Design and Activity of Antimicrobial Peptides against Sporogonic-Stage Parasites Causing Murine Malarias

ABSTRACT Insects produce several types of peptides to combat a broad spectrum of invasive pathogenic microbes, including protozoans. However, despite this defense response, infections are often established. Our aim was to design novel peptides that produce high rates of mortality among protozoa of the genus Plasmodium, the malaria parasites. Using existing antimicrobial peptide sequences as templates, we designed and synthesized three short novel hybrids, designated Vida1 to Vida3. Each has a slightly different predicted secondary structure. The peptides were tested against sporogonic stages of the rodent malaria parasites Plasmodium berghei (in vitro and in vivo) and P. yoelii nigeriensis (in vitro). The level of activity varied for each peptide and according to the parasite stage targeted. Vida3 (which is predicted to have large numbers of β sheets and coils but no α helices) showed the highest level of activity, killing the early sporogonic stages in culture and causing highly significant reductions in the prevalence and intensity of infection of P. berghei after oral administration or injection in Anopheles gambiae mosquitoes. The secondary structures of these peptides may play a crucial role in their ability to interact with and kill sporogonic forms of the malaria parasite.

Cultivation of Plasmodium spp

SUMMARY Cultivation of both human and non-human species of Plasmodium spp., the causal agent of malaria, has been a major research success, leading to a greater understanding of the parasite. Efforts at cultivating the organisms in vitro are complicated by the parasites' alternating between a human host and an arthropod vector, each having its own set of physiological, metabolic, and nutritional parameters. Life cycle stages of the four species that infect humans have been established in vitro. Of these four, P. falciparum remains the only species for which all stages have been cultured in vitro; different degrees of success have been achieved with the other human Plasmodium spp. The life cycle includes the exoerythrocytic stage (within liver cells), the erythrocytic stage (within erythrocytes or precursor reticulocytes), and the sporogonic stage (within the vector). Culture media generally consist of a basic tissue culture medium (e.g., minimal essential medium or RPMI 1640) to which serum and erythrocytes are added. Most of the efforts have been directed toward the stage found in the erythrocyte. This stage has been cultivated in petri plates or other growth vessels in a candle jar to generate elevated CO2 levels or in a more controlled CO2 atmosphere. Later developments have employed continuous-flow systems to reduce the labor-intensive nature of medium changing. The exoerythrocytic and sporogonic life cycle stages have also been cultivated in vitro. A number of avian, rodent, and simian malarial parasites have also been established in vitro. Although cultivation is of great help in understanding the biology of Plasmodium, it does not lend itself to use for diagnostic purposes.

The life-history of Pleistophora gigantea, Thélohan (Glugea gigantea Thél.)

The parasite which I have here described has a life-history containing two distinct stages: a vegetative stage and a sporogonic stage. During the vegetative period multinucleate individuals divide by irregular schizogony producing young uni- or paucinucleate individuals. During the sporogonic period, a number of trophozoites unite to form an aggregation which becomes encysted. In the encysted individuals (the pansporoblasts) there is an increase of nuclear substance and a reduction of plasmatic substance. The nuclei become arranged in pairs and by successive unequal divisions the pansporoblasts produce the sporoblasts which contain a pair of nuclei each. During the formation of the sporoblasts there is an increase of protoplasmatic substance. The sporoblasts become spores by the thickening of their pelliculae.At first the spores contain two nuclei, finally they become mononucleate. This must be explained by the fusion of the two nuclei.This nuclear fusion is probably to be interpreted as a paedogamous autogamy (in accordance with the nomenclature of Hartmann, 1909).