The Dynamic Bacterial Chromosome

Part 1: Dynamics of the Bacterial Chromosome

00:00:03.11 Hi. I'm Lucy Shapiro. I'm a professor at Stanford University. I'm also
00:00:08.22 a part of the department of international studies at Stanford University.
00:00:12.29 And what I study are bacterial cells and how they work. That's going to be
00:00:19.14 the first talk that I'm going to give you. And the second talk, I'm going to
00:00:24.27 talk about why understanding bacterial cells and viruses has become
00:00:32.16 absolutely critical in understanding the way in which we live in a global village.
00:00:38.07 And I'm going to talk about in the second talk emerging infectious diseases
00:00:43.12 and how understanding what goes on inside this tiny world, the world of
00:00:50.12 viruses or the world of bacteria is absolutely vital for dealing with
00:00:56.07 emerging infectious diseases. Now my first talk as you can see from the
00:01:00.01 title is the dynamics of the bacterial chromosome, how it is organized,
00:01:05.18 how it separates during segregation and how it's coordinated during cell division.
00:01:10.13 So we're going to go into the inner workings of the bacterial cell
00:01:15.06 and try to understand how these things happen. I should mention
00:01:19.22 right before I start that the work we've done in understanding this bacterial cell
00:01:24.25 has helped us design an entirely new class of antibiotics. I'm not going to
00:01:30.17 talk about that today but it's something that is the offshoot of all work
00:01:35.18 on either bacterial cells or viruses. So now let's talk about Caulobacter.
00:01:40.17 Caulobacter is a simple bacterial cell. In fact, the name Caulobacter
00:01:46.23 "caulo" means stalk in Greek so that this is a stalk bacterium, you can
00:01:54.21 see the stalk here. What happens is that at every cell division,
00:01:59.13 which you see happening here, you have an asymmetric division.
00:02:04.10 So get a swarmer cell from one part and a stalk cell from the other.
00:02:09.07 The swarmer cell can swim away to find food. The stalk cell sits
00:02:14.05 down on a rock and waits for food to come to it and then it divides again.
00:02:19.27 This swarmer cell, which is shown here going through the cell cycle,
00:02:23.22 will first release the flagellum, which is this wavy thing down here.
00:02:28.21 These are pili--you get rid of both the flagellum and the pili.
00:02:32.16 The circle inside the cell indicates the DNA. And once you differentiate
00:02:39.19 from the cell with the flagellum, which is called a swarmer cell,
00:02:43.04 into a stalk cell, you initiate the replication of the chromosome.
00:02:48.10 As the chromosome is replicating, which is shown here, you're turning
00:02:53.08 on lots of genes, that allow you to build a new flagellum at this pole
00:02:58.07 opposite the stalk, and you turn on all the genes for cell division
00:03:02.15 and chromosome segregation. Now indicated below this cartoon of the cell
00:03:08.13 cycle are all these boxes. And these boxes indicate individual modules
00:03:14.21 that happen at this time during the cell cycle. And these modules
00:03:19.25 have many, many genes within them. So for example, flagellar
00:03:24.06 ejection means that there are like 45 genes that have to be turned on
00:03:28.26 to get rid of it and then to rebuild it. Then as you move through the cell cycle
00:03:34.20 what happens is you initiate DNA replication, flagellar biogenesis,
00:03:39.11 pili biogenesis. And all of these functions have to be coordinated
00:03:44.19 and regulated and be a part of turning on and turning off the replication
00:03:50.18 of the chromosome. So let's now ask, "Well how is this happening?"
00:03:56.09 What about the chromosome is something that we really have to understand?
00:04:02.19 So shown here is a picture of an E. coli cell. That's a bacterial cell--
00:04:09.11 very similar to Caulobacter that's been very extensively studied.
00:04:13.10 And in this particular picture, it's been exploded on an EM grid so
00:04:18.27 that you can actually see the DNA as it's released from the cell.
00:04:23.24 And it's really quite a mess. There's a huge amount of DNA and it
00:04:28.15 doesn't really look like it has any organization. But in fact, when you
00:04:32.26 understand that in this small bacterial cell that is approximately 2 microns--
00:04:39.12 very little, ten times smaller than the smallest eukaryotic cell.
00:04:43.15 The DNA is a thousand times longer than the cell if it were completely
00:04:49.03 stretched out. So we have to compact this DNA very tightly
00:04:53.12 into the cell. And remember that inside the cell you're going to have to
00:04:59.02 duplicate this DNA, make a complete copy of this great big mess
00:05:05.12 and while that's happening you have to transcribe the genes that are
00:05:10.01 allowing you to proceed through the cell cycle. So how is this all happening?
00:05:14.24 Well, one of the first things we did was first ask, "Are there specific
00:05:20.08 parts of that chromosome that sit at specific parts in the cell?"
00:05:25.04 And what I show you here is an experiment in which we have
00:05:29.27 actually lit up a particular pole of the cell. And so up in this
00:05:36.12 picture, you're going to see red dots, and you're going to see
00:05:38.28 green dots. The red dots, indicate the origin of replication. So the
00:05:44.20 bacterial chromosome exists as a circle. And in that circle, you have a
00:05:50.12 origin sequence, where you initiate DNA replication. DNA replication
00:05:55.26 in bacterial cells proceeds bidirectionally, ending at a terminus.
00:06:01.06 And what I'm showing you here is in this little swarmer cell we have
00:06:06.13 a one origin at the pole right down by the flagellum and the terminus
00:06:12.26 winds up at the other pole. So now as we begin replication and go
00:06:18.11 through the cell cycle (this is in synchronized cells) you can see that
00:06:22.18 the very first thing that happens is that the origin duplicates and a
00:06:27.28 new one goes to the opposite pole. Then as you continue through the
00:06:32.08 cell cycle, you see finally you've duplicated the termini, which means
00:06:37.08 you've completed DNA replication. But these cells are dead cells.
00:06:43.15 You know, I synchronized the cells. I do in situ hybridization of a labeled
00:06:49.06 probe. I want to see how these chromosomes are actually able to
00:06:54.20 move a particular locus. How does it move that origin from one
00:06:59.27 pole of the cell to the other? And let me show you the technique
00:07:04.02 that we now use to do this. Here I have a plasmid sitting in the cell.
00:07:11.15 And this plasmid has on it a lacI gene, which is a gene that codes
00:07:18.24 for a protein that can sit on a specific binding site on the chromosome.
00:07:24.00 What we do is we hook that gene up to a fluorescent tag. So it
00:07:31.22 turns on a bright light. Sitting next to it on the plasmid, I in fact have
00:07:37.07 another protein. It encodes for another protein--trpR--that sits next to
00:07:43.15 a different fluorescent tag. So one is shown in blue. The other is shown
00:07:47.28 in yellow. In the meantime, what we've done is we've inserted into
00:07:52.11 the chromosome shown here, repetitive copies of a lacO gene or
00:08:00.16 in fact a tetO gene. And these are the binding sites that we're
00:08:05.26 putting into a specific place on a chromosome so that once we
00:08:10.21 turn on these labeled tagged proteins, we can actually visualize
00:08:17.12 where it is in the cell. So down here, I show the cell. And in one part
00:08:23.23 here--let's just look at tetO. TetO is now being bound by those little
00:08:30.26 lit-up proteins that bind to TetO and we can actually see them
00:08:36.21 in the cell. And if we turn on both, and let's say we tagged two
00:08:42.19 different loci in the chromosome, we can then see two different
00:08:46.13 dots as shown down here. So given that what we started to do was
00:08:53.22 ask, "Where are these positions relative to the origin of replication?"
00:08:58.21 Here's the origin of replication. It's tagged with something yellow.
00:09:03.12 And if it's tagged with yellow, when we look at the cell, which is shown
00:09:07.22 diagrammatically here, we see that the origin is in fact closest to the
00:09:14.10 flagellum, which is what we saw when we did that other experiment.
00:09:18.22 Next, what we did, is we labeled another part of the chromosome
00:09:23.29 with a different color. And we said, "Well, where is that going to go
00:09:27.04 in the cell?" And when we looked at that in the fluorescent microscope,
00:09:31.00 we saw that it was moved away coming in this direction. Then we labeled
00:09:36.24 up yet another. And that's shown here, and that comes next.
00:09:42.23 And then we labeled yet another. And so what we're beginning to see
00:09:47.23 is a linear order of the genes on the chromosome that reflects where
00:09:55.15 they line up in the cell. This was a big surprise. It tells us that the DNA
00:10:02.18 sitting in this cell is not a disorganized mess and individual loci
00:10:09.08 probably know where they are. Now at this point we were just looking at
00:10:14.08 a few loci. What we have to do is look at lots. And so the way
00:10:18.28 in which we looked at lots is we took this mariner transposon
00:10:25.22 that had all these TetO sequences lined up on them, and we
00:10:31.11 jumped this transposon into the chromosome shown here. So that
00:10:37.27 we actually jumped in 114 different sites. One transposon jumped in
00:10:45.20 a particular site in an individual cell. So every cell has just one
00:10:51.27 transposon in a particular site. And then what we did was we mapped
00:10:57.11 and measured where each one was. And as you can see here in this
00:11:03.00 half I show little dots. And we sequenced and measured exactly
00:11:08.03 in each individual cell where that transposon was. On the other side
00:11:12.21 I have yet another group that we measured at a particular site in the cell.
00:11:18.00 And then because each one of these was tagged with a fluorescent tag
00:11:22.08 we were going to be able to see where it was in the living cell. And
00:11:27.17 here's the answer. The answer is that there is a linear relationship
00:11:33.15 between where that TetO array was put in the cell, on the genome,
00:11:40.26 and where it exists in the cell. So therefore, we now know that the chromosome
00:11:47.08 is a highly ordered structure and that every gene has a specific cellular address.
00:11:53.24 The origin is near the flagellum. The terminus is near the top of the cell
00:11:58.16 and every locus in between goes to a specific place. Furthermore,
00:12:05.14 using this technique, we're able to actually follow the movement,
00:12:10.16 let's say of the origin, in real-time in living cells.
00:12:15.15 And up here what I show you is moving now through the cell cycle and looking
00:12:22.28 quite closely at--this is the origin and then as soon as replication
00:12:28.06 starts, the replisome, which is the machine that copies DNA, is laid down
00:12:34.00 on top of it. And as soon as you initiate replication, you see that the
00:12:39.14 dot that lights up the origin travels to the other pole. And I show this
00:12:46.08 down here, and if you can watch this, here's a swarmer cell with the
00:12:51.03 origins at this pole and they are moving right down to the other end
00:12:56.07 of the cell very rapidly. And when we first saw this, we just couldn't
00:13:00.08 believe it. Remember, these cells are tiny and we're looking at them
00:13:03.19 in real-time living cells and watching a newly duplicated segment of
00:13:09.06 DNA harpoon across the cell and hit the other pole. And I show
00:13:14.16 that in three different panels here. In each one you can follow
00:13:19.03 the origin of replication as it moves to the other pole. This allowed us
00:13:25.17 to actually design a computer algorithm that watched the movement
00:13:31.00 of these origins and mapped out for us the rate of movement
00:13:35.14 of the fluorescent tag. So we were able to show that it takes
00:13:41.07 0.2 microns/minute for the new origin, which is this blue line,
00:13:46.00 to move from one pole of the cell to the other, independent of cell length.
00:13:51.24 Now, it has been thought for many, many years--because we really didn't
00:13:56.15 know but it seemed like a logical thing to say--that this great mass
00:14:00.15 of the bacterial chromosome duplicated as a mass and a mess
00:14:05.29 and was attached to the membrane of the cell and the chromosomes
00:14:11.05 only got pulled apart by the growth of the cell. This turns out not to be
00:14:16.15 true. The copying of the DNA and then the movement of the segments
00:14:22.29 as it's being copied occurs simultaneously and is independent
00:14:28.01 of the growth of the cell. And that again is shown here. So now
00:14:33.14 what I've done is just to help you get through this in understanding
00:14:36.23 exactly what I've said. This shows the dynamics of bacterial chromosome
00:14:42.15 segregation. So we start here in G1 phase with our organized chromosome,
00:14:49.01 our origin, our terminus, and this yellow dot is showing a particular locus.
00:14:55.24 And let's follow these. So as this swarmer cell releases the flagellum (and it will
00:15:02.26 grow a stalk in a minute), what happens is the replisome gets formed on this part
00:15:08.01 of the cell. That's that green stuff. And replication begins and as you move,
00:15:14.26 what happens is that the replisome is spewing out DNA in both directions. The origin
00:15:21.22 has been harpooned to the other end of the cell and as you spool out DNA
00:15:26.24 at its pole, it separated pulling to the two poles. Now if you follow your little yellow dot here
00:15:35.09 as this guy is duplicated, what's happening is it is moved to the incipient new cell
00:15:43.15 at exactly the same place, like a mirror image of where it was before and then
00:15:49.21 cell division happens and we're back to the beginning so that when this cell divides
00:15:55.09 the top and the bottom will look very similar. But what could be doing this?
00:16:02.05 So it was thought of for many, many years that the bacterial cell was too small
00:16:10.05 to require proteins that help things move. It was too small to have particular
00:16:17.25 protein complexes in specific places. Its DNA was not wrapped up in a nucleus.
00:16:23.06 So people thought, "Well the cell is so little that proteins and pieces of DNA
00:16:29.03 could be anywhere in the cell in milliseconds by diffusion." In fact, this is not true.
00:16:35.00 Bacterial cells have proteins that control movement. They have localization of
00:16:42.29 complexes at different places in the cell. And one of the candidates for
00:16:47.24 helping this happen is an actin-like protein. Now actin has been well known
00:16:54.10 for many, many years in eukaryotic cells being involved in cytoskeletal functions
00:17:00.01 and movement. Bacterial cells have an actin homolog. And that's called MreB.
00:17:06.29 So we decided to investigate how MreB might help or function in the cellular
00:17:14.23 movement like moving that origin. The MreB actin--this one is showing the polymerization
00:17:23.23 of that actin in another kind of cell. Down here it shows in fact how this actin is
00:17:31.03 organized almost in all bacterial cells, including caulobacter, and it's organized in a spiral.
00:17:36.28 All the way down the length of the cell. And so in order to study this particular
00:17:43.08 protein and how it might work, we of course made mutants, knocked it out.
00:17:48.21 The cells change shape. They become sort of round balls and then ultimately die.
00:17:53.13 So the cell really needs this. But I wanted something that would immediately
00:17:59.19 inactivate that actin in some way. So what we did was go--well first of all we read
00:18:05.21 the literature and found that a group of people led by Dr. Wachi in Japan had
00:18:13.27 discovered a small compound (we'll call it A22) that when applied to E. coli,
00:18:21.07 which is usually a rod, causes it to form a sphere. Now Wachi didn't know
00:18:26.12 how this compound worked, but since we know that if you make a mutation in MreB,
00:18:31.16 in that actin, you get cells to ball up and form a sphere, we figured, "Well let's
00:18:37.11 collaborate with him and try to find out how this works." So here's the compound.
00:18:41.19 It's easy to make. It's a very simple compound and the way in which we determine
00:18:45.25 how this works, is we carried out a screen for mutants that are resistant to this
00:18:52.25 little compound A22. And we found, as you can see here, lots of them and
00:18:59.12 we then showed that of these 20 alleles all of them were able to grow
00:19:06.02 in the presence of A22, which means they're resistant. And then we proved
00:19:11.06 that this was true by replacing the mutant gene with the wild type gene
00:19:15.13 and you go back to sensitivity. And then we prove it in fact in a wild type background
00:19:21.01 with the mutant gene. By sequencing exactly where these genes exist on the
00:19:27.16 chromosome, we were able to show that each and every one of those 20 alleles
00:19:33.25 that are resistant to MreB exists in the ATP-binding domain of the actin monomers.
00:19:43.19 Therefore, A22 is a specific inhibitor of MreB function. Now using this inhibitor,
00:19:51.14 we were able to ask, "Does the origin move if we've knocked out the function of MreB?"
00:19:58.01 But first, we had to prove that MreB does not disrupt DNA replication or initiation.
00:20:05.15 And in fact it doesn't. A control is using a compound hydroxyurea that does
00:20:11.03 stop it, but if you treat it with A22, it does not. So now we're ready for the experiment.
00:20:17.22 And here's the experiment. What you do, as I told you before, if you start with
00:20:24.04 a swarmer cell (the green dot is the origin), as the swarmer cell differentiates into a stalk
00:20:29.15 cell, you have the separation of the origins. One remains at this pole, the other goes
00:20:35.16 to the other side. Then you go through the cell cycle and you wind up with the
00:20:41.13 same organization of the chromosome in both. Now here's the experiment.
00:20:45.21 What we did was we added A22 to these swarmer cells, and asked, "What's going to happen?"
00:20:51.27 Well, what happened really surprised us. DNA replication started and you make two
00:20:58.18 new little origins, but nothing moved to the other end. And as you went through
00:21:03.27 the cell cycle, but of course you were not separating the chromosomes--you didn't
00:21:08.07 divide the cell. And using a cell sorter--FACS--we were able to show that we indeed
00:21:14.25 had just duplicated the DNA. So we had two chromosomes but they had not
00:21:21.16 separated. This tells us now that MreB, actin, is important for the separation
00:21:30.09 of the newly replicated origin of DNA replication. We then went on and
00:21:35.06 showed that MreB binds to some structure at that part of the cell--at that part
00:21:41.20 of the origin. Now what I've shown you is we have coincident DNA replication
00:21:49.10 and segregation. How does the cell use this to coordinate all of its different functions?
00:21:55.05 And it does so in two different ways. So I'm going to tell you two very short
00:21:59.19 stories. One way it does it is it controls the spatial regulation of where you put
00:22:07.24 the cell division site. Now let me show you how this works. So what I'm showing
00:22:12.05 you here is another cartoon of the cell cycle. Shown in green is something called
00:22:17.28 the FtsZ tubulin, which is a protein that is able to go to midcell and
00:22:24.26 contract the cell to carry out cytokinesis. And as you can see here, it's in the
00:22:31.10 center of the cell. Cell division happens, and when it finishes, you still have some
00:22:36.21 of this FtsZ left at that pole. So now let's look back and look at this swarmer cell
00:22:42.29 that has just resulted from cell division and what we have here is the FtsZ left
00:22:49.29 at the pole; at the other pole is the origin. And bound to that origin is a newly
00:22:56.14 discovered protein, which we call MipZ. So we have MipZ bound to this origin. We
00:23:04.04 have the FtsZ tubulin at the other side of the cell. And when the origin with its
00:23:10.04 MipZ moves to the other part of the cell, then the FtsZ monomers go to midcell.
00:23:18.11 So the question that we're faced with is, "Might MipZ directly inhibit FtsZ assembly at
00:23:27.08 the correct cell site?" So here's what this thing looks like. What I show you here is a
00:23:32.05 piece of DNA in which we have the origin shown here, and a protein called ParB lines up
00:23:39.21 all the way around the origin. And then this MipZ protein forms by binding to ParB
00:23:48.10 but now very tightly so that it in fact forms some kind of gradient. And when
00:23:54.03 we look very closely, using the fluorescent microscope labeling either the ParB
00:24:00.09 with GFP or in fact the MipZ protein with YFP, you can see that the ParB is very
00:24:08.15 tight at the poles, whereas the MipZ forms a gradient with the least expression
00:24:15.23 at midcell. So now, what we did was we purified MipZ. We purified the FtsZ tubulin
00:24:24.25 and what I show you here is that in the absence of MipZ, the FtsZ monomers
00:24:33.22 form these long polymers. This is just a higher magnification. But if we then
00:24:39.15 add MipZ to it, these big long monomers get very small and curvy and they're
00:24:45.16 non-functional, telling us that MipZ is an inhibitor of the polymerization of the
00:24:53.14 FtsZ tubulin that will carry out cytokinesis. With this information, we can now
00:25:01.12 draw a model. So here is a cartoon of the Caulobacter cell. This work was done
00:25:07.00 by a postdoc in the lab, Martin Thanbichler and this little blue dot here is the
00:25:12.03 origin of replication. Nothing is happening yet. This is a cell that has just differentiated
00:25:18.09 from a swarmer to a stalk cell. At the other pole of the cell is FtsZ, waiting to
00:25:24.03 do something. Here at the origin we have the ParB, and we have all this MipZ
00:25:29.20 sitting on it. Now as soon as the origin is duplicated, that ParB-MipZ complex
00:25:36.00 lines up on that as well. So we now have it on two. And as it moves across
00:25:42.15 the cell and as the MipZ hits the FtsZ at the other pole, it moves away
00:25:49.29 to the region of lowest concentration. Therefore, the movement of these
00:25:56.05 origins from one pole to another using an inhibitor like MipZ allows you to
00:26:04.06 polymerize FtsZ at the place in the cell where there's lowest concentration of
00:26:10.04 inhibitor and that is the center of the cell. Now the second
00:26:15.06 story I'll tell you, and it's really the last story I'll tell you, is that another
00:26:20.20 way that the cell integrates these various functions--integrates DNA
00:26:27.02 replication and segregation with the progression of the cell cycle--is to
00:26:33.13 control the master regulators, to control the expression of the master
00:26:38.11 regulators. And it does this by using an epigenetic mechanism, which is
00:26:44.07 DNA methylation. So if you have a chromosome of Caulobacter, then a
00:26:51.06 swarmer cell, there are sequences in the DNA--GATC--that get methylated on
00:26:58.04 the adenine. And when you start the cell cycle, you're methylated both on
00:27:05.10 Watson and Crick--on both strands of the DNA. Once you begin replicating though
00:27:11.24 the two newly replicated chromosomes are only hemi-methylated.
00:27:16.13 They are methylated where they started, but the new one is not. And that's
00:27:21.07 going to be the epigenetic control mechanism that's used. So what I
00:27:26.17 show here is the genetic circuitry that is controlled by three master regulators.
00:27:34.00 And how that allows you to move through the cell cycle. The three master
00:27:37.27 regulators are a protein called DnaA. This is made first during the cell cycle.
00:27:43.12 And it controls about 40 genes. When this is turned on, and it's turned on
00:27:48.24 just for a specific time in the cell cycle, it then turns on the expression of
00:27:55.11 another master regulator GcrA, which controls about 50 genes. Then later
00:28:01.07 in the cell cycle, GcrA turns on one of the promoters of the third master
00:28:06.23 regulator CtrA, and CtrA controls about 95 genes. So you have a cascade
00:28:14.12 of master regulators that control all the functions that allow you to proceed
00:28:20.06 through the cell cycle. And what I'm going to show you is that a gene that
00:28:25.19 encodes an enzyme, that actually puts those methylation sites on the DNA
00:28:30.29 is a critical event in making this a cyclical regulatory pathway. And here I show
00:28:40.03 you that the first thing that gets turned on is DnaA, then what happens is GcrA
00:28:47.09 is turned on, then CtrA is turned on, and when it finishes that, CtrA turns on
00:28:54.24 this DNA methyltransferase, which comes back--is turned on at the completion of
00:29:00.15 DNA replication to label up all of the loci. So you go back to a fully methylated
00:29:05.21 chromosome. The critical question is with respect to DnaA in fact is what turns on
00:29:12.22 this cascade? How do you get that DnaA to be turned on? And this was quite
00:29:18.09 a surprise. It turns out that if you look at the chromosome, we have the origin
00:29:24.25 at one part, the terminus at the other. The DnaA gene sits right near the origin.
00:29:32.10 This CtrA gene sits on the other side and a little further down. Now when you
00:29:37.07 begin the very beginning of replication, the origin and the whole rest of the
00:29:42.20 chromosome exists in the fully methylated state, so you're methylated on
00:29:47.06 Watson and Crick. In the fully methylated state, this promoter for the DnaA gene
00:29:55.29 is able to be red. There are methylation sites in that promoter and it can
00:30:01.06 only be red in the fully methylated state. However, the CtrA gene shown here can
00:30:09.15 only be red if it's hemi-methylated. And the cell uses the methylation state of
00:30:16.04 the chromosome as a clock to tell you when to turn on DnaA and when to turn it off,
00:30:22.29 when to turn on CtrA and when to turn it off. So at the very beginning when
00:30:29.08 it is all fully methylated, you can transcribe the DnaA gene but you cannot
00:30:34.28 transcribe the CtrA gene. Once replication is initiated and you copy the DnaA
00:30:41.17 gene, then you get two copies that are each hemi-methylated. Remember
00:30:46.11 I told you that it can only be transcribed if it's in the fully methylated state.
00:30:52.05 So as soon as you get two copies that are hemi-methylated, DnaA is not
00:30:58.08 transcribed. Bingo--it's off. But it's already done its thing. It's allowed
00:31:03.28 DNA replication to start. It's controlled genes that make up the replisome
00:31:08.16 and copy DNA and it's done its thing. Then what happens is as you continue going
00:31:15.05 through the replication of the chromosome, you pass through the CtrA gene.
00:31:20.21 That goes from a fully methylated state, where it can't be transcribed, to a hemi-
00:31:27.11 methylated state, where it can be transcribed. And then it's turned on. So in fact
00:31:33.03 what I've now shown you is that the passage of the replication fork controls
00:31:39.11 methylation state and activity of two promoters of these critical global regulators
00:31:47.22 that allow the progression of the cell cycle. So then to give you a summary of
00:31:55.02 what I have shown you--this again shows you the familiar cell cycle that we've
00:31:59.01 been talking about. And I've shown you that origin movement--and we've watched
00:32:03.25 the origin move--is dependent on MreB actin. A protein complex coordinates
00:32:10.18 DNA replication and the positioning of the cell division site--that was that MipZ
00:32:15.09 protein that forms a gradient. Replication and segregation are simultaneous
00:32:20.11 and finally that whole transcriptional regulatory network is coordinated with
00:32:27.07 DNA replication via the methylation state of the chromosome, an epigenetic
00:32:33.07 mechanism. So what I have shown you is that all these various modules
00:32:39.03 that are turned on and off through the cell cycle are all hooked up and coordinated
00:32:44.21 with the act of replicating the cell's DNA. So I would just like to thank the very,
00:32:52.20 very talented bunch of students and postdocs in the lab who have done the work
00:32:58.07 I've spoken about--Martin Thanbichler did that very pretty work on the MipZ.
00:33:03.18 Justine Collier on the DnaA regulation. Ann Reisenauer on the control of CtrA.
00:33:10.12 Natalie Dye and Zemer Gitai, who's just left the lab to start his own at Princeton,
00:33:16.02 have worked on the MreB actin. Patrick Viollier and Rasmus Jensen on the
00:33:22.14 organization of the chromosome. And I would like to state now that a lot of our
00:33:26.22 work is done in collaboration with a lab that is really a physics lab. And this other
00:33:33.21 lab is led by Harley McAdams and he's a physicist. He's also a professor in the
00:33:39.05 Department of Developmental Biology at Stanford and his students all get their
00:33:44.16 PhD's in physics or electrical engineering. And we work by coordinating our labs
00:33:50.13 completely so that when his students, who are physicists and engineers, start their
00:33:55.24 thesis work they do it in the same lab with all the geneticists and biochemists
00:34:01.22 who are working in my lab. So at this point, we have a completely interdisciplinary
00:34:06.06 crew and it has opened up new areas of investigation, which I don't believe
00:34:11.29 could've been done without that. So with that, I'd like to thank you very much.
00:34:19.28

Part 2: Escalating Infectious Disease Threat

00:00:01.19 Hi I'm back from my second lecture now. This is going to be
00:00:05.04 quite different from the first one you heard. First one was the
00:00:08.18 the science that goes on in my lab. This talk is why I do that
00:00:12.20 science and why we all have to be aware of what's going on
00:00:17.15 in this global village that we live in. It has become clear
00:00:23.22 that the global infectious disease threat is real and something we
00:00:29.00 have to worry about. Today infectious diseases are the leading
00:00:34.02 cause of death worldwide and the third leading cause of death
00:00:39.02 in the United States. Now the diseases that predominate
00:00:43.11 we all know about and have heard about. Of course they include
00:00:46.27 HIV/AIDS. They include acute lower respiratory infections,
00:00:52.05 diarrheal diseases, tuberculosis, malaria but since 1973,
00:00:59.17 at least 29 previously unknown diseases have emerged. Now
00:01:06.27 20 diseases have re-emerged in new places where they never
00:01:11.24 were before and are sitting in new ecosystems. And the question
00:01:17.13 of course is, "Why is this happening?" Where do new diseases
00:01:20.24 come from? Why are we facing such an escalation?
00:01:25.10 And in point of fact, this is due to the very dramatic changes in the
00:01:31.01 society and in the environment in which we live. There's been an
00:01:36.01 explosion population growth, spreading poverty, global warming,
00:01:40.29 and urban migration, and what we're finding is new pathogens in
00:01:47.29 new places and old pathogens in new places. And what do we mean by,
00:01:54.26 "How is this happening?" So if we have urban migration,
00:01:58.25 which means we're moving into our forests, a typical disease that
00:02:03.06 is now blooming all over the place is Lyme disease, carried by a tick.
00:02:07.28 And that was due to deforestation to make way for new homes.
00:02:12.17 And this causes a tick bite that's carrying this bacterial infection
00:02:17.09 is quite devastating. Another that we of course all know about is
00:02:22.00 HIV/AIDS that is growing and spreading in all urban populations.
00:02:28.29 Another interesting one is called Hantavirus. This virus was unknown.
00:02:33.29 It's new to us. And it appears in the American far West in
00:02:39.07 Utah and Arizona. It's carried by mice and rats. This is a very
00:02:44.18 nasty virus with a 60% kill rate. And we're still trying to understand it.
00:02:49.24 Another one that's coming out of the forests in Africa is
00:02:55.21 caused by a virus, and it's Ebola. And that is a very difficult virus
00:03:00.28 to deal with. Now we have also the different kinds of changes in our environment.
00:03:10.01 For example, Mad Cow disease, which you've all read about and
00:03:14.07 heard about, which is caused not by a bacterium, not by a virus,
00:03:19.12 but by a protein that changes conformation and goes into a
00:03:23.28 state that causes a very severe neurological brain defect.
00:03:28.23 And why did this somehow get out of the box? And if you remember
00:03:34.05 in England several years ago, there was just an outbreak
00:03:39.10 of Mad Cow disease, and it turned out to be due to the fact
00:03:43.02 that the production of the foodstuffs that we feed all of our
00:03:48.02 livestock was made differently. We were feeding them various kinds of
00:03:54.22 vegetable, mineral, and animal refuse and this time they would
00:04:00.22 include the nervous structure--the brains and the nerves. And that's
00:04:05.24 where these prions were. And then it wasn't until the mandate
00:04:09.24 came down to change the preparation of food for our livestock that
00:04:14.20 this epidemic was dropped down. And this had to be changed worldwide.
00:04:18.16 So this is another example of us trying to survive in large populations
00:04:24.09 and feed everybody by changing the way in which we carry
00:04:27.22 out what we do. The other thing that's happened is that
00:04:31.04 there's been a resurgence of very drug-resistant tuberculosis.
00:04:36.27 And in South Africa now, there are strains of the bacillus that
00:04:43.11 causes tuberculosis that are resistant to every known antibiotic
00:04:48.12 and it's a particularly virulent strain with a very high kill rate.
00:04:52.14 So the crowding, urban mixing of people and pathogens has given
00:05:00.21 us a bloom of bacteria that are resistant to antibiotics. Now
00:05:08.20 we also know that we have international travel everywhere.
00:05:15.02 It's increased. If we have a disease outbreak in Kuala Lumpur,
00:05:19.09 in a day, the person that gets on that airplane with an infectious
00:05:24.21 disease can be in Chicago in that same day. So that we are
00:05:30.01 rapidly moving all over this globe, and there's an incredibly rapid spread
00:05:34.29 of disease, reminding of us that no country is an island. And we now
00:05:40.00 live in a global village, which means that all the countries on this globe
00:05:45.00 have to now coordinate, collaborate, and help one another
00:05:50.08 to identify diseases and to disseminate things that will help
00:05:55.08 squash down an outbreak of some kind. Now, one other thing that
00:06:00.23 happens when this guy picks up a disease in Kuala Lumpur and
00:06:05.07 winds up in Chicago--now the people in Kuala Lumpur may have
00:06:08.25 been living with this disease for a little while and they've built up immunity.
00:06:11.28 But the people in Chicago have no immunity to this and so it's
00:06:15.24 a bigger problem. Now the part of this that's difficult is that we have
00:06:21.25 asymptomatic travel. You get on a plane and you feel fine but in fact
00:06:27.02 you're infected. And of the big problems with a possible influenza
00:06:32.23 epidemic is if you catch flu bug--influenza--you are asymptomatic
00:06:40.08 for at least two days. And during that period of time, you are
00:06:44.07 infectious. So you don't know what you're transferring. And that is
00:06:48.28 what leads you to pandemic. Now another problem of course is
00:06:54.29 the loss of control of our national borders. And we're really not very
00:07:01.08 good at carrying out our quarantine laws. It's difficult to do this.
00:07:08.04 And we are going to return to this later in my talk because in many diseases--
00:07:14.29 effective quarantine laws are the only thing that we're going to
00:07:19.25 use to protect ourselves. Now every one of these issues is something
00:07:25.21 that is being worked on and understood by many countries
00:07:30.22 coordinated by the World Health Organization. The final issue that
00:07:36.25 is almost making where we live now a perfect storm is that
00:07:41.20 while we have increased globalization, while we have increased
00:07:46.10 population, while we have increased urbanization, and the migration of
00:07:51.04 pathogens into new ecological niches, we have the rise of
00:07:55.15 antibiotic-resistant pathogens. So let me tell you what an
00:07:59.22 antibiotic is. An antibiotic is a small compound either made
00:08:03.24 in a laboratory or made by some living creature and when
00:08:08.01 this compound is made, what it does is kill the pathogen.
00:08:15.16 It kills the bacterial pathogen. Perhaps I should just remind you
00:08:19.12 of the difference between a bacterial pathogen and a viral
00:08:23.07 pathogen. A virus is not a living cell. A virus just has a protein
00:08:29.24 coat and the genetic material sits inside this coat. And it
00:08:34.27 cannot make more of itself. The only way it can make more of itself
00:08:39.24 is if it infects a host cell--one of our cells, one of the cells that are
00:08:45.08 of a rat, or a monkey, or some other animal. And then it gets in there
00:08:49.07 co-opts the machinery of that cell and makes many more of itself.
00:08:53.17 That's a virus. A bacterial cell is this little tiny living cell that can grow
00:09:00.17 and divide and respond to its environment and figure out if it's
00:09:04.22 a pathogen how to get into a host cell and make you very ill.
00:09:09.05 So that's a bacterial cell. Antibiotics are specific for bacterial cells,
00:09:16.00 not viral infections. And what has happened is that we have had
00:09:22.14 a history of various kinds of antibiotics, which were first discovered
00:09:30.11 in 1946 with penicillin. Then soon after that, we had strep and staph
00:09:38.23 infections that would be very sensitive to penicillin. Today, 80%
00:09:44.29 of all strains of staph--staphylococcus--are resistant to penicillin.
00:09:51.03 This was quite a shock to us. In 1950 we had more antibiotics that
00:10:00.25 would infect multiple bugs--streptomycin, chloramphenicol,
00:10:06.07 tetracycline. Then in 1953 there was a Shigella outbreak in Japan
00:10:12.15 and it resulted in the appearance of a strain of dysentery bacillus
00:10:16.22 that was resistant not just to one antibiotic but to many. And that
00:10:22.04 was a red flag. And people started becoming somewhat concerned.
00:10:27.07 Up now to 1982, when we had the last new class of antibiotics--the quinolones--
00:10:34.22 resistance is rising, really, in a frightening manner. Cipro, to which
00:10:41.08 we were eating like candy when there was an anthrax scare in
00:10:45.24 the United States, has caused enormous resistance to that particular drug.
00:10:51.16 In 1998, vancomycin, which is considered by many an antibiotic of
00:10:58.01 last resort for staph infections and other kinds of pathogens,
00:11:01.25 we are now seeing the emergence of resistance to Vancomycin as well.
00:11:06.12 Now what happens when something becomes resistant to an antibiotic
00:11:11.18 is that it turns out that these bugs are very, very smart and what they
00:11:16.15 have learned how to do is if they see a drug coming towards
00:11:21.28 it, like arithrimycin, it figures out how to spit it out. Or if the
00:11:29.00 antibiotic manages to get into the bug, the bacteria have figured out
00:11:34.28 a way of chemically modifying that antibiotic so that it's no longer
00:11:38.23 working. And they also have figured out how to change the target
00:11:43.16 of that antibiotic in that particular cell so it doesn't work any longer.
00:11:47.26 So these bugs are very smart and we're in a war with them and
00:11:53.03 the bugs are winning. And what we need is to understand how to make
00:11:57.22 new and better antibiotics. If I just look at staph infections in the
00:12:03.12 United States, in 1957 100% sensitive to penicillin. 1982-- fewer
00:12:12.04 than 10% of all staph cases could be cured by penicillin.
00:12:16.06 1993-- only vancomycin survived as an effective antibiotic. And today, as I
00:12:23.13 told you, there are strains that are resistant to everything. So
00:12:27.18 one of the questions is, "Why is antibiotic resistance growing so rapidly?"
00:12:32.03 And in fact, what we're seeing is that antibiotics are put into animal
00:12:38.17 feed, into aerosols for fruits and vegetables. Of the 50 million
00:12:43.09 pounds of antibiotics produced annually in the U.S., 40% go into
00:12:49.23 livestock. So how does this resistance arise? Let's say you
00:12:55.06 have 10 to the ninth (10^9) bacterial cells all resistant to a particular antibiotic.
00:13:00.15 One of those 10^9 cells has a mutation that makes it resistant.
00:13:07.12 All the others will be killed by that antibiotic, but that one
00:13:12.00 will happily grow and divide and then you have a bloom of an
00:13:16.18 antibiotic-resistant pathogen. By feeding antibiotics in huge quantities
00:13:23.14 to all of our livestock, we are increasing the chances of that one
00:13:30.05 guy to develop antibiotic resistance. Another reason that things--
00:13:36.10 this resistance--is growing so rapidly is that there are growing numbers
00:13:40.28 of immunocompromised people. And this is really partly due to
00:13:45.10 the wonders of medicine and also to new infectious diseases.
00:13:48.20 Chemotherapy patients have very, very low immunity. They're infected
00:13:53.21 by many different bacteria and they grow and divide and develop
00:13:57.04 resistance. Transplant patients, AIDS patients, even just aging populations--
00:14:04.05 if you're over 65, your immune system is going to hell in a handbasket.
00:14:09.20 And so you have to really realize that you are particular sensitive to
00:14:14.17 bacterial infections and again you become a reservoir for increased
00:14:19.19 antibiotic resistance. There's also the excessive use of antibiotics,
00:14:24.04 over-prescription and then unregulated over-the-counter sales. So these
00:14:29.22 are all very serious problems. Of course, as I told you before,
00:14:34.02 international travel has us all over the place. And so if you get
00:14:38.08 a multi-drug-resistant strain of streptomycin in Spain, you wind up
00:14:46.13 with it in South Africa in four days if somebody is traveling rapidly.
00:14:50.03 So we have complete and rapid dissemination. Now what I'm
00:14:53.26 going to turn to is the story of where new bugs come from.
00:15:00.23 Where do new pathogens come from? And the story I'm going to tell you
00:15:05.22 is one of E. coli 0157. It's a new and pervasive pathogen. It's a food
00:15:12.16 contaminant that is now the leading cause of kidney failure
00:15:15.26 in children. Now the first time I told this story was in a very
00:15:19.28 unusual scenario. It was during Bill Clinton's administration.
00:15:25.27 And he became very worried about genetic engineering--
00:15:30.20 in other words, what we can do in the lab now in building new
00:15:34.01 groups of genes and perhaps altering a pathogen or altering some other
00:15:39.15 normal process. And he was worried. He wanted to know how
00:15:43.11 worried we should be about malevolent forces actually creating
00:15:48.12 new pathogens. And he wanted to understand what was happening.
00:15:53.27 And I was part of a group of six people who were invited to speak
00:15:58.20 with President Clinton and his whole cabinet. And the story I
00:16:03.00 told them really was that genetic engineering, yes we can do
00:16:09.04 in the lab, but the bugs and the various kinds of critters in the
00:16:15.14 natural world are much better genetic engineers than we are and
00:16:19.15 the example is E. coli 0157. Now, this is a picture of a virus
00:16:27.18 and I'm going to show you and tell you how this figured in
00:16:32.06 to the genetic engineering that was carried out by E. coli 0157.
00:16:37.13 So where did it come from? E. coli 0157 was first isolated
00:16:44.08 from a 50-year-old woman in California, who came down with
00:16:49.09 severe gastric distress and bloody diarrhea. She survived but
00:16:54.11 she was quite ill. Then in 1980, 14 children were admitted to a Toronto
00:17:01.03 hospital with the same symptoms. Of these, two children died
00:17:05.24 and the rest were left with severe kidney damage. Again
00:17:10.03 the bug, the bacterium, isolated from one of these kids
00:17:15.18 was the same as that found in that 50-year-old woman and
00:17:20.15 upon analysis, the very surprising development was that this
00:17:27.01 E. coli cell, which is a bug that grows in all of our gastric system
00:17:33.12 and is quite harmless, had picked up a gene--a particular gene--
00:17:40.14 from another bug, a pathogen called Shigella that coded for a toxin.
00:17:46.13 So now we had taken an E. coli cell and put in a gene that made it
00:17:52.04 a pathogen. That is genetic engineering. In 1981, in White City,
00:17:58.08 California 12 people eating at a local hamburger place became
00:18:02.13 ill with the same symptoms. 1982 in Michigan--again a local hamburger
00:18:08.03 place--E. coli 0157 was found in its meat patties. 1993--Jack-in-the-box
00:18:17.01 restaurants in the Northwest--hundreds became ill. Four kids died.
00:18:22.14 And this continues on and on. It was found in 1996 in contaminated
00:18:27.24 apple juice and lettuce. And that turned out because the E. coli cell
00:18:32.15 picked up not only your gene for the toxin but a gene that makes
00:18:35.25 it resistant to acid. So it could grow in an acidic environment,
00:18:39.18 which normal E. coli does not. 1997--there was again a huge
00:18:45.10 recall of contaminated hamburger meat. And in 2007, just a couple
00:18:51.00 months ago, contaminated spinach was found. And that came
00:18:56.02 from the runoff from livestock, which were not too far away.
00:19:01.20 So it turns out that now, there are 25 to 30 outbreaks per year
00:19:09.00 in the United States alone of E. coli 0157 contamination
00:19:13.18 and 5% of our dairy cows carry this pathogen. So how did this
00:19:20.13 happen? How do we think that this occurred? So what I'm showing you
00:19:25.00 here is a bacterial virus. Remember I told you that a virus
00:19:30.08 has a protein head, and that's shown here. That's the head.
00:19:34.12 This is its tail, and there's DNA in this head. And this over here
00:19:39.10 shows you what the virus looks like. This over hear shows you a
00:19:43.26 diagram of this virus. Looks like a moon lander, doesn't it?
00:19:46.29 And what this moon lander does is it lands on a bacterial cell
00:19:51.13 and it injects the DNA, the genetic material, right into the
00:19:57.05 bacterial cell that it hits. And this is how we believe this happened.
00:20:02.12 Okay. In this diagram I show you a Shigella bacterial cell. The blue
00:20:09.03 circle indicates the chromosome--the single chromosome.
00:20:13.10 And the little moon lander up there indicates the virus.
00:20:17.10 So the virus injects its DNA, and that's that little circle in the
00:20:22.13 center of the head, into the cell. Once that DNA gets into the
00:20:27.00 cell, it codes for things that chop up that blue chromosome.
00:20:31.13 You chop up that blue chromosome and the little piece
00:20:35.02 that contains that Shigella gene, then gets put into the head
00:20:40.27 of a new virus. And so this new bacterial virus contains
00:20:46.09 its own DNA and a little bit of that Shigella DNA. And that little bit
00:20:51.24 contains the gene that causes a toxin. Now what happens is
00:20:58.14 that that same virus hits an E. coli cell. And we believe this
00:21:05.04 happened during an epidemic of dysentery in Central America
00:21:10.18 when both E. coli and Shigella were mixed. And this virus injected
00:21:16.04 its DNA into the harmless E. coli. And this piece of DNA got
00:21:21.10 incorporated into the DNA of this E. coli, creating E. coli 0157.
00:21:29.15 That, folks, is genetic engineering. That happens naturally.
00:21:34.05 So now let me tell you a second story. And the second story
00:21:38.25 deals with why it's so difficult when we're first faced with something
00:21:44.20 that we haven't seen before in a given country to decide whether this
00:21:48.24 is a natural outbreak. Is it a malevolent deed? Where is it coming
00:21:54.26 from? And this is an interesting story. Now West Nile virus
00:22:02.00 causes an encephalitis-type disease. But it had never been found
00:22:06.26 in the Western hemisphere. And this is several years ago now.
00:22:12.14 And the way it started was that birds in the Bronx zoo started to die.
00:22:17.25 And they had an encephalitis-type infection. And a vet in the Bronx zoo
00:22:24.02 sent her tissue samples to the CDC--Center for Disease Control--
00:22:28.21 and they were pretty overwhelmed because the CDC never has
00:22:32.17 enough money to do everything they have to do and they sort of
00:22:36.16 said, "Yeah, we'll get to this. Birds are dying." Well, at the same time
00:22:40.27 there was an increasing number of human patients in New York City,
00:22:44.22 which were exhibiting and dying from an encephalitis-type disease.
00:22:49.22 People thought it was the mosquito-born St. Louis encephalitis virus.
00:22:55.04 They didn't really know. Then the chief of Emergency Management
00:23:01.07 in New York City managed to co-opt the entire supply of "Off."
00:23:07.02 "Off" is something that kills mosquitoes and flying bugs, and he
00:23:11.15 just sprayed "Off" all over the city and he stopped the epidemic
00:23:15.21 cold. Meanwhile, the lady vet at the Bronx Zoo was still trying
00:23:21.08 desperately to find out why her birds were dying. Waiting to hear
00:23:25.15 something from the CDC, and the weeks were going on and she
00:23:29.23 happened to go to a wedding on the West Coast and sitting
00:23:34.01 next to her at this wedding was a virologist. And not particularly
00:23:39.15 interested in dancing, they started talking about this odd thing
00:23:43.23 that was happening to her birds at the zoo. And he said, "Look,
00:23:48.01 why don't you send me some of her tissues, and I'll try to figure out what
00:23:51.02 you've got." And that's what happened. He very rapidly identified
00:23:55.17 this as West Nile Virus. Now everyone's initial reaction was,
00:24:01.26 "This couldn't be. We don't have West Nile Virus in the Western
00:24:04.18 hemisphere!" But at about that time, in Fort Collins, the CDC had
00:24:10.06 in fact identified this as well as West Nile Virus. It just took too long.
00:24:18.23 And this was our first experience with trying to rapidly identify
00:24:23.18 something new. Now, interestingly, concurrently while all of this
00:24:28.18 was going on, an Iraqi defector had reported that Saddam Hussein
00:24:34.24 was developing a strain of West Nile Virus as a biological warfare
00:24:39.22 agent and was preparing to release it. This was never confirmed.
00:24:44.12 Was this a BW event? We don't know. Did this come into the United
00:24:50.08 States on a 747 that a mosquito happened to crawl into? We don't
00:24:55.22 know. We don't know the answer, but what's important--what one
00:25:00.20 has to remember--is anything we do to identify a new outbreak
00:25:05.28 will be relevant no matter what the source is--malevolent or natural.
00:25:11.10 The problem is to understand what we've got and to rapidly
00:25:15.19 understand how we can analyze these things and identify the
00:25:20.04 agents. Now, this is changing. And this is changing particularly because
00:25:26.08 of what we found with SARs. Now that's happened fairly recently.
00:25:30.22 SARs is Severe Acute Respiratory Syndrome. It's caused by a
00:25:36.14 corona virus, which is an RNA virus. It's similar to the viruses that
00:25:42.17 cause the common cold. It has a very high potential for natural evolution
00:25:47.21 so it can change itself a lot. Now with SARs--it's an interesting story
00:25:54.21 because this is an infection that first started predominantly in
00:25:59.08 Hong Kong and Beijing and Guangdong Province in China. But
00:26:03.26 very rapidly appeared in Toronto. And what happened there
00:26:08.11 is that we had a highly infectious agent that exemplifies this
00:26:13.08 global village we live in. There was a scientific meeting in Hong Kong.
00:26:17.02 Someone got sick. They wound up in Toronto and it was all
00:26:20.11 over the place. But SARs is an example in which we were much better
00:26:24.26 at identifying the agent rapidly by sequencing. We were also
00:26:30.26 able to realize that the only thing that would be effective
00:26:34.14 was quarantine. And this is interesting because in fact
00:26:40.06 Singapore was very effective in quarantine. They said, "This is
00:26:45.24 what we have to do to stop this." Whereas Hong Kong and Toronto
00:26:49.16 were not. Ultimately it stopped. The dealing with this was very effective.
00:26:58.17 And actually there were very few deaths if you look at it in a global way.
00:27:03.12 But the effect on the economy was enormous. And so this tells
00:27:08.03 us that even a minor outbreak is going to have severe economic
00:27:12.25 implications globally. And what it did do though was help the World
00:27:18.23 Health Organization build a network of reporting, of understanding, of
00:27:25.07 diagnosing outbreaks of diseases everywhere in the world. So
00:27:29.16 that we would know how to respond and deal rapidly with them.
00:27:33.05 Now what I'm going to do is end this talk with a discussion of something
00:27:38.14 that's facing us all right now. And that's Asian bird flu H5N1.
00:27:45.22 This causes influenza. Influenza is with us all the time, various
00:27:50.25 different kinds of strains. This is a particularly frightening
00:27:54.03 one. However, there is no strong evidence as yet of human-
00:27:59.09 to-human transmission. Right now, this is a disease of birds--
00:28:04.26 local fowl, poultry, wild birds. Our concern is that H5N1, which
00:28:13.01 mutates rapidly, will ultimately go from person to person.
00:28:19.16 Now, let me tell you a little bit about this virus because it's relevant.
00:28:24.22 Each virus has a single strand of RNA containing 8 genes.
00:28:31.00 Each gene encodes a single protein. This very high mutagenicity
00:28:36.20 rate, in other words changing the kind of protein that's made
00:28:40.24 from each gene, can happen by reassorting the genes by
00:28:44.22 single-base mutations. And it just changes rapidly. That's
00:28:48.11 why we get flu shots every year. And basically at this time
00:28:54.14 we know that the transmission of H5N1 goes from ducks to
00:29:00.20 either wild birds or to some cats, tigers, lions, leopards, house pets
00:29:06.25 with a fairly easy transmission. However, from wild birds to humans
00:29:14.10 does occur. It's not easy. You need very personal contact.
00:29:19.15 And humans to humans--there's not strong evidence of that yet.
00:29:25.15 Our concern is that it might happen. And so what is H5N1 mean
00:29:31.03 anyway? "H "stands for hemaglutinin and that is a protein that sits
00:29:38.00 on top of that cage that the RNA sits into. The function of
00:29:43.05 that hemaglutinin is to allow the virus to bind to the host cell
00:29:48.23 and allow entry of the RNA to do its bad stuff. "N" stands for
00:29:55.21 neuraminidase. Neuraminidase is another kind of protein
00:29:59.05 that's also sitting on the surface of the cell and it allows
00:30:02.28 newly formed viruses to escape and infect other cells. We have
00:30:09.03 two anti-virals out there now. One is called Tamiflu.
00:30:12.25 The other is Relenza. And the neuraminidase is the target for both
00:30:17.13 of these. And in fact, the best way to use something like Tamiflu
00:30:23.04 is if you've not been infected yet, it will give you 80% protection
00:30:28.12 for awhile. If you've been infected, it will drop the viral load so that
00:30:34.05 you're not as contagious. You'll still get sick but you won't
00:30:37.09 be as sick. Now if we look at the history of flu viruses, the most serious
00:30:46.04 flu pandemic occurred in 1918 and that influenza was H1N1.
00:30:53.20 It killed 40 million people worldwide and H1N1 means a particular
00:31:00.06 derivative of the hemaglutinin and the neuraminidase. 1957
00:31:05.26 flu was H2N2--killed about 2 million people. 1968--H3N2 killed about
00:31:13.19 a million. I know that one very well because I caught that one.
00:31:16.20 And let me tell you--a real flu infection is no fun. Their current
00:31:22.10 Asian bird flu is H5N1 as I've said. Scary thing about this guy
00:31:27.11 is that right now it has a 50% kill rate, which is enormous.
00:31:32.06 And humans have no immunity against H5, whereas we have some
00:31:37.10 against H1, H2, and H3, which has been around for awhile.
00:31:42.14 So what needs to be done? How are we going to deal with this?
00:31:48.10 Let me just tell you first that using something like Tamiflu
00:31:56.10 is best done in my opinion not by sprinkling Tamiflu amongst
00:32:02.21 the 30 million people in the world but rather using it where there's
00:32:07.23 a hot spot. Now that we have an entire network of reporting
00:32:15.16 coming all over the world, keeping their eyes out for hot spots
00:32:20.00 of sudden break outs of H5N1, possibly being passed from human to
00:32:26.02 human, then our Tamiflu has to get there immediately. And then
00:32:31.27 what you do, is you cordon off the area, quarantine, treat with
00:32:36.18 Tamiflu, and start vaccines. Now clearly the vaccine we have now
00:32:41.26 is to the H5N1 that only goes between birds and possibly cats.
00:32:48.06 What we will ultimately need--if this happens--if it mutates to
00:32:53.08 human-to-human is that we then have to get a new vaccine
00:32:59.11 that will be against that particular variant. And a lot of work
00:33:04.04 is going on right now by many small companies and many
00:33:06.27 large pharmaceutical houses to be ready to make this as quickly as
00:33:11.13 possible. Now one thing that is important to realize is that
00:33:18.27 vaccines are made in eggs. I mean zillions of eggs. If you go to one of these
00:33:24.13 vaccine production places, it's astonishing. It's like a football field
00:33:28.05 of eggs. And virus gets injected into these eggs and then a high titer
00:33:34.14 of more viruses made that's impaired. You kill it. You then make
00:33:39.03 the vaccine. The reason that we use eggs is that you get a very high
00:33:43.17 titer. One thing that I can't stress too strongly is that in fact
00:33:49.22 you can't get flu from a flu shot. It's dead. But you certainly can
00:33:56.26 get immunity. But people seem to think that vaccines are an
00:34:03.17 absolute panacea. It's not true. Flu vaccines are 70-90% effective
00:34:10.15 in young healthy people and only 40-60% effective in people over
00:34:17.11 65. So that the flu alone is not going to save us. But there are
00:34:23.20 many things that we can do to help ourselves. One of the things
00:34:28.26 we have to do is that we have to stockpile face masks--the kind
00:34:32.19 you buy in the hardware store for painters--syringes, medical
00:34:38.23 supplies, food, and water. Currently we do not have in the United States
00:34:44.06 enough ventilators if we were to have a pandemic. So it's extremely
00:34:48.05 important that we learn how to deal with large amounts of people
00:34:54.13 becoming ill. So what if we do get a pandemic? What does a
00:34:57.19 country do? And what I'm going to do is end with sort of an
00:35:01.27 economic part and that is in ordinary times economic logic does
00:35:07.07 not dictate pandemic preparedness. We all keep low inventories.
00:35:12.29 We don't want redundancy in reserves. We have lots of offshore
00:35:20.00 drug production because it's cheaper. We don't guarantee the
00:35:24.11 purchase of flu drug as we do with other kinds of weapons.
00:35:27.16 In fact we have just in-time delivery with no surge capacity.
00:35:32.24 Now what does this mean if you have a pandemic? The supply
00:35:38.10 chain is very thin. Every hospital contains only 30 days of drugs.
00:35:42.19 We have, in a pandemic, workers becoming ill. Drug company
00:35:49.22 workers, the production of new vaccines and new drugs will become
00:35:53.09 less. And in fact, we'll have borders closed and embargos. They
00:35:59.13 can't get in. Truck drivers get sick. Things can't be delivered.
00:36:03.13 We then have to say, "Alright, what do we need to do to deal
00:36:09.05 with this?" We need scaled-up manufacturing and stockpiling of
00:36:13.27 vaccines and anti-viral drugs, as well as antibiotics because
00:36:18.18 many people die of flu infection by a secondary bacterial infection.
00:36:24.02 So we need antibiotics. There's a pneumococcal vaccine that is
00:36:29.02 something people should all have that helps. We need better
00:36:32.19 surveillance and epidemiology on a global scale, very accurate
00:36:37.06 reporting of case clusters. We need actual procedures for drug
00:36:42.02 delivery and most important, we need quarantine laws. Not only
00:36:47.09 here in the United States but everywhere in the world. And
00:36:50.09 our population has to understand what these population laws,
00:36:55.17 what these quarantine laws, are before we're faced with the
00:37:00.22 absolute disaster of a pandemic. And you have to know what
00:37:06.24 you're supposed to do in a quarantine, where you'll get your medicines,
00:37:10.21 who will see you. And this can't be done just at a national level.
00:37:15.01 It has to be done in cities and towns where groups of people
00:37:19.03 can work together. That's probably the strongest thing. Now
00:37:23.28 let me just end by turning this back to what are the things that needs
00:37:28.25 to be done with this emerging infectious diseases with the way
00:37:33.16 in which our world has changed and the basic science that's
00:37:37.05 going on. So we have now a real need to increase basic research
00:37:44.14 to understand these viral and bacterial pathogens. We have to
00:37:48.21 identify genes essential for the pathogen's survival. We have to
00:37:53.09 sequence and compare bacterial and viral genomes. We have to
00:37:57.16 identify virulence factors and resistance genes and understand
00:38:03.06 how they work. And as I said in my previous lecture, by understanding
00:38:09.01 how the bacterial cell carries out all of its functions to let them grow
00:38:14.17 and divide, we have identified new targets and designed new
00:38:19.19 antibiotics. That's just one lab. And this has to happen in many
00:38:23.05 many more. The second thing that we have to do is design
00:38:26.12 and stockpile new vaccine strategies and in fact make combination
00:38:31.27 antibiotics where you have a particular drug that kills the bug
00:38:37.19 but in that same pill or shot you've got a compound that prevents
00:38:43.00 the resistance from being expressed. And then finally for epidemic
00:38:47.21 control, we have to develop techniques for very fast--hours, not
00:38:52.28 days, not like what happened with West Nile or even the slowness
00:38:57.13 of SARs, which was much better--to identify causative agents.
00:39:02.08 To do this, we have to exploit viral and bacterial DNA sequence-
00:39:07.01 based technologies. We need, as I keep saying, an increased
00:39:11.02 network of surveillance and reporting protocols and finally
00:39:14.19 return to the historical use of quarantine. If we all work together
00:39:20.07 and if we realize that we are not just independent islands
00:39:24.26 and separate countries, we work together as a global village
00:39:28.25 and help us combat these things. So with that, I'd like to thank
00:39:33.10 you very much.
00:39:35.14

Videos in this Talk

Part 1: Dynamics of the Bacterial Chromosome
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad
Part 2: Escalating Infectious Disease Threat
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad

Speaker: Lucy Shapiro

Total Duration: 1:14:09

Recorded: April 2007

All Talks in Microbiology

Talk Overview

Most bacterial cells have their genes arranged in a single circle of DNA. The circle of DNA plus some attached proteins is referred to as the bacterial chromosome. Up until quite recently, it was thought that the chromosome in the tiny bacterial cell resembled a tangled ball of yarn. It is now known that multiple factors cooperate to condense DNA into a highly dynamic assembly of supercoiled loops. Although there is variability in the lower levels of chromosome structure, the global arrangement of DNA within the cell is conserved, with individual loci arrayed along the long axis of the cell in line with their order on the genetic map. This order is maintained and propagated during DNA replication. Upon duplication of a given segment of the chromosome, it is immediately released from the replisome (the DNA replication machine) and it moves rapidly to its conserved position in the incipient daughter cell compartment. Partitioning of the bacterial chromosome thus takes place while DNA replication is in progress. Furthermore, it is becoming clear that the bacterial cell is highly organized, presenting new challenges and opportunities for the design of new antibiotics.

Many antibiotics, which we have taken for granted since the 1950’s, are now becoming ineffective because bacteria have developed ways of acquiring resistance. The development of new antibiotics is lagging behind the loss of the old ones in this race to combat infectious disease. Simultaneously, there is an increase in infectious diseases around the world due to overpopulation, globalization and urbanization. This results in a lethal combination of emerging diseases and loss of effective antibiotics. Multiple factors have contributed to this escalating scenario. The world is now a global village, there is a loss of control of national borders, there are significant populations of aging and immuno-compromised people, there are drastic changes in global ecology, and migration pathways of animal and insect vectors are changing due to urbanization and global warming. Large networks of epidemiologists and scientists worldwide are now working to coordinate detection, diagnosis and treatment of infectious disease flare-ups in order to contain the threat of epidemics.

Speaker Bio

Lucy Shapiro

Lucy Shapiro holds the Virginia and D.K. Ludwig Chair in Cancer Research and is the Director of the Beckman Center for Genetic and Molecular Medicine at Stanford University. She received her Ph.D. in Molecular Biology from the Albert Einstein College of Medicine. She joined the faculty at Stanford University School of Medicine in 1989 and… Continue Reading