Kamal El Bissati

Disruption of the Plasmodium falciparum PfPMT Gene Results in a Complete Loss of Phosphatidylcholine Biosynthesis via the Serine-Decarboxylase-Phosphoethanolamine-Methyltransferase Pathway and Severe Growth and Survival Defects

Biochemical studies in the human malaria parasite, Plasmodium falciparum, indicated that in addition to the pathway for synthesis of phosphatidylcholine from choline (CDP-choline pathway), the parasite synthesizes this major membrane phospholipid via an alternative pathway named the serine-decarboxylase-phosphoethanolamine-methyltransferase (SDPM) pathway using host serine and ethanolamine as precursors. However, the role the transmethylation of phosphatidylethanolamine plays in the biosynthesis of phosphatidylcholine and the importance of the SDPM pathway in the parasites growth and survival remain unknown. Here, we provide genetic evidence that knock-out of the PfPMT gene encoding the phosphoethanolamine methyltransferase enzyme completely abrogates the biosynthesis of phosphatidylcholine via the SDPM pathway. Lipid analysis in knock-out parasites revealed that unlike in mammalian and yeast cells, methylation of phosphatidylethanolamine to phosphatidylcholine does not occur in P. falciparum, thus making the SDPM and CDP-choline pathways the only routes for phosphatidylcholine biosynthesis in this organism. Interestingly, loss of PfPMT resulted in significant defects in parasite growth, multiplication, and viability, suggesting that this gene plays an important role in the pathogenesis of intraerythrocytic Plasmodium parasites.

Antimalarial activity of the anticancer and proteasome inhibitor bortezomib and its analog ZL3B

BackgroundThe high rate of mortality due to malaria and the worldwide distribution of parasite resistance to the commonly used antimalarial drugs chloroquine and pyrimethamine emphasize the urgent need for the development of new antimalarial drugs. An alternative approach to the long and uncertain process of designing and developing new compounds is to identify among the armamentarium of drugs already approved for clinical treatment of various human diseases those that may have strong antimalarial activity.MethodsProteasome inhibitor bortezomib (Velcade™: [(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl) amino]propyl]amino]butyl] boronic acid), which has been approved for treatment of patients with multiple myeloma, and a second boronate analog Z-Leu-Leu-Leu-B(OH)2 (ZL3B), were tested against four different strains of P. falciparum (3D7, HB3, W2 and Dd2) that are either sensitive or have different levels of resistance to the antimalarial drugs pyrimethamine and chloroquine.ResultsBortezomib and ZL3B are equally effective against drug-sensitive and -resistant parasites and block intraerythrocytic development prior to DNA synthesis, but have no effect on parasite egress or invasion.ConclusionThe identification of bortezomib and its analog as potent antimalarial drugs will set the stage for the advancement of this class of compounds, either alone or in combination therapy, for treatment of malaria, and emphasize the need for large-scale screens to identify new antimalarials within the library of clinically approved compounds.

Effectiveness of a novel immunogenic nanoparticle platform for Toxoplasma peptide vaccine in HLA transgenic mice

We created and produced a novel self-assembling nanoparticle platform for delivery of peptide epitopes that induces CD8(+) and CD4(+)T cells that are protective against Toxoplasma gondii infection. These self-assembling polypeptide nanoparticles (SAPNs) are composed of linear peptide (LP) monomers which contain two coiled-coil oligomerization domains, the dense granule 7 (GRA720-28 LPQFATAAT) peptide and a universal CD4(+)T cell epitope (derived from PADRE). Purified LPs assemble into nanoparticles with icosahedral symmetry, similar to the capsids of small viruses. These particles were evaluated for their efficacy in eliciting IFN-γ by splenocytes of HLA-B*0702 transgenic mice and for their ability to protect against subsequent T. gondii challenge. This work demonstrates the feasibility of using this platform approach with a CD8(+) epitope that binds HLA-B7 and tests the biological activity of potentially protective peptides restricted by human major histocompatibility complex (HLA) class I molecules in HLA transgenic mice.

Toxoplasma gondii HLA-B*0702-restricted GRA7(20-28) peptide with adjuvants and a universal helper T cell epitope elicits CD8(+) T cells producing interferon-γ and reduces parasite burden in HLA-B*0702 mice.

The ability of CD8(+) T cells to act as cytolytic effectors and produce interferon-γ (IFN-γ) was demonstrated to mediate resistance to Toxoplasma gondii in murine models because of the recognition of peptides restricted by murine major histocompatibility complex (MHC) class I molecules. However, no T gondii-specific HLA-B07-restricted peptides were proven protective against T gondii. Recently, 2 T gondii-specific HLA-B*0702-restricted T cell epitopes, GRA7(20-28) (LPQFATAAT) and GRA3(27-35) (VPFVVFLVA), displayed high-affinity binding to HLA-B*0702 and elicited IFN-γ from peripheral blood mononuclear cells of seropositive HLA-B*07 persons. Herein, these peptides were evaluated to determine whether they could elicit IFN-γ in splenocytes of HLA-B*0702 transgenic mice when administered with adjuvants and protect against subsequent challenge. Peptide-specific IFN-γ-producing T cells were identified by enzyme-linked immunosorbent spot and proliferation assays utilizing splenic T lymphocytes from human lymphocyte antigen (HLA) transgenic mice. When HLA-B*0702 mice were immunized with one of the identified epitopes, GRA7(20-28) in conjunction with a universal CD4(+) T cell epitope (PADRE) and adjuvants (CD4(+) T cell adjuvant, GLA-SE, and TLR2 stimulatory Pam(2)Cys for CD8(+) T cells), this immunization induced CD8(+) T cells to produce IFN-γ and protected mice against high parasite burden when challenged with T gondii. This work demonstrates the feasibility of bioinformatics followed by an empiric approach based on HLA binding to test this biologic activity for identifying protective HLA-B*0702-restricted T gondii peptides and adjuvants that elicit protective immune responses in HLA-B*0702 mice.

Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum

The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was unable to grow on physiological concentrations of adenosine, inosine and hypoxanthine. We have now used an episomally complemented pfnt1Delta knockout parasite strain to confirm genetically the functional role of PfNT1 in P. falciparum purine uptake and utilization. Episomal complementation by PfNT1 restored the ability of pfnt1Delta parasites to transport and utilize adenosine, inosine and hypoxanthine as purine sources. The ability of wild-type and pfnt1Delta knockout parasites to transport and utilize the other physiologically relevant purines adenine, guanine, guanosine and xanthine was also examined. Unlike wild-type and complemented P. falciparum parasites, pfnt1Delta parasites could not proliferate on guanine, guanosine or xanthine as purine sources, and no significant transport of these substrates could be detected in isolated parasites. Interestingly, whereas isolated pfnt1Delta parasites were still capable of adenine transport, these parasites grew only when adenine was provided at high, non-physiological concentrations. Taken together these results demonstrate that, in addition to hypoxanthine, inosine and adenosine, PfNT1 is essential for the transport and utilization of xanthine, guanine and guanosine.

Localization of the phosphoethanolamine methyltransferase of the human malaria parasite Plasmodium falciparum to the Golgi apparatus.

Phosphatidylcholine is the most abundant phospholipid in the membranes of Plasmodium falciparum, the agent of severe human malaria. The synthesis of this phospholipid occurs via two routes, the CDP-choline pathway, which uses host choline as a precursor, and the plant-like serine decarboxylase-phosphoethanolamine methyltransferase (SDPM) pathway, which uses host serine as a precursor. Although various components of these pathways have been identified, their cellular locations remain unknown. We have previously reported the identification and characterization of the phosphoethanolamine methyltransferase, Pfpmt, of P. falciparum and shown that it plays a critical role in the synthesis of phosphatidylcholine via the SDPM pathway. Here we provide the first evidence that the transmethylation step of the SDPM pathway occurs in the parasite Golgi apparatus. We show that the level of Pfpmt protein in the infected erythrocyte is regulated in a stage-specific fashion, with high levels detected during the trophozoite stage at the peak of parasite membrane biogenesis. Confocal microscopy revealed that Pfpmt is not cytoplasmic. Immunoelectron microscopy revealed that Pfpmt localizes to membrane structures that extend from the nuclear membrane but that it only partially co-localizes with the endoplasmic reticulum marker BiP. Using transgenic parasites expressing green fluorescent protein targeted to different cellular compartments, a complete co-localization was detected with Rab6, a marker of the Golgi apparatus. Together these studies provide the first evidence that the transmethylation step of the SDPM pathway of P. falciparum occurs in the Golgi apparatus and indicate an important role for this organelle in parasite membrane biogenesis.

γδ Intraepithelial Lymphocyte Migration Limits Transepithelial Pathogen Invasion and Systemic Disease in Mice

BACKGROUND & AIMS Intraepithelial lymphocytes that express the γδ T-cell receptor (γδ IELs) limit pathogen translocation across the intestinal epithelium by unknown mechanisms. We investigated whether γδ IEL migration and interaction with epithelial cells promote mucosal barrier maintenance during enteric infection. METHODS Salmonella typhimurium or Toxoplasma gondii were administered to knockout (KO) mice lacking either the T cell receptor δ chain (Tcrd) or CD103, or control TcrdEGFP C57BL/6 reporter mice. Intravital microscopy was used to visualize migration of green fluorescent protein (GFP)-tagged γδ T cells within the small intestinal mucosa of mice infected with DsRed-labeled S typhimurium. Mixed bone marrow chimeras were generated to assess the effects of γδ IEL migration on early pathogen invasion and chronic systemic infection. RESULTS Morphometric analyses of intravital video microscopy data showed that γδ IELs rapidly localized to and remained near epithelial cells in direct contact with bacteria. Within 1 hour, greater numbers of T gondii or S typhimurium were present within mucosae of mice with migration-defective occludin KO γδ T cells, compared with controls. Pathogen invasion in Tcrd KO mice was quantitatively similar to that in mice with occludin-deficient γδ T cells, whereas invasion in CD103 KO mice, which have increased migration of γδ T cells into the lateral intercellular space, was reduced by 63%. Consistent with a role of γδ T-cell migration in early host defense, systemic salmonellosis developed more rapidly and with greater severity in mice with occludin-deficient γδ IELs, relative to those with wild-type or CD103 KO γδ IELs. CONCLUSIONS In mice, intraepithelial migration to epithelial cells in contact with pathogens is essential to γδ IEL surveillance and immediate host defense. γδ IEL occludin is required for early surveillance that limits systemic disease.

Molecular target validation, antimicrobial delivery, and potential treatment of Toxoplasma gondii infections

Toxoplasma gondii persistently infects over two billion people worldwide. It can cause substantial morbidity and mortality. Existing treatments have associated toxicities and hypersensitivity and do not eliminate encysted bradyzoites that recrudesce. New, improved medicines are needed. Transductive peptides carry small molecule cargos across multiple membranes to enter intracellular tachyzoites and encysted bradyzoites. They also carry cargos into retina when applied topically to eyes, and cross blood brain barrier when administered intravenously. Phosphorodiamidate morpholino oligomers (PMO) inhibit gene expression in a sequence-specific manner. Herein, effect of transductive peptide conjugated PMO (PPMO) on tachyzoite protein expression and replication in vitro and in vivo was studied. Initially, sequence-specific PPMO successfully reduced transfected T. gondii’s fluorescence and luminescence. PPMO directed against T. gondii’s dihydrofolate reductase (DHFR), an enzyme necessary for folate synthesis, limited tachyzoite replication. Rescue with exogenous folate demonstrated DHFR PPMO‘s specificity. PPMO directed against enoyl-ACP reductase (ENR), an enzyme of type II fatty acid synthesis that is structurally distinct in T. gondii from ENR in mammalian cells was investigated. PPMO directed against plant-like Apetela 2 (AP2) domain transcription factor XI-3 (AP2XI-3), not present in human cells, was characterized. ENR and AP2XI-3 PPMO each restricted intracellular parasite replication validating these molecular targets in tachyzoites. DHFR-specific PPMO administered to infected mice diminished parasite burden. Thus, these antisense oligomers are a versatile approach to validate T. gondii molecular targets, reduce essential T. gondii proteins in vitro and in vivo, and have potential for development as curative medicines.

Novel N-Benzoyl-2-hydroxybenzamide Disrupts Unique Parasite Secretory Pathway

ABSTRACT Toxoplasma gondii is a protozoan parasite that can damage the human brain and eyes. There are no curative medicines. Herein, we describe our discovery of N-benzoyl-2-hydroxybenzamides as a class of compounds effective in the low nanomolar range against T. gondii in vitro and in vivo. Our lead compound, QQ-437, displays robust activity against the parasite and could be useful as a new scaffold for development of novel and improved inhibitors of T. gondii. Our genome-wide investigations reveal a specific mechanism of resistance to N-benzoyl-2-hydroxybenzamides mediated by adaptin-3β, a large protein from the secretory protein complex. N-Benzoyl-2-hydroxybenzamide-resistant clones have alterations of their secretory pathway, which traffics proteins to micronemes, rhoptries, dense granules, and acidocalcisomes/plant-like vacuole (PLVs). N-Benzoyl-2-hydroxybenzamide treatment also alters micronemes, rhoptries, the contents of dense granules, and, most markedly, acidocalcisomes/PLVs. Furthermore, QQ-437 is active against chloroquine-resistant Plasmodium falciparum. Our studies reveal a novel class of compounds that disrupts a unique secretory pathway of T. gondii, with the potential to be used as scaffolds in the search for improved compounds to treat the devastating diseases caused by apicomplexan parasites.

T. gondii RP Promoters & Knockdown Reveal Molecular Pathways Associated with Proliferation and Cell-Cycle Arrest

Molecular pathways regulating rapid proliferation and persistence are fundamental for pathogens but are not elucidated fully in Toxoplasma gondii. Promoters of T. gondii ribosomal proteins (RPs) were analyzed by EMSAs and ChIP. One RP promoter domain, known to bind an Apetela 2, bound to nuclear extract proteins. Promoter domains appeared to associate with histone acetyl transferases. To study effects of a RP genes regulation in T. gondii, mutant parasites (Δrps13) were engineered with integration of tetracycline repressor (TetR) response elements in a critical location in the rps13 promoter and transfection of a yellow fluorescent-tetracycline repressor (YFP-TetR). This permitted conditional knockdown of rps13 expression in a tightly regulated manner. Δrps13 parasites were studied in the presence (+ATc) or absence of anhydrotetracycline (-ATc) in culture. -ATc, transcription of the rps13 gene and expression of RPS13 protein were markedly diminished, with concomitant cessation of parasite replication. Study of Δrps13 expressing Myc-tagged RPL22, -ATc, showed RPL22 diminished but at a slower rate. Quantitation of RNA showed diminution of 18S RNA. Depletion of RPS13 caused arrest of parasites in the G1 cell cycle phase, thereby stopping parasite proliferation. Transcriptional differences ±ATc implicate molecules likely to function in regulation of these processes. In vitro, -ATc, Δrps13 persists for months and the proliferation phenotype can be rescued with ATc. In vivo, however, Δrps13 could only be rescued when ATc was given simultaneously and not at any time after 1 week, even when L-NAME and ATc were administered. Immunization with Δrps13 parasites protects mice completely against subsequent challenge with wildtype clonal Type 1 parasites, and robustly protects mice against wildtype clonal Type 2 parasites. Our results demonstrate that G1 arrest by ribosomal protein depletion is associated with persistence of T. gondii in a model system in vitro and immunization with Δrps13 protects mice against subsequent challenge with wildtype parasites.