Modulation of isoniazid susceptibility by flavonoids in Mycobacterium

In the course of a project to identify plant natural products which modulate the susceptibility of different strains of fast-growing mycobacteria to the first-line antituberculotic isoniazid (INH), several flavonoids without significant antimycobacterial activities at the tested concentrations were screened for their ability to decrease the minimum inhibitory concentrations (MICs) of INH. Flavonoids with different substitution patterns, namely epicatechin, isorhamnetin, kaempferol, luteolin, myricetin, quercetin, rutin and taxifolin were tested to examine structure–activity relationships (SARs) of these compounds. Different mycobacterial strains, i.e. Mycobacterium smegmatis (ATCC 14468), M. smegmatis mc²155 (ATCC 700084), M. smegmatis mc²2700, M. phlei (ATCC 11758) and M. fortuitum (ATCC 6841) were used. The strongest synergistic effects were observed in M. smegmatis mc²155 followed by M. phlei, whereas the tendency of INH potentiation by certain flavonoids remained the same within each strain. Myricetin was the most efficient intensifier of INH susceptibility in all tested strains causing a decrease of the MIC of INH up to 64-fold at 16 μg/ml, followed by quercetin. Structure–activity relationships of flavonoids as intensifiers of INH susceptibility in mycobacteria indicate that they overlap with SARs for their radical-scavenging properties, however the potentiation of INH activity cannot only be explained by their radical-scavenging activity alone.     

Mutation of environmental mycobacteria to resist silver nanoparticles also confers resistance to a common antibiotic

Non-tuberculous mycobacteria are a threat to human health, gaining entry to the body through contaminated water systems, where they form persistent biofilms despite extensive attempts at disinfection. Silver is a natural antibacterial agent and in nanoparticle form activity is increased by a high surface area. Silver nanoparticles (AgNPs) have been used as alternative disinfectants in circulating water systems, washing machines and even clothing. However, nanoparticles, like any other antibiotic that has a pervasive durable presence, carry the risk of creating a resistant population. In this study Mycobacterium smegmatis strain mc²155 was cultured in AgNP enriched agar such that only a small population survived. Surviving cultures were isolated and re-exposed to AgNPs and AgNO₃ and resistance to silver was compared to a negative control. After only a single exposure, mutant M. smegmatis populations were resistant to AgNPs and AgNO₃. Further, the silver resistant mutants were exposed to antibiotics to determine if general resistance had been conferred. The minimum inhibitory concentration of isoniazid was four times higher for silver resistant mutants than for strain mc²155. However, core resistance was not conferred to other toxic metal ions. The mutants had lower resistance to CuSO₄ and ZnSO₄ than the mc²155 strain.     

Viability,morphology, and proteome of Mycobacterium smegmatis MSMEG_0319 knockout strain

Mycobacterium tuberculosis Rv0228, a membrane protein, is predicted as a drug target through computational methods. MSMEG_0319 (MS0319) in Mycobacterium smegmatis mc²155 is the ortholog of Rv0228. To study the effect of MS0319 protein on M. smegmatis, an MS0319 gene knockout strain (ΔMS0319) was generated via a homologous recombination technique in this study. The results showed that the lack of MS0319 protein in mc²155 cells led to the loss of viability at nonpermissive temperature. Scanning electron microscopy and transmission electron microscopy observations showed drastic changes in cellular shape especially cell wall disruption in ΔMS0319 cells. Proteomic analysis of ΔMS0319 cells through LC‐MS/MS revealed that 462 proteins had changes in their expressions by lacking MS0319 protein. The M. tuberculosis orthologs of these 462 proteins were found through BLASTp search and functional clustering and metabolic pathway enrichment were performed on the orthologs. The results revealed that most of them were enzymes involved in metabolism of carbohydrates and amino acids, indicating that Rv0228 played an important role in cellular metabolism. All these results suggested Rv0228 as a potential target for development of antituberculosis drugs.     

NADH Dehydrogenase Defects Confer Isoniazid Resistance and Conditional Lethality in Mycobacterium smegmatis

Isoniazid (INH) is a highly effective drug used in the treatment and prophylaxis of Mycobacterium tuberculosis infections. Resistance to INH in clinical isolates has been correlated with mutations in the inhA, katG, and ahpC genes. In this report, we describe a new mechanism for INH resistance in Mycobacterium smegmatis. Mutations that reduce NADH dehydrogenase activity (Ndh; type II) cause multiple phenotypes, including (i) coresistance to INH and a related drug, ethionamide; (ii) thermosensitive lethality; and (iii) auxotrophy. These phenotypes are corrected by expression of one of two enzymes: NADH dehydrogenase and the NADH-dependent malate dehydrogenase of the M. tuberculosis complex. The genetic data presented here indicate that defects in NADH oxidation cause all of the mutant traits and that an increase in the NADH/NAD⁺ ratio confers INH resistance.     

Glycopeptidolipid of Mycobacterium smegmatis J15cs Affects Morphology and Survival in Host Cells

Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc²155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc²155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc²155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells.     

Prime–Boost with Mycobacterium smegmatis Recombinant Vaccine Improves Protection in Mice Infected with Mycobacterium tuberculosis

The development of a new vaccine as a substitute for Bacillus Calmette–Guerin or to improve its efficacy is one of the many World Health Organization goals to control tuberculosis. Mycobacterial vectors have been used successfully in the development of vaccines against tuberculosis. To enhance the potential utility of Mycobacterium smegmatis as a vaccine, it was transformed with a recombinant plasmid containing the partial sequences of the genes Ag85c, MPT51, and HspX (CMX) from M. tuberculosis. The newly generated recombinant strain mc²-CMX was tested in a murine model of infection. The recombinant vaccine induced specific IgG1 or IgG2a responses to CMX. CD4⁺ and CD8⁺ T cells from the lungs and spleen responded ex vivo to CMX, producing IFN-γ, IL17, TNF-α, and IL2. The vaccine thus induced a significant immune response in mice. Mice vaccinated with mc²-CMX and challenged with M. tuberculosis showed better protection than mice immunized with wild-type M. smegmatis or BCG. To increase the safety and immunogenicity of the CMX antigens, we used a recombinant strain of M. smegmatis, IKE (immune killing evasion), to express CMX. The recombinant vaccine IKE-CMX induced a better protective response than mc²-CMX. The data presented here suggest that the expression of CMX antigens improves the immune response and the protection induced in mice when M. smegmatis is used as vaccine against tuberculosis.     

SmoXYB1C1Z of Mycobacterium sp. Strain NBB4: a Soluble Methane Monooxygenase (sMMO)-Like Enzyme,Active on C2 to C4 Alkanes and Alkenes

Monooxygenase (MO) enzymes initiate the aerobic oxidation of alkanes and alkenes in bacteria. A cluster of MO genes (smoXYB1C1Z) of thus-far-unknown function was found previously in the genomes of two Mycobacterium strains (NBB3 and NBB4) which grow on hydrocarbons. The predicted Smo enzymes have only moderate amino acid identity (30 to 60%) to their closest homologs, the soluble methane and butane MOs (sMMO and sBMO), and the smo gene cluster has a different organization from those of sMMO and sBMO. The smoXYB1C1Z genes of NBB4 were cloned into pMycoFos to make pSmo, which was transformed into Mycobacterium smegmatis mc²-155. Cells of mc²-155(pSmo) metabolized C₂ to C₄ alkanes, alkenes, and chlorinated hydrocarbons. The activities of mc²-155(pSmo) cells were 0.94, 0.57, 0.12, and 0.04 nmol/min/mg of protein with ethene, ethane, propane, and butane as substrates, respectively. The mc²-155(pSmo) cells made epoxides from ethene, propene, and 1-butene, confirming that Smo was an oxygenase. Epoxides were not produced from larger alkenes (1-octene and styrene). Vinyl chloride and 1,2-dichloroethane were biodegraded by cells expressing Smo, with production of inorganic chloride. This study shows that Smo is a functional oxygenase which is active against small hydrocarbons. M. smegmatis mc²-155(pSmo) provides a new model for studying sMMO-like monooxygenases.     

Zeta potential as a measure of the surface charge of mycobacterial cells

The surface charge of bacteria is closely related to their envelope structure and interactions with surfaces in natural environments. The aim of this study was to estimate the effect of experimental conditions on the zeta (ζ) potential of mycobacterial cells as a measure of their cell-surface charge. We observed that Mycobacterium smegmatis mc²155 cells at physiological conditions displayed a high and stable ζ potential (?42.9?±?5.9 mV) which increased from the late-exponential phase of growth and at pH levels of >8.0. The optimal conditions for estimating the surface charge of mycobacteria using the ζ potential occurred when cells were harvested during the exponential growth phase (OD₅₉₅ 0.3–0.5) and then dispersed in solutions with pH levels of 7.0–10.0. These optimal conditions of ζ potential measurements were useful for differentiating between the virulent M. tuberculosis H37Rv strain and various non-virulent mycobacterial strains at pH 9.8. This study is the first to use zetametry to estimate the cell-surface charge of M. tuberculosis cells. We expect that the experimental conditions presented in this work will have further applications to estimate the cell-surface charge of other wild-type or genetically modified mycobacterial species and thereby further our understanding of the physicochemical interactions of mycobacteria with external surfaces in natural environments.     

Deciphering the Transcriptional Regulation of Cholesterol Catabolic Pathway in Mycobacteria: IDENTIFICATION OF THE INDUCER OF KstR REPRESSOR*

Cholesterol degradation plays a prominent role in Mycobacterium tuberculosis infection; therefore, to develop new tools to combat this disease, we need to decipher the components comprising and regulating the corresponding pathway. A TetR-like repressor (KstR) regulates the upper part of this complex catabolic pathway, but the induction mechanism remains unknown. Using a biophysical approach, we have discovered that the inducer molecule of KstR in M. smegmatis mc²155 is not cholesterol but 3-oxo-4-cholestenoic acid, one of the first metabolic intermediates. Binding this compound induces dramatic conformational changes in KstR that promote the KstR-DNA interaction to be released from the operator, retaining its dimeric state. Our findings suggest a regulatory model common to all cholesterol degrading bacteria in which the first steps of the pathway are critical to its mineralization and explain the high redundancy of the enzymes involved in these initial steps.     

Antimycobacterial activity in vitro of pigments isolated from Antarctic bacteria

In this study, we describe the antimycobacterial activity of two pigments, violacein, a purple violet pigment from Janthinobacterium sp. Ant5-2 (J-PVP), and flexirubin, a yellow-orange pigment from Flavobacterium sp. Ant342 (F-YOP). These pigments were isolated from bacterial strains found in the land-locked freshwater lakes of Schirmacher Oasis, East Antarctica. The minimum inhibitory concentrations (MICs) of these pigments for avirulent and virulent mycobacteria were determined by the microplate Alamar Blue Assay (MABA) and Nitrate Reductase Assay (NRA). Results indicated that the MICs of J-PVP and F-YOP were 8.6 and 3.6 μg/ml for avirulent Mycobacterium smegmatis mc²155; 5 and 2.6 μg/ml for avirulent Mycobacterium tuberculosis mc²6230; and 34.4 and 10.8 μg/ml for virulent M. tuberculosis H₃₇Rv, respectively. J-PVP exhibited a ~15 times lower MIC for Mycobacterium sp. than previously reported for violacein pigment from Chromobacterium violaceum, while the antimycobacterial effect of F-YOP remains undocumented. Our results indicate these pigments isolated from Antarctic bacteria might be valuable lead compounds for new antimycobacterial drugs used for chemotherapy of tuberculosis.     

A silent mutation in mabA confers isoniazid resistance on Mycobacterium tuberculosis

Drug resistance in Mycobacterium tuberculosis (Mtb) is caused by mutations in restricted regions of the genome. Mutations in katG, the promoter region of the mabA–inhA operon, and inhA are those most frequently responsible for isoniazid (INH) resistance. Several INH‐resistant (INH^r) Mtb clinical isolates without mutations in these regions have been described, however, indicating that there are as yet undetermined mechanisms of INH resistance. We identified the mabA^g609a silent mutation in a significant number of INH^r Mtb clinical isolates without known INH resistance mutations. A laboratory strain, H37Rv, constructed with mabA^g609a, was resistant to INH. We show here that the mabA^g609a mutation resulted in the upregulation of inhA, a gene encoding a target for INH, converting the region adjacent to the mutation into an alternative promoter for inhA. The mabA^g609a silent mutation results in a novel mechanism of INH resistance, filling in a missing piece of INH resistance in Mtb.     

Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro

Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH)-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis genotypes. The first series were selected from multiple parallel 1 ml cultures and the second from single 10 ml cultures. RIF-resistant mutants were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) or by sequencing the rpoB gene. For all strains the mutation rate for RIF resistance was determined with a fluctuation assay. The most striking observation was a shift towards rpoB-S531L (TCG→TTG) mutations in a panel of laboratory-generated INH-resistant mutants selected from the 10-ml cultures (p<0.001). All tested strains showed similar mutation rates (1.33×10⁻⁸ to 2.49×10⁻⁷) except one of the laboratory-generated INH mutants with a mutation rate measured at 5.71×10⁻⁷, more than 10 times higher than that of the INH susceptible parental strain (5.46–7.44×10⁻⁸). No significant, systematic difference in the spectrum of rpoB-mutations between strains of different genotypes was observed. The dramatic shift towards rpoB-S531L in our INH-resistant laboratory mutants suggests that the relative fitness of resistant mutants can dramatically impact the distribution of (subsequent) mutations that accumulate in a M. tuberculosis population, at least in vitro. We conclude that, against specific genetic backgrounds, certain resistance mutations are particularly likely to spread. Molecular screening for these (combinations of) mutations in clinical isolates could rapidly identify these particular pathogenic strains. We therefore recommend that isolates are screened for the distribution of resistance mutations, especially in regions that are highly endemic for (multi)drug resistant tuberculosis.     

Rapid identification and detection of intracellular survival testing of mycobacterium smegmatis mc²155 that contains eis gene from mycobacterium tuberculosis by flow cytometry

Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. The enhanced intracellular survival (eis) gene (Rv2416c) from M. tuberculosis has been identified as a potential factor that can enhance the intracellular survival of Mycobacterium smegmatis in the macrophage cell line. However, the time requirements for intracellular survival testing of Mycobacterium using classical methodologies are still too long. In this study, we used M. smegmatis mc²155 that contains eis to develop and study a rapid method to test intracellular survival using flow cytometry. We demonstrated the success of this technique, which required only a few hours. This assay is rapid, accurate, and reproducible, and it would be valuable for the rapid detection of intracellular survival of mycobacteria.     

Mutational analysis of the Menkes copper P-type ATPase (ATP7A)

The Menkes protein (ATP7A; MNK) is a ubiquitous human copper-translocating P-type ATPase and it has a key role in regulating copper homeostasis. Previously we characterised fundamental steps in the catalytic cycle of the Menkes protein. In this study we analysed the role of several conserved regions of the Menkes protein, particularly within the putative cytosolic ATP-binding domain. The results of catalytic studies have indicated an important role of 1086His in catalysis. Our findings provide a biochemical explanation for the most common Wilson disease-causing mutation (H1069Q in the homologous Wilson copper-translocating P-type ATPase). Furthermore, we have identified a unique role of 1230Asp, within the DxxK motif, in coupling ATP binding and acylphosphorylation with copper translocation. Finally, we found that the Menkes protein mutants with significantly reduced catalytic activity can still undergo copper-regulated exocytosis, suggesting that only the complete loss of catalytic activity prevents copper-regulated trafficking of the Menkes protein.     

Effect of inhA and katG on isoniazid resistance and virulence of Mycobacterium bovis

Isoniazid (INH) resistance of the Mycobacterium tuberculosis Complex (MtbC) is associated with both loss of catalase activity and mutation of the inhA gene. However, the relative contributions of these changes to resistance and to the loss of virulence for guinea-pigs is unknown. In this study, a virulent strain of Mycobacterium bovis, a member of the MtbC., was exposed to increasing concentrations of INH. Two INH-resistant strains were produced which had lost catalase activity. Strain WAg405, which had a higher resistance to INH, also had a mutation in the inhA gene. This demonstrated that loss of catalase activity and mutation of inhA had a cumulative effect on INH resistance. When a functional katG gene was integrated into the genome of WAg405 the INH resistance was greatly reduced. This indicated that most of the resistance had been caused by loss of catalase activity. While the parent INH-sensitive strain was virulent for guinea-pigs, the INH-resistant strains were significantly less virulent. Integration of a functional katG gene into the most resistant strain restored full virulence. This clearly established that katG is a virulence factor for M. bovis and that mutation of the inhA gene has no effect on virulence.     

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis ¹, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms ^2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) ^5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms ⁷.In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin ^8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere ⁹. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc²7000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen ⁹. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.     

Microwave assisted one-pot synthesis of highly potent novel isoniazid analogues

A series of novel isoniazid (INH) analogues were synthesized by microwave assisted one pot reaction of INH, various benzaldehydes and dimedone in water with catalytic amount of DBSA. The synthesized compounds were evaluated for their anti-TB activity against Mycobacterium tuberculosis H37Rv (MTB) and multi-drug resistant Mycobacterium tuberculosis (MDR-TB). Among the 29 compounds, compound N-[9-[2-(benzyloxy)phenyl]-3,3,6,6-tetramethyl-1,8-dioxo-2,3,4,5,6,7,8,9-octahydro-10(1H)-acridinyl]isonicotinamide (12) inhibited MTB with MIC of <0.17 μM and MDR-TB with MIC of 0.69 μM.     

Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

Early detection of drug resistance in Mycobacterium tuberculosis isolates allows for earlier and more effective treatment of patients. The aim of this study was to investigate the performance of the malachite green decolourisation assay (MGDA) in detecting isoniazid (INH) and rifampicin (RIF) resistance in M. tuberculosis clinical isolates. Fifty M. tuberculosis isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and RIF-susceptible samples, were tested. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and agreement of the assay for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%, 100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the reference method. There was a minor discrepancy in the tests of two additional isolates, as they were sensitive to INH by the reference method, but resistant by the MGDA test. The drug susceptibility test results were obtained within eight-nine days. In conclusion, the MGDA test is a reliable and accurate method for the rapid detection of INH and RIF resistance compared with the reference method and the MGDA test additionally requires less time to obtain results.     

Human Menkes X-chromosome disease and the staphylococcal cadmium-resistance ATPase: a remarkable similarity in protein sequences

A search with the proposed amino acid translation product from the new ‘candidate gene’ for human Menkes disease against protein sequence libraries showed a remarkable similarity to that for the cadmium efflux ATPase from Staphylococcus aureus resistance plasmids. The Menkes sequence appears closer to the CadA Cd²⁺ sequence than to P-type ATPases from animal sources. Menkes syndrome is an X-chromosome invariably fatal disease that results from abberant copper metabolism. The gene that is defective in Menkes patients, i.e. the Menkes candidate gene, encodes a P-type ATPase, whose properties satisfactorily explain the phenotype of the disease. P-type ATPases are all cation pumps, either for uptake (e.g. the bacterial Kdp K⁺ ATPase), for efflux (e.g. the muscle sarcoplasmic reticulum Ca²⁺ ATPase), or for cation exchange (e.g. the animal cell Na⁺/K⁺ ATPase). These enzymes have a conserved aspartate residue that is transiently phosphorylated from ATP during the transport cycle, hence the name ‘P-type’ ATPase. The Menkes sequence shares with the staphylococcal CadA ATPase those regions common to all P-type ATPases and also an N-terminal dithiol region that was proposed to be a ‘metal-binding motif’. There are one or two copies of this motif in the available CadA sequences and six copies in the Menkes sequence.