Biology subjects

Borah, K.

Publications and source records attributed to Borah, K..

6 recordsLinked to original sources

bioRxiv2022

Bayesian multi-model-based 13C15N-metabolic flux analysis quantifies carbon-nitrogen metabolism in mycobacteria

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 pathogen Mycobacterium tuberculosis (Mtb), this is particularly important in informing the development of effective drugs targeting Mtbs metabolism. Here we performed 13C15N dual isotopic labelling of mycobacterial steady state cultures and quantified intracellular carbon-nitrogen (CN) and nitrogen (N) fluxes in addition to carbon (C) fluxes and inferred their reaction bidirectionalities. The combination of 13C15N-MFA with a Bayesian multi-model approach allowed us to resolve C and N fluxes simultaneously which was not possible with classical 13C-MFA. We quantified CN fluxes for amino acid and, for the first time, nucleotide biosynthesis. Our analysis identified glutamate as the central CN and N node in mycobacteria, and improved resolution of the anaplerotic node. Our study describes a powerful platform to measure carbon and nitrogen metabolism in any biological system with statistical rigor.

systems biology↗

bioRxiv2021

Murine Norovirus infection results in anti-inflammatory response downstream of amino acids depletion in macrophages

Murine norovirus (MNV) infection results in a late translation shut-off, 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 eIF2 kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signalling during metabolic stress. While viral infection is usually associated with activation of dsRNA binding pattern recognition receptor PKR, we hypothesised 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 characterise cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signalling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation. ImportanceDuring viral infection, host defences are typically characterised by the secretion of pro-inflammatory autocrine and paracrine cytokines, potentiation of the IFN response and induction of the anti-viral response via activation of JAK and Stat signalling. To avoid these and propagate viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signalling. This highlights novel tools in the viral countermeasures tool-kit, and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.

microbiology↗

bioRxiv2021

Metabolic constraints on nitrogen fixation by rhizobia in legume nodules

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which use plant-derived dicarboxylates as energy and electron sources for reduction of atmospheric N2 into ammonia for secretion to plants. Using heterogeneous genome-scale datasets, we reconstructed a model of bacteroid metabolism to investigate the effects of varying dicarboxylate and oxygen supply on carbon and nitrogen allocation. Modelling and 13C metabolic flux analysis in bacteroids indicate that microaerobiosis restricts the decarboxylating arm of the TCA cycle and limits ammonia assimilation into glutamate. Catabolism of dicarboxylates induces a higher oxygen demand but also a higher NADH/NAD+ ratio compared to sugars. Carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules with alanine secretion increasing as oxygen tension decreases. Our results provide a framework for understanding fundamental constraints on rhizobial metabolism during symbiotic nitrogen fixation.

microbiology↗

bioRxiv2021

Metabolic flux partitioning between the TCA cycle and glyoxylate shunt combined with a reversible methyl citrate cycle provide nutritional flexibility for Mycobacterium tuberculosis

The utilisation of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection thereby identifying the Mtb specific metabolic pathways and enzymes required for carbon co-metabolism as potential drug targets. Metabolic flux represents the final integrative outcome of many different levels of cellular regulation that contribute to the flow of metabolites through the metabolic network. It is therefore critical that we have an in-depth understanding of the rewiring of metabolic fluxes in different conditions. Here, we employed 13C-metabolic flux analysis using stable isotope tracers (13C and 2H) and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly on physiologically relevant carbon sources in a steady state chemostat. We demonstrate that Mtb is able to efficiently co-metabolise combinations of either cholesterol or glycerol along with C2 generating carbon substrates. The uniform assimilation of the carbon sources by Mtb throughout the network indicated no compartmentalization of metabolism in these conditions however there were substrate specific differences in metabolic fluxes. This work identified that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle as the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide new insights into the metabolic architecture that affords adaptability of Mtb to divergent carbon substrates. ImportanceEach year more than 1 million people die of tuberculosis (TB). Many more are infected but successfully diagnosed and treated with antibiotics, however antibiotic-resistant TB isolates are becoming ever more prevalent and so novel therapies are urgently needed that can effectively kill the causative agent. Mtb specific metabolic pathways have been identified as an important drug target in TB. However the apparent metabolic plasticity of this pathogen presents a major obstacle to efficient targeting of Mtb specific vulnerabilities and therefore it is critical to define the metabolic fluxes that Mtb utilises in different conditions. Here, we used 13C-metabolic flux analysis to measure the metabolic fluxes that Mtb uses whilst growing on potential in vivo nutrients. Our analysis identified selective use of the metabolic network that included the TCA cycle, glyoxylate shunt and methyl citrate cycle. The metabolic flux phenotypes determined in this study improves our understanding about the co-metabolism of multiple carbon substrates by Mtb identifying a reversible methyl citrate cycle and the glyoxylate shunt as the critical metabolic nodes which underlie the nutritional flexibility of Mtb.

systems biology↗

bioRxiv2019

GSMN-ML- a genome scale metabolic network reconstruction of the obligate human pathogen Mycobacterium leprae

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 pathogens 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 (DPA) 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 predictions of nutrients required for growth of the bacillus in vitro and identify novel drug targets.\n\nAuthor SummaryMycobacterium leprae, the obligate human pathogen is uncultivable in axenic growth medium, and this hinders research on this pathogen, and the pathogenesis of leprosy. The development of novel therapeutics relies on the understanding of growth, survival and metabolism of this bacterium in the host, the knowledge of which is currently very limited. Here we reconstructed a metabolic network of M. leprae-GSMN-ML, a powerful in silico tool to study growth and metabolism of the leprosy bacillus. We demonstrate the application of GSMN-ML to identify the metabolic pathways, and metabolite classes that M. leprae utilizes during intracellular growth.

microbiology↗

bioRxiv2019

A mixed nitrogen diet and compartmentalized utilization for Mycobacterium tuberculosis replicating in host cells: results of a systems-based analysis

Nitrogen metabolism of Mycobacterium tuberculosis (Mtb) is crucial for the survival and virulence of this pathogen inside host macrophages but little is known about the nitrogen sources acquired from the host or their route of assimilation. Here we performed a systems-based analysis of nitrogen metabolism in intracellullar Mtb and developed 15N-Flux Spectral Ratio Analysis (FSRA) to probe the metabolic cross-talk between the host cell and Mtb. We demonstrate that intracellular Mtb acquires nitrogen from multiple amino acids in the macrophage including glutamate, glutamine, aspartate, alanine, glycine and valine, with glutamine being the predominant nitrogen donor. Each nitrogen source is uniquely assimilated into specific intracellular pools indicating compartmentalised metabolism. This was not observed for in vitro-grown Mtb indicating that there is a switch in nitrogen metabolism when the pathogen enters the intracellular environment. These results provide clues about the potential metabolic targets for development of innovative anti-tuberculosis therapies.

microbiology↗