Mass spectrometry-based stable-isotope tracing uncovers metabolic alterations in pyruvate kinase-deficient Aedes aegypti mosquitoes
Graphical abstract
Introduction
Aedes aegypti is the main vector of viruses that cause diseases such as Zika and dengue fever, which present major challenges to the public health (Lessler et al., 2016; Weaver et al., 2018; Messina et al., 2019). Because there are no specific treatments or effective vaccines currently available, the prevention and control of these diseases depends entirely on controlling the mosquito vectors. However, it is extremely difficult to control A. aegypti due to its ability to adapt to human environmental conditions (Lounibos and Kramer, 2016; Achee et al., 2019). Given these challenges and the failure of current methods to effectively control mosquito populations, a comprehensive understanding of mosquito metabolism is needed for the development of better vector mitigation strategies.
As an anautogenous mosquito species, female A. aegypti needs to obtain at least one blood meal to produce eggs. Previous studies have demonstrated that blood-fed A. aegypti utilizes several metabolic pathways to detoxify ammonia, a by-product of amino acid (AA) oxidation (Scaraffia et al., 2005, 2006, 2008, 2010; Isoe and Scaraffia, 2013; Mazzalupo et al., 2016; Petchampai and Scaraffia, 2016; Isoe et al., 2017). Recently, a positional stable-isotope tracer analysis revealed a tight link between glucose and ammonia metabolism in A. aegypti. Specifically, several metabolites are synthesized from the carbon skeleton of glucose to facilitate ammonia detoxification and nitrogen waste disposal in blood-fed mosquitoes (Horvath et al., 2018). The synthesis of these metabolites occurs through multiple metabolic pathways including glycolysis, pentose phosphate pathway (PPP), Krebs cycle, and ammonia fixation, assimilation and excretion pathways (Horvath et al., 2018).
The final and rate-limiting step of glycolysis is catalyzed by pyruvate kinase (PK, EC 2.7.1.40), an enzyme that catalyzes the transfer of a phosphate group from phosphenolpyruvate to ADP, yielding pyruvate and ATP. Recently, two spliced variants of A. aegypti PK, designated as AaPK1 and AaPK2, were identified in the A. aegypti genome. The three-dimensional structure and kinetic properties of recombinant AaPK1 were also reported (Petchampai et al., 2019). In spite of the similarity at the AA sequence and structural levels with the human non-allosteric isoform of PK (PKM1), AaPK1 exhibited allosteric behavior (Petchampai et al., 2019). The allosteric nature of AaPK1 suggests that the enzyme has the ability to respond to different metabolic signals. The unique regulatory property of AaPK1 observed in in vitro assays encouraged us to study the total PK (both PK1 and PK2 isoforms) in vivo, using several techniques including RNA interference (RNAi), high-resolution accurate-mass (HRAM) liquid chromatography-mass spectrometry (HRAM-LC/MS) and HRAM ion chromatography-mass spectrometry (HRAM-IC/MS) methods. Our present data reveal that PK is modulated in the fat body in response to the nutritional status of the females. Survival of PK-deficient mosquitoes is dependent on whether females are maintained on sucrose, water, blood/sucrose or blood/water diets. Depletion of PK by RNAi significantly increased transcript levels of several genes that encode enzymes related to glucose and nitrogen metabolism, and impacted glucose oxidation and ammonia metabolism at specific time points during blood meal digestion. Our data provide evidence that PK plays a key regulatory role in the metabolic homoeostasis of A. aegypti females.
Section snippets
Reagents, chemicals, antibodies, and others
All the primers, and chemicals for PK activity assays were acquired from Millipore Sigma (Burlington, MA, USA). Oligo-(dT)20 primer, reverse transcriptase, and GoTaq® DNA Polymerase were from Promega (Madison, WI, USA). PerfeCTa SYBR Green FastMix was obtained from Quanta BioSciences (Gaithersburg, MD, USA). TRIzol® reagent was from Thermo Fisher Scientific (Waltham, MA, USA). Bovine blood was from Pel-Freez Biologicals (Rogers, AR, USA). [1,2–13C2]-glucose was from Cambridge Isotope
PK mRNA level is up-regulated in response to blood feeding
To assess the transcriptional profiles of PK in response to sucrose and blood feeding, we performed qPCR in mosquito tissues before and after blood feeding (Fig. 1). The expression of PK was up-regulated in response to blood feeding. In the fat body, the level of PK mRNA dramatically increased ~200 fold at 6–12 h PBM and decreased considerably thereafter. At 18–48 h PBM, PK mRNA abundance was relatively constant, but significantly higher (~60–80 fold) than sucrose-fed mosquitoes. At 72–96 h
Discussion
PK is an evolutionary conserved enzyme of the glycolytic pathway (Petchampai et al., 2019; Schormann et al., 2019). In humans, there are four isoforms of PK, which are PKM1, PKM2, PKR, and PKL. With the notable exception of PKM1, all of the PK human isoforms are allosterically regulated by fructose-1,6-bisphosphate (Mattevi et al., 1996; Israelsen and Vander Heiden, 2015). It was also reported that a single phosphate group on the sugar is essential for the activation of human PKL, whereas the
Funding
This work was financially supported by the Corine Adams Baines Professorship Award, COR Research Bridge Funds Award, U.S. National Institutes of Health, National Institute of Allergy and Infectious Diseases Grant R01AI146199 (to PYS), NIH 1S10OD012304-01, NIH U01CA235510, Cancer Prevention and Research Institute of Texas (CPRIT) Grant RP130397, and The University of Texas MD Anderson's NCI Cancer Center Support Grant P30CA016672.
CRediT authorship contribution statement
Natthida Petchampai: Writing - original draft, Formal analysis, Data curation. Jun Isoe: Writing - original draft, Formal analysis, Data curation. Thomas D. Horvath: Writing - original draft, Formal analysis, Data curation. Shai Dagan: Writing - original draft, Formal analysis, Data curation. Lin Tan: Formal analysis, Data curation. Philip L. Lorenzi: Data curation. David H. Hawke: Data curation, Funding acquisition. Patricia Y. Scaraffia: Writing - original draft, Formal analysis, Data
Declaration of competing interest
The authors declare that they have no conflicts of interest with the contents of this article.
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- 1
Present address: Department of Pathology and Immunology, Baylor College of Medicine, and Texas Children's Microbiome Center, Department of Pathology, Texas Children's Hospital, Houston, TX 77030, USA.
- 2
These authors contributed equally to this work.