An intranasal nanoparticle vaccine elicits protective immunity against Mycobacterium tuberculosis.

Mucosal vaccines have the potential to elicit protective immune responses at the point of entry of respiratory pathogens, thus preventing even the initial seed infection. Unlike licensed injectable vaccines, mucosal vaccines comprising protein subunits are only in development. One of the primary challenges associated with mucosal vaccines has been identifying and characterizing safe yet effective mucosal adjuvants that can effectively prime multi-factorial mucosal immunity.

-Acetylation of the virulence factor EsxA is required for mycobacterial cytosolic translocation and virulence.

The virulence factor EsxA and its chaperone EsxB are secreted as a heterodimer (EsxA:B) and are crucial for mycobacterial escape from phagosomes and cytosolic translocation. Current findings support the idea that for EsxA to interact with host membranes, EsxA must dissociate from EsxB at low pH. However, the molecular mechanism by which the EsxA:B heterodimer separates is not clear.

Eicosapentaenoic acid monoglyceride resolves inflammation in an ex vivo model of human peripheral blood mononuclear cell.

Phosphorylation and activation of p38 MAPK and NFκB pathways, along with the resulting overproduction of interleukin IL-1β, IL-6, and tumor necrosis factor a (TNFα) is a hallmark of inflammatory disorders. Omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementations are known to exert anti-inflammatory properties by reduction of keys cytokines and enzymes involved in inflammation. Here, we investigated the anti-inflammatory pathways and mediators modulated by eicosapentaenoic acid monoglyceride (MAG-EPA) on human peripheral blood mononuclear cells (PBMCs) from healthy donors and stimulated, ex vivo, with lipopolysaccharide (LPS).

EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5.

EsxA is required for virulence of Mycobacterium tuberculosis (Mtb) and plays an essential role in phagosome rupture and translocation to the cytosol of macrophages. Recent biochemical studies have demonstrated that EsxA is a membrane-permeabilizing protein. However, evidence that link EsxA membrane-permeabilizing activity to Mtb cytosolic translocation and virulence is lacking.

Imaging cytosolic translocation of Mycobacteria with two-photon fluorescence resonance energy transfer microscopy.

Transition from latency to active tuberculosis requires Mycobacterium tuberculosis (Mtb) to penetrate the phagosomal membrane and translocate to the cytosol of the host macrophage. Quantitative two-photon fluorescence resonance energy transfer (FRET) microscopy is developed to measure cytosolic translocation using Mycobacterium marinum (Mm) as a model organism for Mtb. Macrophages were infected with Mm or non-pathogenic Mycobacterium smegmatis (Ms) as a control, then loaded with a FRET substrate.

Genetic and mass spectrometric tools for elucidating the physiological function(s) of cytochrome P450 enzymes from Mycobacterium tuberculosis.

Tuberculosis remains a leading cause of human mortality. The emergence of strains of Mycobacterium tuberculosis (Mtb), the causative agent, that are resistant to first- and second-line antitubercular drugs urges the development of new therapeutics. The genome of Mtb encodes 20 cytochrome P450 enzymes, at least some of which are potential candidates (CYP121, CYP125, and CYP128) for drug targeting.

Proximal ligand electron donation and reactivity of the cytochrome P450 ferric-peroxo anion.

CYP125 from Mycobacterium tuberculosis catalyzes sequential oxidation of the cholesterol side-chain terminal methyl group to the alcohol, aldehyde, and finally acid. Here, we demonstrate that CYP125 simultaneously catalyzes the formation of five other products, all of which result from deformylation of the sterol side chain. The aldehyde intermediate is shown to be the precursor of both the conventional acid metabolite and the five deformylation products.

Cholesterol catabolism as a therapeutic target in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects 10 million people worldwide and kills 2 million people every year. The uptake and utilization of nutrients by Mtb within the host cell is still poorly understood, although lipids play an important role in Mtb persistence. The recent identification of a large regulon of cholesterol catabolic genes suggests that Mtb can use host sterol for infection and persistence.

A novel intermediate in the reaction of seleno CYP119 with m-chloroperbenzoic acid.

Cytochrome P450-mediated monooxygenation generally proceeds via a reactive ferryl intermediate coupled to a ligand radical [Fe(IV)═O]+• termed Compound I (Cpd I). The proximal cysteine thiolate ligand is a critical determinant of the spectral and catalytic properties of P450 enzymes. To explore the effect of an increased level of donation of electrons by the proximal ligand in the P450 catalytic cycle, we recently reported successful incorporation of SeCys into the active site of CYP119, a thermophilic cytochrome P450.

Reverse type I inhibitor of Mycobacterium tuberculosis CYP125A1.

Cytochrome P450 CYP125A1 of Mycobacterium tuberculosis, a potential therapeutic target for tuberculosis in humans, initiates degradation of the aliphatic chain of host cholesterol and is essential for establishing M. tuberculosis infection in a mouse model of disease. We explored the interactions of CYP125A1 with a reverse type I inhibitor by X-ray structure analysis and UV-vis spectroscopy.

Functional redundancy of steroid C26-monooxygenase activity in Mycobacterium tuberculosis revealed by biochemical and genetic analyses.

One challenge to the development of new antitubercular drugs is the existence of multiple virulent strains that differ genetically. We and others have recently demonstrated that CYP125A1 is a steroid C(26)-monooxygenase that plays a key role in cholesterol catabolism in Mycobacterium tuberculosis CDC1551 but, unexpectedly, not in the M. tuberculosis H37Rv strain.

Structure of cytochrome P450 PimD suggests epoxidation of the polyene macrolide pimaricin occurs via a hydroperoxoferric intermediate.

We present the X-ray structure of PimD, both substrate-free and in complex with 4,5-desepoxypimaricin. PimD is a cytochrome P450 monooxygenase with native epoxidase activity that is critical in the biosynthesis of the polyene macrolide antibiotic pimaricin. Intervention in this secondary metabolic pathway could advance the development of drugs with improved pharmacologic properties.

Structural control of cytochrome P450-catalyzed ω-hydroxylation.

The regiospecific or preferential ω-hydroxylation of hydrocarbon chains is thermodynamically disfavored because the ease of C-H bond hydroxylation depends on the bond strength, and the primary C-H bond of a terminal methyl group is stronger than the secondary or tertiary C-H bond adjacent to it. The hydroxylation reaction will therefore occur primarily at the adjacent secondary or tertiary C-H bond unless the protein structure specifically enforces primary C-H bond oxidation. Here we review the classes of enzymes that catalyze ω-hydroxylation and our current understanding of the structural features that promote the ω-hydroxylation of unbranched and methyl-branched hydrocarbon chains.

Mycobacterium tuberculosis CYP125A1, a steroid C27 monooxygenase that detoxifies intracellularly generated cholest-4-en-3-one.

The infectivity and persistence of Mycobacterium tuberculosis requires the utilization of host cell cholesterol. We have examined the specific role of cytochrome P450 CYP125A1 in the cholesterol degradation pathway using genetic, biochemical and high-resolution mass spectrometric approaches. The analysis of lipid profiles from cells grown on cholesterol revealed that CYP125A1 is required to incorporate the cholesterol side-chain carbon atoms into cellular lipids, as evidenced by an increase in the mass of the methyl-branched phthiocerol dimycocerosates.

The Mycobacterium tuberculosis cytochrome P450 system.

Tuberculosis remains a leading cause of human mortality. The emergence of strains of Mycobacterium tuberculosis, the causative agent, that are resistant to the major frontline antitubercular drugs increases the urgency for the development of new therapeutic agents. Sequencing of the M.

Interaction of Mycobacterium tuberculosis CYP130 with heterocyclic arylamines.

The Mycobacterium tuberculosis P450 enzymes are of interest for their pharmacological development potential, as evidenced by their susceptibility to inhibition by antifungal azole drugs that normally target sterol 14alpha-demethylase (CYP51). Although antifungal azoles show promise, direct screening of compounds against M. tuberculosis P450 enzymes may identify novel, more potent, and selective inhibitory scaffolds.

Reaction of Mycobacterium tuberculosis cytochrome P450 enzymes with nitric oxide.

During the initial growth infection stage of Mycobacterium tuberculosis (Mtb), (*)NO produced by host macrophages inhibits heme-containing terminal cytochrome oxidases, inactivates iron/sulfur proteins, and promotes entry into latency. Here we evaluate the potential of (*)NO as an inhibitor of Mtb cytochrome P450 enzymes, as represented by CYP130, CYP51, and the two previously uncharacterized enzymes CYP125 and CYP142. Using UV-visible absorption, resonance Raman, and stopped-flow spectroscopy, we investigated the reactions of (*)NO with these heme proteins in their ferric resting form.

Covalent heme attachment to the protein in human heme oxygenase-1 with selenocysteine replacing the His25 proximal iron ligand.

To characterize heme oxygenase with a selenocysteine (SeCys) as the proximal iron ligand, we have expressed truncated human heme oxygenase-1 (hHO-1) His25Cys, in which Cys-25 is the only cysteine, in the Escherichia coli cysteine auxotroph strain BL21(DE3)cys. Selenocysteine incorporation into the protein was demonstrated by both intact protein mass measurement and mass spectrometric identification of the selenocysteine-containing tryptic peptide. One selenocysteine was incorporated into approximately 95% of the expressed protein.

Analysis of transient and catalytic desosamine-binding pockets in cytochrome P-450 PikC from Streptomyces venezuelae.

The cytochrome P-450 PikC from Streptomyces venezuelae exhibits significant substrate tolerance and performs multiple hydroxylation reactions on structurally variant macrolides bearing the deoxyamino sugar desosamine. In previously determined co-crystal structures (Sherman, D. H.

Mycobacterium tuberculosis CYP130: crystal structure, biophysical characterization, and interactions with antifungal azole drugs.

CYP130 is one of the 20 Mycobacterium tuberculosis cytochrome P450 enzymes, only two of which, CYP51 and CYP121, have so far been studied as individually expressed proteins. Here we characterize a third heterologously expressed M. tuberculosis cytochrome P450, CYP130, by UV-visible spectroscopy, isothermal titration calorimetry, and x-ray crystallography, including determination of the crystal structures of ligand-free and econazole-bound CYP130 at a resolution of 1.

The roles of Tyr(CD1) and Trp(G8) in Mycobacterium tuberculosis truncated hemoglobin O in ligand binding and on the heme distal site architecture.

The crystal structure of the cyano-met form of Mt-trHbO revealed two unusual distal residues Y(CD1) and W(G8) forming a hydrogen-bond network with the heme-bound ligand [Milani, M., et al. (2003) Proc.

Small-molecule scaffolds for CYP51 inhibitors identified by high-throughput screening and defined by X-ray crystallography.

Sterol 14alpha-demethylase (CYP51), a major checkpoint in membrane sterol biosynthesis, is a key target for fungal antibiotic therapy. We sought small organic molecules for lead candidate CYP51 inhibitors. The changes in CYP51 spectral properties following ligand binding make CYP51 a convenient target for high-throughput screening technologies.

Reaction of Mycobacterium tuberculosis truncated hemoglobin O with hydrogen peroxide: evidence for peroxidatic activity and formation of protein-based radicals.

In this work, we investigated the reaction of ferric Mycobacterium tuberculosis truncated hemoglobin O (trHbO) with hydrogen peroxide. Stopped-flow spectrophotometric experiments under single turnover conditions showed that trHbO reacts with H(2)O(2) to give transient intermediate(s), among which is an oxyferryl heme, different from a typical peroxidase Compound I (oxyferryl heme pi-cation radical). EPR spectroscopy indicated evidence for both tryptophanyl and tyrosyl radicals, whereas redox titrations demonstrated that the peroxide-treated protein product retains 2 oxidizing eq.

Structural bases for heme binding and diatomic ligand recognition in truncated hemoglobins.

Truncated hemoglobins (trHbs) are low-molecular-weight oxygen-binding heme-proteins distributed in eubacteria, cyanobacteria, unicellular eukaryotes, and in higher plants, constituting a distinct group within the hemoglobin (Hb) superfamily. TrHbs display amino acid sequences 20-40 residues shorter than classical (non)vertebrate Hbs and myoglobins, to which they are scarcely related by sequence similarity. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which represents a striking editing of the highly conserved 3-on-3 alpha-helical globin fold, achieved through deletion/truncation of alpha-helices and specific residue substitutions.

Cyanide binding to truncated hemoglobins: a crystallographic and kinetic study.

Cyanide is one of the few diatomic ligands able to interact with the ferric and ferrous heme-Fe atom. Here, the X-ray crystal structure of the cyanide derivative of ferric Mycobacterium tuberculosis truncated hemoglobin-N (M. tuberculosis trHbN) has been determined at 2.