- Welcome everyone to Wednesday
Nite @ the Lab.
I'm Tom Zinnen, I work here at
the UW-Madison Biotechnology Cent
er.
I also work for the Division of
Extension
and Wisconsin 4-H, and on behalf
of those folks
and our other co-organizers, Wisc
onsin Public Television,
the Wisconsin Alumni Association
,
and the UW-Madison Science Alli
ance,
thanks again for coming to
Wednesday Nite @ the Lab.
We do this every Wednesday night,
50 times a year.
Tonight it's my pleasure to intro
duce to you
Caitlin Pepperell, she's a pr
ofessor here
in the departments of medicine a
nd also in
the Department of Medical Microb
iology and Immunology.
She was born in Toronto, Ontario
,
and went to high school there,
and then she went east to go to
McGill University
in Montreal where she studied En
glish literature.
Then she went to Queens Universi
ty in Kingston, Ontario
to get her MD degree.
She did a residency
at the University of Toronto
and then stayed at Toronto at the
university to do
a fellowship, and then did anothe
r fellowship at Stanford.
Then in 2011, she came here to be
on the faculty.
Tonight she's going to talk with
us on the topic
of how do microorganisms evolve t
o become pathogens.
As an old plant pathologist,
that's a pretty interesting ques
tion to ask.
It's going to be interesting to h
ear what you have to say
from the medical side.
Would you please join me in welc
oming Caitlin Pepperell
to Wednesday Nite @ the Lab.
(audience applauds)
- Thank you.
Thank you.
Thanks Tom for the introduction
and the opportunity to give this
talk,
and thank you all for coming.
So yes, I'm gonna talk about
bacterial pathogen emergence
using a little bit of recherche e
xample.
Staphylococcus saprophyticus,
which, as I said in the introduc
tion to this lecture,
I started working on by accident
.
So I'll tell you the story of how
that happened.
But first, I'd like to start with
some definitions
for those of you who are in diffe
rent fields,
starting with pathogen,
what we mean by pathogen and
virulence.
So pathogen is a microorganism
that causes disease,
infectious disease.
It's fashionable nowadays to get
away from
thinking about this in a binary
sense
in terms of pathogens and non-pa
thogens,
and we think of it now,
or conceptualize it more alo
ng a spectrum,
with some organisms having a very
high pathogenic potential,
like the agent of plague and
others being
more occasional pathogens, and we
might put something
for those of you in the plant wor
ld,
I know there are a few of you her
e,
pseudomonas might be in that cat
egory
where it's an occasional pathogen
of humans.
So pathogen refers to the organis
m
and virulence refers to the trai
t.
So virulence is the capacity to
cause disease.
I'm going to be talking about ba
cterial genomics,
so just to introduce you to the
concept of the genome,
which is the total set of genes
encoded via bacterium,
and bacteria being the nifty litt
le creatures that they are,
they have, or can have, one or m
ore main chromosomes
and little accessory
packages called plasmids.
They can have more than one of th
ese,
and they can also deliver protei
ns to their host cell,
and this is just a little schema
tic of the bacterial cell.
This is a very simple phylogeny,
which is a family tree,
so it is a depiction of rela
tionships among organisms,
and here we can see that A an
d B are more closely related
to each other than either of
them is to C.
And I'll be showing you some phy
logenies as well.
I'm also gonna be talking ab
out core and accessory genes
in bacterial genomes; this is how
genes are often drawn or
represented abstractly.
And the concepts behind core a
nd accessory genes
is very simple.
Core genes are always present in
the population,
and accessory genes may or m
ay not be present.
So hopefully I've done a good job
explaining this
and you can keep it in mind as I
go through the story.
So one of the things, overarchin
g questions that
I'm interested in in my resea
rch and that we
work on as a group in my research
group,
is this question of where pathog
ens come from.
So microorganisms are everywhere
,
bacteria, fungi, viruses, parasi
tes, et cetera,
but disease in humans and in o
ther host species
is attributable to a teeny tiny
fraction
of those microorganisms.
So one of the overarching questi
ons
that we're interested in
is the how and why of
how microbes adopt this
particular lifestyle and cause di
sease
in humans and other hosts.
So some of the underlying questio
ns are,
what is the function of virulence
?
So in some cases, virulence or t
he capacity
to cause disease
in some way directly benefits the
microorganism.
In other cases, it's a byproduct
of other things
that are important to the bacte
rium.
We're also interested in mechanis
ms of adaptation
to the pathogenic niche,
and particularly in genome, geno
mic mechanisms,
so how genomes change when micro
organisms,
particularly bacteria in our case
,
adapt and evolve to invade the pa
thogenic niche.
So I'm gonna start with a story a
bout
some mystery nodules, these
are pictured here
and this picture was taken on my
desk shortly after
I started my faculty position
at UW-Madison.
William Aylward is a Professor
of classics here at UW
and he was the deliverer of these
nodules.
He brought them to me to study
because I've done
a lot of work on the evolution of
tuberculosis,
and he thought that these perhap
s represented
calcified tubercles, which are,
it's infected lung tissue with
microbacterium tuberculosis.
And Tom mentioned the field of m
olecular archeology,
Doctor Aylward was really intere
sted in developing
this field here in Madison,
and he thought that perhaps thes
e nodules harbored
some DNA from microbacterium tub
erculosis,
and that by studying this DNA, we
might learn
about the evolution of TB.
So where did the nodules come fro
m?
They came from a cemetery dating
to the late Byzantine era
in Troy, the ancient city of Troy
in Anatolia,
in what is currently Turkey.
And this is a picture of the cem
etery.
The nodules were spotted by a ver
y
sharp-eyed archeologist, she's ac
tually
an archeologist and a physician
to whom I am indebted.
And this is Doctor Kiesewetter
who's pictured here.
So the nodules, as you can see,
they're a couple of centimeter
s in size,
and she picked them out from the
rubble
in the remains around this indiv
idual.
So the person here was dated base
d on archeological
evidence from the cemetery to the
early 13th century.
Doctor Kiesewetter did an analys
is of the people buried
in this cemetery, who eviden
tly had a fairly
difficult and short life on the
whole,
so the life expectancy based
on her analysis
was 32 years for women and 39 for
men.
50% of the decedents in the cemet
ery
had degenerative changes in their
spine and joints,
indicating that they were engage
d in difficult,
probably agrarian labor.
The person whose remains she stud
ied
was sexed as female and aged at a
pproximately age 30.
She lost many of her teeth be
fore her death
at this young age,
indicating again a short and diff
icult life.
And she had a healed arm fractur
e.
Carbon dating of the remains
was consistent
with archeological analysis of th
e cemetery
and dating to the late 12th and
early 13th century A.D.
So I was tasked with making a di
agnosis of the nodules
and the person from whom they de
rived,
and myself and collaborators did
this on the basis
of analysis of DNA that we extr
acted
from the nodules, as well as the
other methods
that are listed here.
Metagenomics refers to analys
is of the mixture
of microbial species present in
a sample
and currently is done, it's a ve
ry active field of research
in analyzing, for example, the
intestinal microbiome
or the bacteria that we harbor
in our gastrointestinal tracts.
We also did a microscopic analys
is of this tissue
and an elemental analysis, so ana
lyze
the elements present in the nodul
es.
In this type of work, in the anci
ent DNA field,
it's really like finding a need
le in a haystack,
so most of what is extracted when
we extract DNA
from archeological remains is con
tamination.
So when we identity bacteria and
other microorganisms,
it's most abundant soil material.
So it's typical to find less th
an 1%
of your target DNA in your sample
.
Here we found a rather astonishi
ng amount
of target DNA.
So there are two numbers listed w
hich refers
to the two different nodules, so
in one of the nodules,
without any enrichment or fil
tering,
12% of the endogenous DNA was hu
man,
31% was from this bacterium Stap
hylococcus saprophyticus,
4% from Gardnerella vaginalis.
So an absolutely stunning amount
of endogenous ancient DNA.
And not tuberculosis,
which is how I accidentally star
ted working on
Staphylococcus saprophyticus.
Based on this really abundant e
ndogenous
ancient bacterial DNA, we were ab
le to assemble
a complete genome of this 800-ye
ar-old
Staphylococcus saprophyticus,
with approximately 300-fold cover
age,
so the genome was covered-- th
at means the genome was covered
300 times approximately in i
ts entirety.
And this was, at the time, and I
believe still is,
the best preserved example of an
ancient bacterial genome,
so it was quite a stunning and lu
cky finding.
What you see here
are scanning electron micrographs
of the nodules,
and what you can see here are coc
ci,
so these are small round bact
erial shapes in abundance,
over here we have extracellular
matrix,
and what we think is the shape
and outline of a white blood cell
.
I showed this picture at a talk
I gave
at the University of Illinois,
and one of the members of the
audience
who works on Staphylococcus aureu
s
told me that this looks just like
an SEM or a micrograph
of a living Staph. aureus biofilm
,
which is a complex bacterial stru
cture.
So this is in fact the 800-ye
ar-old tissue,
and what's been preserved here a
re the shapes
of the bacterial cells,
the shapes of the inflammatory c
ells,
and the shapes of the extruded m
aterial,
which we call extracellular matr
ix, that binds
these cells together.
So the individual cells and
their entire
architecture of the tissue was pr
eserved in this sample.
The diagnosis that I settled
on in the end,
based on the microscopy, the anci
ent DNA analysis,
elemental analysis, metagenomics
, et cetera,
was the diagnosis of chorioamnio
nitis.
This is a severe pregnancy co
mplication,
so it's an ascending infection in
which
typically several species of bac
teria
from the vaginal microbiome asce
nd and infect
the amniotic membrane surrou
nding the fetus,
the placenta, the amniotic fluid,
and the fetus.
As you can imagine, this is asso
ciated
with pregnancy complications with
poor outcomes
for the fetus and can be associa
ted with
major morbidity and mortality in
the mother as well.
And that was almost certainly th
e case here.
We found by looking at differe
nt tissues
from different bones, that she
was likely bacteremic
at the time of her death,
meaning that the Staph. saprophyt
icus
had invaded her blood stream and
caused a septicemia.
We think that the nodular tissue
s represent
calcified placental abscesses
and that the process of calcific
ation that occurred
during this infection is what pr
eserved
the bacterial cells, the tissue a
rchitecture,
and ultimately, the DNA.
So one of the things that happe
ns during
placental development is there is
flux of calcium
through the placenta to allow
mineralization
of the fetal bones, and so placen
tas are prone to calcify,
they calcify spontaneously, and
also as a result
of inflammatory and infectio
us conditions.
So we think that what happened h
ere is that
there was an abrupt inflammatory
calcification
that basically froze this inf
ection in time
and preserved the bacterial cell
s,
inflammatory cells, et cetera.
The placenta is an organ that's j
ointly made
by mother and fetus, and in this
case,
we were actually able to recover
DNA from a male fetus
from this tissue, which was
quite amazing.
So Staph. saprophyticus, this 800
-year-old infection
is actually quite unusual in our
understanding
of this bacterium today.
Staph. saprophyticus is familiar
to clinicians and microbiologist
s
more as a cause of urinary tract
infections
and much milder forms of infe
ction than this woman had.
It is a member of the gastrointe
stinal
and genitourinary microbiomes,
so these complex microbial commun
ities,
from which invaders can emerge a
nd cause things like
urinary tract infections.
It is second only to E. coli
as a cause of UTI
in reproductive aged women,
so it's quite a common cause of
UTI,
and urinary tract infections are
associated with
a very significant global health
burden,
so hundreds of millions of c
ases per year,
and a recent estimate of cost ass
ociated with UTIs
in the U.S. in the billions of do
llars.
So perhaps in most cases, you kno
w,
we think of it as a minor nuisan
ce,
but it's a very major public hea
lth problem
with a lot of costs related to b
oth the care
and treatment of this infection,
and also loss of productivity, et
cetera.
But there's a bigger picture to t
his bacterium.
So it's pictured here streaked o
n a plate,
and the images here show multiple
niches from which
we can obtain this bacterium.
So it's found in meats and cheese
s.
I read a paper recently abou
t isolating
Staph. saprophyticus and other
coagulase-negative Staph.
from a range of different meats
and cheeses in France,
and I'm convinced I must go ther
e
to further pursue this lead.
[laughter]
This bug is in yummy foods.
It's also isolated from--
can be isolated from the environm
ent,
so it can live freely in the env
ironment,
and it's typically been found on
farms
and agricultural settings.
It's also isolated as a pathogen
and a commensal or innocent resi
dent of animals,
and most of the information that
we have is from
agriculturally important animals,
which may just be an issue of
those are the animals that we sam
ple most frequently.
It's found in dairy environments
,
so on belt environments, in milki
ng equipment,
and it does cause disease, it cau
ses mastitis,
which is an infection affect
ing dairy cattle,
so it's also a pathogen of animal
s, including cattle.
Some work has been done to t
ry to understand
why Staph. saprophyticus is a pat
hogen
and what helps it particular
ly adapt to,
most of these studies have been
focused
on the human urinary tract,
and this is a list of some known
virulence genes,
so genes that are thought to ena
ble this organism
to invade the human urinary trac
t.
For urinary tract infections,
one of the most important traits
they need to have
is they need to be able to b
ind to human cells
and to the matrix that connec
ts human cells.
So one of my colleagues who
works on E. coli
that causes UTIs gave me an analo
gy
of the bacteria having to hold o
n to slippery rocks
as a waterfall cascades over them
.
So there's major physical sheer
force applied to bacteria
that try to ascend the urinary tr
act,
and the ones that stick around a
nd win
have to be able to really stick w
ell.
So many of these virulence genes
are adhesions
or they encode adhesions,
which are proteins that allow th
e bacteria to really
rock-climb up the urinary tract.
As part of the analysis of th
is ancient bacterial genome,
we did a phylogenetic analysis,
or looked at relationships among
different
types of Staph. saprophyticus,
and at the time that we had thi
s amazing
ancient bacterial genomes,
there were almost no other fully
sequenced genomes,
so as part of that project, I we
nt knocking on doors
with collaborators around the wor
ld
to try to find some more isolates
of this bacterium.
And the goal of this work was to
place
the ancient genome into a context
and also to understand the evolut
ion of this bacterium
and its genome structure.
And what is pictured here is a co
llection
of genomes that we had at that
time,
and then these are the areas th
at we obtained them from,
and then we've got some cute
little pictures
to show you where they came from
.
So you can see from fish, from m
eat,
this was from a river in Malaysi
a,
and then we had a doggy, and a c
ow,
and our guy from Troy here.
So, consistent with this organis
m being able
to live in multiple niches and e
nvironments.
What's shown over here is a d
ifferent kind of
family tree called a network,
and this shows the relationship
between the plasmids,
so these are those little autono
mous pieces of DNA
that we got from different bacter
ial genomes,
and we looked at how they were r
elated to each other.
And one sort of interesting thin
g that came
out of this analysis of the plas
mids particularly,
is we've got this major clade st
ructure
in the core genome
that is recapitulated on the pla
smids,
suggesting that the bacteria can
't exchange,
from these two clades, can't
exchange plasmids
with each other, which is, at
this point,
an unsolved mystery.
So, in follow-up work, we looked
at a larger sample
of Staph. saprophyticus genomes,
focused on the question of
whether there's any specific a
daptation,
any signature of adaptation in th
e genomes
to the human urinary tract.
And this work was led by Tatum M
ortimer,
who's pictured here as a former
graduate student in my lab
and is now a postdoc at Harvard.
So one of the really interesting
things about
Staph. saprophyticus UTI that
's emerged from
the epidemiological literature i
s that
this infection appears to be
acquired from the environment,
so as opposed to spreading from p
erson to person,
it seems that people acquire it f
rom the environment
and then it causes UTI, and t
hen they shed
the bacteria back into the envir
onment.
So our question was whether this
represented,
human UTIs represented a spil
lover
or whether this could be an adap
tation.
So whether we happened to acq
uire this bug
from the environment, it's reall
y adapted
to do something else,
versus it really has a specif
ic adaptation to humans.
So one clue that supports a spec
ific adaptation
is we found the UTI associated i
solates
were clustered together on t
he phylogeny,
so the bugs that cause UTI are mo
re closely related
to each other than they are
to bugs that were
obtained from different enviro
nments and niches.
Tatum did some further work looki
ng at patterns
of variation to look for signatur
es of adaptation
to human urinary tract infections
.
So one of the things that we did
is a genome scan,
which is pictured here.
And what we're looking for he
re is a dip
in levels of diversity as we m
ove across the genome.
And the reasoning behind this is
fairly simple.
So when you have an advantag
eous mutation,
it spreads through a population q
uickly
and it tends to pull linked DNA n
ext to it
as it rises in frequency,
and so the population at that pa
rticular locus
where the advantageous mutati
on is
will have low diversity rela
tive to other parts
of the genome.
So Tatum found this dip in diver
sity,
this location,
and within this dip, found a muta
tion in a gene
called Aas, that was strongly ass
ociated with
human urinary tract infection.
And this is a protein-coding mut
ation,
so it changed the code of the pr
otein,
as opposed to what we call a
silent mutation.
The Aas gene is one of the v
irulence genes
that I listed at the beginnin
g of the talk.
It is an adhesion, so it binds to
a human material called fibronect
in,
and the mutation was in the fibr
onectin binding region
of the protein.
We looked at patterns of gene ex
change between organisms
in this sample, and we found sev
eral
examples of transfer events where
the bugs
swapped out one version for anot
her,
and each of these transfer event
s reinforce
the association between the
one variant
and human UTI, supporting its rol
e
in mediating adaptation to c
ausing UTI.
Tatum also did some demographic
reconstructions
from the genomic data.
So this is figuring out whet
her populations
have stayed the same size, have g
rown, have shrunk,
and she found evidence of a b
ottleneck,
which is a sudden decrease in the
size of the population
followed by a 15-fold expansion.
So this is consistent with what w
e call a selective sweep,
where an advantageous mutation
sweeps through the population,
decreasing diversity,
and then the population expands
into a new niche.
So we've followed up this work
with Madison, who is in the audie
nce now,
and a graduate student in my lab,
and ongoing studies to follow-up
these observations
are on the larger sample of
165 genomes.
These are from several different
types of sample and host,
and from, you know,
it's not a perfectly represen
tative global sample,
but a quite diverse one.
So we start with the phylogeny,
and it's a much larger sample,
it's a bigger phylogeny.
We've got the same clade structur
e,
so we've got, this is one clade,
and then this whole thing is ano
ther,
so as we've added to the sample,
one of the clades has expanded
to a much greater extent tha
n the other,
suggesting that the places that
we sample
are dominated by this clade.
I have a note here about no geogr
aphic structure,
so what this means is that
we have isolates from Madison, W
isconsin,
that are interspersed with isola
tes from
Brazil and Japan,
so they're more closely rela
ted to isolates
from Brazil and Japan than they
are to
other isolates from Madison, Wis
consin,
which is pretty remarkable.
As I mentioned, clade one is mor
e common,
so this is the one that grew rea
lly dramatically,
and then clade one has a real
ly interesting
sub-clade structure.
So this sub-clade up here is alm
ost exclusively
human UTI isolates.
There's one exception of an isola
te from a dog,
and most of this kind of infecti
on in dogs
represents spillover events from
humans to their pets,
then we have an environmental is
olate here,
but aside from that, we have
all human UTI.
This sub-clade, on the other hand
, contains human UTI,
bovine mastitis isolates,
and food isolates,
so it's consistent with some pe
rhaps different ecologies
in these two sub-clades,
one of which is more strictly as
sociated with humans
and the other with food and agri
cultural animals.
Madison did
another genome scan with thi
s larger sample
to see if the patterns that Tatum
had identified
with a smaller sample would be r
eplicated,
and very nicely, they were,
so this dip here in diversity
corresponds to the dip that Tatum
had identified
in the smaller sample, and suppor
ts the idea that
this region, and particularly the
gene Aas,
is under selection in this sample
.
So one of the possibilities to e
xplain, you know,
why a particular strain caus
es UTI versus
ending up, you know, as a co
mmensal of cattle
is that, perhaps the ones that ca
used UTI
have virulence genes
and the other ones don't.
And there was, if you recall, I
mentioned
that there was that list of
virulence genes
that had been identified pre
viously.
There is no support for that h
ypothesis,
so what we found, what Madiso
n found, is that,
so these are a list of virule
nce genes here
and here,
and these genes tend to be eithe
r
present in the entire sample from
all different niches and environm
ents,
or they're present in a really
small fraction of samples,
so we can't map the presence
of these known virulence genes
onto human UTI and use that as a
n explanation of UTI.
One interesting example is t
his gene dsdA,
which is D-serine deaminase gene
,
and what this does is to detoxify
D-serine, which is found in mamma
lian urine,
and in studies of this gene,
the idea is that this is a reall
y important
protein for bugs that want to liv
e in the urinary tract.
Well, this gene is found in the e
ntire sample
of bugs from all different niches
,
and Madison has looked at other
bacterial species
to see if they encode this gene,
bacteria that don't have anythin
g to do with
the urinary tract, as far as we k
now,
and it's encoded by all kinds of
different bacteria.
So what this suggests to us is t
hat
the gene has multiple functions,
and this is very typical for bact
erial genes,
so I like to think of them as Sw
iss army knives,
they typically do many diffe
rent things.
And so the concept that this is
specific to adaptation to uri
nary tract just
reflects our own biases in h
ow we look at
these genes and proteins, and th
ink about them.
So Madison has done some further
work
to find genes that are associated
with
human urinary tract, and also wit
h bovine mastitis,
and in addition, to look for vari
ants of genes,
so mutations that are associated
with these different niches.
She has found some accessory
gene contents
of some genes, and some varia
nts that are
associated with UTI and that a
re good candidates
for adaptation to the pathogenic
niche.
That's the good news.
The bad news, or slightly more d
ifficult news,
is that the genes that are as
sociated are
poorly characterized,
most of them are hypothetical
proteins,
and they don't have close cous
ins in other bacteria,
so they're...
difficult to know exactly what t
hey do,
and it'll be a lot of work to fi
gure that out.
We've also had a look at the plas
mids
found in the sample,
so those little autonomous b
its of DNA,
and these, what's pictured here,
are assemblies of plasmid genome
s
based on a different kind of seq
uencing technology,
so a type of sequencing that all
ows us to look at
very long stretches of DNA
and resolve these structures.
Madison has found a lot of divers
ity in terms of plasmids,
from bacterium to bacterium,
so between one and five plasmids
harbored by the bacteria,
some of them are quite large, up
to 100,000 base pairs,
some of them have apparently
completely novel gene content on
them,
and she's also found that virulen
ce genes
have been mobilized on these pla
smids.
So the plasmids are giving the b
acteria
an enormous amount of flexibility
,
and there's reflected in a lot o
f diversity
in terms of what the bacteria can
do.
So, to come back to my origina
l question
of where do pathogens come from,
this bug, Staph. saprophyticus,
is found as I said
in humans, animals, indoor, outd
oor environments, and food,
and when we look at human infect
ions,
some of them do appear to be
spillover events,
so those are the organisms that a
re on
the parts of the tree that are no
t pure human UTI,
but many of them appear to reflec
t a specific adaptation
to the human urinary tract.
And this is on the basis of genom
e reorganization,
so it's on the basis of new muta
tions that have arisen,
mutations that are swapped fr
om one bacterium to another,
and entire genes that are swappe
d between bacteria.
So I'll end there with my ack
nowledgments.
This is the Pepperell lab, where
we are
completely serious at all times.
[laughter]
And I'd like to thank the member
s of the Pepperell lab,
particularly Tracy, Lindsey, a
nd Nathan,
who are directly involved in
generating
the data that Madison analyzed.
And my funders at the NIH and NS
F,
and collaborators at UW-Madison a
nd off-campus,
and thank you for your attention
,
and happy to take questions.
[applause]