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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

Chapel SA-3 extract specifically prevents FREP1 from binding P. falciparum rings, gametocytes and ookinetes.(a) The extract from isolate Chapel SA-3 did not affect the interaction between FREP1 and anti-FREP1 polyclonal antibody nor 1st antibody and 2nd antibody determined by ELISA. Treatments: 1: FREP1 (7.5 μg/mL) plus DMSO (1%); 2: FREP1 (7.5 μg/mL) plus Chapel SA-3 extract (100 μg/mL); 3: BSA (7.5 μg/mL) plus DMSO (1%). (b) The extract from isolate Chapel SA-3 did not affect the unrelated molecule-molecule interaction. Treatments: 1: unrelated His-tagged protein (7.5 μg/mL) plus DMSO (1%); 2: unrelated His-tagged protein (7.5 μg/mL) plus Chapel SA-3 (100 μg/mL); 3: BSA (7.5 μg/mL) plus DMSO (1%). (c) The morphology of gametocytes and ookinetes stained by the Giemsa staining. (d) The extract from isolate Chapel SA-3 inhibited the binding between FREP1 protein and P. falciparum parasites shown by IFA. The first and second column detected cell nuclei stained with DAPI and FREP1, respectively. Merging column one and two generated the third column that shows the co-localization of P. falciparum (nuclei) and FREP1 binding. The 4th column shows the bright views of the cells. The uninfected RBC did not bind FREP1 (1st row)’; P. falciparum iRBC rings, gametocytes (GT), and ookinetes (OK) interacted with FREP1 (2nd row); and such interactions were disrupted by Chapel SA-3 (3rd row).
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f3: Chapel SA-3 extract specifically prevents FREP1 from binding P. falciparum rings, gametocytes and ookinetes.(a) The extract from isolate Chapel SA-3 did not affect the interaction between FREP1 and anti-FREP1 polyclonal antibody nor 1st antibody and 2nd antibody determined by ELISA. Treatments: 1: FREP1 (7.5 μg/mL) plus DMSO (1%); 2: FREP1 (7.5 μg/mL) plus Chapel SA-3 extract (100 μg/mL); 3: BSA (7.5 μg/mL) plus DMSO (1%). (b) The extract from isolate Chapel SA-3 did not affect the unrelated molecule-molecule interaction. Treatments: 1: unrelated His-tagged protein (7.5 μg/mL) plus DMSO (1%); 2: unrelated His-tagged protein (7.5 μg/mL) plus Chapel SA-3 (100 μg/mL); 3: BSA (7.5 μg/mL) plus DMSO (1%). (c) The morphology of gametocytes and ookinetes stained by the Giemsa staining. (d) The extract from isolate Chapel SA-3 inhibited the binding between FREP1 protein and P. falciparum parasites shown by IFA. The first and second column detected cell nuclei stained with DAPI and FREP1, respectively. Merging column one and two generated the third column that shows the co-localization of P. falciparum (nuclei) and FREP1 binding. The 4th column shows the bright views of the cells. The uninfected RBC did not bind FREP1 (1st row)’; P. falciparum iRBC rings, gametocytes (GT), and ookinetes (OK) interacted with FREP1 (2nd row); and such interactions were disrupted by Chapel SA-3 (3rd row).

Mentions: To analyze whether Chapel SA-3 prevented anti-FREP1 antibody from binding FREP1 or prevented secondary antibody binding, we coated ELISA plates with the FREP1 and incubated anti-FREP1 with Chapel SA-3, 2nd antibody, and other ELISA reagents sequentially. The DMSO was used as a negative control. Results showed no significant difference (p = 0.45) between the negative control (Fig. 3a, column 1), and the Chapel SA-3 treated sample (Fig. 3a, column 2). The OD405 values of both negative control and Chapel SA-3 treated sample were much higher than background (Fig. 3a, column 3). These results support the fact that Chapel SA-3 does not inhibit the interaction between FREP1 and anti-FREP1 antibody or anti-FREP1 antibody and the secondary antibody. In addition, the effect of Chapel SA-3 on unrelated molecule-molecule interaction was also tested using an anti-His monoclonal antibody binding unrelated His-tagged protein. Coating an unrelated His-tagged protein onto a plate and detecting with anti-His monoclonal antibody showed similar results (p = 0.30) between Chapel SA-3 treated samples (Fig. 3b, column 2) and the control (DMSO treated, Fig. 3b, column 1). The results also showed that the OD405 values of both the DMSO-treated and Chapel SA-3 treated sample were much higher than the background (Fig. 3b, column 3). These data collectively show that Chapel SA-3 specifically disrupts the interaction between FREP1 and parasites.


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)

Chapel SA-3 extract specifically prevents FREP1 from binding P. falciparum rings, gametocytes and ookinetes.(a) The extract from isolate Chapel SA-3 did not affect the interaction between FREP1 and anti-FREP1 polyclonal antibody nor 1st antibody and 2nd antibody determined by ELISA. Treatments: 1: FREP1 (7.5 μg/mL) plus DMSO (1%); 2: FREP1 (7.5 μg/mL) plus Chapel SA-3 extract (100 μg/mL); 3: BSA (7.5 μg/mL) plus DMSO (1%). (b) The extract from isolate Chapel SA-3 did not affect the unrelated molecule-molecule interaction. Treatments: 1: unrelated His-tagged protein (7.5 μg/mL) plus DMSO (1%); 2: unrelated His-tagged protein (7.5 μg/mL) plus Chapel SA-3 (100 μg/mL); 3: BSA (7.5 μg/mL) plus DMSO (1%). (c) The morphology of gametocytes and ookinetes stained by the Giemsa staining. (d) The extract from isolate Chapel SA-3 inhibited the binding between FREP1 protein and P. falciparum parasites shown by IFA. The first and second column detected cell nuclei stained with DAPI and FREP1, respectively. Merging column one and two generated the third column that shows the co-localization of P. falciparum (nuclei) and FREP1 binding. The 4th column shows the bright views of the cells. The uninfected RBC did not bind FREP1 (1st row)’; P. falciparum iRBC rings, gametocytes (GT), and ookinetes (OK) interacted with FREP1 (2nd row); and such interactions were disrupted by Chapel SA-3 (3rd row).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Chapel SA-3 extract specifically prevents FREP1 from binding P. falciparum rings, gametocytes and ookinetes.(a) The extract from isolate Chapel SA-3 did not affect the interaction between FREP1 and anti-FREP1 polyclonal antibody nor 1st antibody and 2nd antibody determined by ELISA. Treatments: 1: FREP1 (7.5 μg/mL) plus DMSO (1%); 2: FREP1 (7.5 μg/mL) plus Chapel SA-3 extract (100 μg/mL); 3: BSA (7.5 μg/mL) plus DMSO (1%). (b) The extract from isolate Chapel SA-3 did not affect the unrelated molecule-molecule interaction. Treatments: 1: unrelated His-tagged protein (7.5 μg/mL) plus DMSO (1%); 2: unrelated His-tagged protein (7.5 μg/mL) plus Chapel SA-3 (100 μg/mL); 3: BSA (7.5 μg/mL) plus DMSO (1%). (c) The morphology of gametocytes and ookinetes stained by the Giemsa staining. (d) The extract from isolate Chapel SA-3 inhibited the binding between FREP1 protein and P. falciparum parasites shown by IFA. The first and second column detected cell nuclei stained with DAPI and FREP1, respectively. Merging column one and two generated the third column that shows the co-localization of P. falciparum (nuclei) and FREP1 binding. The 4th column shows the bright views of the cells. The uninfected RBC did not bind FREP1 (1st row)’; P. falciparum iRBC rings, gametocytes (GT), and ookinetes (OK) interacted with FREP1 (2nd row); and such interactions were disrupted by Chapel SA-3 (3rd row).
Mentions: To analyze whether Chapel SA-3 prevented anti-FREP1 antibody from binding FREP1 or prevented secondary antibody binding, we coated ELISA plates with the FREP1 and incubated anti-FREP1 with Chapel SA-3, 2nd antibody, and other ELISA reagents sequentially. The DMSO was used as a negative control. Results showed no significant difference (p = 0.45) between the negative control (Fig. 3a, column 1), and the Chapel SA-3 treated sample (Fig. 3a, column 2). The OD405 values of both negative control and Chapel SA-3 treated sample were much higher than background (Fig. 3a, column 3). These results support the fact that Chapel SA-3 does not inhibit the interaction between FREP1 and anti-FREP1 antibody or anti-FREP1 antibody and the secondary antibody. In addition, the effect of Chapel SA-3 on unrelated molecule-molecule interaction was also tested using an anti-His monoclonal antibody binding unrelated His-tagged protein. Coating an unrelated His-tagged protein onto a plate and detecting with anti-His monoclonal antibody showed similar results (p = 0.30) between Chapel SA-3 treated samples (Fig. 3b, column 2) and the control (DMSO treated, Fig. 3b, column 1). The results also showed that the OD405 values of both the DMSO-treated and Chapel SA-3 treated sample were much higher than the background (Fig. 3b, column 3). These data collectively show that Chapel SA-3 specifically disrupts the interaction between FREP1 and parasites.

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