Mycobacterium tuberculosis curli pili (MTP) is associated with significant host metabolic pathways in an A549 epithelial cell infection model and contributes to the pathogenicity of Mycobacterium tuberculosis.

INTRODUCTION: A clear understanding of the metabolome of Mycobacterium tuberculosis and its target host cell during infection is fundamental for the development of novel diagnostic tools, effective drugs and vaccines required to combat tuberculosis. The surface-located Mycobacterium tuberculosis curli pili (MTP) adhesin forms initial contact with the host cell and is therefore important for the establishment of infection. OBJECTIVE: The aim of this investigation was to determine the role of MTP in modulating pathogen and host metabolic pathways in A549 epithelial cells infected with MTP proficient and deficient strains of M. tuberculosis. METHODS: Uninfected A549 epithelial cells, and those infected with M. tuberculosis V9124 wild-type strain, Δmtp and the mtp-complemented strains, were subjected to metabolite extraction, two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS) and bioinformatic analyses. Univariate and multivariate statistical tests were used to identify metabolites that were significantly differentially produced in the WT-infected and ∆mtp-infected A549 epithelial cell models, comparatively. RESULTS: A total of 46 metabolites occurred in significantly lower relative concentrations in the Δmtp-infected cells, indicating a reduction in nucleic acid synthesis, amino acid metabolism, glutathione metabolism, oxidative stress, lipid metabolism and peptidoglycan, compared to those cells infected with the WT strain. CONCLUSION: The absence of MTP was associated with significant changes to the host metabolome, suggesting that this adhesin is an important contributor to the pathogenicity of M. tuberculosis, and supports previous findings of its potential as a suitable drug, vaccine and diagnostic target.

Mycobacterium tuberculosis curli pili (MTP) deficiency is associated with alterations in cell wall biogenesis, fatty acid metabolism and amino acid synthesis.

INTRODUCTION: In an effort to find alternative therapeutic interventions to combat tuberculosis, a better understanding of the pathophysiology of Mycobacterium tuberculosis is required. The Mycobacterium tuberculosis curli pili (MTP) adhesin, present on the surface of this pathogen, has previously been shown using functional genomics and global transcriptomics, to play an important role in establishing infection, bacterial aggregation, and modulating host response in vitro and in vivo. OBJECTIVE: This investigation aimed to determine the role of MTP in modulating the metabolism of M. tuberculosis, using mtp gene-knockout mutant and complemented strains. METHODS: Untargeted two-dimensional gas chromatography time-of-flight mass spectrometry, and bioinformatic analyses, were used to identify significant differences in the metabolite profiles among the wild-type, ∆mtp mutant and mtp-complemented strains, and validated with results generated by real-time quantitative PCR. RESULTS: A total of 28 metabolites were found to be significantly altered when comparing the ∆mtp mutant and the wild-type strains indicating a decreased utilisation of metabolites in cell wall biogenesis, a reduced efficiency in the breakdown of fatty acids, and decreased amino acid biosynthesis in the former strain. Comparison of the wild-type to mtp-complement, and ∆mtp to mtp-complemented strains revealed 10 and 16 metabolite differences, respectively. Real-time quantitative PCR results supported the metabolomics findings. Complementation of the ∆mtp mutant resulted in a partial restoration of MTP function. CONCLUSION: The lack of the MTP adhesin resulted in various bacterial cell wall alterations and related metabolic changes. This study highlights the importance of MTP as a virulence factor and further substantiates its potential use as a suitable biomarker for the development of diagnostic tools and intervention therapeutics against TB.

Abnormal tricarboxylic acid cycle metabolites in isovaleric acidaemia.

BACKGROUND: Although a number of abnormal diagnostic metabolites have previously been described in the urine of patients with isovaleric acidaemia (IVA), they do not fully explain the clinical symptoms associated with this disease. METHODS: On the basis of our current understanding of the TCA cycle and IVA, we predicted a number of abnormal methylated TCA cycle metabolites, initiated by methylsuccinic acid. We subsequently obtained characteristic gas chromatography-mass spectrometry elution times and mass spectra of the chemically synthesized predicted compounds and screened the urine of 6 IVA patients and 24 age-matched controls. Further proof for our findings was generated from a series of in vitro enzyme reactions using the chemically synthesized standards as substrates to their respective TCA cycle enzymes. RESULTS: Apart from the previously described methylsuccinic and methylfumaric acid, 3-methylmalic acid, (2R,3S)- and (2R,3R)-methylcitric acid and 2-methyl-cis-aconitic acid were detected in the urine of all 6 IVA patients in increased amounts. Additionally, although not directly determined, the in vitro enzyme reaction using of 3-methylmalic acid and malate dehydrogenase, in conjunction with the detection of 2-ketobutyric acid in the urine of all 6 IVA patients, strongly suggests an additional synthesis of 3-methyloxaloacetic acid by the same cycle. CONCLUSION: Not only do these newly identified metabolites serve as additional diagnostic markers to those previously identified in IVA, but due to the structural arrangements of the (2R,3R)-methylcitric acid and 2-methyl-cis-aconitinic acid-derived 2-methylisocitric acid, inhibition of normal TCA cycle metabolism results at citrate synthase and isocitrate dehydrogenase, respectively. SYNOPSIS: Methylsuccinic acid acts as the initiating substrate to a series of abnormal, potentially harmful, methylated tricarboxylic acid cycle metabolites in isovaleric acidaemia.

Protein kinase C-delta (PKCδ), a marker of inflammation and tuberculosis disease progression in humans, is important for optimal macrophage killing effector functions and survival in mice.

We previously demonstrated that protein kinase C-δ (PKCδ) is critical for immunity against Listeria monocytogenes, Leishmania major, and Candida albicans infection in mice. However, the functional relevance of PKCδ during Mycobacterium tuberculosis (Mtb) infection is unknown. PKCδ was significantly upregulated in whole blood of patients with active tuberculosis (TB) disease. Lung proteomics further revealed that PKCδ was highly abundant in the necrotic and cavitory regions of TB granulomas in multidrug-resistant human participants. In murine Mtb infection studies, PKCδ-/- mice were highly susceptible to tuberculosis with increased mortality, weight loss, exacerbated lung pathology, uncontrolled proinflammatory cytokine responses, and increased mycobacterial burdens. Moreover, these mice displayed a significant reduction in alveolar macrophages, dendritic cells, and decreased accumulation of lipid bodies (lungs and macrophages) and serum fatty acids. Furthermore, a peptide inhibitor of PKCδ in wild-type mice mirrored lung inflammation identical to infected PKCδ-/- mice. Mechanistically, increased bacterial growth in macrophages from PKCδ-/- mice was associated with a decline in killing effector functions independent of phagosome maturation and autophagy. Taken together, these data suggest that PKCδ is a marker of inflammation during active TB disease in humans and required for optimal macrophage killing effector functions and host protection during Mtb infection in mice.

Protein kinase C-delta (PKCδ), a marker of inflammation and tuberculosis disease progression in humans, is important for optimal macrophage killing effector functions and survival in mice.

This corrects the article DOI: 10.1038/mi.2017.68.

Amino-acid depletion induced by abnormal amino-acid conjugation and protein restriction in isovaleric acidemia.

BACKGROUND: Previously, we detected 19 'new' amino-acid conjugates in the urine of patients with isovaleric acidemia. There is currently a poor understanding of the relationship between the clinical symptoms and the excreted metabolites occurring in these patients, owing to insufficient metabolite characterization and quantification. Consequently, controversial treatment protocols exist, particularly pertaining to dietary protein restriction. OBJECTIVE: To determine the effect of the previously identified amino-acid conjugates and conventional dietary protein restriction therapy, on the free amino-acid concentrations in isovaleric acidemia patients, to better explain the clinical symptoms and develop more effective therapy. DESIGN: Free amino-acid quantification via liquid chromatography mass spectrometry (LC-MS-MS) was performed on pre- and post-treatment urine or serum samples collected from six isovaleric acidemia patients, previously investigated for the presence of new induced N-isovaleryl and N-acetyl-amino-acid conjugates. RESULTS: Depleted amino-acid concentrations were detected in varying degrees in all six patients and did not recover after conventional treatment. CONCLUSIONS: The 19 potentially toxic metabolites previously identified and the consequent amino-acid depletions detected in this study, may explain many of the clinical symptoms associated with isovaleric acidemia. Furthermore, the occurrence of amino-acid depletions in these patients, steers away from the controversial dietary protein restriction treatment protocols, and towards dietary leucine restriction alone with essential amino-acid supplementation, in combination with glycine and L-carnitine supplementation.

New sputum metabolite markers implicating adaptations of the host to Mycobacterium tuberculosis, and vice versa.

In this study, a metabolomics research approach was used to identify new tuberculosis (TB) markers from sputum, in an attempt to better characterise the disease as well as the metabolic response of the host to Mycobacterium tuberculosis infection. After GCxGC-TOFMS analyses, various multivariate and univariate statistical methods were implemented to identify those compounds best describing the variation between the TB-positive and TB-negative patient groups. The interpretation of these new metabolite markers led to a number of new hypotheses, including: 1) support of the previously proposed citramalate cycle in M. tuberculosis; 2) the interaction of this cycle with an up-regulated glyoxylate cycle during pulmonary M. tuberculosis infection; 3) the increased utilisation of fatty acids and glutamate as alternative carbon sources by M. tuberculosis during pulmonary infection; 4) an alternative mechanism by which the host produces hydrogen peroxide via glucose oxidation, in order to eliminate the bacterial infection; 5) inhibition of the ETC due to pronounced oxidative stress during an active TB disease state, resulting in increased concentrations of various neurotransmitters and other metabolites previously associated with an inborn error of metabolism (MADD/GA type II); and 6) elevated concentrations of neurotransmitters associated with a number of previously described symptoms of TB.