Dr Khushboo Borah Slater B.Tech, DPhil, L’Oréal-UNESCO Fellow

Metabolic fluxes are at the heart of metabolism and growth in any living system. During tuberculosis (TB) infection, the pathogenic Mycobacterium tuberculosis (Mtb) adapts its nutritional behaviour and metabolic fluxes to survive in human macrophages and cause infection. The infected host cells also undergo metabolic changes. However, our knowledge of the infected host metabolism and identification of the reprogrammed metabolic flux nodes remains limited. In this study, we applied systems-based 13C-metabolic flux analysis (MFA) to measure intracellular carbon metabolic fluxes in Mtb-infected human THP-1 macrophages. We provide a flux map for infected macrophages that quantified significantly increased fluxes through glycolytic fluxes towards pyruvate synthesis and reduced pentose phosphate pathway fluxes when compared to uninfected macrophages. The tri carboxylic acid (TCA) cycle fluxes were relatively low, and amino acid fluxes were reprogrammed upon Mtb infection. The knowledge of host metabolic flux profiles derived from our work expands on how the host cell adapts its carbon metabolism in response to Mtb infection and highlights important nodes that may provide targets for developing new therapeutics to improve TB treatment.

Rapid, reliable, sensitive, portable, and accurate diagnostics are required to control disease outbreaks such as COVID-19 that pose an immense burden on human health and the global economy. Here we developed a loop-mediated isothermal amplification (LAMP)-based electrochemical test for the detection of SARS-CoV-2 that causes COVID-19. The test is based on the oxidation-reduction reaction between pyrophosphates (generated from positive LAMP reaction) and molybdate that is detected by cyclic voltammetry using inexpensive and disposable carbon screen printed electrodes. Our test showed higher sensitivity (detecting as low as 5.29 RNA copies/μL) compared to the conventional fluorescent reverse transcriptase (RT)-LAMP. We validated our tests using human serum and saliva spiked with SARS-CoV-2 RNA and clinical (saliva and nasal-pharyngeal) swab samples demonstrating 100% specificity …

Leprosy, also known as Hansen’s disease, is an ancient chronic infectious disease that remains a major problem in the world today, infecting over 200,000 people each year, particularly affecting resource-limited and the most disadvantaged sections of society in under-developed countries of the world. Mycobacterium leprae, a slow-growing mycobacterium, causes leprosy in humans. Leprosy causes nerve damage and permanent disabilities including blindness and paralysis. People affected by leprosy face stigma and discrimination in society. Although multidrug therapy is available, millions of people are still affected by leprosy, so new vaccine, drug and disease management approaches are urgently needed for control, prevention and treatment of this disease. This chapter is a general review of leprosy, the current treatment and prevention measures and challenges that need to be addressed for complete eradication of this disease.

Metabolic flux is the final output of cellular regulation and has been extensively studied for carbon but much less is known about nitrogen, which is another important building block for living organisms. For the tuberculosis pathogen, this is particularly important in informing the development of effective drugs targeting the pathogen's metabolism. Here we performed 13C15N dual isotopic labeling of Mycobacterium bovis BCG steady state cultures, quantified intracellular carbon and nitrogen fluxes and inferred reaction bidirectionalities. This was achieved by model scope extension and refinement, implemented in a multi‐atom transition model, within the statistical framework of Bayesian model averaging (BMA). Using BMA‐based 13C15N‐metabolic flux analysis, we jointly resolve carbon and nitrogen fluxes quantitatively. We provide the first nitrogen flux distributions for amino acid and nucleotide biosynthesis in …

Tuberculosis (TB) is one of the ten infectious diseases that cause the highest amount of human mortality and morbidity. This infection, which is caused by a single pathogen, Mycobacterium tuberculosis, kills over a million people every year. There is an emerging problem of antimicrobial resistance in TB that needs urgent treatment and management. Tuberculosis treatment is complicated by its complex drug regimen, its lengthy duration and the serious side-effects caused by the drugs required. There are a number of critical issues around drug delivery and subsequent intracellular bacterial clearance. Drugs have a short lifespan in systemic circulation, which limits their activity. Nanomedicine in TB is an emerging research area which offers the potential of effective drug delivery using nanoparticles and a reduction in drug doses and side-effects to improve patient compliance with the treatment and enhance their recovery. Here, we provide a minireview of anti-TB treatment, research progress on nanomedicine and the prospects for future applications in developing innovative therapies.

Metabolism of pathogens in infectious diseases is important for their survival, virulence and pathogenesis. Mycobacterial pathogens successfully scavenge multiple host nutrient sources in the intracellular niche. It is therefore important to identify the intracellular nutrient sources and their metabolic fates in these pathogens. Metabolic phenotype of an organism is defined by metabolic fluxes. We quantified in vivo fluxes of the pathogens and probed host-bacterial metabolic cross talks in tuberculosis (TB) and leprosy using systems-based strategies and techniques of isotopic labelling, metabolic modelling and metabolic flux analysis (MFA). We show that the TB pathogen metabolizes a number of carbon and nitrogen sources in human macrophages and identified vulnerable nodes such as glutamine and serine biosynthesis as potential drug targets. Mycobacterium leprae, the leprosy causing pathogen, uses host cell …

Klebsiella pneumoniae is an important pathogenic bacterium commonly associated with human healthcare and community-acquired infections. In recent years, K. pneumoniae has become a significant threat to global public and veterinary health, due to its high rates of antimicrobial resistance (AMR). Early diagnosis of K. pneumoniae infection and detection of any associated AMR would help to accelerate directed therapy and reduce the risk of the emergence of multidrug-resistant isolates. In this study, we identified three target genes (yhaI, epsL and xcpW) common to K. pneumoniae isolates from both China and Europe, and designed Loop-Mediated Isothermal Amplification (LAMP) assays for the detection of K. pneumoniae in clinical samples. We also designed LAMP assays for the detection of five AMR genes commonly associated with K. pneumoniae. The LAMP assays were validated on a total of 319 type reference strains and clinical isolates of diverse genetic backgrounds, in addition to 40 clinical human sputum samples, and were shown to be reliable, highly specific and sensitive. For the K. pneumoniae specific LAMP assay, the calculated sensitivity, specificity, positive and negative predictive values (comparison with culture and MALDI-TOF MS) were all 100% on clinical isolates and respectively of 100%, 91%, 90% and 100% when tested on clinical sputum samples, while being significantly faster than the reference methods. For the blaKPC and other carbapenemases LAMP assays, the concordance between the LAMP results and the references methods (susceptibility tests) were 100%, on both pure cultures (n=125) and clinical …

Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens causing tuberculosis (TB), a devastating infectious disease responsible for the highest human mortality and morbidity. The emergence of drug-resistant strains of the pathogen has increased the burden of TB tremendously and new therapeutics to overcome the problem of drug resistance are urgently needed. Metabolism of Mtb and its interactions with the host is important for its survival and virulence; this is an important topic of research where there is growing interest in developing new therapies and drugs that target these interactions and metabolism of the pathogen during infection. Mtb adapts its metabolism in its intracellular niche and acquires multiple nutrient sources from the host cell. Carbon metabolic pathways and fluxes of Mtb has been extensively researched for over a decade and is well-defined. Recently, there has been investigations and efforts to measure metabolism of nitrogen, which is another important nutrient for Mtb during infection. This review discusses our current understanding of the central carbon and nitrogen metabolism, and metabolic fluxes that are important for the survival of the TB pathogen.

An Azorhizobium caulinodans phaC mutant (OPS0865) unable to make poly-3-hydroxybutyrate (PHB), grows poorly on many carbon sources and cannot fix nitrogen in laboratory culture. However, when inoculated onto its host plant, Sesbania rostrata, the phaC mutant consistently fixed nitrogen. Upon reisolation from S. rostrata root nodules, a suppressor strain (OPS0921) was isolated that has significantly improved growth on a variety of carbon sources and also fixes nitrogen in laboratory culture. The suppressor retains the original mutation and is unable to synthesize PHB. Genome sequencing revealed a suppressor transition mutation, G to A (position 357,354), 13 bases upstream of the ATG start codon of phaR in its putative ribosome binding site (RBS). PhaR is the global regulator of PHB synthesis but also has other roles in regulation within the cell. In comparison with the wild type, translation from the phaR …

Tuberculosis (TB) is a devastating infectious disease that kills over a million people every year. There is an increasing burden of multi drug resistance (MDR) and extensively drug resistance (XDR) TB. New and improved therapies are urgently needed to overcome the limitations of current treatment. The causative agent, Mycobacterium tuberculosis (Mtb) is one of the most successful pathogens that can manipulate host cell environment for adaptation, evading immune defences, virulence, and pathogenesis of TB infection. Host-pathogen interaction is important to establish infection and it involves a complex set of processes. Metabolic cross talk between the host and pathogen is a facet of TB infection and has been an important topic of research where there is growing interest in developing therapies and drugs that target these interactions and metabolism of the pathogen in the host. Mtb scavenges multiple nutrient sources from the host and has adapted its metabolism to survive in the intracellular niche. Advancements in systems-based omic technologies have been successful to unravel host-pathogen interactions in TB. In this review we discuss the application and usefulness of omics in TB research that provides promising interventions for developing anti-TB therapies.

Murine norovirus (MNV) infection results in a late translation shutoff that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the α subunit of eukaryotic initiation factor 2 (eIF2α) kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signaling during metabolic stress. While viral infection is usually associated with activation of double-stranded RNA (dsRNA) binding pattern recognition receptor PKR, we hypothesized that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterize cellular responses during MNV replication. We demonstrate the activation …

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which catabolize plant-derived dicarboxylates to reduce atmospheric N2 into ammonia. Despite the agricultural importance of this symbiosis, the mechanisms that govern carbon and nitrogen allocation in bacteroids and promote ammonia secretion to the plant are largely unknown. Using a metabolic model derived from genome-scale datasets, we show that carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules. Catabolism of dicarboxylates induces not only a higher oxygen demand but also a higher NADH/NAD+ ratio than sugars. Modeling and 13C metabolic flux analysis indicate that oxygen limitation restricts the decarboxylating arm of the tricarboxylic acid cycle, which limits ammonia assimilation into glutamate. By tightly controlling oxygen supply and providing …

The co‐catabolism of multiple host‐derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady‐state chemostat system. We demonstrate that Mtb efficiently co‐metabolises either cholesterol or glycerol, in combination with two‐carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible …

The raw datasets of oxysterol quantifications from whole cell and mitochondrial fractions of THP-1 monocytes and macrophages, neuronal-like SH-SH5Y cells and human peripheral blood mononuclear cells are presented. Oxysterols were quantified using a new liquid chromatography-mass spectrometry (LC-MS) and multiple reaction monitoring analysis published in the article “A quantitative LC-MS/MS method for analysis of mitochondrial-specific oxysterol metabolism” in Redox Biology [1]. This method showed improved extraction efficiency and recovery of mono and dihydroxycholesterols from cellular matrix. The datasets derived from the three cell lines are included in the appendix. These datasets provide new information about the oxysterol distribution in THP-1 monocytes and macrophages, SH-SY5Y cells and peripheral blood mononuclear cells. These datasets can be used as a guide for oxysterol …

The approval of bedaquiline (BDQ) for the treatment of tuberculosis has generated substantial interest in inhibiting energy metabolism as a therapeutic paradigm. However, it is not known precisely how BDQ triggers cell death in Mycobacterium tuberculosis (Mtb). Using 13C isotopomer analysis, we show that BDQ-treated Mtb redirects central carbon metabolism to induce a metabolically vulnerable state susceptible to genetic disruption of glycolysis and gluconeogenesis. Metabolic flux profiles indicate that BDQ-treated Mtb is dependent on glycolysis for ATP production, operates a bifurcated TCA cycle by increasing flux through the glyoxylate shunt, and requires enzymes of the anaplerotic node and methylcitrate cycle. Targeting oxidative phosphorylation (OXPHOS) with BDQ and simultaneously inhibiting substrate level phosphorylation via genetic disruption of glycolysis leads to rapid sterilization. Our findings …

Oxysterols are critical regulators of inflammation and cholesterol metabolism in cells. They are oxidation products of cholesterol and may be differentially metabolised in subcellular compartments and in biological fluids. New analytical methods are needed to improve our understanding of oxysterol trafficking and the molecular interplay between the cellular compartments required to maintain cholesterol/oxysterol homeostasis. Here we describe a method for isolation of oxysterols using solid phase extraction and quantification by liquid chromatography-mass spectrometry, applied to tissue, cells and mitochondria.

We analysed five monohydroxysterols; 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, 7 ketocholesterol and three dihydroxysterols 7α-24(S)dihydroxycholesterol, 7α-25dihydroxycholesterol, 7α-27dihydroxycholesterol by LC-MS/MS following reverse phase …

Leprosy, caused by Mycobacterium leprae, has plagued humanity for thousands of years and continues to cause morbidity, disability and stigmatization in two to three million people today. Although effective treatment is available, the disease incidence has remained approximately constant for decades so new approaches, such as vaccine or new drugs, are urgently needed for control. Research is however hampered by the pathogen’s obligate intracellular lifestyle and the fact that it has never been grown in vitro. Consequently, despite the availability of its complete genome sequence, fundamental questions regarding the biology of the pathogen, such as its metabolism, remain largely unexplored. In order to explore the metabolism of the leprosy bacillus with a long-term aim of developing a medium to grow the pathogen in vitro, we reconstructed an in silico genome scale metabolic model of the bacillus, GSMN-ML. The model was used to explore the growth and biomass production capabilities of the pathogen with a range of nutrient sources, such as amino acids, glucose, glycerol and metabolic intermediates. We also used the model to analyze RNA-seq data from M. leprae grown in mouse foot pads, and performed Differential Producibility Analysis to identify metabolic pathways that appear to be active during intracellular growth of the pathogen, which included pathways for central carbon metabolism, co-factor, lipids, amino acids, nucleotides and cell wall synthesis. The GSMN-ML model is thereby a useful in silico tool that can be used to explore the metabolism of the leprosy bacillus, analyze functional genomic experimental data, generate …

New approaches are needed to control leprosy, but understanding of the biology of the causative agent Mycobacterium leprae remains rudimentary, principally because the pathogen cannot be grown in axenic culture. Here, we applied 13C isotopomer analysis to measure carbon metabolism of M. leprae in its primary host cell, the Schwann cell. We compared the results of this analysis with those of a related pathogen, Mycobacterium tuberculosis, growing in its primary host cell, the macrophage. Using 13C isotopomer analysis with glucose as the tracer, we show that whereas M. tuberculosis imports most of its amino acids directly from the host macrophage, M. leprae utilizes host glucose pools as the carbon source to biosynthesize the majority of its amino acids. Our analysis highlights the anaplerotic enzyme phosphoenolpyruvate carboxylase required for this intracellular diet of M. leprae, identifying this enzyme …

Nitrogen metabolism of Mycobacterium tuberculosis (Mtb) is crucial for the survival of this important pathogen in its primary human host cell, the macrophage, but little is known about the source(s) and their assimilation within this intracellular niche. Here, we have developed 15N-flux spectral ratio analysis (15N-FSRA) to explore Mtb's nitrogen metabolism; we demonstrate that intracellular Mtb has access to multiple amino acids in the macrophage, including glutamate, glutamine, aspartate, alanine, glycine, and valine; and we identify glutamine as the predominant nitrogen donor. Each nitrogen source is uniquely assimilated into specific amino acid pools, indicating compartmentalized metabolism during intracellular growth. We have discovered that serine is not available to intracellular Mtb, and we show that a serine auxotroph is attenuated in macrophages. This work provides a systems-based tool for exploring the …

Oxysterols are oxidized derivatives of cholesterol that are formed enzymatically or via reactive oxygen species or both. Cholesterol or oxysterols ingested as food are absorbed and packed into lipoproteins that are taken up by hepatic cells. Within hepatic cells, excess cholesterol is metabolised to form bile acids. The endoplasmic reticulum acts as the main organelle in the bile acid synthesis pathway. Metabolised sterols originating from this pathway are distributed within other organelles and in the cell membrane. The alterations to membrane oxysterol:sterol ratio affects the integrity of the cell membrane. The presence of oxysterols changes membrane fluidity and receptor orientation. It is well documented that hydroxylase enzymes located in mitochondria facilitate oxysterol production via an acidic pathway. More recently, the presence of oxysterols was also reported in lysosomes. Peroxisomal deficiencies favour …

Symbiotic nitrogen fixation in rhizobial-legume symbioses is important for agriculture and the establishment of a successful symbiosis depends on the metabolic integration of the rhizobia inside the host nodules. This study aims to understand the metabolic adaptations in a rhizobium during nitrogen fixation. The study uses 13C-metabolic flux analysis to derive carbon fluxes across the metabolic network of the model diazotrophic rhizobium, Azorhizobium caulinodans ORS571...