I am Pascale Cossart from the Pasteur Institute in Paris,
and I am also
a Senior International Research Scholar
for the Howard Hughes Medical Institute.
What I would like to do is to share with you
how we study the bacterial pathogen Listeria Monocytogenes,
to address key issues in infection biology,
but also in fundamental microbiology and in cell biology.
So, my talk will be divided into three parts.
In the first park, I will be rather general
and focus on bacterial pathogenesis,
giving you an overview of the Listeria paradigm.
In the second part, I will show you
how the study of Listeria monocytogenes
has really provided
key new concepts in fundamental microbiology,
and really participated in the renaissance of this discipline.
In the third part, I will focus on the cell biology
of the infectious process
and show you how the study of this infectious process
has unveiled new components, new mechanisms,
which are taking place in the mammalian cells.
So, what is microbiology?
Microbiology is the discipline which addresses
organisms which are not visible with the eye.
So, these organisms are about 50-fold smaller
than mammalian cells.
They are the size of organelles
which are in mammalian cells, like mitochondria.
So, bacteria are very diverse.
Bacteria are very, very rarely pathogenic.
There are many bacteria in the environment,
like Streptomyces,
which is producing the antibiotic streptomycin.
There are bacteria which are in the environment,
but live in symbiosis with, for example,
the roots of the vegetals,
like Sinorhizobium meliloti, which help the bacteria to capture the nitrogen.
There are bacteria which are commensals,
and you all know that we have bacteria in our intestine.
And, finally, there are those which are the pathogenic bacteria,
like Shigella flexneri
or the bacterium that I am going to talk about today,
which is Listeria monocytogenes.
So, pathogenic bacteria can be classified in several categories.
I think there are two main categories of pathogenic bacteria:
those which are acting by producing a single, potent toxin,
like Corynebacterium, which produces diphtheria toxin,
or Clostridium botulinum, which produces botulinum toxin;
but very often pathogenic bacteria
are using multiple factors,
like adhesion, colonization, tissues destruction.
So, all of the bacteria that I'm showing you, here,
so, Pseudomonas, enteropathogenic E. coli, Staphylococci,
are mostly extracellular bacteria,
but there are intracellular bacteria.
And, among the intracellular bacteria,
I see two categories:
those which are intracellular
because they resist killing by professional phagocytes;
and those which are inside mammalian cells
because they are active, they can enter into cells
which are normally not phagocytic.
For example, Listeria monocytogenes,
my favorite organism,
is able to enter into cells which are normally not phagocytic.
So, Listeria was not discovered by Pasteur.
It was not discovered by his competitor Robert Koch.
It was discovered by Murray, in England, in 1926.
I consider that there are several important dates
in the listeriology.
So, in the late '60s, in the '60s,
Mikensis discovered that
recovery from infection by Listeria,
or protection against a secondary infection,
is not due to antibodies,
but is due to a T cell response.
And this was the beginning of the use of Listeria as a tool
to study the induction of a T cell response.
Then, in the late '80s, several groups in the world
started to combine molecular biology,
bacterial genetics, and cell biology
to address the virulence of Listeria.
So, this was the beginning of a new discipline
that we call cellular microbiology,
and this is still going on.
Then, in the beginning of the 2000s...
of this century,
the genome of Listeria monocytogenes was sequenced,
and this was the beginning of a large, large...
of a large number of studies,
which we can call post-genomics.
And very recently,
we have shown that Listeria is inducing epigenetic changes,
and this is really contributing to
what we call epigenetics of infection.
So, the genus Listeria is growing,
has grown recently, become several species,
including two which are pathogenic:
monocytogenes is pathogenic for human and animals;
and Listeria ivanovii is pathogenic only for animals.
So, Listeria monocytogenes is
a Gram-positive bacteria which is ubiquitous in the environment.
It's a saprophyte -- it can live on decaying vegetation --
and it's a pathogen, again, for humans and animals.
It's a bacterium which is motile at low temperature.
And, more importantly,
it can replicate in the cold, at high salt concentration,
at low pH, those conditions which are used,
generally, to keep...
to conserve food in the refrigerator
or in factories.
So, this is the bacterium which can contaminate food products
and it is an economical problem
when food is contaminated,
and, really, in that case,
food is recalled from the market and it has led to
dramatic economical situations.
So, what are the successive steps
of human listeriosis?
Well, very simple...
you ingest a contaminated food product,
the bacteria reach the intestine,
and then the bacteria can cross the intestinal barrier,
reach the liver and the spleen,
and then, via the blood,
it can disseminate to the brain
or, in pregnant women, to the fetus.
So, it's a really dangerous bacteria,
but the disease only occurs in certain cases,
which are immunocompromised individuals, old people,
very... very young babies, or pregnant women.
At the cellular level,
the bacteria enter into cells, as I said previously.
Then, they are trapped in some kind of a vacuole,
which is very rapidly lysed,
the bacteria escape from the vacuole,
and they start moving.
Not only are they moving inside one cell,
but they go can from one cell to the other.
When they arrive to the other cell,
they escape from the two-membrane vacuole
and they start to multiply again,
in the second infected cell.
So, I would like to briefly discuss the entry process.
You see that when the bacterium is entering into a cell,
the membrane has to rearrange,
and the cytoskeleton, which is under the membrane,
has also to rearrange.
We have studied that in good details.
So, first we wanted to know,
what are the molecules of the bacterium
which are important for this entry?
So, we isolated a mutant, a transposon mutant,
which was inserted in the chromosomes upstream of the two genes,
inlA and inlB.
So, this mutant was not invasive.
Then, we deleted each of the two genes,
and we could show that, in fact,
the first gene, inlA, which encodes internalin,
is used for the entry into epithelial cells.
The second gene, when deleted...
the second gene is used for entry
in all human cell types.
So, let's focus on the first one.
The first one is encoding a protein
which is called internalin.
Internalin was shown to interact with a protein
which is called E-cadherin.
E-cadherin is this protein,
which is used for the interaction
that holds epithelial cells together.
So, E-cadherin binds to E-cadherin
and mediates the binding of epithelial cells
to other epithelial cells.
Well, Listeria is using this property of E-cadherin
to enter into cells.
What is interesting is that internalin interacts with human E-cadherin,
but cannot interact with mouse E-cadherin.
And this species specificity is due to a single amino acid at position 16,
as we have been able to show.
So, internalin cannot interact with the mouse E-cadherin,
and this was really a problem for us,
because we wanted to test,
what was the relevance of the interaction
between internalin and E-cadherin?
And we wanted to test the relevance of this interaction
in the mouse model.
So, we decided to create a transgenic mouse,
which would express the human E-cadherin
at the level of the intestine.
Now, I'm going to show you the results of the analysis of the infection
in normal mice and in transgenic mice,
and show you that, in fact,
the interaction between E-cadherin and...
of internalin and human E-cadherin
is absolutely critical for listeriosis.
So, on the left you see
the results of the infection of wild type mice;
on the right you see the infection
with the human E-cadherin transgenic mice.
So, if you infect wild type mice
with 5*10^10 bacteria,
there is 0% mortality.
If you infect with the same amount of bacteria with the mutant,
there is 0% mortality.
But, if you infect, orally, human E-cadherin transgenic mice
with 5*10^10 bacteria,
you see that there is 100% mortality.
If you infect with the internalin mutant,
the delta internalin-A mutant, there is 0% mortality.
This key experiment is really demonstrating
that the interaction between E-cadherin and internalin
is critical for productive Listeria infection,
which leads to lethality.
So, this was a big step,
but of course now the work is
to really understand how Listeria is interacting
with E-cadherin at the level of the intestine.
I'm going just to show you how my colleague,
Mark Lecuit, is pursing this work,
and show you that, in fact,
it's clear that Listeria is associated with human E-cadherin,
which is present on the intestinal villi,
and about 20% is associated at the tip of the villi
and 80% is associated at the border of the villi,
on the side of the villi.
Okay.
I told you about the internalin
interacting with E-cadherin.
I told you that this is an interaction that is species specific.
What about the second gene which is involved in entering?
Well, the second gene is encoding a protein
which is called InlB,
and inlB interacts with a receptor, which is ubiquitous,
which is the hepatocyte growth factor receptor,
which is called Met.
So, InlB interacts with Met in all cells,
including those cells where there is or there is not E-cadherin.
An interesting result is that there is also
a species specificity for InlB,
and, in fact, in contrast to InlA,
which interacts with human E-cadherin
but does not interact with the mouse E-cadherin,
InlB interacts with the human Met
and also interacts with the mouse Met.
And we have been able to show,
together with Mark Lecuit, that, in fact,
InlA and InlB act in concert to cross the placental barrier.
So, we'll now talk more about the entry.
The entry is thus mediated by InlA and InlB.
We have been able to decipher other genes
which are involved in the process of infection.
So, I already mentioned listeriolysin O
as a protein which is
a key protein for the escape from the vacuole.
I would like to focus on the protein
which is able to propel the bacteria inside cells.
So, the phenomenon of actin-based motility
was discovered by Tilney and Portnoy,
and, as shown on this movie from Julie Theriot's lab,
you see that the bacteria are really moving inside cells.
We have been able to show that
this movement is mediated by a protein
which is called ActA,
and you see on the [right] part of this slide that,
in contrast to the wild type bacteria,
this actin mutant is not able to produce these little comet tails
that you see on the movie.
So, this actin mutant does not produce
the protein which is on the surface of the bacteria
and which propels the bacteria inside cells.
So, the bacteria are still alive,
they're multiplying inside cells,
but they're not able to move,
they are not able to spread from cell to cell,
and the mutant is highly attenuated.
What is interesting concerning all these virulence factors
-- I mentioned the listeriolysin O,
which is encoded by hly,
I mentioned ActA, I mentioned InlA and InlB --
is that all these virulence factors
are co-regulated by a single protein, PrfA.
And in my second talk, I will show you that
this PrfA protein is really, really tightly regulated.
So, one big step, as I said in my introduction...
one big step in the listeriology
was the elucidation of the genome of Listeria monocytogenes.
So, the sequence was performed
in the same time that the sequence of Listeria innocua
was performed,
and Listeria innocua is not pathogenic.
So, the goal of this project was to get two genomes
and to be able to compare the genome of the pathogenic
and the genome of the non-pathogenic species
in order to find new virulence factors.
And, in fact, we found some of them...
I'm going just to give you two examples.
Our example... okay...
we found that there were many genes,
and by other techniques we were able to show
that they were regulated, also, by PrfA,
and I would like to focus on the last gene of this slide,
which is Bsh.
This was a surprise.  What is Bsh?
Bsh is a bile salt hydrolase.
We were not expecting to find such a gene in Listeria monocytogenes
and this gene is absent from Listeria innocua,
as you see by the yellow color,
which is not present in Listeria innocua.
So, what is the bile?
The bile is a complex liquid which is made of cholesterol-derived compounds.
And the role of the bile is to
emulsify the lipid from the diet, from the food,
and it really also acts as a antimicrobial agent.
So, the bile is made in the liver, is stored in the gall bladder,
and it goes in the intestine after the meals.
So, the commensals, the intestinal commensals,
have evolved several tricks to
resist the bactericidal action of the bile salts,
including the production of a bile salt hydrolase.
So, we were wondering whether this bile salt hydrolase
was playing a role in the virulence of Listeria,
or at least in the persistence of Listeria
in the intestine.
So, we created a mutant
and we analyzed the behavior of this mutant.
As you can see here,
compared to Listeria monocytogenes wild type,
which is in black,
the mutant, which is in blue,
does not persist in the intestine several hours after the infection.
So, clearly, the BSH is critical for Listeria persistence
in the intestine.
So, similar to commensals,
pathogenic Listeria counteracts the antibacterial activity of bile salts by the BSH,
and this allows the persistence in the intestine
and is critical for virulence.
Another result that we got from the genome
was also a surprise.
At the time,
we and others were studying the role of the peptidoglycan
in the innate immune response,
and it was a surprise that the Listeria peptidoglycan
was stimulating poorly the innate immune receptor NOD.
And we wondered whether this peptidoglycan was maybe...
was maybe modified.
And to make a long story short, the answer is yes.
And we found that, in fact,
there is a deacetylase in the genome of Listeria,
which is acting on the peptidoglycan...
on the peptidoglycan.
So, we created a mutant
and we could show that the deacetylase mutant is much more sensitive to lysozyme,
which is an enzyme
which is cleaving the peptidoglycan.
More than that, we could show that
the growth of the deacetylase mutant
is attenuated in mice.
So, compared so the wild type,
the pgdA mutant is really destroyed very rapidly.
So, in conclusion,
Listeria is about to deacetylate the peptidoglycan
through a peptidoglycan deacetylase
to evade innate immunity.
And the mutant, the pgdA mutant,
is really strongly attenuated in virulence,
and we could really show that the peptidoglycan
is deacetylated in the wild type
and is not deacetylated in the mutant.
So, the conclusion of all of that is
really that Listeria is using a variety of strategies.
I've already mentioned InlA interacting with E-cadherin.
I've already mentioned InlB
interacting with the growth factor receptor which is called Met.
I have mentioned ActA,
which is used for actin polymerization.
I just mentioned the pgdA,
which is a peptidoglycan...
which is a gene involved in peptidoglycan modification
to evade the host immune response.
There are a plethora of strategies used by Listeria,
which are described in the two other talks.
For example, we have shown...
or, it has been shown that ActA
not only is used for actin polymerization,
but also for escape from autophagy.
We have shown that there is the PgdA,
which is modifying the peptidoglycan,
but we have found that there is also an acetyltransferase
which is modifying the peptidoglycan,
again to evade the innate immune response.
This listeriolysin O toxin,
which I briefly mentioned,
which is used for the escape from the vacuole,
seems to have many, many roles during Listeria infection.
It makes pores in the membrane and this induces a series of events
like mitochondrial fragmentation,
deSUMOylation of proteins in the mammalian cells,
also, histone modifications.
So, all of these will be described in my third talk.
So, I think, at this stage, I would like to say that
Listeria monocytogenes has really emerged
as a multifaceted model in different areas,
and as a reference.
For example, in microbiology, I will talk about gene regulation
by noncoding RNAs.
I just mentioned cellular microbiology.
In cell biology, Listeria has been used
to really study actin-based motility, endocytosis,
cell adhesion signaling, mitochondrial dynamics,
and post-translational modification.
Listeria has been one of the first organisms used,
as intensively, for comparative genomics,
for whole-genome transcriptomics,
and now for proteogenomics.
I mentioned briefly that Listeria
is able to target the chromatin and really,
as in the field of epigenetics and infection,
we benefit from studies which have been carried in Listeria.
I also talked about immunology.
I told you that Listeria was shown, not in...
Listeria infection is due to T cell response
and really the immunologists are really using Listeria
as a tool for the induction...
for the study of the induction of the T cell and innate immunity.
And also, Listeria is used as a live vaccine
for cancer therapy.
Finally, I told you that Listeria is able to
cross the human tight barriers,
including the intestinal barrier and the placental barrier,
and there has been very few bacteria
for which it has been shown, like we have shown,
how bacterial proteins are critically
involved in the crossing of the barrier.
