Uncovering the roles of Mycobacterium tuberculosis melH in redox and bioenergetic homeostasis: implications for antitubercular therapy.

Mycobacterium tuberculosis (Mtb), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The melH gene in Mtb and Mycobacterium marinum (Mm) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that melH encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of melH in Mm resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of whiB3, a universal stress sensor. The absence of melH also resulted in reduced intracellular levels of NAD+, NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon source dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of melH in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria. IMPORTANCE: This study unveils the pivotal role played by the melH gene in Mycobacterium tuberculosis and in Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para-hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH. Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.

Uncovering the Roles of Mycobacterium tuberculosis melH in Redox and Bioenergetic Homeostasis: Implications for Antitubercular Therapy.

Mycobacterium tuberculosis ( Mtb ), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The melH gene in Mtb and Mycobacterium marinum ( Mm ) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that melH encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of melH in Mm resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of whiB3 , a universal stress sensor. The absence of melH also resulted in reduced intracellular levels of NAD + , NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon-source-dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of melH in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria. Importance: This study unveils the pivotal role played by the melH gene in Mycobacterium tuberculosis and Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para -hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH . Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH 's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.

A Chemoproteomic Approach to Elucidate the Mechanism of Action of 6-Azasteroids with Unique Activity in Mycobacteria.

By illuminating key 6-azasteroid-protein interactions in both Mycobacterium tuberculosis (Mtb) and the closely related model organism Mycobacterium marinum (Mm), we sought to improve the antimycobacterial potency of 6-azasteroids and further our understanding of the mechanisms responsible for their potentiation of the antituberculosis drug bedaquiline. We selected a newly developed 6-azasteroid analog and an analog reported previously (ACS Infect. Dis. 2019, 5 (7), 1239-1251) to study their phenotypic effects on Mtb and Mm, both alone and in combination with bedaquiline. The 6-azasteroid analog, 17ß-[N-(4-trifluoromethoxy-diphenylmethyl)carbamoyl]-6-propyl-azaandrostan-3-one, robustly potentiated bedaquiline-mediated antimycobacterial activity, with a nearly 8-fold reduction in Mm bedaquiline minimal inhibitory concentration (85 nM alone versus 11 nM with 20 µM 6-azasteroid). This analog displayed minimal inhibitory activity against recombinant mycobacterial 3ß-hydroxysteroid dehydrogenase, a previously identified target of several 6-azasteroids. Dose-dependent potentiation of bedaquiline by this analog reduced mycobacterial intracellular ATP levels and impeded the ability of Mtb to neutralize exogenous oxidative stress in culture. We developed two 6-azasteroid photoaffinity probes to investigate azasteroid-protein interactions in Mm whole cells. Using bottom-up mass spectrometric profiling of the cross-linked proteins, we identified eight potential Mm/Mtb protein targets for 6-azasteroids. The nature of these potential targets indicates that proteins related to oxidative stress resistance play a key role in the BDQ-potentiating activity of azasteroids and highlights the potential impact of inhibition of these targets on the generation of drug sensitivity.

Long-Range Kinetic Effects on the Alternating Ring Opening Metathesis of Bicyclo[4.2.0]oct-6-ene-7-carboxamides and Cyclohexene.

We report an investigation of rates of ruthenium-catalyzed alternating ring opening metathesis (AROM) of cyclohexene with two different Ru-cyclohexylidene carbenes derived from bicyclo[4.2.0]oct-6-ene-7-carboxamides (A monomer) that bear different side chains. These monomers are propylbicyclo[4.2.0]oct-6-ene-7-carboxamide and N-(2-(2-ethoxyethoxy)ethanylbicyclo[4.2.0]oct-6-ene-7-carboxamide. The amide substitution of these monomers directly affects both the rate of the bicyclo[4.2.0]oct-6-ene-7-carboxamide ring opening and the rate of reaction of the resulting carbene with cyclohexene (B monomer). The resulting Ru-cyclohexylidenes underwent reversible ring opening metathesis with cyclohexene. However, the thermodynamic equilibrium disfavored cyclohexene ring opening. Utilization of triphenylphosphine forms a more stable PPh3 ligated complex, which suppresses the reverse ring closing reaction and allowed direct measurements of the forward rate constants for formation of various A-B and A-B-A' complexes through carbene-catalyzed ring-opening metathesis and thus gradient polymer structure-determining steps. The relative rate of the propylbicyclo[4.2.0]oct-6-ene-7-carboxamide ring opening is 3-fold faster than that of the N-(2-(2-ethoxyethoxy)ethanylbicyclo[4.2.0]oct-6-ene-7-carboxamide. In addition, the rate of cyclohexene ring-opening catalyzed by the propyl bicyclooctene is 1.4 times faster than when catalyzed by the ethoxyethoxy bicyclooctene. Also, the subsequent rates of bicyclo[4.2.0]oct-6-ene-7-carboxamide ring opening by propyl-based Ru-hexylidene are 1.6-fold faster than ethoxyethoxy-based Ru-hexylidene. Incorporation of the rate constants into reactivity ratios of bicyclo[4.2.0]amide-cyclohexene provides prediction of copolymerization kinetics and gradient copolymer structures.

Exploring the value of Mycobacterium tuberculosis modified lipoprotein as a potential biomarker for TB detection in children.

BACKGROUND: Current TB diagnostic methods available have been developed for adults and development efforts have neglected the differences in disease and sampling that occur between adults and children. Diagnostic challenges are even greater in HIV co-infected children and infants. METHODS AND RESULTS: We established a sandwich ELISA assay to detect Mycobacterium tuberculosis modified lipoprotein (TLP) ex vivo in plasma. The study population contains plasma samples from 21 patients with active TB and 24 control samples with no TB, collected in the International Maternal Pediatric Adolescent AIDS Clinical Trails (IMPAACT) P1041 study. Retrospective analysis was performed and the results demonstrate that the median plasma levels of TLP in control subjects are 2.7 fold higher than the median plasma values in active TB subjects (p < 0.001). CONCLUSIONS: Plasma levels of TLP are elevated with active TB disease in HIV positive subjects and deserves further exploration as an indicator for TB detection in children.

Controlling Rheology of Fluid Interfaces through Microblock Length of Sequence-Controlled Amphiphilic Copolymers.

Previous studies have demonstrated that films of sequence-controlled amphiphilic copolymers display contact angles that depend on microblock size. This suggests that microblock length may provide a means of tuning surface and interfacial properties. In this work, the interfacial rheology of a series of sequence-controlled copolymers, prepared through the addition of bicyclo[4.2.0]oct-1(8)-ene-8-carboxamide (monomer A) and cyclohexene (monomer B) to generate sequences up to 24 monomeric units composed of (A m B n ) i microblocks, where m, n, and i range from 1 to 6. Interfacial rheometry is used to measure the mechanical properties of an air-water interface with these copolymers. As the microblock size increases, the interfacial storage modulus, G', increases, which may be due to an increase in the size of interfacial hydrophobic domains. Small-angle X-ray scattering shows that the copolymers have a similar conformation in solution, suggesting that any variations in the mechanics of the interface are due to assembly at the interface, and not on solution association or bulk rheological properties. This is the first study demonstrating that microblock size can be used to control interfacial rheology of amphiphilic copolymers. Thus, the results provide a new strategy for controlling the dynamics of fluid interfaces through precision sequence-controlled polymers.

Substituent Effects Provide Access to Tetrasubstituted Ring-Opening Olefin Metathesis of Bicyclo[4.2.0]oct-6-enes.

Herein, we report the origin of unexpected reactivity of bicyclo[4.2.0]oct-6-ene substrates containing an α,ß-unsaturated amide moiety in ruthenium-catalyzed alternating ring-opening metathesis polymerization reactions. Specifically, compared with control substrates bearing an ester, alkyl ketone, nitrile, or tertiary amide substituent, α,ß-unsaturated substrates with a weakly acidic proton showed increased rates of ring-opening metathesis mediated by Grubbs-type ruthenium catalysts. 1H NMR and IR spectral analyses indicated that deprotonation of the α,ß-unsaturated amide substrates resulted in stronger coordination of the carbonyl group to the ruthenium metal center. Principal component analysis identified ring strain and the electron density on the carbonyl oxygen (based on structures optimized by means of ωB97X-D/6311+G(2df,2p) calculations) as the two key contributors to fast ring-opening metathesis of the bicyclo[4.2.0]oct-6-enes; whereas the dipole moment, conjugation, and energy of the highest occupied molecular orbital had little to no effect on the reaction rate. We conclude that alternating ring-opening metathesis polymerization reactions of bicyclo[4.2.0]oct-6-enes with unstrained cycloalkenes require an ionizable proton for efficient generation of alternating polymers.

Corrigendum: Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis.

[This corrects the article .].

Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis.

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3ß-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3ß-HSD activity with the 17ß-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3ß-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

The pursuit of mechanism of action: uncovering drug complexity in TB drug discovery.

Whole cell-based phenotypic screens have become the primary mode of hit generation in tuberculosis (TB) drug discovery during the last two decades. Different drug screening models have been developed to mirror the complexity of TB disease in the laboratory. As these culture conditions are becoming more and more sophisticated, unraveling the drug target and the identification of the mechanism of action (MOA) of compounds of interest have additionally become more challenging. A good understanding of MOA is essential for the successful delivery of drug candidates for TB treatment due to the high level of complexity in the interactions between Mycobacterium tuberculosis (Mtb) and the TB drug used to treat the disease. There is no single "standard" protocol to follow and no single approach that is sufficient to fully investigate how a drug restrains Mtb. However, with the recent advancements in -omics technologies, there are multiple strategies that have been developed generally in the field of drug discovery that have been adapted to comprehensively characterize the MOAs of TB drugs in the laboratory. These approaches have led to the successful development of preclinical TB drug candidates, and to a better understanding of the pathogenesis of Mtb infection. In this review, we describe a plethora of efforts based upon genetic, metabolomic, biochemical, and computational approaches to investigate TB drug MOAs. We assess these different platforms for their strengths and limitations in TB drug MOA elucidation in the context of Mtb pathogenesis. With an emphasis on the essentiality of MOA identification, we outline the unmet needs in delivering TB drug candidates and provide direction for further TB drug discovery.

Enzymatic ß-Oxidation of the Cholesterol Side Chain in Mycobacterium tuberculosis Bifurcates Stereospecifically at Hydration of 3-Oxo-cholest-4,22-dien-24-oyl-CoA.

The unique ability of Mycobacterium tuberculosis (Mtb) to utilize host lipids such as cholesterol for survival, persistence, and virulence has made the metabolic pathway of cholesterol an area of great interest for therapeutics development. Herein, we identify and characterize two genes from the Cho-region (genomic locus responsible for cholesterol catabolism) of the Mtb genome, chsH3 (Rv3538) and chsB1 (Rv3502c). Their protein products catalyze two sequential stereospecific hydration and dehydrogenation steps in the ß-oxidation of the cholesterol side chain. ChsH3 favors the 22S hydration of 3-oxo-cholest-4,22-dien-24-oyl-CoA in contrast to the previously reported EchA19 (Rv3516), which catalyzes formation of the (22R)-hydroxy-3-oxo-cholest-4-en-24-oyl-CoA from the same enoyl-CoA substrate. ChsB1 is stereospecific and catalyzes dehydrogenation of the ChsH3 product but not the EchA19 product. The X-ray crystallographic structure of the ChsB1 apo-protein was determined at a resolution of 2.03 Å, and the holo-enzyme with bound NAD+ cofactor was determined at a resolution of 2.21 Å. The homodimeric structure is representative of a classical NAD+-utilizing short-chain type alcohol dehydrogenase/reductase, including a Rossmann-fold motif, but exhibits a unique substrate binding site architecture that is of greater length and width than its homologous counterparts, likely to accommodate the bulky steroid substrate. Intriguingly, Mtb utilizes hydratases from the MaoC-like family in sterol side-chain catabolism in contrast to fatty acid ß-oxidation in other species that utilize the evolutionarily distinct crotonase family of hydratases.

Gradient Copolymer Prepared from Alternating Ring-Opening Metathesis of Three Monomers.

Bicyclo[4.2.0]oct-6-ene-7-carboxamide is a simple but highly strained olefin monomer which forms an alternating copolymer with cyclohexene in the presence of N-heterocyclic carbene-ruthenium catalyst. [4.2.0] moiety with bulky substituent on C7 that chelate with the ruthenium center of the catalyst propagate more slowly than monomers that cannot chelate. Accordingly, the reactivity ratio of N-propylbicyclo[4.2.0]oct-6-ene-7-carboxamide with cyclohexene is significantly higher than that of N-(2-(2-ethoxyethoxy)ethan)-bicyclo[4.2.0]oct-6-ene-7-carboxamide with cyclohexene. A copolymerization involving the three monomers in a 1:1:2 (propyl:ethylene glycol:cyclohexene) molar ratio formed a gradient copolymer in a one-pot reaction. Surface hydrophobicity, topology, and thermal properties of the gradient copolymer were similar to those of a copolymer comprised of six microblocks prepared through multistep synthesis by alternately employing the same two bicyclo[4.2.0]oct-6-ene-7-carboxamides in each microblock. The properties of the gradient copolymer were distinct from a copolymer comprised of two larger blocks based on the same bicyclo[4.2.0]oct-6-ene-7-carboxamides.

Reductive power generated by Mycobacterium leprae through cholesterol oxidation contributes to lipid and ATP synthesis

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3ß-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3ß-HSD activity with the 17ß-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3ß-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

Cationic Amphiphilic Alternating Copolymers with Tunable Morphology.

A series of ionic amphiphilic alternating copolymers were characterized via SAXS, TEM and DLS to help understand factors that could potentially affect self-assembly, including the degree of polymerization, the length of hydrophobic spacers between ionic units, the distance between charged groups and polymer backbone, solvent envrioment and counterions.

Targeting Multiple Binding Sites on Cholera Toxin B with Glycomimetic Polymers Promotes the Formation of Protein-Polymer Aggregates.

The canonical binding site on the B subunit of cholera toxin (CTB) binds to GM1 gangliosides on host cells. However, the recently discovered noncanonical binding site on CTB with affinity for fucosylated molecules has raised the possibility that both sites can be involved in initiating intoxication. Previously, we showed that blocking CTB binding to human and murine small intestine epithelial cells can be increased by simultaneously targeting both binding sites with multivalent norbornene-based glycopolymers [ACS Infect. Dis. 2020, 6, 5, 1192-1203]. However, the mechanistic origin of the increased blocking efficacy was unclear. Herein, we observed that mixing CTB pentamers and glycopolymers that display fucose and galactose sugars results in the formation of large aggregates, which further inhibits binding of CTB to human granulocytes. Dynamic light scattering analysis, small-angle X-ray scattering analysis, transmission electron microscopy, and turbidimetric assays revealed that the facial directionality of CTB promotes interchain cross-linking, which in turn leads to self-assembly of protein-polymer networks. This cross-linking-induced self-assembly occurs only when the glycopolymer system contains both galactose and fucose. In an assay of the glycopolymer's ability to block CTB binding to human granulocytes, we observed a direct correlation between IC50 and self-assembly size. The aggregation mechanism of inhibition proposed herein has potential utility for the development of low-cost macromolecular clinical therapeutics for cholera that do not have exotic architectures and do not require complex synthetic sequences.

Post-translational Succinylation of Mycobacterium tuberculosis Enoyl-CoA Hydratase EchA19 Slows Catalytic Hydration of Cholesterol Catabolite 3-Oxo-chol-4,22-diene-24-oyl-CoA.

Cholesterol is a major carbon source for Mycobacterium tuberculosis (Mtb) during infection, and cholesterol utilization plays a significant role in persistence and virulence within host macrophages. Elucidating the mechanism by which cholesterol is degraded may permit the identification of new therapeutic targets. Here, we characterized EchA19 (Rv3516), an enoyl-CoA hydratase involved in cholesterol side-chain catabolism. Steady-state kinetics assays demonstrated that EchA19 preferentially hydrates cholesterol enoyl-CoA metabolite 3-oxo-chol-4,22-diene-24-oyl-CoA, an intermediate of side-chain ß-oxidation. In addition, succinyl-CoA, a downstream catabolite of propionyl-CoA that forms during cholesterol degradation, covalently modifies targeted mycobacterial proteins, including EchA19. Inspection of a 1.9 Å resolution X-ray crystallography structure of Mtb EchA19 suggests that succinylation of Lys132 and Lys139 may perturb enzymatic activity by modifying the entrance to the substrate binding site. Treatment of EchA19 with succinyl-CoA revealed that these two residues are hotspots for succinylation. Replacement of these specific lysine residues with negatively charged glutamate reduced the rate of catalytic hydration of 3-oxo-chol-4,22-diene-24-oyl-CoA by EchA19, as does succinylation of EchA19. Our findings suggest that succinylation is a negative feedback regulator of cholesterol metabolism, thereby adding another layer of complexity to Mtb physiology in the host. These regulatory pathways are potential noncatabolic targets for antimicrobial drugs.

Fucose-Galactose Polymers Inhibit Cholera Toxin Binding to Fucosylated Structures and Galactose-Dependent Intoxication of Human Enteroids.

A promising strategy to limit cholera severity involves blockers mimicking the canonical cholera toxin ligand (CT) ganglioside GM1. However, to date the efficacies of most of these blockers have been evaluated in noncellular systems that lack ligands other than GM1. Importantly, the CT B subunit (CTB) has a noncanonical site that binds fucosylated structures, which in contrast to GM1 are highly expressed in the human intestine. Here we evaluate the capacity of norbornene polymers displaying galactose and/or fucose to block CTB binding to immobilized protein-linked glycan structures and also to primary human and murine small intestine epithelial cells (SI ECs). We show that the binding of CTB to human SI ECs is largely dependent on the noncanonical binding site, and interference with the canonical site has a limited effect while the opposite is observed with murine SI ECs. The galactose-fucose polymer blocks binding to fucosylated glycans but not to GM1. However, the preincubation of CT with the galactose-fucose polymer only partially blocks toxic effects on cultured human enteroid cells, while preincubation with GM1 completely blocks CT-mediated secretion. Our results support a model whereby the binding of fucose to the noncanonical site places CT in close proximity to scarcely expressed galactose receptors such as GM1 to enable binding via the canonical site leading to CT internalization and intoxication. Our finding also highlights the importance of complementing CTB binding studies with functional intoxication studies when assessing the efficacy inhibitors of CT.

IpdE1-IpdE2 Is a Heterotetrameric Acyl Coenzyme A Dehydrogenase That Is Widely Distributed in Steroid-Degrading Bacteria.

Steroid-degrading bacteria, including Mycobacterium tuberculosis (Mtb), utilize an architecturally distinct subfamily of acyl coenzyme A dehydrogenases (ACADs) for steroid catabolism. These ACADs are α2ß2 heterotetramers that are usually encoded by adjacent fadE-like genes. In mycobacteria, ipdE1 and ipdE2 (formerly fadE30 and fadE33) occur in divergently transcribed operons associated with the catabolism of 3aα-H-4α(3'-propanoate)-7aß-methylhexahydro-1,5-indanedione (HIP), a steroid metabolite. In Mycobacterium smegmatis, ΔipdE1 and ΔipdE2 mutants had similar phenotypes, showing impaired growth on cholesterol and accumulating 5-OH HIP in the culture supernatant. Bioinformatic analyses revealed that IpdE1 and IpdE2 share many of the features of the α- and ß-subunits, respectively, of heterotetrameric ACADs that are encoded by adjacent genes in many steroid-degrading proteobacteria. When coproduced in a rhodococcal strain, IpdE1 and IpdE2 of Mtb formed a complex that catalyzed the dehydrogenation of 5OH-HIP coenzyme A (5OH-HIP-CoA) to 5OH-3aα-H-4α(3'-prop-1-enoate)-7aß-methylhexa-hydro-1,5-indanedione coenzyme A ((E)-5OH-HIPE-CoA). This corresponds to the initial step in the pathway that leads to degradation of steroid C and D rings via ß-oxidation. Small-angle X-ray scattering revealed that the IpdE1-IpdE2 complex was an α2ß2 heterotetramer typical of other ACADs involved in steroid catabolism. These results provide insight into an important class of steroid catabolic enzymes and a potential virulence determinant in Mtb.

Alternating Ring-Opening Metathesis Polymerization Provides Easy Access to Functional and Fully Degradable Polymers.

Polymers with hydrolyzable groups in their backbones have numerous potential applications in biomedicine, lithography, energy storage and electronics. In this study, acetal and ester functionalities were incorporated into the backbones of copolymers by means of alternating ring-opening metathesis polymerization catalyzed by third-generation Grubbs ruthenium catalyst. Specifically, combining large-ring (7-10 atoms) cyclic acetal or lactone monomers with bicyclo[4.2.0]oct-1(8)-ene-8-carboxamide monomers provided perfectly alternating copolymers with acetal or ester functionality in the backbones and low to moderate molecular weight distribution (D M = 1.2-1.6). Copolymers containing ester and acetal backbones hydrolyzed to significant extent under basic condition (pH 13) and acidic conditions (pH ≤ 5) respectively to yield the expected by-products within 30 hours at moderate temperature. Unlike the copolymer with all-carbon backbone, copolymers with heteroatom-containing backbone exhibited viscoelastic behavior with crossover frequency which decreases as the size of the R group on the acetal increases. In contrast, the glass transition temperature (T g) decreases as the size of the R group decreases. The rate of hydrolysis of the acetal copolymers was also dependent on the R group. Thus, ruthenium-catalyzed alternating ring-opening metathesis copolymerization provides heterofunctional copolymers whose degradation rates, glass transition temperatures, and viscoelastic moduli can be controlled.

Mycobacterium tuberculosis Exploits a Heterohexameric Enoyl-CoA Hydratase Retro-Aldolase Complex for Cholesterol Catabolism.

Cholesterol catabolism plays an important role in Mycobacterium tuberculosis's (Mtb's) survival and persistence in the host. Mtb exploits three ß-oxidation cycles to fully degrade the side chain of cholesterol. Five cistronic genes in a single operon encode three enzymes, 3-oxo-4-pregnene-20-carboxyl-CoA dehydrogenase (ChsE1-ChsE2), 3-oxo-4,17-pregnadiene-20-carboxyl-CoA hydratase (ChsH1-ChsH2), and 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA retro-aldolase (Ltp2), to perform the last ß-oxidation cycle in this pathway. Among these three enzymes, ChsH1-ChsH2 and Ltp2 form a protein complex that is required for the catalysis of carbon-carbon bond cleavage. In this work, we report the structure of the full length ChsH1-ChsH2-Ltp2 complex based on small-angle X-ray scattering and single-particle electron microscopy data. Mutagenesis experiments confirm the requirement for Ltp2 to catalyze the retro-aldol reaction. The structure illustrates how acyl transfer between enzymes may occur. Each protomer of the ChsH1-ChsH2-Ltp2 complex contains three protein components: a chain of ChsH1, a chain of ChsH2, and a chain of Ltp2. Two protomers dimerize at the interface of Ltp2 to form a heterohexameric structure. This unique heterohexameric structure of the ChsH1-ChsH2-Ltp2 complex provides entry to further understand the mechanism of cholesterol catabolism in Mtb.