Malgorzata Korycka-Machala

Polycations increase the permeability of Mycobacterium vaccae cell envelopes to hydrophobic compounds

Polycations [protamine, polymyxin B nonapeptide (PMBN) and polyethyleneimine (PEI)] have been shown to increase the cell wall permeability of Mycobacterium vaccae to highly hydrophobic compounds, as manifested in enhanced intracellular bioconversion of beta-sitosterol to 4-androsten-3,17-dione (AD) and 1,4-androstadien-3,17-dione (ADD), and cell sensitization to erythromycin and rifampicin. The quantity of AD(D) formed per biomass unit was twice as high in the presence of PMBN and PEI, and three times higher with protamine. The sensitization factor, i.e. the MIC(50) ratio of the control bacteria to those exposed to polycations, ranged from 4 to 16, depending on the polycation/antibiotic combination. Non-covalently bound free lipids were extracted from the control and polycation-treated cells and fractionated with the use of chloroform, acetone and methanol. Chloroform- and acetone-eluted fractions (mainly neutral lipids and glycolipids, respectively) showed significant polycation-induced alterations in their quantitative and qualitative composition. The fatty acid profile of neutral lipids was reduced in comparison to control, whereas acetone-derived lipids were characterized by a much higher level of octadecenoic acid (C(18:1)) and a considerably lower content of docosanoic acid (C(22:0)), the marker compound of mycolate-containing glycolipids. Methanol-eluted fractions remained unaltered. Cell-wall-linked mycolates obtained from delipidated cells were apparently unaffected by the action of polycations, as judged from the TLC pattern of mycolic acid subclasses, the mean weight of mycolate preparations and the C(22:0) acid content in the mycolates, determined by GC/MS and pyrolysis GC. The results suggest the involvement of the components of non-covalently bound lipids in the outer layer in the M. vaccae permeability barrier.

Evaluation of NAD+-Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics

ABSTRACT Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD+-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD+-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD+-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD+-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria.

Alterations in lipid composition of Mycobacterium vaccae cell wall outer layer enhance β-sitosterol degradation

The rate of transformation of β-sitosterol to 4-androsten-3,17-dione (AD) by Mycobacterium vaccae increased considerably in the presence of D,L-norleucine and m-fluorophenylalanine. These compounds inhibit the biosynthesis of the complex lipids in the cell wall outermost layer. Non-covalently linked (free) lipids were extracted from control and inhibitor-treated cells, and the fatty acid patterns were determined in the neutral lipid, glycolipid and phospholipid fractions. Profound differences were revealed in both the fatty acid composition and the quantities of the individual components in each analysed fraction, derived from the cells exposed to the action of the inhibitors. Partial disorganization of the outermost layer of M. vaccae cell is assumed to alter the cell wall permeability to β-sitosterol and to enhance its biotransformation.

Evaluation of DNA Primase DnaG as a Potential Target for Antibiotics

ABSTRACT Mycobacteria contain genes for several DNA-dependent RNA primases, including dnaG, which encodes an essential replication enzyme that has been proposed as a target for antituberculosis compounds. An in silico analysis revealed that mycobacteria also possess archaeo-eukaryotic superfamily primases (AEPs) of unknown function. Using a homologous recombination system, we obtained direct evidence that wild-type dnaG cannot be deleted from the chromosome of Mycobacterium smegmatis without disrupting viability, even in backgrounds in which mycobacterial AEPs are overexpressed. In contrast, single-deletion AEP mutants or mutants defective for all four identified M. smegmatis AEP genes did not exhibit growth defects under standard laboratory conditions. Deletion of native dnaG in M. smegmatis was tolerated only after the integration of an extra intact copy of the M. smegmatis or Mycobacterium tuberculosis dnaG gene, under the control of chemically inducible promoters, into the attB site of the chromosome. M. tuberculosis and M. smegmatis DnaG proteins were overproduced and purified, and their primase activities were confirmed using radioactive RNA synthesis assays. The enzymes appeared to be sensitive to known inhibitors (suramin and doxorubicin) of DnaG. Notably, M. smegmatis bacilli appeared to be sensitive to doxorubicin and resistant to suramin. The growth and survival of conditional mutant mycobacterial strains in which DnaG was significantly depleted were only slightly affected under standard laboratory conditions. Thus, although DnaG is essential for mycobacterial viability, only low levels of protein are required for growth. This suggests that very efficient inhibition of enzyme activity would be required for mycobacterial DnaG to be useful as an antibiotic target.

The MM2QM tool for combining docking, molecular dynamics, molecular mechanics, and quantum mechanics.

The use of the MM2QM tool in a combined docking + molecular dynamics (MD) + molecular mechanics (MM) + quantum mechanical (QM) binding affinity prediction study is presented, and the tool itself is discussed. The system of interest is Mycobacterium tuberculosis (MTB) pantothenate synthetase in complexes with three highly similar sulfonamide inhibitors, for which crystal structures are available. Starting from the structure of MTB pantothenate synthetase in the “open” conformation and following the combined docking + MD + MM + QM procedure, we were able to capture the closing of the enzyme binding pocket and to reproduce the position of the ligands with an average root mean square deviation of 1.6 Å. Protein–ligand interaction energies were reproduced with an average error lower than 10%. The discussion on the MD part and a protein flexibility importance is carried out. The presented approach may be useful especially for finding analog inhibitors or improving drug candidates.

Genetic variation of the ABC transporter gene ABCC1 (Multidrug resistance protein 1 – MRP1) in the Polish population

BackgroundMultidrug resistance-associated protein 1 (MRP1), encoded by the ABCC1 gene, is an ATP-binding cassette transporter mediating efflux of organic anions and xenobiotics; its overexpression leads to multidrug resistance. In this study, 30 exons (from 31 in total) of the ABCC1 gene as well as and their flanking intron sequences were screened for genetic variation, using the High Resolution Melting (HRM) method, for 190 healthy volunteers representing the Polish population. Polymorphism screening is an indispensable step in personalized patient therapy. An additional targeted SNP verification study for ten variants was performed to verify sensitivity of the scanning method.ResultsDuring scanning, 46 polymorphisms, including seven novel ones, were found: one in 3’ UTR, 21 in exons (11 of them non-synonymous) and 24 in introns, including one deletion variant. These results revealed some ethnic differences in frequency of several polymorphisms when compared to literature data for other populations. Based on linkage disequilibrium analysis, 4 haplotype blocks were determined for 9 detected polymorphisms and 12 haplotypes were defined. To capture the common haplotypes, haplotype-tagging single nucleotide polymorphisms were identified.ConclusionsTargeted genotyping results correlated well with scanning results; thus, HRM is a suitable method to study genetic variation in this model. HRM is an efficient and sensitive method for scanning and genotyping polymorphic variants. Ethnic differences were found for frequency of some variants in the Polish population compared to others.Thus, this study may be useful for pharmacogenetics of drugs affected by MRP1-mediated efflux.

Direct and Inverted Repeats Elicit Genetic Instability by Both Exploiting and Eluding DNA Double-Strand Break Repair Systems in Mycobacteria

Repetitive DNA sequences with the potential to form alternative DNA conformations, such as slipped structures and cruciforms, can induce genetic instability by promoting replication errors and by serving as a substrate for DNA repair proteins, which may lead to DNA double-strand breaks (DSBs). However, the contribution of each of the DSB repair pathways, homologous recombination (HR), non-homologous end-joining (NHEJ) and single-strand annealing (SSA), to this sort of genetic instability is not fully understood. Herein, we assessed the genome-wide distribution of repetitive DNA sequences in the Mycobacterium smegmatis, Mycobacterium tuberculosis and Escherichia coli genomes, and determined the types and frequencies of genetic instability induced by direct and inverted repeats, both in the presence and in the absence of HR, NHEJ, and SSA. All three genomes are strongly enriched in direct repeats and modestly enriched in inverted repeats. When using chromosomally integrated constructs in M. smegmatis, direct repeats induced the perfect deletion of their intervening sequences ∼1,000-fold above background. Absence of HR further enhanced these perfect deletions, whereas absence of NHEJ or SSA had no influence, suggesting compromised replication fidelity. In contrast, inverted repeats induced perfect deletions only in the absence of SSA. Both direct and inverted repeats stimulated excision of the constructs from the attB integration sites independently of HR, NHEJ, or SSA. With episomal constructs, direct and inverted repeats triggered DNA instability by activating nucleolytic activity, and absence of the DSB repair pathways (in the order NHEJ>HR>SSA) exacerbated this instability. Thus, direct and inverted repeats may elicit genetic instability in mycobacteria by 1) directly interfering with replication fidelity, 2) stimulating the three main DSB repair pathways, and 3) enticing L5 site-specific recombination.

Mycobacterial DNA Replication As a target For Antituberculosis Drug Discovery.

BACKGROUND Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, is a leading infectious disease organism, causing millions of deaths each year. This serious pathogen has been greatly spread worldwide and recent years have observed an increase in the number of multi-drug resistant and totally drug resistant M. tuberculosis strains (WHO report, 2014). The danger of tuberculosis becoming an incurable disease has emphasized the need for the discovery of a new generation of antimicrobial agents. The development of novel alternative medical strategies, new drugs and the search for optimal drug targets are top priority areas of tuberculosis research. FACTORS Key characteristics of mycobacteria include: slow growth, the ability to transform into a metabolically silent - latent state, intrinsic drug resistance and the relatively rapid development of acquired drug resistance. These factors make finding an ideal antituberculosis drug enormously challenging, even if it is designed to treat drug sensitive tuberculosis strains. A vast majority of canonical antibiotics including antituberculosis agents target bacterial cell wall biosynthesis or DNA/RNA processing. Novel therapeutic approaches are being tested to target mycobacterial cell division, twocomponent regulatory factors, lipid synthesis and the transition between the latent and actively growing states. DISCUSSION AND CONCLUSION This review discusses the choice of cellular targets for an antituberculosis therapy, describes putative drug targets evaluated in the recent literature and summarizes potential candidates under clinical and pre-clinical development. We focus on the key cellular process of DNA replication, as a prominent target for future antituberculosis therapy. We describe two main pathways: the biosynthesis of nucleic acids precursors - the nucleotides, and the synthesis of DNA molecules. We summarize data regarding replication associated proteins that are critical for nucleotide synthesis, initiation, unwinding and elongation of the DNA during the replication process. They are pivotal processes required for successful multiplication of the bacterial cells and hence they are extensively investigated for the development of antituberculosis drugs. Finally, we summarize the most potent inhibitors of DNA synthesis and provide an up to date report on their status in the clinical trials.

DNA Ligase C and Prim-PolC participate in base excision repair in mycobacteria

Prokaryotic Ligase D is a conserved DNA repair apparatus processing DNA double-strand breaks in stationary phase. An orthologous Ligase C (LigC) complex also co-exists in many bacterial species but its function is unknown. Here we show that the LigC complex interacts with core BER enzymes in vivo and demonstrate that together these factors constitute an excision repair apparatus capable of repairing damaged bases and abasic sites. The polymerase component, which contains a conserved C-terminal structural loop, preferentially binds to and fills-in short gapped DNA intermediates with RNA and LigC ligates the resulting nicks to complete repair. Components of the LigC complex, like LigD, are expressed upon entry into stationary phase and cells lacking either of these pathways exhibit increased sensitivity to oxidising genotoxins. Together, these findings establish that the LigC complex is directly involved in an excision repair pathway(s) that repairs DNA damage with ribonucleotides during stationary phase.Ligase D is a conserved DNA repair protein complex that repairs double-strand breaks in stationary phase prokaryotes. Here the authors show that orthologous Ligase C has a role in base excision repair during stationary phase.

Naphthalimides Selectively Inhibit the Activity of Bacterial, Replicative DNA Ligases and Display Bactericidal Effects against Tubercle Bacilli

The DNA ligases, enzymes that seal breaks in the backbones of DNA, are essential for all organisms, however bacterial ligases essential for DNA replication use β-nicotinamide adenine dinucleotide as their co-factor, whereas those that are essential in eukaryotes and viruses use adenosine-5′-triphosphate. This fact leads to the conclusion that NAD+-dependent DNA ligases in bacteria could be targeted by their co-factor specific inhibitors. The development of novel alternative medical strategies, including new drugs, are a top priority focus areas for tuberculosis research due to an increase in the number of multi-drug resistant as well as totally drug resistant tubercle bacilli strains. Here, through the use of a virtual high-throughput screen and manual inspection of the top 200 records, 23 compounds were selected for in vitro studies. The selected compounds were evaluated in respect to their Mycobacterium tuberculosis NAD+ DNA ligase inhibitory effect by a newly developed assay based on Genetic Analyzer 3500 Sequencer. The most effective agents (e.g., pinafide, mitonafide) inhibited the activity of M. tuberculosis NAD+-dependent DNA ligase A at concentrations of 50 µM. At the same time, the ATP-dependent (phage) DNA LigT4 was unaffected by the agents at concentrations up to 2 mM. The selected compounds appeared to also be active against actively growing tubercle bacilli in concentrations as low as 15 µM.