The nuclear effector ArPEC25 from the necrotrophic fungus Ascochyta rabiei targets the chickpea transcription factor CaßLIM1a and negatively modulates lignin biosynthesis, increasing host susceptibility.

Fungal pathogens deploy a barrage of secreted effectors to subvert host immunity, often by evading, disrupting, or altering key components of transcription, defense signaling, and metabolic pathways. However, the underlying mechanisms of effectors and their host targets are largely unexplored in necrotrophic fungal pathogens. Here, we describe the effector protein Ascochyta rabiei PEXEL-like Effector Candidate 25 (ArPEC25), which is secreted by the necrotroph A. rabiei, the causal agent of Ascochyta blight disease in chickpea (Cicer arietinum), and is indispensable for virulence. After entering host cells, ArPEC25 localizes to the nucleus and targets the host LIM transcription factor CaßLIM1a. CaßLIM1a is a transcriptional regulator of CaPAL1, which encodes phenylalanine ammonia lyase (PAL), the regulatory, gatekeeping enzyme of the phenylpropanoid pathway. ArPEC25 inhibits the transactivation of CaßLIM1a by interfering with its DNA-binding ability, resulting in negative regulation of the phenylpropanoid pathway and decreased levels of intermediates of lignin biosynthesis, thereby suppressing lignin production. Our findings illustrate the role of fungal effectors in enhancing virulence by targeting a key defense pathway that leads to the biosynthesis of various secondary metabolites and antifungal compounds. This study provides a template for the study of less explored necrotrophic effectors and their host target functions.

Essential role of a Plasmodium berghei heat shock protein (PBANKA_0938300) in gametocyte development.

The continued existence of Plasmodium parasites in physiologically distinct environments during their transmission in mosquitoes and vertebrate hosts requires effector proteins encoded by parasite genes to provide adaptability. Parasites utilize their robust stress response system involving heat shock proteins for their survival. Molecular chaperones are involved in maintaining protein homeostasis within a cell during stress, protein biogenesis and the formation of protein complexes. Due to their critical role in parasite virulence, they are considered targets for therapeutic interventions. Our results identified a putative P. berghei heat shock protein (HSP) belonging to the HSP40 family (HspJ62), which is abundantly induced upon heat stress and expressed during all parasite stages. To determine the role HspJ62, a gene-disrupted P. berghei transgenic line was developed (ΔHspJ62), which resulted in disruption of gametocyte formation. Such parasites were unable to form subsequent sexual stages because of disrupted gametogenesis, indicating the essential role of HspJ62 in gametocyte formation. Transcriptomic analysis of the transgenic line showed downregulation of a number of genes, most of which were specific to male or female gametocytes. The transcription factor ApiAP2 was also downregulated in ΔHspJ62 parasites. Our findings suggest that the downregulation of ApiAP2 likely disrupts the transcriptional regulation of sexual stage genes, leading to impaired gametogenesis. This finding also highlights the critical role that HspJ62 indirectly plays in the development of P. berghei sexual stages and in facilitating the conversion from the asexual blood stage to the sexual stage. This study characterizes the HspJ62 protein as a fertility factor because parasites lacking it are unable to transmit to mosquitoes. This study adds an important contribution to ongoing research aimed at understanding gametocyte differentiation and formation in parasites. The molecule adds to the list of potential drug targets that can be targeted to inhibit parasite sexual development and consequently parasite transmission.

STK35L1 regulates host cell cycle-related genes and is essential for Plasmodium infection during the liver stage of malaria.

Protein kinases of both the parasite and the host are crucial in parasite invasion and survival and might act as drug targets against drug-resistant malaria. STK35L1 was among the top five hits in kinome-wide screening, suggesting its role in malaria's liver stage. However, the role of host STK35L1 in malaria remains elusive. In this study, we found that STK35L1 was highly upregulated during the infection of Plasmodium berghei (P. berghei) in HepG2 cells and mice liver, and knockdown of STK35L1 remarkably suppressed the sporozoites' infection in HepG2 cells. We showed that STAT3 is upregulated and phosphorylated during P. berghei sporozoites' infection, and STAT3 activation is required for both the upregulation of STK35L1 and STAT3. Furthermore, we found that ten cell cycle genes were upregulated in the sporozoite-infected hepatocytes. Knockdown of STK35L1 inhibited the basal expression of these genes except CDKN3 and GTSE1 in HepG2 cells. Thus, we identified STK35L1 as a host kinase that plays an obligatory role in malaria's liver stage and propose that it may serve as a potential drug target against drug-resistant malaria.

Plasmodium berghei-Released Factor, PbTIP, Modulates the Host Innate Immune Responses.

The Plasmodium parasite has to cross various immunological barriers for successful infection. Parasites have evolved mechanisms to evade host immune responses, which hugely contributes to the successful infection and transmission by parasites. One way in which a parasite evades immune surveillance is by expressing molecular mimics of the host molecules in order to manipulate the host responses. In this study, we report a Plasmodium berghei hypothetical protein, PbTIP (PbANKA_124360.0), which is a Plasmodium homolog of the human T-cell immunomodulatory protein (TIP). The latter possesses immunomodulatory activities and suppressed the host immune responses in a mouse acute graft-versus-host disease (GvHD) model. The Plasmodium berghei protein, PbTIP, is expressed on the merozoite surface and exported to the host erythrocyte surface upon infection. It is shed in the blood circulation by the activity of an uncharacterized membrane protease(s). The shed PbTIP could be detected in the host serum during infection. Our results demonstrate that the shed PbTIP exhibits binding on the surface of macrophages and reduces their inflammatory cytokine response while upregulating the anti-inflammatory cytokines such as TGF-ß and IL-10. Such manipulated immune responses are observed in the later stage of malaria infection. PbTIP induced Th2-type gene transcript changes in macrophages, hinting toward its potential to regulate the host immune responses against the parasite. Therefore, this study highlights the role of a Plasmodium-released protein, PbTIP, in immune evasion using macrophages, which may represent the critical strategy of the parasite to successfully survive and thrive in its host. This study also indicates the human malaria parasite TIP as a potential diagnostic molecule that could be exploited in lateral flow-based immunochromatographic tests for malaria disease diagnosis.

Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti.

Babesia microti, an emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human. Stable transfection technique has been reported in many protozoan parasites over the past few years. However, in vivo transient and stable transfection method has not been established for Babesia microti. Here, for the first time, we present a method of transient as well as stable transfection of the Babesia microti (B. microti) in the in vivo conditions. We have identified a novel promoter of B. microti. We also demonstrated that Plasmodium berghei DHFR promoter is recognized and functional in B. microti. We show that BM-CTQ41297 promoter control the expression of two genes, which are present on either side and thus represents a bi-functional promoter in B. microti. The predicted promoter activity values using Promoter 2.0 program is higher for BM- CTQ41297 promoter than strong promoters such as ß-actin, ef-1ß, and many other promoters. Furthermore, we discovered a non-essential locus for the genetic manipulation of the parasite, allowing us to stably integrate foreign genes; GFP, mCherry, into the B. microti. The transfection using an electroporation method and genetic manipulation of B. microti is now achievable and it is possible to obtain transfected viable parasites under in vivo growing conditions. The growth curve analysis of transfected and WT B. microti are similar indicating no defects in the transgenic parasites. This study will enable other researchers in understanding the B. microti biology, host modulation and diverse parasite developmental stages using reverse genetics and holds great potential to identify novel drug targets and vaccine development.

Potent in vivo antimalarial activity of water-soluble artemisinin nano-preparations.

Artemisinin is a remarkable compound whose derivatives and combinations with multiple drugs have been utilized at the forefront of malaria treatment. However, the inherent issues of the parent compound such as poor bioavailability, short serum half-life, and high first-pass metabolism partially limit further applications of this drug. In this study, we enhanced the aqueous phase solubility of artemisinin by encapsulating it in two nanocarriers based on the polymer polycaprolactone (ART-PCL) and lipid-based Large Unilamellar Vesicles (ART-LIPO) respectively. Both nanoformulations exhibit in vitro parasite killing activity against Plasmodium falciparum with the ART-LIPO performing at comparable efficacy to the control drug solubilized in ethanol. These water-soluble formulations showed potent in vivo antimalarial activity as well in the mouse model of malaria at equivalent doses of the parent drug. Additionally, the artemisinin-PCL nanoformulation used in combination with either pyrimethamine or chloroquine increased the survival of the Plasmodium berghei infected mice for more than 34 days and effectively cured the mice of the infection. We highlight the potential for polymer and liposome-based nanocarriers in improving not only the aqueous phase solubility of artemisinin but also concomitantly retaining its therapeutic efficacy in vivo as well.

Multistage antiplasmodial activity of hydroxyethylamine compounds, in vitro and in vivo evaluations.

Malaria, a global threat to the human population, remains a challenge partly due to the fast-growing drug-resistant strains of Plasmodium species. New therapeutics acting against the pathogenic asexual and sexual stages, including liver-stage malarial infection, have now attained more attention in achieving malaria eradication efforts. In this paper, two previously identified potent antiplasmodial hydroxyethylamine (HEA) compounds were investigated for their activity against the malaria parasite's multiple life stages. The compounds exhibited notable activity against the artemisinin-resistant strain of P. falciparum blood-stage culture with 50% inhibitory concentrations (IC50) in the low micromolar range. The compounds' cytotoxicity on HEK293, HepG2 and Huh-7 cells exhibited selective killing activity with IC50 values > 170 µM. The in vivo efficacy was studied in mice infected with P. berghei NK65, which showed a significant reduction in the blood parasite load. Notably, the compounds were active against liver-stage infection, mainly compound 1 with an IC50 value of 1.89 µM. Mice infected with P. berghei sporozoites treated with compound 1 at 50 mg kg-1 dose had markedly reduced liver stage infection. Moreover, both compounds prevented ookinete maturation and affected the developmental progression of gametocytes. Further, systematic in silico studies suggested both the compounds have a high affinity towards plasmepsin II with favorable pharmacological properties. Overall, the findings demonstrated that HEA and piperidine possessing compounds have immense potential in treating malarial infection by acting as multistage inhibitors.

A novel FIKK kinase regulates the development of mosquito and liver stages of the malaria.

Protein phosphorylation is the most important post-translational event in the regulation of various essential signaling pathways in a cell. Here, we show the functional characterization of a FIKK family protein kinase of the rodent malaria parasite (PbMLFK), which is expressed only in mosquito and liver stages and contains two functional C-terminal PEXEL motifs. We demonstrate that this protein plays a role in mosquito and liver stages of parasite growth. The oocysts of PbMLFK-deficient parasites produced 4-fold fewer sporozoites. In the liver of infected mice, PbMLFK-deficient parasites grew 100-fold less than did wild type parasites. We also show that the C-terminal domain of this protein has a functional serine-threonine kinase and that its activity was inhibited by a known PKA inhibitor. Transcriptome analysis of infected host cells suggests that in absence of this protein expression of the 288 host mRNAs are perturbed which are primarily associated with the immune system, cell cycle and metabolism.

Corrigendum: Liver-Stage Specific Response among Endemic Populations: Diet and Immunity.

[This corrects the article on p. 125 in vol. 6, PMID: 25852693.].

A Sporozoite- and Liver Stage-expressed Tryptophan-rich Protein Plays an Auxiliary Role in Plasmodium Liver Stage Development and Is a Potential Vaccine Candidate.

The liver stages of the malaria parasite are clinically silent and constitute ideal targets for causal prophylactic drugs and vaccines. Cellular and molecular events responsible for liver stage development are poorly characterized. Here, we show that sporozoite, liver stage tryptophan-rich protein (SLTRiP) forms large multimers. Mice immunized with a purified recombinant SLTRiP protein gave high antibody titers in both inbred and outbred mice. Immunized mice showed highly significant levels of protection upon challenge with sporozoites and exhibited 10,000-fold fewer parasite 18S-rRNA copy numbers in their livers. The protection offered by immunization with SLTRiP came mainly from T-cells, and antibodies had little role to play despite their high titers. Immunofluorescence assays showed that SLTRiP is expressed in the sporozoite and early to late liver stages of malaria parasites. SLTRiP protein is exported to the cytosol of infected host cells during the early hours of parasite infection. Parasites deficient in SLTRiP were moderately defective in liver stage parasite development. A transcriptome profile of SLTRiP-deficient parasite-infected hepatocytes highlighted that SLTRiP interferes with multiple pathways in the host cell. We have demonstrated a role for SLTRiP in sporozoites and the liver stage of malaria parasites.

Liver-Stage Specific Response among Endemic Populations: Diet and Immunity.

Developing effective anti-malarial vaccine has been a challenge for long. Various factors including complex life cycle of parasite and lack of knowledge of stage specific critical antigens are some of the reasons. Moreover, inadequate understanding of the immune responses vis-à-vis sterile protection induced naturally by Plasmodia infection has further compounded the problem. It has been shown that people living in endemic areas take years to develop protective immunity to blood stage infection. But hardly anyone believes that immunity to liver-stage infection could be developed. Various experimental model studies using attenuated parasite suggest that liver-stage immunity might exist among endemic populations. This could be induced because of the attenuation of parasite in liver by various compounds present in the diet of endemic populations.

Apicoplast triose phosphate transporter (TPT) gene knockout is lethal for Plasmodium.

The C3, C5, C6 type sugar phosphate transporters bring sugars inside apicoplast, thus providing energy, reducing power and elements like carbon to apicoplast. Plasmodium berghei has two C3 type sugar phosphate transporters in the membrane of apicoplast: triose phosphate transporter (TPT) and phosphoenolpyruvate transporter (PPT). Here we report that P. berghei TPT knockout parasites failed to survive. However, PPT knockout parasite behaved similar to the wild type in the blood stages. The absence of PPT in other life stages, leads to defects in the development of parasite and was required at both mosquito as well as liver stages. This study also underlines the essentiality of triose transporters for apicoplast and its downstream pathways.

Lipophilic bisphosphonates are potent inhibitors of Plasmodium liver-stage growth.

Nitrogen-containing bisphosphonates, drugs used to treat bone resorption diseases, also have activity against a broad range of protists, including blood-stage Plasmodium spp. Here, we show that new-generation "lipophilic" bisphosphonates designed as anticancer agents that block protein prenylation also have potent activity against Plasmodium liver stages, with a high (>100) therapeutic index. Treatment of mice with the bisphosphonate BPH-715 and challenge with Plasmodium berghei sporozoites revealed complete protection (no blood-stage parasites after 28 days). There was also activity against blood-stage forms in vitro and a 4-day delay in the prepatent period in vivo. The lipophilic bisphosphonates have activity against a Plasmodium geranylgeranyl diphosphate synthase (GGPPS), as well as low nM activity against human farnesyl and geranylgeranyl diphosphate synthases. The most active inhibitor in vitro and in vivo had enzyme inhibitory activity similar to that of the other, less active compounds but was more lipophilic. Lipophilic bisphosphonates are thus promising leads for novel antimalarials that target liver-stage infection.

Triclosan inhibit the growth of the late liver-stage of Plasmodium.

Annually, approximately two million human deaths are caused worldwide by malaria, most of them being children. Plasmodium falciparum is the leading cause of cerebral malaria, the most severe and fatal form of disease. Moreover, the emergence of resistant strains to the existing drugs has worsened the situation. Currently, primaquine is the only drug available for eliminating liver-stage parasites. Because of the emergence of resistant parasite strains, it becomes necessary to find new targets unique to the malaria parasites. In the Plasmodium species, the discovery of a distinct Type-II fatty-acid synthesis pathway has created an opportunity to target this pathway for the development of new inhibitors of malaria parasite growth. The present study explored the growth inhibition potential of triclosan in the case of liver-stage parasites. Liver-stage of Plasmodium is an excellent target for intervention due to very small parasite load as well as possibility of eliminating parasites before it can cause blood-stage infection. Here we report that triclosan inhibits the development of the Plasmodium liver-stage parasites.

Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite.

The liver stages of malaria are clinically silent but have a central role in the Plasmodium life cycle. Liver stages of the parasite containing thousands of merozoites grow inside hepatocytes for several days without triggering an inflammatory response. We show here that Plasmodium uses a PEXEL/VTS motif to introduce the circumsporozoite (CS) protein into the hepatocyte cytoplasm and a nuclear localization signal (NLS) to enter its nucleus. CS outcompetes NFkappaB nuclear import, thus downregulating the expression of many genes controlled by NFkappaB, including those involved in inflammation. CS also influences the expression of over one thousand host genes involved in diverse metabolic processes to create a favorable niche for the parasite growth. The presence of CS in the hepatocyte enhances parasite growth of the liver stages in vitro and in vivo. These findings have far reaching implications for drug and vaccine development against the liver stages of the malaria parasite.