scholarly journals A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness

2020 ◽  
Author(s):  
Laura A. Mike ◽  
Andrew J. Stark ◽  
Valerie S. Forsyth ◽  
Jay Vornhagen ◽  
Sara N. Smith ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Capsular Polysaccharide ◽  
Tca Cycle ◽  
Central Metabolism ◽  
Healthy Individuals ◽  
Systematic Analysis ◽  
Optimal Balance ◽  
The Tca Cycle ◽  
Cellular Factors

AbstractHypervirulent K. pneumoniae (hvKp) is a distinct pathotype that causes invasive community-acquired infections in healthy individuals. Hypermucoviscosity (hmv) is a major phenotype associated with hvKp characterized by copious capsule production and poor sedimentation. Dissecting the individual functions of CPS production and hmv in hvKp has been stymied by the conflation of these two properties. Although hmv requires capsular polysaccharide (CPS) biosynthesis, other cellular factors may also be required and some fitness phenotypes ascribed to CPS may be distinctly attributed to hmv. To address this challenge, we systematically identified genes that impact capsule and hmv. We generated a condensed, ordered transposon library in hypervirulent strain KPPR1, then evaluated the CPS production and hmv phenotypes of the 3,733 transposon mutants, representing 72% of all open reading frames in the genome. We employed forward and reverse genetic screens to evaluate effects of novel and known genes on CPS biosynthesis and hmv. These screens expand our understanding of core genes that coordinate CPS biosynthesis and hmv, as well as identify central metabolism genes that distinctly impact CPS biosynthesis or hmv, specifically those related to purine metabolism, pyruvate metabolism and the TCA cycle. Six representative mutants, with varying levels of CPS production and hmv, were all significantly out-competed by wildtype in a murine model of disseminating pneumonia. This suggests that an optimal balance between cellular energetics, CPS biosynthesis and hmv are required for maximal fitness. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that these processes are integrated into the metabolic status of the cell. Therefore, hvKp may require certain nutrients to fully elaborate its virulence-associated properties to specifically cause deep tissue infections.Author summaryKlebsiella pneumoniae is a common multi-drug resistant hospital-associated pathogen, however some isolates are capable of causing community-acquired infections in otherwise healthy individuals. The strains causing community-acquired infections have some distinguishing characteristics, which include overproduction of capsule and hypermucoviscosity. Hypermucoviscous strains are very tacky and sediment poorly when centrifuged. Historically, hypermucoviscosity has been attributed to overproduction of capsular polysaccharide, but recent data suggest that other factors contribute to this bacterial phenotype. Moreover, it seems that capsule and hypermucoviscosity may have distinct roles in pathogenesis. In this study, we sought to systematically investigate the genes that contribute to capsule and hypermucoviscosity. We found that in most cases, genes coordinately impact both capsule biosynthesis and hypermucoviscosity. Some metabolic genes linked to the TCA cycle, however, only affect one of these properties. Here, we identify that capsule biosynthesis and hypermucoviscosity are tightly tied to central metabolism and that an optimal balance between metabolism, capsule, and hypermucoviscosity are important for in vivo fitness of K. pneumoniae. These results identify genes that can be further probed to dissect how capsule and hypermucoviscosity are coordinated in response to niche-specific nutrients. Such studies will expand our understanding of the factors that drive the pathobiology of hypervirulent K. pneumoniae.

scholarly journals A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness

2021 ◽  
Vol 17 (3) ◽  
pp. e1009376
Author(s):  
Laura A. Mike ◽  
Andrew J. Stark ◽  
Valerie S. Forsyth ◽  
Jay Vornhagen ◽  
Sara N. Smith ◽  
...  
Keyword(s):  
Capsular Polysaccharide ◽  
Tca Cycle ◽  
Open Reading Frames ◽  
Genetic Screens ◽  
Systematic Analysis ◽  
Capsule Production ◽  
The Tca Cycle ◽  
Cellular Factors ◽  
Transposon Mutants ◽  
The Individual

Hypervirulent K. pneumoniae (hvKp) is a distinct pathotype that causes invasive community-acquired infections in healthy individuals. Hypermucoviscosity (hmv) is a major phenotype associated with hvKp characterized by copious capsule production and poor sedimentation. Dissecting the individual functions of CPS production and hmv in hvKp has been hindered by the conflation of these two properties. Although hmv requires capsular polysaccharide (CPS) biosynthesis, other cellular factors may also be required and some fitness phenotypes ascribed to CPS may be distinctly attributed to hmv. To address this challenge, we systematically identified genes that impact capsule and hmv. We generated a condensed, ordered transposon library in hypervirulent strain KPPR1, then evaluated the CPS production and hmv phenotypes of the 3,733 transposon mutants, representing 72% of all open reading frames in the genome. We employed forward and reverse genetic screens to evaluate effects of novel and known genes on CPS biosynthesis and hmv. These screens expand our understanding of core genes that coordinate CPS biosynthesis and hmv, as well as identify central metabolism genes that distinctly impact CPS biosynthesis or hmv, specifically those related to purine metabolism, pyruvate metabolism and the TCA cycle. Six representative mutants, with varying effect on CPS biosynthesis and hmv, were evaluated for their impact on CPS thickness, serum resistance, host cell association, and fitness in a murine model of disseminating pneumonia. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that hmv and CPS may serve distinct functions during pathogenesis. The integration of hmv and CPS to the metabolic status of the cell suggests that hvKp may require certain nutrients to specifically cause deep tissue infections.

scholarly journals Mitochondrial Mistranslation in Brain Provokes a Metabolic Response Which Mitigates the Age-Associated Decline in Mitochondrial Gene Expression

2021 ◽  
Vol 22 (5) ◽  
pp. 2746
Author(s):  
Dimitri Shcherbakov ◽  
Reda Juskeviciene ◽  
Adrián Cortés Sanchón ◽  
Margarita Brilkova ◽  
Hubert Rehrauer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Response ◽  
Mitochondrial Gene ◽  
Tca Cycle ◽  
Brain Mitochondria ◽  
Mitochondrial Gene Expression ◽  
Rna Seq ◽  
The Tca Cycle ◽  
Neurological Phenotype

Mitochondrial misreading, conferred by mutation V338Y in mitoribosomal protein Mrps5, in-vivo is associated with a subtle neurological phenotype. Brain mitochondria of homozygous knock-in mutant Mrps5V338Y/V338Y mice show decreased oxygen consumption and reduced ATP levels. Using a combination of unbiased RNA-Seq with untargeted metabolomics, we here demonstrate a concerted response, which alleviates the impaired functionality of OXPHOS complexes in Mrps5 mutant mice. This concerted response mitigates the age-associated decline in mitochondrial gene expression and compensates for impaired respiration by transcriptional upregulation of OXPHOS components together with anaplerotic replenishment of the TCA cycle (pyruvate, 2-ketoglutarate).

scholarly journals Accumulation of Succinyl Coenzyme A Perturbs the Methicillin-Resistant Staphylococcus aureus (MRSA) Succinylome and Is Associated with Increased Susceptibility to Beta-Lactam Antibiotics

mBio ◽  
2021 ◽  
Author(s):  
Christopher Campbell ◽  
Claire Fingleton ◽  
Merve S. Zeden ◽  
Emilio Bueno ◽  
Laura A. Gallagher ◽  
...  
Keyword(s):  
Methicillin Resistant Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Tca Cycle ◽  
Cell Wall Biosynthesis ◽  
Central Metabolism ◽  
Beta Lactam ◽  
Beta Lactam Antibiotics ◽  
Lysine Succinylation ◽  
The Tca Cycle ◽  
Increased Susceptibility

mecA -dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene, sucC , increased susceptibility to β-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome.

scholarly journals Bovine Oviduct Epithelial Cell-Derived Culture Media and Exosomes Improve Mitochondrial Health by Restoring Metabolic Flux during Pre-Implantation Development

2020 ◽  
Vol 21 (20) ◽  
pp. 7589
Author(s):  
Tabinda Sidrat ◽  
Abdul Aziz Khan ◽  
Myeon-Don Joo ◽  
Yiran Wei ◽  
Kyeong-Lim Lee ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Embryonic Development ◽  
Metabolic Flux ◽  
Culture Media ◽  
Tca Cycle ◽  
Fine Tuning ◽  
The Tca Cycle ◽  
Oviduct Epithelial Cell

Oviduct flushing is enriched by a wide variety of nutrients that guide the 3–4 days journey of pre-implantation embryo through the oviduct as it develops into a competent blastocyst (BL). However, little is known about the specific requirement and role of these nutrients that orchestrate the early stages of embryonic development. In this study, we aimed to characterize the effect of in vitro-derived bovine oviduct epithelial cell (BOECs) secretion that mimics the in vivo oviduct micro-fluid like environment, which allows successful embryonic development. In this study, the addition of an in vitro derived BOECs-condition media (CM) and its isolated exosomes (Exo) significantly enhances the quality and development of BL, while the hatching ability of BLs was found to be high (48.8%) in the BOECs-Exo supplemented group. Surprisingly, BOECs-Exo have a dynamic effect on modulating the embryonic metabolism by restoring the pyruvate flux into TCA-cycle. Our analysis reveals that Exo treatment significantly upregulates the pyruvate dehydrogenase (PDH) and glutamate dehydrogenase (GLUD1) expression, required for metabolic fine-tuning of the TCA-cycle in the developing embryos. Exo treatment increases the influx into TCA-cycle by strongly suppressing the PDH and GLUD1 upstream inhibitors, i.e., PDK4 and SIRT4. Improvement of TCA-cycle function was further accompanied by higher metabolic activity of mitochondria in BOECs-CM and Exo in vitro embryos. Our study uncovered, for the first time, the possible mechanism of BOECs-derived secretion in re-establishing the TCA-cycle flux by the utilization of available nutrients and highlighted the importance of pyruvate in supporting bovine in vitro embryonic development.

scholarly journals Evidence for reverse flux through pyruvate kinase in skeletal muscle

2009 ◽  
Vol 296 (4) ◽  
pp. E748-E757 ◽  
Author(s):  
Eunsook S. Jin ◽  
A. Dean Sherry ◽  
Craig R. Malloy
Keyword(s):  
Skeletal Muscle ◽  
Glucose Production ◽  
Tca Cycle ◽  
Hepatic Glycogen ◽  
Direct Transfer ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates ◽  
Minced Muscle ◽  
Labeling Pattern

Conversion of lactate to glucose was examined in myotubes, minced muscle tissue, and rats exposed to 2H2O or 13C-enriched substrates. Myotubes or minced skeletal muscle incubated with [U-13C3]lactate released small amounts of [1,2,3-13C3]- or [4,5,6-13C3]glucose. This labeling pattern is consistent with direct transfer from lactate to glucose without randomization in the tricarboxylic acid (TCA) cycle. After exposure of incubated muscle to 2H2O, [U-13C3]lactate, glucose, and glutamine, there was minimal release of synthesized glucose to the medium based on a low level of 2H enrichment in medium glucose but 50- to 100-fold greater 2H enrichment in glucosyl units from glycogen. The 13C enrichment pattern in glycogen from incubated skeletal muscle was consistent only with direct transfer of lactate to glucose without exchange in TCA cycle intermediates. 13C nuclear magnetic resonance (NMR) spectra of glutamate from the same tissue showed flux from lactate through pyruvate dehydrogenase but not flux through pyruvate carboxylase into the TCA cycle. Carbon from an alternative substrate for glucose production that requires metabolism through the TCA cycle, propionate, did not enter glycogen, suggesting that TCA cycle intermediates do not exchange with phospho enolpyruvate. In vivo, the 13C labeling patterns in hepatic glycogen and plasma glucose after administration of [U-13C3]lactate did not differ significantly. However, skeletal muscle glycogen was substantially enriched in [1,2,3-13C3]- and [4,5,6-13C3]glucose units that could only occur through skeletal muscle glyconeogenesis rather than glycogenesis. Lactate serves as a substrate for glyconeogenesis in vivo without exchange into symmetric intermediates of the TCA cycle.

scholarly journals Identification of Genes Induced In Vivo duringKlebsiella pneumoniae CG43 Infection

2001 ◽  
Vol 69 (11) ◽  
pp. 7140-7145 ◽  
Author(s):  
Yi-Chyi Lai ◽  
Hwei-Ling Peng ◽  
Hwan-You Chang
Keyword(s):  
Protein Folding ◽  
Klebsiella Pneumoniae ◽  
Nutrient Uptake ◽  
Capsular Polysaccharide ◽  
The Other ◽  
Deficient Mutant ◽  
Turn On ◽  
In Vivo Selection ◽  
Iron Deprivation

ABSTRACT A novel in vivo expression technology (IVET) was performed to identify Klebsiella pneumoniae CG43 genes that are specifically expressed during infection of BALB/c mice. The IVET employed a UDP glucose pyrophosphorylase (galU)-deficient mutant of K. pneumoniae which is incapable of utilizing galactose and synthesizing capsular polysaccharide, as demonstrated by its low virulence to BALB/c mice and a white nonmucoid colony morphology on MacConkey-galactose agar. By using a functionalgalU gene as the reporter, an IVE promoter could render thegalU mutant virulent while maintaining the white nonmucoid colony phenotype. A total of 20 distinct sequences were obtained through the in vivo selection. Five of them have been identified previously as virulence-associated genes in other pathogens, while another five with characterized functions are involved in regulation and transportation of nutrient uptake, biosynthesis of isoprenoids, and protein folding. No known functions have been attributed to the other 10 sequences. We have also demonstrated that 2 of the 20 IVE genes turn on under iron deprivation, whereas the expression of another five genes was found to be activated in the presence of paraquat, a superoxide generator.

scholarly journals In vivo Selection of Imipenem Resistance Among Ceftazidime-Avibactam-Resistant, Imipenem-Susceptible Klebsiella pneumoniae Isolate With KPC-33 Carbapenemase

2021 ◽  
Vol 12 ◽  
Author(s):  
Chunlei Wang ◽  
Jiankang Zhao ◽  
Zhibo Liu ◽  
Aihua Sun ◽  
Lingxiao Sun ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Clinical Significance ◽  
Capsular Polysaccharide ◽  
Selective Pressure ◽  
Dynamic Change ◽  
Wild Type ◽  
First Report ◽  
Emergence Of Resistance

We describe in vivo evolution of carbapenem and ceftazidime-avibactam resistance by analyzing four longitudinal Klebsiella pneumoniae clinical isolates from a patient with pneumonia following antimicrobial treatment. The patient had fever, cough associated with expectoration, and new infiltration was found on the chest CT. Antimicrobial susceptibility was determined, and whole genome sequencing (WGS) was performed to investigate its dynamic change of resistance phenotype. Population analysis profile was performed to investigate the population of Klebsiella pneumoniae. The infection started with a KPC-2-producing K. pneumoniae (ZRKP01, ceftazidime-avibactam-S/carbapenem-R). Then, after ceftazidime-avibactam treatment, the strain switched to D179Y mutant that is KPC-33 (ZRKP02, ceftazidime-avibactam-R/carbapenem-S), which restored carbapenem susceptibility. However, the restored carbapenem susceptibility in vivo was not stable and the subsequent use of imipenem against KPC-33-producing K. pneumoniae infection resulted in a reversion of KPC-2 producers (ZRKP03 and ZRKP04, ceftazidime-avibactam-S/carbapenem-R). Genetic analysis demonstrated that all four K. pneumoniae isolates belonged to sequence type 11and had identical capsular polysaccharide (KL47), identical porin genes, and same plasmid replicon types. Phylogenetic analysis indicated that four K. pneumoniae isolates showed a high degree of relatedness. Single nucleotide polymorphisms analysis indicated that the number of mutations observed in the KPC-33 isolate was more than in the wild-type KPC-2 isolates and the four KPC-Kp isolates evolved from a longitudinal evolution of K. pneumoniae harboring blaKPC-2 gene. This is the first report to observe the in vivo evolution of wild-type KPC-2 to KPC-33 and then the reversion to its original wild-type KPC-2. Through WGS, we demonstrated the role of selective pressure of antibiotic in the mutation and reversion of blaKPC genes, which leading to the dynamic change of KPC enzymes and the dynamic emergence of resistance to ceftazidime-avibactam and carbapenems.Statement: Recently, studies reported the emergence of ceftazidime-avibactam-resistant strains. The KPC mutations mediating ceftazidime-avibactam resistance are generally associated with the restoration of carbapenem susceptibility. However, clinical significance of this observation is unclear. In this manuscript, we demonstrate the role of selective pressure of antibiotic in the mutation and reversion of blaKPC genes, which leading to the dynamic change of KPC enzymes and the dynamic emergence of resistance to ceftazidime-avibactam and carbapenems. To the best of our knowledge, this is the first report to observe the in vivo evolution of wild-type KPC-2 to KPC-33 and then the reversion to its original wild-type KPC-2. It should be noted that understanding the clinical significance of this observation is of critical importance, and reversion to carbapenem susceptibility would not imply a potential role for carbapenems monotherapy. We hope our study will draw attention to clinicians, so that this agent can be used most effectively for the longest period of time.

scholarly journals Serum Antibody Responses against Carbapenem-Resistant Klebsiella pneumoniae in Infected Patients

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Kasturi Banerjee ◽  
Michael P. Motley ◽  
Elizabeth Diago-Navarro ◽  
Bettina C. Fries
Keyword(s):  
Klebsiella Pneumoniae ◽  
Capsular Polysaccharide ◽  
Serum Antibody ◽  
Antibody Responses ◽  
Capsular Polysaccharides ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Reactive Properties

ABSTRACT Capsular polysaccharide (CPS) heterogeneity within carbapenem-resistant Klebsiella pneumoniae (CR-Kp) strain sequence type 258 (ST258) must be considered when developing CPS-based vaccines. Here, we sought to characterize CPS-specific antibody responses elicited by CR-Kp-infected patients. Plasma and bacterial isolates were collected from 33 hospital patients with positive CR-Kp cultures. Isolate capsules were typed by wzi sequencing. Reactivity and measures of efficacy of patient antibodies were studied against 3 prevalent CR-Kp CPS types (wzi29, wzi154, and wzi50). High IgG titers against wzi154 and wzi50 CPS were documented in 79% of infected patients. Patient-derived (PD) IgGs agglutinated CR-Kp and limited growth better than naive IgG and promoted phagocytosis of strains across the serotype isolated from their donors. Additionally, poly-IgG from wzi50 and wzi154 patients promoted phagocytosis of nonconcordant CR-Kp serotypes. Such effects were lost when poly-IgG was depleted of CPS-specific IgG. Additionally, mice infected with wzi50, wzi154, and wzi29 CR-Kp strains preopsonized with wzi50 patient-derived IgG exhibited lower lung CFU than controls. Depletion of wzi50 antibodies (Abs) reversed this effect in wzi50 and wzi154 infections, whereas wzi154 Ab depletion reduced poly-IgG efficacy against wzi29 CR-Kp. We are the first to report cross-reactive properties of CPS-specific Abs from CR-Kp patients through both in vitro and in vivo models. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae is a rapidly emerging public health threat that can cause fatal infections in up to 50% of affected patients. Due to its resistance to nearly all antimicrobials, development of alternate therapies like antibodies and vaccines is urgently needed. Capsular polysaccharides constitute important targets, as they are crucial for Klebsiella pneumoniae pathogenesis. Capsular polysaccharides are very diverse and, therefore, studying the host’s capsule-type specific antibodies is crucial to develop effective anti-CPS immunotherapies. In this study, we are the first to characterize humoral responses in infected patients against carbapenem-resistant Klebsiella pneumoniae expressing different wzi capsule types. This study is the first to report the efficacy of cross-reactive properties of CPS-specific Abs in both in vitro and in vivo models.

scholarly journals Massively parallel fitness profiling reveals multiple novel enzymes inPseudomonas putidalysine metabolism

2018 ◽  
Author(s):  
Mitchell G. Thompson ◽  
Jacquelyn M. Blake-Hedges ◽  
Pablo Cruz-Morales ◽  
Jesus F. Barajas ◽  
Samuel C. Curran ◽  
...  
Keyword(s):  
Tca Cycle ◽  
Central Metabolism ◽  
It Use ◽  
Catabolic Genes ◽  
Genome Wide ◽  
The Tca Cycle ◽  
A Genome ◽  
Domains Of Life ◽  
Lysine Degradation ◽  
Lysine Metabolism

AbstractDespite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism inPseudomonas putidaremain unresolved. To establish these biochemical links, we leveraged Random Barcode Transposon Sequencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes in both L- and D-lysine metabolism. We first describe three pathway enzymes that catabolize L-2-aminoadipate (L-2AA) to 2-ketoglutarate (2KG), connecting D-lysine to the TCA cycle. One of these enzymes, PP_5260, contains a DUF1338 domain, a family with no previously described biological function. Our work also identified the recently described CoA independent route of L-lysine degradation that metabolizes to succinate. We expanded on previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-oxoacid selectivity. Proteomics of select pathway enzymes revealed that expression of catabolic genes is highly sensitive to particular pathway metabolites, implying intensive local and global regulation. This work demonstrates the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria, as well as a powerful tool for validating previous research.ImportanceP. putidalysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, connecting lysine catabolism to central metabolism inP. putidaremained undefined. Herein we use Random Barcode Transposon Sequencing to fill in the gaps of lysine metabolism inP. putida. We describe a route of 2-oxoadipate (2OA) catabolism in bacteria, which utilizes DUF1338 containing protein PP_5260. Despite its prevalence in many domains of life, DUF1338 containing proteins had no known biochemical function. We demonstrate PP_5260 is a metalloenzyme which catalyzes an unusual 2OA to D-2HG decarboxylation. Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results, and expand the understanding of glutarate hydroxylase CsiD by showing can it use either 2OA or 2KG as a cosubstrate. Our work demonstrates biological novelty can be rapidly identified using unbiased experimental genetics, and that RB-TnSeq can be used to rapidly validate previous results.

scholarly journals In vivo assessment of glutamine anaplerosis into the TCA cycle in human pre-malignant and malignant clonal plasma cells

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Wilson I. Gonsalves ◽  
Jin Sung Jang ◽  
Erik Jessen ◽  
Taro Hitosugi ◽  
Laura A. Evans ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Bone Marrow Aspiration ◽  
Tca Cycle ◽  
Link Type ◽  
The Tca Cycle ◽  
Bone Marrow Plasma

Abstract Background Overexpression of c-Myc is required for the progression of pre-malignant plasma cells in monoclonal gammopathy of undetermined significance (MGUS) to malignant plasma cells in multiple myeloma (MM). c-Myc also increases glutamine anaplerosis into the tricarboxylic acid (TCA) cycle within cancer cells. Whether increased glutamine anaplerosis is associated with the progression of pre-malignant to malignant plasma cells is unknown. Methods Human volunteers (N = 7) and patients with MGUS (N = 11) and MM (N = 12) were prospectively recruited to undergo an intravenous infusion of 13C-labeled glutamine followed by a bone marrow aspiration to obtain bone marrow cells and plasma. Results Despite notable heterogeneity, stable isotope-resolved metabolomics (SIRM) revealed that the mean 13C-labeled glutamine anaplerosis into the TCA cycle was higher in malignant compared to pre-malignant bone marrow plasma cells relative to the remainder of their paired bone marrow mononuclear cells. RNA sequencing demonstrated a higher relative mRNA expression of c-Myc and glutamine transporters such as ASCT2 and SN2 in malignant compared to pre-malignant bone marrow plasma cells. Finally, higher quantitative levels of TCA cycle intermediates in the bone marrow plasma differentiated MM from MGUS patients. Conclusion Measurement of the in vivo activity of glutamine anaplerosis into the TCA cycle provides novel insight into the metabolic changes associated with the transformation of pre-malignant plasma cells in MGUS to malignant plasma cells in MM. Trial registration NCT03384108 and NCT03119883

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