Regulation of mycobacterial infection by macrophage Gch1 and tetrahydrobiopterin.

Inducible nitric oxide synthase (iNOS) plays a crucial role in controlling growth of Mycobacterium tuberculosis (M.tb), presumably via nitric oxide (NO) mediated killing. Here we show that leukocyte-specific deficiency of NO production, through targeted loss of the iNOS cofactor tetrahydrobiopterin (BH4), results in enhanced control of M.tb infection; by contrast, loss of iNOS renders mice susceptible to M.tb. By comparing two complementary NO-deficient models, Nos2-/- mice and BH4 deficient Gch1fl/flTie2cre mice, we uncover NO-independent mechanisms of anti-mycobacterial immunity. In both murine and human leukocytes, decreased Gch1 expression correlates with enhanced cell-intrinsic control of mycobacterial infection in vitro. Gene expression analysis reveals that Gch1 deficient macrophages have altered inflammatory response, lysosomal function, cell survival and cellular metabolism, thereby enhancing the control of bacterial infection. Our data thus highlight the importance of the NO-independent functions of Nos2 and Gch1 in mycobacterial control.

Dictyostelium as a Model to Assess Site-Specific ADP-Ribosylation Events.

The amoeba Dictyostelium discoideum is a single-cell organism that can undergo a simple developmental program, making it an excellent model to study the molecular mechanisms of cell motility, signal transduction, and cell-type differentiation. A variety of human genes that are absent or show limited conservation in other invertebrate models have been identified in this organism. This includes ADP-ribosyltransferases, also known as poly-ADP-ribose polymerases (PARPs), a family of proteins that catalyze the addition of single or poly-ADP-ribose moieties onto target proteins. The genetic tractability of Dictyostelium and its relatively simple genome structure makes it possible to disrupt PARP gene combinations, in addition to specific ADP-ribosylation sites at endogenous loci. Together, this makes Dictyostelium an attractive model to assess how ADP-ribosylation regulates a variety of cellular processes including DNA repair, transcription, and cell-type specification. Here we describe a range of techniques to study ADP-ribosylation in Dictyostelium, including analysis of ADP-ribosylation events in vitro and in vivo, in addition to approaches to assess the functional roles of this modification in vivo.

Redundancy between nucleases required for homologous recombination promotes PARP inhibitor resistance in the eukaryotic model organism Dictyostelium.

ADP-ribosyltransferases promote repair of DNA single strand breaks and disruption of this pathway by Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) is toxic to cells with defects in homologous recombination (HR). Here, we show that this relationship is conserved in the simple eukaryote Dictyostelium and exploit this organism to define mechanisms that drive resistance of the HR-deficient cells to PARPi. Dictyostelium cells disrupted in exonuclease I, a critical factor for HR, are sensitive to PARPi. Deletion of exo1 prevents the accumulation of Rad51 in chromatin induced by PARPi, resulting in DNA damage being channelled through repair by non-homologous end-joining (NHEJ). Inactivation of NHEJ supresses the sensitivity of exo1- cells to PARPi, indicating this pathway drives synthetic lethality and that in its absence alternative repair mechanisms promote cell survival. This resistance is independent of alternate-NHEJ and is instead achieved by re-activation of HR. Moreover, inhibitors of Mre11 restore sensitivity of dnapkcs-exo1- cells to PARPi, indicating redundancy between nucleases that initiate HR can drive PARPi resistance. These data inform on mechanism of PARPi resistance in HR-deficient cells and present Dictyostelium as a convenient genetic model to characterize these pathways.