Community-serving research addressing climate change impacts on vector-borne diseases.

The impacts of climate change on vector-borne diseases are uneven across human populations. This pattern reflects the effect of changing environments on the biology of transmission, which is also modulated by social and other inequities. These disparities are also linked to research outcomes that could be translated into tools for transmission reduction, but are not necessarily actionable in the communities where transmission occurs. The transmission of vector-borne diseases could be averted by developing research that is both hypothesis-driven and community-serving for populations affected by climate change, where local communities interact as equal partners with scientists, developing and implementing research projects with the aim of improving community health. In this Personal View, we share five principles that have guided our research practice to serve the needs of communities affected by vector-borne diseases.

Basophil-Derived IL-4 and IL-13 Protect Intestinal Barrier Integrity and Control Bacterial Translocation during Malaria.

Our previous work demonstrated that basophils regulate a suite of malaria phenotypes, including intestinal mastocytosis and permeability, the immune response to infection, gametocytemia, and parasite transmission to the malaria mosquito Anopheles stephensi. Given that activated basophils are primary sources of the regulatory cytokines IL-4 and IL-13, we sought to examine the contributions of these mediators to basophil-dependent phenotypes in malaria. We generated mice with basophils depleted for IL-4 and IL-13 (baso IL-4/IL-13 (-)) and genotype controls (baso IL-4/IL-13 (+)) by crossing mcpt8-Cre and Il4/Il13fl/fl mice and infected them with Plasmodium yoelii yoelii 17XNL. Conditional deletion was associated with ileal mastocytosis and mast cell (MC) activation, increased intestinal permeability, and increased bacterial 16S levels in blood, but it had no effect on neutrophil activation, parasitemia, or transmission to A. stephensi. Increased intestinal permeability in baso IL-4/IL-13 (-) mice was correlated with elevated plasma eotaxin (CCL11), a potent eosinophil chemoattractant, and increased ileal MCs, proinflammatory IL-17A, and the chemokines MIP-1α (CCL3) and MIP-1ß (CCL4). Blood bacterial 16S copies were positively but weakly correlated with plasma proinflammatory cytokines IFN-γ and IL-12p40, suggesting that baso IL-4/IL-13 (-) mice failed to control bacterial translocation into the blood during malaria infection. These observations suggest that basophil-derived IL-4 and IL-13 do not contribute to basophil-dependent regulation of parasite transmission, but these cytokines do orchestrate protection of intestinal barrier integrity after P. yoelii infection. Specifically, basophil-dependent IL-4/IL-13 control MC activation and prevent infection-induced intestinal barrier damage and bacteremia, perhaps via regulation of eosinophils, macrophages, and Th17-mediated inflammation.

Quantitative phase imaging by gradient retardance optical microscopy.

Quantitative phase imaging (QPI) has become a vital tool in bioimaging, offering precise measurements of wavefront distortion and, thus, of key cellular metabolism metrics, such as dry mass and density. However, only a few QPI applications have been demonstrated in optically thick specimens, where scattering increases background and reduces contrast. Building upon the concept of structured illumination interferometry, we introduce Gradient Retardance Optical Microscopy (GROM) for QPI of both thin and thick samples. GROM transforms any standard Differential Interference Contrast (DIC) microscope into a QPI platform by incorporating a liquid crystal retarder into the illumination path, enabling independent phase-shifting of the DIC microscope's sheared beams. GROM greatly simplifies related configurations, reduces costs, and eradicates energy losses in parallel imaging modalities, such as fluorescence. We successfully tested GROM on a diverse range of specimens, from microbes and red blood cells to optically thick (~ 300 µm) plant roots without fixation or clearing.

Mast cell-derived IL-10 protects intestinal barrier integrity during malaria in mice and regulates parasite transmission to Anopheles stephensi with a female-biased immune response.

Malaria is strongly predisposed to bacteremia, which is associated with increased gastrointestinal permeability and a poor clinical prognosis. We previously identified mast cells (MCs) as mediators of intestinal permeability in malaria and described multiple cytokines that rise with parasitemia, including interleukin (IL)-10, which could protect the host from an inflammatory response and alter parasite transmission to Anopheles mosquitoes. Here, we used the Cre-loxP system and non-lethal Plasmodium yoelii yoelii 17XNL to study the roles of MC-derived IL-10 in malaria immunity and transmission. Our data suggest a sex-biased and local inflammatory response mediated by MC-derived IL-10, supported by early increased number and activation of MCs in females relative to males. Increased parasitemia in female MC IL-10 (-) mice was associated with increased ileal levels of chemokines and plasma myeloperoxidase (MPO). We also observed increased intestinal permeability in female and male MC IL-10 (-) mice relative to MC IL-10 (+) mice but no differences in blood bacterial 16S DNA levels. Transmission success of P. yoelii to A. stephensi was higher in female relative to male mice and from female and male MC IL-10 (-) mice relative to MC IL-10 (+) mice. These patterns were associated with increased plasma levels of pro-inflammatory cytokines in female MC IL-10 (-) mice and increased plasma levels of chemokines and markers of neutrophil activation in male MC IL-10 (-) mice. Overall, these data suggest that MC-derived IL-10 protects intestinal barrier integrity, regulates parasite transmission, and controls local and systemic host immune responses during malaria, with a female bias.

Starving the Beast: Limiting Coenzyme A Biosynthesis to Prevent Disease and Transmission in Malaria.

Malaria parasites must acquire all necessary nutrients from the vertebrate and mosquito hosts to successfully complete their life cycle. Failure to acquire these nutrients can limit or even block parasite development and presents a novel target for malaria control. One such essential nutrient is pantothenate, also known as vitamin B5, which the parasite cannot synthesize de novo and is required for the synthesis of coenzyme A (CoA) in the parasite. This review examines pantothenate and the CoA biosynthesis pathway in the human-mosquito-malaria parasite triad and explores possible approaches to leverage the CoA biosynthesis pathway to limit malaria parasite development in both human and mosquito hosts. This includes a discussion of sources for pantothenate for the mosquito, human, and parasite, examining the diverse strategies used by the parasite to acquire substrates for CoA synthesis across life stages and host resource pools and a discussion of drugs and alternative approaches being studied to disrupt CoA biosynthesis in the parasite. The latter includes antimalarial pantothenate analogs, known as pantothenamides, that have been developed to target this pathway during the human erythrocytic stages. In addition to these parasite-targeted drugs, we review studies of mosquito-targeted allosteric enzymatic regulators known as pantazines as an approach to limit pantothenate availability in the mosquito and subsequently deprive the parasite of this essential nutrient.

Ingested histamine and serotonin interact to alter Anopheles stephensi feeding and flight behavior and infection with Plasmodium parasites.

Blood levels of histamine and serotonin (5-HT) are altered in human malaria, and, at these levels, we have shown they have broad, independent effects on Anopheles stephensi following ingestion by this invasive mosquito. Given that histamine and 5-HT are ingested together under natural conditions and that histaminergic and serotonergic signaling are networked in other organisms, we examined effects of combinations of these biogenic amines provisioned to A. stephensi at healthy human levels (high 5-HT, low histamine) or levels associated with severe malaria (low 5-HT, high histamine). Treatments were delivered in water (priming) before feeding A. stephensi on Plasmodium yoelii-infected mice or via artificial blood meal. Relative to effects of histamine and 5-HT alone, effects of biogenic amine combinations were complex. Biogenic amine treatments had the greatest impact on the first oviposition cycle, with high histamine moderating low 5-HT effects in combination. In contrast, clutch sizes were similar across combination and individual treatments. While high histamine alone increased uninfected A. stephensi weekly lifetime blood feeding, neither combination altered this tendency relative to controls. The tendency to re-feed 2 weeks after the first blood meal was altered by combination treatments, but this depended on mode of delivery. For blood delivery, malaria-associated treatments yielded higher percentages of fed females relative to healthy-associated treatments, but the converse was true for priming. Female mosquitoes treated with the malaria-associated combination exhibited enhanced flight behavior and object inspection relative to controls and healthy combination treatment. Mosquitoes primed with the malaria-associated combination exhibited higher mean oocysts and sporozoite infection prevalence relative to the healthy combination, with high histamine having a dominant effect on these patterns. Compared with uninfected A. stephensi, the tendency of infected mosquitoes to take a second blood meal revealed an interaction of biogenic amines with infection. We used a mathematical model to project the impacts of different levels of biogenic amines and associated changes on outbreaks in human populations. While not all outbreak parameters were impacted the same, the sum of effects suggests that histamine and 5-HT alter the likelihood of transmission by mosquitoes that feed on hosts with symptomatic malaria versus a healthy host.

Longitudinal and Cross-sectional Analyses of Asymptomatic HIV-1/Malaria Co-infection in Kisumu County, Kenya.

Individuals infected with HIV-1 experience more frequent and more severe episodes of malaria and are likely to harbor asymptomatic parasitemia, thus potentially making them more efficient reservoirs of malaria. Two studies (cross-sectional and longitudinal) were designed in sequence between 2015-2018 and 2018-2020, respectively, to test the hypothesis that HIV-1 infected individuals have higher prevalence of asymptomatic parasitemia and gametocytemia than the HIV-1 negatives. This article describes the overall design of the two studies, encompassing data for the longitudinal study and additional data to the previously published baseline data for the cross-sectional study. In the cross-sectional study, HIV-1 positive participants were significantly older, more likely to be male, and more likely to have parasitemia relative to HIV-1 negatives (P < 0.01). In the longitudinal study, 300 participants were followed for 6 months. Of these, 102 were HIV-1 negative, 106 were newly diagnosed HIV-1 positive, and 92 were HIV-1 positive and on antiretroviral therapy, including antifolates, at enrollment. Overall parasitemia positivity at enrollment was 17.3% (52/300). Of these, 44% (23/52) were HIV-1 negative, 52% (27/52) were newly diagnosed HIV-1 positives, and only 4% (2/52) were HIV-1 positive and on treatment. Parasitemia for those on stable antiretroviral therapy was significantly lower (hazard ratio: 0.51, P < 0.001), compared with the HIV-1-negatives. On follow-up, there was a significant decline in parasitemia prevalence (hazard ratio: 0.74, P < 0.001) among the HIV patients newly initiated on antiretroviral therapy including trimethoprim-sulfamethoxasole. These data highlight the impact of HIV-1 and HIV treatment on asymptomatic parasitemia over time.

Initiation of anti-retroviral/Trimethoprim-Sulfamethoxazole therapy in a longitudinal cohort of HIV-1 positive individuals in Western Kenya rapidly decreases asymptomatic malarial parasitemia.

Interactions between malaria and HIV-1 have important public health implications. Our previous cross-sectional studies showed significant associations between HIV-1 positivity and malarial parasitemia with an increased risk of gametocytemia. In this follow-up longitudinal study, we evaluated these associations to determine the magnitude of asymptomatic parasitemia over time, and to examine the effects of initiating Antiretroviral Therapy (ART) together with the broad-spectrum antibiotic Trimethoprim Sulfamethoxazole (TS) on asymptomatic parasitemia. 300 adult volunteers in a malaria holoendemic region in Western Kenya were enrolled and followed for six months. The study groups were composed of 102 HIV-1 negatives, 106 newly diagnosed HIV-1 positives and 92 HIV-1 positives who were already stable on ART/TS. Blood samples were collected monthly and asymptomatic malarial parasitemia determined using sensitive 18S qPCR. Results showed significantly higher malaria prevalence in the HIV-1 negative group (61.4%) (p=0.0001) compared to HIV-1 positives newly diagnosed (36.5%) and those stable on treatment (31.45%). Further, treatment with ART/TS had an impact on incidence of asymptomatic parasitemia. In volunteers who were malaria PCR-negative at enrollment, the median time to detectable asymptomatic infection was shorter for HIV-1 negatives (149 days) compared to the HIV-1 positives on treatment (171 days) (p=0.00136). Initiation of HIV treatment among the newly diagnosed led to a reduction in malarial parasitemia (expressed as 18S copy numbers/µl) by over 85.8% within one week of treatment and a further reduction by 96% after 2 weeks. We observed that while the impact of ART/TS on parasitemia was long term, treatment with antimalarial Artemether/Lumefantrine (AL) among the malaria RDT positives had a transient effect with individuals getting re-infected after short periods. As was expected, HIV-1 negative individuals had normal CD4+ levels throughout the study. However, CD4+ levels among HIV-1 positives who started treatment were low at enrollment but increased significantly within the first month of treatment. From our association analysis, the decline in parasitemia among the HIV-1 positives on treatment was attributed to TS treatment and not increased CD4+ levels per se. Overall, this study highlights important interactions between HIV-1 and malaria that may inform future use of TS among HIV-infected patients in malaria endemic regions.

Longitudinal impact of asymptomatic malaria/HIV-1 co-infection on Plasmodium falciparum gametocyte transcript expression and transmission to Anopheles mosquitoes.

Despite significant developments towards malaria reduction, parasite transmission in the common context of HIV-1 co-infection and treatment for one or both infections has not been fully characterized. This is particularly important given that HIV-1 and malaria chemotherapies have the potential to alter gametocyte burden and mosquito infectivity. In this study, we examined 782 blood samples collected from a longitudinal cohort of 300 volunteers with asymptomatic parasitemia seeking HIV testing or treatment in the endemic region of Kisumu, Kenya, to define the impacts of HIV-1-malaria co-infection, antiretroviral therapy (ART) plus trimethoprim-sulfamethoxazole (TS) and the antimalarials artemether/lumefantrine (AL) on Plasmodium falciparum gametocyte transcript prevalence and parasite transmission to the African malaria mosquito Anopheles gambiae. Volunteers were assigned to three distinct HIV-1 groups: HIV-1 positive on treatment, HIV-1 positive newly diagnosed, and HIV-1 negative. Volunteers were monitored monthly over the course of six months. Using our highly sensitive digital droplet PCR (ddPCR) assay of three gametocyte specific transcript markers, we detected gametocyte transcripts in 51.1% of 18S positive volunteers across all study groups and time points. After correcting for multiple comparisons, the factors of HIV-1 status, time, CD4+ T-cell levels and hematocrit were not predictive of gametocyte prevalence or transmission. However, among those volunteers who were newly diagnosed with HIV-1 and malaria positive by rapid diagnostic test (RDT) at enrollment, the initiation of ART/TS and AL treatment was associated with a significant reduction in gametocyte transcript prevalence in the subsequent month when compared to HIV-1 negative volunteers treated with AL. To assess gametocyte transmissibility, volunteer blood samples were used in standard membrane feeding assays (SFMA) with laboratory-reared A. gambiae, with evidence of transmission confirmed by at least one of 25 dissected mosquitoes per sample positive for at least one midgut oocyst. HIV-1 status, CD4+ T-cell levels and hematocrit were not significantly associated with successful transmission to A. gambiae. Analysis of SMFA blood samples revealed that 50% of transmission-positive blood samples failed to test positive by Plasmodium-specific 18S ribosomal RNA quantitative PCR (qPCR) and 35% failed to test positive for any gametocyte specific transcript marker by droplet digital (ddPCR), documenting that transmission occurred in the absence of molecular parasite/gametocyte detection. Overall, these findings highlight the complexity of HIV-1 malaria co-infection and the need to further define the unpredictable role of asymptomatic parasitemia in transmission to mosquitoes.

Manipulation of pantothenate kinase in Anopheles stephensi suppresses pantothenate levels with minimal impacts on mosquito fitness.

Pantothenate (Pan) is an essential nutrient required by both the mosquito vector and malaria parasite. We previously demonstrated that increasing pantothenate kinase (PanK) activity and co-enzyme A (CoA) biosynthesis led to significantly decreased parasite infection prevalence and intensity in the malaria mosquito Anopheles stephensi. In this study, we demonstrate that Pan stores in A. stephensi are a limited resource and that manipulation of PanK levels or activity, via small molecule modulators of PanK or transgenic mosquitoes, leads to the conversion of Pan to CoA and an overall reduction in Pan levels with minimal to no effects on mosquito fitness. Transgenic A. stephensi lines with repressed insulin signaling due to PTEN overexpression or repressed c-Jun N-terminal kinase (JNK) signaling due to MAPK phosphatase 4 (MKP4) overexpression exhibited enhanced PanK levels and significant reductions in Pan relative to non-transgenic controls, with the PTEN line also exhibiting significantly increased CoA levels. Provisioning of the PTEN line with the small molecule PanK modulator PZ-2891 increased CoA levels while provisioning Compound 7 decreased CoA levels, affirming chemical manipulation of mosquito PanK. We assessed effects of these small molecules on A. stephensi lifespan, reproduction and metabolism under optimized laboratory conditions. PZ-2891 and Compound 7 had no impact on A. stephensi survival when delivered via bloodmeal throughout mosquito lifespan. Further, PZ-2891 provisioning had no impact on egg production over the first two reproductive cycles. Finally, PanK manipulation with small molecules was associated with minimal impacts on nutritional stores in A. stephensi mosquitoes under optimized rearing conditions. Together with our previous data demonstrating that PanK activation was associated with significantly increased A. stephensi resistance to Plasmodium falciparum infection, the studies herein demonstrate a lack of fitness costs of mosquito Pan depletion as a basis for a feasible, novel strategy to control parasite infection of anopheline mosquitoes.

The Basophil IL-18 Receptor Precisely Regulates the Host Immune Response and Malaria-Induced Intestinal Permeability and Alters Parasite Transmission to Mosquitoes without Effect on Gametocytemia.

We have recently demonstrated that basophils are protective against intestinal permeability during malaria and contribute to reduced parasite transmission to mosquitoes. Given that IL-18 is an early cytokine/alarmin in malaria and has been shown to activate basophils, we sought to determine the role of the basophil IL-18R in this protective phenotype. To address this, we infected control [IL18r flox/flox or basoIL-18R (+)] mice and mice with basophils lacking the IL-18R [IL18r flox/flox × Basoph8 or basoIL-18R (-)] with Plasmodium yoelii yoelii 17XNL, a nonlethal strain of mouse malaria. Postinfection (PI), intestinal permeability, ileal mastocytosis, bacteremia, and levels of ileal and plasma cytokines and chemokines were measured through 10 d PI. BasoIL-18R (-) mice exhibited greater intestinal permeability relative to basoIL-18R (+) mice, along with increased plasma levels of proinflammatory cytokines at a single time point PI, day 4 PI, a pattern not observed in basoIL-18R (+) mice. Surprisingly, mosquitoes fed on basoIL-18R (-) mice became infected less frequently than mosquitoes fed on basoIL-18R (+) mice, with no difference in gametocytemia, a pattern that was distinct from that observed previously with basophil-depleted mice. These findings suggest that early basophil-dependent protection of the intestinal barrier in malaria is mediated by IL-18, and that basophil IL-18R-dependent signaling differentially regulates the inflammatory response to infection and parasite transmission.

Basophil Depletion Alters Host Immunity, Intestinal Permeability, and Mammalian Host-to-Mosquito Transmission in Malaria.

Malaria-induced bacteremia has been shown to result from intestinal mast cell (MC) activation. The appearance of MCs in the ileum and increased intestinal permeability to enteric bacteria are preceded by an early Th2-biased host immune response to infection, characterized by the appearance of IL-4, IL-10, mast cell protease (Mcpt)1 and Mcpt4, and increased circulating basophils and eosinophils. Given the functional similarities of basophils and MCs in the context of allergic inflammation and the capacity of basophils to produce large amounts of IL-4, we sought to define the role of basophils in increased intestinal permeability, in MC influx, and in the development of bacteremia in the context of malaria. Upon infection with nonlethal Plasmodium yoelii yoelii 17XNL, Basoph8 × ROSA-DTα mice or baso (-) mice that lack basophils exhibited increased intestinal permeability and increased ileal MC numbers, without any increase in bacterial 16S ribosomal DNA copy numbers in the blood, relative to baso (+) mice. Analysis of cytokines, chemokines, and MC-associated factors in the ileum revealed significantly increased TNF-α and IL-13 at day 6 postinfection in baso (-) mice compared with baso (+) mice. Moreover, network analysis of significantly correlated host immune factors revealed profound differences between baso (-) and baso (+) mice following infection in both systemic and ileal responses to parasites and translocated bacteria. Finally, basophil depletion was associated with significantly increased gametocytemia and parasite transmission to Anopheles mosquitoes, suggesting that basophils play a previously undescribed role in controlling gametocytemia and, in turn, mammalian host-to-mosquito parasite transmission.

Anopheles stephensi Feeding, Flight Behavior, and Infection With Malaria Parasites are Altered by Ingestion of Serotonin.

Approximately 3.4 billion people are at risk of malaria, a disease caused by infection with Plasmodium spp. parasites, which are transmitted by Anopheles mosquitoes. Individuals with severe falciparum malaria often exhibit changes in circulating blood levels of biogenic amines, including reduced serotonin or 5-hydroxytryptamine (5-HT), and these changes are associated with disease pathology. In insects, 5-HT functions as an important neurotransmitter for many behaviors and biological functions. In Anopheles stephensi, we show that 5-HT is localized to innervation in the head, thorax, and midgut, suggesting a gut-to-brain signaling axis that could support the effects of ingested 5-HT on mosquito biology and behavioral responses. Given the changes in blood levels of 5-HT associated with severe malaria and the key roles that 5-HT plays in insect neurophysiology, we investigated the impact of ingesting blood with healthy levels of 5-HT (1.5 µM) or malaria-associated levels of 5-HT (0.15 µM) on various aspects of A. stephensi biology. In these studies, we provisioned 5-HT and monitored fecundity, lifespan, flight behavior, and blood feeding of A. stephensi. We also assessed the impact of 5-HT ingestion on infection of A. stephensi with the mouse malaria parasite Plasmodium yoelii yoelii 17XNL and the human malaria parasite Plasmodium falciparum. Our data show that ingestion of 5-HT associated with severe malaria increased mosquito flight velocity and investigation of visual objects in response to host odor (CO2). 5-HT ingestion in blood at levels associated with severe malaria also increased the tendency to take a second blood meal 4 days later in uninfected A. stephensi. In mosquitoes infected with P. y. yoelii 17XNL, feeding tendency was decreased when midgut oocysts were present but increased when sporozoites were present. In addition to these effects, treatment of A. stephensi with 5-HT associated with severe malaria increased infection success with P. y. yoelii 17XNL compared to control, while treatment with healthy levels of 5-HT decreased infection success with P. falciparum. These changes in mosquito behavior and infection success could be used as a basis to manipulate 5-HT signaling in vector mosquitoes for improved control of malaria parasite transmission.

Coupled small molecules target RNA interference and JAK/STAT signaling to reduce Zika virus infection in Aedes aegypti.

The recent global Zika epidemics have revealed the significant threat that mosquito-borne viruses pose. There are currently no effective vaccines or prophylactics to prevent Zika virus (ZIKV) infection. Limiting exposure to infected mosquitoes is the best way to reduce disease incidence. Recent studies have focused on targeting mosquito reproduction and immune responses to reduce transmission. Previous work has evaluated the effect of insulin signaling on antiviral JAK/STAT and RNAi in vector mosquitoes. Specifically, insulin-fed mosquitoes resulted in reduced virus replication in an RNAi-independent, ERK-mediated JAK/STAT-dependent mechanism. In this work, we demonstrate that targeting insulin signaling through the repurposing of small molecule drugs results in the activation of both RNAi and JAK/STAT antiviral pathways. ZIKV-infected Aedes aegypti were fed blood containing demethylasterriquinone B1 (DMAQ-B1), a potent insulin mimetic, in combination with AKT inhibitor VIII. Activation of this coordinated response additively reduced ZIKV levels in Aedes aegypti. This effect included a quantitatively greater reduction in salivary gland ZIKV levels up to 11 d post-bloodmeal ingestion, relative to single pathway activation. Together, our study indicates the potential for field delivery of these small molecules to substantially reduce virus transmission from mosquito to human. As infections like Zika virus are becoming more burdensome and prevalent, understanding how to control this family of viruses in the insect vector is an important issue in public health.

Mast Cell Chymase/Mcpt4 Suppresses the Host Immune Response to Plasmodium yoelii, Limits Malaria-Associated Disruption of Intestinal Barrier Integrity and Reduces Parasite Transmission to Anopheles stephensi.

An increase in mast cells (MCs) and MCs mediators has been observed in malaria-associated bacteremia, however, the role of these granulocytes in malarial immunity is poorly understood. Herein, we studied the role of mouse MC protease (Mcpt) 4, an ortholog of human MC chymase, in malaria-induced bacteremia using Mcpt4 knockout (Mcpt4-/-) mice and Mcpt4+/+ C57BL/6J controls, and the non-lethal mouse parasite Plasmodium yoelii yoelii 17XNL. Significantly lower parasitemia was observed in Mcpt4-/- mice compared with Mcpt4+/+ controls by day 10 post infection (PI). Although bacterial 16S DNA levels in blood were not different between groups, increased intestinal permeability to FITC-dextran and altered ileal adherens junction E-cadherin were observed in Mcpt4-/- mice. Relative to infected Mcpt4+/+ mice, ileal MC accumulation in Mcpt4-/- mice occurred two days earlier and IgE levels were higher by days 8-10 PI. Increased levels of circulating myeloperoxidase were observed at 6 and 10 days PI in Mcpt4+/+ but not Mcpt4-/- mice, affirming a role for neutrophil activation that was not predictive of parasitemia or bacterial 16S copies in blood. In contrast, early increased plasma levels of TNF-α, IL-12p40 and IL-3 were observed in Mcpt4-/- mice, while levels of IL-2, IL-10 and MIP1ß (CCL4) were increased over the same period in Mcpt4+/+ mice, suggesting that the host response to infection was skewed toward a type-1 immune response in Mcpt4-/- mice and type-2 response in Mcpt4+/+ mice. Spearman analysis revealed an early (day 4 PI) correlation of Mcpt4-/- parasitemia with TNF-α and IFN-γ, inflammatory cytokines known for their roles in pathogen clearance, a pattern that was observed in Mcpt4+/+ mice much later (day 10 PI). Transmission success of P. y. yoelii 17XNL to Anopheles stephensi was significantly higher from infected Mcpt4-/- mice compared with infected Mcpt4+/+ mice, suggesting that Mcpt4 also impacts transmissibility of sexual stage parasites. Together, these results suggest that early MCs activation and release of Mcpt4 suppresses the host immune response to P. y. yoelii 17XNL, perhaps via degradation of TNF-α and promotion of a type-2 immune response that concordantly protects epithelial barrier integrity, while limiting the systemic response to bacteremia and parasite transmissibility.

Malaria-induced bacteremia as a consequence of multiple parasite survival strategies.

Globally, malaria continues to be an enormous public health burden, with concomitant parasite-induced damage to the gastrointestinal (GI) barrier resulting in bacteremia-associated morbidity and mortality in both adults and children. Infected red blood cells sequester in and can occlude the GI microvasculature, ultimately leading to disruption of the tight and adherens junctions that would normally serve as a physical barrier to translocating enteric bacteria. Mast cell (MC) activation and translocation to the GI during malaria intensifies damage to the physical barrier and weakens the immunological barrier through the release of enzymes and factors that alter the host response to escaped enteric bacteria. In this context, activated MCs release Th2 cytokines, promoting a balanced Th1/Th2 response that increases local and systemic allergic inflammation while protecting the host from overwhelming Th1-mediated immunopathology. Beyond the mammalian host, recent studies in both the lab and field have revealed an association between a Th2-skewed host response and success of parasite transmission to mosquitoes, biology that is evocative of parasite manipulation of the mammalian host. Collectively, these observations suggest that malaria-induced bacteremia may be, in part, an unintended consequence of a Th2-shifted host response that promotes parasite survival and transmission. Future directions of this work include defining the factors and mechanisms that precede the development of bacteremia, which will enable the development of biomarkers to simplify diagnostics, the identification of therapeutic targets to improve patient outcomes and better understanding of the consequences of clinical interventions to transmission blocking strategies.

Increased insulin signaling in the Anopheles stephensi fat body regulates metabolism and enhances the host response to both bacterial challenge and Plasmodium falciparum infection.

In vertebrates and invertebrates, the insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) cascade is highly conserved and plays a vital role in many different physiological processes. Among the many tissues that respond to IIS in mosquitoes, the fat body has a central role in metabolism, lifespan, reproduction, and innate immunity. We previously demonstrated that fat body specific expression of active Akt, a key IIS signaling molecule, in adult Anopheles stephensi and Aedes aegypti activated the IIS cascade and extended lifespan. Additionally, we found that transgenic females produced more vitellogenin (Vg) protein than non-transgenic mosquitoes, although this did not translate into increased fecundity. These results prompted us to further examine how IIS impacts immunity, metabolism, growth and development of these transgenic mosquitoes. We observed significant changes in glycogen, trehalose, triglycerides, glucose, and protein in young (3-5 d) transgenic mosquitoes relative to non-transgenic sibling controls, while only triglycerides were significantly changed in older (18 d) transgenic mosquitoes. More importantly, we demonstrated that enhanced fat body IIS decreased both the prevalence and intensity of Plasmodium falciparum infection in transgenic An. stephensi. Additionally, challenging transgenic An. stephensi with Gram-positive and Gram-negative bacteria altered the expression of several antimicrobial peptides (AMPs) and two anti-Plasmodium genes, nitric oxide synthase (NOS) and thioester complement-like protein (TEP1), relative to non-transgenic controls. Increased IIS in the fat body of adult female An. stephensi had little to no impact on body size, growth or development of progeny from transgenic mosquitoes relative to non-transgenic controls. This study both confirms and expands our understanding of the critical roles insulin signaling plays in regulating the diverse functions of the mosquito fat body.

Activation of Anopheles stephensi Pantothenate Kinase and Coenzyme A Biosynthesis Reduces Infection with Diverse Plasmodium Species in the Mosquito Host.

Malaria parasites require pantothenate from both human and mosquito hosts to synthesize coenzyme A (CoA). Specifically, mosquito-stage parasites cannot synthesize pantothenate de novo or take up preformed CoA from the mosquito host, making it essential for the parasite to obtain pantothenate from mosquito stores. This makes pantothenate utilization an attractive target for controlling sexual stage malaria parasites in the mosquito. CoA is synthesized from pantothenate in a multi-step pathway initiated by the enzyme pantothenate kinase (PanK). In this work, we manipulated A. stephensi PanK activity and assessed the impact of mosquito PanK activity on the development of two malaria parasite species with distinct genetics and life cycles: the human parasite Plasmodium falciparum and the mouse parasite Plasmodium yoelii yoelii 17XNL. We identified two putative A. stephensi PanK isoforms encoded by a single gene and expressed in the mosquito midgut. Using both RNAi and small molecules with reported activity against human PanK, we confirmed that A. stephensi PanK manipulation was associated with corresponding changes in midgut CoA levels. Based on these findings, we used two small molecule modulators of human PanK activity (PZ-2891, compound 7) at reported and ten-fold EC50 doses to examine the effects of manipulating A. stephensi PanK on malaria parasite infection success. Our data showed that oral provisioning of 1.3 nM and 13 nM PZ-2891 increased midgut CoA levels and significantly decreased infection success for both Plasmodium species. In contrast, oral provisioning of 62 nM and 620 nM compound 7 decreased CoA levels and significantly increased infection success for both Plasmodium species. This work establishes the A. stephensi CoA biosynthesis pathway as a potential target for broadly blocking malaria parasite development in anopheline hosts. We envision this strategy, with small molecule PanK modulators delivered to mosquitoes via attractive bait stations, working in concert with deployment of parasite-directed novel pantothenamide drugs to block parasite infection in the human host. In mosquitoes, depletion of pantothenate through manipulation to increase CoA biosynthesis is expected to negatively impact Plasmodium survival by starving the parasite of this essential nutrient. This has the potential to kill both wild type parasites and pantothenamide-resistant parasites that could develop under pantothenamide drug pressure if these compounds are used as future therapeutics for human malaria.

Asymptomatic falciparum and Non-falciparum Malarial Parasitemia in Adult Volunteers with and without HIV-1 Coinfection in a Cohort Study in Western Kenya.

Asymptomatic malarial parasitemia represents the largest reservoir of infection and transmission, and the impact of coinfection with HIV-1 on this reservoir remains incompletely described. Accordingly, we sought to determine the prevalence of asymptomatic malarial parasitemia in Kombewa, Western Kenya, a region that is endemic for both malaria and HIV-1. A total of 1,762 dried blood spots were collected from asymptomatic adults in a cross-sectional study. The presence of parasitemia was first determined by a sensitive Plasmodium genus-specific 18S assay, followed by less sensitive species-specific DNA-based quantitative polymerase chain reaction (PCR) assays. The prevalence of asymptomatic malarial parasitemia by 18S genus-specific PCR assay was 64.4% (1,134/1,762). Of the 1,134 malaria positive samples, Plasmodium falciparum was the most prevalent species (57.4%), followed by Plasmodium malariae (3.8%) and Plasmodium ovale (2.6%) as single or mixed infections. As expected, the majority of infections were below the detection limit of microscopy and rapid diagnostic tests. HIV-1 prevalence was 10.6%, and we observed a significant association with malarial parasitemia by χ2 analysis (P = 0.0475). Seventy-one percent of HIV-1 infected volunteers were positive for Plasmodium 18S (132/186), with only 29% negative (54/186). In HIV-1-negative volunteers, the proportion was lower; 64% were found to be positive for 18S (998/1,569) and 36% were negative (571/1,569). Overall, the prevalence of asymptomatic malarial parasitemia in Western Kenya is high, and knowledge of these associations with HIV-1 infection are critically important for malaria elimination and eradication efforts focused on this important reservoir population.