Depletion of Plasmodium berghei plasmoredoxin reveals a non-essential role for life cycle progression of the malaria parasite

Proliferation of the pathogenic Plasmodium asexual blood stages in host erythrocytes requires an exquisite capacity to protect the malaria parasite against oxidative stress. This function is achieved by a complex antioxidant defence system composed of redox-active proteins and low MW antioxidants. Here, we disrupted the P. berghei plasmoredoxin gene that encodes a parasite-specific 22 kDa member of the thioredoxin superfamily. The successful generation of plasmoredoxin knockout mutants in the rodent model malaria parasite and phenotypic analysis during life cycle progression revealed a non-vital role in vivo. Our findings suggest that plasmoredoxin fulfils a specialized and dispensable role for Plasmodium and highlights the need for target validation to inform drug development strategies.     

The physiological roles of autophagy in the mammalian life cycle

Autophagy is primarily an efficient intracellular catabolic pathway used for degradation of abnormal cellular protein aggregates and damaged organelles. Although autophagy was initially proposed to be a cellular stress responder, increasing evidence suggests that it carries out normal physiological roles in multiple biological processes. To date, autophagy has been identified in most organs and at many different developmental stages, indicating that it is not only essential for cellular homeostasis and renovation, but is also important for organ development. Herein, we summarize our current understanding of the functions of autophagy (which here refers to macroautophagy) in the mammalian life cycle.     

Malaria infection of the mosquito Anopheles gambiae activates immune-responsive genes during critical transition stages of the parasite life cycle.

Six gene markers have been used to map the progress of the innate immune response of the mosquito vector, Anopheles gambiae, upon infection by the malaria parasite, Plasmodium berghei. In addition to four previously reported genes, the set of markers included NOS (a nitric oxide synthase gene fragment) and ICHIT (a gene encoding two putative chitin-binding domains separated by a polythreonine-rich mucin region). In the midgut, a robust response occurs at 24 h post-infection, at a time when malaria ookinetes traverse the midgut epithelium, but subsides at later phases of malaria development. In contrast, the salivary glands show no significant response at 24 h, but are activated in a prolonged late phase when sporozoites are released from the midgut into the haemolymph and invade the glands, between 10 and 25 days after blood feeding. Furthermore, the abdomen of the mosquito minus the midgut shows significant activation of immune markers, with complex kinetics that are distinct from those of both midgut and salivary glands. The parasite evidently elicits immune responses in multiple tissues of the mosquito, two of which are epithelia that the parasite must traverse to complete its development. The mechanisms of these responses and their significance for malaria transmission are discussed.     

Plasmodium cysteine repeat modular proteins 1-4: complex proteins with roles throughout the malaria parasite life cycle

The Cysteine Repeat Modular Proteins (PCRMP1-4) of Plasmodium, are encoded by a small gene family that is conserved in malaria and other Apicomplexan parasites. They are very large, predicted surface proteins with multipass transmembrane domains containing motifs that are conserved within families of cysteine-rich, predicted surface proteins in a range of unicellular eukaryotes, and a unique combination of protein-binding motifs, including a >100 kDa cysteine-rich modular region, an epidermal growth factor-like domain and a Kringle domain. PCRMP1 and 2 are expressed in life cycle stages in both the mosquito and vertebrate. They colocalize with PfEMP1 (P. falciparum Erythrocyte Membrane Antigen-1) during its export from P. falciparum blood-stage parasites and are exposed on the surface of haemolymph- and salivary gland-sporozoites in the mosquito, consistent with a role in host tissue targeting and invasion. Gene disruption of pcrmp1 and 2 in the rodent malaria model, P. berghei, demonstrated that both are essential for transmission of the parasite from the mosquito to the mouse and has established their discrete and important roles in sporozoite targeting to the mosquito salivary gland. The unprecedented expression pattern and structural features of the PCRMPs thus suggest a variety of roles mediating host-parasite interactions throughout the parasite life cycle.     

Interactions of the malaria parasite and its mammalian host

A hallmark of Plasmodium development inside its mammalian victim is the remarkable restriction to the host species. Adaptation to an intracellular life style in specific target cells is determined by multiple parasite-host interactions. The first line of crosstalk occurs during intradermal sporozoite injection by an Anopheles mosquito. The following expansion in the liver is highly efficient and leads to successful establishment of the parasite population. During the periodic waves of fevers and chills the parasite destroys and re-infects red blood cells. Recent advances in experimental genetics and imaging techniques begin to expose the complex interactions at the changing parasite-host interfaces. Understanding the cellular and molecular mechanisms of target cell recognition, nutrient acquisition, and hijacking of cellular and immune functions may ultimately explain the elaborate biology of a medically important single cell eukaryote.     

Plasmodium yoelii: axenic development of the parasite mosquito stages

Study of the parasite mosquito stages of Plasmodium and its use in the production of sporozoite vaccines against malaria has been hampered by the technical difficulties of in vitro development. Here, we show the complete axenic development of the parasite mosquito stages of Plasmodium yoelii. While we demonstrate that matrigel is not required for parasite development, soluble factors produced and secreted by Drosophila melanogaster S2 cells appear to be crucial for the ookinete to oocyst transition. Parasites cultured axenically are both morphologically and biologically similar to mosquito-derived ookinetes, oocysts, and sporozoites. Axenically derived sporozoites were capable of producing an infection in mice as determined by RT-PCR; however, the parasitemia was significantly much less than that produced by mosquito-derived sporozoites. Our cell free system for development of the mosquito stages of P. yoelii provides a simplified approach to generate sporozoites that may be for biological assays and genetic manipulations.     

Genome-wide analysis reveals widespread roles for RcREM genes in floral organ development in Rosa chinensis

Members of the REM (Reproductive Meristem) gene family are expressed primarily in reproductive meristems and floral organs. However, their evolution and their functional profiles in flower development remain poorly understood. Here, we performed genome-wide identification and evolutionary analysis of the REM gene family in Rosaceae. This family has been greatly expanded in rose (Rosa chinensis) compared to other species, primarily through tandem duplication. Expression analysis revealed that most RcREM genes are specifically expressed in reproductive organs and that their specific expression patterns are dramatically altered in rose plants with mutations affecting floral organs. Protein-protein interaction analysis indicated that RcREM14 interact with RcAP1 (one of the homology of A class genes in ABCDE model), highlighting the roles of RcREM genes in floral organ identity. Finally, co-expression network analysis indicated that RcREM genes are co-expressed with a high proportion of key genes that regulate flowering time, floral organ development, and cell proliferation and expansion in R. chinensis.     

Variation in apoptosis mechanisms employed by malaria parasites: the roles of inducers, dose dependence and parasite stages

ABSTRACT: BACKGROUND: Plasmodium berghei ookinetes exhibit an apoptotic phenotype when developing within the mosquito midgut lumen or when cultured in vitro. Markers of apoptosis increase when they are exposed to nitric oxide or reactive oxygen species but high concentrations of hydrogen peroxide cause death without observable signs of apoptosis. Chloroquine and other drugs have been used to induce apoptosis in erythrocytic stages of Plasmodium falciparum and to formulate a putative pathway involving cysteine protease activation and mitochondrial membrane permeabilization; initiated, at least in the case of chloroquine, after its accumulation in the digestive vacuole causes leakage of the vacuole contents. The lack of a digestive vacuole in ookinetes prompted the investigation of the effect of chloroquine and staurosporine on this stage of the life cycle. Finally, the suggestion that apoptosis may have evolved as a strategy employed by ookinetes to increase the fitness of surviving parasites was explored by determining whether increasing the ecological triggers parasite density and nutrient depletion induced apoptosis. METHODS: Ookinetes were grown in culture then either exposed to hydrogen peroxide, chloroquine or staurosporine, or incubated at different densities and in different media. The proportion of ookinetes displaying positive markers for apoptosis in treated samples was compared with controls and results were analyzed using analysis of variance followed by a Turkey's test, or a Kruskal-Wallis test as appropriate. RESULTS: Hydrogen peroxide below 50 muM triggered apoptosis but cell membranes were rapidly compromised by higher concentrations, and the mode of death could not be defined. Both chloroquine and staurosporine cause a significant increase in ookinetes with condensed chromatin, caspase-like activity and, in the case of chloroquine, phosphatidylserine translocation and DNA fragmentation (not investigated for staurosporine). However, mitochondrial membrane potential remained intact. No relationship between ookinete density and apoptosis was detected but nutrient depletion significantly increased the proportion of ookinetes with chromatin condensation in four hours. CONCLUSIONS: It is proposed that both a mitochondrial and an amitochondrial apoptotic pathway may be involved, dependent upon the trigger that induces apoptosis, and that pathways may differ between erythrocytic stages and ookinetes, or between rodent and human malaria parasites.     

Distinct malaria parasite sporozoites reveal transcriptional changes that cause differential tissue infection competence in the mosquito vector and mammalian host

The malaria parasite sporozoite transmission stage develops and differentiates within parasite oocysts on the Anopheles mosquito midgut. Successful inoculation of the parasite into a mammalian host is critically dependent on the sporozoite's ability to first infect the mosquito salivary glands. Remarkable changes in tissue infection competence are observed as the sporozoites transit from the midgut oocysts to the salivary glands. Our microarray analysis shows that compared to oocyst sporozoites, salivary gland sporozoites upregulate expression of at least 124 unique genes. Conversely, oocyst sporozoites show upregulation of at least 47 genes (upregulated in oocyst sporozoites [UOS genes]) before they infect the salivary glands. Targeted gene deletion of UOS3, encoding a putative transmembrane protein with a thrombospondin repeat that localizes to the sporozoite secretory organelles, rendered oocyst sporozoites unable to infect the mosquito salivary glands but maintained the parasites' liver infection competence. This phenotype demonstrates the significance of differential UOS expression. Thus, the UIS-UOS gene classification provides a framework to elucidate the infectivity and transmission success of Plasmodium sporozoites on a whole-genome scale. Genes identified herein might represent targets for vector-based transmission blocking strategies (UOS genes), as well as strategies that prevent mammalian host infection (UIS genes).     

The journey of the malaria parasite in the mosquito: hopes for the new century

In this review, Anil Ghosh, Marten Edwards and Marcelo Jacobs-Lorena follow the journey of the Plasmodium parasite in the mosquito vector. At each developmental step, they highlight some of the major unanswered questions currently challenging cell and molecular biologists. A more thorough understanding of Plasmodium-mosquito interactions might lead to the development of mosquitoes unable to support parasite development.     

Changing roles of aurora-B kinase in two life cycle stages of Trypanosoma brucei

Aurora-B kinase is a chromosomal passenger protein essential for chromosome segregation and cytokinesis. In the procyclic form of Trypanosoma brucei, depletion of an aurora-B kinase homologue TbAUK1 inhibited spindle formation, mitosis, cytokinesis, and organelle replication without altering cell morphology. In the present study, an RNA interference knockdown of TbAUK1 or overexpression of inactive mutant TbAUK1-K58R in the bloodstream form also resulted in defects in spindle formation, chromosome segregation, and cytokinesis but allowed multiple rounds of nuclear DNA synthesis, nucleolus multiplication, and continuous replication of kinetoplast, basal body, and flagellum. The typical trypanosome morphology was lost to an enlarged round shape filled with microtubules. It is thus apparent that there are distinctive mechanisms of action of TbAUK1 in regulating cell division between the two developmental stages of trypanosome. While it exerts a tight control on mitosis, organelle replication, and cytokinesis in the procyclic form, it regulates cytokinesis without rigid control over either nuclear DNA synthesis or organelle replication in the bloodstream form. The molecular basis underlining these discrepancies remains to be explored.     

Cell cycle analysis of asexual stages of erythrocytic malaria parasites

Abstract. Intra-erythrocytic Plasmodium species can be stained with the DNA binding dye, Hoechst 33342, and the distribution of DNA content determined for parasite populations by flow cytometric measurement of fluorescence. Analysis of this distribution will determine the parasitaemia (percentage of erythrocytes infected), and the percentages of trophozoite infected red blood cells, polyparasitized (trophozoite) red blood cells, and schizont/segmenter infected red blood cells. This analysis is based on the hypothesis that the asexual parasites cycle with single G₁ period, and effectively, a single S phase with no significant G₂/M period except at schizogony when the genome DNA content is equivalent to 8 N or higher, dependent on the species. Data are presented to support this model.     

Genome-wide analysis reveals the functional and expressional correlation between RhoGAP and RhoGEF in mouse

Rho GTPases play essential roles in various life activities. Rho GTPase-activating protein (RhoGAP) and Rho guanine nucleotide exchange factor (RhoGEF) are the main regulators of Rho GTPases. RhoGAP, RhoGEF and Rho make up a molecular switch and exert crucial roles in signaling pathways. The genome-wide studies can provide us a comprehensive information of special protein family, but the genome-wide information of RhoGAP and RhoGEF families are still lacking in the mammal lineage. Here, we report the correlations between mouse RhoGAPs and RhoGEFs in gene quantities, evolution, molecular function, and their expression levels in heart embryonic development and cardiovascular medicine treatment at genome-wide scale. Besides, we find that the 3D structures of RhoGAP domains between different species are highly conserved, but that of RhoGEF domains are variable between species. Our present study contributes to a better understanding of the complex regulation mechanisms of RhoGAP and RhoGEF families.     

Low levels of mammalian TGF-beta1 are protective against malaria parasite infection, a paradox clarified in the mosquito host

Nitric oxide (NO), derived from catalysis of inducible NO synthase (iNOS), limits malaria parasite growth in mammals. Transforming growth factor (TGF)-beta1 suppresses iNOS in cells in vitro as well as in vivo in mice, but paradoxically severe malaria in humans is associated with low levels of TGF-beta1. We hypothesized that this paradox is a universal feature of infection and occurs in the mosquito Anopheles stephensi, an invertebrate host for Plasmodium that also regulates parasite development with inducible NO synthase (AsNOS). We show that exogenous human TGF-beta1 dose-dependently regulates mosquito AsNOS expression and that parasite killing by low dose TGF-beta1 depends on AsNOS catalysis. Furthermore, induction of AsNOS expression by TGF-beta1 is regulated by NO synthesis. These results suggest that TGF-beta1 plays similar roles during parasite infection in mammals and mosquitoes and that this role is linked to the effects of TGF-beta1 on inducible NO synthesis.     

Identification of Echinococcus granulosus microRNAs and their expression in different life cycle stages and parasite genotypes

The aetiological agent of cystic hydatid disease, the platyhelminth parasite Echinococcus granulosus, undergoes a series of metamorphic events during its complex life cycle. One of its developmental stages, the protoscolex, shows a remarkable degree of heterogeneous morphogenesis, being able to develop either into the vesicular or strobilar direction. Another level of complexity is added by the existence of genotypes or strains that differ in the range of intermediate hosts where they can develop and form fertile cysts. These features make E. granulosus an interesting model for developmental studies. Hence, we focused on the study of the regulation of gene expression by microRNAs (miRNAs), one of the key mechanisms that control development in metazoans and plants and which has not been analysed in E. granulosus yet. In this study, we cloned 38 distinct miRNAs, including four candidate new miRNAs that seem to be specific to Echinococcus spp. Thirty-four cloned sequences were orthologous to miRNAs already described in other organisms and were grouped in 16 metazoan miRNA families, some of them known for their role in the development of other organisms. The expression of some of the cloned miRNAs differs according to the parasite life cycle stage analysed, showing differential developmental expression. We did not detect differences in the expression of the analysed miRNAs between protoscoleces of two parasite genotypes. This work sets the scene for the study of gene regulation mediated by miRNAs in E. granulosus and provides a new approach to study the molecules involved in its developmental plasticity and intermediate host specificity. Understanding the developmental processes of E. granulosus may help to find new strategies for the control of cystic hydatid disease, caused by the metacestode stage of the parasite.