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Drug sensitivity and environmental adaptation of mycobacterial cel wall components
Abstract
The intrinsic resistance of many mycobacterial species to chemotherapy is largely attributable to their impermeable cell wall. The composition of the cell wall of a particular species appears to be influenced by the environmental niche that the species occupies. The complex regulatory and biosynthetic pathways involved in cell wall biosynthesis and construction offer useful chemotherapeutic targets against mycobacteria.
... The resistance of Mycobacteria is related to the high lipid content in their 'waxy' cell wall, which hampers the penetration of hydrophilic compounds [89,135]. Differences in lipid composition explain differences in resistance between mycobacterial species [16,49], but beyond this little is known about exact mechanisms or variations within species [16,135]. ...
... The resistance of Mycobacteria is related to the high lipid content in their 'waxy' cell wall, which hampers the penetration of hydrophilic compounds [89,135]. Differences in lipid composition explain differences in resistance between mycobacterial species [16,49], but beyond this little is known about exact mechanisms or variations within species [16,135]. ...
Disinfection of empty animal houses has routinely been incorporated in all-in all-out farm systems for decades. The scientific evidence for the efficacy of disinfecting farm buildings is sparse, both as regards general production parameters and the elimination of specific micro-organisms. Information on chemical disinfectants can often be obtained only from the companies that market or produce them. Three aspects determine the outcome of disinfection: the detrimental factor (chemical and/or physical disinfection), the micro-organism, and the environment. The ideal farm disinfectant is unaffected by organic matter, it works at low temperatures, is non-corrosive to materials, and breaks down to harmless substances. Routine farm disinfection only involves chemical disinfectants, albeit these have been combined with heat to increase the effectiveness. Micro-organisms differ with regard to susceptibility to detrimental conditions, ranging from the very resistant prions to the relatively sensitive enveloped viruses. For bacteria, much is known about their phase and resistance, e.g., stationary vs. growth phase. This is closely interrelated with the environment in which factors such as the amount and type of organic matter, humidity, temperature, pH, and UV-light are important. Most official disinfection tests, which have been implemented by only a few countries, are unrealistic suspension tests. These often yield best-case results because bacteria in suspensions are more susceptible to detrimental conditions than under real-life conditions on surfaces. With regard to viruses and fungi even less evidence on the effectiveness of disinfection exists. This review outlines conditions that influence disinfection efficacy and elaborates on applied research which is useful for monitoring the disinfection of empty animal houses. Suggestions are given for future research areas which can generate scientific evidence for recommended procedures.
... DNA extraction from clinical samples can be a difficult task, when there are low levels of genomic DNA and/or it is degraded. The mycobacterial cell wall complex has a waterproof structure, which hinders their lysis and subsequent release of the genetic material [67][68][69]. The efficiency of cell lysis, the ratio of DNA/RNA extracted, and residual extraction reagents will influence this process [69][70][71]. ...
... Other problems that may occur are, contaminants and inhibitors of PCR, the partial degradation of the DNA molecules and the duration of the protocols [65,72]. Procedures commonly used for the isolation of nucleic acids do not obtain good results for M. leprae DNA extraction [68,73,74] and pretreatment steps with: freezing/thawing [75], bead beater with pearls [27,35] proteinase K [8,30,66,76] minimum 16 hours incubation [33], tris-HCL or tris-EDTA [44], detergents such as triton X-100 [22,77], and SDS [8,66,[76][77][78], alone or in combination should be included. Additionally, DNA extraction with phenol-chloroform mixture [66,79] can be used to obtain good quality DNA although it has several steps of sample handling, that can reduce performance and increase cross contamination [80]. ...
Leprosy is a chronic infectious disease caused by an obligatory intracellular mycobacteria Mycobacterium
leprae, which presents tropism for Schwann cells and skin macrophages. Leprosy is a public health problem and early diagnosis is essential to avoid incapacities. The disease´s clinical presentation varies from few to widespread lesions and its diagnosis continues to be a challenge due to the low sensibility of the conventional methods, based
on bacillary counts of skin smears and histopathology. Molecular techniques, especially the methods to identify M. leprae DNA based on polymerase chain reaction (PCR) have emerged as a support of the conventional methods for the analysis of clinical samples in difficult to diagnose cases, such as pure neural leprosy, indeterminate and paucibacillary leprosy. The technique has also proved useful in the study of leprosy transmission and monitoring résistance to the WHO recommended Multidrug treatment. Different biological samples can be analysed and there is no consensus in the molecular diagnostic techniques respect of the most efficient nucleic acid extraction method, most appropriate methodology and genetic target for PCR. These methods provide a very valuable option for confirmation of difficult clinical cases with scarce bacilli but requires a well-equipped laboratory and the high cost makes it inaccessible to be used as a routine diagnostic tool in most endemic countries.
... DNA extraction from clinical samples can be a difficult task, when there are low levels of genomic DNA and/or it is degraded. The mycobacterial cell wall complex has a waterproof structure, which hinders their lysis and subsequent release of the genetic material676869. The efficiency of cell lysis, the ratio of DNA/RNA extracted, and residual extraction reagents will influence this process697071. Other problems that may occur are, contaminants and inhibitors of PCR, the partial degradation of the DNA molecules and the duration of the protocols [65,72]. ...
... Other problems that may occur are, contaminants and inhibitors of PCR, the partial degradation of the DNA molecules and the duration of the protocols [65,72]. Procedures commonly used for the isolation of nucleic acids do not obtain good results for M. leprae DNA extraction [68,73,74] and pretreatment steps with: freezing/thawing [75], bead beater with pearls [27,35] proteinase K [8,30,66,76] minimum 16 hours incubation [33], tris-HCL or tris-EDTA [44], detergents such as triton X-100 [22,77], and SDS [8,66,767778, alone or in combination should be included. Additionally, DNA extraction with phenol-chloroform mixture [66,79] can be used to obtain good quality DNA although it has several steps of sample handling, that can reduce performance and increase cross contamination [80]. ...
Leprosy is a chronic infectious disease caused by an obligatory intracellular mycobacteria Mycobacterium leprae, which presents tropism for Schwann cells and skin macrophages. Leprosy is a public health problem and early diagnosis is essential to avoid incapacities. The disease´s clinical presentation varies from few to widespread lesions and its diagnosis continues to be a challenge due to the low sensibility of the conventional methods, based on bacillary counts of skin smears and histopathology. Molecular techniques, especially the methods to identify M. leprae DNA based on polymerase chain reaction (PCR) have emerged as a support of the conventional methods for the analysis of clinical samples in difficult to diagnose cases, such as pure neural leprosy, indeterminate and paucibacillary leprosy. The technique has also proved useful in the study of leprosy transmission and monitoring résistance to the WHO recommended Multidrug treatment. Different biological samples can be analysed and there is no consensus in the molecular diagnostic techniques respect of the most efficient nucleic acid extraction method, most appropriate methodology and genetic target for PCR. These methods provide a very valuable option for confirmation of difficult clinical cases with scarce bacilli but requires a well-equipped laboratory and the high cost makes it inaccessible to be used as a routine diagnostic tool in most endemic countries.
... Mycobacterium tuberculosis is an important pathogen of man that displays an intrinsic resistance to many standard antimicrobials (1,2). An important factor in this resistance is the formidable permeability barrier imposed by the mycobacterial cell wall (3)(4)(5)(6)(7)(8). This barrier is formed by a lipid-rich complex heteropolymer composed of covalently linked subunits of peptidoglycan, arabinogalactan, and mycolic acids (3). ...
... Mycolic acids are very long chain two-branched, three-hy-droxy fatty acids that range from 75 to 88 carbons in total length in the tubercle bacilli (3,(11)(12)(13). Several unique structural features of these molecules may play a direct role in allowing the formation and determining the properties of the assymetrical lipid bilayer, which is characteristic of mycobacteria and closely related genera (4). The primary division of mycolic acids into subclasses is dependent upon the presence or the absence of oxygen-containing functional groups in the longer (mero) chain (11). ...
The proportion of mycolic acid containing trans-substituents at the proximal position of the meromycolate chain is an important determinant of fluidity of the mycobacterial
cell wall and is directly related to the sensitivity of mycobacterial species to hydrophobic antibiotics. MMAS-1, an enzyme
encoded in the gene cluster responsible for the biosynthesis of methoxymycolates, was overexpressed in Mycobacterium tuberculosis and shown to result in the overproduction of trans-cyclopropane and trans-olefin-containing oxygenated mycolic acids. MMAS-1 converted a cis-olefin into a trans-olefin with concomitant introduction of an allylic methyl branch in a precursor to both the methoxy and ketone-containing
mycolic acids. In addition to an increase in the amount of trans-mycolate, MMAS-1 expression resulted in a substantial increase in the amount of ketomycolate produced relative to methoxymycolate.
Thus MMAS-1 may act at a complex branch point where expression of this enzyme directly affects the cis- to trans-ratio and indirectly affects the keto to methoxy ratio. Overexpression of MMAS-1 resulted in a substantially slower growth
rate at moderately elevated temperature, decreased thermal stability of the cell wall as measured by differential scanning
calorimetry, and an increased permeability to chenodeoxycholate. These results provide experimental evidence for the intermediacy
of trans-olefinic mycolate precursors in trans-cyclopropane formation and suggest that increasing the proportion of the polar ketomycolate subclass may exert a significant
fluidizing effect on the cell wall.
... The ongoing emergence and spread of Mycobacterium tuberculosis (Mtb) strains that are resistant to existing antimycobacterial drugs have highlighted the urgent need to develop new drugs with novel mechanisms of action (Raviglione et al., 2012;Zignol et al., 2016;Zumla et al., 2013). Chemotherapeutic therapies for TB remain very difficult and complicated, primarily due to the slow rate of growth of the bacilli and the presence of a dense, greasy, and relatively drug-impermeable cell wall (Barry & Mdluli, 1996). In recent years, an estimated 110,000 people with rifampicin-resistant TB (RR-TB) were documented, with higher occurrences of MDR/RR-TB in Asia and the Russian Federation accounting for nearly half of the reported cases globally (Bj€ orkelid et al., 2013;World Health Organization, 2019). ...
The growing occurrence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (Mtb) strains underscores an urgent need for new antibiotics. The development of more bioactive antibiotics against drug-resistant organisms with a different mode of action could be a game-changer for the cure and eradication of tuberculosis (TB). Pantothenate Kinase (PanK) and CTP synthetase (PyrG) are both essential for RNA, DNA, and Lipids biosynthesis pathways. Given the extensive knowledge on these biosynthesis pathways inhibition of Mtb growth and survival, these enzymes present a fascinating opportunity for anti-mycobacterial drug discovery. Recently, it was experimentally established that the active metabolite 11426026 of compound 7947882 (a prodrug activated by EthA monooxygenase, 5-methyl-N-(4-nitrophenyl) thiophene-2-carboxamide) inhibits the activities of PyrG and PanK to indicate novel multitarget therapy aimed at discontinuing Mtb growth. However, the molecular mechanisms of their selective inhibition remain subtle. In this work, molecular dynamics simulations were employed to investigate the inhibitory mechanism as well as the selectivity impact of the active metabolite inhibitor of these enzymes. Computational modeling of the studied protein-ligand systems reveals that the active metabolite can potentially inhibit both PanK and PyrG, thereby creating a pathway as a double target approach in tuberculosis treatment.
Communicated by Ramaswamy H. Sarma
... Cgl2857 is a membrane protein, that is highly conserved in Corynebacterineae and required for synthesis of fulllength lipomannans and lipoarabinomannans, abundant components of the multilaminate cell wall 43 . C. glutamicum synthesizes a complex cell wall, which can confer intrinsic resistance to adverse environmental conditions 44 . Although synonymous mutations do not alter the encoded protein, they can influence gene expression 45 . ...
Synthetic methylotrophy has recently been intensively studied to achieve methanol-based biomanufacturing of fuels and chemicals. However, attempts to engineer platform microorganisms to utilize methanol mainly focus on enzyme and pathway engineering. Herein, we enhanced methanol bioconversion of synthetic methylotrophs by improving cellular tolerance to methanol. A previously engineered methanol-dependent Corynebacterium glutamicum is subjected to adaptive laboratory evolution with elevated methanol content. Unexpectedly, the evolved strain not only tolerates higher concentrations of methanol but also shows improved growth and methanol utilization. Transcriptome analysis suggests increased methanol concentrations rebalance methylotrophic metabolism by down-regulating glycolysis and up-regulating amino acid biosynthesis, oxidative phosphorylation, ribosome biosynthesis, and parts of TCA cycle. Mutations in the O-acetyl-l-homoserine sulfhydrylase Cgl0653 catalyzing formation of l-methionine analog from methanol and methanol-induced membrane-bound transporter Cgl0833 are proven crucial for methanol tolerance. This study demonstrates the importance of tolerance engineering in developing superior synthetic methylotrophs. Wang et al. improve the methanol tolerance for the synthetic methylotroph, Corynebacterium glutamicum. They generate 3 new strains by directed evolution and use biochemical, transcriptomic, and genetic approaches to characterize the pathways underlying the enhanced methanol metabolism. Their findings are important for biomanufacturing purposes.
... M. tuberculosis is a resilient microorganism that can persist silently through long chemotherapeutic courses and years of dormancy within the host. The standard chemotherapeutic treatments remain very challenging, substantiated by the slow growth of M. tuberculosis and the presence of a thick and drug-impermeable waxy cell envelope (7). In this context, new chemical entities that kill actively growing as well as persistent bacilli are needed. ...
Menaquinone (MK) or vitamin K 2 is an important metabolite that controls the redox/energy status of Mycobacterium tuberculosis . Although the major steps of MK biosynthesis have been delineated, the regulatory mechanisms of this pathway have not been adequately explored. Bashiri et al. now demonstrate that MenD, catalyzing the first committed step of MK production, is allosterically inhibited by a downstream cytosolic metabolite in the MK biosynthesis pathway.
... Moreover, the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB largely contributes to the difficulty in curing TB patients (1). The standard chemotherapeutic treatments remain very complicated and challenging, substantiated by the slow rate of growth of the bacilli and the existence of a thick and waxy drug-impermeable cell envelope (2). This latter structure consists of a complex skeleton where peptidoglycan, arabinogalactan, and mycolic acids are covalently linked together, and in which noncovalently associated lipids are interspersed (3). ...
The prevalence of pulmonary infections due to non-tuberculous mycobacteria such as Mycobacterium abscessus, has been increasing and surpassing tuberculosis (TB) in some industrialized countries. Due to intrinsic resistance to most antibiotics that drastically limits conventional chemotherapeutic treatment options, new anti-M. abscessus therapeutics are urgently needed against this emerging pathogen. Extensive screening of a library of benzimidazole derivatives that were previously shown to be active against Mycobacterium tuberculosis led to the identification of a lead compound exhibiting very potent in vitro activity against a wide panel of M. abscessus clinical strains. Designated EJMCh-6, this compound, a 2-(2-cyclohexylethyl)-5,6-dimethyl-1H-benzo[d]imidazole), also exerted a very strong activity against intramacrophage-residing M. abscessus. Moreover, treatment of infected zebrafish embryos with EJMCh-6 correlated with a significantly increased embryo survival and a decrease in the bacterial burden as compared to untreated fish. Insights into the mechanism of action were inferred from the generation of spontaneous benzimidazole-resistant strains and the identification of a large set of missense mutations in MmpL3; the mycolic acid transporter in mycobacteria. Over-expression of the mutated mmpL3 alleles in a susceptible M. abscessus strain was associated with high resistance levels to EJMCh-6 and to other known MmpL3 inhibitors. Mapping the mutations conferring resistance on a MmpL3 three-dimensional homology model defined a potential EJMCh-6-binding cavity. These data emphasize a yet unexploited chemical structure class against M. abscessus with promising translational developments for the treatment of M. abscessus lung diseases.
... Moreover, the emergence of multi drug-resistant (MDR) and extensively drug-resistant (XDR) TB largely contributes to the difficulty in curing TB patients (1). The standard chemotherapeutic treatments remain very complicated and challenging, substantiated by the slow rate of growth of the bacilli and the existence of a thick and waxy drug-impermeable cell envelope (2). This latter consists of a complex skeleton where peptidoglycan, arabinogalactan and mycolic acids are covalently linked together and in which noncovalently associated lipids are interspersed (3). ...
Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat due to the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against M. tuberculosis Herein, we show that PIPD1 and related analogs exert in vitro bactericidal activity against the M. tuberculosis strain mc26230 and also against a panel of MDR and XDR clinical isolates of M. tuberculosis, suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of SNPs in mmpL3, encoding an inner membrane-associated mycolic acid flippase in M. tuberculosis Results from functional assays with spheroplasts derived from a M. smegmatis strain lacking the endogenous mmpL3 gene, but harboring the M. tuberculosis mmpL3 homolog, indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate without altering the proton motive force across the inner membrane. Using a predictive structural model of MmpL3 from M. tuberculosis, docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in M. smegmatis MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria.
... Mycobacteria possess a thick cell wall (31,32), a millimolar concentration of mycothiol (33,34), detoxification enzymes such as catalase (KatG) (35,36)and superoxide dismutases (SodA and SodC) (37,38), and DNA-binding proteins such as Lrs2 (39). However, no oxidative stress regulators homologous to SoxR, OxyR and OhrR in mycobacteria have been described to date. ...
Cyclic di-GMP (c-di-GMP) is a global signaling molecule that widely modulates diverse cellular processes. However, whether or not the c-di-GMP signal participates in regulation of bacterial antioxidant defense is unclear, and the involved regulators remain to be explored. In this study, we characterized HpoR as a novel c-di-GMP effective transcription factor and found a link between the c-di-GMP signal and the antioxidant regulation in Mycobacterium smegmatis. H2O2 stress induces c-di-GMP accumulation in M. smegmatis. High level of c-di-GMP triggers expression of a redox gene cluster, designated as hpoR operon, which is required for the mycobacterial H2O2 resistance. HpoR acts as an inhibitor of the hpoR operon and recognizes a 12-bp motif sequence within the upstream regulatory region of the operon. c-di-GMP specifically binds with HpoR at a ratio of 1:1. Low concentrations of c-di-GMP stimulate the DNA-binding activity of HpoR, whereas high concentrations of the signal molecule inhibit the activity. Strikingly, high level of c-di-GMP de-represses the intracellular association of HpoR with the regulatory region of the hpoR operon in M. smegmatis and enhances the mycobacterial H2O2 resistance. Therefore, we report a novel c-di-GMP effective regulator in mycobacteria, which extends the second messenger's function to bacterial antioxidant defense.
... The emergence of multi drug-resistant (MDR) and extensively drug-resistant (XDR) TB contribute to the difficulties in treating this bacterial infection (1). Chemotherapeutic treatments against TB remain very challenging and complicated, essentially because of the slow rate of growth of the bacilli and the presence of a thick, greasy and relatively drug-impermeable cell wall (2). This mycobacterial cell wall consists of a complex skeleton comprising covalently linked macromolecules such as peptidoglycan, arabinogalactan and mycolic acids, in which noncovalently associated glycolipids are interspersed (3). ...
An increasing prevalence of cases of drug-resistant tuberculosis requires the development of more efficacious chemotherapies. We previously reported the discovery of a new class of Cyclipostins and Cyclophostin (CyC) analogs exhibiting potent activity against Mycobacterium tuberculosis both in vitro and in infected macrophages. Competitive labeling/enrichment assays combined with MS have identified several serine or cysteine enzymes in lipid and cell wall metabolism as putative targets of these CyC compounds. These targets included members of the antigen 85 (Ag85) complex (i.e. Ag85A, Ag85B, and Ag85C), responsible for biosynthesis of trehalose dimycolate (TDM) and mycolylation of arabinogalactan. Herein, we used biochemical and structural approaches to validate the Ag85 complex as a pharmacological target of the CyC analogs. We found that CyC7β, CyC8β, and CyC17 bind covalently to the catalytic Ser124 residue in Ag85C, inhibit mycolyltransferase activity, i.e. the transfer of a fatty acid molecule onto trehalose, and reduce triacylglycerol synthase activity, a property previously attributed to Ag85A. Supporting these results, an X-ray structure of Ag85C in complex with CyC8β disclosed that this inhibitor occupies Ag85C's substrate-binding pocket. Importantly, metabolic labeling of M. tuberculosis cultures revealed that the CyC compounds impair both TDM synthesis and mycolylation of arabinogalactan. Overall, our study provides compelling evidence that CyC analogs can inhibit the activity of the Ag85 complex in vitro and in mycobacteria, opening the door to a new strategy for inhibiting Ag85. The high-resolution crystal structure obtained will further guide the rational optimization of new CyC scaffolds with greater specificity and potency against M. tuberculosis.
... Corynebacterium diphtheriae (Diphtheria). These bacteria synthesize a complex and highly distinctive cell wall which confers intrinsic resistance to host antibacterial factors, antibiotics and adverse environmental conditions (1). ...
Mycobacterium tuberculosis
and related Corynebacterineae synthesize a family of lipomannans (LM) and lipoarabinomannans (LAM) that are abundant components of the multilaminate cell wall and essential virulence factors in pathogenic species. Here we describe a new membrane protein, highly conserved in all Corynebacterineae, that is required for synthesis of full-length LM and LAM. Deletion of theCorynebacterium glutamicum NCgl2760gene resulted in a complete loss of mature LM/LAM and the appearance of a truncated LM (t-LM). Complementation of the mutant with theNCgl2760gene fully restored LM/LAM synthesis. Structural studies, including monosaccharide analysis, methylation linkage analysis, and mass spectrometry of native LM species, indicated that the ΔNCgl2760t-LM comprised a series of short LM species (8-27 residues long) containing an α1-6-linked mannose backbone with greatly reduced α1-2-mannose side chains and no arabinose caps. The structure of the ΔNCgl2760t-LM was similar to that of the t-LM produced by aC. glutamicummutant lacking themptAgene, encoding a membrane α1-6-mannosyltransferase involved in extending the α1-6-mannan backbone of LM intermediates. Interestingly, NCgl2760 lacks any motifs or homology to other proteins of known function. Attempts to delete theNCgl2760orthologue inMycobacterium smegmatiswere unsuccessful, consistent with previous studies indicating that theM. tuberculosisorthologue,Rv0227c, is an essential gene. Together, these data suggest that NCgl2760/Rv0227c plays a critical role in the elongation of the mannan backbone of mycobacterial and corynebacterial LM, further highlighting the complexity of lipoglycan pathways of Corynebacterineae.
... The bacterial derived natural biosurfactant such as rhamnolipids have been studied for their antibacterial activity against a wide range of pathogenic bacteria including Mycobacterium tuberculosis (Abalos et al., 2001). Since mycobacteria posses uniquely impermeable cell wall, therefore, a large arsenal of chemically synthesized antibacterial molecules remains ineffective against this pathogen (Barry and Mdluli, 1996). However, owing to the biosurfactant nature the bacterial derived rhamnolipids (RLs) might be the effective antibacterial candidates against mycobacteria. ...
Rhamnolipids (RLs) are the bacterial derived biosurfactants and known for a wide range of industrial and therapeutic applications. They exhibit potent anti-bacterial activity against various gram positive, gram negative and acid fast bacteria including Mycobacterium tuberculosis. Since, Pseudomonas is one of the largest known genuses containing a variety of rhamnolipid producing strains. Therefore, in this study, we selectively isolated the Pseudomonas aeruginosa strains from the rhizospheric soil of the Indian plants of medicinal value, e.g. Azadirachta Indica and Ficus spp., and evaluated them for their natural ability to produce antibacterial rhamnolipids. The bacteria were identified on the basis of 16s rRNA sequencing and biochemical characterization. Among 33 of P. aeruginosa isolates from different soil samples, four isolates showed potent inhibitory activity against methicillin resistant Staphylococcus aureus (MRSA) and fast grower mycobacterial spp. The inhibitory potential of the isolates was found to be correlated with their ability to produce RLs in the medium. The industrial viability of the strains was assessed on the basis of cytotoxicity determining alternative allele, exoS/exoU and cell mediated cytotoxicity against murine macrophages J774.1. The newly isolated strains harbor exoS allele and exhibits lower cell mediated cytotoxicity on macrophage cell line as compared to the clinical strains PA-BAA-427 and PA-27853 used as a control in this study.Evaluation of antimycobacterial rhamnolipid production from non-cytotoxic strains of Pseudomonas aeruginosa isolated from rhizospheric soil of medicinal plants
... This is consistent with what has long been known -that MAC in general are resistant to standard antituberculous drugs (326)(327)(328). MAC can prevent these agents from penetrating the mycobacterial cell and can rapidly develop mutations that confer drug resistance (329)(330)(331)(332)(333). MAC infections in immunocompetent hosts are difficult to eradicate; prolonged treatment is required, and relapse either on treatment or off treatment is common. ...
Mycobacterium avium subspecies paratuberculosis (MAP) is a member of the M avium complex (MAC). It differs genetically from other MAC in having 14 to 18 copies of IS900 and a single cassette of DNA involved in the biosynthesis of surface carbohydrate. Unlike other MAC, MAP is a specific cause of chronic inflammation of the intestine in many animal species, including primates. The disease ranges from pluribacillary to paucimicrobial, with chronic granulomatous inflammation like leprosy in humans. MAP infection can persist for years without causing clinical disease. The herd prevalence of MAP infection in Western Europe and North America is reported in the range 21% to 54%. These subclinically infected animals shed MAP in their milk and onto pastures. MAP is more robust than tuberculosis, and the risk that is conveyed to human populations in retail milk and in domestic water supplies is high. MAP is harboured in the ileocolonic mucosa of a proportion of normal people and can be detected in a high proportion of full thickness samples of inflamed Crohn's disease gut by improved culture systems and IS900 polymerase chain reaction if the correct methods are used. MAP in Crohn's disease is present in a protease-resistant nonbacillary form, can evade immune recognition and probably causes an immune dysregulation. As with other MAC, MAP is resistant to most standard antituberculous drugs. Treatment of Crohn's disease with combinations of drugs more active against MAC such as rifabutin and clarithromycin can bring about a profound improvement and, in a few cases, apparent disease eradication. New drugs as well as effective MAP vaccines for animals and humans are needed. The problems caused by MAP constitute a public health issue of tragic proportions for which a range of remedial measures are urgently needed.
... Therefore the development of this intrinsic resistance could be also considered as an effective and useful resolution for multidrug-resistant M. tuberculosis against toxic compounds. Mycobacterial cell wall was known as the primary permeability barrier responsible for resistance to antibiotics [53]. The higher thickness of the cell wall was observed in multidrugresistant M. tuberculosis [54] and therefore some proteins involved in the construction of the cell envelope of M. tuberculosis will be discussed below. ...
Reports in recent years indicate that the increasing emergence of resistance to drugs be using to TB treatment. The resistance to them severely affects to options for effective treatment. The emergence of multi-drug-resistant tuberculosis has increased interest in understanding the mechanism of drug resistance in M. tuberculosis and the development of new therapeutics, diagnostics and vaccines. In this study, a label-free quantitative proteomics approach has been used to analyze proteome of multidrug-resistant and susceptible clinical isolates of M. tuberculosis and identify differences in protein abundance between the two groups. With this approach, we were able to identify a total of 1,583 proteins. The majority of identified proteins have predicted roles in lipid metabolism, intermediary metabolism, cell wall and cell processes. Comparative analysis revealed that 68 proteins identified by at least two peptides showed significant differences of at least twofolds in relative abundance between two groups. In all protein differences, the increase of some considering proteins such as NADH dehydroge-nase, probable aldehyde dehydrogenase, cyclopropane mycolic acid synthase 3, probable arabinosyltransferase A, putative lipoprotein, uncharacterized oxidoreductase and six membrane proteins in resistant isolates might be involved in the drug resistance and to be potential diagnostic protein targets. The decrease in abundance of proteins related to secretion system and immunogenicity (ESAT-6-like proteins, ESX-1 secretion system associated proteins, O-antigen export system and MPT63) in the multidrug-resistant strains can be a defensive mechanism undertaken by the resistant cell.
... La aplicación de las técnicas de biología molecular va a depender en gran medida de la cantidad y calidad de los ácidos nucleicos extraídos de las muestras biológicas (Taylor y Reischl, 1996; Barry y Mdluli, 1996). Resultado de ello, es que con la mayoría de procedimientos comúnmente usados para el aislamiento de ácidos nucleicos no se obtengan buenos resultados (Barry y Mdluli, 1996; Kolk y col., 1992; Fries y col., 1991). Por ello, para la lisis celular y extracción exitosa del ADN de M. leprae, se deben introducir algunos cambios en las primeras fases de la extracción, como: congelación/descongelación (Via y col., 1995), incubaciones más largas de las convencionales de 16 horas en adelante (Torres y col., 2003), agitación con perlas (Plikaytis y col., 1990), añadir proteinasa K (de Wit y col., 1991), Tris-HCL, Tris-EDTA (Yoon y col., 1993) o detergentes como Tritón X-100 (Williams y col.,1990; ...
... Isolation of nucleic acid (DNA) from mycobacteria is difficult due to its complex cell wall structure. 1 Therefore, most of the simple and commonly used DNA extraction methods result in poor quality and low yield of DNA, which is also affected by type of sample used. 2 However, the sensitivity of molecular diagnosis is largely dependent on the efficiency of cell lysis and extraction of DNA. 3 Several methods for mycobacterial cell wall lysis and DNA extraction have been used like simple boiling in distilled water, disruption by glass bead or sonication, enzymatic lysis, chemical lysis or combination of these techniques. 4 The objective of this study was to compare three methods of extracting M. tuberculosis DNA from various types of extrapulmonary specimens. ...
Sensitivity of the molecular diagnostic tests of extrapulmonary tuberculosis largely depends upon the efficiency of DNA extraction methods. The objective of our study was to compare three methods of extracting DNA of Mycobacterium tuberculosis for testing by polymerase chain reaction. All three methods; heating, heating with sonication and addition of lysis buffer with heating and sonication were implicated on 20 extrapulmonary samples. PCR positivity was 2 (10%), 4 (20%) and 7 (35%) in the samples extracted by heating, heat+sonication and heat+sonication+lysis buffer method respectively. Of the extraction methods evaluated, maximum PCR positive results were achieved by combined heat, sonication and lysis buffer method which can be applied in routine clinical practice. DOI: http://dx.doi.org/10.3329/imcj.v6i1.14711 Ibrahim Med. Coll. J. 2012; 6(1): 9-11
... M ycobacterium tuberculosis remains a leading cause of morbidity, tuberculosis (TB), and mortality in the world. M. tuberculosis is intrinsically resistant to most classical antibiotics, partly because of its impermeable cell wall (1)(2)(3)(4)(5)(6). Due to selective mutations in M. tuberculosis, almost one-third of new TB patients are now infected with first-line drug-resistant strains, monoresistant strains, or multidrug-resistant (MDR) strains. ...
Mycobacterium tuberculosis is wrapped in complex waxes, impermeable to most antibiotics. Comparing M. bovis BCG and M. tuberculosis mutants, lacking phthiocerol dimycocerosates (PDIM) and/or phenolic glycolipids, with wild-type strains, we observed that glycopeptides strongly inhibited PDIM deprived mycobacteria. Vancomycin together with a drug targeting lipids synthesis inhibited multidrug-resistant (MDR) and extensively-drug resistant (XDR) clinical isolates. Our study puts glycopeptides in the pipeline of potential anti-TB agents and might provide a new antimycobacterial drug-screening strategy.
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... Tuberculosis is an infection that has plagued mankind for millennia and still remains a major worldwide health problem, causing almost 2 million deaths annually, mainly located in the developing world (World Health Organization 2008, www.who.int/tb/en/). TDM and TMM are based on a common trehalose core, acylated mainly with mycolic acids (Chatterjee 1997) that are long chain (C60-C90) cyclopropanated lipids; essential for M. tuberculosis, they are relevant for bacterial outer membrane structure, virulence, and persistence within the host (Barry and Mdluli 1996). Due to the relevance of SL-1 and TDM trehalose-based lipids to mycobacterial viability and virulence, several inhibitors of the enzymes involved in their biosynthesis have been designed over the years. ...
This report gives an overview of relevant trehalose processing enzymes and trehalose mimics as bioactive compounds.
... However, it is possible that these enzymes might play a role in the construction of mycobacterial cell wall through lipid metabolism. Mycobacterial cell wall is known as the primary permeability barrier responsible for resistance to antibiotics [50] e dlaT gene (Rv2215) and glnA1 genes were encoding for dihydrolipoyllysine acetyltransferase (DlaT) component of pyruvate dehydrogenase complex and glutamine synthase, respectively. ey were found to be up-regulated in RH resistant isolates. ...
Tuberculosis (TB) is the leading cause of infectious disease related mortality worldwide. Infection of Mycobacterium tuberculosis (Mtb) leads to nearly 3 million deaths every year due to tuberculosis. Rifampicin and Isoniazid (RH) are the key drugs to being used for the treatment of tuberculosis. Reports in recent years indicate that the increasing emergence of resistance to these drugs. of resistance to these drugs severely affects options for effective treatment. of current vaccine for tuberculosis has variable protective efficacy and there is no commercially available serodiagnostic test for this disease with acceptable sensitivity and specificity for routine laboratory use, especially in case of multidrug resistance. In order to develop a new diagnostic tool for detection of Mtb, multidrug resistant Mtb as well and improve the tuberculosis vaccine, it is necessary to indentify novel antigenic candidates, especially in identification of multidrug resistant associated protein antigens. Here, we present a 2-D gel-based proteomic survey of the changes in RH resistant Mtb. The proteins extracted from RH resistant and susceptible Mtb clinical isolates were analyzed by two-dimensional diffierential in gel electrophoresis (2D-DIGE). Protein intensities of 41 spots were found to be regulated in RH resistant isolates. A total of 28 proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Twelve proteins of interest are NADH-dependent enoyl-[acyl-carrier-protein] reductase, 60 kDa chaperonin 2, Chaperone protein DnaK, 3-oxoacyl-(Acylcarrier-protein0 reductase, Probable acetyl-CoA acyltransferase FadA2, two Acetyl/propionyl-CoA carboxylase, alpha subunit, Universal stress protein Rv1636/MT1672, Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, Glutamine synthetase 1 and two uncharacterized proteins (Rv2557 and Rv1505c).
... The dominant abundance has arabinogalactan polysaccharide, which is linked with large 2-alkyl 3-hydroxy branched-chain fatty acids called mycolic acids. This covalently assembled complex is responsible for the cell surface hydrophobicity and impermeability [110][111][112]. The cell hydrophobic character helps mycolata to uptake the hydrophobic substrate from the environment without the production of surfactant and enables the use of such bacteria in bioremediation technologies [34][35][36]. ...
Bacterial strains living in the environment must cope with the toxic compounds originating from humans production. Surface
bacterial structures, cell wall and cytoplasmic membrane, surround each bacterial cell and create selective barriers between the cell
interior and the outside world. They are a first site of contact between the cell and toxic compounds. Organic pollutants are able to
penetrate into cytoplasmic membrane and affect membrane physiological functions. Bacteria had to evolve adaptationmechanisms
to counteract the damage originated from toxic contaminants and to prevent their accumulation in cell. This review deals with
various adaptation mechanisms of bacterial cell concerning primarily the changes in cytoplasmic membrane and cell wall. Cell
adaptation maintains the membrane fluidity status and ratio between bilayer/nonbilayer phospholipids as well as the efflux of toxic
compounds, protein repair mechanisms, and degradation of contaminants. Low energy consumption of cell adaptation is required
to provide other physiological functions. Bacteria able to survive in toxic environment could help us to clean contaminated areas
when they are used in bioremediation technologies.
... Tal resistência é provocada em grande parte pela constituição da parede celular micobacteriana, que funciona como uma barreira física a muitos fármacos. A parede celular é formada por componentes altamente lipofílicos e os principais são: (1) ácidos graxos de cadeia longa, denominados ácidos micólicos, e (2) polissacarídeos singulares, denominados arabinogalactanos (Brenan, Nikaido, 1995;Barry, Mdluli, 1996). ...
Associada à disseminação da infecção causada pelo HIV, a tuberculose (TB) é considerada, atualmente, problema mundial de saúde pública devido às proporções que vem assumindo. A resistência micobacteriana aos fármacos utilizados na terapêutica é a principal causa da reincidência da TB. Diante deste quadro alarmante, o desenvolvimento de novos e seletivos fármacos anti-TB se faz urgente e necessário. A biossíntese de ácidos graxos é um processo bioquímico realizado por procariotos e eucariotos, o qual fornece precursores essenciais à montagem de componentes celulares importantes, tais como fosfolipídeos, lipoproteínas, lipopolissacarídeos, ácidos micólicos e envelope celular. As diferenças bioquímicas e funcionais entre o mecanismo biossintético de ácidos graxos em bactérias e mamíferos tornam-no alvo relevante ao planejamento de novos antibacterianos, mais seletivos e menos tóxicos. As enoil-ACP redutases são enzimas cruciais à etapa de alongamento de ácidos graxos, considerados produtos intermediários na biossíntese de ácidos micólicos - os principais componentes da parede celular micobacteriana. Portanto, tais enzimas são tidas como alvos moleculares no planejamento racional de novos tuberculostáticos. Avanços recentes no processo de descoberta de novos agentes anti-TB, particularmente os inibidores da enoil-ACP redutase, serão discutidos nesta revisão.
... The mycobacterial cell wall is an incredibly complex structure, with multiple layers that collectively constitute a waxy, durable coat around the cell, which serves as the major permeability barrier to drug action1234. Considering this, the cell wall and related components are attractive for the mining of new drug targets, and remain relatively unexploited for drug discovery in the case of certain bacterial pathogens [2,5,6]. Peptidoglycan (PG or the murein sacculus) is a rigid layer that constricts the cell membrane and the cell within, providing mechanical stability to counteract imbalances of cytoplasmic turgour pressure, and plays an important role in determining cell size and shape78910. ...
Mycobacteria comprise diverse species including non-pathogenic, environmental organisms, animal disease agents and human pathogens, notably Mycobacterium tuberculosis. Considering that the mycobacterial cell wall constitutes a significant barrier to drug penetration, the aim of this study was to conduct a comparative genomics analysis of the repertoire of enzymes involved in peptidoglycan (PG) remodelling to determine the potential of exploiting this area of bacterial metabolism for the discovery of new drug targets.
We conducted an in silico analysis of 19 mycobacterial species/clinical strains for the presence of genes encoding resuscitation promoting factors (Rpfs), penicillin binding proteins, endopeptidases, L,D-transpeptidases and N-acetylmuramoyl-L-alanine amidases. Our analysis reveals extensive genetic multiplicity, allowing for classification of mycobacterial species into three main categories, primarily based on their rpf gene complement. These include the M. tuberculosis Complex (MTBC), other pathogenic mycobacteria and environmental species. The complement of these genes within the MTBC and other mycobacterial pathogens is highly conserved. In contrast, environmental strains display significant genetic expansion in most of these gene families. Mycobacterium leprae retains more than one functional gene from each enzyme family, underscoring the importance of genetic multiplicity for PG remodelling. Notably, the highest degree of conservation is observed for N-acetylmuramoyl-L-alanine amidases suggesting that these enzymes are essential for growth and survival.
PG remodelling enzymes in a range of mycobacterial species are associated with extensive genetic multiplicity, suggesting functional diversification within these families of enzymes to allow organisms to adapt.
... Methoxy mycolic acid synthase mmaA3 (Rv0643c) is an important constituent of mycobacterial cell wall complex, involved in mycolic acid modification and helps in growth, survival and pathogenicity of mycobacteria. Mycobacterial cell wall is the primary permeability barrier responsible for intrinsic resistance to many antibiotics [38,39]. It has been widely explored by many researchers [40][41][42] and defined as a potential antibiotic target. ...
... In this study, we investigated how Mtb infection shapes the quality of the memory CD4 + T cells during latency by comparing the immune phenotype and function of long-lived antigen-specific memory CD4 + T cells in healthy BCG-vaccinated individuals who were either uninfected (BCG) or infected with Mtb (LTBI). We used Mtb cell wall (CW) antigens for stimulation of PBMCs from these two groups as the cell wall plays a critical role in Mtb intracellular survival, persistence, and pathogenicity, and provides a rich source of diverse antigens for immune recognition [37,38]. ...
... Trehalose is a nonreducing disaccharide of glucose, which is used by many lower organisms such as mycobacteria for key functions such as energy storage, signaling, protein-protection and bacterial cell wall components (Elbein 1974;Takayama and Armstrong 1976;Crowe et al. 1984). In mycobacteria, trehalose is also part of a toxic lipid in the cell wall known as trehalose-6,6′-dimycolate or cord factor, which has been identified as the main virulence factor of tuberculosis (Barry and Mdluli 1996;Barry et al. 1998;Daffe and Draper 1998). Accordingly, considerable attention has been directed toward the possibility that enzymes involved in the production of trehalose may serve as drug targets. ...
Trehalose synthase (TreS) catalyzes the reversible conversion of maltose to trehalose in mycobacteria as one of three biosynthetic pathways to this non-reducing disaccharide. Given the importance of trehalose to survival of mycobacteria there has been considerable interest in understanding the enzymes involved in its production; indeed the structures of the key enzymes in the other two pathways have already been determined. Herein we present the first structure of TreS from Mycobacterium smegmatis, thereby providing insights into the catalytic machinery involved in this intriguing intramolecular reaction. This structure, which is of interest both mechanistically and as a potential pharmaceutical target, reveals a narrow and enclosed active site pocket within which substrate intramolecular rearrangements can occur. We also present the structure of a complex of TreS with acarbose, revealing a hitherto unsuspected oligosaccharide binding site within the C-terminal domain. This may well provide an anchor point for the association of TreS with glycogen, thereby enhancing its role in glycogen biosynthesis and degradation.
... In this study, we investigated how Mtb infection shapes the quality of the memory CD4 + T cells during latency by comparing the immune phenotype and function of long-lived antigen-specific memory CD4 + T cells in healthy BCG-vaccinated individuals who were either uninfected (BCG) or infected with Mtb (LTBI). We used Mtb cell wall (CW) antigens for stimulation of PBMCs from these two groups as the cell wall plays a critical role in Mtb intracellular survival, persistence, and pathogenicity, and provides a rich source of diverse antigens for immune recognition [37,38]. ...
Two billion people worldwide are estimated to be latently infected with Mycobacterium tuberculosis (Mtb) and are at risk for developing active tuberculosis since Mtb can reactivate to cause TB disease in immune-compromised hosts. Individuals with latent Mtb infection (LTBI) and BCG-vaccinated individuals who are uninfected with Mtb, harbor antigen-specific memory CD4(+) T cells. However, the differences between long-lived memory CD4(+) T cells induced by latent Mtb infection (LTBI) versus BCG vaccination are unclear. In this study, we characterized the immune phenotype and functionality of antigen-specific memory CD4(+) T cells in healthy BCG-vaccinated individuals who were either infected (LTBI) or uninfected (BCG) with Mtb. Individuals were classified into LTBI and BCG groups based on IFN-γ ELISPOT using cell wall antigens and ESAT-6/CFP-10 peptides. We show that LTBI individuals harbored high frequencies of late-stage differentiated (CD45RA(-)CD27(-)) antigen-specific effector memory CD4(+) T cells that expressed PD-1. In contrast, BCG individuals had primarily early-stage (CD45RA(-)CD27(+)) cells with low PD-1 expression. CD27(+) and CD27(-) as well as PD-1(+) and PD-1(-) antigen-specific subsets were polyfunctional, suggesting that loss of CD27 expression and up-regulation of PD-1 did not compromise their capacity to produce IFN-γ, TNF-α and IL-2. PD-1 was preferentially expressed on CD27(-) antigen-specific CD4(+) T cells, indicating that PD-1 is associated with the stage of differentiation. Using statistical models, we determined that CD27 and PD-1 predicted LTBI versus BCG status in healthy individuals and distinguished LTBI individuals from those who had clinically resolved Mtb infection after anti-tuberculosis treatment. This study shows that CD4(+) memory responses induced by latent Mtb infection, BCG vaccination and clinically resolved Mtb infection are immunologically distinct. Our data suggest that differentiation into CD27(-)PD-1(+) subsets in LTBI is driven by Mtb antigenic stimulation in vivo and that CD27 and PD-1 have the potential to improve our ability to evaluate true LTBI status.
In this work, we review some physical methods of macroscopic experiments, which have been recently argued to be promising for the acquisition of valuable characteristics of biomolecular structures and interactions. The methods we focused on are electron paramagnetic resonance spectroscopy, Raman spectroscopy, and differential scanning calorimetry. They were chosen since it can be shown that they are able to provide a mutually complementary picture of the composition of cellular envelopes (with special attention paid to mycobacteria), transitions between their molecular patterning, and the response to biologically active substances (reactive oxygen species and their antagonists—antioxidants—as considered in our case study).
Background:
2-Phenylethanol is a specific aromatic alcohol with a rose-like smell, which has been widely used in the cosmetic and food industries. At present, 2-phenylethanol is mainly produced by chemical synthesis. The preference of consumers for "natural" products and the demand for environmental-friendly processes have promoted biotechnological processes for 2-phenylethanol production. Yet, high 2-phenylethanol cytotoxicity remains an issue during the bioproduction process.
Results:
Corynebacterium glutamicum with inherent tolerance to aromatic compounds was modified for the production of 2-phenylethanol from glucose and xylose. The sensitivity of C. glutamicum to 2-phenylethanol toxicity revealed that this host was more tolerant than Escherichia coli. Introduction of a heterologous Ehrlich pathway into the evolved phenylalanine-producing C. glutamicum CALE1 achieved 2-phenylethanol production, while combined expression of the aro10. Encoding 2-ketoisovalerate decarboxylase originating from Saccharomyces cerevisiae and the yahK encoding alcohol dehydrogenase originating from E. coli was shown to be the most efficient. Furthermore, overexpression of key genes (aroGfbr, pheAfbr, aroA, ppsA and tkt) involved in the phenylpyruvate pathway increased 2-phenylethanol titer to 3.23 g/L with a yield of 0.05 g/g glucose. After introducing a xylose assimilation pathway from Xanthomonas campestris and a xylose transporter from E. coli, 3.55 g/L 2-phenylethanol was produced by the engineered strain CGPE15 with a yield of 0.06 g/g xylose, which was 10% higher than that with glucose. This engineered strain CGPE15 also accumulated 3.28 g/L 2-phenylethanol from stalk hydrolysate.
Conclusions:
In this study, we established and validated an efficient C. glutamicum strain for the de novo production of 2-phenylethanol from corn stalk hydrolysate. This work supplied a promising route for commodity 2-phenylethanol bioproduction from nonfood lignocellulosic feedstock.
Mycobacterium tuberculosis is the causative agent of tuberculosis, a resilient microbe that can survive within the long courses of medications as well as several years of dormancy in the host system. Although tuberculosis is a preventable and curable disease, however, the rise of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) have made remedy more difficult and a major concern to public health. In Mycobacterium tuberculosis, DprE1 is a key isomerase enzyme that has a vital function in cell wall synthesis. Moreover, DprE1 has also been recognized as a crucial target for drug development against tuberculosis. In the present study, molecular docking, dynamics simulations, and thermodynamic free energy calculations were used to analyze the ability of aminoarylbenzosuberene (AAB) molecules compared to six co-crystallized DprE1 inhibitors. This study identified a promising molecule AAB4 with better stability and binding affinity than the co-crystallized inhibitors. Moreover, AAB4 molecule could also inhibit (Y314C)-DprE1 protein (a TCA1 inhibitor resistance mutant). Thus, the molecule AAB4 could be used as potential lead against tuberculosis and would be an easy pick for in-vitro and in-vivo studies.
Carbohydrate Chemistry provides review coverage of all publications relevant to the chemistry of monosaccharides and oligosaccharides in a given year. The amount of research in this field appearing in the organic chemical literature is increasing because of the enhanced importance of the subject, especially in areas of medicinal chemistry and biology. In no part of the field is this more apparent than in the synthesis of oligosaccharides required by scientists working in glycobiology. Clycomedicinal chemistry and its reliance on carbohydrate synthesis is now very well established, for example, by the preparation of specific carbohydrate- based antigens, especially cancer-specific oligosaccharides and glycoconjugates. Coverage of topics such as nucleosides, amino-sugars, alditols and cyclitols also covers much research of relevance to biological and medicinal chemistry. Each volume of the series brings together references to all published work in given areas of the subject and serves as a comprehensive database for the active research chemist Specialist Periodical Reports provide systematic and detailed review coverage in major areas of chemical research. Compiled by teams of leading authorities in the relevant subject areas, the series creates a unique service for the active research chemist, with regular, in-depth accounts of progress in particular fields of chemistry. Subject coverage within different volumes of a given title is similar and publication is on an annual or biennial basis.
In recent years there has been increasing evidence of the importance of carbohydrates and glycoconjugates in biomedical applications, and the use of synthetic ligands based on carbohydrates as drugs has received much attention. Focussing on drug discovery from key targets and placing an emphasis on the multi-disciplinary approaches necessary to challenge these issues, this book comprehensively covers the new and recent discoveries in the area of carbohydrate drug discovery.
Carbohydrates in Drug Design and Discovery is split into five sections, beginning with a introduction and perspective on the current market. The book then goes on to discuss new synthetic methods in glycobiology, the use of glycobiology in chemical biology and glycobiology in drug discovery.
Providing a worldwide perspective on this broad area, and providing examples of therapeutics already developed using these methods, this book provides a comprehensive introduction, discussion and update on this fast developing field for medicinal chemists and biochemists working in industry and academia.
L-Arabinose is an important component of mycobacterial cell wall. L-Arabinose is involved in the synthesis of arabinogalactan, lipoarabinomannan, and other sugar compounds, which suggests that it can modulate cell wall permeability and drug resistance. However, whether L-arabinose affects mycobacterial antibiotic resistance and the underlying regulatory mechanism remains unclear. In this study, we characterized a new transcription factor of Mycobacterium smegmatis, AraR, that responds to L-arabinose and regulates mycobacterial sensitivity to isoniazid (INH). AraR specifically recognizes two conserved 15-bp motifs within the upstream regulatory region of the arabinose (araR) operon. AraR functions as a transcriptional repressor that negatively regulates araR expression. In contrast to the effect of AraR, overexpression of the araR operon contributes to the mycobacterial INH resistance. L-arabinose acts as an effector and derepresses transcriptional inhibition by AraR. The araR-deficient strain is more resistant to INH than the wild-type strain, whereas the araR-overexpressing strain is more sensitive to INH. Addition of L-arabinose to the medium can significantly increase the resistance to INH of the wild-type strain, but not of the araR knockout strain. Therefore, we identified a new L-arabinose-responding transcription factor and revealed its effect on the bacterial antibiotic resistance. These findings can provide new insights in the regulatory mechanisms mediated by sugar molecules and their relationship with drug resistance in mycobacteria.
There is a paramount need for expanding the drug armamentarium to counter the growing problem of drug-resistant tuberculosis. Salicyl-AMS, an inhibitor of salicylic acid adenylation enzymes, is a first-in-class antibacterial lead compound for the development of tuberculosis drugs targeting the biosynthesis of salicylic acid-derived siderophores. In this study, we determined the Ki of salicyl-AMS for inhibition of the salicylic acid adenylation enzyme MbtA from Mycobacterium tuberculosis (MbtAtb), designed and synthesized two new salicyl-AMS analogues to probe structure–activity relationships (SAR), and characterized these two analogues alongside salicyl-AMS and six previously reported analogues in biochemical and cell-based studies. The biochemical studies included determination of kinetic parameters (Kiapp, konapp, koff, and tR) and analysis of the mechanism of inhibition. For these studies, we optimized production and purification of recombinant MbtAtb, for which Km and kcat values were determined, and used the enzyme in conjunction with an MbtAtb-optimized, continuous, spectrophotometric assay for MbtA activity and inhibition. The cell-based studies provided an assessment of the antimycobacterial activity and post-antibiotic effect of the nine MbtAtb inhibitors. The antimycobacterial properties were evaluated using a strain of non-pathogenic, fast-growing Mycobacterium smegmatis that was genetically engineered for MbtAtb-dependent susceptibility to inhibitors. This convenient model system greatly facilitated the cell-based studies by bypassing the methodological complexities associated with the use of pathogenic, slow-growing M. tuberculosis. Collectively, these studies provide new information on the mechanism of inhibition of MbtAtb by salicyl-AMS and eight analogues, afford new SAR insights for these inhibitors, and highlight several suitable candidates for future preclinical evaluation.
Mycolic acids are high molecular weight α-alkyl-branched Β-hydroxy fatty acids found in the cell wall lipids of acid-fast bacteria. Mycobacterial mycolic acids are among the longest chain (contain between 60 and 90 carbon atoms) and usually occur as complex mixtures of structurally related molecules having oxygen functions such as carboxy, epoxy, keto or methoxy groups in addition to the 3-hydroxy unit and combinations of cis or trans double bonds or cyclopropane rings, methyl branches also being encountered. Mycolic acid from other members of the suborder Corynebacterineae contains between 22 and 78 carbon atoms and desaturation is the only form of functionalization found in them. A wide array of chemical methods is used for mycolic acid analysis. Thin-layer chromatography (TLC) provides a simple method for distinguishing mycobacteria from related genera that produces mycolic acids. Both one- and two-dimensional TLC methodology, employing a variety of solvents, has been used. Molecular heterogeneity of individual mycolic acid classes can be assessed without pyrolysis by gas-liquid chromatography (GLC) or combined gas chromatography-mass spectrometry (GC-MS) of trimethylsilyl ether derivatives of mycolates. Profiling of mycolic acid is a potential tool in distinguishing between acid-fast bacteria including Mycobacterium and related genera of the suborder Corynebacterineae. The GLC pattern is regarded as a fingerprint characteristic for certain species. High-performance liquid chromatography enables separation of p-bromophenacyl esters of mycolate into classes, and homologous components are resolved by reverse-phase chromatography. The generated mycolate profiles are characteristic for many mycobacterial species and these have been used diagnostically.
Reports in recent years indicate that the increasing emergence of resistance to drugs be using to TB treatment. The resistance to them severely affects to options for effective treatment. The emergence of multidrug-resistant tuberculosis has increased interest in understanding the mechanism of drug resistance in M. tuberculosis and the development of new therapeutics, diagnostics and vaccines. In this study, a label-free quantitative proteomics approach has been used to analyze proteome of multidrug-resistant and susceptible clinical isolates of M. tuberculosis and identify differences in protein abundance between the two groups. With this approach, we were able to identify a total of 1,583 proteins. The majority of identified proteins have predicted roles in lipid metabolism, intermediary metabolism, cell wall and cell processes. Comparative analysis revealed that 68 proteins identified by at least two peptides showed significant differences of at least twofolds in relative abundance between two groups. In all protein differences, the increase of some considering proteins such as NADH dehydrogenase, probable aldehyde dehydrogenase, cyclopropane mycolic acid synthase 3, probable arabinosyltransferase A, putative lipoprotein, uncharacterized oxidoreductase and six membrane proteins in resistant isolates might be involved in the drug resistance and to be potential diagnostic protein targets. The decrease in abundance of proteins related to secretion system and immunogenicity (ESAT-6-like proteins, ESX-1 secretion system associated proteins, O-antigen export system and MPT63) in the multidrug-resistant strains can be a defensive mechanism undertaken by the resistant cell.
Glutaraldehyde-resistant Mycobacterium chelonae have been isolated from endoscope washer disinfectors and endoscope rinse water. The mechanism of glutaraldehyde resistance is not well understood. Two spontaneous, glutaraldehyde-resistant mutants of the sensitive type strain, NCTC 946, were investigated. The colony morphology of the two mutants differed from that of the the type strain: colonies of the former were dry and waxy whereas those of the latter were smooth and shiny. Increased resistance to glutaraldehyde of the mutants was matched by small increases in the MICs of rifampicin and ethambutol but not isoniazid. Both mutants showed increased surface hydrophobicity. No changes were identified in the extractable fatty acids or the mycolic acid components of the cell wall but a reduction in each of the resistant strains in the arabinogalactan/arabinomannan portion of the cell wall was detected.
The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification
reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important
constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity.
Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms
at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently
mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form
TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary
site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity.
These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the
adjacent Pro216–Phe228 loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence
of three isoforms and will allow for future work in developing inhibitors.
The history and prevalence of tuberculosis and the role of bedaquiline in multidrug-resistant (MDR) tuberculosis are reviewed.
Tuberculosis continues to cause significant morbidity and mortality worldwide. Increasing rates of drug-resistant tuberculosis are a significant concern and pose serious implications for current and future treatment of the disease. In December 2012, the Food and Drug Administration approved bedaquiline as part of the treatment regimen for pulmonary MDR tuberculosis. Bedaquiline's unique mechanism of action presents an alternative approach to current antimycobacterial killing. By directly inhibiting adenosine triphosphate (ATP) synthase, bedaquiline is effective against both replicating and dormant mycobacteria. Pulmonary cavitary lesions can contain heterogeneous populations. This potential mix of semireplicating and hypometabolic mycobacteria is more difficult to eliminate with conventional antitubercular drugs, thus increasing the risk of resistance. No in vitro cross-resistance between bedaquiline and currently available antitubercular agents has been observed thus far. Because bedaquiline targets a completely different enzyme, cross-resistance with other conventional agents remains unlikely. Enhanced sterilizing capacity via synergistic depletion of ATP further exhibits the promising potential of bedaquiline with pyrazinamide. A course of bedaquiline requires 24 weeks of therapy in combination with other antitubercular drugs.
The approval of bedaquiline represents a major milestone in MDR tuberculosis therapy. Bedaquiline should be considered in patients who have not responded to a regimen containing four second-line drugs and pyrazinamide and patients with documented evidence of MDR tuberculosis resistant to fluoroquinolones. The exact role of bedaquiline cannot be determined until further efficacy and safety data are obtained through ongoing Phase III trials.
CR3 is a key receptor for Mycobacterium tuberculosis on mononuclear phagocytes. CR3-mediated phagocytosis may provide a comparatively safe route of entry and targeting towards a preferred intracellular niche. To test the hypothesis that CR3 may constitute a pathogen-favorable receptor, novel experimental approaches are required, especially ones that can address the in vivo situation. Measures of disease progression in vivo may provide insights that are not evident from simple in vitro studies. For instance, the advantages conferred by entry via CR3 may be subtle and may result only in a more rapid attainment of replicative growth, rather than simple survival, or in minor disturbances in macrophage cytokine production. In vivo, however, such subtle effects could mean the difference between the pathogen being above or below a critical threshold number of organisms that can overwhelm the immune response and the difference between an effective Thl immune response or an ineffective Th2 response.
This report aims at giving a broad overview on biological roles of trehalose, on trehalose processing enzymes and on the use of trehalose mimetics as biological probes and/or enzyme inhibitors for therapeutical purposes.
Phosphatidylinositol mannosides (PIM), lipomannan (LM) and lipoarabinomannan (LAM) are essential components of the cell wall and plasma membrane of Mycobacteria, including the human pathogen Mycobacterium tuberculosis, as well as related Corynebacterineae. We have previously shown that the lipoprotein, LpqW, regulates PIM and LM/LAM biosynthesis in mycobacteria. Here, we provide direct evidence that LpqW regulates the activity of key mannosyltransferases in the periplasmic leaflet of the cell membrane. Inactivation of the Corynebacterium glutamicum lpqW orthologue, NCgl1054, resulted in a slow growth phenotype and a global defect in lipoglycan biosynthesis. The NCg11054 mutant lacked LAMs and was defective in the elongation of the major PIM species, AcPIM2, as well as a second glycolipid, termed Gl-X (mannose-α1-3-glucuronic acid-α1-diacylglycerol), that function as membrane anchors for LM-A and LM-B, respectively. Elongation of AcPIM2 and Gl-X was found to be dependent on expression of polyprenol phosphomannose (ppMan) synthase. However, the NCgl1054 mutant synthesized normal levels of ppMan, indicating that LpqW is not required for synthesis of this donor. A spontaneous suppressor strain was isolated in which lipoglycan synthesis in the NCgl1054 mutant was partially restored. Genome-wide sequencing indicated that a single amino-acid substitution within the ppMan-dependent mannosyltransferase MptB could bypass the need for LpqW. Further evidence of an interaction is provided by the observation that MptB activity in cell-free extracts was significantly reduced in the absence of LpqW. Collectively, our results suggest that LpqW may directly activate MptB, highlighting the role of lipoproteins in regulating key cell wall biosynthetic pathways in these bacteria.
The cellular mycolate synthetase activity of Mycobacterium tuberculosis H37Ra was previously shown to be very sensitive to isoniazid (Wang, L., and K. Takayama. 1972. Antimicrob. Agents Chemother. 2: 438-441). We have now examined the question of how isoniazid inhibits the synthesis of mycolic acids. The saponifiable 14-C-labeled lipids of control and isoniazid-treated cells (1.0 mug/ml, 60 min) were compared on a Sephadex LH-20 column, and it appeared that the synthesis of the intermediate-sized fatty acids was partially inhibited. These fatty acids were fractionated as their methyl esters by Sephadex LH-20 column chromatograp-y and gas-liquid (6% Dexsil) chromatography. Mass sectral analysis of the fractionated lipids revealed several series of fatty acids: fraction II, C39-C56; fraction III, C27-C40. The long-chain fatty acids in three kinds of isoniazid-treated cells were examined: (a) long-term exposure (48 hr, 0.5 mug/ml), (b) short-term exposure (60 min, 1.0 mug/ml), and (c) variable exposure at low concentration (0-90 min, 0.2 mug/ml). Both long- and short-term exposure experiments showed that isoniazid inhibited the synthesis of saturated fatty acids greater than C26 and of unsaturated fatty acids greater than C24. The variable-exposure experiment at low isoniazid concentration showed that the syntheses of mycolic acids and long-chain fatty acid fractions II and III were inhibited to the same extent. These fatty acids may thus be precursors of mycolic acids.
The lipopolysaccharides of mycobacteria, lipoarabinomannan (LAM) and lipomannan (LM), of key importance in host-pathogen interaction, were recently shown to contain a phosphatidylinositol "anchoring domain." We now have established that LAM and LM are based on the phosphatidylinositol mannosides, the characteristic glycophospholipids of mycobacteria. Digestion of the arabinose-free LM with an endo-alpha 1----6-mannosidase yielded evidence for the presence of the 1-(sn-glycerol-3-phospho)-D-myo-inositol-2,6-bis-alpha-D-mannopyranoside unit, indistinguishable from that derived from phosphatidylinositol dimannoside. This same inositol substitution pattern was shown to be present in LAM by methylation analysis before and after dephosphorylation. Positions C-2 and C-6 of the inositol unit of LAM are occupied by mannosyl residues and C-1 by a phosphoryl group. Partial acid hydrolysis of per-O-methylated LAM and comparison by gas chromatography-mass spectrometry of the resulting derivatized oligosaccharides with like products from phosphatidylinositol hexamannoside demonstrated that the C-6 of inositol is the point of attachment of the mannan core of LAM, which consists of an alpha 1----6-linked backbone with considerable alpha-1----2 side chains. Thus, a structural and presumably biosynthetic relationship is established between some of the membranous mannosylphosphatidylinositols described some 25 years ago and the newly emerging, biologically active lipopolysaccharides of mycobacteria.
The peptidoglycan-bound arabinogalactan of a virulent strain of Mycobacterium tuberculosis was per-O-methylated, partially hydrolyzed with acid, and the resulting oligosaccharides reduced and O-pentadeute-rioethylated. The per-O-alkylated oligoglycosyl alditol fragments were separated by high pressure liquid chromatography and the structures of 43 of these constituents determined by 1H NMR and gas chromatography/mass spectrometry. The arabinogalactan was shown to consist of a galactan containing alternating 5-linked beta-D-galactofuranosyl (Galf) and 6-linked beta-D-Galf residues. The arabinan chains are attached to C-5 of some of the 6-linked Galf residues. The arabinan is comprised of at least three major structural domains. One is composed of linear 5-linked alpha-D-arabinofuranosyl (Araf) residues; a second consists of branched 3,5-linked alpha-D-Araf units substituted with 5-linked alpha-D-Araf residues at both branched positions. The non-reducing terminal region of the arabinan was characterized by a 3,5-linked alpha-D-Araf residue substituted at both branched positions with the disaccharide beta-D-Araf-(1----2)-alpha-D-Araf. 13C NMR of intact soluble arabinogalactan established the presence of both alpha- and beta-Araf residues in this domain. This non-reducing terminal motif apparently provides the structural basis of the dominant immunogenicity of arabinogalactan within mycobacteria. A rhamnosyl residue occupies the reducing terminus of the galactan core and may link the arabinogalactan to the peptidoglycan. Evidence is also presented for the presence of minor structural features involving terminal mannopyranosyl units. Models for most of the heteropolysaccharide are proposed which should increase our understanding of a molecule responsible for much of the immunogenicity, pathogenicity, and peculiar physical properties of the mycobacterial cell.
In order to define the permeability barrier to hydrophilic molecules in mycobacteria, we used as a model a smooth, beta-lactamase-producing strain of Mycobacterium chelonei. The rates of hydrolysis of eight cephalosporins by intact and sonicated cells were measured, and the permeability coefficient (P) was calculated from these rates by the method of Zimmermann and Rosselet (W. Zimmermann and A. Rosselet, Antimicrob. Agents Chemother. 12:368-372, 1977). P ranged from (0.9 +/- 0.3) x 10(-8) (benzothienylcephalosporin) to (10 +/- 3.3) x 10(-8) cm/s (cephaloridine); i.e., the P values were lower than those reported for Pseudomonas aeruginosa and Escherichia coli by 1 and 3 orders of magnitude, respectively. The permeability barrier was shown to reduce drastically the stream of drug molecules entering the cell, allowing the rather low level of beta-lactamase (0.1 U/mg of protein with penicillin G) to decrease radically the concentration of the drug at the target; this explains the poor in vitro activities of the beta-lactams against M. chelonei. We also estimated P for small, hydrophilic molecules (glucose, glycerol, glycine, leucine), by studying their uptake kinetics. The values found, ranging from 15 x 10(-8) to 490 x 10(-8) cm/s, were consistent again with a very low permeability of M. chelonei cell wall. The permeation of cephalosporins was not very dependent on the hydrophobicity of the molecules or on the temperature, suggesting a hydrophilic pathway of penetration for these molecules.
Characteristic waxes, based on methoxy and keto long-chain diols, members of the phthiocerol family, have been isolated from representatives of Mycobacterium bovis, M. kansasii, M. marinum, M. microti and M. tuberculosis. M. kansasii produced essentially di-esters of the ketodiol phthiodiolone A, but the remaining species also had waxes based on the methoxy-diols phthiocerol A and phthiocerol B. Gas chromatography of derivatives of the components of the waxes showed that the phthiocerol A components from M. bovis, M. microti and M. microti and M. tuberculosis were qualitatively similar, being mainly C34 and C36, but potentially significant differences were seen in the proportions of the components from M. bovis. The phthiocerols A from M. marinum were C28 and C30 and the phthiodiolones A from M. kansasii were C25 and C27. The multimethyl-branched acids from the waxes of M. bovis were quantitatively different from those of M. microti and M. tuberculosis but all these mycocerosic acids ranged in size from C23 or C24 to C32, with C29 or C30 being the major component in most cases. M. marinum and M. kansasii strains had mainly C26 or C27 and C29 or C30 multimethyl-branched acids, respectively.
Whole cells of Mycobacterium avium, characterized by their negative response in the nine biochemical tests used for mycobacterial
identification in our laboratory, turned positive for nitrate reductase, Tween-80 hydrolysis, beta-glucosidase, acid phosphatase,
alkaline phosphatase, penicillinase, and trehalase after their wall portion was removed to yield spheroplasts. This suggested
that the negative results in most of the biochemical procedures were caused by the exclusion mechanism at the wall level.
Preliminary transmission and scanning electron microscopic studies showed differences at wall level between laboratory-maintained
opaque, dome-shaped (SmD) and host-recycled smooth, transparent (SmT) colony type variants of M. avium and suggested the presence
of an outer regularly structured layer in SmT variants. Comparative ultrastructural studies utilizing different polysaccharide
coloration methods confirmed the presence of an outer polysaccharide layer in SmT variants which was probably related to their
enhanced pathogenicity for experimental animals and drug resistance as compared to that of SmD variants. These findings are
discussed with respect to multiple drug resistance, virulence, and gene expression of M. avium.
The mycobacterial cell wall contains large amounts of unusual lipids, including mycolic acids that are covalently linked to the underlying arabinogalactan-peptidoglycan complex. Hydrocarbon chains of much of these lipids have been shown to be packed in a direction perpendicular to the plane of the cell surface. In this study, we examined the dynamic properties of the organized lipid domains in the cell wall isolated from Mycobacterium chelonae grown at 30 degrees C. Differential scanning calorimetry showed that much of the lipids underwent major thermal transitions between 30 degree C and 65 degrees C, that is at temperatures above the growth temperature, a result suggesting that a significant portion of the lipids existed in a structure of extremely low fluidity in the growing cells. Spin-labeled fatty acid probes were successfully inserted into the more fluid part of the cell wall. Our model of the cell wall suggests that this domain corresponds to the outermost leaflet, a conclusion reinforced by the observation that labeling of intact cells produced electron spin resonance spectra similar to those of the isolated cell wall. Use of stearate labeled at different positions showed that the fluidity within the outer leaflet increased only slightly as the nitroxide group was placed farther away from the surface. These results are consistent with the model of mycobacterial cell wall containing an asymmetric lipid bilayer, with an internal, less fluid mycolic acid leaflet and an external, more fluid leaflet composed of lipids containing shorter chain fatty acids. The presence of the low-fluidity layer will lower the permeability of the cell wall to lipophilic antibiotics and chemotherapeutic agents and may contribute to the well-known intrinsic resistance of mycobacteria to such compounds.
The major mycolic acid produced by Mycobacterium tuberculosis contains two cis-cyclopropanes in the meromycolate chain. The gene whose product cyclopropanates the proximal double bond was cloned by homology
to a putative cyclopropane synthase identified from the Mycobacterium leprae genome sequencing project. This gene, named cma2, was sequenced and found to be 52% identical to cma1 (which cyclopropanates the distal double bond) and 73% identical to the gene from M. leprae. Both cma genes were found to be restricted in distribution to pathogenic species of mycobacteria. Expression of cma2 in Mycobacterium smegmatis resulted in the cyclopropanation of the proximal double bond in the α series of mycolic acids. Coexpression of both cyclopropane synthases resulted in cyclopropanation of both centers, producing
a molecule structurally similar to the M. tuberculosis α-dicyclopropyl mycolates. Differential scanning calorimetry of purified cell walls and mycolic acids demonstrated that cyclopropanation
of the proximal position raised the observed transition temperature by 3°C. These results suggest that cyclopropanation contributes
to the structural integrity of the cell wall complex.
Mycolic acids represent a major constituent of the mycobacterial cell wall complex, which provides the first line of defense against potentially lethal environmental conditions. Slow-growing pathogenic mycobacteria such as Mycobacterium tuberculosis modify their mycolic acids by cyclopropanation, whereas fast-growing saprophytic species such as Mycobacterium smegmatis do not, suggesting that this modification may be associated with an increase in oxidative stress experienced by the slow-growing species. We have demonstrated the transformation of the distal cis double bond in the major mycolic acid of M. smegmatis to a cis-cyclopropane ring upon introduction of cosmid DNA from M. tuberculosis. This activity was localized to a single gene (cma1) encoding a protein that was 34% identical to the cyclopropane fatty acid synthase from Escherichia coli. Adjacent regions of the DNA sequence encode open reading frames that display homology to other fatty acid biosynthetic enzymes, indicating that some of the genes required for mycolic acid biosynthesis may be clustered in this region. M. smegmatis overexpressing the cma1 gene product significantly resist killing by hydrogen peroxide, suggesting that this modification may be an important adaptation of slow-growing mycobacteria to oxidative stress.
To persist in macrophages and in granulomatous caseous lesions, pathogenic mycobacteria must be equipped to withstand the action of toxic oxygen metabolites. In Gram-negative bacteria, the OxyR protein is a critical component of the oxidative stress response. OxyR is both a sensor of reactive oxygen species and a transcriptional activator, inducing expression of detoxifying enzymes such as catalase/hydroperoxidase and alkyl hydroperoxidase. We have characterized the responses of various mycobacteria to hydrogen peroxide both phenotypically and at the levels of gene and protein expression. Only the saprophytic Mycobacterium smegmatis induced a protective oxidative stress response analogous to the OxyR response of Gram-negative bacteria. Under similar conditions, the pathogenic mycobacteria exhibited a limited, nonprotective response, which in the case of Mycobacterium tuberculosis was restricted to induction of a single protein, KatG. We have also isolated DNA sequences homologous to oxyR and ahpC from M. tuberculosis and Mycobacterium avium. While the M. avium oxyR appears intact, the oxyR homologue of M. tuberculosis contains numerous deletions and frameshifts and is probably nonfunctional. Apparently the response of pathogenic mycobacteria to oxidative stress differs significantly from the inducible OxyR response of other bacteria.
Ethambutol is known to rapidly inhibit biosynthesis of the arabinan component of the mycobacterial cell wall core polymer, arabinogalactan (K. Takayama and J. O. Kilburn, Antimicrob. Agents Chemother. 33:1493-1499, 1989). This effect was confirmed, and it was also shown that ethambutol inhibits biosynthesis of the arabinan of lipoarabinomannan, a lipopolysaccharide noncovalently associated with the cell wall core. In contrast to cell wall core arabinan, which is completely inhibited by ethambutol, synthesis of the arabinan of lipoarabinomannan was only partially affected, demonstrating a differential effect on arabinan synthesis in the two locales. Further studies of the effect of ethambutol on cell wall biosynthesis revealed that the synthesis of galactan in the cell wall core is strongly inhibited by the drug. In addition, ethambutol treatment resulted in the cleavage of arabinosyl residues present in the mycobacterial cell wall; more than 50% of the arabinan in the cell wall core was removed from the wall 1 h after addition of the drug to growing mycobacterial cultures. In contrast, galactan was not released from the cell wall during ethambutol treatment. The natural function of the arabinosyl-releasing enzyme remains unknown, but its action in combination with inhibition of synthesis during ethambutol treatment results in severe disruption of the mycobacterial cell wall. Accordingly, ethambutol-induced damage to the cell wall provides a ready molecular explanation for the known synergetic effects of ethambutol with other chemotherapeutic agents. Nevertheless, the initial direct effect of ethambutol remains to be elucidated.
The MIC of isoniazid, peroxidase-catalase expression, and the presence of the katG gene for 102 Mycobacterium tuberculosis isolates from patients in Sβo Paulo were compared. Fifty-three isoniazid-resistant and 49 isoniazid-sensitive isolates were
analyzed by polymerase chain reaction (PCR) for the presence of katG sequences. All isoniazid-sensitive and 43 (81 %) isoniazid-resistant isolates expressed catalase (P = .001). None of isoniazid-sensitive and 4 (7%) of 53 isoniazid-resistant isolates lacked katG sequences. Among 6 isolates with MICs > 50 ILg/mL, 5 (83%) did not express catalase and 2 lacked katG sequences; only 1 had complete gene deletion shown by Southern blot analysis. These findings indicate a correlation between
loss of catalase and isoniazid resistance among highly resistant isolates, but these isolates were a small proportion of resistant
clinical M. tuberculosis isolates from Sao Paulo.
Despite major advances in our understanding of the structure of mycobacterial cell walls, little is known of their biogenesis, and yet they are the site of action of many anti-tuberculosis drugs and implicated in much of the pathology of tuberculosis and leprosy. A family of monoglycosyl polyprenylphosphates was isolated from Mycobacterium smegmatis, containing arabinose, ribose, and mannose. The isoprenoid nature of the lipid components was established by 1H NMR, and fast atom bombardment mass spectroscopy (FAB-MS) demonstrated the presence of C50 decaprenyl-P derivatives and smaller amounts of the C35 octahydroheptaprenyl-P products. The configuration of the mycobacterial decaprenol was established as mono-trans, octa-cis, pointing to carriers of unusual structure. Combined gas chromatography (GC)/MS, FAB-MS/MS, and 1H NMR allowed characterization of one of the primary components as beta-D-arabinofuranosyl-1-monophosphodecaprenol. Pulse-chase metabolic labeling of cells with D-[14C]glucose indicated that the decaprenyl-P-arabinose is an active intermediate in the biosynthesis of the arabinan of cell wall arabinogalactan and arabinomannan. The identification of polyprenyl-P-ribose suggests the existence of ribose-containing polysaccharides in the cell walls of M. smegmatis or/and of a novel epimerase in the D-arabinose biosynthetic pathway. Ethambutol, a powerful anti-tuberculosis drug known to inhibit arabinogalactan and arabinomannan biosynthesis, results in the rapid accumulation of decaprenyl-P-arabinose, indicating that the drug interferes with either the transfer of arabinose from the donor or, alternatively, the synthesis of the arabinose acceptor itself.
Our understanding of fatty acid biosynthesis in Escherichia coli has increased greatly in recent years. Since the discovery that the intermediates of fatty acid biosynthesis are bound to the heat-stable protein cofactor termed acyl carrier protein, the fatty acid synthesis pathway of E. coli has been studied in some detail. Interestingly, many advances in the field have aided in the discovery of analogous systems in other organisms. In fact, E. coli has provided a paradigm of predictive value for the synthesis of fatty acids in bacteria and plants and the synthesis of bacterial polyketide antibiotics. In this review, we concentrate on four major areas of research. First, the reactions in fatty acid biosynthesis and the proteins catalyzing these reactions are discussed in detail. The genes encoding many of these proteins have been cloned, and characterization of these genes has led to a better understanding of the pathway. Second, the function and role of the two essential cofactors in fatty acid synthesis, coenzyme A and acyl carrier protein, are addressed. Finally, the steps governing the spectrum of products produced in synthesis and alternative destinations, other than membrane phospholipids, for fatty acids in E. coli are described. Throughout the review, the contribution of each portion of the pathway to the global regulation of synthesis is examined. In no other organism is the bulk of knowledge regarding fatty acid metabolism so great; however, questions still remain to be answered. Pursuing such questions should reveal additional regulatory mechanisms of fatty acid synthesis and, hopefully, the role of fatty acid synthesis and other cellular processes in the global control of cellular growth.
Isoniazid (isonicotinic acid hydrazide, INH) is one of the most widely used antituberculosis drugs, yet its precise target
of action on Mycobacterium tuberculosis is unknown. A missense mutation within the mycobacterial inhA gene was shown to confer
resistance to both INH and ethionamide (ETH) in M. smegmatis and in M. bovis. The wild-type inhA gene also conferred INH and
ETH resistance when transferred on a multicopy plasmid vector to M. smegmatis and M. bovis BCG. The InhA protein shows significant
sequence conservation with the Escherichia coli enzyme EnvM, and cell-free assays indicate that it may be involved in mycolic
acid biosynthesis. These results suggest that InhA is likely a primary target of action for INH and ETH.
The isoniazid susceptibility of Mycobacterium tuberculosis is mediated by the product of the katG gene which encodes the heme-containing enzyme catalase-peroxidase. In this study, the chromosomal location of katG has been established and its nucleotide sequence has been determined so that the primary structure of catalase-peroxidase could be predicted. The M. tuberculosis enzyme is an 80,000-dalton protein containing several motifs characteristic of peroxidases and shows strong similarity to other bacterial catalase-peroxidases. Expression of the katG gene in M. tuberculosis, M. smegmatis, and Escherichia coli was demonstrated by Western blotting (immunoblotting). Homologous genes were detected in other mycobacteria, even those which are naturally insensitive to isoniazid.
Our understanding of fatty acid biosynthesis in Escherichia coli has increased greatly in recent years. Since the discovery that the intermediates of fatty acid biosynthesis are bound to the heat-stable protein cofactor termed acyl carrier protein, the fatty acid synthesis pathway of E. coli has been studied in some detail. Interestingly, many advances in the field have aided in the discovery of analogous systems in other organisms. In fact, E. coli has provided a paradigm of predictive value for the synthesis of fatty acids in bacteria and plants and the synthesis of bacterial polyketide antibiotics. In this review, we concentrate on four major areas of research. First, the reactions in fatty acid biosynthesis and the proteins catalyzing these reactions are discussed in detail. The genes encoding many of these proteins have been cloned, and characterization of these genes has led to a better understanding of the pathway. Second, the function and role of the two essential cofactors in fatty acid synthesis, coenzyme A and acyl carrier protein, are addressed. Finally, the steps governing the spectrum of products produced in synthesis and alternative destinations, other than membrane phospholipids, for fatty acids in E. coli are described. Throughout the review, the contribution of each portion of the pathway to the global regulation of synthesis is examined. In no other organism is the bulk of knowledge regarding fatty acid metabolism so great; however, questions still remain to be answered. Pursuing such questions should reveal additional regulatory mechanisms of fatty acid synthesis and, hopefully, the role of fatty acid synthesis and other cellular processes in the global control of cellular growth.
Mycobacteria show a high degree of intrinsic resistance to most antibiotics and chemotherapeutic agents. The low permeability of the mycobacterial cell wall, with its unusual structure, is now known to be a major factor in this resistance. Thus hydrophilic agents cross the cell wall slowly because the mycobacterial porin is inefficient in allowing the permeation of solutes and exists in low concentration. Lipophilic agents are presumably slowed down by the lipid bilayer which is of unusually low fluidity and abnormal thickness. Nevertheless, the cell wall barrier alone cannot produce significant levels of drug resistance, which requires synergistic contribution from a second factor, such as the enzymatic inactivation of drugs.
Nocardomycolic acids from Nocardia rubra were fully separated and characterized by a combination of argentation thin-layer chromatography and gas chromatography — mass spectrometry (GCMS). The occurrence of 20 or more different molecular species of mycolic acids was demonstrated. GCMS analysis of each subclass of mycolic acids after separation on AgNO3 thin-layer chromatography revealed that in general the major species consisted of the even-carbon mycolic acids ranging from C38 to C52. However, the most abundant species differed by the subclasses; C44 being in saturated, C46 in monoenoic and C46 in dienoic mycolic acids, respectively. All these acids were shown to possess C12 or C14 alkyl branch at 2 position, while double bonds were located in longer straight chain alkyl unit.By using this method, distinctive changes in mycolic acid composition by growth temperature were observed. The ratios of saturated, monoenoic to dienoic mycolic acids in a mixture of certain carbon numbered mycolic acids varied greatly, according to the shift of growth temperature. The mass fragmentographic analysis, monitoring M-15 ions derived from the loss of methyl group from the molecular ions showed the lower temperature (15°C) grown cells contained more unsaturated (especially dienoic) mycolic acids, while the higher temperature (40°C) grown cells contained more saturated mycolic acids in both extractable and cell-wall bound lipids. These changes in mycolic acid composition occurred shortly after shifting up the growth temperature from 20°C to 43°C at a logarithmic stage of the bacterial growth.
The inactivation of the enoyl-reductase InhA from Mycobacterium tuberculosis by reactive intermediates formed during the oxidn. of isoniazid and ethionamide was studied. Both drugs can generate electrophilic intermediates capable of reacting with a nucleophilic group of InhA, leading to its inactivation. After inactivation of InhA by isoniazid, one mol. of isoniazid per InhA is covalently bound to the enzyme. Mapping studies suggest that Cys243 is the residue modified in the course of the inactivation. [on SciFinder (R)]
The enzymic oxidn. of isoniazid by the catalase-peroxidase of Mycobacterium tuberculosi was studied. The reaction products suggest two reaction pathways involving both electrophilic and radical species. Labeling expts. are consistent with a reaction mechanism involving the formation of an acyldiimide intermediate followed by decompn. to the corresponding aldehyde and peracid. The results suggest that isoniazid is oxidized by the catalase-peroxidase to a reactive species that may act by inactivating an essential enzyme of M. tuberculosis. [on SciFinder (R)]
Isoniazid inhibited C(24) and C(26) monounsaturated fatty acid synthesis in Mycobacterium tuberculosis H37Ra. Time courses of this inhibition and that of mycolic acid synthesis were similar.
The cell wall of mycobacteria is an efficient permeability barrier that makes mycobacteria naturally resistant to most antibiotics.
Liposome swelling assays and planar bilayer experiments were used to investigate the diffusion process of hydrophilic molecules
through the cell wall of Mycobacterium chelonae and identify the main hydrophilic pathway. A 59-kilodalton cell wall protein
formed a water-filled channel with a diameter of 2.2 nanometers and an average single-channel conductance equal to 2.7 nanosiemens
in 1 M potassium chloride. These results suggest that porins can be found in the cell wall of a Gram-positive bacterium. A
better knowledge of the hydrophilic pathways should help in the design of more effective antimycobacterial agents.
Tuberculosis is responsible for one in four of all avoidable adult deaths in developing countries. Increased frequency and accelerated fatality of the disease among individuals infected with human immunodeficiency virus has raised worldwide concern that control programmes may be inadequate, and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in several recent fatal outbreaks in the United States. Isonicotinic acid hydrazide (isoniazid, INH) forms the core of antituberculosis regimens; however, clinical isolates that are resistant to INH show reduced catalase activity and a relative lack of virulence in guinea-pigs. Here we use mycobacterial genetics to study the molecular basis of INH resistance. A single M. tuberculosis gene, katG, encoding both catalase and peroxidase, restored sensitivity to INH in a resistant mutant of Mycobacterium smegmatis, and conferred INH susceptibility in some strains of Escherichia coli. Deletion of katG from the chromosome was associated with INH resistance in two patient isolates of M. tuberculosis.
The structure of the porin from Rhodobacter capsulatus was determined at a resolution of 1.8 A. The analysis started from a closely related crystal structure that had been solved at a medium resolution of 3 A using multiple isomorphous replacement and solvent flattening. The new structure contains the complete sequence of 301 amino acid residues. Refinement of the model is under way; the present R-factor is 22% with good geometry. Except for the lengths of several loops, the resulting chain fold corresponds to the medium resolution model. The membrane channel is lined by a large number of ionogenic side chains with characteristic segregation of differently charged groups.
The effect of growth temperature on mycolic acid composition in eight strains of Mycobacterium smegmatis was investigated by gas chromatography/mass spectrometry. A change in growth temperature from 45 to 20 degrees C caused a shift in the subclass and molecular species composition of mycolic acids. The relative amount of alpha'-mycolic acids to alpha-mycolic acids decreased, and that of hydroxy mycolic acids increased at lower temperatures. Moreover, the proportion of shorter-chain species of alpha-mycolic acids increased, and those of longer-chain species of alpha-mycolic and hydroxy mycolic acids decreased. This observation seems to be due to the changes of the chain length of meromycolates because the alpha-alkyl chain unit of mycolic acids was not affected. The ratio of odd to even carbon-numbered alpha-mycolates decreased as the growth temperature was lowered. In contrast, the molecular species composition of alpha'-mycolic acid was not influenced by the growth temperature.
Ethambutol at 3.0 micrograms/ml inhibited the transfer of label from D-[14C]glucose into the D-arabinose residue of arabinogalactan
in whole cells of a drug-susceptible strain of Mycobacterium smegmatis. This inhibition began almost immediately after exposure
of the cells to the drug. When drug-resistant M. smegmatis was used in a similar experiment, no such drug inhibition was detected.
A much higher concentration of ethambutol (greater than 50 micrograms/ml) was required to show this inhibition. The drug also
inhibited synthesis of arabinose-containing oligosaccharides when a cell-free enzyme system was used. These results suggest
that the site of action of ethambutol is somewhere on the pathway between the conversion of D-glucose to D-arabinose and the
transfer of arabinose into arabinogalactan. The primary mode of action of ethambutol appears to be inhibition of arabinogalactan
synthesis.
Crude extracts of Mycobacterium tuberculosis H37Ra, an isonicotinic acid hydrazide (isoniazid) (INH)-susceptible strain which
has peroxidase activity, catalyzed the production of catechol from phenol in the presence of INH and H2O2 as shown by the
development of the 444-nm absorption peak of oxidized catechol product. Extracts of the INH-resistant strain of M. tuberculosis
H37Ra, which has no peroxidase, did not catalyze the reaction. The rate of development of the 444-nm peak increased proportionately
with increased superoxide dismutase concentrations. The hydroxyl radical (. OH) scavengers dimethylsulfoxide and mannitol
inhibited the reaction. Isonicotinamide, isonicotinic acid, and nicotinic acid could not replace INH.
Oxidation of isonicotinic acid hydrazide (isoniazid) by horseradish peroxidase at the expense of H2O2 yielded reactive species
which were able to reduce nitroblue tetrazolium and bleach p-nitrosodimethylaniline. Nicotinic acid hydrazide oxidation did
not cause these effects. At slightly alkaline pH, oxidation of isonicotinic acid hydrazide by horseradish peroxidase proceeded
at the expense of molecular O2, and the reaction was oxygen consuming. The addition of H2O2 abolished O2 consumption. Bovine
liver catalase enhanced the rate of nitroblue tetrazolium reduction and decreased the maximal velocity of the reaction proportionately
to catalase concentration. During oxidation of isonicotinic acid hydrazide by horseradish peroxidase-H2O2, splitting of the
heme group of horseradish peroxidase took place as shown by the disappearance of the Soret and minor bands in the visible
region of the spectrum.
Cells of Mycobacterium tuberculosis H37Ra were pre exposed to 0.5 μg isoniazid (INH) per ml for 60 min and washed, and the nature of restoration of cellular mycolate synthetase activity was studied upon reincubation. The restoration of this enzyme activity was a slow and gradual process involving an 8 hour lag period and requiring about 24 hours' recirculation. The washed control cells also had an 8 hour lag period, but complete restoration occurred after about 10 to 11 hours. By subtracting the washing effect, the actual restoration time (a reversal of the INH effect) was about 14 hours. The INH inhibition of mycolate synthetase activity became irreversible when the exposure time was 10 hours or longer. Rifampin at 1.0 μg per ml was shown to inhibit protein synthesis in M. tuberculosis, but its effect on the mycolate synthetase activity was relatively moderate. Because rifampin did not effectively inhibit the restoration of mycolate synthetase activity, it was concluded that this restoration phenomenon did not involve protein synthesis.
When an actively growing culture of the H37Ra strain of Mycobacterium tuberculosis was exposed to isoniazid at a concentration of 0.5 μg/ml, the cells began to lose their ability to synthesize mycolic acids
immediately. After 60 min, the cells had completely lost this ability. The synthesis of the three mycolate components—α-mycolate,
methoxymycolate, and β-mycolate—was inhibited. The viability of the isoniazid-treated cells was unaffected up to about 60
min of exposure, after which time there was a gradual decline in the viability to about 18% after 180 min. Correspondingly,
growth of the drug-treated cells slowed down and stopped after 24 hr. The inhibition of the synthesis of mycolic acids was
reversible if the drug was removed before the loss of viability set in. Incubation of the viable cells in the absence of the
drug for 24 hr restored the mycolate synthesis. These results strongly suggest that the inhibition of the synthesis of the
mycolic acids is closely associated with the primary mechanism of action of isoniazid on the tubercle bacilli. The sequence
of events which leads to the loss of viability of cells exposed to isoniazid is described.
E. coli incorporates increasing proportions of saturated and long-chain fatty acids into phospholipids as growth temperature is increased. It was found that this compositional variation results in the biosynthesis of phospholipids that have identical viscosities at the temperature of growth of the cells. This "homeoviscous adaptation" can also be observed in E. coli membrane preparations. Viscosities were determined by use of the electron spin resonance spin-label technique.
Representative strains of some species of Mycobacterium were degraded by both acid and alkaline methanolysis. Two-dimensional thin-layer chromatography was used to determine the patterns of mycolic acids and other long-chain components in these methanolysates. Patterns composed of alpha-, methoxy- and ketomycolates were found in Mycobacterium asiaticum, Mycobacterium bovis, Mycobacterium gastri, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium marinum and Mycobacterium tuberculosis; a representative of Mycobacterium thermoresistibile also contained lower molecular weight alpha'-mycolates in addition to these three acids. In representatives of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium nonchromogenicum, "Mycobacterium novum", Mycobacterium paratuberculosis, Mycobacterium scrofulaceum, Mycobacterium terrae, Mycobacterium xenopi, and Mycobacterium sp. MNC 165 alpha- and ketomycolates were accompanied by omega-carboxymycolates and 2-eicosanol and homologous alcohols which are derived from wax-ester mycolates. Mycobacterium fortuitum and "Mycobacterium giae" contained alpha'- and epoxymycolates and both serovars of Mycobacterium simiae had a very characteristic pattern of alpha-, alpha'- and ketomycolic acids. Comparison with data for other mycobacteria showed the chemotaxonomic significance of these mycolic acid patterns.
The nocardomycolic acid compositions of extractable and the cell wall-bound lipids from five strains of Nocardia asteroides (A-23007, A-23094, B-23006, B-23095, and IFO 3384) were compared by using gas chromatography-mass spectrometry. The molecular species composition of mycolic acid differed significantly among the strains of N. asteroides. The A-23007 strain possessed the shortest species, centering at C(44(46)), and the A-23094 and IFO-3384 strains followed, each centering at C(52). The B-23006 and B-23095 strains possessed the longest species, centering at C(56) or C(54), thus indicating that N. asteroides strains accommodate a heterogeneous group in respect to carbon numbers of mycolic acids. The doublebond isomers of mycolic acids from the representative strain IFO 3384 were fully separated and analyzed by argentation thin-layer chromatography, followed by gas chromatography-mass spectrometry. The reference strain (IFO 3384) possessed up to four double bonds on the straight chain of mycolic acids ranging from C(46) to C(60). All of the species possessed a C(14) alkyl branch at C-2. The more highly unsaturated subclasses consisted of the longer-chain mycolic acids. Marked changes in mycolic acid composition were induced by altering the growth temperature of strain IFO 3384. The cells grown at the higher temperature (50 degrees C) contained more saturated mycolic acids, whereas those grown at the lower temperature (17 degrees C) had more polyunsaturated (up to tetraenoic) mycolic acids, although a significant difference in carbon chain length was not detected. These changes in the degree of unsaturation of mycolic acids occurred shortly after shifting the growth temperature from 17 to 50 degrees C at logarithmic stages of the bacterial growth, thus indicating that N. asteroides can adapt to changes in the environmental temperature by altering the structure of mycolic acids of the cell walls.
Mycobacterium microti walls contained three types of mycolic acids, very similar to those found in Mycobacterium tuberculosis. An alpha-mycolate with two cyclopropane rings, a methoxymycolate with one cyclopropane ring and a methoxyl group, and a ketomycolate with one cyclopropane ring and a keto group were partially characterized. The mycolates made up 34% (by weight) of the peptidoglycan-arabinogalactan-mycolate wall skeleton. Young exponential phase cultures and organisms harvested from mouse lungs contained high proportions of ketomycolates; older cultures had roughly equal proportions of keto- and methoxymycolates. The proportion of alpha-mycolates increased slightly with age of culture, but was always less than one-third of the total.
Molecular species of two major subclasses of mycolic acids from Mycobacterium phlei, α-mycolic acids (M1) and dicarboxy mycolic acids (M3), were separated gas-chromatographically and identified mass-spectrometrically. The mycolic acid compositions of extractable and cell wall bound lipids were markedly influenced by growth temperature. Increasing growth temperature from 20°C to 50°C resulted in an increase in longer chain species of both mycolic acid subclasses with a concomitant decrease in shorter chain homologues. The most abundant molecular species were C76 and C58 of M1 and M3 in the 20°C grown cells, while the 50°C grown cells contained C80 in M1 and C⁶², in M³, most abundantly. Changes in mycolic acid composition occurred rapidly after growth temperature was raised from 20°C to 50oC with an increase in C⁶² and a concomitant decrease in C⁵⁸. Mass fragmentographic analysis revealed that an increase in total carbon numbers of mycolic acids was caused by the elongation of straight chain alkyl unit, without any changes in α-branch. Changes in the molecular species composition of secondary alcohols presumably derived from the ester mycolic acids were also observed and an increase in longer species (C20-ol-2) with a concomitant decrease in shorter ones (C18-ol-2) was noted as the temperature rose. An increase in the growth temperature also resulted in a decrease in unsaturated fatty acids in extractable lipids.
These observations suggest that mycobacteria alter the molecular species composition of mycolic acid subclasses and phospholipids, in response to growth temperature, to maintain a suitable membrane function.
Mycobacteria, members of which cause tuberculosis and leprosy, produce cell walls of unusually low permeability, which contribute to their resistance to therapeutic agents. Their cell walls contain large amounts of C60-C90 fatty acids, mycolic acids, that are covalently linked to arabinogalactan. Recent studies clarified the unusual structures of arabinogalactan as well as of extractable cell wall lipids, such as trehalose-based lipooligosaccharides, phenolic glycolipids, and glycopeptidolipids. Most of the hydrocarbon chains of these lipids assemble to produce an asymmetric bilayer of exceptional thickness. Structural considerations suggest that the fluidity is exceptionally low in the innermost part of bilayer, gradually increasing toward the outer surface. Differences in mycolic acid structure may affect the fluidity and permeability of the bilayer, and may explain the different sensitivity levels of various mycobacterial species to lipophilic inhibitors. Hydrophilic nutrients and inhibitors, in contrast, traverse the cell wall presumably through channels of recently discovered porins.
The molecular mechanisms of resistance to streptomycin, rifampin, and isoniazid in 53 Mycobacterium tuberculosis clinical isolates were examined. Twenty-five of 44 streptomycin-resistant strains had mutations in the rpsL gene and 5 of these had rrs gene perturbations. The region of the rpoRgene that is associated with resistance to rifampin
was altered in 28 of29 rifampin-resistant strains. Mutations in known genetic markers of isoniazid resistance were detected
in 25 of 42 isoniazid-resistant isolates: 20 strains had katG gene alterations and 5 had perturbations in the inhAoperon. Of the 20 multiply resistant strains with reduced sensitivity to streptomycin, rifampin, and isoniazid, 11 had mutations
in genetic markers associated with resistance to each of these three drugs. These studies suggest that the primary mechanism
of multiple drug resistance in tuberculosis is the accumulation of mutations in individual drug target genes.
The cell wall of Mycobacterium smegmatis mc2155 was shown to be an effective permeability barrier to hydrophilic compounds. Permeability coefficients to beta-lactams ranged from 10 x 10(-7) to 0.5 x 10(-7) cms-1. Cell wall proteins were solubilized with EDTA and Genapol and were tested for channel-forming activity by reconstitution into lipid bilayers. Proteins were able to induce a voltage-gated cation-selective channel. The mycobacterial porin channel appeared to be water-filled since the single-channel conductance followed the mobility sequence of hydrated ions in the aqueous phase. On the basis of the Renkin equation and the single-channel conductance, the channel diameter was estimated to be around 3 nm. Model calculations showed that cation selectivity may be caused by four negative point-charges at the channel mouth. The permeability properties of the cell wall of intact cells were in good agreement with those of the reconstituted channel. Negatively charged cephalosporins, cefamandole and cephalothin, diffused at a 10- to 20-fold lower rate than the zwitterionic cephaloridine. The mycobacterial porin represents a major hydrophilic pathway of the cell wall of M. smegmatis.
To develop and demonstrate the utility of automated DNA sequencing strategies for rapid and unambiguous identification of Mycobacterium species and mutations associated with antimicrobial resistance in Mycobacterium tuberculosis. DESIGN AND SPECIMENS: A 360-base pair segment of the gene (hsp65) encoding a 65-kd heat shock protein was characterized from 91 isolates assigned to 24 Mycobacterium species by traditional biochemical techniques. Areas of seven genes recently shown to contain mutations associated with antimicrobial resistance in M tuberculosis strains were also sequenced in a sample of 128 resistant organisms. Early positive BACTEC 460 cultures and acid-fast, bacterium-positive sputum specimens from patients with tuberculosis were also studied.
Automated DNA sequencing identified species-specific polymorphism in the target segment of hsp65, successfully identified organisms to the species level in smear-positive sputum samples, and unambiguously characterized seven genes associated with antimicrobial resistance in M tuberculosis.
Rapid identification of M tuberculosis and other Mycobacterium species is possible by automated DNA sequencing of a portion of hsp65. The technique is also feasible for analysis of some smear-positive sputum specimens. Unambiguous characterization of target segments of genes harboring mutations associated with antimicrobial resistance in M tuberculosis is possible from primary patient specimens. Taken together, the data demonstrate the feasibility of mycobacterial species identification and potential to identify mutations associated with antimicrobial resistance in less than 48 hours.
Resistance to isoniazid in Mycobacterium tuberculosis can be mediated by substitution of alanine for serine 94 in the InhA protein, the drug's primary target. InhA was shown to catalyze the beta-nicotinamide adenine dinucleotide (NADH)-specific reduction of 2-trans-enoyl-acyl carrier protein, an essential step in fatty acid elongation. Kinetic analyses suggested that isoniazid resistance is due to a decreased affinity of the mutant protein for NADH. The three-dimensional structures of wild-type and mutant InhA, refined to 2.2 and 2.7 angstroms, respectively, revealed that drug resistance is directly related to a perturbation in the hydrogen-bonding network that stabilizes NADH binding.
Mycobacteria show a high degree of intrinsic resistance to most antibiotics and chemotherapeutic agents. The low permeability of the mycobacterial cell wall, with its unusual structure, is now known to be a major factor in this resistance. Thus hydrophilic agents cross the cell wall slowly because the mycobacterial porin is inefficient in allowing the permeation of solutes and exists in low concentration. Lipophilic agents are presumably slowed down by the lipid bilayer which is of unusually low fluidity and abnormal thickness. Nevertheless, the cell wall barrier alone cannot produce significant levels of drug resistance, which requires synergistic contribution from a second factor, such as the enzymatic inactivation of drugs.
The history of the disease tuberculosis is briefly discussed. Now human societal failures have potentiated the evolution of drug-resistant strains of the tubercle bacillus in the United States and around the world. Until recently, this evolutionary change largely posed a threat to the health and survival of the individual in whom inadequate therapy promoted the drug resistance. However, the human immunodeficiency virus epidemic threatens to promote wholesale transmission of multidrug-resistant tuberculosis with the potential for immense morbidity and mortality. Reinforced treatment and control programs for tuberculosis are vital.
Isoniazid resistance in Mycobacterium tuberculosis has been associated with total deletion of the katG gene, which codes for catalase-peroxidase production. To determine whether this is a common mechanism of drug resistance,
9 isolates of isoniazid-resistant and 1 of isoniazid-sensitive M. tuberculosis were analyzed by polymerase chain reaction amplification of a 237-bp sequence of the katG gene. Amplification was observed in the isoniazid-sensitive isolate and in 8 resistant isolates; in only 1 isoniazid-resistant
isolate was there no amplification of the expected band, suggesting gene deletion. DNA sequencing showed that 8 of the 9 isolates
had point mutations, deletions, or insertions of 1–3 bases. Evidence corroborating the presence of mutations in the katG gene was obtained by single-strand conformation polymorphism analysis in these 8 isolates. Thus, mutations as well as insertions
and deletions in the katG gene can account for inactive catalase peroxidase, leading to isoniazid resistance; gene deletion occurs only infrequently,
in ∼11% of cases.
Humans are the main reservoir of the tubercle bacilli in nature. Among the one third of the world's population estimated to be infected by tubercle bacilli, most of the infected individuals carry the infection throughout their lives without developing the disease. However, it progresses into disease in about l0 million people per year, who then become transmitters of the tubercle bacilli to the non-infected. Chemoprophylaxis of the newly infected offers the only possibility for fully stopping transmission, since it stops infection from developing into full-blown disease. However, practical difficulties in applying chemoprophylaxis to all those in need makes it clear that eradication of tuberculosis is not in the realm of immediate possibilities. The most efficient way to stop transmission is by treatment of pulmonary cases, but this may result in development of drug resistance. Transmission of drug-resistant tubercle bacilli has been recognized since the early days of chemotherapy and has been the basis for programs of surveillance aimed at evaluating the danger of such an event. These primary drug resistance (PDR) surveillance programs were established very early in several countries (in the early sixties in the United States and France). In both countries, the annual risk of tuberculosis caused by resistant tubercle bacilli remained low, but it was clear that higher incidences might be encountered in high-risk groups, or in cases of non-compliance with medication. In industrialized countries where PDR was satisfactorily surveyed and prevented, the prevailing conclusion that emerged from current experience was that PDR would not significantly affect tuberculosis control programs. This optimistic view was recently challenged by reports of tuberculosis outbreaks caused by multiple-drug-resistant tubercle bacilli. Because these reports may constitute the first warning of an emerging epidemiological problem requiring adequate public health measures, it was thought desirable to review current knowledge on the modes of action of antituberculosis drugs and the mechanisms of drug resistance in tubercle bacilli – which is the main objective of the present review article.
Mycobacterial cell wall functions as an effective permeability barrier, making these bacteria resistant to most antibacterial agents. It has been assumed that this low permeability was due to the presence of a large amount of unusual lipids in the cell wall, but it was not known how these lipids are able to produce such an exceptional barrier. We report here the first experimental evidence on the physical arrangement of these lipids based on X-ray diffraction studies of purified Mycobacterium chelonae cell wall, a result suggesting that the hydrocarbon chains of the cell-wall lipids are arranged predominantly in a direction perpendicular to the cell wall surface, probably producing an asymmetric bilayer structure.
Isoniazid resistance in Mycobacterium tuberculosis is associated with lack of catalase-peroxidase activity. A recent study showed that some isoniazid-resistant M. tuberculosis strains have a complete deletion of the gene (katG) encoding this enzyme. To examine what proportion of clinical isolates of M. tuberculosis have katG deletion, katG sequences in 80 randomly selected isolates from New York City were analyzed. Polymerase chain reaction was used to amplify
a 282-bp segment of M. tuberculosis katG and showed that 35 (90%) of 39 isoniazid-sensitive and 31 (76%) of 41 isoniazid-resistant strains contained katG sequences (P > .1). Ten multidrug and high-level isoniazid-resistant strains with identical restriction fragment length polymorphism patterns
were also analyzed. All were found to have katG sequences. These findings suggest that mechanisms other than complete deletion of katG are involved in isoniazid resistance among most clinical isolates of M. tuberculosis from New York City.
Isoniazid-resistant isolates of Mycobacterium tuberculosis were transformed with a plasmid vector carrying the functional catalase-peroxidase (katG) gene. Expression of katG restored full drug susceptibility in isolates initially resistant to concentrations ranging from 3.2 to > 50 micrograms ml-1. Transformation with the corresponding katG gene from Escherichia coli resulted in low-level expression of catalase and peroxidase activities and conferred partial isoniazid sensitivity.