Chalkophore mediated respiratory oxidase flexibility controls M. tuberculosis virulence.

Oxidative phosphorylation has emerged as a critical therapeutic vulnerability of M. tuberculosis, but it is unknown how M. tuberculosis and other pathogens maintain respiration during infection. M. tuberculosis synthesizes diisonitrile lipopeptide chalkophores that chelate copper tightly, but their role in host-pathogen interactions is also unknown. We demonstrate that M. tuberculosis chalkophores maintain the function of the heme-copper bcc:aa3 respiratory oxidase under copper limitation. Chalkophore deficient M. tuberculosis cannot survive, respire to oxygen, or produce ATP under copper deprivation in culture. M. tuberculosis lacking chalkophore biosynthesis is attenuated in mice, a phenotype that is severely exacerbated by loss of the CytBD alternative respiratory oxidase (encoded by cydAB), revealing a multilayered flexibility of the respiratory chain that maintains oxidative phosphorylation during infection. Taken together, these data demonstrate that chalkophores counter host inflicted copper deprivation and highlight that protection of cellular respiration is a critical virulence function in M. tuberculosis.

The Rip1 intramembrane protease contributes to iron and zinc homeostasis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis is exposed to a variety of stresses during a chronic infection, as the immune system simultaneously produces bactericidal compounds and starves the pathogen of essential nutrients. The intramembrane protease, Rip1, plays an important role in the adaptation to these stresses, at least partially by the cleavage of membrane-bound transcriptional regulators. Although Rip1 is known to be critical for surviving copper intoxication and nitric oxide exposure, these stresses do not fully account for the regulatory protein's essentiality during infection. In this work, we demonstrate that Rip1 is also necessary for growth in low-iron and low-zinc conditions, similar to those imposed by the immune system. Using a newly generated library of sigma factor mutants, we show that the known regulatory target of Rip1, SigL, shares this defect. Transcriptional profiling under iron-limiting conditions supported the coordinated activity of Rip1 and SigL and demonstrated that the loss of these proteins produces an exaggerated iron starvation response. These observations demonstrate that Rip1 coordinates several aspects of metal homeostasis and suggest that a Rip1- and SigL-dependent pathway is necessary to thrive in the iron-deficient environments encountered during infection. IMPORTANCE Metal homeostasis represents a critical point of interaction between the mammalian immune system and potential pathogens. While the host attempts to intoxicate microbes with high concentrations of copper or starve the invader of iron and zinc, successful pathogens have acquired mechanisms to overcome these defenses. Our work identifies a regulatory pathway consisting of the Rip1 intramembrane protease and the sigma factor, SigL, that is essential for the important human pathogen, Mycobacterium tuberculosis, to grow in low-iron or low-zinc conditions such as those encountered during infection. In conjunction with Rip1's known role in resisting copper toxicity, our work implicates this protein as a critical integration point that coordinates the multiple metal homeostatic systems required for this pathogen to survive in host tissue.

Diisonitrile Lipopeptides Mediate Resistance to Copper Starvation in Pathogenic Mycobacteria.

Bacterial pathogens and their hosts engage in intense competition for critical nutrients during infection, including metals such as iron, copper, and zinc. Some metals are limited by the host, and some are deployed by the host as antimicrobials. To counter metal limitation, pathogens deploy high-affinity metal acquisition systems, best exemplified by siderophores to acquire iron. Although pathogen strategies to resist the toxic effects of high Cu have been elucidated, the role of Cu starvation and the existence of Cu acquisition systems are less well characterized. In this study, we examined the role of diisonitrile chalkophores of pathogenic mycobacteria, synthesized by the enzymes encoded by the virulence-associated nrp gene cluster, in metal acquisition. nrp gene cluster expression is strongly induced by starvation or chelation of Cu but not starvation of Zn or excess Cu. Mycobacterium tuberculosis and Mycobacterium marinum strains lacking the nrp-encoded nonribosomal peptide sythetase, the fadD10 adenylate-forming enzyme, or the uncharacterized upstream gene ppe1 are all sensitized to Cu, but not Zn, starvation. This low Cu sensitivity is rescued by genetic complementation or by provision of a synthetic diisonitrile chalkophore. These data demonstrate that diisonitrile lipopeptides in mycobacteria are chalkophores that facilitate survival under Cu-limiting conditions and suggest that Cu starvation is a relevant stress for M. tuberculosis in the host. IMPORTANCE Bacterial pathogens and their hosts engage in intense competition for nutrients, including metals. Mycobacterium tuberculosis, the cause of tuberculosis, lives within host macrophages and is subject to diverse stresses, including metal excess and metal limitation. In this study, we demonstrated that the nrp gene cluster, required for M. tuberculosis virulence and which directs synthesis of diisonitrile lipopeptides, mediates copper acquisition. Copper, but not zinc, deprivation strongly induces diisonitrile biosynthesis, and M. tuberculosis strains lacking the nrp gene, or the associated genes fadD10 or ppe1, are all sensitized to copper chelation or copper deprivation. These results establish a copper binding, or chalkophore, system in M. tuberculosis and indicate that resistance to copper restriction plays an important role in the ability of this global pathogen to cause infection.

Distinctive roles of translesion polymerases DinB1 and DnaE2 in diversification of the mycobacterial genome through substitution and frameshift mutagenesis.

Antibiotic resistance of Mycobacterium tuberculosis is exclusively a consequence of chromosomal mutations. Translesion synthesis (TLS) is a widely conserved mechanism of DNA damage tolerance and mutagenesis, executed by translesion polymerases such as DinBs. In mycobacteria, DnaE2 is the only known agent of TLS and the role of DinB polymerases is unknown. Here we demonstrate that, when overexpressed, DinB1 promotes missense mutations conferring resistance to rifampicin, with a mutational signature distinct from that of DnaE2, and abets insertion and deletion frameshift mutagenesis in homo-oligonucleotide runs. DinB1 is the primary mediator of spontaneous -1 frameshift mutations in homo-oligonucleotide runs whereas DnaE2 and DinBs are redundant in DNA damage-induced -1 frameshift mutagenesis. These results highlight DinB1 and DnaE2 as drivers of mycobacterial genome diversification with relevance to antimicrobial resistance and host adaptation.

Integrated sensing of host stresses by inhibition of a cytoplasmic two-component system controls M. tuberculosis acute lung infection.

Bacterial pathogens that infect phagocytic cells must deploy mechanisms that sense and neutralize host microbicidal effectors. For Mycobacterium tuberculosis, the causative agent of tuberculosis, these mechanisms allow the bacterium to rapidly adapt from aerosol transmission to initial growth in the lung alveolar macrophage. Here, we identify a branched signaling circuit in M. tuberculosis that controls growth in the lung through integrated direct sensing of copper ions and nitric oxide by coupled activity of the Rip1 intramembrane protease and the PdtaS/R two-component system. This circuit uses a two-signal mechanism to inactivate the PdtaS/PdtaR two-component system, which constitutively represses virulence gene expression. Cu and NO inhibit the PdtaS sensor kinase through a dicysteine motif in the N-terminal GAF domain. The NO arm of the pathway is further controlled by sequestration of the PdtaR RNA binding response regulator by an NO-induced small RNA, controlled by the Rip1 intramembrane protease. This coupled Rip1/PdtaS/PdtaR circuit controls NO resistance and acute lung infection in mice by relieving PdtaS/R-mediated repression of isonitrile chalkophore biosynthesis. These studies identify an integrated mechanism by which M. tuberculosis senses and resists macrophage chemical effectors to achieve pathogenesis.

Palmitoylation of Hedgehog proteins.

Hedgehog (Hh) proteins are secreted signaling proteins that contain amide-linked palmitate at the N-terminus and cholesterol at the C-terminus. Palmitoylation of Hh proteins is critical for effective long- and short-range signaling. The palmitoylation reaction occurs during transit of Hh through the secretory pathway, most likely in the lumen of the ER. Attachment of palmitate to Hh proteins is independent of cholesterol modification and autoprocessing and is catalyzed by Hhat (Hedgehog acyltransferase). Hhat is a member of the membrane bound O-acyltransferase (MBOAT) family, a subgroup of multipass membrane proteins that catalyze transfer of fatty acyl groups to lipids and proteins. Several classes of secreted proteins have recently been shown to be substrates for MBOAT acyltransferases, including Hh proteins and Spitz (palmitoylated by Hhat), Wg/Wnt proteins (modified with palmitate and/or palmitoleate by Porcupine) and ghrelin (octanoylated by ghrelin O-acyltransferase). These findings highlight protein fatty acylation as a mechanism that not only influences membrane binding of intracellular proteins but also regulates the signaling range and efficacy of secreted proteins.

Identification of conserved regions and residues within Hedgehog acyltransferase critical for palmitoylation of Sonic Hedgehog.

BACKGROUND: Sonic hedgehog (Shh) is a palmitoylated protein that plays key roles in mammalian development and human cancers. Palmitoylation of Shh is required for effective long and short range Shh-mediated signaling. Attachment of palmitate to Shh is catalyzed by Hedgehog acyltransferase (Hhat), a member of the membrane bound O-acyl transferase (MBOAT) family of multipass membrane proteins. The extremely hydrophobic composition of MBOAT proteins has limited their biochemical characterization. Except for mutagenesis of two conserved residues, there has been no structure-function analysis of Hhat, and the regions of the protein required for Shh palmitoylation are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we undertake a systematic approach to identify residues within Hhat that are required for protein stability and/or enzymatic activity. We also identify a second, novel MBOAT homology region (residues 196-234) that is required for Hhat activity. In total, ten deletion mutants and eleven point mutants were generated and analyzed. Truncations at the N- and C-termini of Hhat yielded inactive proteins with reduced stability. Four Hhat mutants with deletions within predicted loop regions and five point mutants retained stability but lost palmitoylation activity. We purified two point mutants, W378A and H379A, with defective Hhat activity. Kinetic analyses revealed alterations in apparent K(m) and V(max) for Shh and/or palmitoyl CoA, changes that likely explain the catalytic defects observed for these mutants. CONCLUSIONS/SIGNIFICANCE: This study has pinpointed specific regions and multiple residues that regulate Hhat stability and catalysis. Our findings should be applicable to other MBOAT proteins that mediate lipid modification of Wnt proteins and ghrelin, and should serve as a model for understanding how secreted morphogens are modified by palmitoyl acyltransferases.

Hhat is a palmitoylacyltransferase with specificity for N-palmitoylation of Sonic Hedgehog.

Palmitoylation of Sonic Hedgehog (Shh) is critical for effective long- and short-range signaling. Genetic screens uncovered a potential palmitoylacyltransferase (PAT) for Shh, Hhat, but the molecular mechanism of Shh palmitoylation remains unclear. Here, we have developed and exploited an in vitro Shh palmitoylation assay to purify Hhat to homogeneity. We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. Other palmitoylated proteins (e.g. PSD95 and Wnt) are not substrates for Hhat, and Porcupine, a putative Wnt PAT, does not palmitoylate Shh. Neither autocleavage nor cholesterol modification is required for Shh palmitoylation. Both the Shh precursor and mature protein are N-palmitoylated by Hhat, and the reaction occurs during passage through the secretory pathway. This study establishes Hhat as a bona fide Shh PAT and serves as a model for understanding how secreted morphogens are modified by distinct PATs.

Structure, function, and mechanism of the phenylacetate pathway hot dog-fold thioesterase PaaI.

The structure and biochemical function of the hot dog-fold thioesterase PaaI operative in the aerobic phenylacetate degradation pathway are examined. PaaI showed modest activity with phenylacetyl-coenzyme A, suggestive of a role in coenzyme A release from this pathway intermediate in the event of limiting downstream pathway enzymes. Minimal activity was observed with aliphatic acyl-coenzyme A thioesters, which ruled out PaaI function in the lower phenylacetate pathway. PaaI was most active with ring-hydroxylated phenylacetyl-coenzyme A thioesters. The x-ray crystal structure of the Escherichia coli thioesterase is reported and analyzed to define the structural basis of substrate recognition and catalysis. The contributions of catalytic and substrate binding residues, thus, identified were examined through steady-state kinetic analysis of site-directed mutant proteins.

Palmitoylation of the EGFR ligand Spitz by Rasp increases Spitz activity by restricting its diffusion.

Lipid modifications such as palmitoylation or myristoylation target intracellular proteins to cell membranes. Secreted ligands of the Hedgehog and Wnt families are also palmitoylated; this modification, which requires the related transmembrane acyltransferases Rasp and Porcupine, can enhance their secretion, transport, or activity. We show here that rasp is also essential for the developmental functions of Spitz, a ligand for the Drosophila epidermal growth factor receptor (EGFR). In cultured cells, Rasp promotes palmitate addition to the N-terminal cysteine residue of Spitz, and this cysteine is required for Spitz activity in vivo. Palmitoylation reduces Spitz secretion and enhances its plasma membrane association, but does not alter its ability to activate the EGFR in vitro. In vivo, overexpressed unpalmitoylated Spitz has an increased range of action but reduced activity. These data suggest a role for palmitoylation in restricting Spitz diffusion, allowing its local concentration to reach the threshold required for biological function.

Crystal structure and functional analysis of lipoamide dehydrogenase from Mycobacterium tuberculosis.

We report the 2.4 A crystal structure for lipoamide dehydrogenase encoded by lpdC from Mycobacterium tuberculosis. Based on the Lpd structure and sequence alignment between bacterial and eukaryotic Lpd sequences, we generated single point mutations in Lpd and assayed the resulting proteins for their ability to catalyze lipoamide reduction/oxidation alone and in complex with other proteins that participate in pyruvate dehydrogenase and peroxidase activities. The results suggest that amino acid residues conserved in mycobacterial species but not conserved in eukaryotic Lpd family members modulate either or both activities and include Arg-93, His-98, Lys-103, and His-386. In addition, Arg-93 and His-386 are involved in forming both "open" and "closed" active site conformations, suggesting that these residues play a role in dynamically regulating Lpd function. Taken together, these data suggest protein surfaces that should be considered while developing strategies for inhibiting this enzyme.

Crystal structure of PapA5, a phthiocerol dimycocerosyl transferase from Mycobacterium tuberculosis.

Polyketide-associated protein A5 (PapA5) is an acyltransferase that is involved in production of phthiocerol and phthiodiolone dimycocerosate esters, a class of virulence-enhancing lipids produced by Mycobacterium tuberculosis. Structural analysis of PapA5 at 2.75-A resolution reveals a two-domain structure that shares unexpected similarity to structures of chloramphenicol acetyltransferase, dihydrolipoyl transacetylase, carnitine acetyltransferase, and VibH, a non-ribosomal peptide synthesis condensation enzyme. The PapA5 active site includes conserved histidine and aspartic acid residues that are critical to PapA5 acyltransferase activity. PapA5 catalyzes acyl transfer reactions on model substrates that contain long aliphatic carbon chains, and two hydrophobic channels were observed linking the PapA5 surface to the active site with properties consistent with these biochemical activities and substrate preferences. An additional alpha helix not observed in other acyltransferase structures blocks the putative entrance into the PapA5 active site, indicating that conformational changes may be associated with PapA5 activity. PapA5 represents the first structure solved for a protein involved in polyketide synthesis in Mycobacteria.

Mycobacterial polyketide-associated proteins are acyltransferases: proof of principle with Mycobacterium tuberculosis PapA5.

Mycobacterium tuberculosis (Mt) produces complex virulence-enhancing lipids with scaffolds consisting of phthiocerol and phthiodiolone dimycocerosate esters (PDIMs). Sequence analysis suggested that PapA5, a so-called polyketide-associated protein (Pap) encoded in the PDIM synthesis gene cluster, as well as PapA5 homologs found in Mt and other species, are a subfamily of acyltransferases. Studies with recombinant protein confirmed that PapA5 is an acyltransferase [corrected]. Deletion analysis in Mt demonstrated that papA5 is required for PDIM synthesis. We propose that PapA5 catalyzes diesterification of phthiocerol and phthiodiolone with mycocerosate. These studies present the functional characterization of a Pap and permit inferences regarding roles of other Paps in the synthesis of complex lipids, including the antibiotic rifamycin.

Structure of Escherichia coli uridine phosphorylase at 2.0 A.

The 2.0 A crystal structure has been determined for Escherichia coli uridine phosphorylase (UP), an essential enzyme in nucleotide biosynthesis that catalyzes the phosphorolytic cleavage of the C-N glycosidic bond of uridine to ribose-1-phosphate and uracil. The structure determination of two independent monomers in the asymmetric unit revealed the residue composition and atomic details of the apo configurations of each active site. The native hexameric UP enzyme was revealed by applying threefold crystallographic symmetry to the contents of the asymmetric unit. The 2.0 A model reveals a closer structural relationship to other nucleotide phosphorylase enzymes than was previously appreciated.

Structural and biochemical analysis of the Obg GTP binding protein.

The Obg nucleotide binding protein family has been implicated in stress response, chromosome partitioning, replication initiation, mycelium development, and sporulation. Obg proteins are among a large group of GTP binding proteins conserved from bacteria to man. Members of the family contain two equally and highly conserved domains, a C-terminal GTP binding domain and an N-terminal glycine-rich domain. Structural analysis of Bacillus subtilis Obg revealed respective domain architectures and how they are coupled through the putative switch elements of the C-terminal GTPase domain in apo and nucleotide-bound configurations. Biochemical analysis of bacterial and human Obg proteins combined with the structural observation of the ppGpp nucleotide within the Obg active sight suggest a potential role for ppGpp modulation of Obg function in B. subtilis.

Structural analysis of Saccharomyces cerevisiae myo-inositol phosphate synthase.

The New York Structural Genomics Research Consortium has targeted highly conserved but uncharacterized enzyme families for structure determination. As part of this effort, the 2.65-A crystal structure has been determined for Saccharomyces cerevisiae myo-inositol 1-phosphate synthase (MIP), an essential enzyme that catalyzes critical steps in inositol biosynthesis. The structure determination of four independent monomers in the asymmetric unit (240 kDa) reveals atomic details and residue composition for the partially closed NAD-containing active sites in apo-configuration. The structure further reveals extensive interactions involved in tetrameric assembly of the enzyme complex.