Bio21 - Research Publications
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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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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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ItemLipidomic Profiling of Adipose Tissue Reveals an Inflammatory Signature in Cancer-Related and Primary Lymphedema(PUBLIC LIBRARY SCIENCE, 2016-05-16)Cancer-related and primary lymphedema (LE) are associated with the production of adipose tissue (AT). Nothing is known, however, about the lipid-based molecules that comprise LE AT. We therefore analyzed lipid molecules in lipoaspirates and serum obtained from LE patients, and compared them to lipoaspirates from cosmetic surgery patients and healthy control cohort serum. LE patient serum analysis demonstrated that triglycerides, HDL- and LDL-cholesterol and lipid transport molecules remained within the normal range, with no alterations in individual fatty acids. The lipidomic analysis also identified 275 lipid-based molecules, including triacylglycerides, diacylglycerides, fatty acids and phospholipids in AT oil and fat. Although the majority of lipid molecules were present in a similar abundance in LE and non-LE samples, there were several small changes: increased C20:5-containing triacylglycerides, reduced C10:0 caprinic and C24:1 nervonic acids. LE AT oil also contained a signature of increased cyclopropane-type fatty acids and inflammatory mediators arachidonic acid and ceramides. Interestingly C20:5 and C22:6 omega-3-type lipids are increased in LE AT, correlating with LE years. Hence, LE AT has a normal lipid profile containing a signature of inflammation and omega-3-lipids. It remains unclear, however, whether these differences reflect a small-scale global metabolic disturbance or effects within localised inflammatory foci.
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ItemLeishmania carbon metabolism in the macrophage phagolysosome- feast or famine?(F1000 Research Ltd, 2015)A number of medically important microbial pathogens target and proliferate within macrophages and other phagocytic cells in their mammalian hosts. While the majority of these pathogens replicate within the host cell cytosol or non-hydrolytic vacuolar compartments, a few, including protists belonging to the genus Leishmania, proliferate long-term within mature lysosome compartments. How these parasites achieve this feat remains poorly defined. In this review, we highlight recent studies that suggest that Leishmania virulence is intimately linked to programmed changes in the growth rate and carbon metabolism of the obligate intra-macrophage stages. We propose that activation of a slow growth and a stringent metabolic response confers resistance to multiple stresses (oxidative, temperature, pH), as well as both nutrient limitation and nutrient excess within this niche. These studies highlight the importance of metabolic processes as key virulence determinants in Leishmania.
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ItemAn Efficient Single Phase Method for the Extraction of Plasma Lipids(MDPI AG, 2015-06)Lipidomic approaches are now widely used to investigate the relationship between lipid metabolism, health and disease. Large-scale lipidomics studies typically aim to quantify hundreds to thousands of lipid molecular species in a large number of samples. Consequently, high throughput methodology that can efficiently extract a wide range of lipids from biological samples is required. Current methods often rely on extraction in chloroform:methanol with or without two phase partitioning or other solvents, which are often incompatible with liquid chromatography electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS). Here, we present a fast, simple extraction method that is suitable for high throughput LC ESI-MS/MS. Plasma (10 μL) was mixed with 100 μL 1-butanol:methanol (1:1 v/v) containing internal standards resulting in efficient extraction of all major lipid classes (including sterols, glycerolipids, glycerophospholipids and sphingolipids). Lipids were quantified using positive-ion mode LC ESI-MS/MS. The method showed high recovery (>90%) and reproducibility (%CV < 20%). It showed a strong correlation of all lipid measures with an established chloroform:methanol extraction method (R2 = 0.976). This method uses non-halogenated solvents, requires no drying or reconstitution steps and is suitable for large-scale LC ESI-MS/MS-based lipidomic analyses in research and clinical laboratories.
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ItemReversing diet-induced metabolic dysregulation by diet switching leads to altered hepatic de novo lipogenesis and glycerolipid synthesis(NATURE PORTFOLIO, 2016-06-07)In humans, low-energy diets rapidly reduce hepatic fat and improve/normalise glycemic control. Due to difficulties in obtaining human liver, little is known about changes to the lipid species and pathway fluxes that occur under these conditions. Using a combination of stable isotope, and targeted metabolomic approaches we investigated the acute (7-9 days) hepatic effects of switching high-fat high-sucrose diet (HFD) fed obese mice back to a chow diet. Upon the switch, energy intake was reduced, resulting in reductions of fat mass and hepatic triacyl- and diacylglycerol. However, these parameters were still elevated compared to chow fed mice, thus representing an intermediate phenotype. Nonetheless, glucose intolerance and hyperinsulinemia were completely normalized. The diet reversal resulted in marked reductions in hepatic de novo lipogenesis when compared to the chow and HFD groups. Compared with HFD, glycerolipid synthesis was reduced in the reversal animals, however it remained elevated above that of chow controls, indicating that despite experiencing a net loss in lipid stores, the liver was still actively esterifying available fatty acids at rates higher than that in chow control mice. This effect likely promotes the re-esterification of excess free fatty acids released from the breakdown of adipose depots during the weight loss period.
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ItemDExSI: a new tool for the rapid quantitation of 13C-labelled metabolites detected by GC-MS(OXFORD UNIV PRESS, 2018-06-01)SUMMARY: Stable isotope directed metabolomics is increasingly being used to measure metabolic fluxes in microbial, plant and animal cells. Incorporation of 13C/15N isotopes into a wide range of metabolites is typically determined using gas chromatography-mass spectrometry (GC/MS) or other hyphenated mass spectrometry approaches. The DExSI (Data Extraction for Stable Isotope-labelled metabolites) pipeline is an interactive graphical software package which can be used to rapidly quantitate isotopologues for a wide variety of metabolites detected by GC/MS. DExSI performs automated metabolite annotation, mass and positional isotopomer abundance determination and natural isotope abundance correction. It provides a range of output options and is suitable for high throughput analyses. AVAILABILITY AND IMPLEMENTATION: DExSI is available for non-commercial use from: https://github.com/DExSI/DExSI/. For Microsoft Windows 7 or higher (64-bit). CONTACT: malcolmm@unimelb.edu.au or michael.dagley@unimelb.edu.au. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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ItemPhospholipase A2 activity during the replication cycle of the flavivirus West Nile virus(PUBLIC LIBRARY SCIENCE, 2018-04)Positive-sense RNA virus intracellular replication is intimately associated with membrane platforms that are derived from host organelles and comprised of distinct lipid composition. For flaviviruses, such as West Nile virus strain Kunjin virus (WNVKUN) we have observed that these membrane platforms are derived from the endoplasmic reticulum and are rich in (at least) cholesterol. To extend these studies and identify the cellular lipids critical for WNVKUN replication we utilized a whole cell lipidomics approach and revealed an elevation in phospholipase A2 (PLA2) activity to produce lyso-phosphatidylcholine (lyso-PChol). We observed that the PLA2 enzyme family is activated in WNVKUN-infected cells and the generated lyso-PChol lipid moieties are sequestered to the subcellular sites of viral replication. The requirement for lyso-PChol was confirmed using chemical inhibition of PLA2, where WNVKUN replication and production of infectious virus was duly affected in the presence of the inhibitors. Importantly, we could rescue chemical-induced inhibition with the exogenous addition of lyso-PChol species. Additionally, electron microscopy results indicate that lyso-PChol appears to contribute to the formation of the WNVKUN membranous replication complex (RC); particularly affecting the morphology and membrane curvature of vesicles comprising the RC. These results extend our current understanding of how flaviviruses manipulate lipid homeostasis to favour their own intracellular replication.
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ItemTalaromyces marneffei simA Encodes a Fungal Cytochrome P450 Essential for Survival in Macrophages(AMER SOC MICROBIOLOGY, 2018)Fungi are adept at occupying specific environmental niches and often exploit numerous secondary metabolites generated by the cytochrome P450 (CYP) monoxygenases. This report describes the characterization of a yeast-specific CYP encoded by simA ("survival in macrophages"). Deletion of simA does not affect yeast growth at 37°C in vitro but is essential for yeast cell production during macrophage infection. The ΔsimA strain exhibits reduced conidial germination and intracellular growth of yeast in macrophages, suggesting that the enzymatic product of SimA is required for normal fungal growth in vivo. Intracellular ΔsimA yeast cells exhibit cell wall defects, and metabolomic and chemical sensitivity data suggest that SimA may promote chitin synthesis or deposition in vitro. In vivo, ΔsimA yeast cells subsequently lyse and are degraded, suggesting that SimA may increase resistance to and/or suppress host cell biocidal effectors. The results suggest that simA synthesizes a secondary metabolite that allows T. marneffei to occupy the specific intracellular environmental niche within the macrophage. IMPORTANCE This study in a dimorphic fungal pathogen uncovered a role for a yeast-specific cytochrome P450 (CYP)-encoding gene in the ability of T. marneffei to grow as yeast cells within the host macrophages. This report will inspire further research into the role of CYPs and secondary metabolite synthesis during fungal pathogenic growth.