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Hi, my name is Norma Andrews.
I'm a professor at Yale University, and what I'm going to do in this third part
of my lecture is to talk to you about insights that we have obtained in my lab
on the strategies used by both Trypanosoma cruzi and Leishmania to invade host cells
and then to survive inside the compartments generated inside these host cells.
Trypanosoma cruzi, as already detailed in the first part of this lecture,
is the causative agent of Chagas' disease,
a serious endemic disease in South and Central America
which is shown here in this infected child. And this is what is seen
in the blood of these patients during the acute phase of the disease
is this large number of these very highly motile parasites circulating in the blood.
This infective stage is the form that is deposited by the insect after the blood meal,
so it is contamination of the wound created by the blood meal
that transmits the parasite through the feces of the insect,
and this is the way they gain access to the mammalian host.
What is really the main characteristic of Trypanosoma cruzi is that
it is capable of invading and replicating inside a large number of different cell types.
This is the mechanism that we have studied for several years in my laboratory.
Our insights into this mechanism started with these scanning electron micrographs
taken by Edith Robbins at NYU.
What is shown here is this infective stage, trypomastigote
attached to a host cell. And what happens after this,
is that these parasites initiate this very dramatic process of entering the host cell.
You can see that these are large parasites, and what is surprising...
what surprised us in these first observations is this entry mechanism
was very different from phagocytosis.
And our assumption at the time was that these parasites should enter by phagocytosis
because they are so large, so they would, in principle, need actin polymerization
on the side of the host cell to really expand the membrane
to be able to accommodate a particle so large.
But these images were suggesting that something quite different was going on.
Just to illustrate here, this is what is seen in cells
when they are undergoing phagocytosis.
These are images of large particles being taken up by macrophages,
showing how you see this profusion of pseudopods
and this extension of membrane that gradually envelops the particle.
What happens with Trypanosoma cruzi entry is something quite different.
We were sure that this was the case when we just did these simple experiments
of staining infected cells. So, these cells were just exposed
to the parasites for very brief periods of time of just 10 minutes,
and then these parasites that are inside the cell...
The parasites had been stained with antibodies against surface proteins
(which is shown in red) and then the cell is stained with phalloidin,
in green, which is a protein that binds to polymerized actin.
And, you can see here how different it is,
the patterns seen with these parasites inside host cells
and what is seen in macrophages when they take up for example yeast particles,
in which you see this classical ring of actin around the particle,
and we don't see it around parasites that have successfully entered host cells.
Although the microfilaments of the cells are still present,
they don't seem to be altered significantly by this invasion process.
Drugs that disrupt the actin cytoskeleton
also had no inhibitory effect on Trypanosoma cruzi entry.
But, to our surprise, this invasion process was highly sensitive
to drugs that disrupt microtubules,
which is the second important cytoskeletal element in cells,
which actually are elements of the cytoskeleton
that provide the tracks where organelles move.
This observation and this second important observation that markers for lysosomes
(which is shown here in this immunofluorescence)
is that when we stain cells also exposed for brief periods of time
to infective stages of Trypanosoma cruzi,
we can see that these markers for lysosomes
(in this case, antibodies against Lamp-1, a major glycoprotein of lysosomes)
can be seen in this very tight vacuole that forms
around the parasites as they enter the cells.
This also looked very different from the classical maturation of phagosomes
that was known to take several minutes.
Lysosomes were known to meet particles after they were internalized,
and in this case, here, this appeared to be happening much faster
during the invasion process.
What was important about this observation is that we know
that lysosomes move on microtubules.
So these two findings that drugs that disrupt microtubules
inhibit Trypanosoma cruzi entry,
and the fact that you see markers for lysosomes
associated with the vacuole surrounding these parasites,
really indicated to us that probably the parasite had very unique mechanisms
to really recruit these organelles early in the invasion process.
This is what is shown in these images here. On the top is again immunofluorescence
in which antibodies against the parasite were added to these cells
before they were permeabilized.
Only the parasites outside the cell got stained in red.
Then these cells were permeabilized with detergent
and we added antibodies against lysosomal proteins,
so we can see here that the second parasite inside the cell
is actually stained by lysosomal markers
indicating that it's already inside the cell.
The important finding was that we see this dramatic pattern
of tight association of lysosomes,
which are inside the cell, just immediately underneath this parasite
attached to the surface,
indicating that there was a localized signaling process
recruiting these lysosomes exactly to the site of the invasion.
And this is illustrated perhaps better here in this transmission electron microscopy,
in which the lysosomes were loaded with horseradish peroxidase,
so this allows us to do this cytochemistry reaction and visualize these lysosomes,
and you can see that the parasite is still completely outside the cell,
but these lysosomes were recruited to the site of the invasion.
The next step in this process is that you can see images like this,
in which this part of the parasite is still out of the cell,
but the portion that is inside is surrounded by markers of lysosomes.
And the same thing is seen here.
This is a section through a parasite in the process of entering cells.
It's actually hard to distinguish the vacuole because this is a very tight vacuole,
but there are two membranes here, and this material that is coming from lysosomes
is actually delivered to this very tight vacuole
that forms around the parasite as it enters cells.
In this movie here, I'm just going to show you,
this is one fibroblast, in which the outline
of the cell is around here, and there are parasites attached.
And what was done here, is that this cell was preloaded
with albumin complexed to colloidal
gold so the lysosomes in this movie appear as these small black dots.
And when we play this movie, we can see that actually, it's possible
to visualize microtubules as these elevated regions
and it is possible to see lysosomes actually moving in these very large steps
towards the site where this parasite is going to start to invade the cell.
So then, analyzing these movies...
taking the position of each lysosome in each time frame,
it is possible to construct these diagrams in which it becomes very clear
that just in regions slightly removed from the invasion site,
microtubules are showing
the known behavior which is this saltatory movement which is bidirectional.
But, when it comes close to the site where an invasion event is going to happen,
there are these dramatic directional steps
which can be seen perhaps easier here with these arrows
indicating that this directional movement is happening
in this area of influence of the parasite,
but before invasion starts.
This is really what led us to then study in detail the signaling process that was involved
in this process. And what we learned is that the parasites produce an agonist
that interacts with receptors in the host cell, activates phospholipase C
and the production of IP3, which is a mediator
for the release of calcium from intracellular stores.
There is an elevation in intracellular calcium,
which is what this movie up here is showing.
This cell was loaded with a calcium-sensitive dye and exposed to the parasites.
And we can see how these cells continuously flash
as they are being stimulated by the parasite,
showing transient elevations in the cytosolic, free calcium.
We showed in a series of experiments that this calcium elevation is necessary
for this recruitment of lysosomes to the site of parasite invasion.
What we learned through this process
(this is a very good example of the unexpected findings
that can happen) By studying this exotic parasite from South America,
we actually understood something quite basic
about the behavior of conventional lysosomes in mammalian cells.
When we decided to take the trypanosomes out of the picture
and just look at the effect of elevations in cytosolic calcium,
we learned that actually many different cell types...
Lysosomes, which were originally viewed,
(or is still very consistently viewed)
as terminal compartments of the endocytic pathway...
Actually, these organelles can be transformed
into secretory and regulated secretory organelles,
just by elevating the cytosolic calcium concentration.
And I'm not going to get into this in this lecture, but we have learned, in my lab,
that this process is very important in the physiology of mammalian cells.
For, for example, the repair of mechanical wounds on the plasma membrane,
and also for phagocytosis, as a mechanism
for adding more membrane at the sites of phagocytosis.
What I'm going to show you,
in collaboration with Sandy Simon at Rockefeller University,
is how we have been able, recently, to directly visualize
this process of lysosomal exocytosis.
For this, we use this powerful technique of total internal reflection microscopy.
Which, what is indicated here, in this technique, the cells are illuminated
from the bottom with a laser beam, and the laser is directed to the cells at an angle
in which most of the light is reflected away,
so the small amount of light that penetrates
these cells does so at a very narrow field which is called the evanescent field.
And it's called the evanescent field
because it has this very interesting property of decaying
exponentially away from the coverslip where the cells are lying.
If you just add a fluorescent marker to your vesicles of interest,
it is possible to visualize these vesicles becoming brighter and brighter
as they travel through this evanescent field
and then the moment of fusion is quite obvious
because there is high intensity fluorescence and then diffusion of the label.
In these studies, Jyoti Jaiswal and Sandy Simon observed
something quite interesting that we didn't know when we started these studies.
Lysosomes were known to concentrate in cells in the perinuclear area,
which is shown here is this classical epifluoresence microscopy.
And what this technique of TIRF microscopy showed is that,
in addition to this perinuclear population,
there is quite a significant number of lysosomes
that can be seen in this very membrane-proximal region,
which is just 80 or 100 nanometers close to the plasma membrane.
This was an important observation because when these fusion events were analyzed...
I'm going to show you one example, here, in which this is an embryonic fibroblast.
Again, like in the previous pictures, the lysosomes were loaded
by chasing a fluorescent molecule, dextran,
throughout the endocytic pathway, and then accumulating in lysosomes.
And this movie is going to show, when it starts playing, now
this cell being stimulated by a calcium ionophore.
And we're going to see these puffs of fluorescence,
which reflect the luminal dextran being released.
I'd like you to focus on this lysosome here that is getting brighter and brighter,
and we're going to see the moment in which this
lysosome fuses with the plasma membrane,
releasing its contents.
When Jyoti Jaiswal and Sandy Simon analyzed these events,
what became clear is that, in cells either stimulated with a calcium ionophore
or these two more physiological agonists, thrombin and bombesin,
the large majority of the lysosomes that were seen fusing with the plasma membrane
were originating from this membrane-proximal population.
Very few, shown here in red, were recruited from deeper regions of the cell,
meaning that they were first invisible and they entered the illuminated field.
So, this gave us a very important piece of information
that this peripheral population of lysosomes
(that had really not been described before) actually is functional and is actually
responsible for the majority of these fusion events that are modulated by calcium.
This got us interested in the molecular machinery controlling lysosomal exocytosis,
and shown here in this diagram from a review of Ed Sheckman,
it is quite understood now
because of the work of Ed Sheckman and several others that this class of molecules,
the synaptotagmins, are very important in coupling calcium
to membrane fusion events.
So what is indicated here are the SNARE molecules, in which you have
the v-SNAREs on the membranes of vesicles
and the t-SNAREs on the acceptor membranes.
And these molecules form these tight, coiled-coil bundles,
which are believed to be responsible for promoting membrane fusion.
This process can be made sensitive to calcium when synaptotagmin molecules interact
with the SNARE proteins after binding calcium,
and they bind calcium through these two domains
that they have in their cytoplasmic region,
which are the C2 domains.
These calcium sensor molecules associated with membranes
were very interesting candidates
for also mediating lysosomal exocytosis, which
we learned was a process clearly controlled by elevations of calcium in the cytosol.
We got interested in one specific isoform, synaptotagmin VII
because it is ubiquitously expressed,
it is evolutionarily conserved (so it is one of the isoforms conserved in Drosophila
and other lower organisms, including C. elegans)
and it has a very wide distribution, which could be consistent
with a lysosomal localization.
This was confirmed in several studies in our lab.
I'm just showing here one example here of immunofluorescence,
in which antibodies specific to synaptotagmin VII in red
were used to stain a cell that is also infected with Trypanosoma cruzi.
And we can see that in addition to the lysosomes, that over here, (stained in green
with antibodies against Lamp-1) we can see a very good overlap,
which is shown in the yellow image down here,
showing that the synaptotagmin VII localizes to lysosomes in mammalian cells and is
also delivered to the vacuole surrounding Trypanosoma cruzi right after invasion
just like other lysosomal markers. And then we went on to show that
deficiency in this molecule, synaptotagmin VII can inhibit not only lysosomal exocytosis
induced by calcium, but also invasion of these cells by Trypanosoma cruzi.
Additional work allowed us to identify the partners of this fusion reaction:
the SNARE molecules (in which VAMP7 present on lysosomes
interacts with syntaxin 4) and SNAP-23.
And synaptotagmin VII associates with these molecules in a calcium dependent manner,
which led us to propose that it is also similar
to what has been shown in synaptic vesicles
in neuronal cells that this would be how this process becomes calcium regulated.
These lysosomal fusion events actually have proven
to be critical not only for Trypanosoma cruzi
invasion, but they're interestingly in more recent work from the lab.
We understood that this event is very important for retaining
these highly motile parasites inside host cells.
So, after Trypanosomes invade, as I already showed you,
there is during the invasion process,
there is this gradual fusion of lysosomes,
and we know that lysosomes are associated with
these microtubules tracks through the molecular motors.
And we think that this process is very important for keeping
these parasites inside the host cell
for completion of the cycle.
Part of the evidence for this is just indicated in this diagram here,
that when we prevent lysosomal fusion with different procedures,
these parasites can still enter cells by invaginating the plasma membrane,
most probably propelled by their very active motility.
But very interestingly, this entry can be reversible, so if there is no lysosomal fusion
and there's no tethering to the microtubule network,
these parasites actually can exit the cell,
and this invasion process actually can be reversible.
So this is an interesting concept that I believe will prove
to be true for other motile pathogens,
in which they have not only to be able to invade cells
and to create an intracellular compartment,
but they have to be able to be retained inside host cells.
What this image here is showing, is a summary of what we learned.
The signaling process induced by trypanosomes in host cells
triggers this calcium elevation in the cytosol,
which drives this fusion of lysosomes at the site of invasion
This provides the parasites with membrane of host cell origin,
in which they form this initial compartment.
In the case of Trypanosoma cruzi, they escape from this compartment very soon,
a few hours after invasion in some cell types,
and complete the process in the cytoplasm,
as I already discussed in the first part of this lecture.
We were interested in, in addition to this entry process,
in understanding how the parasites survive in these lysosome like compartments
which are so degradative and, in principle, should be promoting destruction
and not survival of these pathogens.
For this question, we turned to Leishmania
which I also already introduced in the second segment of this lecture,
which is a parasite transmitted... it's closely related to Trypanosoma cruzi.
It is transmitted by sand flies, and when these infective stages
are introduced into the mammalian host,
they invade macrophages and they replicate inside these lysosome-like compartments.
This is here showing a picture of a patient with a cutaneous form of the disease
which is a consequence of the inflammatory process
that develops when these parasites are replicating inside macrophages.
So, what is interesting about Leishmania is that they are adapted, (as shown here,
examples of several species of Leishmania) in which in all of them,
the vacuole that is formed around this parasite
and where they replicate really has markers of lysosomes.
There are many studies in which this was shown,
mostly from Jean-Claude Antoine in the Pasteur Institute in Paris,
to be lysosomal compartments, so the question became
how do they survive inside these degradative compartments.
So, this leads us to a very interesting pathway in mammalian cells,
which is the mechanism by which cells acquire iron.
Mammalian cells have receptors for transferrin,
which is the carrier protein for the oxidized form of iron, Fe3+.
This is very understood that the receptors for transferrin are endocytosed
and inside these early endosomes, when the pH drops,
the iron is released from transferrin
and then transferrin is recycled back out of the cell,
and the iron that is released gets translocated into the cytosol.
The way this happens is by reduction into Fe2+ by reductases inside the endosome,
and then there are specific transporters,
which in the case of early endosomes, this transporter is known as Nramp2.
And it is responsible for translocation of a large fraction
of the iron trafficking through this pathway into the cytoplasm,
where it becomes accessible to the metabolic processes of the mammalian cell.
What was a subject of discussion for a long time, but is recently becoming more clear
is that another transporter, Nramp1, works in a similar manner as Nramp2,
but it is deeper in the endocytic pathway, so it is found mostly in lysosomes.
What is very interesting about Nramp1 is that it is widely known
as a susceptibility gene for infectious diseases.
So, for quite some time, it has been clear that mutations in Nramp1
promote susceptibility to several pathogens
like Leishmania, salmonella, and mycobacteria,
which replicate inside the endocytic pathway.
This creates some very interesting questions and also illustrates the fact
that this mechanism of gradually depleting the endocytic pathway in iron
is a mechanism of resistance against pathogens
because this severely limits the access of these intracellular organisms to this key
metal that is needed for many important enzymatic reactions.
What we decided to do is that, to understand how Leishmania acquires iron
in these compartments, since it was clear that this is the type of compartment
where they replicate and unlike bacteria,
nothing was known about how Leishmania could acquire iron intracellularly.
In my lab, this work was done by a very talented investigator, Chau Huynh
who asked this question: How does Leishmania acquire iron
in these very specialized lysosome-like compartments
of which an example is shown here, these highly expanded compartments
that are typical of Leishmania amazonensis.
What Chau found, just doing homology searches in the recently completed Leishmania
genome, he found a protein which he called LIT1 for Leishmania Iron Transporter 1
that showed significant identity of 30% and 54% similarity
with a known iron transporter from Arabidopsis.
This is the structure of this transporter. It has 8 transmembrane domains,
and these residues which have been shown to be critical
in iron transport are highly conserved
in the Leishmania protein.
Chau postulated that this would be a member of this ZIP family of metal transporters,
and what he did initially was to produce antibodies
specific against this Leishmania protein.
And interestingly, he could not detect this protein by immunofluorescence
on parasites that had recently invaded cells.
He could not see it either in the extracellular stages of Leishmania.
So what is shown here in blue is just the parasite...
the DNA of the host cell and of the parasites,
and we can see that there is no staining with the antibody which is shown in green.
But if he just waited 24 hours... So this parasite still has not started replicating,
but you can see that the compartment has greatly expanded,
and they are going to start to replicate soon.
At this stage, there is clearly a signal with the antibodies against this LIT1 protein,
and in this enlarged portion here, we can see that the pattern is what we would expect.
It is distributed around the surface of these intracellular parasites.
What Chau went on to do is to really obtain evidence
that this could function as an iron transporter.
He first did experiments in which he introduced this LIT1 gene from Leishmania
into strains of yeast that are defective in iron transport.
What is shown here is that if you grow these strains in iron-rich media,
they can still grow,
although they lack two main pathways of iron transport.
But, when iron is chelated in the medium, they cannot grow.
But, just introducing the LIT1 gene allows us to rescue the growth of these strains,
really providing strong evidence that LIT1 can function as an iron transporter.
What is shown down here is the direct demonstration that LIT1
functions as an iron transporter in Leishmania.
So, Chau created a null mutant (a knockout mutant) lacking LIT1
and compared it with parasites that had been complemented with this gene.
This curve up here is uptake of radioactive iron by these complemented parasites,
and you can see that it's very different from the very low levels
observed in these knockout forms.
This knockout was constructed by this standard technique in the field
of homologous recombination.
LIT1 is actually encoded by two genes, LIT1A and LIT1B,
but these genes are close enough that they can be eliminated
(deleted) in one step of homologous recombination
by replacement with a selectable marker
and in Leishmania this only requires that it's done in two steps
with two different selectable markers, because these are diploid organisms.
Once these parasites are generated, Chau could show here,
again by immunofluorescence,
that this protein is detected on the surface of the parasites only in the wildtype strain,
but it is not present in the mutant.
What was interesting was the behavior of these forms inside macrophages.
This here shows how Leishmania amazonensis grows inside macrophages,
forming these extremely enlarged compartments
that contain the lysosomal protein, Lamp-1,
which is shown in green.
Over here in blue is DNA staining showing the parasites replicating
associated with the membrane of these vacuoles.
When the same timeframe was analyzed in cells
infected with these parasites lacking the LIT1 protein,
we could see that the initial stages were very similar.
The parasites actually can infect the cells, they can expand initially the vacuole,
but then they don't divide.
This is indicated here by the DAPI staining showing that the number of parasites
remains similar throughout several hours that this infection was followed.
This is the quantification of the process,
really showing that we see wild-type parasites increasing in numbers,
as they replicate inside the host cells.
And in black, how these knockout parasites do not grow,
but if the LIT1 gene is reintroduced into this knockout strain, growth is rescued.
This was the formal demonstration that actually LIT1 is an essential requirement
for intracellular growth in macrophages,
really linking this pathway of iron acquisition as a critical requirement for Leishmania
to actually make it in this iron-poor, deprived compartment
of the late endosomes/lysosomes.
With the help of David Sacks at the NIH, we also analyzed the capacity
of these knockout parasites to cause pathology in mice,
and this cutaneous form of Leismaniasis can be reproduced in mice
by injecting the parasites into the footpad.
This has a very clear pattern when the animals are injected with wildtype organisms
that a lesion develops in the footpad.
Consistent with what was observed in the macrophages in culture,
these parasites did not form lesions in the mice
even after being injected in large numbers
and being followed for several months in the animals.
The only observation was that, despite the complete absence of pathology,
these mutant parasites lacking the LIT1 protein could be recovered
from the tissues of these mice, indicating persistence.
This is a very important and interesting point which I would say
is a very critical question for future research,
which is How does Leishmania survive and persist in host tissues
even after effective immune responses are developed?
It is known in physiological conditions, when small numbers of parasites are injected
that the lesions usually heal but the parasites persist,
and it is not known in what cell type
these parasites are persisting and how they survive and prevent elimination
when their main host cell (at least the major host cell that is well known)
is the macrophage.
This is the point that I hope that we're going to get to understand better in the future
when we start to go deeper into what are the requirements for Leishmania survival
in the mammalian host.
I would like to acknowledge a large number of very talented people
(post-docs and students)
that over the years have contributed to the work that I mentioned here,
and also I would like to acknowledge important collaborators in this work
and also the sources of funding which were mostly from the NIH,
the Burroughs Wellcome Fund, and the Human Frontiers Science Program.
Thank you.