Profiling the Essential Nature of Lipid Metabolism in Asexual Blood and Gametocyte Stages of Plasmodium falciparum.

During its life cycle, Plasmodium falciparum undergoes rapid proliferation fueled by de novo synthesis and acquisition of host cell lipids. Consistent with this essential role, Plasmodium lipid synthesis enzymes are emerging as potential drug targets. To explore their broader potential for therapeutic interventions, we assayed the global lipid landscape during P. falciparum sexual and asexual blood stage (ABS) development. Using liquid chromatography-mass spectrometry, we analyzed 304 lipids constituting 24 classes in ABS parasites, infected red blood cell (RBC)-derived microvesicles, gametocytes, and uninfected RBCs. Ten lipid classes were previously uncharacterized in P. falciparum, and 70%-75% of the lipid classes exhibited changes in abundance during ABS and gametocyte development. Utilizing compounds that target lipid metabolism, we affirmed the essentiality of major classes, including triacylglycerols. These studies highlight the interplay between host and parasite lipid metabolism and provide a comprehensive analysis of P. falciparum lipids with candidate pathways for drug discovery efforts.

Origin of robustness in generating drug-resistant malaria parasites.

Biological robustness allows mutations to accumulate while maintaining functional phenotypes. Despite its crucial role in evolutionary processes, the mechanistic details of how robustness originates remain elusive. Using an evolutionary trajectory analysis approach, we demonstrate how robustness evolved in malaria parasites under selective pressure from an antimalarial drug inhibiting the folate synthesis pathway. A series of four nonsynonymous amino acid substitutions at the targeted enzyme, dihydrofolate reductase (DHFR), render the parasites highly resistant to the antifolate drug pyrimethamine. Nevertheless, the stepwise gain of these four dhfr mutations results in tradeoffs between pyrimethamine resistance and parasite fitness. Here, we report the epistatic interaction between dhfr mutations and amplification of the gene encoding the first upstream enzyme in the folate pathway, GTP cyclohydrolase I (GCH1). gch1 amplification confers low level pyrimethamine resistance and would thus be selected for by pyrimethamine treatment. Interestingly, the gch1 amplification can then be co-opted by the parasites because it reduces the cost of acquiring drug-resistant dhfr mutations downstream in the same metabolic pathway. The compensation of compromised fitness by extra GCH1 is an example of how robustness can evolve in a system and thus expand the accessibility of evolutionary trajectories leading toward highly resistant alleles. The evolution of robustness during the gain of drug-resistant mutations has broad implications for both the development of new drugs and molecular surveillance for resistance to existing drugs.

Tricks in Plasmodium's molecular repertoire--escaping 3'UTR excision-based conditional silencing of the chloroquine resistance transporter gene.

In the human malaria parasite Plasmodium falciparum, the major determinant of chloroquine resistance, P. falciparum chloroquine resistance transporter (pfcrt), likely plays an essential role in asexual blood stages, thus precluding conventional gene targeting approaches. We attempted to conditionally silence the expression of its ortholog in Plasmodium berghei (pbcrt) through Flp recombinase-mediated excision of the 3'untranslated region (UTR) during mosquito passage. However, parasites maintained pbcrt expression despite 3'UTR excision. Characterisation of these pbcrt mRNAs, by 3'rapid amplification of cDNA ends, identified several replacement 3'UTR sequences. Our observations demonstrate the astounding genetic plasticity of this parasite when faced with the loss of an essential gene.

Normalization of the lymph node T cell stromal microenvironment in lpr/lpr mice is associated with SU5416-induced reduction in autoantibodies.

The vascular-stromal elements of lymph nodes can play important roles in regulating the activities of the lymphocytes within. During model immune responses, the vascular-stromal compartment has been shown to undergo proliferative expansion and functional alterations. The state of the vascular-stromal compartment and the potential importance of this compartment in a spontaneous, chronic model of autoimmunity have not been well studied. Here, we characterize the vascular expansion in MRL-lpr/lpr lymph nodes and attempt to ask whether inhibiting this expansion can interfere with autoantibody generation. We show that characteristics of vascular expansion in enlarging MRL-lpr/lpr lymph nodes resemble that of the VEGF-dependent expansion that occurs in wild-type mice after model immunization. Surprisingly, treatment with SU5416, an inhibitor of VEGF and other receptor tyrosine kinases, did not have sustained effects in inhibiting vascular growth, but attenuated the anti-dsDNA response and altered the phenotype of the double negative T cells that are expanded in these mice. In examining for anatomic correlates of these immunologic changes, we found that the double negative T cells are localized within ectopic follicles around a central B cell patch and that these T cell-rich areas lack the T zone stromal protein ER-TR7 as well as other elements of a normal T zone microenvironment. SU5416 treatment disrupted these follicles and normalized the association between T zone microenvironmental elements and T cell-rich areas. Recent studies have shown a regulatory role for T zone stromal elements. Thus, our findings of the association of anti-dsDNA responses, double negative T cell phenotype, and altered lymphocyte microenvironment suggest the possibility that lymphocyte localization in ectopic follicles protects them from regulation by T zone stromal elements and functions to maintain autoimmune responses. Potentially, altering the lymphocyte microenvironment that is set up by the vascular-stromal compartment can be a means by which to control undesired autoimmune responses.

Diversity-Oriented Synthesis Yields a Novel Lead for the Treatment of Malaria.

Here, we describe the discovery of a novel antimalarial agent using phenotypic screening of Plasmodium falciparum asexual blood-stage parasites. Screening a novel compound collection created using diversity-oriented synthesis (DOS) led to the initial hit. Structure-activity relationships guided the synthesis of compounds having improved potency and water solubility, yielding a subnanomolar inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable in human plasma. This compound is available via the molecular libraries probe production centers network (MLPCN) and is designated ML238.

Quantitative assessment of Plasmodium falciparum sexual development reveals potent transmission-blocking activity by methylene blue.

Clinical studies and mathematical models predict that, to achieve malaria elimination, combination therapies will need to incorporate drugs that block the transmission of Plasmodium falciparum sexual stage parasites to mosquito vectors. Efforts to measure the activity of existing antimalarials on intraerythrocytic sexual stage gametocytes and identify transmission-blocking agents have, until now, been hindered by a lack of quantitative assays. Here, we report an experimental system using P. falciparum lines that stably express gametocyte-specific GFP-luciferase reporters, which enable the assessment of dose- and time-dependent drug action on gametocyte maturation and transmission. These studies reveal activity of the first-line antimalarial dihydroartemisinin and the partner drugs lumefantrine and pyronaridine against early gametocyte stages, along with moderate inhibition of mature gametocyte transmission to Anopheles mosquitoes. The other partner agents monodesethyl-amodiaquine and piperaquine showed activity only against immature gametocytes. Our data also identify methylene blue as a potent inhibitor of gametocyte development across all stages. This thiazine dye almost fully abolishes P. falciparum transmission to mosquitoes at concentrations readily achievable in humans, highlighting the potential of this chemical class to reduce the spread of malaria.

Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches.

Malarial parasites have evolved resistance to all previously used therapies, and recent evidence suggests emerging resistance to the first-line artemisinins. To identify antimalarials with novel mechanisms of action, we have developed a high-throughput screen targeting the apicoplast organelle of Plasmodium falciparum. Antibiotics known to interfere with this organelle, such as azithromycin, exhibit an unusual phenotype whereby the progeny of drug-treated parasites die. Our screen exploits this phenomenon by assaying for "delayed death" compounds that exhibit a higher potency after two cycles of intraerythrocytic development compared to one. We report a primary assay employing parasites with an integrated copy of a firefly luciferase reporter gene and a secondary flow cytometry-based assay using a nucleic acid stain paired with a mitochondrial vital dye. Screening of the U.S. National Institutes of Health Clinical Collection identified known and novel antimalarials including kitasamycin. This inexpensive macrolide, used for agricultural applications, exhibited an in vitro IC(50) in the 50 nM range, comparable to the 30 nM activity of our control drug, azithromycin. Imaging and pharmacologic studies confirmed kitasamycin action against the apicoplast, and in vivo activity was observed in a murine malaria model. These assays provide the foundation for high-throughput campaigns to identify novel chemotypes for combination therapies to treat multidrug-resistant malaria.

Taking Charge: Feeding Malaria via Anion Channels.

The human malaria parasite Plasmodium falciparum increases red blood cell membrane permeability during infection to allow for import of nutrients and other solutes. Nguitragool et al. (2011) have now identified parasite-encoded CLAG3 proteins as key components of the import channel located on the erythrocyte membrane.

Fibroblast-type reticular stromal cells regulate the lymph node vasculature.

The lymph node vasculature is essential to immune function, but mechanisms regulating lymph node vascular maintenance and growth are not well understood. Vascular endothelial growth factor (VEGF) is an important mediator of lymph node endothelial cell proliferation in stimulated lymph nodes. It is expressed basally in lymph nodes and up-regulated upon lymph node stimulation, but the identity of VEGF-expressing cells in lymph nodes is not known. We show that, at homeostasis, fibroblast-type reticular stromal cells (FRC) in the T zone and medullary cords are the principal VEGF-expressing cells in lymph nodes and that VEGF plays a role in maintaining endothelial cell proliferation, although peripheral node addressin (PNAd)(+) endothelial cells are less sensitive than PNAd(-) endothelial cells to VEGF blockade. Lymphotoxin beta receptor (LTbetaR) blockade reduces homeostatic VEGF levels and endothelial cell proliferation, and LTbetaR stimulation of murine fibroblast-type cells up-regulates VEGF expression, suggesting that LTbetaR signals on FRC regulate lymph node VEGF levels and, thereby, lymph node endothelial cell proliferation. At the initiation of immune responses, FRC remain the principal VEGF mRNA-expressing cells in lymph nodes, suggesting that FRC may play an important role in regulating vascular growth in stimulated nodes. In stimulated nodes, VEGF regulates the proliferation and expansion of both PNAd(+) and PNAd(-) endothelial cells. Taken together, these data suggest a role for FRC as paracrine regulators of lymph node endothelial cells and suggest that modulation of FRC VEGF expression may be a means to regulate lymph node vascularity and, potentially, immune function.

In vitro evaluations of antimalarial drugs and their relevance to clinical outcomes.

Plasmodium falciparum resistance to the former first-line antimalarials chloroquine and sulfadoxine/pyrimethamine has reached critically high levels in many malaria-endemic regions. This has spurred the introduction of several new artemisinin-based combination therapies (ACTs) that display excellent potency in treating drug-resistant malaria. Monitoring for the emergence of drug resistant P. falciparum is important for maximising the clinically effective lifespan of ACTs. Here, we provide a commentary on the article by Kaddouri et al., published in this issue of the International Journal of Parasitology, which documents the levels of susceptibility to ACT drugs and chloroquine in P. falciparum isolates from Mali. These authors report that some isolates approached a proposed in vitro threshold of resistance to monodesethyl-amodiaquine (the principal effective metabolite of amodiaquine, an important ACT partner drug), and establish baseline levels of susceptibility to the ACT drugs dihydroartemisinin and lumefantrine. The majority of clinical isolates manifested in vitro resistance to chloroquine. The authors also show good concordance between field-based assays employing a non-radioactive lactate dehydrogenase-based method of determining in vitro drug IC(50) values and the well-established [(3)H]hypoxanthine-based radioactive method. This work illustrates a good example of drug resistance surveillance, whose global coordination is being championed by the World Antimalarial Resistance Network. Our current opinion also more generally discusses the complexities inherent to conducting in vitro investigations with P. falciparum patient isolates and correlating these findings with treatment outcome data.

Advances in understanding the genetic basis of antimalarial drug resistance.

The acquisition of drug resistance by Plasmodium falciparum has severely curtailed global efforts to control malaria. Our ability to define resistance has been greatly enhanced by recent advances in Plasmodium genetics and genomics. Sequencing and microarray studies have identified thousands of polymorphisms in the P. falciparum genome, and linkage disequilibrium analyses have exploited these to rapidly identify known and novel loci that influence parasite susceptibility to antimalarials such as chloroquine, quinine, and sulfadoxine-pyrimethamine. Genetic approaches have also been designed to predict determinants of in vivo resistance to more recent first-line antimalarials such as the artemisinins. Transfection methodologies have defined the role of determinants including pfcrt, pfmdr1, and dhfr. This knowledge can be leveraged to develop more efficient methods of surveillance and treatment.

Regulation of lymph node vascular growth by dendritic cells.

Lymph nodes grow rapidly and robustly at the initiation of an immune response, and this growth is accompanied by growth of the blood vessels. Although the vessels are critical for supplying nutrients and for controlling cell trafficking, the regulation of lymph node vascular growth is not well understood. We show that lymph node endothelial cells begin to proliferate within 2 d of immunization and undergo a corresponding expansion in cell numbers. Endothelial cell proliferation is dependent on CD11c+ dendritic cells (DCs), and the subcutaneous injection of DCs is sufficient to trigger endothelial cell proliferation and growth. Lymph node endothelial cell proliferation is dependent on vascular endothelial growth factor (VEGF), and DCs are associated with increased lymph node VEGF levels. DC-induced endothelial cell proliferation and increased VEGF levels are mediated by DC-induced recruitment of blood-borne cells. Vascular growth in the draining lymph node includes the growth of high endothelial venule endothelial cells and is functionally associated with increased cell entry into the lymph node. Collectively, our results suggest a scenario whereby endothelial cell expansion in the draining lymph node is induced by DCs as part of a program that optimizes the microenvironment for the ensuing immune response.

Requirements for follicular exclusion and competitive elimination of autoantigen-binding B cells.

Results from several mouse tolerance models indicate that autoreactive B cells in peripheral lymphoid organs develop an anergic phenotype, migrate to the boundary between the T cell zone and the B cell follicle (T/B boundary), and undergo rapid cell death. We have used B cells from mice that are double-transgenic for soluble hen egg lysozyme (HEL) and an Ig that recognizes HEL with a high affinity to characterize the mechanisms underlying the migration and elimination of autoreactive B cells. In contrast to the situation for acutely activated B cells, we find that anergic B cells have reduced levels of CXCR5, the receptor for the follicular chemokine, CXCL13, and this contributes to their exclusion from follicles. CCR7 expression is required for follicular exclusion of anergic cells, although up-regulation of the receptor does not appear to be necessary. By TUNEL analysis, we observe that excluded anergic cells die in situ at the T/B boundary. We also show that this elimination occurs via a Fas-independent mechanism. Using CCR7(-/-)Ig(HEL)-transgenic B cells we find that localization to the T/B boundary is not a necessary event to achieve the competitive elimination of autoantigen-binding B cells. These findings characterize the mechanism for follicular exclusion of autoantigen-binding B cells and they indicate that B cells compete for survival by mechanisms that are separate from competition for the follicular niche.

Chemokine requirements for B cell entry to lymph nodes and Peyer's patches.

B cell entry to lymph nodes and Peyer's patches depends on chemokine receptor signaling, but the principal chemokine involved has not been defined. Here we show that the homing of CXCR4-/- B cells is suppressed in CCL19 (ELC)- and CCL21 (SLC)-deficient paucity of lymph node T cells mice, but not in wild-type mice. We also find that CXCR4 can contribute to T cell homing. Using intravital microscopy, we find that B cell adhesion to high endothelial venules (HEVs) is disrupted when CCR7 and CXCR4 are predesensitized. In Peyer's patches, B cell entry is dependent on CXCR5 in addition to CCR7/CXCR4. CXCL12 (SDF1) is displayed broadly on HEVs, whereas CXCL13 (BLC) is found selectively on Peyer's patch follicular HEVs. These findings establish the principal chemokine and chemokine receptor requirements for B cell entry to lymph nodes and Peyer's patches.

Balanced responsiveness to chemoattractants from adjacent zones determines B-cell position.

B lymphocytes re-circulate between B-cell-rich compartments (follicles or B zones) in secondary lymphoid organs, surveying for antigen. After antigen binding, B cells move to the boundary of B and T zones to interact with T-helper cells. Despite the importance of B--T-cell interactions for the induction of antibody responses, the mechanism causing B-cell movement to the T zone has not been defined. Here we show that antigen-engaged B cells have increased expression of CCR7, the receptor for the T-zone chemokines CCL19 and CCL21, and that they exhibit increased responsiveness to both chemoattractants. In mice lacking lymphoid CCL19 and CCL21 chemokines, or with B cells that lack CCR7, antigen engagement fails to cause movement to the T zone. Using retroviral-mediated gene transfer we demonstrate that increased expression of CCR7 is sufficient to direct B cells to the T zone. Reciprocally, overexpression of CXCR5, the receptor for the B-zone chemokine CXCL13, is sufficient to overcome antigen-induced B-cell movement to the T zone. These findings define the mechanism of B-cell relocalization in response to antigen, and establish that cell position in vivo can be determined by the balance of responsiveness to chemoattractants made in separate but adjacent zones.