Mycobacterium abscessus Infections in Cystic Fibrosis Individuals: A Review on Therapeutic Options.

Mycobacterium abscessus is an opportunistic pathogen that mainly colonizes and infects cystic fibrosis patients' lungs. M. abscessus is naturally resistant to many antibiotics such as rifamycin, tetracyclines and ß-lactams. The current therapeutic regimens are not very effective and are mostly based on repurposed drugs used against Mycobacterium tuberculosis infections. Thus, new approaches and novel strategies are urgently needed. This review aims to provide an overview of the latest ongoing findings to fight M. abscessus infections by analyzing emerging and alternative treatments, novel drug delivery strategies, and innovative molecules.

PEGylated mucus-penetrating nanocrystals for lung delivery of a new FtsZ inhibitor against Burkholderia cenocepacia infection.

C109 is a potent but poorly soluble FtsZ inhibitor displaying promising activity against Burkholderia cenocepacia, a high-risk pathogen for cystic fibrosis (CF) sufferers. To harness C109 for inhalation, we developed nanocrystal-embedded dry powders for inhalation suspension consisting in C109 nanocrystals stabilized with D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) embedded in hydroxypropyl-ß-cyclodextrin (CD). The powders could be safely re-dispersed in water for in vitro aerosolization. Owing to the presence of a PEG shell, the rod shape and the peculiar aspect ratio, C109 nanocrystals were able to diffuse through artificial CF mucus. The promising technological features were completed by encouraging in vitro/in vivo effects. The formulations displayed no toxicity towards human bronchial epithelial cells and were active against planktonic and sessile B. cenocepacia strains. The efficacy of C109 nanosuspensions in combination with piperacillin was confirmed in a Galleria mellonella infection model, strengthening their potential for combined therapy of B. cenocepacia lung infections.

Various Evolutionary Trajectories Lead to Loss of the Tobramycin-Potentiating Activity of the Quorum-Sensing Inhibitor Baicalin Hydrate in Burkholderia cenocepacia Biofilms.

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant bacteria. As potentiators do not interfere with essential processes, it has been hypothesized that they are less likely to induce resistance. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop reduced susceptibility toward one such adjuvant, baicalin hydrate (BH). Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to TOB and TOB+BH compared to the start population, and the potentiating effect of BH toward TOB was lost. Whole-genome sequencing was performed to probe which changes were involved in the reduced effect of BH, and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter). No changes in the MIC or MBC of TOB or changes in the number of persister cells were observed. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that B. cenocepacia J2315 biofilms rapidly lose susceptibility toward the antibiotic-potentiating activity of BH and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as mechanisms.

Phenylacetyl Coenzyme A, Not Phenylacetic Acid, Attenuates CepIR-Regulated Virulence in Burkholderia cenocepacia.

During phenylalanine catabolism, phenylacetic acid (PAA) is converted to phenylacetyl coenzyme A (PAA-CoA) by a ligase, PaaK, and then PAA-CoA is epoxidized by a multicomponent monooxygenase, PaaABCDE, before further degradation through the tricarboxylic acid (TCA) cycle. In the opportunistic pathogen Burkholderia cenocepacia, loss of paaABCDE attenuates virulence factor expression, which is under the control of the LuxIR-like quorum sensing (QS) system, CepIR. To further investigate the link between CepIR-regulated virulence and PAA catabolism, we created knockout mutants of the first step of the pathway (PAA-CoA synthesis by PaaK) and characterized them in comparison to a paaABCDE mutant using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and virulence assays. We found that while loss of PaaABCDE decreased virulence, deletion of the paaK genes resulted in a more virulent phenotype than that of the wild-type strain. Deletion of either paaK or paaABCDE led to higher levels of released PAA but no differences in levels of internal accumulation compared to the wild-type level. While we found no evidence of direct cepIR downregulation by PAA-CoA or PAA, a low-virulence cepR mutant reverted to a virulent phenotype upon removal of the paaK genes. On the other hand, removal of paaABCDE in the cepR mutant did not impact its attenuated phenotype. Together, our results suggest an indirect role for PAA-CoA in suppressing B. cenocepacia CepIR-activated virulence.IMPORTANCE The opportunistic pathogen Burkholderia cenocepacia uses a chemical signal process called quorum sensing (QS) to produce virulence factors. In B. cenocepacia, QS relies on the presence of the transcriptional regulator CepR which, upon binding QS signal molecules, activates virulence. In this work, we found that even in the absence of CepR, B. cenocepacia can elicit a pathogenic response if phenylacetyl-CoA, an intermediate of the phenylacetic acid degradation pathway, is not produced. Instead, accumulation of phenylacetyl-CoA appears to attenuate pathogenicity. Therefore, we have discovered that it is possible to trigger virulence in the absence of CepR, challenging the classical view of activation of virulence by this QS mechanism. Our work provides new insight into the relationship between metabolism and virulence in opportunistic bacteria. We propose that in the event that QS signaling molecules cannot accumulate to trigger a pathogenic response, a metabolic signal can still activate virulence in B. cenocepacia.

New insight into structure-activity of furan-based salicylate synthase (MbtI) inhibitors as potential antitubercular agents.

Starting from the analysis of the hypothetical binding mode of our previous furan-based hit (I), we successfully achieved our objective to replace the nitro moiety, leading to the disclosure of a new lead exhibiting a strong activity against MbtI. Our best candidate 1 h displayed a Ki of 8.8 µM and its antimycobacterial activity (MIC99 = 250 µM) is conceivably related to mycobactin biosynthesis inhibition. These results support the hypothesis that 5-phenylfuran-2-carboxylic derivatives are a promising class of MbtI inhibitors.

Quorum Sensing as Antivirulence Target in Cystic Fibrosis Pathogens.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder which leads to the secretion of a viscous mucus layer on the respiratory epithelium that facilitates colonization by various bacterial pathogens. The problem of drug resistance has been reported for all the species able to colonize the lung of CF patients, so alternative treatments are urgently needed. In this context, a valid approach is to investigate new natural and synthetic molecules for their ability to counteract alternative pathways, such as virulence regulating quorum sensing (QS). In this review we describe the pathogens most commonly associated with CF lung infections: Staphylococcus aureus, Pseudomonas aeruginosa, species of the Burkholderia cepacia complex and the emerging pathogens Stenotrophomonas maltophilia, Haemophilus influenzae and non-tuberculous Mycobacteria. For each bacterium, the QS system(s) and the molecules targeting the different components of this pathway are described. The amount of investigations published in the last five years clearly indicate the interest and the expectations on antivirulence therapy as an alternative to classical antibiotics.

Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ.

To streamline the elucidation of antibacterial compounds' mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing. Here, we used Illumina sequencing of transposon insertions to track the competitive fitness of a Burkholderia cenocepacia library containing essential gene knockdowns. Using this method, we characterized a novel benzothiadiazole derivative, 10126109 (C109), with antibacterial activity against B. cenocepacia, for which whole-genome sequencing of low-frequency spontaneous drug-resistant mutants had failed to identify the drug target. By combining the identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring green fluorescent protein (GFP) cell division protein fusions revealed that C109 prevents divisome formation by altering the localization of the essential cell division protein FtsZ. In agreement with this, C109 inhibited both the GTPase and polymerization activities of purified B. cenocepacia FtsZ. C109 displayed antibacterial activity against Gram-positive and Gram-negative cystic fibrosis pathogens, including Mycobacterium abscessus C109 effectively cleared B. cenocepacia infection in the Caenorhabditis elegans model and exhibited additive interactions with clinically relevant antibiotics. Hence, C109 is an enticing candidate for further drug development.

The Crystal Structure of Burkholderia cenocepacia DfsA Provides Insights into Substrate Recognition and Quorum Sensing Fatty Acid Biosynthesis.

Burkholderia cenocepacia is a major concern among respiratory tract infections in cystic fibrosis patients. This pathogen is particularly difficult to treat because of its high level of resistance to the clinically relevant antimicrobial agents. In B. cenocepacia, the quorum sensing cell-cell communication system is involved in different processes that are important for bacterial virulence, such as biofilm formation and protease and siderophore production. Targeting the enzymes involved in this process represents a promising therapeutic approach. With the aim of finding effective quorum sensing inhibitors, we have determined the three-dimensional structure of B. cenocepacia diffusible factor synthase A, DfsA. This bifunctional crotonase (dehydratase/thioesterase) produces the characteristic quorum sensing molecule of B. cenocepacia, cis-2-dodecenoic acid or BDSF, starting from 3-hydroxydodecanoyl-acyl carrier protein. Unexpectedly, the crystal structure revealed the presence of a lipid molecule in the catalytic site of the enzyme, which was identified as dodecanoic acid. Our biochemical characterization shows that DfsA is able to use dodecanoyl-acyl carrier protein as a substrate, demonstrating that dodecanoic acid, the product of this reaction, is released very slowly from the DfsA active site, therefore acting as a DfsA inhibitor. This molecule shows an unprecedented conformational arrangement inside the DfsA active site. In contrast with previous hypotheses, our data illustrate how DfsA and closely related homologous enzymes can recognize long hydrophobic substrates without large conformational changes or assistance by additional regulator molecules. The elucidation of the substrate binding mode in DfsA provides the starting point for structure-based drug discovery studies targeting B. cenocepacia quorum sensing-assisted virulence.

The Redox State Regulates the Conformation of Rv2466c to Activate the Antitubercular Prodrug TP053.

Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin-fold with a Cys(19)-Pro(20)-Trp(21)-Cys(22) motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle x-ray scattering showed that the fully reduced form of Rv2466c adopts a "closed" compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an "open" conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical, and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.

Mechanism of action of 5-nitrothiophenes against Mycobacterium tuberculosis.

On using the streptomycin-starved 18b strain as a model for nonreplicating Mycobacterium tuberculosis, we identified a 5-nitrothiophene compound as highly active but not cytotoxic. Mutants resistant to 5-nitrothiophenes were found be cross-resistant to the nitroimidazole PA-824 and unable to produce the F420 cofactor. Furthermore, 5-nitrothiophenes were shown to be activated by the F420-dependent nitroreductase Ddn and to release nitric oxide, a mechanism of action identical to that described for nitroimidazoles.

Biochemistry of lipolytic enzymes secreted by Penicillium solitum and Cladosporium cladosporioides.

Two distinct extracellular lipases were obtained from Penicillium solitum 194A, isolated from domestic compost, and Cladosporium cladosporioides 194B, isolated from dairy wastewater. These alkaline enzymes had molecular masses of 42 and 30 kDa, respectively. The P. solitum 194A lipase differed in mass from previously reported enzyme, indicating that it is a novel lipase, and indicating that penicillia can secrete lipase isoenzymes. The C. cladosporioides lipase was more active on esters of medium-chain acids, whereas the P. solitum lipase was more active on longer chained substrates. The C. cladosporioides enzyme displayed higher thermal stability than the P. solitum lipase, preserving full activity up to 48 °C and showing a T50 (10 min) of 60 °C. Their different catalytic properties and good protein stability should make these enzymes suitable for biotechnological applications. Furthermore, the combined use of these two fungal strains may prove to be valuable in lipid-rich waste management.

Benzothiozinone derivatives with anti-tubercular Activity-Further side chain investigation.

A series of novel benzothiozinone (BTZ) derivatives were designed, prepared and evaluated for antituberculosis activity. Specifically, the BTZ pharmacophore is retained and the previous heterocyclic ring linker is replaced by alkynyl or vinyl linker, the resulting compounds displayed about 5-fold improved antimycobacterial activity. We further revealed that the linker attached tail group affects the compound metabolic stability, potency and other drug like properties. This work led to the discovery of two compounds (A1 and A11) with acceptable low MICs and improved metabolic stability. The representative compound A11 demonstrated bactericidal efficacy in an acute TB infection mouse model.

Structural basis for specific inhibition of salicylate synthase from Mycobacterium abscessus.

Blocking iron uptake and metabolism has been emerging as a promising therapeutic strategy for the development of novel antimicrobial compounds. Like all mycobacteria, M. abscessus (Mab) has evolved several countermeasures to scavenge iron from host carrier proteins, including the production of siderophores, which play a crucial role in these processes. In this study, we solved, for the first time, the crystal structure of Mab-SaS, the first enzyme involved in the biosynthesis of siderophores. Moreover, we screened a small, focused library and identified a compound exhibiting a potent inhibitory effect against Mab-SaS (IC50 ≈ 2 µM). Its binding mode was investigated by means of Induced Fit Docking simulations, performed on the crystal structure presented herein. Furthermore, cytotoxicity data and pharmacokinetic predictions revealed the safety and drug-likeness of this class of compounds. Finally, the crystallographic data were used to optimize the model for future virtual screening campaigns. Taken together, the findings of our study pave the way for the identification of potent Mab-SaS inhibitors, based on both established and unexplored chemotypes.

The novel drug candidate VOMG kills Mycobacterium abscessus and other pathogens by inhibiting cell division.

AIMS: The incidence of lung infections is increasing worldwide in individuals suffering from cystic fibrosis and chronic obstructive pulmonary disease. Mycobacterium abscessus is associated with chronic lung deterioration in these populations. The intrinsic resistance of M. abscessus to most conventional antibiotics jeopardizes treatment success rates. To date, no single drug has been developed targeting M. abscessus specifically. The objective of this study was to characterize VOMG, a pyrithione-core drug-like small molecule, as a new compound active against M. abscessus and other pathogens. METHODS: A multi-disciplinary approach including microbiological, chemical, biochemical and transcriptomics procedures was used to validate VOMG as a promising anti-M. abscessus drug candidate. RESULTS: To the authors' knowledge, this is the first study to report the in-vitro and in-vivo bactericidal activity of VOMG against M. abscessus and other pathogens. Besides being active against M. abscessus biofilm, the compound showed a favourable pharmacological (ADME-Tox) profile. Frequency of resistance studies were unable to isolate resistant mutants. VOMG inhibits cell division, particularly the FtsZ enzyme. CONCLUSIONS: VOMG is a new drug-like molecule active against M. abscessus, inhibiting cell division with broad-spectrum activity against other microbial pathogens.

Structural and energetic basis of protein kinetic destabilization in human phosphoglycerate kinase 1 deficiency.

Protein kinetic destabilization is a common feature of many human genetic diseases. Human phosphoglycerate kinase 1 (PGK1) deficiency is a rare genetic disease caused by mutations in the PGK1 protein, which often shows reduced kinetic stability. In this work, we have performed an in-depth characterization of the thermal stability of the wild type and four disease-causing mutants (I47N, L89P, E252A, and T378P) of human PGK1. PGK1 thermal denaturation is a process under kinetic control, and it is described well by a two-state irreversible denaturation model. Kinetic analysis of differential scanning calorimetry profiles shows that the disease-causing mutations decrease PGK1 kinetic stability from ~5-fold (E252A) to ~100000-fold (L89P) compared to that of wild-type PGK1, and in some cases, mutant enzymes are denatured on a time scale of a few minutes at physiological temperature. We show that changes in protein kinetic stability are associated with large differences in enthalpic and entropic contributions to denaturation free energy barriers. It is also shown that the denaturation transition state becomes more nativelike in terms of solvent exposure as the protein is destabilized by mutations (Hammond effect). Unfolding experiments with urea further suggest a scenario in which the thermodynamic stability of PGK1 at least partly determines its kinetic stability. ATP and ADP kinetically stabilize PGK1 enzymes, and kinetic stabilization is nucleotide- and mutant-selective. Overall, our data provide insight into the structural and energetic basis underlying the low kinetic stability displayed by some mutants causing human PGK1 deficiency, which may have important implications for the development of native state kinetic stabilizers for the treatment of this disease.

BEL ß-trefoil: a novel lectin with antineoplastic properties in king bolete (Boletus edulis) mushrooms.

A novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, also called porcino, cep or penny bun). The lectin was structurally characterized i.e its amino acid sequence and three-dimensional structure were determined. The new protein is a homodimer and each protomer folds as ß-trefoil domain and therefore we propose the name Boletus edulis lectin (BEL) ß-trefoil to distinguish it from the other lectin that has been described in these mushrooms. The lectin has potent anti-proliferative effects on human cancer cells, which confers to it an interesting therapeutic potential as an antineoplastic agent. Several crystal forms of the apoprotein and of complexes with different carbohydrates were studied by X-ray diffraction. The structure of the apoprotein was solved at 1.12 Å resolution. The interaction of the lectin with lactose, galactose, N-acetylgalactosamine and T-antigen disaccharide, Galß1-3GalNAc, was examined in detail. All the three potential binding sites present in the ß-trefoil fold are occupied in at least one crystal form and are described in detail in this paper. No important conformational changes are observed in the lectin when comparing its co-crystals with carbohydrates with those of the ligand-free protein.

Improving Protection to Prevent Bacterial Infections: Preliminary Applications of Reverse Vaccinology against the Main Cystic Fibrosis Pathogens.

Reverse vaccinology is a powerful tool that was recently used to develop vaccines starting from a pathogen genome. Some bacterial infections have the necessity to be prevented then treated. For example, individuals with chronic pulmonary diseases, such as Cystic Fibrosis, are prone to develop infections and biofilms in the thick mucus that covers their lungs, mainly caused by Burkholderia cepacia complex, Haemophilus influenzae, Mycobacterium abscessus complex, Pseudomonas aeruginosa and Staphylococcus aureus. These infections are complicated to treat and prevention remains the best strategy. Despite the availability of vaccines against some strains of those pathogens, it is necessary to improve the immunization of people with Cystic Fibrosis against all of them. An effective approach is to develop a broad-spectrum vaccine to utilize proteins that are well conserved across different species. In this context, reverse vaccinology, a method based on computational analysis of the genome of various microorganisms, appears as one of the most promising tools for the identification of putative targets for broad-spectrum vaccine development. This review provides an overview of the vaccines that are under development by reverse vaccinology against the aforementioned pathogens, as well as the progress made so far.

Structural Study of a New MbtI-Inhibitor Complex: Towards an Optimized Model for Structure-Based Drug Discovery.

MbtI from Mycobacterium tuberculosis (Mtb) is a Mg2+-dependent salicylate synthase, belonging to the chorismate-utilizing enzyme (CUE) family. As a fundamental player in iron acquisition, MbtI promotes the survival and pathogenicity of Mtb in the infected host. Hence, it has emerged in the last decade as an innovative, potential target for the anti-virulence therapy of tuberculosis. In this context, 5-phenylfuran-2-carboxylic acids have been identified as potent MbtI inhibitors. The first co-crystal structure of MbtI in complex with a member of this class was described in 2020, showing the enzyme adopting an open configuration. Due to the high mobility of the loop adjacent to the binding pocket, large portions of the amino acid chain were not defined in the electron density map, hindering computational efforts aimed at structure-driven ligand optimization. Herein, we report a new, high-resolution co-crystal structure of MbtI with a furan-based derivative, in which the closed configuration of the enzyme allowed tracing the entirety of the active site pocket in the presence of the bound inhibitor. Moreover, we describe a new crystal structure of MbtI in open conformation and in complex with the known inhibitor methyl-AMT, suggesting that in vitro potency is not related to the observed enzyme conformation. These findings will prove fundamental to enhance the potency of this series via rational structure-based drug-design approaches.

Targeting Siderophore-Mediated Iron Uptake in M. abscessus: A New Strategy to Limit the Virulence of Non-Tuberculous Mycobacteria.

Targeting pathogenic mechanisms, rather than essential processes, represents a very attractive approach for the development of new antimycobacterial drugs. In this context, iron acquisition routes have recently emerged as potentially druggable pathways. However, the importance of siderophore biosynthesis in the virulence and pathogenicity of M. abscessus (Mab) is still poorly understood. In this study, we investigated the Salicylate Synthase (SaS) of Mab as an innovative molecular target for the development of inhibitors of siderophore production. Notably, Mab-SaS does not have any counterpart in human cells, making it an interesting candidate for drug discovery. Starting from the analysis of the binding of a series of furan-based derivatives, previously identified by our group as inhibitors of MbtI from M. tuberculosis (Mtb), we successfully selected the lead compound 1, exhibiting a strong activity against Mab-SaS (IC50 ≈ 5 µM). Computational studies characterized the key interactions between 1 and the enzyme, highlighting the important roles of Y387, G421, and K207, the latter being one of the residues involved in the first step of the catalytic reaction. These results support the hypothesis that 5-phenylfuran-2-carboxylic acids are also a promising class of Mab-SaS inhibitors, paving the way for the optimization and rational design of more potent derivatives.

Side Chain-Modified Benzothiazinone Derivatives with Anti-Mycobacterial Activity.

Tuberculosis (TB) is a leading infectious disease with serious antibiotic resistance. The benzothiazinone (BTZ) scaffold PBTZ169 kills Mycobacterium tuberculosis (Mtb) through the inhibition of the essential cell wall enzyme decaprenylphosphoryl-ß-D-ribose 2'-oxidase (DprE1). PBTZ169 shows anti-TB potential in animal models and pilot clinical tests. Although highly potent, the BTZ type DprE1 inhibitors in general show extremely low aqueous solubility, which adversely affects the drug-like properties. To improve the compounds physicochemical properties, we generated a series of BTZ analogues. Several optimized compounds had MIC values against Mtb lower than 0.01 µM. The representative compound 37 displays improved solubility and bioavailability compared to the lead compound. Additionally, compound 37 shows Mtb-killing ability in an acute infection mouse model.