Legionella shows a diverse secondary metabolism dependent on a broad spectrum Sfp-type phosphopantetheinyl transferase

Several members of the genus Legionella cause Legionnaires' disease, a potentially debilitating form of pneumonia. Studies frequently focus on the abundant number of virulence factors present in this genus. However, what is often overlooked is the role of secondary metabolites from Legionella. Following whole genome sequencing, we assembled and annotated the Legionella parisiensis DSM 19216 genome. Together with 14 other members of the Legionella, we performed comparative genomics and analysed the secondary metabolite potential of each strain. We found that Legionella contains a huge variety of biosynthetic gene clusters (BGCs) that are potentially making a significant number of novel natural products with undefined function. Surprisingly, only a single Sfp-like phosphopantetheinyl transferase is found in all Legionella strains analyzed that might be responsible for the activation of all carrier proteins in primary (fatty acid biosynthesis) and secondary metabolism (polyketide and non-ribosomal peptide synthesis). Using conserved active site motifs, we predict some novel compounds that are probably involved in cell-cell communication, differing to known communication systems. We identify several gene clusters, which may represent novel signaling mechanisms and demonstrate the natural product potential of Legionella

Legionella shows a diverse secondary metabolism dependent on a broad spectrum Sfp-type phosphopantetheinyl transferase

Several members of the genus Legionella cause Legionnaires' disease, a potentially debilitating form of pneumonia. Studies frequently focus on the abundant number of virulence factors present in this genus. However, what is often overlooked is the role of secondary metabolites from Legionella. Following whole genome sequencing, we assembled and annotated the Legionella parisiensis DSM 19216 genome. Together with 14 other members of the Legionella, we performed comparative genomics and analysed the secondary metabolite potential of each strain. We found that Legionella contains a huge variety of biosynthetic gene clusters (BGCs) that are potentially making a significant number of novel natural products with undefined function. Surprisingly, only a single Sfp-like phosphopantetheinyl transferase is found in all Legionella strains analyzed that might be responsible for the activation of all carrier proteins in primary (fatty acid biosynthesis) and secondary metabolism (polyketide and non-ribosomal peptide synthesis). Using conserved active site motifs, we predict some novel compounds that are probably involved in cell-cell communication, differing to known communication systems. We identify several gene clusters, which may represent novel signaling mechanisms and demonstrate the natural product potential of Legionella

The Endogenous tryptophan metabolite and NAD⁺ precursor quinolinic acid confers resistance of gliomas to oxidative stress

Quinolinic acid is a product of tryptophan degradation and may serve as a precursor for NAD⁺, an important enzymatic cofactor for enzymes such as the DNA repair protein PARP. Pathologic accumulation of quinolinic acid has been found in neurodegenerative disorders including Alzheimer and Huntington disease, where it is thought to be toxic for neurons by activating the N-methyl-D-aspartate (NMDA) receptor and inducing excitotoxicity. Although many tumors including gliomas constitutively catabolize tryptophan, it is unclear whether quinolinic acid is produced in gliomas and whether it is involved in tumor progression. Here, we show that quinolinic acid accumulated in human gliomas andwas associated with a malignant phenotype. Quinolinic acid was produced by microglial cells, as expression of the quinolinic acid-producing enzyme 3- hydroxyanthranilate oxygenase (3-HAO) was confined to microglia in glioma tissue. Human malignant glioma cells, but not nonneoplastic astrocytes, expressed quinolinic acid phosphoribosyltransferase (QPRT) to use quinolinic acid for NAD⁺ synthesis and prevent apoptosis when de novo NAD⁺ synthesis was blocked. Oxidative stress, temozolomide, and irradiation induced QPRT in glioma cells. QPRT expression increased with malignancy. In recurrent glioblastomas after radiochemotherapy, QPRT expression was associated with a poor prognosis in two independent datasets. Our data indicate that neoplastic transformation in astrocytes is associated with a QPRT-mediated switch in NAD⁺ metabolism by exploiting microglia-derived quinolinic acid as an alternative source of replenishing intracellular NAD⁺ pools. The elevated levels of QPRT expression increase resistance to oxidative stress induced by radiochemotherapy, conferring a poorer prognosis. These findings have implications for therapeutic approaches inducing intracellular NAD⁺ depletion, such as alkylating agents or direct NAD⁺ synthesis inhibitors, and identify QPRT as a potential therapeutic target in malignant gliomas.10 page(s

METABOLIC PATHWAYS

The kynurenine pathway (KP) is the principal route of L-Tryptophan (TRP) catabolism leading to the production of kynurenine (KYN), the neuroprotectants, kynurenic acid (KYNA) and picolinic acid (PIC), and the excitotoxic neurotoxin, quinolinic acid (QUIN). The enzymes indoleamine 2,3-dioxygenase-1 (IDO-1), indoleamine 2, 3-dioxygenase-2 (IDO-2) and tryptophan 2,3-dioxygenase (TDO-2) initiate the first step of the KP. Downstream enzymes include kynureninase (KYNU), 3-hydroxyanthranilate 3,4-dioxygenase (3-HAAO), kynurenine hydroxylase (KMO) and 2-amino-3-carboxymuconate semialdehyde decarboxylase (ACMSD). Kynurenine aminotransferase-I (KAT-I) is one of the enzymes responsible for synthesising KYNA. Mounting evidence directly implicates that IDO-1 induction in various tumours is a crucial mechanism facilitating tumour immune evasion and persistence. However, the involvement of the downstream machinery of the KP in brain tumour progression remains unexplored. A complete characterisation of the KP in brain tumours and the role of the KP in maintaining homeostasis between neuroprotection and neurodegeneration in glioma has not yet been investigated. Here we report the first comprehensive characterisation of the KP in cultured human glioma cells and GBM patient plasma. Our qRT-PCR data revealed that interferon-gamma (IFN-γ) (100 IU/ml) stimulation significantly potentiated the expression of IDO-1 IDO-2, KYNU, 3-HAAO, KMO and significantly down-regulated ACMSD and KAT-I expression in cultured human glioma cells. HPLC analysis revealed that IFN-γ stimulation significantly increased KP activity (KYN/TRP ratio), and significantly lowered the KYNA/KYN neuroprotective ratio in human cultured glioma cells. Our HPLC and GCMS data revealed that KP activation was significantly higher and the concentrations of TRP, KYNA, QUIN and PIC and the KYNA/KYN ratio were significantly lower in GBM patient plasma (n = 18) compared to controls. These results provide further evidence for the involvement of the KP in glioma pathophysiology and highlights a potential role of KP products as novel and highly attractive therapeutic targets to evaluate for the treatment of brain tumours