Biochemistry and Pharmacology - Research Publications
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ItemMtrP, a putative methyltransferase in Corynebacteria, is required for optimal membrane transport of trehalose mycolates(ELSEVIER, 2020-05-01)Pathogenic bacteria of the genera Mycobacterium and Corynebacterium cause severe human diseases such as tuberculosis (Mycobacterium tuberculosis) and diphtheria (Corynebacterium diphtheriae). The cells of these species are surrounded by protective cell walls rich in long-chain mycolic acids. These fatty acids are conjugated to the disaccharide trehalose on the cytoplasmic side of the bacterial cell membrane. They are then transported across the membrane to the periplasm where they act as donors for other reactions. We have previously shown that transient acetylation of the glycolipid trehalose monohydroxycorynomycolate (hTMCM) enables its efficient transport to the periplasm in Corynebacterium glutamicum and that acetylation is mediated by the membrane protein TmaT. Here, we show that a putative methyltransferase, encoded at the same genetic locus as TmaT, is also required for optimal hTMCM transport. Deletion of the C. glutamicum gene NCgl2764 (Rv0224c in M. tuberculosis) abolished acetyltrehalose monocorynomycolate (AcTMCM) synthesis, leading to accumulation of hTMCM in the inner membrane and delaying its conversion to trehalose dihydroxycorynomycolate (h2TDCM). Complementation with NCgl2764 normalized turnover of hTMCM to h2TDCM. In contrast, complementation with NCgl2764 derivatives mutated at residues essential for methyltransferase activity failed to rectify the defect, suggesting that NCgl2764/Rv0224c encodes a methyltransferase, designated here as MtrP. Comprehensive analyses of the individual mtrP and tmaT mutants and of a double mutant revealed strikingly similar changes across several lipid classes compared with WT bacteria. These findings indicate that both MtrP and TmaT have nonredundant roles in regulating AcTMCM synthesis, revealing additional complexity in the regulation of trehalose mycolate transport in the Corynebacterineae.
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ItemIdentification of novel lipid modifications and intermembrane dynamics in Corynebacterium glutamicum using high-resolution mass spectrometry(ELSEVIER, 2018-07)The complex cell envelopes of Corynebacterineae contribute to the virulence of pathogenic species (such as Mycobacterium tuberculosis and Corynebacterium diphtheriae) and capacity of nonpathogenic species (such as Corynebacterium glutamicum) to grow in diverse niches. The Corynebacterineae cell envelope comprises an asymmetric outer membrane that overlays the arabinogalactan-peptidoglycan complex and the inner cell membrane. Dissection of the lipid composition of the inner and outer membrane fractions is important for understanding the biogenesis of this multilaminate wall structure. Here, we have undertaken the first high-resolution analysis of C. glutamicum inner and outer membrane lipids. We identified 28 lipid (sub)classes (>233 molecular species), including new subclasses of acylated/acetylated trehalose mono/dicorynomycolic acids, using high-resolution LC/MS/MS coupled with mass spectral library searches in MS-DIAL. All lipid subclasses exhibited polarized distributions across the inner and outer membrane fractions generated by differential solvent extraction. Strikingly, deletion of the TmaT protein, which is required for transport of trehalose corynomycolates across the inner membrane, led to the accumulation of triacylglycerols in the inner membrane and to suppressed synthesis of phosphatidylglycerol and alanylated lipids. These analyses indicate unanticipated connectivity in the synthesis and/or transport of different lipid classes in C. glutamicum.
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ItemAnalysis of Ca2+ mediated signaling regulating Toxoplasma infectivity reveals complex relationships between key molecules(WILEY, 2017-04)Host cell invasion, exit and parasite dissemination is critical to the pathogenesis of apicomplexan parasites such as Toxoplasma gondii and Plasmodium spp. These processes are regulated by intracellular Ca2+ signaling although the temporal dynamics of Ca2+ fluxes and down-stream second messenger pathways are poorly understood. Here, we use a genetically encoded biosensor, GFP-Calmodulin-M13-6 (GCaMP6), to capture Ca2+ flux in live Toxoplasma and investigate the role of Ca2+ signaling in egress and motility. Our analysis determines how environmental cues and signal activation influence intracellular Ca2+ flux, allowing placement of effector molecules within this pathway. Importantly, we have identified key interrelationships between cGMP and Ca2+ signaling that are required for activation of egress and motility. Furthermore, we extend this analysis to show that the Ca2+ Dependent Protein Kinases-TgCDPK1 and TgCDPK3-play a role in signal quenching before egress. This work highlights the interrelationships of second messenger pathways of Toxoplasma in space and time, which is likely required for pathogenesis of all apicomplexan species.
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ItemNo Preview AvailableFunction of hTim8a in complex IV assembly in neuronal cells provides insight into pathomechanism underlying Mohr-Tranebjaerg syndrome (vol 8, e48828, 2020)(ELIFE SCIENCES PUBLICATIONS LTD, 2020-03-18)
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ItemA Family of Dual-Activity Glycosyltransferase-Phosphorylases Mediates Mannogen Turnover and Virulence in Leishmania Parasites(CELL PRESS, 2019-09-11)Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.
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ItemLeishmania mexicana can utilize amino acids as major carbon sources in macrophages but not in animal models(WILEY, 2018-04)Leishmania parasites target macrophages in their mammalian hosts and proliferate within the mature phagolysosome compartment of these cells. Intracellular amastigote stages are dependent on sugars as a major carbon source in vivo, but retain the capacity to utilize other carbon sources. To investigate whether amastigotes can switch to using other carbon sources, we have screened for suppressor strains of the L. mexicana Δlmxgt1-3 mutant which lacks the major glucose transporters LmxGT1-3. We identified a novel suppressor line (Δlmxgt1-3s2 ) that has restored growth in rich culture medium and virulence in ex vivo infected macrophages, but failed to induce lesions in mice. Δlmxgt1-3s2 amastigotes had lower rates of glucose utilization than the parental line and primarily catabolized non-essential amino acids. The increased mitochondrial metabolism of this line was associated with elevated levels of intracellular reactive oxygen species, as well as increased sensitivity to inhibitors of the tricarboxylic acid (TCA) cycle, including nitric oxide. These results suggest that hardwired sugar addiction of Leishmania amastigotes contributes to the intrinsic resistance of this stage to macrophage microbicidal processes in vivo, and that these stages have limited capacity to switch to using other carbon sources.
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ItemImmunometabolism ofLeishmaniagranulomas(WILEY, 2020-11)Leishmania are parasitic protists that cause a spectrum of diseases in humans characterized by the formation of granulomatous lesions in the skin or other tissues, such as liver and spleen. The extent to which Leishmania granulomas constrain or promote parasite growth is critically dependent on the host T-helper type 1/T-helper type 2 immune response and the localized functional polarization of infected and noninfected macrophages toward a classically (M1) or alternatively (M2) activated phenotype. Recent studies have shown that metabolic reprograming of M1 and M2 macrophages underpins the capacity of these cells to act as permissive or nonpermissive host reservoirs, respectively. In this review, we highlight the metabolic requirements of Leishmania amastigotes and the evidence that these parasites induce and/or exploit metabolic reprogramming of macrophage metabolism. We also focus on recent studies highlighting the role of key macrophage metabolic signaling pathways, such as mechanistic target of rapamycin, adenosine monophosphate-activated protein kinase and peroxisome proliferator receptor gamma in regulating the pathological progression of Leishmania granulomas. These studies highlight the intimate connectivity between Leishmania and host cell metabolism, the need to investigate these interactions in vivo and the potential to exploit host cell metabolic signaling pathways in developing new host-directed therapies.
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ItemThe Key Glycolytic Enzyme Phosphofructokinase Is Involved in Resistance to Antiplasmodial Glycosides(AMER SOC MICROBIOLOGY, 2020-12-08)Plasmodium parasites rely heavily on glycolysis for ATP production and for precursors for essential anabolic pathways, such as the methylerythritol phosphate (MEP) pathway. Here, we show that mutations in the Plasmodium falciparum glycolytic enzyme, phosphofructokinase (PfPFK9), are associated with in vitro resistance to a primary sulfonamide glycoside (PS-3). Flux through the upper glycolysis pathway was significantly reduced in PS-3-resistant parasites, which was associated with reduced ATP levels but increased flux into the pentose phosphate pathway. PS-3 may directly or indirectly target enzymes in these pathways, as PS-3-treated parasites had elevated levels of glycolytic and tricarboxylic acid (TCA) cycle intermediates. PS-3 resistance also led to reduced MEP pathway intermediates, and PS-3-resistant parasites were hypersensitive to the MEP pathway inhibitor, fosmidomycin. Overall, this study suggests that PS-3 disrupts core pathways in central carbon metabolism, which is compensated for by mutations in PfPFK9, highlighting a novel metabolic drug resistance mechanism in P. falciparumIMPORTANCE Malaria, caused by Plasmodium parasites, continues to be a devastating global health issue, causing 405,000 deaths and 228 million cases in 2018. Understanding key metabolic processes in malaria parasites is critical to the development of new drugs to combat this major infectious disease. The Plasmodium glycolytic pathway is essential to the malaria parasite, providing energy for growth and replication and supplying important biomolecules for other essential Plasmodium anabolic pathways. Despite this overreliance on glycolysis, no current drugs target glycolysis, and there is a paucity of information on critical glycolysis targets. Our work addresses this unmet need, providing new mechanistic insights into this key pathway.
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ItemProtein kinase A negatively regulates Ca2+ signalling in Toxoplasma gondii(PUBLIC LIBRARY SCIENCE, 2018-09)The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating stages and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca2+ signalling activates motility, but how this is switched off after invasion is complete to allow for replication to begin is not understood. Here, we show that the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A catalytic subunit 1 (PKAc1) of Toxoplasma is responsible for suppression of Ca2+ signalling upon host cell invasion. We demonstrate that PKAc1 is sequestered to the parasite periphery by dual acylation of PKA regulatory subunit 1 (PKAr1). Upon genetic depletion of PKAc1 we show that newly invaded parasites exit host cells shortly thereafter, in a perforin-like protein 1 (PLP-1)-dependent fashion. Furthermore, we demonstrate that loss of PKAc1 prevents rapid down-regulation of cytosolic [Ca2+] levels shortly after invasion. We also provide evidence that loss of PKAc1 sensitises parasites to cyclic GMP (cGMP)-induced Ca2+ signalling, thus demonstrating a functional link between cAMP and these other signalling modalities. Together, this work provides a new paradigm in understanding how Toxoplasma and related apicomplexan parasites regulate infectivity.
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ItemEvidence That Intracellular Stages of Leishmania major Utilize Amino Sugars as a Major Carbon Source(PUBLIC LIBRARY SCIENCE, 2010-12)Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the host cell in order to replicate. Here we present evidence that intracellular amastigote stages of Leishmania exploit amino sugars in the phagolysosome of mammalian macrophages as a source of carbon and energy. L. major parasites are capable of using N-acetylglucosamine and glucosamine as primarily carbon sources and contain key enzymes required for conversion of these sugars to fructose-6-phosphate. The last step in this pathway is catalyzed by glucosamine-6-phosphate deaminase (GND), which was targeted to glycosomes via a canonical C-terminal targeting signal when expressed as a GFP fusion protein. Mutant parasites lacking GND were unable to grow in medium containing amino sugars as sole carbohydrate source and rapidly lost viability, concomitant with the hyper-accumulation of hexosamine-phosphates. Expression of native GND, but not a cytosolic form of GND, in Δgnd parasites restored hexosamine-dependent growth, indicating that toxicity is due to depletion of glycosomal pools of ATP. Non-lethal increases in hexosamine phosphate levels in both Δgnd and wild type parasites was associated with a defect in promastigote metacyclogenesis, suggesting that hexosamine phosphate levels may influence parasite differentiation. Promastigote and amastigote stages of the Δgnd mutant were unable to replicate within macrophages and were either completely cleared or exhibited reduced lesion development in highly susceptible Balb/c mice. Our results suggest that hexosamines are a major class of sugars in the macrophage phagolysosome and that catabolism of scavenged amino sugars is required to sustain essential metabolic pathways and prevent hexosamine toxicity.