Jason K. Sello

Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin.

BACKGROUND Many pathogenic bacteria secrete iron-chelating siderophores as virulence factors in the iron-limiting environments of their vertebrate hosts to compete for ferric iron. Mycobacterium tuberculosis mycobactins are mixed polyketide/nonribosomal peptides that contain a hydroxyaryloxazoline cap and two N-hydroxyamides that together create a high-affinity site for ferric ion. The mycobactin structure is analogous to that of the yersiniabactin and vibriobactin siderophores from the bacteria that cause plague and cholera, respectively. RESULTS A ten-gene cluster spanning 24 kilobases of the M. tuberculosis genome, designated mbtA-J, contains the core components necessary for mycobactin biogenesis. The gene products MbtB, MbtE and MbtF are proposed to be peptide synthetases, MbtC and MbtD polyketide synthases, MbtI an isochorismate synthase that provides a salicylate activated by MbtA, and MbtG a required hydroxylase. An aryl carrier protein (ArCP) domain is encoded in mbtB, and is probably the site of siderophore chain initiation. Overproduction and purification of the mbtB ArCP domain and MbtA in Escherichia coli allowed validation of the mycobactin initiation hypothesis, as sequential action of PptT (a phosphopantetheinyl transferase) and MbtA (a salicyl-AMP ligase) resulted in the mbtB ArCP domain being activated as salicyl-S-ArCP. CONCLUSIONS Mycobactins are produced in M. tuberculosis using a polyketide synthase/nonribosomal peptide synthetase strategy. The mycobactin gene cluster has organizational homologies to the yersiniabactin and enterobactin synthetase genes. Enzymatic targets for inhibitor design and therapeutic intervention are suggested by the similar ferric-ion ligation strategies used in the siderophores from Mycobacteria, Yersinia and E. coli pathogens.

Regulation of genes in Streptomyces bacteria required for catabolism of lignin-derived aromatic compounds

The major utilization pathway for lignin-derived aromatic compounds in microorganisms is the β-ketoadipate pathway. Through this pathway, the aromatic compounds protocatechuate and catechol are converted to acetyl coenzyme A and succinyl coenzyme A. The enzymes of the protocatechuate branch of this pathway are encoded by the pca genes. Here, we describe a gene cluster in Streptomyces coelicolor containing the pca structural genes and a regulatory gene required for the catabolism of protocatechuate. We found that transcription of the structural genes in S. coelicolor is induced by protocatechuate and p-hydroxybenzoate. We also observed inducible transcription of pca structural genes in the ligninolytic strain Streptomyces viridosporus ATCC 39115. Disruption of a gene encoding a putative MarR family transcription factor that is divergently transcribed from the pca structural genes resulted in constitutive transcription of the structural genes. Thus, the transcription factor encoded by this gene is an apparent negative regulator of pca gene transcription in S. coelicolor. Our findings suggest how Streptomyces bacteria could be engineered for and used in biotechnology for the utilization and degradation of lignin and lignin-derived aromatic compounds.

Diversity-oriented synthesis of cyclic acyldepsipeptides leads to the discovery of a potent antibacterial agent

A class of cyclic acyldepsipeptide antibiotics collectively known as the enopeptins has recently attracted much attention because of their activity against multidrug-resistant bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. These antibiotics are further distinguished by their novel mechanism of action in which they bind and deregulate the tightly controlled activity of the cytoplasmic protease ClpP. Although the natural products have poor pharmacological properties, a synthetic derivative called acyldepsipeptide 4 (ADEP 4) showed remarkable antibacterial activity both in vitro and in mouse models of bacterial infections. A novel route to the ADEP 4 peptidolactone core structure, featuring the Joullié-Ugi three-component reaction, was developed. This multicomponent reaction and a related multicomponent reaction, the Ugi four-component reaction, were used to prepare analogs that were designed using the principles of conformational analysis. These cyclic acyldepsipeptides were tested for their activity against drug-resistant, clinical isolates of Staphylococci and Enterococci. One ADEP 4 analog in which the pipecolate was replaced by 4-methyl pipecolate exhibited in vitro antibacterial activity against Enterococci that was fourfold higher than the parent compound.

Study of PcaV from Streptomyces coelicolor yields new insights into ligand-responsive MarR family transcription factors

MarR family proteins constitute a group of >12 000 transcriptional regulators encoded in bacterial and archaeal genomes that control gene expression in metabolism, stress responses, virulence and multi-drug resistance. There is much interest in defining the molecular mechanism by which ligand binding attenuates the DNA-binding activities of these proteins. Here, we describe how PcaV, a MarR family regulator in Streptomyces coelicolor, controls transcription of genes encoding β-ketoadipate pathway enzymes through its interaction with the pathway substrate, protocatechuate. This transcriptional repressor is the only MarR protein known to regulate this essential pathway for aromatic catabolism. In in vitro assays, protocatechuate and other phenolic compounds disrupt the PcaV–DNA complex. We show that PcaV binds protocatechuate in a 1:1 stoichiometry with the highest affinity of any MarR family member. Moreover, we report structures of PcaV in its apo form and in complex with protocatechuate. We identify an arginine residue that is critical for ligand coordination and demonstrate that it is also required for binding DNA. We propose that interaction of ligand with this arginine residue dictates conformational changes that modulate DNA binding. Our results provide new insights into the molecular mechanism by which ligands attenuate DNA binding in this large family of transcription factors.

Restriction of the Conformational Dynamics of the Cyclic Acyldepsipeptide Antibiotics Improves Their Antibacterial Activity

The cyclic acyldepsipeptide (ADEP) antibiotics are a new class of antibacterial agents that kill bacteria via a mechanism that is distinct from all clinically used drugs. These molecules bind and dysregulate the activity of the ClpP peptidase. The potential of these antibiotics as antibacterial drugs has been enhanced by the elimination of pharmacological liabilities through medicinal chemistry efforts. Here, we demonstrate that the ADEP conformation observed in the ADEP-ClpP crystal structure is fortified by transannular hydrogen bonding and can be further stabilized by judicious replacement of constituent amino acids within the peptidolactone core structure with more conformationally constrained counterparts. Evidence supporting constraint of the molecule into the bioactive conformer was obtained by measurements of deuterium-exchange kinetics of hydrogens that were proposed to be engaged in transannular hydrogen bonds. We show that the rigidified ADEP analogs bind and activate ClpP at lower concentrations in vitro. Remarkably, these compounds have up to 1200-fold enhanced antibacterial activity when compared to those with the peptidolactone core structure common to two ADEP natural products. This study compellingly demonstrates how rational modulation of conformational dynamics may be used to improve the bioactivities of natural products.

A Retrograde Trafficking Inhibitor of Ricin and Shiga-Like Toxins Inhibits Infection of Cells by Human and Monkey Polyomaviruses

ABSTRACT Polyomaviruses are ubiquitous pathogens that cause severe disease in immunocompromised individuals. JC polyomavirus (JCPyV) is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), whereas BK polyomavirus (BKPyV) causes polyomavirus-induced nephropathy and hemorrhagic cystitis. Vaccines or antiviral therapies targeting these viruses do not exist, and treatments focus on reducing the underlying causes of immunosuppression. We demonstrate that retro-2cycl, an inhibitor of ricin and Shiga-like toxins (SLTs), inhibits infection by JCPyV, BKPyV, and simian virus 40. Retro-2cycl inhibits retrograde transport of polyomaviruses to the endoplasmic reticulum, a step necessary for productive infection. Retro-2cycl likely inhibits polyomaviruses in a way similar to its ricin and SLT inhibition, suggesting an overlap in the cellular host factors used by bacterial toxins and polyomaviruses. This work establishes retro-2cycl as a potential antiviral therapy that broadly inhibits polyomaviruses and possibly other pathogens that use retrograde trafficking. IMPORTANCE The human polyomaviruses JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) cause rare but severe diseases in individuals with reduced immune function. During immunosuppression, JCPyV disseminates from the kidney to the central nervous system and destroys oligodendrocytes, resulting in the fatal disease progressive multifocal leukoencephalopathy. Kidney transplant recipients are at increased risk of BKPyV-induced nephropathy, which results in kidney necrosis and loss of the transplanted organ. There are currently no effective therapies for JCPyV and BKPyV. We show that a small molecule named retro-2cycl protects cells from infection with JCPyV and BKPyV by inhibiting intracellular viral transport. Retro-2cycl treatment reduces viral spreading in already established infections and may therefore be able to control infection in affected patients. Further optimization of retro-2cycl may result in the development of an effective antiviral therapy directed toward pathogens that use retrograde trafficking to infect their hosts. The human polyomaviruses JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) cause rare but severe diseases in individuals with reduced immune function. During immunosuppression, JCPyV disseminates from the kidney to the central nervous system and destroys oligodendrocytes, resulting in the fatal disease progressive multifocal leukoencephalopathy. Kidney transplant recipients are at increased risk of BKPyV-induced nephropathy, which results in kidney necrosis and loss of the transplanted organ. There are currently no effective therapies for JCPyV and BKPyV. We show that a small molecule named retro-2cycl protects cells from infection with JCPyV and BKPyV by inhibiting intracellular viral transport. Retro-2cycl treatment reduces viral spreading in already established infections and may therefore be able to control infection in affected patients. Further optimization of retro-2cycl may result in the development of an effective antiviral therapy directed toward pathogens that use retrograde trafficking to infect their hosts.

Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery

Significance Caseinolytic peptidase P (ClpP) normally collaborates with ATPases associated with diverse activities (AAA+) partner proteins, such as ClpX and ClpC, to carry out energy-dependent degradation of proteins within cells. The ClpP enzyme from Mycobacterium tuberculosis is required for survival of this human pathogen, is a validated drug target, and is unusual in consisting of discrete ClpP1 and ClpP2 rings. We solved the crystal structure of ClpP1P2 bound to peptides that mimic binding of protein substrates and small molecules that mimic binding of a AAA+ partner and cause unregulated rogue proteolysis. These studies explain why two different ClpP rings are required for peptidase activity and provide a foundation for the rational development of drugs that target ClpP1P2 and kill M. tuberculosis. Caseinolytic peptidase P (ClpP), a double-ring peptidase with 14 subunits, collaborates with ATPases associated with diverse activities (AAA+) partners to execute ATP-dependent protein degradation. Although many ClpP enzymes self-assemble into catalytically active homo-tetradecamers able to cleave small peptides, the Mycobacterium tuberculosis enzyme consists of discrete ClpP1 and ClpP2 heptamers that require a AAA+ partner and protein–substrate delivery or a peptide agonist to stabilize assembly of the active tetradecamer. Here, we show that cyclic acyldepsipeptides (ADEPs) and agonist peptides synergistically activate ClpP1P2 by mimicking AAA+ partners and substrates, respectively, and determine the structure of the activated complex. Our studies establish the basis of heteromeric ClpP1P2 assembly and function, reveal tight coupling between the conformations of each ring, show that ADEPs bind only to one ring but appear to open the axial pores of both rings, provide a foundation for rational drug development, and suggest strategies for studying the roles of individual ClpP1 and ClpP2 rings in Clp-family proteolysis.

Antibacterial Activity of and Resistance to Small Molecule Inhibitors of the ClpP Peptidase

There is rapidly mounting evidence that intracellular proteases in bacteria are compelling targets for antibacterial drugs. Multiple reports suggest that the human pathogen Mycobacterium tuberculosis and other actinobacteria may be particularly sensitive to small molecules that perturb the activities of self-compartmentalized peptidases, which catalyze intracellular protein turnover as components of ATP-dependent proteolytic machines. Here, we report chemical syntheses and evaluations of structurally diverse β-lactones, which have a privileged structure for selective, suicide inhibition of the self-compartmentalized ClpP peptidase. β-Lactones with certain substituents on the α- and β-carbons were found to be toxic to M. tuberculosis. Using an affinity-labeled analogue of a bioactive β-lactone in a series of chemical proteomic experiments, we selectively captured the ClpP1P2 peptidase from live cultures of two different actinobacteria that are related to M. tuberculosis. Importantly, we found that the growth inhibitory β-lactones also inactivate the M. tuberculosis ClpP1P2 peptidase in vitro via formation of a covalent adduct at the ClpP2 catalytic serine. Given the potent antibacterial activity of these compounds and their medicinal potential, we sought to identify innate mechanisms of resistance. Using a genome mining strategy, we identified a genetic determinant of β-lactone resistance in Streptomyces coelicolor, a non-pathogenic relative of M. tuberculosis. Collectively, these findings validate the potential of ClpP inhibition as a strategy in antibacterial drug development and define a mechanism by which bacteria could resist the toxic effects of ClpP inhibitors.

Genome Sequence of Amycolatopsis sp. Strain ATCC 39116, a Plant Biomass-Degrading Actinomycete

We announce the availability of a high-quality draft of the genome sequence of Amycolatopsis sp. strain 39116, one of few bacterial species that are known to consume the lignin component of plant biomass. This genome sequence will further ongoing efforts to use microorganisms for the conversion of plant biomass into fuels and high-value chemicals.

The Gene Encoding RNase III in Streptomyces coelicolor Is Transcribed during Exponential Phase and Is Required for Antibiotic Production and for Proper Sporulation

Phenotypic analysis of a constructed RNase III null mutant of Streptomyces coelicolor revealed that RNase III is required for both antibiotic production and proper formation of sporulation septa. Transcriptional analysis of the gene encoding RNase III indicated that it is transcribed exclusively during exponential phase as part of a tricistronic message.