Mitochondrial integrity and homeostasis in the midgut are key factors controlling mosquito fitness and anti-pathogen resistance. Targeting genes that regulate mitochondrial dynamics represents a potential strategy for limiting mosquito-borne diseases. AMP-activated protein kinase (AMPK) is a key cellular energy sensor found in nearly all eukaryotic cells. When activated, AMPK inhibits anabolic pathways that consume ATP and activates catabolic processes that synthesize ATP. In this study, we overexpressed a truncated and constitutively active α-subunit of AMPK under the control of the midgut-specific carboxypeptidase promotor in the midgut of female Anopheles stephensi. As expected, AMPK overexpression in homozygous transgenic mosquitoes was associated with changes in nutrient storage and metabolism, decreasing glycogen levels at 24 h post-blood feeding when transgene expression was maximal, and concurrently increasing circulating trehalose at the same time point. When transgenic lines were challenged with Plasmodium falciparum, we observed a significant decrease in the prevalence and intensity of infection relative to wild type controls. Surprisingly, we did not observe a significant difference in the survival of adult mosquitoes fed either sugar only or both sugar and bloodmeals throughout adult life. This may be due to the limited period that the transgene was activated before homeostasis was restored. However, we did observe a significant decrease in egg production, suggesting that manipulation of AMPK activity in the mosquito midgut resulted in the re-allocation of resources away from egg production. In summary, this work identifies midgut AMPK activity as an important regulator of metabolism, reproduction, and innate immunity in An. stephensi, a highly invasive and important malaria vector species.

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.

Cytokine production is a core function of effector T cells, yet the mechanisms that regulate cytokine output during an immune response remain incompletely understood. Here, we identify citrate compartmentalization as a cellular mechanism by which CD8 ⁺ T cells couple cytokine production to glucose availability. Under glucose-replete conditions, citrate transport from the mitochondria to the cytosol by the citrate carrier SLC25A1 suppresses calcium-dependent transcription factor activity in effector T cells. Either reducing glucose availability or blocking the exchange of citrate across the mitochondrial membrane raises free cytosolic calcium, thereby driving nuclear localization of Nuclear Factor of Activated T cells (NFAT)-family transcription factors and sustaining cytokine production. As a calcium-chelating metabolite, we show that citrate buffers free cytosolic calcium, thereby linking calcium-dependent signaling to mitochondrial fuel oxidation. We also identify signatures of this regulatory mechanism across hundreds of human cancer cell lines, where there are negative associations between citrate-derived metabolites and calcium-dependent transcriptional programs, and within the spatial organization of human tumors. These findings identify cytosolic citrate as a broadly conserved metabolic rheostat coupling glucose availability to calcium signaling. By adding calcium signaling to the known functions regulated by SLC25A1, our work reveals a mechanism by which mitochondria adaptively tune cytokine expression and other calcium-dependent programs in response to local metabolic conditions, such as nutrients that are available within a tissue or tumor.

Heterogeneity is a hallmark of biological systems, where cell-to-cell variability supports adaptation to changing environments, but also enables maladaptive states such as drug resistance. Many sources of non-genetic variation, particularly bioenergetics and metabolism, remain difficult to measure in living cells and connect to functional outcomes. Here, we introduce MARBL (Methionine Analogues for Ratiometric Bioenergetics in Live cells), a method that encodes translationally-coupled energetic responses to metabolic stress as an internally normalized signal within the surface proteome of living cells. Applying MARBL to primary immune cells reveals that differences in baseline translational activity can underlie apparent metabolic vulnerabilities, underscoring the importance of ratiometric measurements. We demonstrate that MARBL can enrich pathogenic from non-pathogenic TH17 cells based on resilience to bioenergetic stress, which functionally distinguishes cells that produce IFNγ upon restimulation. Overall, MARBL offers a versatile platform to profile metabolic resilience in living cells and link bioenergetic state to cellular function.