H. pylori
00:11:16;15 and instead of just seeing the surface part
00:11:19;17 of the stomach,
00:11:21;06 we can now look at the entirety of the gastric glands
00:11:23;27 and figure out where the bacteria are specifically located
00:11:27;14 and growing on the cell surface.
00:11:30;02 And so, using a technology
00:11:32;08 called immunofluorescence microscopy,
00:11:35;01 what we can do is
00:11:37;06 label specific biological components of cells
00:11:41;01 and be able to visualize
00:11:43;08 where the bacteria are in relation to the epithelial cells
00:11:46;00 that make up our tissue.
00:11:48;07 So, what... I want to show you now one of these
00:11:51;02 immunofluorescence sections.
00:11:53;10 So, in red what you're seeing is
00:11:55;05 the outline of each of these epithelial cells
00:11:57;09 that make up the glands
00:11:58;29 and the blue is the nucleus of each of these cells.
00:12:01;26 The top part here is that mucus layer
00:12:03;23 and what you can see is that
00:12:06;02 there are a lot of bacteria, labeled here in green,
00:12:08;14 found in this overlying mucus layer.
00:12:10;09 And you can also see that there are bacteria
00:12:12;14 directly attached to the cells.
00:12:14;07 But wait, there's more...
00:12:16;11 when we looked deeper into the tissue,
00:12:18;19 we found that there was actually this population of H. pylori
00:12:21;25 residing deep in the gastric glands,
00:12:23;24 and this particular zone of the stomach
00:12:26;02 was really intriguing to us
00:12:28;17 because this is thought to be where precursor cells reside.
00:12:30;25 Precursor cells are slightly differentiated from stem cells,
00:12:34;26 but still have a lot of potential
00:12:37;04 to form into all the other cells
00:12:39;15 that make up the gastric glands.
00:12:41;16 And so, as you can imagine,
00:12:43;10 these bacteria are directly adhered to these precursor cells
00:12:46;23 and they could possibly be
00:12:50;00 injecting a lot of these toxins into the cells
00:12:52;06 and causing them to die.
00:12:54;09 And in fact what we have found from
00:12:57;21 more recent studies in the lab is that
00:13:00;03 the bacteria specifically located in the glands
00:13:03;06 actually are able to activate the stem cells
00:13:06;14 and cause them to divide more.
00:13:08;13 And increased cell division is one of the
00:13:11;05 really, really important steps
00:13:13;17 towards cancer development,
00:13:15;05 because uncontrolled cell growth is really
00:13:17;17 the hallmark of cancer.
00:13:19;01 So, we really wanted to understand
00:13:21;02 how this population is established
00:13:23;03 #NAME?
00:13:25;29 But before we actually proceeded on
00:13:28;07 with more experiments, we asked ourselves,
00:13:30;29 so, all of this was observed in the mouse
00:13:33;09 and H. pylori is actually a
00:13:35;22 human-specific pathogen,
00:13:37;16 meaning it's not found in any other environmental reservoir,
00:13:40;08 any other animals,
00:13:43;16 so how relevant is this to humans?
00:13:45;16 Could this observation be just an
00:13:47;04 artifact of being in the mouse?
00:13:49;12 So that's the question we asked:
00:13:51;06 What about humans?
00:13:52;28 Is there a way that we can actually
00:13:55;09 obtain human stomachs
00:13:56;28 so that we could figure out
00:13:58;13 whether H. pylori was found deep in the glands?
00:14:00;28 Well, we were really fortunate
00:14:02;24 to be able to establish a collaboration
00:14:04;15 with a team of clinicians and scientists in Mexico,
00:14:07;11 and it was really important that we went to
00:14:10;06 a place like Mexico
00:14:11;25 as opposed to going to a hospital in the U.S.
00:14:13;26 because even though H. pylori
00:14:15;29 infects half of the world's population,
00:14:17;29 it doesn't infect each country equally,
00:14:21;10 so places like Japan, China, and Mexico
00:14:25;02 have a much higher prevalence of H. pylori
00:14:27;09 than in the U.S.
00:14:28;20 So, by going to Mexico
00:14:30;06 we were increasing our chances
00:14:31;25 of finding H. pylori-positive tissues.
00:14:35;05 And luckily they were able the
00:14:37;02 get tissues from patients
00:14:39;03 that were undergoing gastric bypass surgery.
00:14:41;00 So, usually these pieces of their stomachs
00:14:43;21 were just discarded after surgery,
00:14:45;24 but we asked them if they could send it to us
00:14:48;21 so that we could use our technique
00:14:51;06 to see whether we could find bacteria
00:14:52;21 in these stomachs.
00:14:54;08 And, to our delight,
00:14:56;07 we were able to observe that there are
00:14:58;21 indeed bacteria in these human stomachs.
00:15:01;11 So, what you're seeing here is,
00:15:03;01 again, H. pylori in this mucus layer
00:15:05;13 that overlies the tissue,
00:15:07;12 but more importantly we saw that the bacteria
00:15:10;01 are also found deep in the glands,
00:15:12;09 just like we had seen in the mouse.
00:15:14;17 So, this was really exciting because
00:15:16;22 it verified and confirmed for us that
00:15:18;19 this discovery that we had made in the mouse
00:15:20;24 is completely relevant to humans.
00:15:22;25 And so we proceeded to ask the question
00:15:26;02 of whether there were factors
00:15:28;05 that control H. pylori's ability to find the epithelium,
00:15:30;19 because if we could figure out
00:15:32;19 what it's sensing to find the epithelium,
00:15:34;29 then maybe we could prevent it from actually getting there
00:15:37;09 and causing disease.
00:15:39;10 So, to study this,
00:15:42;20 we had previous grad students
00:15:45;02 that actually were working on this problem,
00:15:46;29 and one of these grad students, Mike Howitt,
00:15:49;23 actually serendipitously discovered
00:15:51;23 a new protein in H. pylori
00:15:53;29 that seemed to control the bacteria's ability
00:15:56;04 to find this specific niche in the stomach.
00:15:58;06 So, this protein he called as ChePep
00:16:01;04 and what you can see very dramatically in these images
00:16:04;23 is that, while the wild type is found in the glands,
00:16:06;18 just like in those previous images I showed you,
00:16:09;01 the mutant strain that doesn't have this protein
00:16:11;27 is no longer able to localize to the glands.
00:16:14;21 It's only found in the overlying mucus layer.
00:16:18;07 So Mike was really, really puzzled by this,
00:16:20;22 but super excited too,
00:16:22;18 because he had just discovered this new protein
00:16:25;06 and it seems to control the bacteria's
00:16:28;03 ability to find this niche,
00:16:29;24 so he wanted to know,
00:16:31;11 just what is the protein doing for the bacteria?
00:16:33;29 And through his studies,
00:16:36;05 he figured out that this ChePep protein
00:16:38;18 is actually a member of the chemotaxis pathway in H. pylori.
00:16:42;15 So, chemotaxis is a signaling process
00:16:45;10 that allows bacteria, and other organisms too,
00:16:47;24 to be able to sense environmental chemical gradients
00:16:50;26 and that allows bacteria
00:16:53;02 to move towards environments
00:16:55;01 that are beneficial for them
00:16:56;19 or, conversely, move away from environments
00:16:59;03 that are dangerous for them.
00:17:01;04 So, in H. pylori, chemotaxis
00:17:03;26 starts with the fact that it has four chemoreceptors,
00:17:06;17 these proteins that essentially act as H. pylori's noses,
00:17:10;01 and these are highlighted in green, here,
00:17:12;09 in the bacterium.
00:17:13;19 And when one of these receptors
00:17:15;09 senses something in the environment,
00:17:16;24 it relays a signal down to the flagella tails
00:17:19;07 so that the bacteria know
00:17:21;13 whether to swim towards whatever it's smelling or not,
00:17:24;11 and ChePep turns out to be
00:17:26;08 a regulator of this whole process.
00:17:28;03 So, essentially, the ChePep mutant
00:17:30;20 was a H. pylori bacterium with a stuffy nose
00:17:33;15 -- it couldn't smell properly.
00:17:35;20 So, we knew then,
00:17:37;18 from these studies,
00:17:39;01 that smelling the environment properly in the stomach
00:17:41;10 was necessary to get the bacteria to the cells.
00:17:44;12 But at this point, I had joined the lab,
00:17:46;02 and I wanted to know,
00:17:47;25 what were those signals in the environment
00:17:49;20 that the bacteria were actually detecting
00:17:51;20 to find its way to the cells.
00:17:53;18 And so... in order to study chemotaxis,
00:17:56;23 we actually had to develop a novel system,
00:17:59;28 which we call here the microgradient assay.
00:18:02;21 And it essentially...
00:18:05;02 what we did was we repurposed
00:18:07;05 another technique called the microinjection system,
00:18:09;14 which normally is injecting small amounts of things into cells,
00:18:12;12 but instead of injecting into cells,
00:18:14;16 what we used this microinjection system is...
00:18:17;03 we load a needle, a fine-tipped needle,
00:18:19;17 with our chemical of interest,
00:18:21;10 and we can control when it goes into a live culture of bacteria,
00:18:24;18 which is put in the center of a dish,
00:18:27;08 you can see that a gradient forms,
00:18:30;10 locally,
00:18:32;02 in this live culture of bacteria.
00:18:34;10 And underneath this dish is a microscope
00:18:37;27 that lets us record the behavior of the bacteria
00:18:40;03 as it's exposed to this chemical gradient.
00:18:42;17 And so one of the most important signals
00:18:45;07 that we have long known that H. pylori
00:18:47;28 is able to detect
00:18:49;19 is acid, as I had mentioned earlier.
00:18:52;12 So, acid is a repellent
00:18:54;24 and we wanted to test whether
00:18:57;16 our assay would be able to show us
00:18:59;29 a repellent response from H. pylori
00:19:01;24 when it is exposed to a chemical gradient
00:19:03;23 of hydrochloric acid.
00:19:05;10 So, what you're going to see in these movies here
00:19:08;09 is the response of the wild type strain,
00:19:10;12 which is the normal strain that can sense,
00:19:12;18 versus a strain that is
00:19:15;22 a chemotaxis mutant, which is unable to sense.
00:19:19;01 And you'll see that a needle will come in
00:19:22;00 on the left side of the video,
00:19:23;14 and when this happens a gradient of acid is formed,
00:19:26;03 and we can watched the behaviors of the bacteria
00:19:28;19 as they're exposed to this acidic gradient.
00:19:31;09 And what you'll notice right away is that
00:19:33;18 the wild type strain runs away very quickly
00:19:36;02 -- within a matter of five seconds,
00:19:38;18 most of the bacteria have moved out of the field of view --
00:19:42;06 but the chemotaxis null mutant, which can't sense,
00:19:45;20 remains in that field of view.
00:19:47;17 And that demonstrates to us that...
00:19:49;23 there are two things it actually shows us:
00:19:51;16 1) H. pylori is sensing acid as a repellent
00:19:54;25 and, perhaps a little more importantly in our case,
00:19:57;11 2) our assay works,
00:20:00;16 which rarely kind of happens in science.
00:20:02;16 But this was really great because this now allows us
00:20:04;27 to be able to visualize chemotactic responses
00:20:08;00 in real time.
00:20:10;00 And so, in addition to the acid that is thought to help
00:20:13;18 repel the bacteria away from the lumen,
00:20:15;12 I hypothesized that there could be
00:20:17;24 chemicals coming from cells
00:20:19;17 that actually attract the bacteria,
00:20:21;08 which helps them navigate towards the cells.
00:20:24;11 So, to test this idea,
00:20:25;28 I took advantage of a relatively new technology
00:20:28;25 called organoids.
00:20:31;07 So, organoids essentially allow me to
00:20:34;25 grow a stomach in a dish.
00:20:36;20 This is a way for us to take the most relevant cell type,
00:20:39;09 and in this case human stomach cells,
00:20:42;23 and be able to create these organoid cultures
00:20:46;08 in the lab
00:20:47;29 that recapitulates some of the features of the stomach.
00:20:50;26 So, a lot of these organoids look like spheres,
00:20:52;23 and this is just an image of
00:20:54;16 a cross-section through one of these spheres
00:20:56;07 #NAME?
00:20:58;08 So what I did was I took these organoids,
00:21:00;04 which are kind of like our stomachs,
00:21:01;26 but in a dish,
00:21:03;29 and I took the cells
00:21:06;00 and washed them extensively
00:21:07;27 to get rid of anything that was sitting with the cells
00:21:10;19 and then I added fresh media,
00:21:12;29 which then was allowed to condition
00:21:15;17 with the cells.
00:21:17;10 So, the idea was if the cells were releasing any compounds,
00:21:20;00 I would be able to collect this in our microinjection needle
00:21:23;08 and then test to see whether the bacteria
00:21:25;16 had any response to this conditioned media.
00:21:29;11 I needed to first make sure, though,
00:21:31;09 that the media I was using
00:21:32;29 didn't have any compounds that would elicit a response,
00:21:35;14 and as you can see from this video,
00:21:37;03 the bacteria could care less
00:21:38;23 -- they don't detect anything.
00:21:40;17 But then what about the media exposed to the stomach cells?
00:21:45;05 As you can see, right away the bacteria
00:21:47;22 are incredibly attracted to this gradient
00:21:50;21 of the media that had been collected from cells.
00:21:53;09 In fact, they look like they're
00:21:55;07 a total huge bee swarm
00:21:57;11 that's flooding the needle tip.
00:21:59;06 And within a few seconds,
00:22:00;25 you can already see that the bacteria
00:22:02;19 are just swimming so quickly
00:22:04;22 to try to just get to this area,
00:22:06;15 because they're simply loving whatever is coming out of this needle tip.
00:22:09;27 And this was incredibly exciting for me and my lab
00:22:13;02 because this was the first time that
00:22:14;29 we were able to visualize H. pylori
00:22:17;28 actually detecting and moving towards things
00:22:19;21 that were coming from cells.
00:22:21;17 It had long been hypothesized that
00:22:23;22 there were things coming from cells that the bacteria
00:22:25;05 could be attracted to,
00:22:26;16 but this was our first piece of evidence
00:22:28;01 really showing this.
00:22:30;05 And so, at this point,
00:22:32;05 this was a really huge milestone for me,
00:22:33;20 you know, this opened up a whole slew of questions
00:22:36;27 and I was so excited to dig deeper,
00:22:39;10 to understand, how are the bacteria doing this?
00:22:41;00 What are they sensing?
00:22:43;07 But one of the strengths of our technique here
00:22:45;09 is that we can actually quantify these videos,
00:22:47;06 even though you don't think it
00:22:49;12 probably needs to be quantified
00:22:50;23 because it's so obvious,
00:22:51;28 as scientists we do need to quantify things periodically.
00:22:54;21 So what we could do is take these videos,
00:22:56;26 convert them into black and white images,
00:22:58;26 and then I quantified t
00:23:02;23 he number of black pixels within a certain radius away from the needle tip,
00:23:05;23 and this pixel density is my proxy for bacterial number.
00:23:08;13 So, for those two movies that I just showed you,
00:23:10;23 this is what that data looks like.
00:23:12;24 On the y axis is the pixel density
00:23:15;01 and it's plotted over time from
00:23:17;19 4 seconds pre-injection
00:23:19;15 to 30 seconds post-injection,
00:23:21;01 so you can see that in red,
00:23:22;25 wild type response to the conditioned media
00:23:24;28 are very, very strong.
00:23:26;17 There are three different movies represented here
00:23:28;05 in these plots.
00:23:30;00 So, just for simplicity, for future graphs,
00:23:32;28 I want to just show the data at
00:23:34;15 4 seconds pre-injection
00:23:36;02 and 15 seconds post-injection
00:23:37;29 as bar graphs
00:23:39;16 so I can show more conditions
00:23:41;12 without obscuring the entire graph.
00:23:42;27 So, this is the corresponding bar graph
00:23:44;29 to the data I just showed you,
00:23:46;14 so any time you see a significant increase
00:23:49;04 in the blue bar compared to the red bar,
00:23:51;01 that means that there's an attraction happening.
00:23:53;16 So, at this point, I really wanted to know,
00:23:56;21 what are the compounds that the bacteria are sensing
00:23:58;28 in order to reach the cells?
00:24:01;25 And to do this, I decided that,
00:24:04;00 maybe, if I could figure out what nose the bacteria
00:24:08;08 needed to sense this attractant,
00:24:10;14 then I could at least hone in onto
00:24:12;19 a specific candidate that the bacteria
00:24:15;12 could be actually detecting from the cells.
00:24:17;06 So I made mutants
00:24:19;22 in each of those four chemoreceptors,
00:24:22;06 then tlpA/B/C/D receptors,
00:24:24;28 which were the noses for H. pylori.
00:24:27;02 And like wild type,
00:24:29;23 the tlpA, C, and D receptor mutants
00:24:32;16 were still able to sense,
00:24:34;22 meaning...
00:24:36;01 that told me that these proteins were not necessary
00:24:37;22 for that attraction.
00:24:39;09 But the mutant lacking TlpB
00:24:41;02 was no longer able to detect this,
00:24:43;21 so this told me that H. pylori
00:24:46;05 needs TlpB in order to sense the cells.
00:24:49;08 So, what does TlpB do?
00:24:51;00 I decided to look into the literature
00:24:52;20 to see if anyone else had described
00:24:54;26 any other compounds that were
00:24:56;23 being detected by TlpB
00:24:58;06 and I really only found two studies,
00:24:59;23 and both of them referred to the fact
00:25:01;18 that it was sensing certain compounds
00:25:03;22 as a repellent,
00:25:05;11 and no descriptions of attractants
00:25:07;07 were really published in the literature.
00:25:09;05 And so I realized that what I was seeing,
00:25:12;08 this attraction through TlpB,
00:25:13;26 was a novel compound that
00:25:15;29 no one had described before.
00:25:17;19 And so I was going to have to proceed
00:25:19;11 by doing some more fine experiments
00:25:21;07 to be able to identify these compounds
00:25:23;15 that the bacteria were sensing,
00:25:25;01 but I actually got really, really lucky,
00:25:27;01 because at this point,
00:25:29;11 this paper came out that
00:25:31;21 described the structure of the TlpB receptor.
00:25:34;11 So, what you're seeing here in green
00:25:36;10 is the crystal structure of the protein TlpB.
00:25:39;16 And what this tells us is
00:25:42;16 not only how the protein looks,
00:25:44;16 but what the authors actually found was that,
00:25:47;17 in one of these pockets of the TlpB receptor
00:25:50;26 was a molecule of urea
00:25:53;22 that was very tightly bound, actually.
00:25:55;24 And it was so tightly bound that
00:25:58;09 they thought it was just part of the protein complex
00:26:00;28 that enabled the receptor to function,
00:26:02;29 but urea is actually a really interesting candidate
00:26:06;11 in the context of my story,
00:26:08;24 because we know,
00:26:10;25 when cells undergo metabolism,
00:26:12;22 and particularly when they
00:26:14;14 use and degrade amino acids for energy,
00:26:17;04 they need to convert the metabolic byproducts
00:26:22;05 into urea, because urea is actually
00:26:24;02 a very stable, non-toxic compound.
00:26:26;25 And we know that all cells in the body have to go through metabolism
00:26:30;25 because they also need energy to function too,
00:26:33;19 so urea is a ubiquitous molecule in our body
00:26:37;06 and it's known to be in the gastric juices.
00:26:39;10 So that made us think:
00:26:41;20 could it be that perhaps H. pylori
00:26:43;23 is sensing urea through its TlpB receptor?
00:26:46;27 And so I tested this idea
00:26:48;26 by taking the conditioned media
00:26:50;16 that had elicited that very cool response
00:26:53;06 and I treated it with an enzyme
00:26:55;23 that I was able to purchase called urease,
00:26:58;08 and urease is an enzyme that can
00:27:00;29 degrade urea into other components
00:27:04;04 and it effectively destroys all urea
00:27:06;15 that comes its way.
00:27:08;29 And so I wanted then to test,
00:27:10;05 what happens then when I expose bacteria
00:27:12;04 to that urease-treated conditioned media.
00:27:15;14 I found that that caused the bacteria
00:27:17;29 to no longer respond,
00:27:20;03 so that suggested that H. pylori
00:27:22;02 may be sensing urea in that conditioned media
00:27:24;26 in order to find the epithelium.
00:27:27;14 So to test that idea directly,
00:27:29;09 that H. pylori may be sensing, actually,
00:27:31;10 the urea through TlpB,
00:27:34;16 I put a pure solution of urea into our needle
00:27:37;19 and then watched the bacteria as they're exposed
00:27:40;11 to this urea gradient.
00:27:42;16 And what you can appreciate and see right away is that
00:27:45;12 the bacteria are just simply loving the urea,
00:27:47;19 they are coming in masses and droves,
00:27:50;09 and unlike that other video,
00:27:52;12 you can no longer see that entire swarm
00:27:55;06 within the field of view.
00:27:56;07 This concentration of urea,
00:27:57;17 which is thought to be the concentration in the stomach,
00:27:59;29 is so strong it attracts
00:28:02;15 almost the entire culture of bacteria
00:28:04;24 to this very small area around the needle tip.
00:28:07;28 And most importantly,
00:28:09;24 this was through the TlpB receptor,
00:28:11;22 because the mutant that doesn't have that protein
00:28:14;05 is not able to sense any of it.
00:28:16;13 And so I was really struck by
00:28:19;05 how large this response was,
00:28:21;05 and I asked the question of,
00:28:22;24 well, how low can it go?
00:28:24;19 How little urea can this bacterium actually detect?
00:28:29;02 With 1 millimolar (mM) urea,
00:28:30;24 we saw that really, really massive response,
00:28:33;14 and I decided then to test
00:28:35;10 lower and lower concentrations just to see,
00:28:37;23 how low can the bacteria still detect urea?
00:28:41;02 So I went ten times lower
00:28:42;26 and a hundred times,
00:28:45;02 and at a hundred times lower, about 10-15 micromolar,
00:28:48;23 the bacteria were still able to sense the urea.
00:28:51;27 But what I want to first point out, though,
00:28:54;28 is that the urea coming out of the needle tip
00:28:57;25 diffuses actually really rapidly into the solution,
00:29:01;07 and that means that it's getting diluted
00:29:03;11 by the time it comes out to 60 microns,
00:29:05;17 which is where we're still seeing that swarm radius.
00:29:08;12 So that means that the bacteria out here
00:29:10;26 are likely to be sensing urea
00:29:12;25 that's actually much less
00:29:15;27 than that 10-15 micromolar that we have in the needle tip.
00:29:18;19 And so I really wanted to know,
00:29:20;27 what was that threshold concentration?
00:29:23;16 And so I continued testing even lower concentrations of urea
00:29:26;27 in the needle,
00:29:28;14 but instead of the gradient forming normally,
00:29:30;26 I pulsed in the solution that I loaded into this needle tip,
00:29:34;12 and in this case it was 50 nanomolar,
00:29:37;06 and I could see that the bacteria actually are
00:29:41;02 transiently responding to this incredibly low concentration,
00:29:46;05 and you can see that more clearly in these bacterial tracings.
00:29:49;07 50 nanomolar is almost a million times lower
00:29:52;11 than that 1 millimolar solution
00:29:54;04 that elicited that large response,
00:29:55;27 and this was really, really surprising to us,
00:29:58;17 because, with 50 nanomolar,
00:30:01;01 this is essentially like sensing
00:30:03;04 three molecules of urea
00:30:05;12 in a billion molecules of water.
00:30:07;08 How is it able to still smell that in the environment?
00:30:10;06 When, in the actual stomach environment,
00:30:12;26 there's a million times more urea molecules
00:30:16;01 to begin with.
00:30:17;14 You would think that this would completely
00:30:20;09 just overload and saturate the TlpB receptor.
00:30:23;21 You could kind of think of this like
00:30:26;10 going into a perfume shop.
00:30:27;27 Imagine walking in and sniffing your first perfume...
00:30:31;13 it smells great...
00:30:33;12 but you want to test another perfume
00:30:35;07 and see what that smells like.
00:30:36;16 If you were to try to smell a second perfume
00:30:38;10 right after smelling the first one,
00:30:40;22 you wouldn't be able to smell the second one,
00:30:42;05 because your nose receptors have been
00:30:44;02 completely overwhelmed by that first perfume.
00:30:46;11 Luckily, we actually figured out that,
00:30:48;25 of all things, coffee beans
00:30:51;09 can actually reset your sense of smell.
00:30:53;22 So, after sensing that...
00:30:55;21 smelling that first perfume,
00:30:57;09 you can take a whiff of coffee beans
00:30:59;04 and that will actually help you
00:31:01;18 be able to smell the next perfume.
00:31:03;20 And so we wondered,
00:31:05;29 could H. pylori actually have something
00:31:07;20 like coffee beans to help it
00:31:09;11 reset its sense of smell?
00:31:11;24 And, thinking a bit more about its biology,
00:31:14;29 we actually realized that
00:31:17;00 perhaps there is such a thing,
00:31:19;01 and this is actually a urease enzyme.
00:31:22;08 So, H. pylori's urease
00:31:25;27 actually acts just like the urease I had
00:31:29;03 used earlier in my experiments
00:31:31;01 in that the urease effectively
00:31:33;22 destroys any urea that it comes into contact with.
00:31:37;19 And what's really striking is actually that
00:31:39;27 H. pylori dedicates a lot of its energy
00:31:42;19 into creating massive amounts of this enzyme.
00:31:45;11 So, why would it want an enzyme,
00:31:47;15 and so much of it,
00:31:49;12 to destroy urea?
00:31:51;07 Well, if we think about the environment again
00:31:53;28 that H. pylori is exposed to
00:31:55;23 when it enters the stomach,
00:31:57;08 it has the cells here at a neutral pH,
00:32:00;12 buffered by this mucus layer,
00:32:01;18 but when the bacteria come into the stomach
00:32:03;17 they're first exposed to that gastric juice,
00:32:05;22 which has a pH of 2.
00:32:07;16 And not being an acidophile,
00:32:09;21 it can't survive in this acidic environment
00:32:12;01 for very long
00:32:13;23 without having some proper protective shield around it.
00:32:17;02 And, effectively, what urease does
00:32:19;22 is create this shield.
00:32:21;15 By having this enzyme,
00:32:23;05 which degrades urea into
00:32:25;10 [ammonia] and bicarbonate,
00:32:27;19 which are basic compounds that can neutralize the acid,
00:32:30;04 the bacteria have this
00:32:32;20 really, really cool protective shield around it
00:32:34;29 as it's temporarily going through
00:32:37;03 this really acidic gastric juice,
00:32:39;01 and that then will allow it to find its way to the cells,
00:32:41;29 which then, at that point,
00:32:43;28 it's in a safe space.
00:32:45;05 But what we realized is that,
00:32:47;03 because it has this urease enzyme
00:32:49;24 degrading urea constantly around the bacterium,
00:32:52;06 the concentration of urea around the bacteria
00:32:54;26 is actually really, really low,
00:32:57;05 even though the concentration of urea in the stomach
00:33:01;03 is at 1 millimolar.
00:33:03;07 And so we actually were wondering, then,
00:33:05;18 whether the urease could be
00:33:08;05 acting like the coffee beans
00:33:10;17 and basically keeping the gradient of urea so steep
00:33:14;14 that that will still then allow
00:33:16;29 the TlpB receptor to sense molecules of urea
00:33:19;29 in the environment without being overwhelmed.
00:33:22;10 And so I proceeded to test this idea
00:33:24;13 by making a urease mutant
00:33:27;08 and then seeing whether the urease mutant
00:33:29;16 is able to sense urea or not.
00:33:32;05 And when I tested the urease mutant,
00:33:33;28 I found that it couldn't.
00:33:35;27 So, this suggested to us that
00:33:38;17 the urease really is playing an important role
00:33:40;28 in allowing the TlpB receptor
00:33:43;27 to actually detect urea in the environment,
00:33:47;12 and that perhaps, without this enzyme,
00:33:49;29 the receptor is then constantly saturated
00:33:53;20 and unable to respond properly.
00:33:56;22 And so to test this in kind of in a different way,
00:34:01;25 we decided that, what if we didn't take away the urease enzyme,
00:34:04;18 but instead overwhelm the system
00:34:07;08 with more urea.
00:34:08;29 So, that's what I did in this experiment.
00:34:11;12 Again, at the left,
00:34:14;10 you see the response to the 1 millimolar
00:34:16;04 -- it's very high, meaning that there was an attraction --
00:34:18;15 but if I go and add ten times more,
00:34:21;07 a hundred times more,
00:34:22;20 even a thousand times more urea
00:34:24;18 into the needle tip,
00:34:26;07 what would happen?
00:34:27;24 I saw that these high, high concentrations of urea
00:34:30;19 actually inhibited the response of the bacteria.
00:34:34;04 So, no longer were they attracted,
00:34:36;10 they actually just swam normally,
00:34:38;18 and I didn't see that swarm around the needle tip.
00:34:41;17 And what this told me was that,
00:34:43;13 at these high, high concentrations,
00:34:46;03 what I'm doing is essentially overloading the system,
00:34:50;09 I'm saturating both the urease and the TlpB receptor,
00:34:53;28 and so that's preventing the bacteria
00:34:55;29 from being able to detect
00:34:57;17 any other molecules in the environment,
00:34:59;17 because it's not detecting a gradient any more.
00:35:02;20 And so we did a final experiment
00:35:04;26 where we thought that maybe,
00:35:07;16 with a mutant that doesn't have urease,
00:35:10;05 we can actually help it out by
00:35:13;20 adding some urease to the outside of the culture.
00:35:15;19 So that's what I did on this experiment.
00:35:17;20 The urease mutant, on the left here,
00:35:19;26 you can see doesn't respond at all,
00:35:22;15 just like I showed you previously in the graphs,
00:35:24;15 but in this culture to the right,
00:35:26;18 what I've done is I've added urease enzyme
00:35:29;04 to the culture of the urease mutant.
00:35:31;15 And what you're seeing now is that
00:35:33;11 the bacteria are starting to swarm around the needle tip,
00:35:36;09 and that told us that,
00:35:38;19 indeed, urease is important for sensing
00:35:41;25 because it generates and helps maintain
00:35:44;19 this steep urea gradient around the bacteria,
00:35:47;00 which is necessary in order for this receptor
00:35:50;09 that's so sensitive
00:35:51;28 to be able to detect new molecules in the environment.
00:35:55;05 So, now I'd like to take us back to the stomach.
00:35:56;28 Imagine when an H. pylori arrives
00:36:00;06 in this very harsh, acidic environment.
00:36:02;06 It's faced with many chemical gradients
00:36:03;18 that we now know exist,
00:36:04;29 and in one of these gradients
00:36:06;20 is a hydrochloric acid gradient,
00:36:08;12 which H. pylori definitely knows to swim away from.
00:36:12;08 But in addition to hydrochloric acid,
00:36:14;09 my research has shown that there
00:36:16;05 are gradients also coming from cells.
00:36:18;24 One of these molecules is urea,
00:36:20;11 which H. pylori has also evolved a way
00:36:23;03 to sensitively detect this gradient
00:36:25;15 in the environment
00:36:27;00 in order to find its way to the gastric epithelium.
00:36:29;20 And so what does this mean
00:36:31;05 for its life on the epithelium?
00:36:33;02 This means that it can then
00:36:35;08 find its way into these particular niches
00:36:37;27 where it's going to be protected
00:36:39;23 from that very harsh, acidic lumenal space.
00:36:42;26 And for us, as we're reaching the state
00:36:46;01 where antibiotics will no longer be effective
00:36:49;15 for treating bacterial infections like H. pylori,
00:36:53;01 we now have insight
00:36:55;20 into these novel pathways in its biology
00:36:59;21 so that we can create drugs
00:37:02;14 against these new targets,
00:37:03;23 such as chemotaxis components,
00:37:06;02 and that will allow us to
00:37:08;17 really be able to develop some more effective drugs
00:37:10;17 to prevent it from successfully colonizing the stomach
00:37:14;12 and establishing an infection.
00:37:16;26 And so, with that,
00:37:18;24 I'd like to thank my advisor,
00:37:21;00 Manuel Amieva,
00:37:22;14 and all the members of our lab, both past and present.
00:37:24;16 A lot of the work has really
00:37:27;06 come together, also,
00:37:30;08 with the collaboration with various labs
00:37:32;07 across the world,
00:37:33;29 and as well as funding sources
00:37:35;25 that have really enabled me to do this in my graduate studies.
00:37:39;26 And I'd really like to thank also
00:37:42;27 the Young Scientist Series through the iBiology program
00:37:46;19 for really giving me this chance to share my work with you today.
00:37:49;04 And lastly thank you all for your attention.

This Talk

Speaker: Julie Huang

Audience:

Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: May 2016

Talk Overview

Most microorganisms cannot survive in the hostile acidic environment of the stomach, but somehow Helicobacter pylori calls the stomach its home. Infection with H. pylori can cause stomach ulcers in humans and is a risk factor for the development of stomach cancer, the third-leading cause of cancer deaths worldwide. Dr. Julie Huang tells us the story of how H. pylori thrives by navigating towards more neutral environments within the stomach using a chemoreceptor called TlpB. Understanding how H. pylori finds this niche opens new routes for developing novel approaches to preventing stomach ulcers and cancer.

This talk is part of the Young Scientist Seminars, a video series produced that features young scientists giving talks about their research and discoveries.

Speaker Bio

Julie Huang

Dr. Julie Huang completed her Bachelor degree at the California Institute of Technology. Given her scientific merits, she was granted a Fulbright scholarship in 2009 to work at the Max Planck Institute for Marine Microbiology in Germany. Afterwards, Huang joined the Microbiology and Immunology Doctoral Program at Stanford University, where she worked with Dr. Manuel… Continue Reading