Hi. I'm Joseph DeRisi and I'm an associate professor in the department of biochemistry and biophysics.
And I'm a Howard Hughes investigator at the University of California in San Francisco.
And it is my privilege today to tell you about our research
on the parasites that cause the human disease malaria.
So, our research goals are very simple.
If you saw my previous segment on the background and overview of malaria the disease,
you know what a devastating condition this is for humanity.
So it is our goal to develop strategies that are affordable and sustainable
for the treatment of the disease malaria.
And we are going to go about this in two different ways.
First of all, through a basic science approach using the power of genomics
to understand more about the basic molecular physiology of the parasite Plasmodium falciparum.
The causative agent of the most deadly form of human malaria.
Second of all, a more near term approach is to take a drug discovery pathway,
To find new anti-malarial chemotherapeutics for the treatment of this disease.
So, let me first review the malaria life cycle.
Shown here is the a cartoon simplification of the life cycle of Plasmodium falciparum.
It begins with the bite of a mosquito.
The anopheles mosquito
that releases sporozoites into the bloodstream.
These travel to the liver, whereupon they invade hepatocytes.
It is here that they will undergo multiple rounds of replication, and then burst forth from the liver,
in a form called merozoites.
It is this form that will then infect red blood cells in a 48-hour,
asexual cycle that will repeat itself, essentially indefinitely.
This is where all the clinical manifestations of the disease malaria occur.
Now, with some infrequent rate, some parasites will develop into a sexual form called gametocytes.
And it is these gametocytes that can be picked up by a new mosquito,
taking a blood meal. And the entire process begins again.
And so, traditionally, researchers have focused on the asexual cycle of the parasite,
because this is where all the clinical manifestations occur.
And where drugs have been developed in the past to fight this disease in patients.
The genome of Plasmodium falciparum was completely sequenced in 2002
by Sanger, Tiger, and the Stanford Genome Centers.
It provides much of the foundation that my lab uses to research this parasite.
It was found, when it was completely sequenced,
it was to contain 14 linear chromosomes covering 22.8 megabases of DNA.
This genome codes for approximately 5,400 genes.
So in comparison, this is roughly the same size genome in number of genes
as the model organism yeast, Saccharomyces cerevisiae
Of which we know so much about.
This is in contrast to Plasmodium falciparum, which we know very little.
The genome was also found to contain a 35 Kb circular plastid genome,
and a 6 Kb linear mitochondrial genome. Now, one of the most
remarkable features of this genome is its very high percentage of AT relative to GC.
And a very high coding density.
Also remarkable in this genome,
is the fact that 60% of all the genes found in the genome have essentially no known function.
This is an extraordinary high percentage.
We are sequencing more and more genomes every year.
And with that comes more and more knowledge about every gene.
Yet in Plasmodium falciparum, the vast dominant portion of the genes,
still have no known function.
Now, to understand the work that we are doing,
I'd like to tell you about the asexual blood stage.
And this is where all the clinical manifestations of the disease occurs.
It begins of the release of merozoites into the blood stream,
which then infect red blood cells.
This is the so-called ring stage of the parasite.
After the ring stage, follows a metabolic stage of the parasite called
the trophozoite phase.
This is then followed by multiple rounds of replication, and the so-called schizont phase.
After the schizont phase,
the erythrocyte ruptures, releasing merozoites,
and this 48-hour cycle repeats itself, essentially indefinitely.
And so what we really wanted to know is,
what genes are expressed during the asexual blood stage,
and when.
Why?
Because the genes that are expressed could provide clues
as to the biochemical mechanisms that are occurring during this phase.
And may highlight the pathways or targets
that can be used for anti-malarial chemotherapeutic intervention.
And so, to know more about what genes are used,
and when throughout the asexual blood phase of the parasite,
we decided to take a DNA microarray approach.
That is, we want to make a DNA glass chip on which we print
all the genes of Plasmodium falciparum,
for the purpose of measuring the expression level of all genes in the genome simultaneously.
So, a DNA-microarray is nothing but a glass slide,
on to which is printed DNA from different genes,
so each spot shown on this array here, is a different gene from the genome.
This entire chip measures only 18 millimeters across in diameter.
So we can use this as a hybridization target.
Now, if you had a very powerful microscope, like an atomic force microscope,
and zoomed in on one of these spots,
this is what you would see.
What we would see here, in this ASM microscope,
taken courtesy of Hewlett-Packard,
in the late 1990s,
is all the strands of the DNA of this microarray laying flat on the glass in a monolayer.
So this provides an excellent hybridization target,
for subsequent assays like gene expression monitoring.
Now, how are these chips made, and how do we go about this process?
So, what we did is we built machines in the lab
that can make these so-called DNA-microarrays,
these microarray printers are easily constructed,
and can be built from simple directions.
And so if you are so inclined, I invite you to go visit our webpage at
derisilab.ucsf.edu/core
and download the directions for building your own DNA-microarray printer.
This would enable labs to print essentially unlimited chips
on their own time in their own labs,
without having to rely on commercial sources for such reagents.
So how does a typical microarray experiment actually occur?
So, with respect to malaria, an experiment might go down something like this.
You start with two different phases of the blood phase
So here, this is an early trophozoite, and this is a late schizont.
You can isolate parasites from each of these phases,
by growing them in a synchronous fusion.
You can then isolate their RNA, which is the manifestation of their DNA,
the gene expression that occurs,
converted to cDNA using reverse-transcriptase.
And then labeled, the subsequent cDNA, with fluorescent dyes.
You can label one in red, and one in green.
And then we can co-hybridize that to the array.
And so, what we see is the relative abundance level of each gene expressed
as a ratio of colors on each spot, between the red and the green.
And this is how a typical microarray expression works.
But, there is one complication:
I don't simply want to measure the difference between a trophozoite and a schizont.
What I'd like to do is measure the gene expression levels of every gene in the genome,
every hour, throughout the 48-hour life-cycle,
to capture the dynamic nature of the transcriptional network as it plays out in this parasite.
And so, what we decided to do is isolate RNA from each stage,
every single hour, for two days essentially.
And to do this, we had to grow a very large culture of parasites.
So we utilized a 5-liter bioreactor system
that we could fill with human blood harvested from volunteers,
infect with parasites,
and then keep in very controlled conditions:
temperature, pH, gas, agitation, and so on.
And so this synchronous culture, growing as shown here,
we were able to take out samples on the hour, every hour.
And this shows the relative synchrony of the parasites as they are growing in this fashion.
And so, what did we see when we did this experiment?
We pulled out RNA every hour, on the hour,
we hybridized it to our DNA microarrays
that represents the whole Plasmodium falciparum genome,
and we took a look at some genes.
So, here is an example gene.
This is a gene called erythrocyte binding antigen 175
, or EBA175, a very well characterized gene in Plasmodium falciparum.
And what we can see is, early in the cycle, there is very high expression,
left over from the late schizonts as they are releasing merozoites,
and then expression just continues to crash out,
and it reaches a low point during the trophozoite phase,
and then immediately rockets up and reaches its highest point,
during the point of invasion.
When merozoites are again being made and released into the blood stream.
Now Peak to trough, this gene exhibits a 256-fold regulation.
And this is typical of invasion type genes.
Now what we also notice, is if we looked at other genes in the Plasmodium falciparum genome,
and looked at their expression profile,
we noted that they almost all have one trough, and one peak.
That is, most genes in the genome that are expressed at decently high levels,
throughout the asexual lifecycle, exhibit periodic expression.
They are induced and repressed approximately only once per cycle.
So, that enabled us to use some new tools to analyze this expression.
What we did, is we took all the expression profiles,
and did a fast Fourier transform
to understand how periodic these signals are from each gene.
And what this histogram with this power specter is showing you
is that most genes are very periodic.
If we randomized the data and recalculated the fast Fourier transform with permuted profiles,
we get a distribution that is like this, which is very different from this.
So this indicates, that by and large, most genes in the genome have a wave-like expression profile
throughout the asexual blood cycle.
So, if we then extract the phase of expression from the fast fourier transform
of all these gene expression profiles, and plot the expression by phase,
we end up with something like this.
So shown here, are all of the genes that have measurable expression levels
throughout the asexual blood cycle on the Y axis,
approximately 2,700 genes. And across the X axis,
are all the time points from 0-48 hours.
And what you can see here, by this red stripe that proceeds down this way,
is a very smooth cascade of expression.
That is, the genes are being induced once and only once per cycle,
in a clean cascade that starts with the beginning of the ring stage,
ends with the schizont stage, and then wraps around to the beginning.
So, what we observed in this very smooth cascade of gene transcription during the asexual
cell cycle of the Plasmodium falciparum parasite,
was a certain metabolic logic to the genes induced and when they were induced.
So if we take a closer look at this logic,
we can basically understand what the parasite is doing during this time.
So, beginning with invasion,
we see genes immediately induced that have to do with both building ribosomes,
translation machinery, and transcription complexes.
This is intuitive because these are the building blocks of all eucaryotic cells,
and are required to make more of the same.
Now following that induction of these genes,
what we see are genes involved in both glycolysis,
the main energy production system of the cell,
and ribonucleotide synthesis.
That is, making more substrates to make more RNA, the manifestation of genes themselves.
This is then followed with some phase delay,
by genes involved in making deoxyribonucleotides. The building blocks of DNA.
And not surprisingly, we see that this is directly in phase
with genes involved in DNA replication.
Now, I wonder if you can basically see this here,
but it appears as though the genes and when they are induced, resemble a just in time production process.
That things are made when they're needed, in the amount they are needed, and then they are shut off.
This is how many commercial car factories work.
Now, one of the problems with this, is a supply of a certain part doesn't get there in time,
or just in time. And this can cause, to, errors during the system,
or collapse of the whole system.
After this point in the cascade, we see genes induced that have to do with the TCA cycle,
and the proteosome, and then quickly following that,
we find that genes are induced that have functions related to plastid,
and merozoite formation.
So what we can see here is the beginning of the process of building new parasites
for reinvasion of new cells.
This concludes with, genes explicitly involved in the invasion phase.
Actin-myosin motility genes,
are known to be involved in the process of physically invading new erythrocytes.
And we see these in genes induced right at the end of the cycle.
And this finally concludes with early ring transcripts,
that is beginning the whole cycle all over again, with newly infected erythrocytes.
And so what I hope I've shown you here, is that Plasmodium falciparum gene expression,
appears as a single cascade that proceeds throughout the 48 hours,
and represents a certain metabolic logic,
reminiscent of a just-in-time production facility.
Now, was this just a fluke?
Is this just the way Plasmodium falciparum looks like in the lab, in this one particular strain?
We sought to replicate this experiment
two more time with different strains of Plasmodium falciparum, that come from different parts of the world.
And shown here, are all three side by side.
And all though there are small differences in the synchrony of the parasites,
What I think that you can see quite clearly here is that,
for all practical purposes, all three transcriptomes are nearly identical.
That is, it appears as if Plasmodium, from different parts of the world,
express exactly the same transcriptome cascade as their relatives in other parts of the world.
And so, what this leaves us with, is many outstanding questions.
Like all very interesting discovery based science,
more questions are generated than answered.
And this leads us to the next slide, which will discuss these.
To what extent is the transcriptome, that we observed, rigid?
How do we know that there are response genes,
genes that can change this transcriptome, in relation to environmental perturbations?
It's been found by others in the field,
that very few transcription factors are coded for in the genome.
At least apparent transcription factors that
are recognizable using common bionformatic techniques.
So could it be that Plasmodium falciparum possesses a very streamlined,
stripped-down, transcriptional complement?
It's possible.
What are the cis-acting determinants of the expression phase?
It is known that the cis-acting region upstream of genes determine their phase of expression.
But we don't know what are the elements inside
these promoter regions that are responsible for driving phase of expression.
And, a very related question,
what are the transcription factors that bind those putative cis-acting elements?
And what are their roles in each of the phase-specific expression profiles?
To date, very little work has been done on transcription factor biology in Plasmodium falciparum.
Yet, it is quite clear that if one can design an anti-malarial chemotherapeutic
to disrupt the regulatory process, because the transcriptome appears to be rather rigid
in its style of expression. This could potentially result in catastrophe for the parasite.
That is, perhaps this apparent rigid transcriptome is the Achilles heal of Plasmodium falciparum.
And anything that we do that would misregulte, or cause a perturbation,
to the regulatory cascade as we observe it, may kill the parasite easily.
And that brings us to our final question:
Can we disrupt this transcriptome?
Now, if you are interested in the data that I've told you about today,
and you'd like to browse, either the whole genome, or gene by gene,
all the data which I've showed is available on our website at malaria.ucsf.edu.
And I encourage you to log on, pick out your favorite genes,
or just browse the genome for functions and expression profiles.
And try to understand basically what the parasite is doing.
Many, many projects can emanate from such data, and I encourage you to take a look.
Thank you.