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CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research.

ABSTRACT: The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. falciparum NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.

SUBMITTER: Boltryk SD

PROVIDER: S-EPMC8355313 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research.

Boltryk Sylwia D SD Passecker Armin A Alder Arne A Carrington Eilidh E van de Vegte-Bolmer Marga M van Gemert Geert-Jan GJ van der Starre Alex A Beck Hans-Peter HP Sauerwein Robert W RW Kooij Taco W A TWA Brancucci Nicolas M B NMB Proellochs Nicholas I NI Gilberger Tim-Wolf TW Voss Till S TS

Nature communications 20210810 1

The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. fa ...[more]

PMID: 34376675

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Project description:BackgroundMalaria caused by Plasmodium falciparum remains a serious global public health challenge especially in Africa. Interventions that aim to reduce malaria transmission by targeting the gametocyte reservoir are key to malaria elimination and/or eradication. However, factors that are associated with gametocyte carriage have not been fully explored. Consequently, identifying predictors of the infectious reservoir is fundamental in the elimination campaign.MethodsWe cultured P. falciparum NF54 gametocytes (to stage V) and prepared crude gametocyte extract. Samples from a total of 687 participants (aged 6 months to 67 years) representing two cross-sectional study cohorts in Kilifi, Kenya were used to assess IgG antibody responses by ELISA. We also analyzed IgG antibody responses to the blood-stage antigen AMA1 as a marker of asexual parasite exposure. Gametocytemia and asexual parasitemia data quantified by microscopy and molecular detection (QT-NASBA) were used to determine the relationship with antibody responses, season, age, and transmission setting. Multivariable logistic regression models were used to study the association between antibody responses and gametocyte carriage. The predictive power of the models was tested using the receiver operating characteristic (ROC) curve.ResultsMultivariable logistic regression analysis showed that IgG antibody response to crude gametocyte extract predicted both microscopic (OR=1.81 95% CI: 1.06-3.07, p=0.028) and molecular (OR=1.91, 95% CI: 1.11-3.29, p=0.019) P. falciparum gametocyte carriage. Antibody responses to AMA1 were also associated with both microscopic (OR=1.61 95% CI: 1.08-2.42, p=0.020) and molecular (OR=3.73 95% CI: 2.03-6.74, p<0.001) gametocytemia. ROC analysis showed that molecular (AUC=0.897, 95% CI: 0.868-0.926) and microscopic (AUC=0.812, 95% CI: 0.758-0.865) multivariable models adjusted for gametocyte extract showed very high predictive power. Molecular (AUC=0.917, 95% CI: 0.891-0.943) and microscopic (AUC=0.806, 95% CI: 0.755-0.858) multivariable models adjusted for AMA1 were equally highly predictive.ConclusionIn our study, it appears that IgG responses to crude gametocyte extract are not an independent predictor of gametocyte carriage after adjusting for AMA1 responses but may predict gametocyte carriage as a proxy marker of exposure to parasites. Serological responses to AMA1 or to gametocyte extract may facilitate identification of individuals within populations who contribute to malaria transmission and support implementation of transmission-blocking interventions.

Project description:BackgroundFor future eradication strategies of malaria it is important to control the transmission of gametocytes from humans to the anopheline vector which causes the spread of the disease. Sensitive, non-invasive methods to detect gametocytes under field conditions can play a role in monitoring transmission potential.MethodsMicroscopically Plasmodium falciparum-positive patients from Jimma, Ethiopia donated finger-prick blood, venous blood, saliva, oral mucosa and urine samples that were spotted on filter paper or swabs. All samples were taken and stored under equal, standardized conditions. RNA was extracted from the filter paper and detected by real-time QT-NASBA. Pfs16-mRNA and Pfs25-mRNA were measured with a time to positivity to detect gametocyte specific mRNA in different gametocyte stages. They were compared to 18S-rRNA, which is expressed in all parasite stages. Results were quantified via a known dilution series of artificial RNA copies.ResultsNinety-six samples of 16 uncomplicated malaria patients were investigated. 10 (66.7%) of the slides showed gametocyte densities between 0.3-2.9 gametocytes/?l. For all RNA-targets, molecular detection in blood samples was most sensitive; finger-prick sampling required significantly smaller amounts of blood than venous blood collection. Detection of asexual 18S-rRNA in saliva and urine showed sensitivities of 80 and 67%, respectively. Non-invasive methods to count gametocytes proved insensitive. Pfs16-mRNA was detectable in 20% of urine samples, sensitivities for other materials were lower. Pfs25-mRNA was not detectable in any sample.ConclusionsThe sensitivity of non-invasively collected material such as urine, saliva or mucosa seems unsuitable for the detection of gametocyte-specific mRNA. Sensitivity in asymptomatic carriers might be generally even lower. Finger-prick testing revealed the highest absolute count of RNA copies per ?L, especially for Pfs25-mRNA copies. The method proved to be the most effective and should preferably be applied in future transmission control and eradication plans. A rapid test for gametocyte targets would simplify efforts.

Project description:BackgroundMalaria transmission depends on the presence of gametocytes in the peripheral blood of infected human hosts. Understanding malaria infectious reservoirs enables transmission-blocking interventions to target the most important hosts for the disease. This study characterized the distribution of gametocyte carriage as a baseline for the clinical evaluation of a Pfs25-based transmission-blocking vaccine candidate in Bagamoyo, Tanzania.MethodsA malaria survey was conducted in five locations from May to August 2022. A total of 467 participants-192 children (5-12 years), 65 adolescents (13-17 years) and 210 adults (18-45 years)-were enrolled. Malaria was detected using three methods: rapid diagnostic tests, light microscopy and quantitative polymerase chain reaction. The geometric mean of the gametocyte density, and weighted arithmetic mean of the gametocytes sex ratio were estimated.ResultsOverall, 23.5% (110/467) of the participants tested positive for malaria parasites, with the majority of positives (> 92%) being Plasmodium falciparum. The overall gametocytaemia was 5.6%, with a percent positivity of 6.8% (13/192), 6.2% (4/65) and 4.3% (9/210), in children, adolescents, and adults, respectively. The geometric mean gametocyte density (gametocytes/μL) was greater in adults (124.6) than in children (71.7) and adolescents (50.5). Regression analysis revealed that gametocytes were more likely to be present among male participants than among female participants [ORa: 2.79 (95% CI: 1.19 - 6.59) p = 0.019]. The gametocyte sex ratio in children and adult gametocyte carriers was similar but greater than that in adolescents.ConclusionThe observed gametocyte densities and distribution across age groups suggest the need for malaria transmission-blocking interventions to target all populations in heterogeneous transmission settings. The implication of targeting only children may leave residual malaria transmission and reinfection from the left-out groups.

Dataset Information

CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research.

Publications

CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research.

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