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Targeting mosquito FREP1 with a fungal metabolite blocks malaria transmission.

Niu G, Wang B, Zhang G, King JB, Cichewicz RH, Li J - Sci Rep (2015)

Bottom Line: The inhibition specificity was confirmed by immunofluorescence assays.Therefore, disruption of the interaction between FREP1 and parasites effectively reduces Plasmodium infection in mosquitoes.Targeting FREP1 with small molecules is thus an effective novel approach to block malaria transmission.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA.

ABSTRACT
Inhibiting Plasmodium development in mosquitoes will block malaria transmission. Fibrinogen-related protein 1 (FREP1) is critical for parasite infection in Anopheles gambiae and facilitates Plasmodium invasion in mosquitoes through interacting with gametocytes and ookinetes. To test the hypothesis that small molecules that disrupt this interaction will prevent parasites from infecting mosquitoes, we developed an ELISA-based method to screen a fungal extract library. We obtained a candidate fungal extract of Aspergillus niger that inhibited the interaction between FREP1 and P. falciparum infected cells by about 92%. The inhibition specificity was confirmed by immunofluorescence assays. Notably, feeding mosquitoes with the candidate fungal extract significantly inhibited P. falciparum infection in the midgut without cytotoxicity or inhibition of the development of P. falciparum gametocytes or ookinetes. A bioactive natural product that prevents FREP1 from binding to gametocytes or ookinetes was isolated and identified as P-orlandin. Importantly, the nontoxic orlandin significantly reduced P. falciparum infection intensity in mosquitoes. Therefore, disruption of the interaction between FREP1 and parasites effectively reduces Plasmodium infection in mosquitoes. Targeting FREP1 with small molecules is thus an effective novel approach to block malaria transmission.

No MeSH data available.


Related in: MedlinePlus

Identification of fungal extracts that disrupt FREP1-iRBC lysate interaction and interfere with P. falciparum infection in mosquitoes.(a) Screening fungal extracts that inhibit FREP1 binding to iRBC lysate by ELISA. Each rectangle represents an individual fungal extract and the color represents the inhibition rate. Three fungal extracts that had high inhibition rates are marked with circles. The assays were repeated twice and the graph was generated by R-Project software. The fungal extract (marked with red circle) had the highest inhibition rate. (b) Validation of the identified fungal extracts by ELISA using four replicates. (c) The number of oocyst in mosquito midguts co-ingesting P. falciparum gametocytes with 20 μg/mL candidate fungal extracts. (d–f) The candidate fungal extract (#6D from isolate Chapel SA-3) significantly inhibited P. falciparum infection in mosquito midguts and the inhibition displayed a dose-dependent pattern. The assays were repeated independently three times. The results showed significantly fewer oocysts developed in An. gambiae midguts treated with Chapel SA-3 extract than treated with DMSO (p < 0.05). N: the number of mosquitoes for each treatment; μ: the mean oocysts per midgut; PR: infection prevalence in mosquitoes.
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f2: Identification of fungal extracts that disrupt FREP1-iRBC lysate interaction and interfere with P. falciparum infection in mosquitoes.(a) Screening fungal extracts that inhibit FREP1 binding to iRBC lysate by ELISA. Each rectangle represents an individual fungal extract and the color represents the inhibition rate. Three fungal extracts that had high inhibition rates are marked with circles. The assays were repeated twice and the graph was generated by R-Project software. The fungal extract (marked with red circle) had the highest inhibition rate. (b) Validation of the identified fungal extracts by ELISA using four replicates. (c) The number of oocyst in mosquito midguts co-ingesting P. falciparum gametocytes with 20 μg/mL candidate fungal extracts. (d–f) The candidate fungal extract (#6D from isolate Chapel SA-3) significantly inhibited P. falciparum infection in mosquito midguts and the inhibition displayed a dose-dependent pattern. The assays were repeated independently three times. The results showed significantly fewer oocysts developed in An. gambiae midguts treated with Chapel SA-3 extract than treated with DMSO (p < 0.05). N: the number of mosquitoes for each treatment; μ: the mean oocysts per midgut; PR: infection prevalence in mosquitoes.

Mentions: Since FREP1 promotes Plasmodium invasion through binding to P. falciparum gametocytes or ookinetes24, we hypothesize that small molecules interfering with FREP1-parasite interaction might be good candidates to inhibit parasite infection in mosquitoes. Using the ELISA assay, we screened a library of crude extracts from various fungi. A fungal extract was judged to have completely inhibited the binding between FREP1 and iRBC if the ELISA signals were decreased to the level of the uninfected RBC control (gray color in Fig. 2a). After screening a plate of fungal extracts (N = 88), we found three fungal extracts (marked with circles in Fig. 2a) that exhibited >75% inhibition rates. The inhibitory activities of the three candidate extracts against FREP1-iRBC lysate interaction were reconfirmed in biologically replicates (N = 4) before further testing occurred. Dimethyl sulfoxide (DMSO) was used as a negative control. The results (Fig. 2b) show that candidate extracts #6D, #6G, and #8C inhibited 92%, 64%, and 75% of FREP1-iRBC lysate interaction, respectively, compared to the negative control. Next, we determined their effects on P. falciparum infection in mosquitoes. For each test, a 5 μL sample of fungal extract (2 mg/mL) in DMSO was added into 495 μL human blood containing 0.2% sexual stage V gametocytes. The results from standard membrane feeding assays (SMFA) indicated that all three candidates reduced the number of oocysts in mosquitoes (Fig. 2c) compared to the DMSO control. Notably, the in vivo infection inhibition rates of three candidates (Fig. 2c) matched their in vitro inhibition rates on FREP1-iRBC lysate interaction (Fig. 2b). Because fungal extract #6D had ~92% inhibition rate (marked with red circle in Fig. 2a) and was most effective in reducing P. falciparum infection in mosquito midguts, we focused on this sample to demonstrate the principle of our proposed approach. Extract #6D was obtained from an isolate designated “Chapel SA-3” and it is noted as such throughout this manuscript.


Targeting mosquito FREP1 with a fungal metabolite blocks malaria transmission.

Niu G, Wang B, Zhang G, King JB, Cichewicz RH, Li J - Sci Rep (2015)

Identification of fungal extracts that disrupt FREP1-iRBC lysate interaction and interfere with P. falciparum infection in mosquitoes.(a) Screening fungal extracts that inhibit FREP1 binding to iRBC lysate by ELISA. Each rectangle represents an individual fungal extract and the color represents the inhibition rate. Three fungal extracts that had high inhibition rates are marked with circles. The assays were repeated twice and the graph was generated by R-Project software. The fungal extract (marked with red circle) had the highest inhibition rate. (b) Validation of the identified fungal extracts by ELISA using four replicates. (c) The number of oocyst in mosquito midguts co-ingesting P. falciparum gametocytes with 20 μg/mL candidate fungal extracts. (d–f) The candidate fungal extract (#6D from isolate Chapel SA-3) significantly inhibited P. falciparum infection in mosquito midguts and the inhibition displayed a dose-dependent pattern. The assays were repeated independently three times. The results showed significantly fewer oocysts developed in An. gambiae midguts treated with Chapel SA-3 extract than treated with DMSO (p < 0.05). N: the number of mosquitoes for each treatment; μ: the mean oocysts per midgut; PR: infection prevalence in mosquitoes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4593950&req=5

f2: Identification of fungal extracts that disrupt FREP1-iRBC lysate interaction and interfere with P. falciparum infection in mosquitoes.(a) Screening fungal extracts that inhibit FREP1 binding to iRBC lysate by ELISA. Each rectangle represents an individual fungal extract and the color represents the inhibition rate. Three fungal extracts that had high inhibition rates are marked with circles. The assays were repeated twice and the graph was generated by R-Project software. The fungal extract (marked with red circle) had the highest inhibition rate. (b) Validation of the identified fungal extracts by ELISA using four replicates. (c) The number of oocyst in mosquito midguts co-ingesting P. falciparum gametocytes with 20 μg/mL candidate fungal extracts. (d–f) The candidate fungal extract (#6D from isolate Chapel SA-3) significantly inhibited P. falciparum infection in mosquito midguts and the inhibition displayed a dose-dependent pattern. The assays were repeated independently three times. The results showed significantly fewer oocysts developed in An. gambiae midguts treated with Chapel SA-3 extract than treated with DMSO (p < 0.05). N: the number of mosquitoes for each treatment; μ: the mean oocysts per midgut; PR: infection prevalence in mosquitoes.
Mentions: Since FREP1 promotes Plasmodium invasion through binding to P. falciparum gametocytes or ookinetes24, we hypothesize that small molecules interfering with FREP1-parasite interaction might be good candidates to inhibit parasite infection in mosquitoes. Using the ELISA assay, we screened a library of crude extracts from various fungi. A fungal extract was judged to have completely inhibited the binding between FREP1 and iRBC if the ELISA signals were decreased to the level of the uninfected RBC control (gray color in Fig. 2a). After screening a plate of fungal extracts (N = 88), we found three fungal extracts (marked with circles in Fig. 2a) that exhibited >75% inhibition rates. The inhibitory activities of the three candidate extracts against FREP1-iRBC lysate interaction were reconfirmed in biologically replicates (N = 4) before further testing occurred. Dimethyl sulfoxide (DMSO) was used as a negative control. The results (Fig. 2b) show that candidate extracts #6D, #6G, and #8C inhibited 92%, 64%, and 75% of FREP1-iRBC lysate interaction, respectively, compared to the negative control. Next, we determined their effects on P. falciparum infection in mosquitoes. For each test, a 5 μL sample of fungal extract (2 mg/mL) in DMSO was added into 495 μL human blood containing 0.2% sexual stage V gametocytes. The results from standard membrane feeding assays (SMFA) indicated that all three candidates reduced the number of oocysts in mosquitoes (Fig. 2c) compared to the DMSO control. Notably, the in vivo infection inhibition rates of three candidates (Fig. 2c) matched their in vitro inhibition rates on FREP1-iRBC lysate interaction (Fig. 2b). Because fungal extract #6D had ~92% inhibition rate (marked with red circle in Fig. 2a) and was most effective in reducing P. falciparum infection in mosquito midguts, we focused on this sample to demonstrate the principle of our proposed approach. Extract #6D was obtained from an isolate designated “Chapel SA-3” and it is noted as such throughout this manuscript.

Bottom Line: The inhibition specificity was confirmed by immunofluorescence assays.Therefore, disruption of the interaction between FREP1 and parasites effectively reduces Plasmodium infection in mosquitoes.Targeting FREP1 with small molecules is thus an effective novel approach to block malaria transmission.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA.

ABSTRACT
Inhibiting Plasmodium development in mosquitoes will block malaria transmission. Fibrinogen-related protein 1 (FREP1) is critical for parasite infection in Anopheles gambiae and facilitates Plasmodium invasion in mosquitoes through interacting with gametocytes and ookinetes. To test the hypothesis that small molecules that disrupt this interaction will prevent parasites from infecting mosquitoes, we developed an ELISA-based method to screen a fungal extract library. We obtained a candidate fungal extract of Aspergillus niger that inhibited the interaction between FREP1 and P. falciparum infected cells by about 92%. The inhibition specificity was confirmed by immunofluorescence assays. Notably, feeding mosquitoes with the candidate fungal extract significantly inhibited P. falciparum infection in the midgut without cytotoxicity or inhibition of the development of P. falciparum gametocytes or ookinetes. A bioactive natural product that prevents FREP1 from binding to gametocytes or ookinetes was isolated and identified as P-orlandin. Importantly, the nontoxic orlandin significantly reduced P. falciparum infection intensity in mosquitoes. Therefore, disruption of the interaction between FREP1 and parasites effectively reduces Plasmodium infection in mosquitoes. Targeting FREP1 with small molecules is thus an effective novel approach to block malaria transmission.

No MeSH data available.


Related in: MedlinePlus