Mass spectrometry-based stable-isotope tracing uncovers metabolic alterations in pyruvate kinase-deficient Aedes aegypti mosquitoes

https://doi.org/10.1016/j.ibmb.2020.103366Get rights and content

Highlights

  • Pyruvate kinase (PK) activity in fat body varies in response to nutritional changes.

  • PK silencing enhances survival in females maintained under specific nutritional regimens.

  • PK-deficient females increase transcript levels of specific genes.

  • PK knockdown reduces the abundance of metabolites involved in several pathways.

Abstract

A recent in vitro characterization of a recombinant pyruvate kinase (PK) from Aedes aegypti mosquitoes demonstrated that the enzyme is uniquely regulated by multiple allosteric effectors. Here, we further explored PK gene and protein expression, and enzymatic activity in key metabolic tissues of mosquitoes maintained under different nutritional conditions. We also studied the metabolic effects of PK depletion using several techniques including RNA interference and mass spectrometry-based stable-isotope tracing. Transcriptional analysis showed a dynamic post-feeding PK mRNA expression pattern within and across mosquito tissues, whereas corresponding protein levels remained stable throughout the time course analyzed. Nevertheless, PK activity significantly differed in the fat body of sucrose-, blood-fed, and starved mosquitoes. Genetic silencing of PK did not alter survival in blood-fed females maintained on sucrose. However, an enhanced survivorship was observed in PK-deficient females maintained under different nutritional regimens. Our results indicate that mosquitoes overcame PK deficiency by up-regulating the expression of genes encoding NADP-malic enzyme-1, phosphoenolpyruvate carboxykinase-1, phosphoglycerate dehydrogenase and glutamate dehydrogenase, and by decreasing glucose oxidation and metabolic pathways associated with ammonia detoxification. Taken together, our data demonstrate that PK confers to A. aegypti a metabolic plasticity to tightly regulate both carbon and nitrogen metabolism.

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.

References (39)

  • P.Y. Scaraffia et al.

    Proline can be utilized as an energy substrate during flight of Aedes aegypti females

    J. Insect Physiol.

    (2003)
  • P.Y. Scaraffia et al.

    Differential ammonia metabolism in Aedes aegypti fat body and midgut tissues

    J. Insect Physiol.

    (2010)
  • P.Y. Scaraffia et al.

    Analysis of whole body ammonia metabolism in Aedes aegypti using [15N]-labeled compounds and mass spectrometry

    Insect Biochem. Mol. Biol.

    (2006)
  • P. von Dungern et al.

    Enzymatic analysis of uricotelic protein catabolism in the mosquito Aedes aegypti

    J. Insect Physiol.

    (2001)
  • J. Yang et al.

    What is the metabolic role of phosphoenolpyruvate carboxykinase?

    J. Biol. Chem.

    (2009)
  • G. Zhou et al.

    Metabolic fate of [14C]-labeled meal protein amino acids in Aedes aegypti mosquitoes

    J. Insect Physiol.

    (2004)
  • G. Zhou et al.

    Utilization of pre-existing energy stores of female Aedes aegypti mosquitoes during the first gonotrophic cycle

    Insect Biochem. Mol. Biol.

    (2004)
  • N.L. Achee et al.

    Alternative strategies for mosquito-borne arbovirus control

    PLoS Neglected Trop. Dis.

    (2019)
  • R. Bakszt et al.

    The crystal structure of Toxoplasma gondii pyruvate kinase 1

    PLoS One

    (2010)
  • Cited by (6)

    • Lateral oviduct-secreted proteins in the brown planthopper

      2022, Journal of Proteomics
      Citation Excerpt :

      In addition, a great variety of identified lateral oviduct-secreted proteins revealed their multifunctional roles. For example, glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme, plays an important role in sperm motility and is specific to male germ cells [13,14]; a fatty acid synthase is involved in molting cycles and lipid synthesis and waterproofs the cuticle [15,16]; pyruvate kinase is correlated with nitrogen and carbohydrate metabolism in hypothermia [17,18]; nucleoside diphosphate kinase (NDK) is highly expressed in salivary glands and is essential for insect-plant interactions [19,20]; phosphoglycerate kinase (PGK), a cytosolic protein, regulates the internalization of Spiroplasma citri [21]; ribosomal proteins such as 40S ribosomal proteins and 60S acidic ribosomal proteins might be related to the control of development, molting and metamorphosis of insects [22,23]; actin has also been found in salivary secretions; and vitellogenin, a protein related to oocyte development with highly expressed transcripts in fat body and gelling saliva in BPHs, might be brought in oviducts during egg transportation [24,25]. Moreover, there were also some proteins involved in cellular processes, such as bromodomain-containing protein, which is associated with transcription and proliferation of cellular genes [26]; guanine nucleotide-binding protein (GNBP), a mediator located on the cytoplasmic cell membrane [27]; Y-box-binding protein (YB), a multifunctional protein that plays a role in the survival of insects in hostile environments [28]; and ATP synthase, which is embedded in follicular epithelial cells and oocytes [29,30].

    • Unraveling mosquito metabolism with mass spectrometry-based metabolomics

      2021, Trends in Parasitology
      Citation Excerpt :

      Genetic inhibition of PK by RNAi disrupts Glc and ammonia metabolism in mosquito whole body. PK silencing delays several physiological processes and reduces [13C]-metabolite abundance (i.e., G6P, F6P, Pyr, Lac, α-KG, citrate, isocitrate, Ala, Glu, Gln, Pro, and UA) mainly at 6 h after feeding, by decreasing the flux of Glc to glycolysis, PPP, Krebs cycle, and pathways associated with ammonia detoxification [38] (Figure 3). The application of metabolomics with cutting-edge genome-editing technologies [39] can further facilitate the discovery of metabolic interactions and regulators that control multiple metabolic pathways in mosquitoes.

    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.

    View full text