Suzanne L. Fleck

Regulation of infectivity of Plasmodium to the mosquito vector

Publisher Summary This chapter discusses the complex factors regulating the infectivity of the gametocyte to the mosquito vector and describes the patterns of parasite biology that individually advantageous to particular host parasite combinations. The chapter discusses the gametocytogenesis, including the factors that regulate the differentiation of the erythrocytic stage parasites into sexual or asexual parasites, and the development of the gametocytes into either male or female cells. The biology of the mature gametocyte, including its cell biology, distribution and longevity/viability in the peripheral bloodstream, and the biology of the gametocyte-infected host and the regulation of mosquito infection by host factors that interact either directly with the parasite or indirectly upon the parasite following its ingestion by the mosquito vector are presented in the chapter. Gametogenesis, its molecular regulation with respect to the inducers required to trigger the gametocyte to leave its arrested state, the secondary signal pathways that regulate the constituent events of microgametogenesis (exflagellation), and the impact of external regulatory molecules (usually inhibitors) on gamete formation are described. The chapter also explains fertilization, its efficiency and role as a developmental regulator and outlines the post-fertilization development, including the mechanisms that regulate the survival or destruction of the parasite before its successful establishment under the midgut basal lamina where the oocyst then forms.

A Single Malaria Merozoite Serine Protease Mediates Shedding of Multiple Surface Proteins by Juxtamembrane Cleavage

Erythrocyte invasion by the malaria merozoite is accompanied by the regulated discharge of apically located secretory organelles called micronemes. Plasmodium falciparum apical membrane antigen-1 (PfAMA-1), which plays an indispensable role in invasion, translocates from micronemes onto the parasite surface and is proteolytically shed in a soluble form during invasion. We have previously proposed, on the basis of incomplete mass spectrometric mapping data, that PfAMA-1 shedding results from cleavage at two alternative positions. We now show conclusively that the PfAMA-1 ectodomain is shed from the merozoite solely as a result of cleavage at a single site, just 29 residues away from the predicted transmembrane-spanning sequence. Remarkably, this cleavage is mediated by the same membrane-bound parasite serine protease as that responsible for shedding of the merozoite surface protein-1 (MSP-1) complex, an abundant, glycosylphosphatidylinositol-anchored multiprotein complex. Processing of MSP-1 is essential for invasion. Our results indicate the presence on the merozoite surface of a multifunctional serine sheddase with a broad substrate specificity. We further demonstrate that translocation and shedding of PfAMA-1 is an actin-independent process.

A Subtilisin-like Protein in Secretory Organelles of Plasmodium falciparum Merozoites

In the vertebrate host, the malaria parasite invades and replicates asexually within circulating erythrocytes. Parasite proteolytic enzymes play an essential but poorly understood role in erythrocyte invasion. We have identified a Plasmodium falciparum gene, denoted pfsub-1, encoding a member of the subtilisin-like serine protease family (subtilases). Thepfsub-1 gene is expressed in asexual blood stages ofP. falciparum, and the primary gene product (PfSUB-1) undergoes post-translational processing during secretory transport in a manner consistent with its being converted to a mature, enzymatically active form, as documented for other subtilases. In the invasive merozoite, the putative mature protease (p47) is concentrated in dense granules, which are secretory organelles located toward the apical end of the merozoite. At some point following merozoite release and completion of erythrocyte invasion, p47 is secreted from the parasite in a truncated, soluble form. The subcellular location and timing of secretion of p47 suggest that it is likely to play a role in erythrocyte invasion. PfSUB-1 is a new potential target for antimalarial drug development.

Heterogeneity in patterns of malarial oocyst infections in the mosquito vector

Oocyst prevalence and intensity have been recorded in 349 laboratory infections of Anopheles stephensi with Plasmodium berghei. Intensity and prevalence of infection are shown to be predictably related. The structure and heterogeneity in the infections has been analysed with the objective of describing the biological mechanisms by which the observed negative binomial oocyst distributions are generated. The analysis has revealed that the most likely processes lie within the population dynamic events of malaria within the mosquito, namely gametogenesis, fertilization and mortality. The distribution is similar in all Plasmodium-mosquito combinations examined so far, whether they are of laboratory (P. gallinaceum in Aedes aegypti) or field (P. vivax in An. albimanus and P. falciparum in An. gambiae s.l. and An. funestus) origin. Further we conclude that there is competition between parasites in the vector. Oocyst frequency distribution analysis shows that under natural conditions of transmission intensity, and even under the best laboratory conditions, significant numbers (> 10%) of fully susceptible mosquitoes will not be infected under conditions where the mean infection is as high as 250 oocysts. Failure to infect is not therefore an absolute indicator of refractoriness. In assessing transmission data it is shown that sample sizes should not be less than 50, and ideally 100 mosquitoes, if reliable data are to be obtained. In field it is suggested that difficulties in determining the low natural intensity of oocyst infections indicate that prevalence estimates are a useful and accessible parameter to measure.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of atovaquone (566C80) on the maturation and viability of Plasmodium falciparum gametocytes in vitro

Atovaquone (566C80), a hydroxynaphthoquinone, was investigated for activity against Plasmodium falciparum gametocytes (NF54 strain) in vitro. After 96 h of continuous exposure to the drug at 1.4 x 10(-7) M (a concentration achievable in humans 14 d after administration of a therapeutic dose of 10 mg/kg) reductions of 75%, 54% and 20% in the number of gametocyte stages 2, 3 and 4, respectively, were achieved. A small increase (14%) in stage 5 gametocytes was seen. At the same concentration, atovaquone showed greater activity against the asexual stages of P. falciparum, reductions of 93%, 96% and 43% in the number of rings, schizonts and trophozoites, respectively, being achieved. These data are consistent with inhibition of maturation of trophozoites. The observed effect on maturation of gametocytes is similarly consistent with blockade of gametocyte recruitment from merozoites produced by the preceding schizogony, or to stasis of intraerythrocytic sexual development before the formation of stage 2 gametocytes.

Characterization of the modes of action of anti-Pbs21 malaria transmission-blocking immunity: ookinete to oocyst differentiation in vivo

The impact of immune sera, and peripheral blood cells (PBC) from mice immunized with Plasmodium berghei ookinetes; and of purified immunoglobulin or Fab fragments from anti-Pbs21 monoclonal antibody 13.1, upon establishment of oocyst infections in the mosquito was studied. Infections were initiated either from gametocyte-infected mice, or membrane feeders which contained either gametocytes or mature ookinetes. PBC from ookinete-immunized mice presented with non-immune serum failed to show any transmission-blocking activity. Anti-ookinete serum, intact anti-Pbs21 monoclonal antibody 13.1 or its Fab fragments, all inhibited oocyst formation significantly. When gametocyte-infected mice or gametocytes in membrane feeds were used, inhibition did not directly correlate with antibody concentration. In membrane feeders that contained ookinetes and antibody, concentration-dependent inhibition usually occurred. The efficacy of purified 13.1 IgG was dependent upon the ookinete concentration. The ookinete plasmalemma and cytoplasm were significantly disturbed after 12 h in bloodmeals that contained antibody 13.1, but not in the isotype controls. These changes may have caused the observed failure of the ookinete to migrate as rapidly as the controls from the destructive environment of the bloodmeal.

The complete development in vitro of the vertebrate phase of the mammalian malarial parasite Plasmodium berghei

All three vertebrate stages of the rodent malarial parasite Plasmodium berghei berghei were grown in vitro in the absence of the vertebrate host. The parasite was introduced into culture from infected mosquitoes and 2 in vitro culture methods were used sequentially to complete the vertebrate phases of development in hepatoma and erythrocyte host cells. The resultant blood infection produced mature schizonts and male and female gametocytes. The protocol, which is now being extended to the human pathogen P. falciparum, may assist future studies on this important group of parasites.

Antigenic and sequence diversity at the C-terminus of the merozoite surface protein-1 from rodent malaria isolates, and the binding of protective monoclonal antibodies.

Merozoite surface protein-1 (MSP-1) is a major candidate in the development of a vaccine against malaria. Immunisation with a recombinant fusion protein containing the two Plasmodium yoelii MSP-1 C-terminal epidermal growth factor-like domains (MSP-1(19)) can protect mice against homologous but not heterologous challenge, and therefore, antigenic differences resulting from sequence diversity in MSP-1(19) may be crucial in determining the potential of this protein as a vaccine. Representative sequence variants from a number of distinct P. yoelii isolates were expressed in Escherichia coli and the resulting recombinant proteins were screened for binding to a panel of monoclonal antibodies (Mabs) capable of suppressing a P. yoelii YM challenge infection in passive immunisation experiments. The sequence polymorphisms affected the binding of the antibodies to the recombinant proteins. None of the Mabs recognised MSP-1(19) of P. yoelii yoelii 2CL or 33X or P. yoelii nigeriensis N67. The epitopes recognised by the Mabs were further distinguished by their reactivity with the other fusion proteins. The extent of sequence variation in MSP-1(19) among the isolates was extensive, with differences detected at 35 out of the 96 positions compared. Using the 3-dimensional structure of the Plasmodium falciparum MSP-1(19) as a model, the locations of the amino acid substitutions that may affect Mab binding were identified. The DNA sequence of MSP-1(19) from two Plasmodium vinckei isolates was also cloned and the deduced amino acid sequence compared with that in other species.

Versatile Field's stain.

Focal infiltrate None A 1025 3601 3-51 B 804 2687 3-34 C 79

The chemotherapy of rodent malaria. LIII. 'Fenozan B07' (Fenozan-50F), a difluorinated 3,3′-spirocyclopentane 1,2,4-trioxane: comparison with some compounds of the artemisinin series.

Fenozan B07, a difluorinated 3,3-spirocyclopentane, 1,2,4-trioxane, is a novel, second-generation antimalarial endoperoxide which is a potent blood schizontocide against strains of rodent malaria that are highly resistant to a wide spectrum of classical antimalarials. Like compounds of the artemisinin series, its action is limited to the intra-erythrocytic stages, both asexual and sexual, and it is devoid of causal prophylactic activity. Both Fenozan B07 and the artemisinins are potent gametocytocides. In contrast to arteether, in a model using synchronous infection with Plasmodium vinckei petteri, Fenozan B07 inhibits the development of all asexual stages except preschizonts, as well as gametocytes. The activity of the artemisinin series in rodent-malaria models is limited to the rings and young trophozoites. The combined effect of Fenozan B07 with artesunate against P. v. petteri was only additive. A slight degree of potentiation was found in mice infected with asynchronous, drug-sensitive P. berghei but the combination was only additive against CQ-resistant P. yoelli ssp. NS. On the other hand, a significant degree of synergism was observed when mice infected with the artemisinin-resistant ART line of P. yoelii ssp. NS received combinations of Fenozan B07 with artemisinin. The conclusion is drawn from these and other data that there are significant differences between the blood schizontocidal actions of Fenozan B07 and the artemisinins. The basis of these differences remains to be determined.