ABSTRACT
Antibodies to AgTRIO, a mosquito salivary protein, partially reduce the initial Plasmodium burden in mice. We therefore silenced AgTRIO in mosquitoes and determined the relative contribution of AgTRIO to the ability of Anopheles gambiae to transmit Plasmodium berghei to mice. RNA interference-mediated silencing of AgTRIO in A. gambiae resulted in a 60% reduction in AgTRIO expression. The decrease in AgTRIO expression did not alter the burden of Plasmodium sporozoites in mosquito salivary glands. When experimentally injected into mice, sporozoites from AgTRIO-silenced mosquitoes colonized the liver less effectively than sporozoites from control mosquitoes. Silencing of AgTRIO did not decrease the infectivity of sporozoites in vitro or influence the expression of genes associated with Plasmodium cell adhesion or traversal activity. AgTRIO decreased the expression of proinflammation cytokines by splenocytes in vitro. Moreover, in vivo, AgTRIO decreased the expression of TNF-α when coinjected with sporozoites into the skin and there was more TNF-α expression at the bite site of AgTRIO knockdown mosquitoes than at the bite site of control mosquitoes. AgTRIO therefore influences the local environment in the vertebrate host, which facilitates Plasmodium sporozoite infection in mice.
INTRODUCTION
Malaria is a mosquito-borne infectious disease that results in substantial morbidity and mortality throughout the world (1). Mosquito control and antimalarial drugs significantly decrease Plasmodium infection rates and disease severity in areas of endemicity (2, 3). At the same time, the emergence of drug-resistant Plasmodium and pesticide-resistant Anopheles mosquitoes makes treatment and prevention increasingly difficult (2). Therefore, a highly effective vaccine is needed. The circumsporozoite protein (CSP) is prominently expressed during the preerythrocytic phase of the Plasmodium life cycle. CSP is a major component of the most advanced current vaccine being tested (4), but only incomplete protection has been demonstrated in clinical trials (4–7).
Malaria begins when Plasmodium-infected female Anopheles mosquitoes target a vertebrate host for a blood meal. During the probing process, components of mosquito saliva influence coagulation, vasodilation, and other host responses, which facilitate feeding (8–11). At the same time, arthropod saliva proteins can also generate an environment in the host that is more conducive for vector-borne infectious disease agents. This has been demonstrated in diverse model systems, including sand flies and Leishmania (12–15), ticks and Borrelia (16), and Aedes and Culex mosquitoes and arboviruses (17–19).
During the maturation of Plasmodium within mosquitoes, the salivary protein saglin interacts with thrombospondin-related adhesive protein and contributes to the invasion of salivary glands, demonstrating that interactions between the protozoa and mosquito occur (20). Subsequently, the infectivity of sporozoites transmitted by mosquito bite appears to be more efficient than the infectivity of intravenously injected sporozoites (21), implying that interrelationships between saliva and Plasmodium facilitate the initial stage of infection. Indeed, passive immunization of mice with a high-titer antiserum against salivary gland extracts reduces the Plasmodium level during the initial stage of mosquito-borne infection (22), and antibodies directed against a specific protein in saliva, AgTRIO (AGAP001374), contribute to this effect (22). AgTRIO is expressed in salivary glands of Anopheles gambiae (23) and is significantly induced in salivary glands when Anopheles mosquitoes are infected with Plasmodium (22). Moreover, elevated AgTRIO expression levels are related to insecticide resistance in A. gambiae with a mutation in acetylcholinesterase (24), and silencing AgTRIO does not influence probing or blood feeding by Anopheles gambiae mosquitoes (25). In this study, we now use RNA interference (RNAi) silencing of AgTRIO in mosquitoes to examine the relative contribution of AgTRIO in the establishment of Plasmodium infection in mice.
RESULTS
RNAi-mediated silencing of AgTRIO in A. gambiae results in a 60% decrease in AgTRIO transcript abundance.Based on our finding that immunization of mice with AgTRIO partially reduces Plasmodium infection (22), we hypothesized that AgTRIO plays a role during Plasmodium infection. AgTRIO was therefore silenced in mosquitoes to determine the importance of native AgTRIO in the pathogenesis of malaria. To determine the role of AgTRIO during Plasmodium transmission to hosts, we used RNAi to knock down the expression of AgTRIO in Plasmodium-infected mosquitoes and then examined the impact of AgTRIO during transmission. In order to diminish the expression of AgTRIO in mosquito salivary glands, we synthesized double-stranded RNA (dsRNA) based on the AgTRIO sequence (dsAgTRIO) and used dsRNA of Gaussia luciferase (dsGluc) as the control. A. gambiae mosquitoes were fed on Plasmodium berghei-infected mice, and 13 to 14 days later, the P. berghei-infected mosquitoes received either dsAgTRIO or dsGluc via injection. Seven days after dsRNA injection, the salivary glands were collected to assess the expression of AgTRIO (Fig. 1A). By real-time PCR (RT-PCR), the median transcript abundance of AgTRIO in dsAgTRIO-injected mosquitoes was decreased 60% compared to the control group (Fig. 1B). To determine whether the level of AgTRIO protein was also decreased after dsAgTRIO injection, the salivary glands were collected from both groups and probed with AgTRIO rabbit antiserum (22). AgTRIO protein in the dsAgTRIO-injected group was decreased compared to the dsGluc-injected control group (Fig. 1C). By densitometry analysis, the abundance of AgTRIO was 75% less in the dsAgTRIO group than in the dsGluc group. Using RNAi silencing, the abundance of AgTRIO was significantly decreased compared to the control group.
AgTRIO expression in salivary glands is decreased by RNAi-mediated silencing. (A) Diagram of the timing of mosquito rearing and RNAi knockdown. At 14 days after Anopheles gambiae fed on Plasmodium berghei-infected mice, the infected mosquitoes were selected based on the positive green fluorescent protein (GFP) signal of the salivary glands. Then, dsRNA of AgTRIO (dsAgTRIO, AgTRIO knockdown) or the control Gaussia luciferase (dsGluc) was injected into the infected mosquitoes. At 7 days after dsRNA injection, the mosquitoes were collected to determine AgTRIO expression. (B) Expression level of AgTRIO by RT-PCR. The salivary glands were collected from individual mosquitoes injected with dsAgTRIO (n = 11) or dsGluc (n = 14) from two independent experiments. Each point represents one mosquito (P = 0.021, Mann-Whitney U test). (C) AgTRIO silencing was confirmed by immunoblotting. The salivary glands were collected from dsAgTRIO- or dsGluc-injected mosquitoes and probed with AgTRIO antiserum or salivary gland (SG) extract antiserum.
Plasmodium levels are not altered in the salivary glands of AgTRIO-knockdown mosquitoes.The expression of AgTRIO is significantly increased in female mosquitoes after P. berghei infection compared to uninfected female mosquitoes (22). The influence of AgTRIO depletion on sporozoite numbers in the salivary glands was therefore examined. We collected salivary glands to determine the AgTRIO expression levels (Fig. 1B) and performed RT-PCR to analyze the burden of sporozoites by measuring the expression levels of P. berghei 18S rRNA (Pb18S) and circumsporozoite protein (CSP) mRNA in salivary glands. There was no difference in Pb18S and CSP levels between the AgTRIO-knockdown and control groups (Fig. 2). These data demonstrate that the degree of Plasmodium infection was not significantly altered by the level of AgTRIO in mosquitoes.
AgTRIO knockdown did not affect the Plasmodium burden in mosquito salivary glands. GFP P. berghei-infected mosquitoes were injected with dsAgTRIO (AgTRIO knockdown) or dsGluc (control) on day 14 after infection. After 7 days, the salivary glands were collected from individual mosquitoes, which were used to determine the expression level of AgTRIO in Fig. 1. Using the same samples, the Plasmodium burden in salivary glands was determined by the expression of P. berghei 18S (A) or the gene encoding the circumsporozoite protein (CSP) (B) by RT-PCR after being normalized with the expression of actin. The mosquitoes were collected from two independent experiments. Each dot represents one mosquito. Means ± the standard deviations (SD) are shown; P values were determined using the Mann-Whitney U test.
Sporozoites from AgTRIO-knockdown mosquitoes caused a lower level of hepatic infection in C57BL/6 mice.Immunization of mice with AgTRIO resulted in a partial reduction in pathogen number during the initial stage of Plasmodium infection (22). We therefore examined how silencing AgTRIO in A. gambiae influenced Plasmodium infection of mice. To control for potential differences in sporozoite burden per mosquito, we collected the sporozoites from AgTRIO-silenced or control mosquitoes and then injected 300 sporozoites into C57BL/6 mice by intradermal injection. Immunoblots demonstrated that the amount of AgTRIO was less in the collection of sporozoites from AgTRIO-knockdown mosquitoes than from the control mosquitoes (see Fig. S1 in the supplemental material). Based on the densitometry analysis, the estimated concentration of AgTRIO in salivary gland extracts from control mosquitoes was ∼1 μg/ml. The liver burden was then determined by measuring the expression level of Pb18S after normalization with HNF-4α (22). There was a significant decrease in the Plasmodium burden in the liver when the mice were administered sporozoites from AgTRIO-silenced mosquitoes compared to the controls (Fig. 3, P = 0.018). To determine whether AgTRIO can facilitate the infection of Plasmodium, we expressed and purified recombinant AgTRIO using a baculovirus expression system (Fig. S2). Then, we collected the sporozoites from AgTRIO knockdown mosquitoes and injected the sporozoites with 1 or 10 μg/ml of recombinant AgTRIO. The liver Plasmodium burden was restored to levels similar to controls when AgTRIO was added back to the sporozoites collected from AgTRIO silenced mosquitoes (Fig. 3). Based on these findings, the production of AgTRIO in mosquito salivary glands contributes to the establishment of the initial stage of Plasmodium infection in mice.
AgTRIO is required for optimal P. berghei infection. P. berghei-infected mosquitoes were injected with dsAgTRIO or dsGluc on day 14. The sporozoites were collected from the salivary glands 7 days later. A total of 300 sporozoites from dsAgTRIO (AgTRIO knockdown)- or dsGluc (control)-injected mosquitoes were intradermally injected into the left ears of individual C57BL/6 mice. After 40 h, the livers were dissected, and the Plasmodium infection was determined by RT-PCR. Three independent experiments were performed, and the pooled results are presented. To determine whether AgTRIO protein restored the infectivity of the AgTRIO-knockdown group, the 300 sporozoites from AgTRIO-knockdown mosquitoes were intradermally injected, along with 1 or 10 μg/ml of AgTRIO, into C57BL/6 mice. After 40 h, the livers were dissected, and the Plasmodium infection level was determined by RT-PCR. Two independent experiments were performed and the pooled results are presented. Each dot represents one mouse. Means ± the SD are shown; P values were determined using the Mann-Whitney U test.
AgTRIO did not directly affect the infectivity of sporozoites.In order to determine whether AgTRIO directly interacts with sporozoites, we probed sporozoites with AgTRIO antiserum. Reactivity of the sporozoites with the antiserum was not evident (data not shown). Moreover, recombinant AgTRIO was not observed to bind to sporozoites in an indirect immunofluorescence assay (data not shown). Cell traversal and invasion are two processes that are associated with Plasmodium infection (26–29). In order to examine whether AgTRIO influences Plasmodium infectivity, we collected the sporozoites from the salivary glands of AgTRIO-knockdown or control mosquitoes and then examined these functions. The sporozoites were cocultured with murine fibroblasts (Fig. 4A) or hepatocytes (Fig. 4B) to determine whether there was any difference in cell traversal. No difference was observed. We further determined whether AgTRIO affects cell invasion when sporozoites were incubated with hepatocytes for 24 h to form exoerythrocytic forms (Fig. 4C). Levels of cell invasion by sporozoites isolated from AgTRIO-knockdown and control mosquitoes were similar. Finally, we determined whether AgTRIO influenced expression of sporozoite genes associated with cell adhesion or cell traversal (26). There was no difference in cell adhesion- or traversal-related gene expression between sporozoites collected from these two groups of mosquitoes (Fig. 4D). To examine the gliding activity of sporozoites, the sporozoites were collected from control or AgTRIO-silenced mosquitoes and then placed on glass for 1 h. The gliding trails were stained with anti-CSP antibody. There were 54 and 61% of sporozoites from control mosquitoes with gliding trails, and there were 50 and 66% of sporozoites from AgTRIO-silenced mosquitoes with trails. Sporozoites from AgTRIO-silenced mosquitoes did not show any alteration in gliding activity in vitro compared to sporozoites from control mosquitoes. Collectively, these data suggest that AgTRIO does not directly impact sporozoites.
AgTRIO silencing does not decrease the infectivity of sporozoite in vitro. Sporozoites were collected from dsAgTRIO (AgTRIO knockdown)- or dsGluc (control)-injected mosquitoes. Sporozoites were incubated with murine dermal fibroblasts (A) or murine hepatocytes (B) with fluorescently labeled dextran for 3 h. The traversal of cells was verified by determining the positive fluorescence-labeled cells by fluorescence-activated cell sorting (FACS). The data are representative of more than three experiments. Means ± the SD are shown; nonsignificance was determined using the Mann-Whitney U test. (C) GFP-positive sporozoites were incubated with murine hepatocytes for 24 h to generate exoerythrocytic forms (EEFs). Productive invasion was determined by examining GFP-positive hepatocytes by flow cytometry. The data are representative of two independent experiments. Means ± the SD are shown; P values were determined using the Mann-Whitney U test. The monoclonal antibody against CSP (anti-CSP, 10 μg/ml) was used as a positive control to inhibit cell traversal or productive invasion. (D) RNA collected from sporozoites of the dsAgTRIO (AgTRIO knockdown)- or dsGluc (control)-injected mosquitoes was used to analyze the gene expression of different cell adhesion and invasion genes by real-time PCR. Sporozoites were pooled from five to eight mosquitoes from each experiment, and four independent experiments were used for the comparison. Means ± the SD are shown (n = 3); nonsignificance (N.S.) was determined using the Mann-Whitney U test.
AgTRIO modified the inflammatory response in vitro and in vivo.Since AgTRIO does not directly interact with sporozoites, we determined whether AgTRIO alters the local inflammatory response in the host following a mosquito bite. Based on the analysis of AgTRIO levels in mosquito salivary gland extract (Fig. S1), we used 1 μg/ml of recombinant AgTRIO in our studies. To determine the effect of AgTRIO during inflammation, splenocytes were collected from C57BL/6 mice and stimulated with lipopolysaccharide (LPS) in the presence of 1 μg/ml recombinant AgTRIO or a control antigen (bovine serum albumin [BSA]). After 24 h, the cells were collected, and the expression of different cytokines was determined by real-time PCR. There was significantly less TNF-α in the AgTRIO-treated group than in the controls (Fig. 5A). The IFNα, IFNβ, IL-2, IL-4, IL-10, and IL-6 levels were similar in both groups (Fig. 5A). To directly test whether AgTRIO affects the inflammatory response in the skin, 300 sporozoites collected from wild-type mosquitoes were incubated with 1 or 10 μg/ml of AgTRIO or BSA and then intradermally injected into the ears of mice. After 3 h, the skin was collected, and the expression of different cytokines was examined by real-time PCR. There was no difference when sporozoites were incubated in 1 μg/ml AgTRIO or BSA (Fig. S3). There was a significant reduction in TNF-α mRNA in the tissue containing sporozoites combined with 10 μg/ml AgTRIO (Fig. 5B). To further determine the effects of AgTRIO during mosquito bite, we then silenced the expression of AgTRIO in uninfected mosquitoes by dsRNA. The control and AgTRIO knockdown mosquitoes were then allowed to take a blood meal on the ears of mice. After 3 h, the skin bitten by one mosquito was collected by punch biopsy. By RT-PCR, the TNF-α expression levels were significantly increased in the group of AgTRIO knockdown mosquitoes compared to the control group (Fig. 5C). These data further suggest that AgTRIO influences the local inflammatory response to sporozoites in vivo.
AgTRIO modulates the cytokine response in vitro and after mosquito bites in vivo. (A) LPS-activated splenocytes were incubated with 1 μg/ml BSA or AgTRIO for 24 h. The expression of different cytokines was measured by RT-PCR. Means ± the SD are shown (n = 7). *, P < 0.05 (Mann-Whitney U test). (B) A total of 300 sporozoites (SPZ) from wild type-infected mosquitoes were collected and then intradermally injected into the ears of mice with 10 μg/ml of AgTRIO or BSA. After 3 h, the injection site was removed by punch biopsy, and the expression of cytokines was determined by RT-PCR. Means ± the SD are shown (n = 5). *, P = 0.05 (Mann-Whitney U test). (C) The control or AgTRIO-knockdown uninfected mosquitoes were freely allowed to take a blood meal on the ears of mice. After 3 h, the bite site was removed by punch biopsy, and the expression of TNF-α was determined by RT-PCR. Means ± the SD are shown (n = 5). *, P < 0.05 (Mann-Whitney U test).
DISCUSSION
Diverse vector saliva proteins facilitate the ability of pathogens to establish the initial stage of infection in a vertebrate host, including bacteria (30, 31), viruses (32–34), and parasites (35, 36). The effects of various saliva proteins are distinct and can be associated with changes in the microenvironment or a direct effect on the pathogen (36, 37). The influence of mosquito saliva on Plasmodium infection is unclear (38–40). In general, mosquito exposure does not appear to offer any protection against Plasmodium infection (39). Nevertheless, high doses of hyperimmune antiserum against salivary gland extracts can dampen the initial levels of Plasmodium infection in mice (22). However, antisera directed toward individual saliva proteins impact sporozoites differently. As examples, AgTRIO antiserum reduces the initial Plasmodium burden in mice (22), while conversely, GILT antiserum enhances Plasmodium infection (41). The ability of AgTRIO antiserum to diminish the early stage of murine Plasmodium infection suggests that AgTRIO facilitates Plasmodium infection. To understand whether mosquito saliva with diminished AgTRIO had an altered ability to influence Plasmodium infection of a vertebrate host, we silenced AgTRIO in mosquitoes using RNAi. AgTRIO knockdown resulted in a 60% decrease in AgTRIO mRNA and AgTRIO protein in mosquitoes. AgTRIO depletion did not alter Plasmodium levels in mosquitoes but resulted in a diminished ability of sporozoites from these mosquitoes to establish early infection in mice. It is currently unknown whether the complete deletion of AgTRIO has any deleterious effects in the mosquito.
AgTRIO could influence Plasmodium directly or indirectly to impact the initial stage of infection. AgTRIO did not appear to alter sporozoite properties associated with viability or infectivity, such as gliding motility, or cell traversal or invasion. Moreover, AgTRIO did not directly bind to sporozoites. These data suggested that AgTRIO did not exert a direct effect on Plasmodium. We therefore determined whether AgTRIO had an indirect influence on sporozoites. For example, recent studies demonstrated that the neutrophilic influx caused by Aedes aegypti saliva contributed to arboviral replication and dissemination in the host (42) and that an A. aegypti salivary protein, LTRIN, facilitated Zika virus transmission by interfering with lymphotoxin-β receptor signaling (19). It has been shown that Anopheles stephensi feeding on mice results in increased macrophage inflammatory protein 2 (MIP-2) and tumor necrosis factor alpha (TNF-α) in the skin (43). Moreover, sporozoites and sporozoite lysates activate innate immune receptors and induce the expression of interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), and TNF-α (44). Furthermore, the administration of a high dose of TNF-α can partially protect rhesus monkeys from Plasmodium infection (45). Our data demonstrated that AgTRIO can diminish TNF-α in the host after mosquito bites and also when splenocytes are stimulated with LPS. This indicates that AgTRIO may work as an immunomodulator to decrease the inflammation reaction in the bite site, thereby favoring early Plasmodium infection by altering the local host environment. Since AgTRIO can decrease the response of LPS-stimulated splenocytes, the impact of AgTRIO on innate immune signaling can be explored, and the mechanism(s) by which AgTRIO changes the inflammatory response at the mosquito bite site can now be delineated.
In summary, the silencing of AgTRIO in mosquitoes reduced the ability of saliva to facilitate sporozoite infection of the vertebrate host. Indeed, since the RNA silencing of AgTRIO was incomplete, absolute deletion of the AgTRIO gene could potentially result in an even greater effect. It is possible that the immunomodulating effects of AgTRIO on the host could be influenced by the immune status of the mammalian host. Moreover, the results of our laboratory studies need to be extended to populations of P. falciparum-infected mosquitoes that are collected in nature. A further understanding of the positive and negative effects of specific salivary components on Plasmodium survival at the bite site during the early stage of infection will increase our understanding of the pathogenesis of malaria. This may lead to new strategies to help prevent the effective movement of sporozoites from the arthropod vector to the vertebrate host, as well as establishment of the initial phases of mammalian infection with Plasmodium.
MATERIALS AND METHODS
Ethics statement.All animal experiments were performed under protocols based on the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Yale University Institutional Animal Care and Use Committee (protocol permit 2017-07941). All Plasmodium infections were performed in biosafety level 2 animal facilities, according to Yale University regulations.
Animals.A. gambiae mosquitoes (4arr strain, MRA-121, MR4) were raised at 27°C and 80% humidity under a 12/12-h light/dark cycle and maintained with 10% sucrose under standard laboratory conditions in the insectary of Yale University. Swiss Webster and C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA).
P. berghei infection.P. berghei (ANKA GFPcon 259cl2 [MRA-865] or NK65 RedStar [MRA-905]; ATCC, Manassas, VA) was maintained by serial passage in 6- to 8-week-old female Swiss Webster or C57BL/6 mice as previously mentioned (22). Briefly, the Swiss Webster or C57BL/6 mice were infected with P. berghei-infected red blood cells via intraperitoneal injection. A. gambiae mosquitos then took a blood meal from the infected mice, when the parasitemia was approximately 5%. At 17 to 24 days after P. berghei infection, the mosquitoes were sorted using the fluorescent signal of the salivary glands. The salivary glands from infected mosquitoes were used to harvest the sporozoites for further experiments.
AgTRIO dsRNA synthesis and microinjection.The AgTRIO gene (AGAP001374) fragment between base pairs 112 and 650 of the coding sequence was amplified from the plasmid pET21b-TRIO by using PCR (22). AgTRIO dsRNAs were further synthesized from the AgTRIO PCR-amplified fragments using a T7 MEGAscript RNAi kit (Ambion). The primer sequences are listed in Table S1 in the supplemental material. Gaussia luciferase dsRNA was used as the control for dsRNA injection. On days 13 to 14 after P. berghei infection, 1,500 ng of dsRNA was injected into the thoraces of female mosquitoes under ice-anesthesia. The injection was performed using a nanoinjector (Nanoject II; Drummond) with a glass capillary needle, as previously described (46, 47).
Intradermal injection of sporozoites.The sporozoites were collected from salivary glands and then passed through 30-gauge syringes 10 to 15 times. The cell debris was removed by filtering through a 40-μm-pore-size filter mesh. The Swiss Webster or C57BL/6 mice were anesthetized with 100 mg/kg of ketamine and 10 mg/kg of xylazine. Then, 300 sporozoites in a volume of 300 nl were injected into the left ear of individual mice using glass micropipettes with an 80-μm-diameter beveled opening (48) and a Nanoject II Auto-Nanoliter injector (Drummond). To determine the burden of Plasmodium infection, the mice were sacrificed 40 h after intradermal injection, and the livers were collected for RNA extraction.
Gene expression and Plasmodium load.An RNeasy minikit (Qiagen) was used to extract RNA from mosquito salivary glands or murine skin. TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) was used to purify the total RNA from murine livers. All extraction was performed according to the manufacturer’s protocols. An iScript RT-qPCR kit (Bio-Rad, Hercules, CA) was used to generate cDNA from RNA. Using iTaq SYBR green Supermix (Bio-Rad), real-time PCR was performed on a CFX96 real-time platform (Bio-Rad). The burden of Plasmodium in livers after sporozoite infection was determined by assessing the expression level of P. berghei 18S rRNA, normalized to M. musculus hnf4α (22). The primers used for the expression of sporozoite genes are listed in Table S1.
Cell traversal and invasion assay.Mouse hepatocytes (Hepa1-6) or skin fibroblasts (M. dunni, clone III8C; ATCC) were plated in a 96-well plate (5 × 104 cells/well) 1 day before the assay. P. berghei ANKA sporozoites (2 × 103/well) collected from AgTRIO-silenced or control mosquitoes were incubated on ice for 1 h with or without 10 μg/ml CSP monoclonal antibody (3D11), which was used to inhibit cell traversal, as the positive control. Sporozoites were transferred onto hepatocytes, endothelial cells, or fibroblasts with fluorescently labeled dextran (10,000 molecular weight, 0.2 mg/ml; Molecular Probes). After 3 h of incubation in 37°C and 5% CO2, the cells were washed twice with phosphate-buffered saline (PBS) and collected after being trypsinized. The percentage of traversed cells (dextran+) was determined by flow cytometric analysis (Stratedigm, Inc., San Jose, CA). For invasion assays, the hepatocytes were cocultured with GFP+ sporozoites as mentioned above. Twenty-four hours after coculture, the cells were harvested and the percentage of GFP+ sporozoites was determined by flow cytometric analysis (Stratedigm). This depicts the formation of exoerythrocytic forms.
Gliding activity of sporozoites in vitro.The sporozoites were collected as described above. The sporozoites were plated on Lab-Tek 8 chamber slides (Thermo Fisher Scientific) with Dulbecco modified Eagle medium with 10% fetal bovine serum. The sporozoites were allowed to glide for 1 h at 37°C. After incubation, the medium was removed, and the sporozoites and trails were fixed with 4% paraformaldehyde for 30 min. The wells were washed with PBS for three times and then blocked with 1% BSA for 30 min. After blocking, the trails were stained by anti-CSP monoclonal antibody (clone 3D11, 10 μg/ml) at 37°C for 1 h, followed by incubation with goat-anti mouse Alexa Fluor 555 (1:500; Thermo Fisher Scientific) for 1 h at 37°C. The trails were visualized by an EVOS FL Auto cell imaging system (Thermo Fisher Scientific).
Immunostaining of sporozoite.The immunostaining was performed as described previously (41). Sporozoites were fixed with 4% paraformaldehyde and then blocked with 1% BSA in PBS. The sporozoites were then incubated with 10 μg/ml rabbit anti-AgTRIO IgG or anti-OVA IgG, which was generated by immunization, as described previously (22). The slides were washed three times in PBS and then incubated with a goat anti-rabbit Alexa Fluor 555 secondary antibody (Thermo Fisher Scientific, 1:500). The slides were washed three times with PBS and mounted with Prolong Gold Antifade containing DAPI (4′,6′-diamidino-2-phenylindole; Thermo Fisher Scientific). To determine whether AgTRIO was bound to the sporozoites, the sporozoites were incubated with 1 μg/ml of BSA or AgTRIO on ice for 1 h. The sporozoites were then fixed as mentioned above, followed by incubation with a fluorescein isothiocyanate-labeled anti-His tag monoclonal antibody (3D5; Thermo Fisher Scientific) on ice for 1 h. Sporozoites were viewed using the EVOS FL Auto cell imaging system.
Western blotting.One salivary gland was collected from either AgTRIO-silenced or control mosquitos. Each salivary gland was placed in Laemmli sample buffer (Bio-Rad) and separated by SDS-PAGE using 4 to 20% Mini-Protean TGX gels (Bio-Rad). Proteins were transferred onto a 0.45-μm-pore-size polyvinylidene difluoride membrane and then probed with AgTRIO rabbit antiserum (1:1,000) or anti-salivary gland extract rabbit antiserum as the primary antibody (22). Horseradish peroxidase-labeled goat anti-rabbit antibody (Invitrogen) was used as the secondary antibody (1:5,000), and the images were developed with a LI-COR Odyssey imaging system.
AgTRIO protein synthesis and purification.The AgTRIO sequence was designed for optimal expression using baculovirus and then subcloned into pFastBac1. The plasmid was transfected into Sf9 cells with the transfection reagent Cellfectin II (Thermo Fisher Scientific), and then the baculovirus was collected. The Sf9 cells were infected with baculovirus to express AgTRIO, and AgTRIO protein expression was confirmed by Western blotting. The protein (43 kDa) was purified from the cell lysate using a Ni-NTA resin column and then filtered through a 0.22-μm-pore-size filter. The purity was confirmed by SDS-PAGE gel (Fig. S2) and Western blot (data not shown) analyses. The protein concentration was determined using a BCA protein assay with BSA as the standard (Thermo Fisher). All expression and purification experiments were performed by GenScript USA, Inc. Based on our previous studies related to the concentration of mosquito saliva protein (41), 1 μg/ml (24 μM) or 10 μg/ml (240 μM) AgTRIO was used to study the effect of AgTRIO on inflammation in vitro and in vivo.
Cytokines releasing from splenocytes after stimulation with LPS.The spleens were collected from female C57BL/6 mice after euthanization, and the splenocytes were collected by mashing the spleens through a 70-μm-pore-size cell strainer after the lysis of red blood cells using RBC lysis buffer (Sigma). A total of 5 × 106 splenocytes were plated on 24-well plates. The splenocytes were incubated with 1 μg/ml LPS plus 1 μg/ml AgTRIO or BSA for 24 h. After incubation, the cells were collected using an RNeasy kit (Qiagen) to harvest RNA, and then RT-PCR was performed as described above. The primers used are listed in Table S1 in the supplemental material.
Skin cytokine responses during sporozoite infection or mosquito bites.Sporozoites were collected as mentioned above. Here, we used outbred Swiss Webster female mice to limit the genetic effects on the inflammatory response during the blood meal of mosquitoes. A total of 300 sporozoites with 10 μg/ml AgTRIO or BSA were intradermally injected to the ears of Swiss Webster mice. At 3 h after injection, the skin at the injection site was removed by punch biopsy. The RNA was collected by using RNeasy kits (Qiagen), and then the expression of cytokines was determined by RT-PCR. To assess the cytokine response after mosquito bites, dsRNA was injected into uninfected mosquitoes to generate AgTRIO-knockdown mosquitoes. At 7 days after dsRNA injection, the mosquitoes were fed on the ears of Swiss Webster mice. At 3 h after the mosquito bite, the bite sites were removed by punch biopsy and processed as described above.
Statistical analysis.The analysis, graphs and statistics of all data were performed in Prism 7.0 software (GraphPad Software, Inc., San Diego, CA).
ACKNOWLEDGMENTS
These studies were supported by funding from the National Institutes of Health (T32 AR007016-44) and the Dermatology Foundation to M.F. E.F. is an Investigator of the Howard Hughes Medical Institute. This work was supported by the Bloomberg Philanthropies.
We thank Kathleen DePonte and Ming-Jie Wu for their technical assistance.
FOOTNOTES
- Received 25 April 2019.
- Returned for modification 28 May 2019.
- Accepted 3 July 2019.
- Accepted manuscript posted online 8 July 2019.
Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00326-19.
- Copyright © 2019 American Society for Microbiology.
