26 research outputs found
New Assays to Characterise Growth-Related Phenotypes of Plasmodium falciparum Reveal Variation in Density-Dependent Growth Inhibition between Parasite Lines
The growth phenotype of asexual blood stage malaria parasites
can influence their virulence and also their ability to survive
and achieve transmission to the next host, but there are few
methods available to characterise parasite growth parameters in
detail. We developed a new assay to measure growth rates at
different starting parasitaemias in a 96-well format and applied
it to characterise the growth of Plasmodium falciparum lines
3D7-A and 3D7-B, previously shown to have different invasion
rates and to use different invasion pathways. Using this simple
and accurate assay we found that 3D7-B is more sensitive to high
initial parasitaemia than 3D7-A. This result indicates that
different parasite lines show variation in their levels of
density-dependent growth inhibition. We also developed a new
assay to compare the duration of the asexual blood cycle between
different parasite lines. The assay is based on the tight
synchronisation of cultures to a 1 h parasite age window and the
subsequent monitoring of schizont bursting and formation of new
rings by flow cytometry. Using this assay we observed
differences in the duration of the asexual blood cycle between
parasite lines 3D7 and HB3. These two new assays will be useful
to characterise variation in growth-related parameters and to
identify growth phenotypes associated with the targeted deletion
of specific genes or with particular genomic, transcriptomic or
proteomic patterns. Furthermore, the identification of
density-dependent growth inhibition as an intrinsic parasite
property that varies between parasite lines expands the
repertoire of measurable growth-related phenotypic traits that
have the potential to influence the outcome of a malarial blood
infection
Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes
The product of the Plasmodium falciparum genes clag3.1 and
clag3.2 plays a fundamental role in malaria parasite biology by
determining solute transport into infected erythrocytes.
Expression of the two clag3 genes is mutually exclusive, such
that a single parasite expresses only one of the two genes at a
time. Here we investigated the properties and mechanisms of
clag3 mutual exclusion using transgenic parasite lines with
extra copies of clag3 promoters located either in stable
episomes or integrated in the parasite genome. We found that the
additional clag3 promoters in these transgenic lines are
silenced by default, but under strong selective pressure
parasites with more than one clag3 promoter simultaneously
active are observed, demonstrating that clag3 mutual exclusion
is strongly favored but it is not strict. We show that silencing
of clag3 genes is associated with the repressive histone mark
H3K9me3 even in parasites with unusual clag3 expression
patterns, and we provide direct evidence for heterochromatin
spreading in P. falciparum. We also found that expression of a
neighbor ncRNA correlates with clag3.1 expression. Altogether,
our results reveal a scenario where fitness costs and
non-deterministic molecular processes that favor mutual
exclusion shape the expression patterns of this important gene
family
Revisiting the initial steps of sexual development in the malaria parasite Plasmodium falciparum
Human to vector transmission of malaria requires that
some blood-stage parasites abandon asexual growth and convert
into non-replicating sexual forms called gametocytes. The
initial steps of gametocytogenesis remain largely
uncharacterized. Here, we study this part of the malaria life
cycle in Plasmodium falciparum using PfAP2-G, the master
regulator of sexual conversion, as a marker of commitment. We
demonstrate the existence of PfAP2-G-positive sexually committed
parasite stages that precede the previously known committed
schizont stage. We also found that sexual conversion can occur
by two different routes: the previously described route in which
PfAP2-G-expressing parasites complete a replicative cycle as
committed forms before converting into gametocytes upon
re-invasion, or a direct route with conversion within the same
cycle as initial PfAP2-G expression. The latter route is linked
to early PfAP2-G expression in ring stages. Reanalysis of
published single-cell RNA-sequencing (RNA-seq) data confirmed
the presence of both routes. Consistent with these results,
using plaque assays we observed that, in contrast to the
prevailing model, many schizonts produced mixed plaques
containing both asexual parasites and gametocytes. Altogether,
our results reveal unexpected features of the initial steps of
sexual development and extend the current view of this part of
the malaria life cycle
New Assays to Characterise Growth-Related Phenotypes of Plasmodium falciparum Reveal Variation in Density-Dependent Growth Inhibition between Parasite Lines
The growth phenotype of asexual blood stage malaria parasites
can influence their virulence and also their ability to survive
and achieve transmission to the next host, but there are few
methods available to characterise parasite growth parameters in
detail. We developed a new assay to measure growth rates at
different starting parasitaemias in a 96-well format and applied
it to characterise the growth of Plasmodium falciparum lines
3D7-A and 3D7-B, previously shown to have different invasion
rates and to use different invasion pathways. Using this simple
and accurate assay we found that 3D7-B is more sensitive to high
initial parasitaemia than 3D7-A. This result indicates that
different parasite lines show variation in their levels of
density-dependent growth inhibition. We also developed a new
assay to compare the duration of the asexual blood cycle between
different parasite lines. The assay is based on the tight
synchronisation of cultures to a 1 h parasite age window and the
subsequent monitoring of schizont bursting and formation of new
rings by flow cytometry. Using this assay we observed
differences in the duration of the asexual blood cycle between
parasite lines 3D7 and HB3. These two new assays will be useful
to characterise variation in growth-related parameters and to
identify growth phenotypes associated with the targeted deletion
of specific genes or with particular genomic, transcriptomic or
proteomic patterns. Furthermore, the identification of
density-dependent growth inhibition as an intrinsic parasite
property that varies between parasite lines expands the
repertoire of measurable growth-related phenotypic traits that
have the potential to influence the outcome of a malarial blood
infection
Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes
The product of the Plasmodium falciparum genes clag3.1 and
clag3.2 plays a fundamental role in malaria parasite biology by
determining solute transport into infected erythrocytes.
Expression of the two clag3 genes is mutually exclusive, such
that a single parasite expresses only one of the two genes at a
time. Here we investigated the properties and mechanisms of
clag3 mutual exclusion using transgenic parasite lines with
extra copies of clag3 promoters located either in stable
episomes or integrated in the parasite genome. We found that the
additional clag3 promoters in these transgenic lines are
silenced by default, but under strong selective pressure
parasites with more than one clag3 promoter simultaneously
active are observed, demonstrating that clag3 mutual exclusion
is strongly favored but it is not strict. We show that silencing
of clag3 genes is associated with the repressive histone mark
H3K9me3 even in parasites with unusual clag3 expression
patterns, and we provide direct evidence for heterochromatin
spreading in P. falciparum. We also found that expression of a
neighbor ncRNA correlates with clag3.1 expression. Altogether,
our results reveal a scenario where fitness costs and
non-deterministic molecular processes that favor mutual
exclusion shape the expression patterns of this important gene
family
Identification of Antimalarial Compounds That Require CLAG3 for Their Uptake by Plasmodium falciparum-Infected Erythrocytes
During the intraerythrocytic asexual cycle malaria parasites acquire nutrients and other solutes through a broad selectivity channel localized at the membrane of the infected erythrocyte termed the plasmodial surface anion channel (PSAC). The protein product of the Plasmodium falciparum clonally variant clag3.1 and clag3.2 genes determines PSAC activity. Switches in the expression of clag3 genes, which are regulated by epigenetic mechanisms, are associated with changes in PSAC-dependent permeability that can result in resistance to compounds toxic for the parasite, such as blasticidin S. Here, we investigated whether other antimalarial drugs require CLAG3 to reach their intracellular target and consequently are prone to parasite resistance by epigenetic mechanisms. We found that the bis-thiazolium salts T3 (also known as albitiazolium) and T16 require the product of clag3 genes to enter infected erythrocytes. P. falciparum populations can develop resistance to these compounds via the selection of parasites with dramatically reduced expression of both genes. However, other compounds previously demonstrated or predicted to enter infected erythrocytes through transport pathways absent from noninfected erythrocytes, such as fosmidomycin, doxycycline, azithromycin, lumefantrine, or pentamidine, do not require expression of clag3 genes for their antimalarial activity. This suggests that they use alternative CLAG3-independent routes to access parasites. Our results demonstrate that P. falciparum can develop resistance to diverse antimalarial compounds by epigenetic changes in the expression of clag3 genes. This is of concern for drug development efforts because drug resistance by epigenetic mechanisms can arise quickly, even during the course of a single infection
Identification of Antimalarial Compounds That Require CLAG3 for Their Uptake by Plasmodium falciparum-Infected Erythrocytes
During the intraerythrocytic asexual cycle malaria parasites acquire nutrients and other solutes through a broad selectivity channel localized at the membrane of the infected erythrocyte termed the plasmodial surface anion channel (PSAC). The protein product of the Plasmodium falciparum clonally variant clag3.1 and clag3.2 genes determines PSAC activity. Switches in the expression of clag3 genes, which are regulated by epigenetic mechanisms, are associated with changes in PSAC-dependent permeability that can result in resistance to compounds toxic for the parasite, such as blasticidin S. Here, we investigated whether other antimalarial drugs require CLAG3 to reach their intracellular target and consequently are prone to parasite resistance by epigenetic mechanisms. We found that the bis-thiazolium salts T3 (also known as albitiazolium) and T16 require the product of clag3 genes to enter infected erythrocytes. P. falciparum populations can develop resistance to these compounds via the selection of parasites with dramatically reduced expression of both genes. However, other compounds previously demonstrated or predicted to enter infected erythrocytes through transport pathways absent from noninfected erythrocytes, such as fosmidomycin, doxycycline, azithromycin, lumefantrine, or pentamidine, do not require expression of clag3 genes for their antimalarial activity. This suggests that they use alternative CLAG3-independent routes to access parasites. Our results demonstrate that P. falciparum can develop resistance to diverse antimalarial compounds by epigenetic changes in the expression of clag3 genes. This is of concern for drug development efforts because drug resistance by epigenetic mechanisms can arise quickly, even during the course of a single infection
Revisiting the initial steps of sexual development in the malaria parasite Plasmodium falciparum
Human to vector transmission of malaria requires that
some blood-stage parasites abandon asexual growth and convert
into non-replicating sexual forms called gametocytes. The
initial steps of gametocytogenesis remain largely
uncharacterized. Here, we study this part of the malaria life
cycle in Plasmodium falciparum using PfAP2-G, the master
regulator of sexual conversion, as a marker of commitment. We
demonstrate the existence of PfAP2-G-positive sexually committed
parasite stages that precede the previously known committed
schizont stage. We also found that sexual conversion can occur
by two different routes: the previously described route in which
PfAP2-G-expressing parasites complete a replicative cycle as
committed forms before converting into gametocytes upon
re-invasion, or a direct route with conversion within the same
cycle as initial PfAP2-G expression. The latter route is linked
to early PfAP2-G expression in ring stages. Reanalysis of
published single-cell RNA-sequencing (RNA-seq) data confirmed
the presence of both routes. Consistent with these results,
using plaque assays we observed that, in contrast to the
prevailing model, many schizonts produced mixed plaques
containing both asexual parasites and gametocytes. Altogether,
our results reveal unexpected features of the initial steps of
sexual development and extend the current view of this part of
the malaria life cycle