Evolution matters: Chloroplasts as
targets for antiparasitic drugs
Summary malaria II
• Severe forms of malaria include: severe anemia in kids, and cerebral and
renal malaria in adults
• Severe pathogenesis is related to adherence of infected RBC to entothelia
• Adherence is mediated by knobs on the RBC surface made up by parasite
proteins (PFEMP1)
• PFEMP1 undergoes antigenic variation
• Chloroquine accumulates in the food vacuole and prevents heme
polymerization (detoxification), resistance is linked to mutations in a
transport protein in the food vacuole membrane
• Emerging drug resistance is a tremendous challenge to the management
of malaria, a constant stream of new drugs with novel modes of action is
required to stay ahead of the pathogen
Evolution matters: Chloroplasts as
targets for antiparasitic drugs
• One of the most hotly pursued new targets for
drugs is the chloroplast of Apicomplexa, or
apicoplast
• The surprising discovery of a parasite
chloroplast
• What does the chloroplast do?
• How does endosymbiosis work?
The convergence of three
(initially) independent
observation
• Apicomplexa appear to have three genomes
• Apicomplexa are susceptible to certain
antibiotics that usually only affect bacteria
• Apicomplexa have a subcellular structure in
the vicinity of the nucleus that is surrounded
by multiple membranes
How many genomes do we have?
• DNA can be separated by
gradient centrifugation
• The image shows yeast
DNA the bright lower
band are the nuclear
chromosomes the higher
band is the mitochondrial
genome
• Apicomplexa have a
nuclear, a mitochondrial
and a chloroplast genome
www.cc.ysu.edu/~helorime/cscl.html
The third genome looks like a chloroplast
genome and localizes outside of the nucleus
Small dot: apicoplast genome, large spot
nuclear genome; red label: specific probe
for apicoplast genome
The chloroplast genome is localized in an
organelle with four membranes
What do we know
about chloroplasts?
• Home of photosynthesis
• Have their own genome
• Surrounded by two
membranes
• Evolved by
endosymbiosis from a
cyanobacteria
The apicoplast is the product of
secondary endosymbiosis
Apicomplexa are not plants – but they ate a plant
What does the apicoplast do?
What does the apicoplast do?
What does the apicoplast do?
Modified after Ralph et al., Nat. Rev. Microbiol. 2: 203,
Fleige et al., Eukaryot. Cell 6:984
The apicoplast makes fatty acids,
isoprenoids and heme
• Fatty acids
• Isoprene
• Major component of
• Precursor for many
lipids that form cellular
important molecules
membranes and serve
including cholesterol
as energy reservoir
and many hormones
• Heme
• Prosthetic group for
important enzymes
that catalyze
oxidation/reduction
reactions
Due to the evolutionary history of the
apicoplast its pathways are “bacterial”
Mammals and bacteria (and
plastids)synthesize isoprenes
differently
• Isoprenoid synthesis is rich
in proven drug targets
• Animals use the
mevalonate pathways
• Bacteria and plastids use
the deoxy-xylulose-P
pathway
• These pathways use
different enzymes,
different intermediates and
are susceptible to different
drugs
Mammals and bacteria (and
plastids)synthesize isoprenes
differently
• Apicomplexa have
bacterial type
isoprene synthesis
• The enzymes for this
pathway are
localized in the
apicoplast
The apicoplast isoprenoid pathway is
essential for parasite survival
DLytB
wild type
Fos
mutant
What does the apicoplast do?
• Apicoplast is home to several biosynthetic pathways
that are specific to the parasite
• Genetic studies show that some of these pathways are
essential to parasite growth
• Certain antibiotics that target these pathways in
bacteria also inhibit parasites and some show promise
in clinical trials
• Current work is focused on identifying the best target in
the apicoplast and to discovery more potent drugs to
inhibit them
How does endosymbiosis work?
Massive gene transfer from
endosymbiont to nucleus, why?
The difference between a docile new
endosymbiont and hostile take over is control
Gene transfer is a hallmark
of endosymbiosis
• Gene transfer is not only found for the
apicoplast but also for the primary
chloroplast in plants or our
mitochondrion
• Gene transfer moves control of gene
expression (and therefore essentially
everything the symbiont does) to the
host
• Also note that organellar genomes are
only maternally inherited. Transferring
to the nucleus makes organellar genes
accessible to sexual recombination
• What is the problem with transfering
genes?
Nuclear encoded plastid proteins feature
a bipartite targeting signal
• Apicoplast proteins that are encoded in
the nucleus carry an address tag at the
beginning (N-terminus) of the protein
• Think of it as a molecular zip code
• Conceptually similar tags are found on
many organelle protein (e.g. those that
are secreted outside or have to go to the
mitochondrion or chloroplast)
• The apicoplast zip has two elements: the
first is a secretory signal peptide resulting
into insertion into the endoplasmic
reticulum, the second is responsible for
the rest of the journey
• The tag is removed upon arrival
Import across four membranes
Import across four membranes
Proteins related to protein of the chloroplast Tic
are present in the apicoplast membranes
ApTic20
Apicoplast
merge
Van Dooren et al.,(2008) PNAS 105:13574
Mutants in apicoplast Tic genes kill
parasites and block protein import
Wild type
Mutant
Gene “on”
Gene “off”
Van Dooren et al.,(2008) PNAS 105:13574
That is one how do
you cross the rest?
TIC20
TIC22
Secondary chloroplasts have an ER
cleanup system (ERAD) but no ER
Chlorarachnion reptans
Ludwig & Gibbs J Phycol 1989
Sommer et al. Mol Biol Evol. 2007
ERAD: a clean up system for the ER
• Many proteins travel through the
endoplasmic reticulum – sometimes
things get messed up and they don’t fold
as they should
• Such proteins can cause problems and are
pulled back out of the ER into the
cytoplasm where they are chucked into
the cells trash can for proteins – the
proteasome
• A protein machine called ERAD (ER
associated degradation) forms a pore in
the membrane and pulls misfolded
proteins back out
Apicomplexa have two ERAD systems:
one in the ER & cytoplasm
HA
ER
83.m00015
HA
46.m01752
Plastid
And a second set in the apicoplast
HA
76.m03417
The Apicoplast ERAD
system is derived from
the red algal
endosymbiont
A simple & elegant way
to engineer protein import
A simple & elegant way
to engineer protein import
A simple & elegant way
to engineer protein import
A simple & elegant way
to engineer protein import
Import across four membranes
How do host and endosymbiont
divide in a coordinated fashion?
Conclusions
• Apicoplast metabolic pathways
are essential and provide
excellent highly divergent targets
for drug development
• Multiple phylogentically
independent translocons enable
import over four
• Plastid division is controlled by
the host and depends on “host
factors”: the mitotic spindle, and
a plastid fission protein