Targeting plasmodium α-tubulin-1 to block malaria transmission to mosquitoes

Abstract

Plasmodium ookinetes use an invasive apparatus to invade mosquito midguts, and tubulins are the major structural proteins of this apical complex. We examined the role of tubulins in malaria transmission to mosquitoes. Our results demonstrate that the rabbit polyclonal antibodies (pAb) against human α-tubulin significantly reduced the number of P. falciparum oocysts in Anopheles gambiae midguts, while rabbit pAb against human β-tubulin did not. Further studies showed that pAb, specifically against P. falciparum α-tubulin-1, also significantly limited P. falciparum transmission to mosquitoes. We also generated mouse monoclonal antibodies (mAb) using recombinant P. falciparum α-tubulin-1. Out of 16 mAb, two mAb, A3 and A16, blocked P. falciparum transmission with EC₅₀ of 12 μg/ml and 2.8 μg/ml. The epitopes of A3 and A16 were determined to be a conformational and linear sequence of EAREDLAALEKDYEE, respectively. To understand the mechanism of the antibody-blocking activity, we studied the accessibility of live ookinete α-tubulin-1 to antibodies and its interaction with mosquito midgut proteins. Immunofluorescent assays showed that pAb could bind to the apical complex of live ookinetes. Moreover, both ELISA and pull-down assays demonstrated that insect cell-expressed mosquito midgut protein, fibrinogen-related protein 1 (FREP1), interacts with P. falciparum α-tubulin-1. Since ookinete invasion is directional, we conclude that the interaction between Anopheles FREP1 protein and Plasmodium α-tubulin-1 anchors and orients the ookinete invasive apparatus towards the midgut PM and promotes the efficient parasite infection in the mosquito.

Keywords: FREP1; invasive apparatus; malaria transmission-blocking vaccine; mosquito; ookinete; pathogen-host interaction.

Figure 1

Tubulin proteins are highly conserved between human and Plasmodium and the effects of polyclonal anti-tubulin antibodies on oocyst development. (A) Sequence alignment of human α-tubulin (HA) and P. falciparum α-tubulin-1 (PfA). Clustal X Colour Scheme was used to show amino acids. (B) Sequence alignment of human β-tubulin (Hβ) and P. falciparum β-tubulin-1 (Pfβ). Clustal X Colour Scheme was used to show amino acids. (C) Standard membrane feeding assays were used to measure Intensity and prevalence of oocysts in the presence of purified rabbit anti-human α-tubulin polyclonal Ab (labeled with α, 0.01 mg/ml ) and purified polyclonal Ab against human β-tubulin (labeled with β, 0.01 mg/ml). A non-related purified rabbit polyclonal Ab (anti-V5, labeled with control, 0.01 mg/ml) was used as the negative control. Mann-Whitney test was used to calculate P-value. N, number of mosquitoes.

Figure 2

Anti-α-tubulin-1 antisera block P. falciparum transmission to An. gambiae. Infection intensity and prevalence of oocyst development in the presence of anti-α-tubulin-1 (titer: 1x10⁵), anti-HSP70 (control, titer: 1x10⁵), and the control (pre-immune serum) by standard membrane feeding assays. P values were calculated using the Mann-Whitney test. N, number of mosquitoes.

Figure 3

Purified rabbit pAb recognizes P. falciparum α-tubulin-1 and inhibits the P. falciparum transmission to An. gambiae. (A) Proteins in cell lysates were separated by SDS-PAGE and (B) analyzed by immunoblotting showing the purified rabbit pAb recognize P. falciparum α-tubulin-1. Lanes, M: protein ladder; 2: P. falciparum lysate; 3: α-tubulin-1 expressed Hi5 lysate; 4: Hi5 lysate; 5: HEK 293 cell lysate. 6: Whole blood lysate. (C) The number of oocysts (infection intensity) in individual mosquitoes and prevalence in the presence of purified rabbit anti-P. falciparum α-tubulin-1 Ab at different concentrations (0,17, 50, 150, and 450 μg/ml) by SMFA. N, number of mosquitoes. P values were calculated by Mann-Whitney test.

Figure 4

Different mAb show different reactions. (A) ELISA show the reactions of 16 mAb to samples. The data exhibit means and standard deviations from three replicates. A3 and A16 bind to Pf-iRBC lysate significantly. (B) Binding assay of four mAb confirm the interaction A3 and A16 with Pf-iRBC. A3 has some degree of cross-reaction with human kidney cell (HEK293) lysate. The data display means and standard deviations from three replicates. (C): Specificity analyses of two anti-α-tubulin-1 mAb by Western blotting show that A3 and A16 recognize P. falciparum α-tubulin-1 specifically.

Figure 5

MAb A3 and A16 inhibit P. falciparum infection in An. gambiae. (A) SMFA show that A3 and A16 significantly inhibit P. falciparum transmission to mosquitoes. (B) The transmission-blocking activities of A3 depend on the antibody’s concentration. (C) A16 shows concentration-dependent transmission-blocking activity. Prov (%): prevalence. Each dot represents the number of oocysts from one individual mosquito. Red lines show the average number of oocysts per midgut. P values were calculated by Mann-Whitney tests.

Figure 6

The customized peptide arrays map epitopes of A3 and A16. (A) No specific linear epitopes were detected for A3. (B) The epitope of A16 was detected. Strong monoclonal antibody response against a single epitope-like spot pattern formed by adjacent peptides with the consensus motif REDLAALEKD on α-tubulin-1.

Figure 7

Anti-α-tubulin Ab binds live P. falciparum ookinetes at their apical end. (A) Enriching ookinetes through differential density centrifugation using 65% Percoll. Arrows point ookinetes. (B) IFA assays localized α-tubulin-1 on living ookinetes. The co-localization of P. falciparum (nuclei, blue color) and α-tubulin-1 (red). A closeup of individual ookinetes shows Ab bound to the apical end of living P. falciparum ookinetes. A closeup of the apical complex shows anti-α-tubulin Ab bound to the apical polar ring of living ookinetes. (C) Ookinetes stained with CF568 dye-conjugated anti-V5 antibodies as a negative control showed no binding. Ookinetes fixed by methanol stained with anti-α-tubulin Ab, showing that Ab could stain α-tubulin-1 tubulin inside the permeable cells.

Figure 8

Confocal immunofluorescence assays confirm P. falciparum α-tubulin-1 protein on the live ookinete surface. (A) α-tubulin-1 was detected on the live ookinete surface and not on the gametocyte surface. (B) Higher magnification showing the ookinete invasion apparatus of the ookinete (protrusion) and the strongest signals (red dots) evenly distributed at the apical polar rings (pointed by white arrows). * is the apical end. (C) Two rings at the apical region displayed the highest pixel density of red color.

Figure 9

P. falciparum α-tubulin-1 interacts with An. gambiae FREP1. (A) ELISA showed that P. falciparum α-tubulin-1 bound to An. gambiae FREP1. A bar exhibits the actual value of A₄₀₅ from one replicate, and there are three replicates per treatment. (B) P. falciparum α-tubulin-1 pulled down FREP1 from mosquito midgut lysate, which was detected by a western blotting assay. .

Figure 10

Model of the FREP1-α-tubulin-1 interaction that mediates Plasmodium infection of the mosquito midgut. Adhesion) The interaction between FREP1 in the peritrophic matrix (PM) and parasite α-tubulin-1 exposed on the cell surface at ookinete apical ends orientates invasive apparatus opening toward the PM. Invasion) Enzymes released from the parasitic apical opening disrupt midgut integrity. Together, these actions facilitate an ookinete to penetrate the midgut physical barrier for invasion.