Plasmodium sexual stage antigens

The sexual stages of the malaria parasite are highly specialized cells adapted to withstand major environmental changes during their development. They also induce immune responses that may affect the outcome of the infection in the mosquito. In this review Pietro Alano considers the nature and the role of the antigens expressed by Plasmodium sexual stages.     

Structure of a gametocyte protein essential for sexual development in Plasmodium falciparum

Malaria transmission is dependent on the development of sexual forms of Plasmodium falciparum, called gametocytes, in the vertebrate host. Pfg27 is an abundantly expressed sexual stage-specific protein that is essential for gametocytogenesis in P. falciparum. We describe the crystal structure of Pfg27, which reveals a novel fold composed of two pseudo dyad-related repeats of the helix-turn-helix motif. Structurally equivalent helices of each repeat either form a dimer interface or interact with RNA in vitro. One side of the dimer presents an unprecedented juxtaposition of four polyproline (PXXP) motifs. Preliminary binding data indicate that these sites are capable of binding Src homology-3 (SH3) modules. Molecular modeling suggests that the dimer can accommodate two SH3 modules simultaneously, potentially enabling molecular crosstalk between SH3-containing proteins. The structural and initial biochemical evidence suggests that Pfg27 may serve as a platform for RNA and SH3 binding.     

Genome plasticity and sexual differentiation in Plasmodium

Spontaneous subtelomeric deletions of Plasmodium chromosomes have been observed both in natural infections and in laboratory maintained parasites. In the latter case, functions dispensable for asexual parasite multiplication and encoded at the extremities of the chromosomes are easily lost. In particular, spontaneous subtelomeric deletions have been characterised which affect gametocytogenesis both in Plasmodium berghei maintained in laboratory animals and in Plasmodium falciparum propagated in in vitro cultures. In order to identify these genetic determinants, and, potentially, other genes located subtelomerically, we designed a transfection system able to induce and select for controlled, site-specific subtelomeric deletions.     

Nematode development: new tricks for old genes

The evolution of novel features requires evolutionary change in gene function. New work in nematodes shows how conserved genes can nonetheless contribute to innovative traits.     

Naturally Occurring Triggers that Induce Apoptosis-Like Programmed Cell Death in Plasmodium berghei Ookinetes

Several protozoan parasites have been shown to undergo a form of programmed cell death that exhibits morphological features associated with metazoan apoptosis. These include the rodent malaria parasite, Plasmodium berghei. Malaria zygotes develop in the mosquito midgut lumen, forming motile ookinetes. Up to 50% of these exhibit phenotypic markers of apoptosis; as do those grown in culture. We hypothesised that naturally occurring signals induce many ookinetes to undergo apoptosis before midgut traversal. To determine whether nitric oxide and reactive oxygen species act as such triggers, ookinetes were cultured with donors of these molecules. Exposure to the nitric oxide donor SNP induced a significant increase in ookinetes with condensed nuclear chromatin, activated caspase-like molecules and translocation of phosphatidylserine that was dose and time related. Results from an assay that detects the potential-dependent accumulation of aggregates of JC-1 in mitochondria suggested that nitric oxide does not operate via loss of mitochondrial membrane potential. L-DOPA (reactive oxygen species donor) also caused apoptosis in a dose and time dependent manner. Removal of white blood cells significantly decreased ookinetes exhibiting a marker of apoptosis in vitro. Inhibition of the activity of nitric oxide synthase in the mosquito midgut epithelium using L-NAME significantly decreased the proportion of apoptotic ookinetes and increased the number of oocysts that developed. Introduction of a nitric oxide donor into the blood meal had no effect on mosquito longevity but did reduce prevalence and intensity of infection. Thus, nitric oxide and reactive oxygen species are triggers of apoptosis in Plasmodium ookinetes. They occur naturally in the mosquito midgut lumen, sourced from infected blood and mosquito tissue. Up regulation of mosquito nitric oxide synthase activity has potential as a transmission blocking strategy.     

Ectopic Expression of a Neospora caninum Kazal Type Inhibitor Triggers Developmental Defects in Toxoplasma and Plasmodium

Regulated proteolysis is known to control a variety of vital processes in apicomplexan parasites including invasion and egress of host cells. Serine proteases have been proposed as targets for drug development based upon inhibitor studies that show parasite attenuation and transmission blockage. Genetic studies suggest that serine proteases, such as subtilisin and rhomboid proteases, are essential but functional studies have proved challenging as active proteases are difficult to express. Proteinaceous Protease Inhibitors (PPIs) provide an alternative way to address the role of serine proteases in apicomplexan biology. To validate such an approach, a Neospora caninum Kazal inhibitor (NcPI-S) was expressed ectopically in two apicomplexan species, Toxoplasma gondii tachyzoites and Plasmodium berghei ookinetes, with the aim to disrupt proteolytic processes taking place within the secretory pathway. NcPI-S negatively affected proliferation of Toxoplasma tachyzoites, while it had no effect on invasion and egress. Expression of the inhibitor in P. berghei zygotes blocked their development into mature and invasive ookinetes. Moreover, ultra-structural studies indicated that expression of NcPI-S interfered with normal formation of micronemes, which was also confirmed by the lack of expression of the micronemal protein SOAP in these parasites. Our results suggest that NcPI-S could be a useful tool to investigate the function of proteases in processes fundamental for parasite survival, contributing to the effort to identify targets for parasite attenuation and transmission blockage.     

Embryology and disorders of sexual development

Recent consensus is that individuals with atypical male or female phenotype are to be considered to have a "disorder of sexual development." The goal is to eliminate previous terminology that included the terms intersex, hermaphrodite, or pseudohermaphrodite. However, the teaching of embryology, and particularly teaching about the development of the reproductive system, still has not made the change to the new terminology. If those who teach embryology to health-care professionals remain unaware of the controversies associated with the old terminology and continue to use it, they will perpetuate a nomenclature that can be destructive. Any terminology must be used carefully to avoid dehumanizing the individual to a disease or a medical state. We should be able to state clearly the variations in morphology that exist, attend to the immediate health of the individual, and avoid any attempt to stigmatize gender-atypical individuals.     

Functional characterisation of sexual stage specific proteins in Plasmodium falciparum

The various stages of the malaria parasites in the vertebrate host and in the mosquito vector offer numerous candidates for vaccine and drug development. However, the biological complexity of the parasites and the interaction with the immune system of the host continue to frustrate all such efforts thus far. While most of the targets for drug and vaccine design have focused on the asexual stages, the sexual stages of the parasite are critical for transmission and maintenance of parasites among susceptible vertebrate hosts. Sexual stage parasites undergo a series of morphological and biochemical changes during their development, accompanied by a co-ordinated cascade of a distinct expression pattern of sexual stage specific proteins. Mechanisms underlying the developmental switch from asexual parasite to sexual parasite still remain elusive. Methods that can break the malaria transmission cycle thus occupy a central place in the overall malaria control strategies. This paper provides a review of genes expressed in sexually differentiated Plasmodium. In the past few years, a molecular approach based on targeted gene disruption has revealed fascinating biological roles for many of the sexual stage gene products. In addition, we will briefly discuss other functional genomic approaches employed to study not only sexual but also other aspects of host-parasite biology.     

Altering sexual development inArabidopsis

The reproductive system determines the way in which gametes develop and interact to form a new organism. Therefore, it exerts the primary level of control of genotypic frequencies in plant populations, and plays a fundamental role in plant breeding. A basic understanding of plant reproductive development will completely transform current breeding strategies used for seed production. Apomixis is an asexual form of reproduction in which embryogenesis occurs in a cell lineage lacking both meiosis and fertilization, and that culminates in the formation of viable progeny genetically identical to the mother plant. The transfer of apomixis into sexual crops will allow the production of self-perpetuating improved hybrids, and the fixation of any desired heterozygous genotype. The initiation of apomictic development invariably takes place at early stages of ovule ontogeny, before the establishment of the megagametophytic phase. The developmental versatility associated with megagametophyte formation suggests that the genetic and molecular regulation of apomixis is intimately related to the regulation of sexuality. Differences between the initiation of sexual and apomictic development may be determined by regulatory genes that act during megasporogenesis, and that control events leading to the formation of unreduced female gametophytes. To test this hypothesis, we are isolating and characterizing genes that act during megasporogenesis inArabidopsis thaliana and investigating their potential role in the induction of apomixis. We are using a recently established transposon-based enhancer detection and gene trap insertional mutagenesis system that allows the identification of genes based on their expression patterns. An initial screen of transposants has yielded over 20 lines conferring restricted GUS expression during early ovule development. We have obtained the sequence of genomic fragments flanking the transposon insertion. Several have homology to genes playing important roles in plant and animal development. They include cell cycle regulators, enzymes involved in callose hydrolysis, leucine-rich repeat protein kinase receptors, and expressed sequence tags (ESTs) of unknown function. Independently, a genetic screen allows the identification of female sterile mutants defective in megasporogenesis. Results from these experiments will improve our basic understanding of reproductive development in plants, and will set the basis for a sustained effort in plant germ line biotechnology, a first step toward a flexible transfer of apomixis into a large variety of sexual crops.     

Analysis of the sporogonic development of Plasmodium falciparum and Plasmodium berghei in anopheline mosquitoes

Basic knowledge of the sporogonic development of malarial parasites is crucial when evaluating the sporontocidal activity of antimalarial drugs or when determining why certain vectors are refractory to a particular parasite while others are competent vectors. We have developed a model which we have used to i) assess the sporogonic development of Plasmodium berghei ANKA in Anopheles stephensi and A. freeborni mosquitoes and ii) determine the effect of chloroquine on the sporogony of P. falciparum NF-54 in A. stephensi. Criteria used to assay sporogonic development include: i) number of oocysts present, ii) percentage of mosquitoes with oocysts, iii) time of release of sporozoites from the oocysts into the hemolymph, iv) time and degree of sporozoite invasion of salivary glands, and v) transmission (P. berghei) into vertebrate hosts. Parasite development in the mosquito is evaluated every other day, commencing on ca. day 7 post-feed (PF) and continuing until ca. day 22 PF. These detailed observations allow us to delineate the chronology of sporogonic development.     

Platform for Plasmodium vivax vaccine discovery and development

Plasmodium vivax is the most prevalent malaria parasite on the American continent. It generates a global burden of 80-100 million cases annually and represents a tremendous public health problem, particularly in the American and Asian continents. A malaria vaccine would be considered the most cost-effective measure against this vector-borne disease and it would contribute to a reduction in malaria cases and to eventual eradication. Although significant progress has been achieved in the search for Plasmodium falciparum antigens that could be used in a vaccine, limited progress has been made in the search for P. vivax components that might be eligible for vaccine development. This is primarily due to the lack of in vitro cultures to serve as an antigen source and to inadequate funding. While the most advanced P. falciparum vaccine candidate is currently being tested in Phase III trials in Africa, the most advanced P. vivax candidates have only advanced to Phase I trials. Herein, we describe the overall strategy and progress in P. vivax vaccine research, from antigen discovery to preclinical and clinical development and we discuss the regional potential of Latin America to develop a comprehensive platform for vaccine development.     

Peripuberal sexual development of Pantaneiro stallions

Pantaneiro horses are a breed native to flood plains of Brazil, where they thrive with little human interference. The aims of this study were to characterize age-associated changes in testicular size, serum testosterone and sexual behavior and to determine age at puberty as well as to evaluate daily sperm production in the Pantaneiro stallion. After weaning the males were kept in bachelor groups away from the females, in a separated area in the natural environment of flood plains. Infantile period ended at approximately 14.4 m.o. of age. Sexual interest and breeding capability first appeared between the ages of 15.6 to 27.5 m.o. The transition from prepuberal to postpuberal histological stage, occurred at approximately 27.8 m.o. of age. All animals were in the postpuberal period at 38.5 m.o. of age. Daily sperm production estimated with testicle homogenate, at 38.5 m.o. of age was low (1.4603 +/- 0.48 x 10(9) sperm) and serum testosterone levels were similar to adult levels by 27.8 m.o. In conclusion the 2 y old Pantaneiro stallions showed full sexual behavior and had adult testosterone levels, but at this age, the stage of development of the seminiferous epithelium was variable and the efficiency of sperm production was still low.