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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

Insect cell-expressed FREP1 protein and its interaction with iRBC lysate detected by ELISA.(a) The FREP1 was expressed and secreted from High Five cells, determined by 12% SDS-PAGE (left) and western blot assay (right). This result also demonstrated that anti-FREP1 antibody could specifically recognize FREP1. Lanes: 1,3: cell lysate; 2,4: culture medium. (b) ELISA signals were significantly different between Plasmodium falciparum-infected red blood cell (iRBC) lysate and uninfected RBC lysate. The lysate of iRBC and uninfected human RBC were used to coat the ELISA plate, followed by sequential incubation with recombinant insect cell expressed FREP1 protein, 1st antibody, alkaline phosphatase-conjugated 2nd antibody. The samples were developed by the addition of 100 μL of pNPP and OD405 reading. The retained FREP1 in iRBC lysate-treated wells was significantly higher than in uninfected RBC lysate (p < 0.0002). (c) When the heat inactivated FREP1 protein (InactFREP1) replaced the functional FREP1, the binding between FREP1 and iRBC lysate disappeared (p < 0.001).
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f1: Insect cell-expressed FREP1 protein and its interaction with iRBC lysate detected by ELISA.(a) The FREP1 was expressed and secreted from High Five cells, determined by 12% SDS-PAGE (left) and western blot assay (right). This result also demonstrated that anti-FREP1 antibody could specifically recognize FREP1. Lanes: 1,3: cell lysate; 2,4: culture medium. (b) ELISA signals were significantly different between Plasmodium falciparum-infected red blood cell (iRBC) lysate and uninfected RBC lysate. The lysate of iRBC and uninfected human RBC were used to coat the ELISA plate, followed by sequential incubation with recombinant insect cell expressed FREP1 protein, 1st antibody, alkaline phosphatase-conjugated 2nd antibody. The samples were developed by the addition of 100 μL of pNPP and OD405 reading. The retained FREP1 in iRBC lysate-treated wells was significantly higher than in uninfected RBC lysate (p < 0.0002). (c) When the heat inactivated FREP1 protein (InactFREP1) replaced the functional FREP1, the binding between FREP1 and iRBC lysate disappeared (p < 0.001).

Mentions: Because FREP1 expressed in E. coli forms inclusion bodies and lacks post-translational modification, we used an insect cell expression system to generate functional FREP1 that is similar to endogenous FREP1. The endotoxin-free pIB-FREP1 plasmid was used to express FREP1 in insect cells using protein-free medium. We found that FREP1 was specifically detected in the culture medium of the insect High Five cells transfected with pIB-FREP1 DNA but not in the cell lysate (Fig. 1a), confirming that FREP1 is a secreted protein. The western blotting result also demonstrates that the anti-FREP1 polyclonal antibody24 can specifically recognize FREP1.


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)

Insect cell-expressed FREP1 protein and its interaction with iRBC lysate detected by ELISA.(a) The FREP1 was expressed and secreted from High Five cells, determined by 12% SDS-PAGE (left) and western blot assay (right). This result also demonstrated that anti-FREP1 antibody could specifically recognize FREP1. Lanes: 1,3: cell lysate; 2,4: culture medium. (b) ELISA signals were significantly different between Plasmodium falciparum-infected red blood cell (iRBC) lysate and uninfected RBC lysate. The lysate of iRBC and uninfected human RBC were used to coat the ELISA plate, followed by sequential incubation with recombinant insect cell expressed FREP1 protein, 1st antibody, alkaline phosphatase-conjugated 2nd antibody. The samples were developed by the addition of 100 μL of pNPP and OD405 reading. The retained FREP1 in iRBC lysate-treated wells was significantly higher than in uninfected RBC lysate (p < 0.0002). (c) When the heat inactivated FREP1 protein (InactFREP1) replaced the functional FREP1, the binding between FREP1 and iRBC lysate disappeared (p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Insect cell-expressed FREP1 protein and its interaction with iRBC lysate detected by ELISA.(a) The FREP1 was expressed and secreted from High Five cells, determined by 12% SDS-PAGE (left) and western blot assay (right). This result also demonstrated that anti-FREP1 antibody could specifically recognize FREP1. Lanes: 1,3: cell lysate; 2,4: culture medium. (b) ELISA signals were significantly different between Plasmodium falciparum-infected red blood cell (iRBC) lysate and uninfected RBC lysate. The lysate of iRBC and uninfected human RBC were used to coat the ELISA plate, followed by sequential incubation with recombinant insect cell expressed FREP1 protein, 1st antibody, alkaline phosphatase-conjugated 2nd antibody. The samples were developed by the addition of 100 μL of pNPP and OD405 reading. The retained FREP1 in iRBC lysate-treated wells was significantly higher than in uninfected RBC lysate (p < 0.0002). (c) When the heat inactivated FREP1 protein (InactFREP1) replaced the functional FREP1, the binding between FREP1 and iRBC lysate disappeared (p < 0.001).
Mentions: Because FREP1 expressed in E. coli forms inclusion bodies and lacks post-translational modification, we used an insect cell expression system to generate functional FREP1 that is similar to endogenous FREP1. The endotoxin-free pIB-FREP1 plasmid was used to express FREP1 in insect cells using protein-free medium. We found that FREP1 was specifically detected in the culture medium of the insect High Five cells transfected with pIB-FREP1 DNA but not in the cell lysate (Fig. 1a), confirming that FREP1 is a secreted protein. The western blotting result also demonstrates that the anti-FREP1 polyclonal antibody24 can specifically recognize FREP1.

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