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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

The pure candidate compound specifically prevents FREP1 from binding iRBC, gametocytes, and ookinetes.(a) The candidate compound specifically inhibited the binding of FREP1 protein to P. falciparum parasites as demonstrated by IFA. The first and second column detected FREP1 and parasite nuclei respectively. Merging column one and two generated the third column, which shows the co-localization of P. falciparum (nuclei) and FREP1 binding. The fourth column shows the bright views of the cells. No FREP1 signals were detected without adding FREP1 (1st row, arrows points to gametocytes or ookinetes). Incubating FREP1 with P. falciparum gametocytes and ookinetes supplemented with 1% DMSO showed the co-localization of FREP1 and gametocytes or ookinetes (2nd row). Addition of the candidate compound (40 μg/mL) reduced the interaction between FREP1 and gametocytes/ookinetes (3rd row). The fourth row shows the bright field. Of note, many DAPI-positive dots that do not match with red spots are merazoites (free parasites), suggesting FREP1 does not bind merazoites well. (b) The intensity of red fluorescence indicated that the compound significantly prevented FREP1 from binding to gametocytes or ookinetes. (c) The candidate compound did not affect the ELISA reaction to detect FREP1, supporting the compound specifically interferes with FREP1-iRBC lysate interaction. Treatments: 1: FREP1 (7.5 μg/mL) plus DMSO (1%); 2: FREP1 (7.5 μg/mL) plus the candidate compound (40 μg/mL); 3: BSA (7.5 μg/mL) plus DMSO (1%).
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f7: The pure candidate compound specifically prevents FREP1 from binding iRBC, gametocytes, and ookinetes.(a) The candidate compound specifically inhibited the binding of FREP1 protein to P. falciparum parasites as demonstrated by IFA. The first and second column detected FREP1 and parasite nuclei respectively. Merging column one and two generated the third column, which shows the co-localization of P. falciparum (nuclei) and FREP1 binding. The fourth column shows the bright views of the cells. No FREP1 signals were detected without adding FREP1 (1st row, arrows points to gametocytes or ookinetes). Incubating FREP1 with P. falciparum gametocytes and ookinetes supplemented with 1% DMSO showed the co-localization of FREP1 and gametocytes or ookinetes (2nd row). Addition of the candidate compound (40 μg/mL) reduced the interaction between FREP1 and gametocytes/ookinetes (3rd row). The fourth row shows the bright field. Of note, many DAPI-positive dots that do not match with red spots are merazoites (free parasites), suggesting FREP1 does not bind merazoites well. (b) The intensity of red fluorescence indicated that the compound significantly prevented FREP1 from binding to gametocytes or ookinetes. (c) The candidate compound did not affect the ELISA reaction to detect FREP1, supporting the compound specifically interferes with FREP1-iRBC lysate interaction. Treatments: 1: FREP1 (7.5 μg/mL) plus DMSO (1%); 2: FREP1 (7.5 μg/mL) plus the candidate compound (40 μg/mL); 3: BSA (7.5 μg/mL) plus DMSO (1%).

Mentions: We determined the specificity of the active compound in preventing FREP1 from binding to asexual stage parasites, gametocytes, or ookinetes using IFA. The P. falciparum gametocytes and ookinetes were fixed on glass coverslips using 4% paraformaldehyde in PBS. The FREP1 mixed with 40 μg/mL of the compound was incubated with the cells, followed by standard procedure of IFA. The DMSO (1%, v/v) without the compound was used as a positive control, and BSA replacing FREP1 as a negative control. Because asexual stage parasites are irrelevant to Plasmodium infection in mosquitoes, we focused on gametocytes and ookinetes. The fluorescence intensity values of gametocytes and ookinetes in three samples were: 3.97 ± 0.65 for negative control (Fig. 7a, 1st row), 14.7 ± 2.73 for positive control (Fig. 7a, 2nd row), and 6.01 ± 1.28 for the experimental group (Fig. 7a, 3rd row). Of note, many DAPI-positive dots that do not match with red spots were merazoites (free parasites), suggesting FREP1 does not bind merazoites. Apparently, the compound significantly (p < 0.001) prevented FREP1 from binding to ookinetes (Fig. 7b). It is worth noting that the inhibition rate measured by IFA was consistent with the inhibition rate measured through ELISA with the same concentration of the compound (Fig. 6b).


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)

The pure candidate compound specifically prevents FREP1 from binding iRBC, gametocytes, and ookinetes.(a) The candidate compound specifically inhibited the binding of FREP1 protein to P. falciparum parasites as demonstrated by IFA. The first and second column detected FREP1 and parasite nuclei respectively. Merging column one and two generated the third column, which shows the co-localization of P. falciparum (nuclei) and FREP1 binding. The fourth column shows the bright views of the cells. No FREP1 signals were detected without adding FREP1 (1st row, arrows points to gametocytes or ookinetes). Incubating FREP1 with P. falciparum gametocytes and ookinetes supplemented with 1% DMSO showed the co-localization of FREP1 and gametocytes or ookinetes (2nd row). Addition of the candidate compound (40 μg/mL) reduced the interaction between FREP1 and gametocytes/ookinetes (3rd row). The fourth row shows the bright field. Of note, many DAPI-positive dots that do not match with red spots are merazoites (free parasites), suggesting FREP1 does not bind merazoites well. (b) The intensity of red fluorescence indicated that the compound significantly prevented FREP1 from binding to gametocytes or ookinetes. (c) The candidate compound did not affect the ELISA reaction to detect FREP1, supporting the compound specifically interferes with FREP1-iRBC lysate interaction. Treatments: 1: FREP1 (7.5 μg/mL) plus DMSO (1%); 2: FREP1 (7.5 μg/mL) plus the candidate compound (40 μg/mL); 3: BSA (7.5 μg/mL) plus DMSO (1%).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: The pure candidate compound specifically prevents FREP1 from binding iRBC, gametocytes, and ookinetes.(a) The candidate compound specifically inhibited the binding of FREP1 protein to P. falciparum parasites as demonstrated by IFA. The first and second column detected FREP1 and parasite nuclei respectively. Merging column one and two generated the third column, which shows the co-localization of P. falciparum (nuclei) and FREP1 binding. The fourth column shows the bright views of the cells. No FREP1 signals were detected without adding FREP1 (1st row, arrows points to gametocytes or ookinetes). Incubating FREP1 with P. falciparum gametocytes and ookinetes supplemented with 1% DMSO showed the co-localization of FREP1 and gametocytes or ookinetes (2nd row). Addition of the candidate compound (40 μg/mL) reduced the interaction between FREP1 and gametocytes/ookinetes (3rd row). The fourth row shows the bright field. Of note, many DAPI-positive dots that do not match with red spots are merazoites (free parasites), suggesting FREP1 does not bind merazoites well. (b) The intensity of red fluorescence indicated that the compound significantly prevented FREP1 from binding to gametocytes or ookinetes. (c) The candidate compound did not affect the ELISA reaction to detect FREP1, supporting the compound specifically interferes with FREP1-iRBC lysate interaction. Treatments: 1: FREP1 (7.5 μg/mL) plus DMSO (1%); 2: FREP1 (7.5 μg/mL) plus the candidate compound (40 μg/mL); 3: BSA (7.5 μg/mL) plus DMSO (1%).
Mentions: We determined the specificity of the active compound in preventing FREP1 from binding to asexual stage parasites, gametocytes, or ookinetes using IFA. The P. falciparum gametocytes and ookinetes were fixed on glass coverslips using 4% paraformaldehyde in PBS. The FREP1 mixed with 40 μg/mL of the compound was incubated with the cells, followed by standard procedure of IFA. The DMSO (1%, v/v) without the compound was used as a positive control, and BSA replacing FREP1 as a negative control. Because asexual stage parasites are irrelevant to Plasmodium infection in mosquitoes, we focused on gametocytes and ookinetes. The fluorescence intensity values of gametocytes and ookinetes in three samples were: 3.97 ± 0.65 for negative control (Fig. 7a, 1st row), 14.7 ± 2.73 for positive control (Fig. 7a, 2nd row), and 6.01 ± 1.28 for the experimental group (Fig. 7a, 3rd row). Of note, many DAPI-positive dots that do not match with red spots were merazoites (free parasites), suggesting FREP1 does not bind merazoites. Apparently, the compound significantly (p < 0.001) prevented FREP1 from binding to ookinetes (Fig. 7b). It is worth noting that the inhibition rate measured by IFA was consistent with the inhibition rate measured through ELISA with the same concentration of the compound (Fig. 6b).

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