CsoR is a novel Mycobacterium tuberculosis copper-sensing transcriptional regulator

Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. Here, we identify a new copper-specific repressor (CsoR) of a copper-sensitive operon (cso) in Mycobacterium tuberculosis (Mtb) that is representative of a large, previously uncharacterized family of proteins (DUF156). Electronic and X-ray absorption spectroscopies reveal that CsoR binds a single-monomer mole equivalent of Cu(I) to form a trigonally coordinated (S(2)N) Cu(I) complex. The 2.6-A crystal structure of copper-loaded CsoR shows a homodimeric antiparallel four-helix bundle architecture that represents a novel DNA-binding fold. The Cu(I) is coordinated by Cys36, Cys65' and His61' in a subunit bridging site. Cu(I) binding negatively regulates the binding of CsoR to a DNA fragment encompassing the operator-promoter region of the Mtb cso operon; this results in derepression of the operon in Mtb and the heterologous host Mycobacterium smegmatis. Substitution of Cys36 or His61 with alanine abolishes Cu(I)- and CsoR-dependent regulation in vivo and in vitro. Potential roles of CsoR in Mtb pathogenesis are discussed.     

GarA is an essential regulator of metabolism in Mycobacterium tuberculosis

Alpha‐ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α‐ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non‐pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient‐dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways.     

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages

Mutations in the leucine‐rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol‐3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2‐dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.     

ESX‐1‐induced apoptosis is involved in cell‐to‐cell spread of Mycobacterium tuberculosis

Apoptosis modulation is a procedure amply utilized by intracellular pathogens to favour the outcome of the infection. Nevertheless, the role of apoptosis during infection with Mycobacterium tuberculosis, the causative agent of human tuberculosis, is subject of an intense debate and still remains unclear. In this work, we describe that apoptosis induction in host cells is clearly restricted to virulent M. tuberculosis strains, and is associated with the capacity of the mycobacteria to secrete the 6 kDa early secreted antigenic target ESAT‐6 bothunder in vitro and in vivo conditions. Remarkably, only apoptosis‐inducing strains are able to propagate infection into new cells, suggesting that apoptosis is used by M. tuberculosis as a colonization mechanism. Finally, we demonstrate that in vitro modulation of apoptosis affects mycobacterial cell‐to‐cell spread capacity, establishing an unambiguous relationship between apoptosis and propagation of M. tuberculosis. Our data further indicate that BCG and MTBVAC vaccines are inefficient in inducing apoptosis and colonizing new cells, correlating with the strong attenuation profile of these strains previously observed in vitro and in vivo.     

Mycobacterium tuberculosis KatG is a peroxynitritase

Mycobacterium tuberculosis resides within the highly oxidative environment of the human macrophage and previous reports have indicated that these mycobacteria are susceptible to reactive nitrogen intermediates including peroxynitrite. This work provides evidence that the Mycobacterium tuberculosis hemoprotein KatG acts as an efficient peroxynitritase exhibiting a kapp of 1.4 x 10(5) M-1s-1 for peroxynitrite decomposition at pH 7.4 and 37 degrees C. The ability of KatG to act as a peroxynitritase adds to its growing list of enzymatic activities and may in part explain the ability of Mycobacterium tuberculosis to persist in macrophages.     

Protein splicing of SufB is crucial for the functionality of the Mycobacterium tuberculosis SUF machinery

The SufBCD complex is an essential component of the SUF machinery of [Fe-S] cluster biogenesis in many organisms. We show here that in Mycobacterium tuberculosis the formation of this complex is dependent on the protein splicing of SufB, suggesting that this process is a potential new target for antituberculous drugs.     

Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells

The survival and persistence of Mycobacterium tuberculosis depends on its capacity to manipulate multiple host defense pathways, including the ability to actively inhibit the death by apoptosis of infected host cells. The genetic basis for this anti-apoptotic activity and its implication for mycobacterial virulence have not been demonstrated or elucidated. Using a novel gain-of-function genetic screen, we demonstrated that inhibition of infection-induced apoptosis of macrophages is controlled by multiple genetic loci in M. tuberculosis. Characterization of one of these loci in detail revealed that the anti-apoptosis activity was attributable to the type I NADH-dehydrogenase of M. tuberculosis, and was mainly due to the subunit of this multicomponent complex encoded by the nuoG gene. Expression of M. tuberculosis nuoG in nonpathogenic mycobacteria endowed them with the ability to inhibit apoptosis of infected human or mouse macrophages, and increased their virulence in a SCID mouse model. Conversely, deletion of nuoG in M. tuberculosis ablated its ability to inhibit macrophage apoptosis and significantly reduced its virulence in mice. These results identify a key component of the genetic basis for an important virulence trait of M. tuberculosis and support a direct causal relationship between virulence of pathogenic mycobacteria and their ability to inhibit macrophage apoptosis.     

Functional studies of the Mycobacterium tuberculosis iron-dependent regulator

The iron-dependent regulator (IdeR) protein in Mycobacterium tuberculosis, and its better characterized homologue, the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae, are iron-dependent regulatory proteins that control gene expression in response to iron availability in bacteria. IdeR regulates several genes required for iron uptake and storage including those involved in the synthesis of transition metal chelators called siderophores that are linked to the M. tuberculosis virulence. In this study, the metal ion and binding affinities for IdeR binding to an fxbA operator duplex DNA were estimated using fluorescence assays. The Fe(2+), Co(2+), and Ni(2+) affinities of the two metal ion binding sites in IdeR that are involved in the activation of the regulator DNA binding process in vitro were independently estimated. Binding to the two metal ion binding sites is apparently cooperative and the two affinities differ significantly. Occupation of the first metal ion binding site causes dimerization of IdeR, and the metal ion affinity is about 4 microM for Ni(2+) and much less for Fe(2+) and Co(2+). Binding of the second metal ion fully activates IdeR for binding to the fxbA operator. The equilibrium metal ion dissociation constants for IdeR-fxbA operator binding are approximately 9 microM for Fe(2+), 13 microM for Ni(2+), and 23 microM for Co(2+). Interestingly, the natural IdeR cofactor, Fe(2+), shows high affinities toward both binding sites. These results provide insight into the possible roles for each metal binding site in IdeR activation.     

Grb10 interacts with Bim L and inhibits apoptosis

Bim is a proapoptotic member of the Bcl-2 family and is primarily involved in the regulation of the intrinsic apoptotic pathway. However, the detail of regulation of Bim’s proapoptotic activity has not been clarified yet. Using Bim L as bait, we screened a human fetal cDNA library for interacting proteins and identified Grb10 as an interactor. This interaction was verified by co-immunoprecipitation and intracellular co-localization studies. The potential segment of Bim L that binds Grb10 was identified via a yeast mating test. Grb10 interacted with the DBD (dynein binding domain) of Bim and inhibited apoptosis triggered by overexpression of DBD containing Bim isoforms. The putative phosphorylation sites on DBD of Bim play a role for the anti-proapoptotic activity of Grb10. Our results suggest that Grb10 interacts with Bim L and inhibits its proapoptotic activity in a phosphorylation-dependant manner.     

Mycobacterium tuberculosis carbon and nitrogen metabolic fluxes

Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens causing tuberculosis (TB), a devastating infectious disease responsible for the highest human mortality and morbidity. The emergence of drug-resistant strains of the pathogen has increased the burden of TB tremendously and new therapeutics to overcome the problem of drug resistance are urgently needed. Metabolism of Mtb and its interactions with the host is important for its survival and virulence; this is an important topic of research where there is growing interest in developing new therapies and drugs that target these interactions and metabolism of the pathogen during infection. Mtb adapts its metabolism in its intracellular niche and acquires multiple nutrient sources from the host cell. Carbon metabolic pathways and fluxes of Mtb has been extensively researched for over a decade and is well-defined. Recently, there has been investigations and efforts to measure metabolism of nitrogen, which is another important nutrient for Mtb during infection. This review discusses our current understanding of the central carbon and nitrogen metabolism, and metabolic fluxes that are important for the survival of the TB pathogen.     

Mycobacterium tuberculosis oriC sequestration by MtrA response regulator

The regulators of Mycobacterium tuberculosis DNA replication are largely unknown. Here, we demonstrate that in synchronously replicating M. tuberculosis, MtrA access to origin of replication (oriC) is enriched in the post‐replication (D) period. The increased oriC binding results from elevated MtrA phosphorylation (MtrA～P) as evidenced by reduced expression of dnaN, dnaA and increased expression of select cell division targets. Overproduction of gain‐of‐function MtrA_Y102C advanced the MtrA oriC access to the C period, reduced dnaA and dnaN expression, interfered with replication synchrony and compromised cell division. Overproduction of wild‐type (MtrA+) or phosphorylation‐defective MtrA_D56N did not promote oriC access in the C period, nor affected cell cycle progression. MtrA interacts with DnaA signaling a possibility that DnaA helps load MtrA on oriC. Therefore, oriC sequestration by MtrA～P in the D period may normally serve to prevent untimely initiations and that DnaA–MtrA interactions may facilitate regulated oriC replication. Finally, despite the near sequence identity of MtrA in M. smegmatis and M. tuberculosis, the M. smegmatis oriC is not MtrA‐target. We conclude that M. tuberculosis oriC has evolved to be regulated by MtrA and that cell cycle progression in this organisms are governed, at least in part, by oscillations in the MtrA～P levels.     

CTL-mediated killing of intracellular Mycobacterium tuberculosis is independent of target cell nuclear apoptosis

Two subsets of human CTL have been defined based upon phenotype and function: CD4(-) CD8(-) double-negative (DN) CTL lyse susceptible targets via Fas-Fas ligand interaction and CD8(+) CTL via the granule exocytosis pathway. CD8(+) CTL, but not DN CTL, can mediate an antimicrobial activity against Mycobacterium tuberculosis-infected target cells that is dependent on cytotoxic granules that contain granulysin. We investigated the role of nuclear apoptosis for the antimicrobial effector function of CD1-restricted CTL using the caspase inhibitor N:-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. We found that DN CTL-induced target cell lysis was completely dependent on caspase activation, whereas the cytolytic activity of CD8(+) CTL was caspase independent. However, both DN and CD8(+) CTL-induced nuclear apoptosis required caspase activation. More important, the antimicrobial effector function of CD8(+) CTL was not diminished by inhibition of caspase activity. These data indicate that target cell nuclear apoptosis is not a requirement for CTL-mediated killing of intracellular M. tuberculosis.     

REDD2-mediated inhibition of mTOR promotes dendrite retraction induced by axonal injury

Dendritic defects occur in neurodegenerative diseases accompanied by axonopathy, yet the mechanisms that regulate these pathologic changes are poorly understood. Using Thy1-YFPH mice subjected to optic nerve axotomy, we demonstrate early retraction of retinal ganglion cell (RGC) dendrites and selective loss of mammalian target of rapamycin (mTOR) activity, which precede soma loss. Axonal injury triggered rapid upregulation of the stress-induced protein REDD2 (regulated in development and DNA damage response 2), a potent inhibitor of mTOR. Short interfering RNA-mediated REDD2 knockdown restored mTOR activity and rescued dendritic length, area and branch complexity in a rapamycin-dependent manner. Whole-cell recordings demonstrated that REDD2 depletion leading to mTOR activation in RGCs restored their light response properties. Lastly, we show that REDD2-dependent mTOR activity extended RGC survival following axonal damage. These results indicate that injury-induced stress leads to REDD2 upregulation, mTOR inhibition and dendrite pathology causing neuronal dysfunction and subsequent cell death.During normal neural development there is selective elimination of dendritic and axonal branches without loss of the neuron itself.¹ This developmental pruning refines neuronal processes and ensures precise connectivity. Most of our current knowledge about structural changes in dendrites stems from studies of dendritic remodeling during development.^2,3 In contrast, little is known about how dendritic arbors are affected by trauma or disease in the adult central nervous system (CNS). Defects in dendritic arborization and connectivity are being recognized as one of the first stages of neurodegeneration. Indeed, dendritic abnormalities and loss of synapses have been reported in neuropsychiatric disorders such as schizophrenia and depression, as well as in neurodegenerative conditions including Alzheimer''s disease, stroke and glaucoma.^4,5 Despite the fact that dendritic defects are likely to have devastating consequences on neuronal function and survival, the mechanisms that regulate dendrite degeneration in mature CNS neurons are poorly understood.Recent studies have identified the mammalian target of rapamycin (mTOR) as a critical component of dendritic tree development.^{6, 7, 8, 9} A substantial reduction in the number of dendritic branches and arbor shrinkage were observed in developing hippocampal neurons when mTOR was inhibited.^6,7 In addition, mTOR has been recently implicated in the regulation of dendritic spine morphology, synaptogenesis and synaptic plasticity.^10,11 The emerging developmental role of mTOR in the regulation of dendritic dynamics prompted us to put forward the hypothesis that dysregulation of mTOR function might contribute to dendritic pathology in adult neurons following injury.Many of the signals that impinge upon mTOR activity act through the tuberous sclerosis complex (TSC1/2), a negative regulator of mTOR function. For instance, stress signals such as hypoxia and energy depletion activate TSC1/2 through the REDD (regulated in development and DNA damage response) proteins,^{12, 13, 14} leading to the loss of mTOR activity. REDD2, a member of this family also known as DDIT4L or RTP801L, is an attractive target because in addition to being a potent mTOR inhibitor, it is implicated in stress responses leading to cell death.^15,16 Although REDD2 is enriched in skeletal muscle and has been shown to inhibit mTOR signaling in response to leucine and stretch,¹⁷ its expression and function in the nervous system is currently unknown.We used a model of acute optic nerve lesion in vivo to ask whether axonal damage had a direct effect on retinal ganglion cell (RGC) dendrite morphology and, if so, to identify the molecular mechanisms that regulate this injury-induced response. Our data demonstrate that axonal damage leads to substantial retraction of RGC dendritic arbors before soma loss. Optic nerve lesion led to selective REDD2 upregulation in RGCs, which coincided with the loss of mTOR activity. Short interfering RNA (siRNA)-mediated knockdown of REDD2 restored mTOR function in injured neurons and fully rescued their dendritic arbors, increasing dendritic length, field area and branch complexity. REDD2 depletion also abrogated pathologic RGC hyperexcitability and restored the light response properties of these neurons. Collectively, these data identify the REDD2-mTOR signaling pathway as a critical regulator of dendritic arbor morphology in adult central neurons undergoing axonal damage.     

Mycobacterium tuberculosis Rv3651 is a triple sensor‐domain protein

The genome of the human pathogen Mycobacterium tuberculosis (Mtb) encodes ～4,400 proteins, but one third of them have unknown functions. We solved the crystal structure of Rv3651, a hypothetical protein with no discernible similarity to proteins with known function. Rv3651 has a three‐domain architecture that combines one cG MP‐specific phosphodiesterases, a denylyl cyclases and F hlA (GAF) domain and two P er‐A RNT‐S im (PAS) domains. GAF and PAS domains are sensor domains that are typically linked to signaling effector molecules. Unlike these sensor‐effector proteins, Rv3651 is an unusual sensor domain‐only protein with highly divergent sequence. The structure suggests that Rv3651 integrates multiple different signals and serves as a scaffold to facilitate signal transfer.     

Mycobacterium tuberculosis malate synthase is a laminin-binding adhesin

Mycobacterium tuberculosis (M. tb) uses the glyoxalate bypass for intracellular survival in vivo. These studies provide evidence that the M. tb malate synthase (MS) has adapted to function as an adhesin which binds to laminin and fibronectin. This binding is achieved via the unique C-terminal region of the M. tb MS. The ability to function as an adhesin necessitates extracellular localization. We provide evidence that despite the absence of a Sec-translocation signal sequence the M. tb MS is secreted/excreted, and is anchored on the cell wall by an undefined mechanism. The MS of Mycobacterium smegmatis is cytoplasmic but the M. tb MS expressed in M. smegmatis localizes to the cell wall and enhances the adherence of the bacteria to lung epithelial A549 cells. Antibodies to the C-terminal laminin/fibronectin-binding domain interfere with the binding of the M. tb MS to laminin and fibronectin and reduce the adherence of M. tb to A549 cells. Coupled to the earlier evidence of in vivo expression of M. tb MS during active but not latent infection in humans, these studies show that a housekeeping enzyme of M. tb contributes to its armamentarium of virulence promoting factors.