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Partners in and out of the lab

August 31, 2019


(lively music) – [Christina] We go beyond
the journal articles and into the laboratory and the field, as scientists share their discoveries in a way everyone can understand. It’s science, explained. You are listening to Science Sound Off on Texas A&M Health Talk. (lively music) Welcome to Science Sound
Off; I’m Christina Sumners, and with me today are
two assistant professors from the Texas A&M College of Medicine, Robert Watson and Kristin Patrick. They’re also husband and wife. Welcome, Dr. Watson and Dr. Patrick. – [Dr. Patrick] Thanks for having us. – [Dr. Watson] Thanks,
we’re excited to be here. – [Christina] Well, maybe
could we just start by talking a little bit about yourselves? What is your background
and what is your lab like? – [Dr. Watson] Since it’s
the Patrick Watson Lab, I think Kristin should go first. – [Dr. Patrick] Well, for
alphabetical order’s sake. I am an RNA biologist by training, so I’m interested in fundamental
aspects of gene expression, how our cells go from
DNA to RNA to protein. Since we’ve been here for about
three and a half years now, at A&M, we’ve been working
together to combine my interest in fundamental basic science, with Robby’s interest, as
he’ll tell you in a second, in the innate immune response. We’re interested in how
gene expression changes when your cells become infected. – [Dr. Watson] My background
is host pathogen interactions, and my training basically
is looking at how intracellular bacteria or
bacteria interact with our cells and with our immune system, and how they can get around
these things and make us sick. We look at the molecular
details of those interactions, and we understand what the
host developed in order to kind of kill the bacteria and
control bacterial infection, and what specific adaptations
the bacteria have evolved, co-evoled with humans
with, to make us sick and to establish an infection. That’s my interest and
that’s how we’re combining our interests together into one super lab. – [Dr. Patrick] I like that, super lab. – [Christina] Fantastic, so maybe … You just had a paper published
in the Cell Systems Journal. Maybe you could tell us
a little bit about that. What did y’all find? – [Dr. Patrick] Yeah, so this
paper is a long time coming. I’ll give you a little bit of background. Robby and I came up for
the concept of the screen, this paper is based on, what, like seven or eight years ago now? It was a while. – [Dr. Watson] Three plus, yeah, probably seven or eight years ago. – [Dr. Patrick] Over a bottle of Malbec in Mendoza Argentina, and we were just talking
about ways that we can combine our science, and during my post doc at University of California San Francisco, I did this yeast genetic screen. To explain what a yeast genetic screen is, in the most basic terms,
it’s basically taking two mutant yeast and then, who each have a defect in one
gene, and then combining them so you end up with a double mutant yeast. Based on how that yeast
grows, you can identify relationships between genes. To give you an example,
you have a computer, right? We all use a laptop computer. If you have a problem with your R button and a problem with your touch pad, now your computer is a
little bit janky, right? You have trouble typing,
you have some typos, you can’t really use the mouse, but you can still get around it. If you had a problem with your touch pad, and your external mouse broke, now you have a pretty much
broken computer, right? You can’t use your cursor at all. It’s the same way when we
study mutations in yeast. If you have mutations in
two things that are similar, you end up with a very sick yeast, because now the yeast has two
defects in similar pathways, whereas if you have two mutations in totally different things, maybe the yeast are a
little bit sick, but not, but they can get over it. We use that principal now in this paper, to identify how these things called bacterial effector proteins, that are virulence factors, how
they interact with the cell. I think Robby can give
you a little bit more information about what
these effector proteins are. – [Dr. Watson] Yeah,
so that was her system, and I was a post doc at UCSF too. We were actually right down the hall from, two labs down in the same floor, walk back and forth all the time. I was interested again,
in how bacterial pathogens can make us sick, basically,
and interact with our cells to make us sick. I was working at the time,
working on tuberculosis, but I also have a background in working on enteric pathogens, things
that cause diarrhea, such as salmonella campylobacter, and all of these pathogens
are what are called intracellular pathogens. That means they live intracellularly, or inside our host cells. All of these intracellular pathogens, and many of these pathogens, encode these things
called secretion systems. What these things are, are
basically, nano syringes. They inject what Kristin
called effector proteins, these proteins inside the cell, and these reprogram the cell such that, they either can gain intracellular access or establish a little home for themselves. These things are really key for
establishing a niche, right, and for virulence and to make us sick. We are really interested in how these function during infection, but we actually, just looking at them, we have no idea what they are. You just look at the
sequence of the protein, you know that they’re secreted by these different analogate systems, but you have no idea
actually their function, and so what do you do? Well, you can do a lot of things, but one of the things we want to do is use this yeasts system
and make the assumption that they target specific pathways inside that are conserved
between yeast and mammals, and use this system to see if we can get some idea of what they do. – [Dr. Patrick] Yeah,
and being here at A&M was the perfect place to
start this project because while Robby had a background
in salmonella pathogenesis, we also have two researchers
in our department, Dr. James Samuel, and
Dr. Paul de Figueiredo, who study different
intracellular bacteria– – [Dr. Watson] I’m glad you
know how to say that name. I can never say Paul’s name, last name. – [Dr. Patrick] You better edit that. – [Dr. Watson] Why?
(Dr. Patrick laughs) – [Dr. Patrick Jim and
Paul’s lab are interested in a bacteria called coxiella burnetti and brucella melitensis, respectively. These are two intracellular bacteria. They’re not really widely studied, but both are zoonoses. We can get them both by
exposure to farm animals, like sheep, or cows, or pigs. They both have these secretion systems that Robby talked about, and hundreds and hundreds
of these proteins that we literally know nothing about. It was great; we did this
work as a collaboration, and we screened a set of
proteins that were from coxiella, a set of proteins from brucella, and a set of proteins from salmonella, and asked the question, merely: How does expressing these
proteins in mutant yeast make the yeast grow differently? Just by using growth of
yeast as a measurement, we were able to identify
pathways that were targeted by these different effector proteins. – [Christina] Interesting. Going back up for a second here. Salmonella, it’s a pretty
common foodborne pathogen. What made you target
that one specifically? – [Dr. Watson] I was a graduate student in Jorge Galan’s lab at Yale University, and he’s a very well known
host pathogen person, a bacterial patho-geneticist professor. He actually discovered
the type three secretion, or these nano syringes, in salmonella, so I have a background in
having handled and experienced in dealing with salmonella. In addition to that, because
he was one of the first researchers to find the secretion system, and really understand what
a lot of these effectors do, we actually included some
known effector proteins, kind of as a control. He was the first study to
actually pioneer this field, and because of his work
and a lot of other people that I don’t have time to
give credit for in the field, some of these kind of effector proteins that we use in the screen, were used, were well known of what they do, because of his work we
included in the screen, to get an idea, to use as a control, so that we can know that
our screen was working. – Yeah.
– That was kind of how salmonella, most of salmonella. In addition to that, there’s
actually a lot of unknown effectors proteins in salmonella. We use it because of my background and because it’s a good positive control. It told us that the screen was
working but we could also … We actually ended up identifying
a salmonella protein, a protein of unknown function,
actually it turned out, in the screen, and so we actually
included some unknown ones that their role in infection
was totally unknown. – [Dr. Patrick] Yeah, and to
give you a real life example of one of those functions, one of the top, or the strongest positive
controls in our screen, was this protein from
salmonella called SPTP. Its name is not important,
but what it does is. When it’s secreted by
salmonella into our cells, it reprograms the actin cytoskeleton. Essentially, for salmonella
to get into a cell, a non-phagocytic cell specifically,
in our intestinal tract, it needs to make these big
rearrangements of actin, so the cell actually engulfs the pathogen, but once it’s inside
the cell it needs to fix the actin cytoskeleton, otherwise the cell’s
going to get really upset. It uses this protein SPTP, which has a bunch of
biochemical functions, but the end result is that it reprograms the actin cytoskeleton. We put in SPTP into our
screen, and when we asked, “Well, what pathways in the
cell, what yeast mutants “were especially sensitive to having SPTP “expressed inside them?” we saw it was a lot of mutants involved in the actin cytoskeleton. That was, sort of we
call, proof of principal. That told us that the screen was working and that it was recapitulating
biology that we already knew. – [Christina] I think that
might surprise a lot of people, that yeast can really tell
us something about ourselves. – [Dr. Patrick] Yeah, the
power of yeast genetics is a term that people have
been using for a while, but it really is strong, just because we have an
entire yeast deletion library. We have mutant yeast that
have a defect in every single non-essential gene, and actually, a bunch of mutations in
essential genes as well now. We can really query an
entire eucaryotic cell. Yeah, it is surprising,
but it’s pretty remarkable, I think, that actin cytoskeleton
and endocytic trafficking, and how our DNA is replicated,
that’s all the same. That hasn’t changed throughout evolution, so a lot of these pathways
targeted by pathogens can be queried in yeast, and
that is a super simple model. It grows fast, it’s cheap, a lot of things that
are very difficult to do in a mammalian system. Although, now it’s becoming easier because of CRISPR-Cas9 technology. Up until then, you couldn’t really genetically
reprogram a mammalian cell, so yeast were really the
workhorse of all genetics. – [Dr. Watson] I think it’s amazing. It says something about,
also these pathogens. All of these pathogens
that we’re looking at have co-evolved with their host, whether it’s an animal
or certainly humans, for hundreds of thousands of years. These proteins that are
injected by these syringes, have really, it hits conserved pathways. I think that our yeast screen shows that really these pathways are conserved, as Kristin was mentioning,
really conserved, whether it’s how vesicles
traffic inside a cell, move things around, whether
it’s the cytoskeletal elements, but a lot of these signaling pathways and the things that these pathogens target are conserved from mammalians
all the way down into yeast. Our screen told us that, basically. It really, I think, tells
us the power of the yeast. Now, I would say not to
undermine the study or whatever, but there are limitations. These aren’t immune cells. Immune cells have evolved to detect and respond to a pathogen, right? Not to any old bacteria or
virus that’s in the environment, but really the amazing thing
about our immune system is it can distinguish between what is a pathogen and
what is not a pathogen. There are these signaling
pathways and these sensors that can detect these
pathogen associated patterns. That’s one thing that’s missing,
and we’re thinking about some other screens possibly, that can incorporate some of these ideas, because that’s one thing that
a yeast do not have, right? They don’t have an immune system. If a pathogen targets
something and doesn’t want … Inhibits an immune function, basically, that’s something that yeast will not have and something that will not be discovered by this particular screen. That’s something we have to keep in mind, that we’re thinking about that a protein, that virulence factor that
target these kinds of pathways. The yeast won’t uncover
their function in this way. – [Christine] That makes sense. What are some of the next
steps now, that you have? – [Dr. Patrick] The biggest finding, the biggest novel finding of this screen was that one of the proteins
that really forms the tip of this nano syringe that
Robby referenced earlier, is called SSEC, and it’s
expressed once salmonella has been inside our cells
for a couple of hours. Salmonella lives inside an
endosome, which is like a bag that protects it from
the rest of the cell. It’s a way that it can
hide out inside the cell, and before it’s ready
to infect other cells. One of the main things
salmonella needs to do when it’s inside your
cell, is to avoid fusion with lysosomes. Lysosomes, if you remember
from high school biology, or even further back, remember your learning
about the organelles, the endoplasmic reticulum,
and the golgi apparatus. Well, the lysosome is the bag of enzymes that degrades things. Anything that is damaged in your cell, or even a pathogen that’s
invading your cell, needs to go to a lysosome
so the cell can destroy it. Salmonella obviously doesn’t
want to go to a lysosome, because that would be a sad place for it. It’s evolved ways to avoid
fusion with lysosomes. Obviously, that’s a major
virulence phenomenon. If we can figure out a way to make salmonella go to a lysosome, then that would be a way that
our cells could destroy it. It’s an important mechanism to study, and we found that this protein, SSEC, this part of the translocon
pore, it’s called, actually interacts with
this complex in our cells called the retromer. We found this in yeast, and
the retromer is a complex that traffics endosomes around your cell, and it basically either takes
them to the golgi apparatus or it takes them to the plasma membrane, but it takes them away from lysosomes. It’s the exact thing that
salmonella wants to co-opt. We found in yeast that
these retromer mutants looked exactly like cells expressing SSEC through out genetic profiling. We thought that was really interesting, and then we wanted to follow
up on that in mammalian cells, and ask, “Well, does that
happen in real life?” We infected HeLa cells as
well as human macrophages. We infected HeLa cells as
well as human macrophages, with salmonella, or we expressed SSEC in some of our experiments, and we found that there are
defects in cell traffic, in endosomal trafficking in
the presence of this protein. Then if you take away the
retromer in a human cell, then salmonella has a replication defect. Basically, what we showed
is that yeast told us that the retromer was being
targeted by this SSEC protein, and then when we looked in human cells, we found that the retromer
was an important thing that the salmonella needed
to recruit to its vacuole and interact with in order to maintain its ability to replicate. We have some beautiful
biochemistry in this paper that was done by a collaborator
here at main campus at A&M, Dr. Pingwei Li, and they showed that this salmonella protein,
SSEC, directly interacts with two components of this
retromer complex in human cells. They can show in vitro
when you just express these three proteins, that they interact. That’s a really, like the
best, the gold standard for showing that proteins interact. We’re pretty confident that
that is what’s going on in the cell, and now we’re
doing additional experiments to figure out what are the
consequences of that interaction and what happens if we can
mutate that interaction, what happens to salmonella. – [Dr. Watson] I think what
we’re finding, I think, our studies and some of a lot
of other people’s studies, are actually finding that
a lot of pathogens target the retromer, in fact. It has been shown that both
chlamydia and legionella, they’re both intracellular
bacterial pathogens, they also can target and
manipulate this thing that we’re calling the retromer, and exploit it for their
benefit, to establish a nice little home for
themselves inside a cell. It seems like it’s actually
emerging that this is a common theme in host pathogen, and this is a very common … It seems like pathogens
have evolved to target this for their benefit, this
retromer for their benefit. That’s a really interesting,
I think, a really good area, and we’re going to be able to
really compare and contrast these different lifestyles
of these pathogens, and see how they manipulate the retromer in order to establish this
niche we’re talking about. – [Dr. Patrick] Again, the
exciting thing is that this, our interest in salmonella and its interaction with the retromer, came from this yeast screen. Really, people weren’t
looking at the retromer as a major component of the salmonella containing
vacuole, previously. That was the most exciting,
I think, thing to come out. What’s next for us is we’d
like to expand on this screen and screen other effector proteins. The cool thing that
that will allow us to do is to start to compare and contrast the effectors themselves. We tried to do this with a set of effectors that we screened. We screened 36 different effectors, but I think it’s just
not quite enough yet. Imagine if we had hundreds of effectors, and we had multiple effectors
that interfaced with the actin cytoskeleton, or
interfaced with the retromer. Then we can start to draw
these really interesting evolutionary comparisons
between effector proteins from completely different
bacterial pathogens. That requires us to scale
up the screen considerably, and we’ve been struggling with this, because the screen itself
was actually done at UCSF with the help and guidance
of my post doc advisor, Nevan Krogan, who basically
gave us access to this robot. You can imagine if you
have a yeast library of about five thousand mutants. The way this is done is that you plate, it’s actually in 15 36 format. Picture a rectangle of agar, which is basically science jello. We have a science jello
rectangle with 1,536 different yeast colonies on it. They’re tiny, tiny little colonies. Each colony has a
different mutation in it. It’s on three plates. What we do is we have a plate that has our bacterial
effector expressing strain, and then we cross that,
we basically, the robot, takes its arm and it mixes
those cells on one plate, and it moves them over onto the other one, and mixes it onto the other one, and it does this in a very,
very precise way, right? These 15 36 colonies, you can see them, but if I were to try to
touch one with a by-pet, and then touch the exact
same one on the other plate, god, there’s a 90% chance
I’d get the wrong one, right? You need the precision of a robot. This robot, unfortunately,
is pretty expensive. The screen itself is pretty laborious. It’s about two weeks you’re on the robot, eight, ten hours a day. I did this seven months
pregnant, by the way. I’m going to put that out there. We had to get it done before
the baby came, and we did. It’s not the greatest screen. It’s been very powerful, but
now thinking of doing it more, and here at A&M we want to
see if we can improve upon it. Again, in a glorious collaborative effort, we’ve started working with Craig Caplan in the biology department, who has done this type of
yeast screening in the past, this agar based yeast
screening, but is now developing a way to do it in liquid, where instead of having all the colonies individual on a plate, you combine them, and instead of measuring yeast growth just by taking a picture
of the colony size, you use sequencing to sequence
a barcode that’s unique in each of the yeast mutants. Because sequencing costs have
become so low in recent years, and because we have access to
these great DNA sequencing, and RNA sequencing capacities
here, we can now hopefully do this screen in liquid
and just sequence out all the barcodes of the different mutants, and if you have few of a barcode, which is just a little unique sequence, is what a barcode means, if you have very few of those
reads in your sequencing, that would mean that yeast
doesn’t grow very well, and if you had a lot of reads
from particular barcode, that would mean that a yeast grows better. It’s really a proxy for growth. Now we’re trying to adapt
that technology to our screen, and we’re doing that as we speak. Hopefully, we’ll be able to scale up in a way that
does not require this robot, and does not require being on
your feet for 10 hours a day, and is also probably equally
sensitive and as powerful. – [Dr. Watson] I think
building upon the yeast screen and this format, what’s
the next step, right? One of the things I just told you about is that we’re missing … This is not a mammalian cell, right? We’re missing pathways evolve
specifically mammalian cells, we’re missing innate immune pathways and those kinds of things. Actually, beyond that, we’re
actually thinking about now doing a screen in human cells. One of my best friends,
Cole Dovey, actually, he’s at Standford. He’s a post doc at Stanford. I should say Dr. Cole. (mumbles) He’s in Dr. Ca-ret’s lab. What Dr. Caret has actually,
the reason he’s in business is he actually was one of
the first people to use these cells called HAP1s;
they’re actually haploid. They only have half a copy
of genetic information. We all have two copies. The reason why yeast is so powerful and it’s easy to make a
knock-out, a deletion in one gene, is because they’re only haploid also. These mammalian cells
only have a half a copy, and therefore only one copy of each gene. It’s really easy to use
this CRISPR-Cas9 technology that everybody’s talking about, to knock out individual genes. In the same way that we’re going to do population based yeast,
we’re actually going to use yeast HAP1 cells, and do a very similar type
screen in these cells, using CRISPR-Cas9 and
expressing effector proteins, and looking at seeing which mutants really stick upon expression
of these effectors, or which ones are more resistant, which mutants are more resistant. Beyond that, I think we want to take, and that’s the next step. Beyond that, we actually
really want to use, we use macrophages, TB. Tuberculosis actually infects macrophages. They’re an innate immune cell. We’re going to actually
develop this screen probably in macrophage. That’s kind of not imminent, but I think that’s what we’re shooting for because that’s really the all … Everything we want is in that cell. We can screen any kind of
protein that we want, basically, in a macrophage.
– Yeah. We’re really just developing multiple genetic screening platforms
to manipulate a host, whether it be a yeast,
or a haploid human cell, or a macrophage. Say, if we get rid of certain
genes, how does the cell respond to expression
of an effector protein? You can do this high throughput, in a way where we can look at almost all the genes in the cell. We call it a hypothesis
generating experiment. It’s not going to tell us
exactly mechanistically, what each of these effectors do, but you will literally go from, this is just an amino acid sequence, I know nothing about this protein, to now saying, “Oh look, it looks like a lot
of these mutants coming up, “have to do with endocytic trafficking, “or have to do with chromative remodeling, “or have to do with pre MRNA splicing.” Then as an investigator, you
can follow up on those things. We think it would be a really good resource for the community at large, and we’re looking for some outside of A&M collaborations as well, to bring in other bacteria. It’s also just a really
neat way for a pathogen that we don’t know much about. There’s a bunch of emerging
bacterial pathogens that are really poorly studied, that we can start to identify
how they target out cells using these completely
unbiased approaches. – [Christine] Well, we’ll
look forward to following up with you guys as you continue
on these experiments. Thank you so much to both of
you for being with us today here on Science Sound Off. – [Dr. Patrick] Thank you; it was fun. – [Dr. Wilson] Thank you for
having us; it was really fun. – [Christine] Thank you for joining us, and we’ll see you next time. (lively music) – [Narrator] Thank you for joining us on Texas A&M Health Talk, a
production of the Texas A&M University Health Science Center. Visit us on the web at
vitalrecord.tamhsc.edu, where you’ll find answers to
all of your health questions. Until next time, stay healthy. (lively music)

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