Satish Mishra

Malaria parasites must undergo complex developmental transitions to complete their life cycle and transmit between vertebrate and mosquito hosts. These transitions are tightly regulated by protein phosphorylation events, yet the specific kinases involved remain poorly characterized. Here, we investigate the role of a previously uncharacterized tyrosine kinase-like protein, TKL3, in the rodent malaria parasite Plasmodium berghei . We show that TKL3 is expressed in blood stages, schizonts, gametocytes, zygotes, and ookinetes and localizes to the cytoplasm. Targeted disruption of the TKL3 gene impairs asexual blood-stage growth, male gamete fertility, and consequently compromises ookinete development. Although oocyst numbers are significantly reduced in mosquitoes, the sporozoites that do form are morphologically normal, retain hepatocyte infectivity, and complete liver-stage development. However, TKL3 knockout sporozoites showed delayed blood-stage patency due to reduced asexual replication. These findings identify TKL3 as an important regulator of parasite growth and transmission, providing new insights into kinases involved in the Plasmodium life cycle.

Malaria parasites undergo extensive replication in hepatocytes before initiating the pathogenic blood stage of infection. Although this clinically silent phase is essential for parasite expansion, the molecular mechanisms regulating late liver stage maturation remain incompletely understood. Here, we characterize a previously unannotated protein (PBANKA_0620800), which we designate liver stage maturation protein (LSMP). LSMP was expressed during schizont, sporozoite, and liver stages and localized predominantly to the parasite cytosol. Using reverse genetic approaches, we generated LSMP KO parasites and examined their development across the parasite life cycle. Loss of LSMP did not affect asexual blood‐stage replication or mosquito‐stage development, including ookinete formation, oocyst development, and sporozoite formation. LSMP KO sporozoites invaded hepatocytes normally but exhibited pronounced defects during late liver stage development, including reduced nuclear replication, impaired merozoite formation, and delayed rupture of the parasitophorous vacuole membrane. Together, our findings demonstrate that LSMP is important for malaria liver stage maturation.

Autophagy is a highly conserved degradative and recycling pathway essential for maintaining cellular homeostasis. Although its molecular machinery is well characterized in yeast and mammalian systems, it is less studied in the early‐diverging apicomplexan parasite Plasmodium , the causative agent of malaria. Plasmodium possesses a reduced yet functional repertoire of autophagy‐related (ATG) proteins, suggesting adaptations of this pathway to parasite‐specific biology. Among these, ATG8, a ubiquitin‐like protein, has emerged as a central marker and key effector of plasmodial autophagy. Its branched localization and association with the relict plastid (apicoplast) membrane indicate roles beyond canonical degradative autophagy, particularly in organelle maintenance and biogenesis. ATG7, an essential E1‐like enzyme, activates ATG8 and facilitates its lipidation, thereby regulating organelle turnover and development. This process is further supported by a conserved conjugation system involving ATG3 (E2‐like enzyme) and the ATG12, ATG5, ATG16 complex, functioning as a ligase to enable ATG8 membrane association. ATG4, a cysteine protease, is critical for recycling lipidated ATG8 and maintaining its cytosolic pool, while the homolog Otu can partially compensate for its function. ATG18 also plays an important role in apicoplast biogenesis and maintenance. Collectively, these findings highlight both canonical and non‐canonical roles of autophagy proteins in Plasmodium , driving metabolic reprogramming, intracellular remodeling, and stage‐specific differentiation, and support their potential as targets for new antimalarial therapies.

DNA ligases are a fundamental class of enzymes required for DNA replication and repair. They catalyze the formation of phosphodiester bonds, specifically at single-strand breaks in double-stranded DNA. The nuclear genome of malaria parasites encodes a single DNA ligase that is likely involved in nuclear and organellar DNA replication and repair. DNA ligase I from Plasmodium falciparum ( Pf Lig1) has been biochemically characterized and shown to possess nick-sealing activity. However, its localization and function in the three genome-containing compartments—the nucleus, apicoplast, and mitochondrion—of the malaria parasites remain unknown. Here, we found that Lig1 is located primarily in the nucleus in both human and rodent malaria parasites throughout the parasite life cycle. Furthermore, we detected its presence in organelles via a chromatin immunoprecipitation-PCR assay. Our attempts to disrupt Plasmodium berghei Lig1 ( Pb Lig1) in the blood stages have failed, indicating that the gene is likely essential. Next, we used an Flp/FRT-based conditional mutagenesis system that silences gene function in sporozoites. We demonstrated that Pb Lig1 is essential for parasite liver-stage development. Sporozoites lacking Pb Lig1 invade hepatocytes but arrest growth during mid-liver-stage development. Pb Lig1 cKO parasites undergo limited nuclear division and present a reduced DNA content that fails to increase beyond mid-liver stage of development. These data suggest that Lig1 is an essential enzyme for parasite blood- and liver-stage development. IMPORTANCE: Unlike mammalian cells that possess multiple DNA ligases, the malaria parasite’s nuclear genome encodes a single DNA ligase. This single DNA ligase is likely involved in both DNA replication and DNA repair. However, the importance of parasite DNA ligase remains largely unknown. Here, we show that Plasmodium Lig1 is primarily found within the nucleus, but it also exhibits a distribution across parasite organelles. Knockout of Pb Lig1 in sporozoites abolishes parasite liver-stage development, preventing the formation of hepatic merozoites and ultimately blocking the transition from the liver to the blood stage of infection. More specifically, Pb Lig1 is essential for nuclear division during hepatic schizogony. These findings enhance our understanding of the role of DNA ligase I in malaria parasite liver-stage development.

The 6‐kb linear repeat genome of the mitochondrion (mtDNA) of the malaria parasite is among the smallest known in nature, but is well‐conserved in comparison with its apicoplast and nuclear genomes. Except for the presence of base excision repair (BER) and two double‐strand break repair (DSBR) proteins in mitochondria, the mechanisms for preservation of mtDNA integrity during traversal of the parasite through different cell types and environments in the mosquito vector and mammalian host are not characterized. We identified two putative organellar exonucleases in Plasmodium falciparum , Pf Exo _mit1 and Pf Exo _mit2 , with homologs present only within certain alveolates. Immunofluorescence localization and chromatin immunoprecipitation experiments using antibodies generated against recombinant proteins showed that they are localized to the mitochondrion. Pf Exo _mit1 and Pf Exo _mit2 demonstrated specificity for different DNA substrates; Pf Exo _mit1 cleaved ssDNA in both polarities, while Pf Exo _mit2 was a bipolar exonuclease on dsDNA with 3′‐5′ exonuclease activity on ssDNA. The mismatch repair (MMR) protein Pf MutS, which carries an additional endonuclease domain, was localized in the mitochondria and interacted with Pf Exo _mit2 in pull‐down assays. Pf Exo _mit2 also interacted with the mitochondria‐targeted DSBR protein Pf Rad51, suggesting that it is a component of both MMR and DSBR pathways. When Exo _mit1 expression in the rodent parasite P. berghei was silenced in sporozoites via conditional mutagenesis, Pb Exo _mit1 conditional knockout sporozoites invaded hepatocytes and developed in the liver, but could not transition to the blood stage. Pb Exo _mit1 localized to the mitochondria in liver stages as well, indicating that its ssDNA exonuclease function in mtDNA processing in the liver impacted establishment of blood‐stage infection.

Protozoan parasites of the genus Plasmodium cause malaria and involve infection of multiple hosts and cell types during the life cycle. Producing sexually fit gametocytes is essential for transmitting the Plasmodium parasite into an anopheline mosquito vector. After the uptake of malaria parasites, male gametocytes undergo three rounds of DNA replication to produce eight nucleated flagellar gametes. Here, we report that the actin-like proteins Alp5a and Alp5b are involved in DNA segregation during male gametogenesis. The Plasmodium -specific Alp5a and Alp5b can be superimposed on human Arp2 and Arp3, localize to the nucleus, and interact with each other. Alp5a and Alp5b are individually dispensable for the development of P. berghei blood stages, but are simultaneously indispensable for parasite viability. Consistent with genetic studies, the inhibitory activity of the Arp2/3 complex inhibitor in Plasmodium supports an essential role for this complex during the blood stage. Deletion of Alp5a or Alp5b had no impact on actin nucleation, parasite growth, or gametocytemia during the blood stage. The knockout parasites were able to invade the mosquito midgut and form oocysts; however, these oocysts were significantly smaller in size and failed to mature, ultimately leading to their death. Genetic crosses revealed defects in male gamete integrity. We found that the reduced oocyst development was due to impaired DNA segregation during male gametogenesis. Our study provides molecular insights into the fundamental requirements of the Alps in Plasmodium , which are essential for malaria transmission.

Malaria parasites develop within a specialized vacuole delimited by the parasitophorous vacuolar membrane (PVM). The exit of Plasmodium from host cells is a well-regulated process involving several molecules that disrupt the PVM to continue the life cycle. While several protein classes play a role in Plasmodium PVM rupture and merozoite egress, the first class of enzymes one would consider to act on membranes is lipases. However, the role of a lipase in PVM disruption remains unknown. Here, we characterized the role of UIS28 (upregulated in infective sporozoite gene 28) in Plasmodium berghei. Bioinformatic studies indicated that UIS28 is a class 3 lipase containing a fungal lipase-like domain. We found that UIS28 localizes to the PVM in infected hepatocytes. To understand the role of UIS28 in PVM disruption, we validated its lipase activity and found that it breaks down lipids into glycerol. Parasites lacking UIS28 develop normally in the blood and mosquito stages and mature fully into hepatic merozoites but show a defect in egress from host hepatocytes. We quantified the PVM rupture and reported that mutant parasites exhibit delayed egress due to impaired PVM disruption. Together, our results demonstrate that UIS28 is a lipase involved in the disruption of the PVM and the successful egress of hepatic merozoites. Author Summary: An essential step in the life cycle of malaria parasites is their egress from hepatocytes, allowing them to transition from the liver stage to the blood stage. Liver stage parasites reside in a PV, which is surrounded by a PVM. Parasites must breakdown the PVM before they can infect other cells. This complex, multistep process is orchestrated by the parasite and involves several molecules to disrupt the PVM. However, little is known about these molecules. Here, we validated that UIS28 is indeed a lipase. We show that UIS28 is expressed in sporozoites and in the liver stage, and that it localizes to the PVM of parasites within hepatocytes. Disruption of UIS28 gene does not affect blood and mosquito stage development. However, mutant parasites show a defect in the disruption of the PVM and egress of hepatic merozoites. For the first time, we demonstrate the role of a lipase in PVM rupture during the release of liver-stage merozoites.

Malonyl-CoA-acyl carrier protein transacylase (MCAT) catalyzes the transfer of a malonyl moiety from malonyl–CoA to acyl carrier protein during the initiation step of type II fatty acid synthesis (FASII). The Plasmodium FASII pathway was found to be essential for late liver-stage development in rodent malaria parasites. Here, we generated a novel genetically attenuated parasite (GAP) by disrupting Plasmodium MCAT. Deleting MCAT in rodent malaria parasites did not affect asexual blood-stage propagation and mosquito-stage development. MCAT KO sporozoites failed to initiate blood-stage infection in mice. Hepatic MCAT KO parasites showed impaired nuclear division and apicoplast biogenesis. This led to a defect in hepatic merozoite formation and attenuation of parasites during late liver stages. Vaccination of mice with MCAT KO sporozoites exhibited sterilizing immunity against homologous and heterologous species challenge. Further, MCAT KO-immunized mice were able to clear blood stage infection after iRBCs challenge. These findings highlight that late-liver arresting MCAT KO sporozoite is a promising GAP vaccine candidate for inducing pre-erythrocytic, stage, and species-transcending protection in mice.