Search

Abstract While significant advances have been made in understanding Plasmodium falciparum gametocyte biology and its relationship with malaria parasite transmission, the gametocyte sex ratio contribution to this process still remains a relevant research question. The present review discusses the biology of sex determination in P. falciparum, the underlying host and parasite factors, the sex specific susceptibility to drugs, the effect of sex ratio dynamics on malaria parasite transmission and the development of gametocyte sex specific diagnosis tools. Despite the inherent differences across several studies and approaches, the emerging picture highlights a potentially relevant contribution of the P. falciparum gametocyte sex ratio in the modulation of malaria parasite transmission. The increasing availability of molecular methods to measure gametocyte sex ratio will enable evaluation of important parameters, such as the impact of drug treatment on gametocyte sex ratio in vitro and in vivo as well as the changes of gametocyte sex ratios in natural infections, key steps towards elucidating how these parameters affect parasite infectiousness to the mosquito vectors.

Abstract Background The presence of Plasmodium falciparum gametocytes in peripheral blood is essential for human to mosquito parasite transmission. The detection of submicroscopic infections with gametocytes and the estimation of the gametocyte sex ratio are crucial to assess the human host potential ability to infect mosquitoes and transmit malaria parasites. Aim and objectives The aim of this work was to develop sensitive and cheap Real Time qPCR assays for large-scale epidemiological surveys, based on detection and amplification of gametocyte sex specific transcripts selected from the literature: the female-specific pfs25 and pf glycerol kinase (pfGK) and the male-specific pfs230p and pf13 transcripts. Methods RTqPCR assays were used to test the gametocyte- and sex-specific expression of the target genes using asexual stages of the gametocyteless parasite clone F12 and FACS purified male and female gametocytes of the PfDynGFP/P47mCherry line. Assays were performed on 50 blood samples collected during an epidemiological survey in the Soumousso village, Burkina Faso, West-Africa, and amplification of the human housekeeping gene 18S rRNA was employed to normalize RNA sample variability. Results SYBR Green assays were developed that showed higher sensitivity compared to Taqman assays at a reduced cost. RTqPCR results confirmed that expression of pfs25 and pfs230p are female and male-specific, respectively, and introduced two novel markers, the female-specific pfGK and the male-specific pf13. A formula was derived to calculate the ratio of male to female gametocytes based on the ratio of male to female transcript copy number. Use of these assays in the field samples showed, as expected, a higher sensitivity of RTqPCR compared to microscopy. Importantly, similar values of gametocyte sex-ratio were obtained in the field samples based on the four different target combinations. Conclusion Novel, sensitive, cheap and robust molecular assays were developed for the detection and quantification of female and male P. falciparum gametocytes. In particular, the RTqPCR assays based on the female-specific pfs25 and the newly described male gametocyte-specific pf13 transcripts, including normalization by the human 18S, reliably assess presence and abundance of female and male gametocytes and enable to determine their sex-ratio in human subjects in endemic areas.

Abstract Background Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. Results This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca2+ levels. Conclusions This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca2+ oscillations suggests a potential role of NAADP in regulating Ca2+ signalling of P. falciparum.

Abstract Background Gametocytes, the blood stages responsible for Plasmodium falciparum transmission, contain electron dense organelles, traditionally named osmiophilic bodies, that are believed to be involved in gamete egress from the host cell. In order to provide novel tools in the cellular and molecular studies of osmiophilic body biology, a P. falciparum transgenic line in which these organelles are specifically marked by a reporter protein was produced and characterized. Methodology A P. falciparum transgenic line expressing an 80-residue N-terminal fragment of the osmiophilic body protein Pfg377 fused to the reporter protein DsRed, under the control of pfg377 upstream and downstream regulatory regions, was produced. Results The transgenic fusion protein is expressed at the appropriate time and stage of sexual differentiation and is trafficked to osmiophilic bodies as the endogenous Pfg377 protein. These results indicate that a relatively small N-terminal portion of Pfg377 is sufficient to target the DsRed reporter to the gametocyte osmiophilic bodies. Conclusions This is the first identification of a P. falciparum aminoacid sequence able to mediate trafficking to such organelles. To fluorescently tag such poorly characterized organelles opens novel avenues in cellular and imaging studies on their biogenesis and on their role in gamete egress.

Abstract Background In Ethiopia, malaria is caused by both Plasmodium falciparum and Plasmodium vivax. Drug resistance of P. falciparum to sulfadoxine-pyrimethamine (SP) and chloroquine (CQ) is frequent and intense in some areas. Methods In 100 patients with uncomplicated malaria from Dilla, southern Ethiopia, P. falciparum dhfr and dhps mutations as well as P. vivax dhfr polymorphisms associated with resistance to SP and P. falciparum pfcrt and pfmdr1 mutations conferring CQ resistance were assessed. Results P. falciparum and P. vivax were observed in 69% and 31% of the patients, respectively. Pfdhfr triple mutations and pfdhfr/pfdhps quintuple mutations occurred in 87% and 86% of P. falciparum isolates, respectively. Pfcrt T76 was seen in all and pfmdr1 Y86 in 81% of P. falciparum. The P. vivax dhfr core mutations N117 and R58 were present in 94% and 74%, respectively. Conclusion These data point to an extraordinarily high frequency of drug-resistance mutations in both P. falciparum and P. vivax in southern Ethiopia, and strongly support that both SP and CQ are inadequate drugs for this region.

Background: The presence of Plasmodium falciparum gametocytes in peripheral blood is essential for human to mosquito parasite transmission. The detection of submicroscopic infections with gametocytes and the estimation of the gametocyte sex ratio are crucial to assess the human host potential ability to infect mosquitoes and transmit malaria parasites., Aim and Objectives: The aim of this work was to develop sensitive and cheap Real Time qPCR assays for large-scale epidemiological surveys, based on detection and amplification of gametocyte sex specific transcripts selected from the literature: the female-specific pfs25 and pf glycerol kinase (pfGK) and the male-specific pfs230p and pf13 transcripts., Methods: RTqPCR assays were used to test the gametocyte- and sex-specific expression of the target genes using asexual stages of the gametocyteless parasite clone F12 and FACS purified male and female gametocytes of the PfDynGFP/P47mCherry line. Assays were performed on 50 blood samples collected during an epidemiological survey in the Soumousso village, Burkina Faso, West-Africa, and amplification of the human housekeeping gene 18S rRNA was employed to normalize RNA sample variability., Results: SYBR Green assays were developed that showed higher sensitivity compared to Taqman assays at a reduced cost. RTqPCR results confirmed that expression of pfs25 and pfs230p are female and male-specific, respectively, and introduced two novel markers, the female-specific pfGK and the male-specific pf13. A formula was derived to calculate the ratio of male to female gametocytes based on the ratio of male to female transcript copy number. Use of these assays in the field samples showed, as expected, a higher sensitivity of RTqPCR compared to microscopy. Importantly, similar values of gametocyte sex-ratio were obtained in the field samples based on the four different target combinations., Conclusion: Novel, sensitive, cheap and robust molecular assays were developed for the detection and quantification of female and male P. falciparum gametocytes. In particular, the RTqPCR assays based on the female-specific pfs25 and the newly described male gametocyte-specific pf13 transcripts, including normalization by the human 18S, reliably assess presence and abundance of female and male gametocytes and enable to determine their sex-ratio in human subjects in endemic areas.

Background: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca 2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs., Results: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca 2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca 2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca 2+ levels., Conclusions: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca 2+ oscillations suggests a potential role of NAADP in regulating Ca 2+ signalling of P. falciparum.

Background: Transmission of the malaria parasite Plasmodium falciparum from humans to the mosquito vector requires differentiation of a sub-population of asexual forms replicating within red blood cells into non-dividing male and female gametocytes. The nature of the molecular mechanism underlying this key differentiation event required for malaria transmission is not fully understood., Methods: Whole genome sequencing was used to examine the genomic diversity of the gametocyte non-producing 3D7-derived lines F12 and A4. These lines were used in the recent detection of the PF3D7_1222600 locus (encoding PfAP2-G), which acts as a genetic master switch that triggers gametocyte development., Results: The evolutionary changes from the 3D7 parental strain through its derivatives F12 (culture-passage derived cloned line) and A4 (transgenic cloned line) were identified. The genetic differences including the formation of chimeric var genes are presented., Conclusion: A genomics resource is provided for the further study of gametocytogenesis or other phenotypes using these parasite lines.

Background: Plasmodium falciparum sexual development plays a fundamental role in the transmission and spread of malaria. The ability to generate gametocytes can be lost during culture in vitro, often associated with the loss of a subtelomeric region of chromosome 9. Gametocytogenesis starts with erythrocyte invasion by a sexually committed merozoite, but the first available specific marker of sexual differentiation appears only from 24 h post invasion., Methods: Specific antibodies and gene fusions were produced to study the timing of expression and the sub-cellular localization of the P. falciparum Gametocyte EXported Protein-5 (PfGEXP5), encoded in the subtelomeric region of chromosome 9. Expression patterns were examined in wild-type parasites and in parasite lines mutated in the Apetala2-G (AP2-G) transcription factor, governing a cascade of early sexual stage specific genes., Results: PfGEXP5 is highly expressed in early sexual stages and it is actively exported to the infected erythrocyte cytoplasm from as early as 14 h post-invasion in haemozoin-free, ring stage-like parasites. The pattern of PfGEXP5 expression and export is similar in wild-type parasites and in independent AP2-G defective parasite lines unable to produce gametocytes., Conclusions: PfGEXP5 represents the earliest post-invasion sexual stage marker described to date. This provides a tool that can be used to identify sexually committed ring stage parasites in natural infections. This early gametocyte marker would enable the identification and mapping of malaria transmission reservoirs in human populations and the study of gametocyte sequestration dynamics in infected individuals. The fact that regulation of PfGEXP5 does not depend on the AP2-G master regulator of parasite sexual development suggests that, after sexual commitment, differentiation progresses through multiple checkpoints in the early phase of gametocytogenesis.

Background: Ability of Plasmodium falciparum gametocytes to become extracellular during gametogenesis in the mosquito midgut is a key step of the parasite life cycle. Reliable and quantitative measurement of the efficiency of gamete egress is currently constrained by the fact that this phenomenon is usually observed and quantified in vitro either by live microscopy, by statistically limited ultrastructural analysis or by surface antibody-based protocols which can interfere with this fast and complex cellular process., Methods: A protocol was developed based on fluorescent wheat germ agglutinin (WGA) surface staining of erythrocytes containing mature P. falciparum gametocytes. After a single centrifugation step and within minutes from the induction of gametogenesis, the activated gametes can be inspected for presence or absence of the fluorescent WGA staining of the host erythrocyte membrane and scored respectively as intracellular or emerged from the erythrocyte., Results: Gametogenesis and gamete egress from WGA surface stained, infected erythrocytes occur with normal kinetics and efficiencies. Quantitative measurements of gamete egress can be obtained in live and in paraformaldehyde-fixed cells, which validates this protocol as a suitable tool both for live imaging studies and for higher throughput applications. The protocol was used here to provide functional information on the ability of gametes to egress through a single exit point induced in the host red blood cell membrane, and to re-analyse the phenotype of Pfg377- and osmiophilic body-defective gametes, suggesting that such parasite components are not directly involved in disruption and shedding of the erythrocyte membrane in female gamete egress., Conclusions: The development of a reliable, fast, non-invasive and quantitative protocol to finely describe and to measure efficiency of P. falciparum gamete egress is a significant improvement in the tools for functional studies on this key process of the parasite life cycle. This protocol can be used to investigate the molecular mechanisms underlying gamete egress and its adaptation to high throughput applications will enable identification of transmission blocking inhibitors.