Biology subjects

Mohmmed, A.

Publications and source records attributed to Mohmmed, A..

12 recordsLinked to original sources

bioRxiv2025

Plasmodium falciparum aquaglyceroporin (PfAQP) orchestrates glycerol-dependent lipid metabolism, membrane biogenesis, metabolomic flux, parasite growth and stress adaptation

Aquaglyceroporins (AQPs) are integral membrane channel proteins that facilitate transport of water and other solutes such as glycerol, thereby playing vital roles in cellular homeostasis. Here, we functionally characterized Plasmodium falciparum aquaglyceroporin (PfAQP) through localization, inducible knockdown, and comprehensive metabolomic analyses revealing its key role in lipid homeostasis and metabolomic flux. PfAQP localizes to the parasite plasma membrane and food vacuole membrane, at critical interfaces for nutrient and metabolite exchange. A glmS-ribozyme mediated inducible knock-down of PfAQP severely impairs parasite development from trophozoite to schizont, leading to marked suppression in parasite growth. Ultra-Expansion Microscopy (UExM) analyses show profound defects in membrane biogenesis, along with disruption in cytokinesis under PfAQP knock-down conditions. Detailed lipidomic analyses suggests that PfAQP disruption cause altered lipid homeostasis, characterized by reduced membrane phospholipid biosynthesis, impaired lipid scavenging, and accumulation of storage lipids. Metabolomic analyses further indicate metabolic dysregulation characterized by reduction in amino acid levels and dysregulated mitochondrial metabolism, including impaired tricarboxylic acid (TCA) cycle. Strikingly, PfAQP expression gets transiently upregulated under nutrient starvation condition, and its ablation drastically compromise parasite recovery, highlighting its role in adaptive responses. Overall, these results establish PfAQP as a critical regulator of glycerol-dependent metabolic pathways, orchestrating membrane biogenesis, energy metabolism, and stress resilience--functions indispensable for parasite growth and survival.

microbiology↗

bioRxiv2025

LFA-1 Interaction with GBP-130 on Plasmodium falciparum-infected Red Blood Cells mediates NK Cell Activation and Parasite Control

Natural Killer (NK) cells contribute to early immunity against Plasmodium falciparum by recognizing and eliminating infected red blood cells (iRBCs), a process mediated in part by the integrin LFA-1. However, the cognate parasite ligand for LFA-1 has remained unknown. Here, we identify Glycophorin Binding Protein-130 (PfGBP-130) as a surface-expressed ligand on iRBCs that binds the I-domain of LFA-1 (LFA-1 I). Using an LFA-1 I-Fc fusion protein, we demonstrate stage-specific binding to iRBCs, and LC-MS/MS analysis of immunopreciptates of I-Fc bound to iRBC revealed PfGBP-130 as a high-confidence interactor. Recombinant PfGBP-130 binds NK and THP-1 cells in an LFA-1-dependent manner. Co-culture assays show that PfGBP-130 promotes NK cell activation, degranulation, and facilitates contact-dependent killing of iRBCs. Neutralizing antibodies against PfGBP-130 significantly impair these responses. Our findings establish PfGBP-130 as the LFA-1 ligand on iRBCs, providing new insight into NK cell-mediated immunity in malaria and identifying a potential target for host-directed interventions.

immunology↗

bioRxiv2024

Importin alpha inhibitors act against the differentiated stages of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii

Protozoan parasites of the phylum Apicomplexa, including Plasmodium falciparum and Toxoplasma gondii, cause widespread disease in humans. New drugs and protein targets are required for the treatment of these diseases, particularly therapies targeting multiple stages of the parasite life cycles. Nuclear import, carried out by the transporters importin (IMP) and {beta} subunits, is a valid target for the discovery of lead compounds against these protozoan parasites: small molecules were identified that inhibit interactions between IMP and nuclear localisation signals in vitro and also inhibit the growth of the rapidly-dividing stages of P. falciparum and T. gondii (asexual stages and tachyzoites) in culture. In this report, we add another small molecule (Bay 11-7082) to the panel of inhibitors of IMP and test the ability of these inhibitors to first, inhibit nuclear transport in the rapidly dividing stages and, next, the maturation of differentiated stages of both parasites. We show that GW5074 and CAPE inhibit nuclear transport in the P. falciparum blood stages, while Bay 11-7085 inhibits nuclear transport in T. gondii tachyzoites. Interestingly, CAPE strongly inhibits gametocyte maturation, the sexual stages of P. falciparum, and Bay 11-7085 weakly inhibits bradyzoite differentiation, the latent stages of T. gondii. As differentiation of both these stages is dependent on activation of gene expression, triggered by the nuclear translocation of transcription factors, our work provides a "proof of concept" that targeting nuclear import is a viable strategy for the development of therapeutics against multiple stages of apicomplexan parasites, some of them recalcitrant to existing drugs.

molecular biology↗

bioRxiv2024

A dynamin-like protein in the human malaria parasite plays an essential role in mitochondrial homeostasis and fission during asexual blood stages

Malaria parasite harbors a single mitochondrion and its proper segregation during the parasite multiplication is crucial for propagation of the parasite within the host. Mitochondrial fission machinery consists of a number of proteins that associate with mitochondrial membrane during segregation. Here, we have identified a dynamin-like protein in P. falciparum, PfDyn2, and deciphered its role in mitochondrial division, segregation and homeostasis. GFP targeting approach combined with high resolution microscopy studies showed that the PfDyn2 associates with mitochondrial membrane to form a clip/hairpin loop like structure around it at specific sites during mitochondrial division. The C-terminal degradation tag mediated inducible knock-down (iKD) of PfDyn2 resulted in significant inhibition of parasite growth. PfDyn2-iKD hindered mitochondrial development and functioning, decreased mtDNA replication, and induced mitochondrial oxidative-stress, ultimately causing parasite death. Further, treatment of parasites with dynamin specific inhibitors disrupted the recruitment of PfDyn2 on the mitochondria, blocked mitochondrial development, and induced oxidative stress. Regulated overexpression of a phosphorylation mutant of PfDyn2 (Ser-612-Ala) had no effect on the recruitment of PfDyn2 on the mitochondria; normal mitochondrial division and parasite growth showed that phosphorylation/dephosphorylation of this conserved serine residue (Ser612) may not be responsible for regulating recruitment of PfDyn2 to the mitochondrion. Overall, we show essential role of PfDyn2 in mitochondrial dynamics and fission as well as in maintaining its homeostasis during asexual cycle of the parasite.

cell biology↗

bioRxiv2022

Characterization of Plasmodium falciparum RAP domain proteins-RAP291 and RAP070, and their association with ribosomal RNAs

Plasmodium genomes encode multiple RAP (RNA-binding domain abundant in Apicomplexan) domain proteins that contain a conserved module of 56 to 73 amino acids. Here, we characterized two of the P. falciparum RAP domain proteins; PfRAP291 & PfRAP070, for their expression and role at asexual blood stages. RNA binding assays and high-throughput CLIP-seq analysis showed that these proteins mainly bind ribosome associated RNAs. Blue-native PAGE and protein-protein interaction studies suggested association of these proteins with MSP-1 complex. Anti-PfRAP291 and anti-PfRAP070 antibodies showed moderate inhibitions in in-vitro merozoite invasion assays. Together, these results suggest multiple roles of these proteins; PfRAP291 and PfRAP070, in merozoite invasion and in ribosome regulation during asexual stages of the parasite.

microbiology↗

bioRxiv2022

Small-molecule inhibitors targeting prokaryotic ClpQ protease in Plasmodium display anti-malarial efficacies against blood and liver stages

The ATP-dependent ClpQY system is a prokaryotic proteasome-like machinery in the mitochondrion of malaria parasite, Plasmodium. This protease system is identified as a validated target for malaria intervention. Here we have identified drug-like small chemical compounds, pyrimidotriazine derivatives, as specific inhibitors of P. falciparum ClpQ (PfClpQ) having effective parasiticidal efficacies on asexual and liver stages. Screening of the Malaria Box compound library, using a robust in vitro activity assay, identified a hit compound targeting the PfClpQ protease with IC50 at ~750nM. Further, Structure-Activity Relationship (SAR) analysis, using a small library of derivatives of the hit compound, identified three potential lead compounds, ICGEB-L1, ICGEB-L2 and ICGEB-L3, which inhibited enzyme activity with IC50 in 100-200nM ranges. The three lead compounds effectively inhibited in vitro asexual stage parasite growth in similar concentration ranges with ~100-fold selectivity over mammalian cell line; in addition, these compounds effectively inhibited in vitro growth and maturation of hepatocytic stage parasites with EC50 in low nano-molar ranges. These compounds were found to effectively block the development of trophozoite stages to schizont stage, concurrently arresting development and segregation of parasite mitochondria, in asexual as well as hepatocytic stages of the parasite. These morphological and developmental effects of the drugs on parasites mimic the data from genetic ablation of PfClpQ. Importantly, all three compounds showed significant in vivo anti-parasitic efficacies on blood stages of P. berghei mouse malaria model. Overall these results suggest that these PfClpQ targeting compounds are promising candidates to develop new anti-malarials.

microbiology↗

bioRxiv2022

Dual role of an essential HtrA2/Omi protease in the human malaria parasite: maintenance of mitochondrial homeostasis and induction of apoptosis like cell death under cellular stress

HtrA family of serine protease is known to play role in mitochondrial homeostasis as well as in the programmed cell death. Mitochondrial homeostasis and metabolism are crucial for survival and propagation of malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin like protease activity that can be inhibited by its specific inhibitor, ucf-101. Transgenic parasite line was generated, using HA-glmS C-terminal tagging approach, for localization as well as for inducible knockdown of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, ucf-101 treatment had no effect on the parasite growth, suggesting non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase like protease as well as parasite cell death, suggesting involvement of protease activity of PfHtrA2 in apoptosis like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show specific role of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating the stress induced cell death.

cell biology↗

bioRxiv2021

Involvement of translocon complex in hemoglobin import from infected erythrocyte cytoplasm into the Plasmodium parasite

Haemoglobin degradation is crucial for the growth and survival of Plasmodium falciparum in human erythrocytes. Although the process of Hb degradation has been studied in great detail, the mechanisms of Hb uptake remain ambiguous to date. Here, we characterized Heme Detoxification Protein (PfHDP), a protein localized in the parasitophorous vacuole, parasite food vacuole and infected erythrocyte cytosol for its role in Hb uptake. Immunoprecipitation of PfHDP-GFP fusion protein from a transgenic line using anti-GFP antibody and of Plasmodium parasite extract using anti-human Hb antibodies respectively, showed the association of PfHDP/Hb with each other as well as with the members of PTEX translocon complex. Some of these associations such as PfHDP/Hb and PfHDP/Pfexp-2 interactions were confirmed by in vitro protein-protein interaction tools. To know the roles of PfHDP and translocon complex in Hb import into the parasites, we next studied the Hb uptake by the parasite in PfHDP knock-down line using the GlmS ribozyme strategy. PfHDP knock-down significantly reduced the Hb uptake in these parasites in comparison to the wild type parasites. Further, the transient knock-down of one of the members of the translocon complex; PfHSP101 showed considerable reduction in Hb uptake. Morphological analysis of PfHDP-HA-GlmS transgenic parasites in the presence of GlcN showed food vacuole abnormalities and parasite stress, thereby causing a growth defect in the development of these parasites. Together, we implicate the translocon complex in the trafficking of PfHDP/Hb complex in the parasite and suggest a role for PfHDP in the uptake of Hb and parasite development. The study thus reveals new insights into the function of PfHDP, making it an extremely important target for developing new antimalarials.

molecular biology↗

bioRxiv2021

Artemisinin acts by inhibiting Plasmodium falciparum Ddi1, a retropepsin, resulting into the accumulation of ubiquitinated proteins

Reduced sensitivity of the human malaria parasite, Plasmodium falciparum, to Artemisinin and its derivatives (ARTs) threatens the global efforts towards eliminating malaria. ARTs have been shown to cause ubiquitous cellular and genetic insults, which results in the activation of the unfolded protein response (UPR) pathways. The UPR restores protein homeostasis, which otherwise would be toxic to cellular survival. Here, we interrogated the role of DNA-damage inducible protein 1 (PfDdi1), a unique proteasome-interacting retropepsin in mediating the actions of the ARTs. We demonstrate that PfDdi1 is an active A2 family protease that hydrolyzes ubiquitinated substrates. We further show that treatment with ARTs lead to the accumulation of ubiquitinated proteins in the parasites and blocks the destruction of the ubiquitinated substrates by PfDdi1. Besides, whereas the PfDdi1 is predominantly localised in the cytoplasm, exposure of the parasites to ARTs leads to DNA fragmentation and increased recruitment of the PfDdi1 into the nucleus. Furthermore, Ddi1 knock-out Saccharomyces cerevisiae cells are more suceptible to ARTs and the PfDdI1 protein robustly restores the corresponding functions in the knock-out cells. Together, these results show that ARTs act by inducing DNA and protein damage, and impairing the damage recovery by inhibiting the activity of PfDdi1, an essential ubiquitin-proteasome retropepsin.

cell biology↗

bioRxiv2021

A Plasmodium falciparum lysophospholipase regulates fatty acid acquisition for membrane biogenesis to enable schizogonic asexual division

Phospholipid metabolism is crucial for membrane biogenesis and homeostasis during the intracellular life cycle of Plasmodium falciparum. To generate large amounts of phospholipids required during blood stages, the parasite massively scavenge, recycle and reassemble host lipids. P. falciparum possesses an unusual large number of lysophospholipases. However, their functional roles and importance remain to be elucidated. Here, we functionally characterized one of P. falciparum lysophospholipase (PfLPL3) (Gene ID PF3D7_1476800), to reveal its critical role in parasite propagation during asexual blood stages. We generated a transgenic parasite line using GFP-glmS C-terminal tagging approach, for localization as well as inducible knockdown of PfLPL3. PfLPL3 displayed a dynamic localization throughout asexual stages, mainly localizing in the host parasite interface: parasitophorous vacuole space and expanding into the tubulovesicular network within the host cell. Inducible knock-down of PfLPL3 hindered normal intraerythrocytic cycle, specifically causing disruption in parasite development from trophozoites to schizont, as well as reduction in number of merozoites progenies. Thus, down-regulation of PfLPL3 significantly inhibited parasite growth suggesting its critical role for proper parasite propagation during blood stages. Detailed lipidomic analyses showed that PfLPL3 generates fatty-acids for the synthesis of neutral lipids DAG and TAG, whilst controlling the timely synthesis of phospholipids that are crucial for membrane biogenesis required for merozoite development during asexual cycle. Setting up an in vitro activity based screening of Malaria Box allowed identification of specific inhibitors of PfLPL3 having potent parasitical efficacies. These compounds are pertinent both as anti-malarial drug candidates and chemical tools specifically targeting membrane biogenesis during asexual blood stages.

microbiology↗

bioRxiv2019

A lysophospholipase plays role in generation of neutral-lipids required for hemozoin formation in malaria parasite

Phospholipid metabolism is crucial for membrane dynamics in malaria parasites during entire cycle in the host cell. Plasmodium falciparum harbours several members of phospholipase family, which play key role in phospholipid metabolism. Here we have functionally characterized a parasite lysophospholipase (PfLPL1) with a view to understand its role in lipid homeostasis. We used a regulated fluorescence affinity tagging, which allowed endogenous localization and transient knock-down of the protein. PffLPL1localizes to dynamic vesicular structures that traffic from parasite periphery, through the cytosol to get associated as a multi-vesicular neutral lipid rich body next to the food-vacuole during blood stages. Down-regulation of the PfLPL1 disrupted parasite lipid-homeostasis leading to significant reduction of neutral lipids in lipid-bodies. This hindered conversion of heme to hemozoin, leading to food-vacuole abnormalities, which in turn disrupted parasite development cycle and significantly inhibited parasite growth. Detailed lipidomic analyses of inducible knock-down parasites confirmed role of PfLPL1 in generation of neutral lipid through recycling of phospholipids. Our study thus suggests a specific role of PfLPL1 to generate neutral lipids in the parasite, which are essential for parasite survival.\n\nImportancePresent study was undertaken with a view to understand the functional role of a unique lipase (lysophopholipase, PfLPL1) of the human malaria parasite. We utilized genetic approaches for GFP tagging as well as to knock-down the target protein in the parasite. Our studies showed that PfLPL1 associates closely with the lysosome like organelle in the parasite, the food-vacuole. During the blood stage parasite cycle, the food-vacuole is involved in degradation of host haemoglobin and conversion of heme to hemozoin. Genetic knock-down approaches and detailed lipidomic studies confirmed that PfLPL1 protein plays key role in generation of neutral lipid stores in the parasite; neutral lipids are essentially required for hemozoin formation in the parasite, a vital function of the food-vacuole. Overall, this study identified specific role of PfLPL1 in the parasite which is essential for parasite survival.

microbiology↗

bioRxiv2019

ApicoTFdb: The comprehensive web repository of apicomplexan transcription factors and regulators.

Despite significant progress in apicomplexans genome sequencing and genomics, the current list of experimentally validated TFs in these genomes is incomplete and mainly consists of AP2 family of proteins, with only a limited number of non-AP2 family TFs and TAFs. We have performed systematic bioinformatics aided prediction of TFs and TAFs in apicomplexan genomes, and developed ApicoTFdb database which consists of experimentally validated as well as computationally predicted TFs and TAFs in 14 apicomplexan species. The predicted TFs are manually curated to complement the existing annotations. The current version of the database includes 1310 TFs, out of which 833 are novel and computationally predicted TFs, representing 22 distinct families across 14 apicomplexan species. The predictions include TFs of TUB, NAC, BSD, CCAAT, HTH, Cupin/Jumonji, winged-helix, and FHA family proteins, not reported earlier in the genomes. Apart from TFs, ApicoTFdb also classifies TAFs into three main subclasses-TRs, CRRs and RNARs, representing 3047 TAFs in 14 apicomplexan species are analyzed in this study. The database is equipped with a set of useful tools for comparative analysis of a user-defined list of the proteins. ApicoTFdb will be useful to the researchers interested in less-studied gene regulatory mechanisms mediating the complex life cycle of the apicomplexan parasites. The database will aid the discovery of novel drug targets to much needed combat the growing drug resistance in the parasites.

bioinformatics↗