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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

A pure compound was isolated from the Chapel SA-3 isolate’s extract that inhibited the FREP1-iRBC lysate interaction and P. falciparum infection in mosquitoes.(a) A bioactive pure compound showed a single peak in HPLC profile (PDA detection 190–400nm). (b) The candidate compound showed greater inhibition of FREP1 binding to iRBC lysate as the compound concentration increased. (c) The candidate pure compound (8 μg/mL) significantly inhibited P. falciparum infection. (d) The candidate pure compound’s inhibition of P. falciparum infection in mosquitoes displayed a dose-dependent pattern. N: the number of mosquitoes for each treatment; μ: the average number of oocysts per midgut; PR: infection prevalence in mosquitoes.
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f6: A pure compound was isolated from the Chapel SA-3 isolate’s extract that inhibited the FREP1-iRBC lysate interaction and P. falciparum infection in mosquitoes.(a) A bioactive pure compound showed a single peak in HPLC profile (PDA detection 190–400nm). (b) The candidate compound showed greater inhibition of FREP1 binding to iRBC lysate as the compound concentration increased. (c) The candidate pure compound (8 μg/mL) significantly inhibited P. falciparum infection. (d) The candidate pure compound’s inhibition of P. falciparum infection in mosquitoes displayed a dose-dependent pattern. N: the number of mosquitoes for each treatment; μ: the average number of oocysts per midgut; PR: infection prevalence in mosquitoes.

Mentions: An extract consisting of 53.8 grams of crude natural-product-containing residue was prepared from an A. niger culture and fractioned by HP20ss flash column chromatography. The active fractions, which inhibited the interaction of FREP1-iRBC lysate were combined and subjected to HPLC as described in the Methods. One bioactive compound was purified. The purity of the fungal metabolite was confirmed by HPLC, wherein it eluted as a single peak at 6.8 min (PDA detection at 190–400 nm, Fig. 6a). The concentration-dependent inhibitory effects of the purified metabolite were analyzed over a range of 0 to 120 μg/mL for an ability to disrupt the interaction of FREP1-iRBC lysate using ELISA. Using the linear regression between logarithmic transformed dose and inhibition rates, we determined the IC50 of this compound to be 40 μg/mL (Fig. 6b). Next, we determined whether the pure compound was able to inhibit P. falciparum infection in mosquito midguts. Due to the much lower levels of endogenous FREP1 in mosquito midguts compared to the FREP1 in the ELISA assay, we tested the activity of the compound at a concentration well below the ELISA assay-derived IC50 value. For the experiment, 5 μL of pure compound (800 μg/mL) in DMSO was added into 495 μL blood containing 0.2% stage V P. falciparum gametocytes, and the mosquitoes were fed with SMFA. DMSO (5 μL aliquots) were used as vehicle-only controls. After dissecting the mosquitoes, significantly (p < 0.001) fewer oocysts were observed in the compound treated mosquito midguts than in the control samples (Fig. 6c). The results (Fig. 6d) also demonstrate that inhibition of P. falciparum infection occurred in a dose-dependent manner. As little as 3 μg/mL of the pure compound was capable of significantly reducing P. falciparum infection load in mosquitoes (p < 0.006), and reducing the number of oocyst by 56.7% and the infection prevalence rate by 35.3%.


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)

A pure compound was isolated from the Chapel SA-3 isolate’s extract that inhibited the FREP1-iRBC lysate interaction and P. falciparum infection in mosquitoes.(a) A bioactive pure compound showed a single peak in HPLC profile (PDA detection 190–400nm). (b) The candidate compound showed greater inhibition of FREP1 binding to iRBC lysate as the compound concentration increased. (c) The candidate pure compound (8 μg/mL) significantly inhibited P. falciparum infection. (d) The candidate pure compound’s inhibition of P. falciparum infection in mosquitoes displayed a dose-dependent pattern. N: the number of mosquitoes for each treatment; μ: the average number of oocysts per midgut; PR: infection prevalence in mosquitoes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: A pure compound was isolated from the Chapel SA-3 isolate’s extract that inhibited the FREP1-iRBC lysate interaction and P. falciparum infection in mosquitoes.(a) A bioactive pure compound showed a single peak in HPLC profile (PDA detection 190–400nm). (b) The candidate compound showed greater inhibition of FREP1 binding to iRBC lysate as the compound concentration increased. (c) The candidate pure compound (8 μg/mL) significantly inhibited P. falciparum infection. (d) The candidate pure compound’s inhibition of P. falciparum infection in mosquitoes displayed a dose-dependent pattern. N: the number of mosquitoes for each treatment; μ: the average number of oocysts per midgut; PR: infection prevalence in mosquitoes.
Mentions: An extract consisting of 53.8 grams of crude natural-product-containing residue was prepared from an A. niger culture and fractioned by HP20ss flash column chromatography. The active fractions, which inhibited the interaction of FREP1-iRBC lysate were combined and subjected to HPLC as described in the Methods. One bioactive compound was purified. The purity of the fungal metabolite was confirmed by HPLC, wherein it eluted as a single peak at 6.8 min (PDA detection at 190–400 nm, Fig. 6a). The concentration-dependent inhibitory effects of the purified metabolite were analyzed over a range of 0 to 120 μg/mL for an ability to disrupt the interaction of FREP1-iRBC lysate using ELISA. Using the linear regression between logarithmic transformed dose and inhibition rates, we determined the IC50 of this compound to be 40 μg/mL (Fig. 6b). Next, we determined whether the pure compound was able to inhibit P. falciparum infection in mosquito midguts. Due to the much lower levels of endogenous FREP1 in mosquito midguts compared to the FREP1 in the ELISA assay, we tested the activity of the compound at a concentration well below the ELISA assay-derived IC50 value. For the experiment, 5 μL of pure compound (800 μg/mL) in DMSO was added into 495 μL blood containing 0.2% stage V P. falciparum gametocytes, and the mosquitoes were fed with SMFA. DMSO (5 μL aliquots) were used as vehicle-only controls. After dissecting the mosquitoes, significantly (p < 0.001) fewer oocysts were observed in the compound treated mosquito midguts than in the control samples (Fig. 6c). The results (Fig. 6d) also demonstrate that inhibition of P. falciparum infection occurred in a dose-dependent manner. As little as 3 μg/mL of the pure compound was capable of significantly reducing P. falciparum infection load in mosquitoes (p < 0.006), and reducing the number of oocyst by 56.7% and the infection prevalence rate by 35.3%.

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