An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death.

Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases.

Structure of the Mycobacterium tuberculosis type VII secretion system chaperone EspG5 in complex with PE25-PPE41 dimer.

The growth or virulence of Mycobacterium tuberculosis bacilli depends on homologous type VII secretion systems, ESX-1, ESX-3 and ESX-5, which export a number of protein effectors across membranes to the bacterial surface and environment. PE and PPE proteins represent two large families of highly polymorphic proteins that are secreted by these ESX systems. Recently, it was shown that these proteins require system-specific cytoplasmic chaperones for secretion. Here, we report the crystal structure of M. tuberculosis ESX-5-secreted PE25-PPE41 heterodimer in complex with the cytoplasmic chaperone EspG(5). EspG(5) represents a novel fold that is unrelated to previously characterized secretion chaperones. Functional analysis of the EspG(5) -binding region uncovered a hydrophobic patch on PPE41 that promotes dimer aggregation, and the chaperone effectively abolishes this process. We show that PPE41 contains a characteristic chaperone-binding sequence, the hh motif, which is highly conserved among ESX-1-, ESX-3- and ESX-5-specific PPE proteins. Disrupting the interaction between EspG(5) and three different PPE target proteins by introducing different point mutations generally affected protein secretion. We further demonstrate that the EspG(5) chaperone plays an important role in the ESX secretion mechanism by keeping aggregation-prone PE-PPE proteins in their soluble state.

Specific chaperones for the type VII protein secretion pathway.

Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG(5), which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG(5) interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG(1) interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG(5) and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG(5) and EspG(1) interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway.

The pMy vector series: A versatile cloning platform for the recombinant production of mycobacterial proteins in Mycobacterium smegmatis.

Structural and biophysical characterization of molecular mechanisms of disease-causing pathogens, such as Mycobacterium tuberculosis, often requires recombinant expression of large amounts highly pure protein. For the production of mycobacterial proteins, overexpression in the fast-growing and non-pathogenic species Mycobacterium smegmatis has several benefits over the standard Escherichia coli expression strains. However, unlike for E. coli, the range of expression vectors currently available is limited. Here we describe the development of the pMy vector series, a set of expression plasmids for recombinant production of single proteins and protein complexes in M. smegmatis. By incorporating an alternative selection marker, we show that these plasmids can also be used for co-expression studies. All vectors in the pMy vector series are available in the Addgene repository (www.addgene.com).

Structural Variability of EspG Chaperones from Mycobacterial ESX-1, ESX-3, and ESX-5 Type VII Secretion Systems.

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central ß-sheet and two α-helical bundles. In addition, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3, and EspG5 chaperones using small-angle X-ray scattering reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3-PE5-PPE4 complex based on the small-angle X-ray scattering analysis.

Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis.

Mycobacteria are characterized by their impermeable outer membrane, which is rich in mycolic acids1. To transport substrates across this complex cell envelope, mycobacteria rely on type VII (also known as ESX) secretion systems2. In Mycobacterium tuberculosis, these ESX systems are essential for growth and full virulence and therefore represent an attractive target for anti-tuberculosis drugs3. However, the molecular details underlying type VII secretion are largely unknown, due to a lack of structural information. Here, we report the molecular architecture of the ESX-5 membrane complex from Mycobacterium xenopi determined at 13 Šresolution by electron microscopy. The four core proteins of the ESX-5 complex (EccB5, EccC5, EccD5 and EccE5) assemble with equimolar stoichiometry into an oligomeric assembly that displays six-fold symmetry. This membrane-associated complex seems to be embedded exclusively in the inner membrane, which indicates that additional components are required to translocate substrates across the mycobacterial outer membrane. Furthermore, the extended cytosolic domains of the EccC ATPase, which interact with secretion effectors, are highly flexible, suggesting an as yet unseen mode of substrate interaction. Comparison of our results with known structures of other bacterial secretion systems demonstrates that the architecture of type VII secretion system is fundamentally different, suggesting an alternative secretion mechanism.

A standardized production pipeline for high profile targets from Mycobacterium tuberculosis.

PURPOSE: Tuberculosis is still a major threat to global health. New tools and strategies to produce disease-related proteins are quintessential for the development of novel vaccines and diagnostic markers. EXPERIMENTAL DESIGN: To obtain recombinant proteins from Mycobacterium tuberculosis (Mtb) for use in clinical applications, a standardized procedure was developed that includes subcloning, protein expression in Mycobacterium smegmatis and protein purification using chromatography. The potential for the different protein targets to serve as diagnostic markers for tuberculosis was established using multiplex immunoassays. RESULTS: Twelve soluble proteins from Mtb, including one protein complex, were purified to near-homogeneity following recombinant expression in M. smegmatis. Protein purity was assessed both by size exclusion chromatography and MS. Multiplex serological testing of the final protein preparations showed that all but one protein displayed a clear antibody response in serum samples from 278 tuberculosis patients. CONCLUSION AND CLINICAL RELEVANCE: The established workflow comprises a simple, cost-effective, and scalable pipeline for production of soluble proteins from Mtb and can be used to prioritize immunogenic proteins suitable for use as diagnostic markers.

A novel class of self-sufficient cytochrome P450 monooxygenases in prokaryotes.

The Bacillus cytochrome P450 BM3 integrates an entire P450 system in one polypeptide and represents a convenient prokaryotic model for microsomal P450s. This self-sufficient class II P450 is also present in actinomycetes and fungi. By genome analysis we have identified additional homologues in the pathogenic species Bacillus anthracis and Bacillus cereus, and in Ralstonia metallidurans. This analysis also revealed a novel class of putative self-sufficient P450s, P450 PFOR, comprising a class I P450 that is related to Rhodococcus erythropolis CYP116, and a phthalate family oxygenase reductase (PFOR) module. P450 PFOR genes are found in a Rhodococcus strain, three pathogenic Burkholderia species and in the R. metallidurans strain that possesses a P450 BM3 homologue. Co-evolution of P450 and reductase domains is apparent in both types of self-sufficient enzymes. The new class of P450 enzymes is of potential interest for various biotechnological applications.

Stress-related Pseudomonas genes involved in production of bacteriocin LlpA.

Pseudomonas sp. BW11M1 produces a novel type of bacteriocin that inhibits the growth of Pseudomonas putida GR12-2R3 and some phytopathogenic fluorescent Pseudomonas. A collection of mutants was screened for altered bacteriocin production phenotypes. Strongly reduced bacteriocin production was found to be caused by inactivation of the recA gene or the spoT gene. Conversely, in a recJ mutant, the bacteriocin was constitutively overproduced. The same phenotype was observed for a mutant hit in a gene of unknown function. The predicted gene product belongs to a distinct subgroup of prokaryotic helicase-like proteins within the SWI/SNF family of regulatory proteins. One mutant that also exhibited a bacteriocin overproducer phenotype was deficient in the production of the peptidoglycan-associated lipoprotein OprL. This study shows that various environmental stress response pathways are involved in controlling expression of the Pseudomonas sp. BW11M1 bacteriocin.

The thiocarbamate-inducible Rhodococcus enzyme ThcF as a member of the family of alpha/beta hydrolases with haloperoxidative side activity.

Purified thiocarbamate-inducible ThcF of Rhodococcus erythropolis NI86/21, overexpressed in Escherichia coli, displayed several characteristics of the HASH family of enzymes that groups prokaryotic proteins of the alpha/beta hydrolase superfamily possessing serine-dependent hydrolase and/or haloperoxidase activity. Kinetic analysis of bromination and ester hydrolysis revealed a low affinity of ThcF for model substrates. Sulfoxidation of thiocarbamates was demonstrated but probably represents a side activity due to peroxoacid generation by the enzyme. The thcF-linked thcG gene, encoding a LAL-type regulator, triggers expression of thcF in Rhodococcus. The tandem gene organization thcG-thcF is conserved in the thiocarbamate-degrading strain Rhodococcus sp. B30. It is proposed that HASH enzymes may be involved in the metabolism of plant-derived compounds.

Novel lectin-like bacteriocins of biocontrol strain Pseudomonas fluorescens Pf-5.

Bacteriocin LlpA, produced by Pseudomonas sp. strain BW11M1, is a peculiar antibacterial protein due to its homology to mannose-binding lectins mostly found in monocots (A. H. A. Parret, G. Schoofs, P. Proost, and R. De Mot, J. Bacteriol. 185:897-908, 2003). Biocontrol strain Pseudomonas fluorescens Pf-5 contains two llpA-like genes, named llpA1(Pf-5) and llpA2(Pf-5). Recombinant Escherichia coli cells expressing llpA1(Pf-5) or llpA2(Pf-5) acquired bacteriocin activity and secreted a 31-kDa protein cross-reacting with LlpA(BW11M1) antibodies. Antibacterial activity of the recombinant proteins was evidenced by gel overlay assays. Analysis of the antimicrobial spectrum indicated that LlpA1(Pf-5) and LlpA2(Pf-5) are able to inhibit P. fluorescens strains, as well as the related mushroom pathogen Pseudomonas tolaasii. LlpA-type bacteriocins are characterized by a domain structure consisting of tandem monocot mannose-binding lectin (MMBL) domains. Molecular phylogeny of these MMBL domains suggests that the individual MMBL domains within an LlpA protein have evolved separately toward a specific, as yet unknown, function or, alternatively, were acquired from different ancestral sources. Our observations are consistent with earlier observations, which hinted that MMBL-like bacteriocins represent a new family of antibacterial proteins, probably with a novel mode of action.

Plant lectin-like bacteriocin from a rhizosphere-colonizing Pseudomonas isolate.

Rhizosphere isolate Pseudomonas sp. strain BW11M1, which belongs to the Pseudomonas putida cluster, secretes a heat- and protease-sensitive bacteriocin which kills P. putida GR12-2R3. The production of this bacteriocin is enhanced by DNA-damaging treatment of producer cells. We isolated a TnMod mutant of strain BW11M1 that had lost the capacity to inhibit the growth of strain GR12-2R3. A wild-type genomic fragment encompassing the transposon insertion site was shown to confer the bacteriocin phenotype when it was introduced into Escherichia coli cells. The bacteriocin structural gene was identified by defining the minimal region required for expression in E. coli. This gene was designated llpA (lectin-like putidacin) on the basis of significant homology of its 276-amino-acid product with mannose-binding lectins from monocotyledonous plants. LlpA is composed of two monocot mannose-binding lectin (MMBL) domains. Several uncharacterized bacterial genes encoding diverse proteins containing one or two MMBL domains were identified. A phylogenetic analysis of the MMBL domains present in eukaryotic and prokaryotic proteins assigned the putidacin domains to a new bacterial clade within the MMBL-containing protein family. Heterologous expression of the llpA gene also conveyed bacteriocin production to several Pseudomonas fluorescens strains. In addition, we demonstrated that strain BW11M1 and heterologous hosts secrete LlpA into the growth medium without requiring a cleavable signal sequence. Most likely, the mode of action of this lectin-like bacteriocin is different from the modes of action of previously described Pseudomonas bacteriocins.

Overexpression, purification and crystallization of bacteriocin LlpA from Pseudomonas sp. BW11M1.

LlpA is a bacteriocin produced by Pseudomonas sp. BW11M1 that shows remarkable similarity to a family of mannose-binding plant lectins. A His-tagged version of LlpA was recombinantly produced in Escherichia coli and purified to homogeneity. Single crystals were grown by vapour diffusion and belong to space group P2(1)2(1)2, with unit-cell parameters a = 150.5, b = 154.5, c = 34.2 A. The crystals diffract to at least 2.2 A using synchrotron radiation.

The N-terminal coiled coil of the Rhodococcus erythropolis ARC AAA ATPase is neither necessary for oligomerization nor nucleotide hydrolysis.

Deletion mutants of the Rhodococcus erythropolis ARC AAA ATPase were generated and characterized by biochemical analysis and electron microscopy. Based on sequence comparisons the ARC protein was divided into three consecutive regions, the N-terminal coiled coil, the central ARC-specific inter domain and the C-terminal AAA domain. When the ARC AAA domain was expressed separately it formed aggregates of undefined structure. However, when the AAA domain was expressed in conjunction with the preceeding inter domain, but without the N-terminal coiled coil, high-molecular weight-complexes were formed (ARC-DeltaCC) which showed an N-ethylmaleimide-sensitive ATPase activity. In 2D crystallization experiments the ARC-DeltaCC particles yielded crystals nearly identical to those formed by the wild-type ARC complexes. Thus, the N-terminal coiled coil, which was proposed to have a role in the assembly of and/or interaction between the eukaryotic AAA ATPases in the 26S proteasome, is neither essential for assembly nor for ATP hydrolysis of the ARC ATPase. The N-terminal domain of related AAA ATPases mediates the interaction with substrates or co-factors, suggesting a regulatory function for the N-terminal coiled coil of the ARC ATPase. Surprisingly, the mutant ARC protein ARC-DeltaAAA consisting of the N-terminal coiled coil and the central inter domain, but deleted for the C-terminal AAA domain, was shown to form a dodecameric complex with sixfold symmetry. This suggests an important role of the inter domain for the ordered assembly of the ARC ATPase.