Edited by: Luiz Gustavo Gardinassi, Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás (IPTSP – UFG), Brazil
Reviewed by: Nazzy Pakpour, California State University, East Bay, United States; Yuemei Dong, Johns Hopkins University, United States
*Correspondence: Jun Li,
†These authors have contributed equally to this work
This article was submitted to Parasite and Host, a section of the journal Frontiers in Cellular and Infection Microbiology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Malaria transmission relies on parasite-mosquito midgut interaction. The interactive proteins are hypothesized to be ideal targets to block malaria transmission to mosquitoes. We chose 76 genes that contain signal peptide-coding regions and are upregulated and highly abundant at sexual stages. Forty-six of these candidate genes (60%) were cloned and expressed using the baculovirus expression system in insect cells. Six of them, e.g., PF3D7_0303900, PF3D7_0406200 (Pfs16), PF3D7_1204400 (Pfs37), PF3D7_1214800, PF3D7_1239400, and PF3D7_1472800 were discovered to interact with blood-fed mosquito midgut lysate. Previous works showed that among these interactive proteins, knockout the orthologs of Pfs37 or Pfs16 in
Malaria is a deadly infectious disease spread by mosquitoes. It affects half of the world’s population and claims nearly a half-million lives annually. Although the global malaria cases have decreased since 2010, the ten most burdened countries in Africa still had 3.5 million more cases than the previous year (
Since
The interaction between parasites and the mosquito midgut is a crucial determinant of successful infections in a mosquito. Quite some studies have shown mosquito proteins are essential for the ookinete formation in the mosquito midgut and their penetration of the peritrophic matrix (PM) and midgut epithelial cells. Mosquito proteins AnAPN1 and FREP1 have been identified to play roles in malaria transmission. AnAPN1 localizes on the apical surfaces of
Parasitic proteins involved in gametogenesis, fertilization of macro- and micro-gametes, zygote-to-ookinete transformation, and penetration of the midgut endothelium are also critical for malaria transmission (
Besides targeted by transmission-blocking vaccines, these interactive proteins can also be targeted by small molecules to block malaria transmission. The fungal secondary metabolite
Therefore, this study focused on sexual stage parasitic proteins that interact with mosquito midguts. These proteins should be at the cytoplasmic membrane or secreted from the parasites. The identification of these proteins will help us to understand the molecular mechanisms of malaria transmission and provide targets for vaccines and drugs to control malaria transmission.
The sequences of
The information of the signal peptide for each protein was obtained from PlasmoDB. The data of the gene expression of the published RNA-Seq data (
Total RNA was extracted from the
The 3-5-day old adult female mosquitoes were fed with fresh blood using a membrane feeding device. Starting 16 h post blood meal and lasting more than 4 h, ~100 midguts were dissected from blood-engorged mosquitoes. The blood was carefully removed from the midguts. The midguts were then placed in a 1.5 mL plastic tube containing lysis buffer (50 mM Tris-HCl, 0.15 mM NaCl, 0.2% Tween-20, pH7.8) and grounded with a micro pestle (Sigma-Aldrich). Subsequently, the tissues were lysed in native cell lysis buffer (Clontech) with ultrasonication for 10 m on ice with 30 s pulse and 10 s sonication. The debris was removed by centrifugation (10,000 g for 5 m) at 4°C. The protein concentration in the supernatant was measured by the Bradford method.
About 50 µL of 1 mg/mL midgut proteins were used to coat each well on a 96 well plate at 4°C overnight. The plate was then blocked with 100 µL of 2% BSA in PBS for 2 h at RT. About 100 ng of recombinant candidate proteins in 50 µl PBS were added to each well. Each corresponding recombinant protein was heat-inactivated for 15 m at 65°C as a control. The plates were incubated for 1 h at RT. Then, 50 µl anti-His mouse monoclonal antibody (Sigma, 1:1, 000 dilutions in blocking buffer) was added and incubated at RT for 1 h, followed by incubation with 50 µl of goat anti-mouse IgG-Alkaline Phosphatase conjugate (Sigma, 1:10, 000 dilutions in blocking buffer) for 1 h at RT. Finally, 50 µL of
These six recombinant proteins were further verified by an alternative ELISA. About 10 unhomogenized midguts (collected 18 h after bloodmeal) in a 1.5 mL plastic tube were suspended in 50 µl PBS containing 100 ng of a recombinant candidate protein and incubated for 1 h at RT. The PBS buffer with chloramphenicol acetyltransferase (CAT), which was expressed with the baculovirus system in the High Five cells, was used as a blank control. Then, the midguts were collected through centrifugation (500 g for 5 m), washed with 100 µl PBS three times, and homogenized in 100 µl lysis buffer (50 mM Tris-HCl, 0.15 mM NaCl, 0.2% Tween-20, pH7.8) by a micro pestle (Sigma-Aldrich) and ultrasonication as described above. After removing the debris by centrifugation (10,000 g for 5 m) at 4°C, 50 µl of the supernatant was used to coat ELISA, followed by incubation with anti-His mouse monoclonal antibody, goat anti-mouse IgG-Alkaline Phosphatase conjugate, and pNPP as described above. The plate was washed three times with PBST (1xPBS containing 0.2% Tween 20) between two incubations. The signal was detected with an Epoch Microplate Spectrophotometer at A405 nm (BioTek, Winooski, VT). Statistical analysis was conducted by multiple t-test using the Two-stage linear step-up procedure (
Mosquito midguts were isolated from 3-5-day-old naïve mosquitoes. The mosquito midguts were also isolated from mosquitoes 18 hr post bloodmeal. The midguts were cut in half, and the content was rinsed with 200 μL 1xPBS three times. Midguts were incubated sequentially with the recombinant Pfs16 protein (experimental group) or CAT (control group) expressed in High Five cells culture supernatant (10 μg/mL) for 1 h at RT, the rabbit antibodies against Pfs16 protein in PBS (1μg/mL) for 1 h at RT, Alexa Fluor 594-conjugated goat against rabbit antibodies (ThermoFisher, 1:500 dilutions in PBS) for 1 h in the dark at RT. The sample midguts were washed with 1XPBS 5 min for 3 times between each incubation. Finally, the treated midguts were examined under a fluorescence microscope. All midguts were exposed with the same activated light intensity, and images were taken with the same exposure time. The fluorescence pixel in images was measured with Adobe Photoshop 2020.
We selected
Expression profile of the
The features of selected candidate proteins.
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Expression based on RNA-seq data |
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Ring | ET | LT | ST | GII | GV | Ookinete | ||||||
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1.14 | 3.09 | 29.91 | 12.51 | 117.95 | 475.94 | 190.3 | 50S ribosomal protein L9, apicoplast, putative | yes | 197 | apicoplast | yes |
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5.25 | 2.87 | 40.18 | 34.21 | 604.92 | 704.38 | 144.83 | longevity-assurance (LAG1) protein, putative | yes | 355 | apicoplast | yes |
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6.5 | 1.67 | 66.31 | 30.45 | 269.35 | 163.88 | 121.91 | conserved Plasmodium protein, unknown function | yes | 60 | apicoplast | |
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15.91 | 7.27 | 41.39 | 17.78 | 165.71 | 260.26 | 150.4 | superoxide dismutase [Fe] (SOD2) | no | 266 | apicoplast | yes |
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4.97 | 6.87 | 41.46 | 6.04 | 325.31 | 367.53 | 121.77 | conserved Plasmodium protein, unknown function | no | 170 | apicoplast | |
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2.32 | 0 | 14.88 | 2.41 | 401.49 | 193.37 | 98.58 | early transcribed membrane protein 8 (ETRAMP8) | yes | 170 | apicoplast | yes |
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32.74 | 7.98 | 73.41 | 35.48 | 238.38 | 1960.34 | 307.28 | ribosomal protein L35, apicoplast, putative | no | 191 | apicoplast | |
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6.48 | 2.34 | 83.13 | 39.8 | 424.56 | 486.84 | 150.68 | conserved protein, unknown function | yes | 305 | apicoplast | |
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2.36 | 0.53 | 13.9 | 9.68 | 60.51 | 359.01 | 126.53 | conserved Plasmodium protein, unknown function | yes | 191 | apicoplast | yes |
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20.15 | 5.42 | 8.46 | 2.58 | 113.51 | 78.63 | 321.75 | dihydrolipoamide acyltransferase component E2 | no | 640 | apicoplast | |
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8.5 | 1.91 | 53.69 | 7.74 | 120.29 | 822.3 | 117.75 | conserved protein, unknown function | yes | 79 | apicoplast | yes |
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6.08 | 11.91 | 35.52 | 13 | 175.17 | 379.06 | 66.1 | apicoplast ribosomal protein S15 precursor, putative | yes | 269 | apicoplast | yes |
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4.58 | 2.07 | 78.58 | 31.02 | 213.01 | 276.4 | 163.35 | apicoplast ribosomal protein S14p/S29e precursor, | no | 172 | apicoplast | yes |
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4.51 | 1.68 | 71.14 | 23.82 | 145.41 | 355.88 | 130.75 | HSP20-like chaperone, putative | yes | 363 | apicoplast | yes |
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3.8 | 0.86 | 0.7 | 1.15 | 178.99 | 4730.33 | 85.86 | conserved protein, unknown function (Pfs37) | yes | 178 | cell surface | yes |
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5.41 | 1.74 | 1.28 | 0 | 269.16 | 3109.6 | 2284.3 | PH domain-containing protein, putative | yes | 292 | cell surface | yes |
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3.24 | 1.02 | 1.55 | 0 | 260.54 | 2094.45 | 148.73 | sexual stage-specific protein G37, putative | yes | 349 | cell surface | yes |
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5.14 | 7.23 | 32.17 | 13.49 | 78.81 | 178.26 | 280.73 | CPW-WPC family protein | no | 550 | cell surface | |
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5.84 | 0.78 | 0.95 | 0 | 803.15 | 5476.06 | 624.45 | MOLO1 domain-containing protein, putative | yes | 261 | crystalloid | yes |
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3.89 | 0.5 | 0.27 | 0 | 108.54 | 2480.37 | 580.03 | crystalloid-specific PH domain-containing protein | no | 305 | crystalloid | yes |
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4.12 | 0.46 | 0.76 | 1.88 | 264.59 | 1771.22 | 202.82 | LIMP protein, putative | no | 109 | crystalloid | yes |
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9.18 | 3.14 | 13.98 | 6.92 | 668.75 | 586.6 | 155.1 | conserved Plasmodium protein, unknown function | no | 178 | cytoplasm | |
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2.08 | 1.13 | 3.07 | 6.07 | 931.97 | 6706.56 | 740.02 | secreted ookinete protein, putative (PSOP6) | no | 135 | cytoplasm | yes |
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2.41 | 0.81 | 7.32 | 1.1 | 305.88 | 5919.52 | 334.91 | conserved Plasmodium protein, unknown function | no | 187 | cytoplasm | yes |
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4.66 | 1.29 | 7.26 | 5.23 | 194.98 | 1926.68 | 276.13 | conserved protein, unknown function | no | 315 | cytoplasm | yes |
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0.94 | 0.85 | 0.7 | 0 | 170.17 | 1061.44 | 107.42 | conserved protein, unknown function | no | 179 | cytoplasm | yes |
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12.38 | 14.2 | 48.88 | 19.14 | 62.95 | 129.63 | 216.7 | conserved protein, unknown function | yes | 150 | cytoplasm | |
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2.56 | 1.15 | 5.96 | 0 | 1072.11 | 4267.03 | 499.5 | conserved protein, unknown function | yes | 132 | cytoplasm | yes |
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4.46 | 3.62 | 14.12 | 1.63 | 1388.9 | 8222.2 | 808.08 | conserved Plasmodium protein, unknown function | no | 126 | cytoplasm | yes |
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2.2 | 0.4 | 0.97 | 0.53 | 63.24 | 1233.72 | 154.13 | plasmepsin VIII, putative | no | 385 | cytoplasm | yes |
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11.58 | 29.21 | 142.95 | 45.35 | 566.79 | 191.19 | 238.5 | DER1-like protein, putative (Derlin) | yes | 263 | endoplasmic reticulum | yes |
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6.86 | 8.4 | 20.98 | 1.92 | 116.99 | 318.5 | 70.83 | steroid dehydrogenase, putative | yes | 321 | membrane | |
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216.87 | 522.9 | 1153.39 | 744.61 | 431324.27 | 52190.48 | 16181.79 | sexual stage-specific protein precursor (Pfs16) | yes | 157 | membrane | yes |
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3.08 | 0.56 | 4.23 | 5.23 | 849.57 | 4697.28 | 741.87 | MOLO1 domain-containing protein, putative | yes | 275 | membrane | |
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5.48 | 3.29 | 23.54 | 9.99 | 1668.26 | 3629.97 | 1111.5 | CPW-WPC family protein | yes | 185 | membrane | yes |
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11.9 | 5.13 | 6.47 | 9.42 | 150.05 | 2537.45 | 477.67 | conserved Plasmodium protein, unknown function | yes | 218 | membrane | |
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3.09 | 0.7 | 22.49 | 27.47 | 86.12 | 209.96 | 69.37 | conserved Plasmodium protein, unknown function | yes | 292 | membrane | |
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10.63 | 18.78 | 40.9 | 11.86 | 133.55 | 243.47 | 108.27 | transmembrane protein 147, putative | yes | 260 | membrane | yes |
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21.82 | 50.13 | 807.63 | 136.34 | 89715.85 | 2928.45 | 3224.83 | early transcribed membrane protein 10.3 (ETRAMP10.3) | yes | 108 | membrane | |
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18.62 | 4.66 | 44.37 | 89.53 | 576.08 | 1043.52 | 694.99 | ookinete surface protein P28 (Pfs28) | no | 218 | membrane | |
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9.35 | 1.64 | 8.8 | 8.52 | 4217.91 | 23349.99 | 3477.26 | ookinete surface protein P25 (Pfs25) | yes | 217 | membrane | |
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3.3 | 0.52 | 1.71 | 0 | 282.14 | 685.68 | 356.55 | conserved Plasmodium protein, unknown function | no | 291 | membrane | yes |
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4.95 | 1.67 | 3.19 | 6.77 | 90.42 | 64.18 | 1725.74 | cell traversal protein for ookinetes and sporozoites (CelTOS) | no | 182 | microneme | yes |
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7.25 | 1.26 | 54.23 | 146.41 | 94.81 | 198.7 | 10377.13 | secreted ookinete adhesive protein, putative (SOAP) | no | 202 | microneme | |
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4.44 | 0.5 | 0.41 | 0 | 1039.54 | 10083.58 | 1662.31 | secreted ookinete protein, putative (PSOP13) | no | 203 | nucleus | |
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4.58 | 0.94 | 10.11 | 2.86 | 1190.93 | 3185.75 | 369.83 | plasmepsin VI | yes | 432 | osmiophilic body | |
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5.66 | 0.46 | 5.31 | 1.88 | 1108.51 | 2803.96 | 869.86 | conserved Plasmodium protein, unknown function | yes | 109 | osmiophilic body | yes |
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3.83 | 1.15 | 65.56 | 4.65 | 6183 | 5110.23 | 2927.77 | male development gene 1 (MDV1) | no | 221 | osmiophilic body | yes |
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3.64 | 0.21 | 6.7 | 3.32 | 189 | 203.55 | 1188.67 | gamete egress and sporozoite traversal protein (GEST) | no | 248 | osmiophilic body | yes |
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14.94 | 13.25 | 79.5 | 43.91 | 4159.99 | 1545.12 | 780.59 | conserved Plasmodium protein, unknown function | yes | 234 | unknown | |
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6.07 | 6.28 | 66.04 | 28.19 | 86.63 | 103.68 | 107.44 | acyl-CoA synthetase (ACS9) | no | 885 | unknown | |
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5.43 | 0 | 11.58 | 4.17 | 555.38 | 3358.77 | 1091.71 | phosphatidylethanolamine-binding protein, putative | no | 197 | unknown | Yes |
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4.72 | 3.7 | 2.87 | 0.75 | 110.98 | 263.42 | 251.48 | conserved Plasmodium protein, unknown function | yes | 275 | unknown | Yes |
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2.36 | 0.71 | 1.16 | 0 | 218.42 | 3456.57 | 358.06 | conserved Plasmodium protein, unknown function | no | 71 | unknown | yes |
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3.39 | 0 | 3.92 | 1.76 | 113.35 | 1323.48 | 1237.32 | conserved Plasmodium protein, unknown function | yes | 118 | unknown | yes |
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7.76 | 10.4 | 21.47 | 6.12 | 104.32 | 538.55 | 227.69 | conserved protein, unknown function | no | 269 | unknown | |
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23.74 | 4.24 | 2.55 | 0.9 | 202.94 | 2764.89 | 251.14 | conserved protein, unknown function | no | 228 | unknown | |
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12.22 | 6.61 | 57.56 | 33.19 | 1237.56 | 2048.45 | 289.69 | CPW-WPC family protein | no | 254 | unknown | yes |
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4.03 | 0.55 | 0.74 | 0 | 157.87 | 1484 | 442.85 | CPW-WPC family protein | yes | 289 | unknown | yes |
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4.38 | 2.11 | 4.08 | 0 | 682.37 | 1631.31 | 286.22 | cysteine-rich secretory protein, putative | yes | 193 | unknown | yes |
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13.07 | 4.71 | 14.76 | 3.18 | 61.39 | 89.27 | 106.78 | conserved protein, unknown function | yes | 64 | unknown | |
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5.58 | 1.03 | 0.93 | 0 | 105.54 | 2523.89 | 2355.77 | conserved protein, unknown function | no | 446 | unknown | |
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7.47 | 2.13 | 1.45 | 0 | 301.55 | 4881.44 | 354.62 | conserved Plasmodium protein, unknown function | no | 143 | unknown | |
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23.35 | 8.62 | 26.54 | 34.18 | 1085.19 | 398.79 | 415.29 | conserved protein, unknown function | no | 307 | unknown | yes |
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3.86 | 0 | 4.26 | 0 | 995.55 | 7107.6 | 1081.7 | conserved protein, unknown function | no | 175 | unknown | |
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9.99 | 9.56 | 4.46 | 1.1 | 258.57 | 5263.68 | 705.22 | conserved Plasmodium protein, unknown function | yes | 186 | unknown | yes |
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3.72 | 2.04 | 7.27 | 1.18 | 108.64 | 259.86 | 120.4 | secreted ookinete protein, putative (PSOP17) | no | 349 | unknown | |
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1.74 | 0.98 | 6.23 | 2.37 | 150.63 | 3971.38 | 628.74 | conserved Plasmodium protein, unknown function | no | 260 | unknown | |
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7.01 | 0.97 | 0.79 | 0 | 675.09 | 3205.13 | 533.55 | conserved Plasmodium protein, unknown function | no | 104 | unknown | yes |
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5.04 | 1.05 | 7.14 | 1.41 | 5387.24 | 2344.32 | 112.06 | conserved Plasmodium protein, unknown function | yes | 145 | unknown | yes |
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49.77 | 16.63 | 27.17 | 14.07 | 336.07 | 2473.53 | 292.96 | conserved protein, unknown function | yes | 263 | unknown | |
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3.91 | 0.45 | 3.9 | 0.92 | 109.52 | 128.45 | 80.61 | 6-cysteine protein (P48/45) | yes | 448 | unknown | |
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4.48 | 0.92 | 10.95 | 4.45 | 181.53 | 1539.74 | 108.79 | conserved protein, unknown function | yes | 277 | unknown | yes |
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5.94 | 7.41 | 37.22 | 6.1 | 1131 | 1842.23 | 378.5 | CPW-WPC family protein | no | 371 | unknown | yes |
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5.57 | 1.26 | 2.73 | 0 | 373.24 | 687.62 | 312.91 | conserved protein, unknown function | yes | 121 | unknown | yes |
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6.74 | 0 | 1.99 | 0 | 501.1 | 3660.75 | 692.51 | conserved Plasmodium protein, unknown function | no | 126 | unknown | yes |
ET, early trophozoite; LT, late trophozoite; ST, Schizont; GII, stage II gametocyte; GV, stage V gametocyte; C&E, successfully cloned and expressed.
Because not all membrane proteins have signal peptides, we also investigated the parasitic proteins that had TM while lacking signal peptides. About 949
About 39 of the 76 candidate proteins contained TM. These 76 candidate proteins were localized in different subcellular compartments, although subcellular locations of 35% of these proteins (n=27) are still unknown (
The subcellular location of the candidate proteins. ER, endoplasmic reticulum; OB, osmiophilic body.
Candidate genes were PCR-cloned and the recombinant proteins were expressed in High Five insect cells using the baculovirus expression system in the serum-free medium. A monoclonal antibody against the 6xHis tag at the C-terminus of recombinant proteins was used to quantify the recombinant protein concentration. We have finally cloned and expressed 46 genes successfully at ELISA detectable level for further analysis (
The insect-cell expressed recombinant parasitic proteins and their interaction with midguts.
To determine if
These six recombinant proteins were further verified by an alternative ELISA using unhomogenized midguts to pull down the recombinant proteins following by homogenizing and detection. The results (
The interaction between the candidate proteins and unhomogenized mosquito midguts.
To understand the function of candidate proteins, we searched their orthologs in
P
Gene ID( |
Pb ID( |
Coverage% | Identity% | The function of gene products | # of TM | location | knock out | Phenotype | ||||
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AS | GAM | OK | OC | SP | ||||||||
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96 | 45 | unknown | 0 | cytoplasm | fail/mutable | |||||
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97 | 35 | unknown | 1 | osmiophilic body | succeed | ND | ||||
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97 | 64 | sexual stage-specific protein G37 (Pfs37) | 7 | cell surface | succeed | ND | ATTN | ATTN | ATTN | |
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35 | 26 | sexual stage-specific protein (Pfs16) | 2 | membrane | succeed | ND | ND | ND | ATTN | ND |
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96 | 53 | phosphatidylethanolamine-binding protein | 0 | unknown | fail/mutable | |||||
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72 | 60 | HSP20-like chaperone | 1 | cytoplasm | fail/nonmutable |
AS, asexual stage; GAM, gametocyte; OK, ookinete; OC, oocyst; SP, sporozoite.
ND, no difference; ATTN, the number of the knocked parasite was attenuated compared with wild type strain. “Fail/succeed” was based on individual gene knockout. Mutable/nonmutable was based on transposon mutagenesis (
Next, we selected Pfs16 to study the antibody effects on malaria transmission. Pfs16 was found in the
The anti-Pfs16 polyclonal antibody was generated in rabbits immunized with
The functional analysis of Pfs16 on
Finally, we examined the binding tissues of Pfs16. The naïve mosquito midgut contains endothelia and the basal lamina, while a peritrophic matrix was formed after a blood meal. The mosquito midguts were isolated from the naïve and blood-fed mosquitoes and incubated with insect cell-expressed recombinant Pfs16 protein or the non-specific insect cell-expressed protein (CAT) as the control. The rabbit polyclonal antibody against Pfs16 was used to detect the bound Pfs16. Results showed that the fluorescence from naïve mosquitoes incubated with CAT was the weakest (
Recombinant Pfs16 protein bound to blood-fed mosquito midguts.
Mosquito midgut is the first place for
About 76 parasitic proteins with signal peptides and abundant at the sexual stage of parasites were selected for investigation. Many of these candidate proteins reportedly play essential roles in malaria transmission. For instance, the CelTOS is essential for ookinetes to rupture the midgut epithelial cell membrane (
The 76 candidate proteins locate at different subcellular locations, such as apicoplast, osmiophilic body, and microneme. Previous studies have shown that the abundant proteins in apicoplast have been often the targets to develop malaria drugs (
In the meantime, our bioinformatics analysis might miss some genes if they lack signal peptides or their expression data were incomplete. For instance, mosquito AgP47Rec was recently reported to interact with parasitic protein PF3D7_0134800 (P47) (
Out of the 76 candidate genes, 30 were not cloned into the plasmid due to incorrect annotation or were cloned into plasmids with low expression in insect cells. This is because some proteins such as SOAP rapturing insect cells and some membrane proteins are hard to clone and express in insect cells. Notably, forty-six genes (60.5%) were successfully cloned and highly expressed with a baculovirus expression system in High Five cells. By screening these proteins
Knockout of the Pbg16 gene did not change parasites at the sexual stage, and the formation of ookinetes was normal. The amino acid sequences between Pfs16 and Pbg16 are strikingly different, with only 35% Pfs16 was covered by Pbg16, and covered regions shared 26% identical amino acids. Therefore, we generated polyclonal antibodies to examine Pfs16 function in malaria transmission. Our data show that the ingested antibodies against Pfs16 significantly inhibited parasite transmission to mosquitoes. This result supports that Pfs16 is accessible to extracellular proteins such as antibodies. Pfs16 was observed as a gene at the onset of
Successful parasite invasion of mosquitoes begins with parasites’ interaction with mosquito midguts. Through bioinformatics analysis followed by protein interaction assays, we identified six midgut-binding parasitic proteins. Moreover, we showed that antibodies against Pfs16 inhibited malaria transmission. Further investigation of these proteins will improve our understanding of the molecular mechanisms of malaria transmission and provide targets to break malaria transmission.
The original contributions presented in the study are included in the article/
GN and YC conducted experiments, interpreted the data, and wrote the manuscript draft. XW conducted bioinformatics and statistical analysis. YK expressed Pfs16 proteins in insect cells. JL conceived concepts, designed the project, and wrote the manuscript. All authors contributed to the article and approved the submitted version.
This work is supported by NIAID (No. 1R01AI125657) and NSF Career Award (No. 1453287). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at:
Specific primers for candidate genes.
The expression data of genes that contain transmembrane domains and lack signal peptides.
The heatmap of 1,079 parasitic proteins containing signal peptides.
The expression of parasitic proteins based on microarray showing the abundance of candidate genes at sexual stages.