Chronic oxidative stress and lipid imbalance drive metabolic disorders such as obesity and non-alcoholic fatty liver disease, yet few therapies target the upstream redox imbalance in key tissues. Human carbonic anhydrase III (hCA III), a redox-associated enzyme enriched in liver and adipose tissue, has long remained pharmacologically elusive due to its low catalytic activity and lack of modulators. Here, we identify fragment-like nicotinic acid derivatives as non-sulfonamide hCA III modulators and evaluate their associated cellular effects. Using an esterase activity assay, we screened 25 analogues and identified two fragment-like hits, compound 17 (2-thioethyl) and compound 22 (6-morpholino), with IC50 values of 487 and 361 µM, respectively. Orthogonal thermal shift analysis supported compound-protein interaction, and selected hits were subsequently evaluated in HepG2 cells. Both compounds were associated with reduced CA3 mRNA expression after treatment at 1 µM, while their cellular phenotypes diverged, with compound 22 increasing ROS under oxidative stress conditions and compound 17 affecting mitochondrial membrane potential. Taken together, these findings identify tractable nicotinic acid-derived fragment hits and associated cellular phenotypes that warrant further mechanistic investigation. These fragment-like hits provide a practical starting point for studying the redox-linked biology of hCA III.

Fusarium endophytes damage cereal crops and contaminate produce with mycotoxins. Those fungi overcome the main chemical defence of host via detoxification by a malonyl-CoA-dependent enzyme homologous to xenobiotic metabolizing arylamine N-acetyltransferase (NAT). In Fusarium verticillioides (teleomorph Gibberella moniliformis, GIBMO), this N-malonyltransferase activity is attributed to (GIBMO)NAT1, and the fungus has two additional isoenzymes, (GIBMO)NAT3 (N-acetyltransferase) and (GIBMO)NAT2 (unknown function). We present the crystallographic structure of (GIBMO)NAT1, also modelling other fungal NAT homologues. Monomeric (GIBMO)NAT1 is distinctive, with access to the catalytic core through two "tunnel-like" entries separated by a "bridge-like" helix. In the quaternary arrangement, (GIBMO)NAT1 monomers interact in pairs along an extensive interface whereby one entry of each monomer is covered by the N-terminus of the other monomer. Although monomeric (GIBMO)NAT1 apparently accommodates acetyl-CoA better than malonyl-CoA, dimerization changes the active site to allow malonyl-CoA to reach the catalytic triad (Cys110, His158 and Asp173) via the single uncovered entry, and anchor its terminal carboxyl-group via hydrogen bonds to Arg109, Asn157 and Thr261. Lacking a terminal carboxyl-group, acetyl-CoA cannot form such stabilizing interactions, while longer acyl-CoAs enter the active site but cannot reach catalytic Cys. Other NAT isoenzymes lack such structural features, with (GIBMO)NAT3 resembling bacterial NATs and (GIBMO)NAT2 adopting a structure intermediate between (GIBMO)NAT1 and (GIBMO)NAT3. Biochemical assays confirmed differential donor substrate preference of (GIBMO)NAT isoenzymes, with phylogenetic analysis demonstrating evolutionary separation. Given the role of (GIBMO)NAT1 in enhancing Fusarium pathogenicity, unravelling the structure and function of this enzyme may benefit research into more targeted strategies for pathogen control.

Arylamine N-Acetyltransferases in Health and Disease, pp. 3-41 (2018) Free AccessChapter 1.1: Drug Metabolism and Pharmacogenetics Then and NowEdith Sim and Nicola LaurieriEdith SimDepartment of Pharmacology, University of Oxford, Oxford, United KingdomFaculty of Science Engineering and Computing, Kingston University, Kingston on Thames, United Kingdom and Nicola LaurieriDepartment of Pharmacology, University of Oxford, Oxford, United KingdomDepartment of Emergency and Organ Transplantation, Medical School, University of Bari 'A. Moro', Bari, Italyhttps://doi.org/10.1142/9789813232013_0001Cited by:4 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: Arylamine N-acetyltransferases (NAT, EC 2.3.1.5) were amongst the first enzymes known to result in a genetically determined response to a drug, in this case isoniazid. We now know there are two functional NAT genes in humans and these encode (HUMAN)NAT1 and (HUMAN)NAT2 enzymes. The NAT genes are each encoded at polymorphic loci. This chapter explores the early studies on phenotypic variation in NAT activity and discusses how genotyping of (HUMAN)NAT1 and (HUMAN)NAT2 has changed since NAT pharmacogenetics were first described 50 years ago. The relationship between clinical and molecular studies on genes and proteins has provided current understanding of the NAT enzymes and their genes in humans, where evidence indicates (HUMAN)NAT1 has an endogenous role in addition to a drug metabolising role. NATs in other animal species, bacteria and fungi, are also introduced and polymorphisms in other species are described. Enzymic and later structural studies established that the acetylation reaction involved the transfer of an acetyl group to a cysteine sulphydryl group, activated as part of a juxtaposed catalytic traid with aspartate and histidine in all NAT enzymes with only one exception in bacteria to date. The body of research on NATs has laid the foundation for an exciting period to come encompassing whole genome analyses and understanding epigenetic control to allow exploitation of NAT biology in order to understand and perhaps treat disease. Keywords: ToxicityDrug MetabolismTuberculosisIsoniazidAcetylationCatalytic TriadAcetyl CoAArylamineGene Polymorphism FiguresReferencesRelatedDetailsCited By 4Synthesis and evaluation of new 2-oxo-1,2-dihydroquinoline-3-carboxamides as potent inhibitors against acetylcholinesterase enzymeMuhammed Alzweiri, Kamal Sweidan, Obada abu Saleh and Tamam Al-Helo4 July 2022 | Medicinal Chemistry Research, Vol. 31, No. 9Moonlighting in drug metabolismPhilip G. Board and M. W. Anders28 December 2020 | Drug Metabolism Reviews, Vol. 53, No. 1N-acetyltransferase: the practical consequences of polymorphic activity in manStephen C. Mitchell4 June 2019 | Xenobiotica, Vol. 50, No. 1Humans and Chimpanzees Display Opposite Patterns of Diversity in Arylamine N-Acetyltransferase GenesChristelle Vangenot, Pascal Gagneux, Natasja G de Groot, Adrian Baumeyer and Médéric Mouterde et al.1 July 2019 | G3 Genes|Genomes|Genetics, Vol. 9, No. 7 Arylamine N-Acetyltransferases in Health and DiseaseMetrics History KeywordsToxicityDrug MetabolismTuberculosisIsoniazidAcetylationCatalytic TriadAcetyl CoAArylamineGene PolymorphismPDF download

Arylamine N-Acetyltransferases in Health and Disease, pp. 109-132 (2018) No AccessChapter 1.5: Arylamine N-Acetyltransferases in Normal and Abnormal Embryonic DevelopmentLesley A. Stanley and Edith SimLesley A. StanleyInvestigative Toxicology, Linlithgow, Scotland, United Kingdom and Edith SimDepartment of Pharmacology, University of Oxford, Oxford, United KingdomFaculty of Science Engineering and Computing, Kingston University, Kingston on Thames, United Kingdomhttps://doi.org/10.1142/9789813232013_0005Cited by:2 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: During embryogenesis (HUMAN)NAT1 is expressed in oocytes, all foetal tissues and placenta. Wild-type mouse embryos express (MOUSE) Nat2 (the orthologue of (HUMAN)NAT1, which encodes a functionally similar protein) in the developing neural tube, heart and hindbrain. In reporter mice, (MOUSE)Nat2 is transcribed in the developing eye, neuroendocrine system, heart, intestine, kidney, limb buds and vibrissae. (MOUSE)Nat2 deletion has limited developmental consequences; however, attempts to generate mice overexpressing (HUMAN)NAT1 have failed. The role of (HUMAN)NAT1 in birth defects, whose multifactorial nature and rarity necessitate enormous epidemiological studies, remains ambiguous. Either low or high activity may be detrimental: high activity may lower intracellular folate, possibly exacerbating a pre-existing low folate phenotype, while moderately reduced activity may improve cellular folate profiles. Thus, (HUMAN)NAT1 may participate in an integrated network of proteins which maintains cellular folate homeostasis. Its tight regulation is therefore not surprising, nor is the difficulty of overexpressing it in transgenic mice. Keywords: Arylamine N-acetyltransferaseHuman N-acetyltransferase Type 1Embryonic DevelopmentTransgenicMice, KnockoutGenes, ReporterFolic AcidCongenital AbnormalitiesNeural Tube DefectsCleft Palate FiguresReferencesRelatedDetailsCited By 2Human arylamine N-acetyltransferase 2 genotype-dependent protein expression in cryopreserved human hepatocytesRaúl A. Salazar-González, Mark A. Doll and David W. Hein5 May 2020 | Scientific Reports, Vol. 10, No. 1N-acetyltransferase: the practical consequences of polymorphic activity in manStephen C. Mitchell4 June 2019 | Xenobiotica, Vol. 50, No. 1 Arylamine N-Acetyltransferases in Health and DiseaseMetrics History KeywordsArylamine N-acetyltransferaseHuman N-acetyltransferase Type 1Embryonic DevelopmentTransgenicMice, KnockoutGenes, ReporterFolic AcidCongenital AbnormalitiesNeural Tube DefectsCleft PalatePDF download

Xenobiotic metabolising N -acetyltransferases (NATs) perform biotransformation of drugs and carcinogens. Human NAT1 is associated with endogenous metabolic pathways of cells and is a candidate drug target for cancer. Human NAT2 is a well-characterised polymorphic xenobiotic metabolising enzyme, modulating susceptibility to drug-induced toxicity. Human NATs are difficult to express to high purification yields, complicating large-scale production for high-throughput screens or use in sophisticated enzymology assays and crystallography. We undertake comparative functional investigation of the NAT homologues of ten non-human primates, to characterise their properties and evaluate their suitability as models of human NATs. Considering the amount of generated recombinant protein, the enzymatic activity and thermal stability, the NAT homologues of non-human primates are demonstrated to be a much more effective resource for in vitro studies compared with human NATs. Certain NAT homologues are proposed as better models, such as the NAT1 of macaques Macaca mulatta and M . sylvanus , the NAT2 of Erythrocebus patas , and both NAT proteins of the gibbon Nomascus gabriellae which show highest homology to human NATs. This comparative investigation will facilitate in vitro screens towards discovery and optimisation of candidate pharmaceutical compounds for human NAT isoenzymes, while enabling better understanding of NAT function and evolution in primates.

Isoniazid is still, 60 years after its introduction, a main front line drug for treating tuberculosis. Isoniazid, a hydrazine compound, is metabolized through N-acetylation by the arylamine N-acetyltransferase (NAT) enzyme in humans and its metabolism was important in establishing the early observations on pharmacogenetics since its metabolism to N-acetylisoniazid was identified as being genetically controlled. The incidence of adverse side effects to isoniazid is also linked to its metabolism. These side effects include liver toxicity, neuropathy and a condition resembling the autoimmune disorder Systemic Lupus Erythematosus (SLE). The latter side effect shares similarities with side effects to hydralazine, an anti-hypertensive, which is also a hydrazine and, like isoniazid, induces SLE-like symptoms in a sub group of patients who are almost exclusively slow NAT acetylators. The complement system in humans is essential for immune complex clearance and the chemical mechanism by which isoniazid and hydralazine interact with the activation of the complement cascade has been established, demonstrating their interference with the activation of the thiol ester in complement component C4 such that immune complexes become deposited at inappropriate tissue sites in the small blood vessels, kidneys and joints, thereby generating a SLE-like condition. The relevance of immunohistocompatibitility types relating to the polymorphic C4 type is also explored. ABBREVIATIONS ADPR: Adenosine Diphosphate Ribose; HLA: Human Leukocyte Antigen; NAD: Nicotinamide Adenine Dinucleotide; INH: Isoniazid; InhA: enoyl-[acyl-carrier-protein]-reductase; SLE: Systemic Lupus Erythematosis; TB: Tuberculosis INTRODUCTION Isoniazid (INH) was first introduced for the treatment of tuberculosis (TB) in 1952. It has revolutionized treatment of TB, usually in combination with other drugs [1]. Nevertheless there is a growing search for new anti-tubercular therapies following the availability of genomic information and identification of possible new anti-tubercular targets [2,3] with new treatments reaching the clinical trials stage as part of a growing pipeline of novel anti-tuberculars [4]. Although drug resistance is a growing and real problem, INH is still a front line treatment in combined therapies [5]. The mechanism of action of isoniazid is important as it is one way of identifying new drug treatments [6,7] and resulted in the identification of the agent ethionamide [8]. With INH, the drug is activated by oxidation by KatG (Figure 1), inside the mycobacterial cells and the resulting activated moiety then forms an adduct with NAD+ [8].There is now a consensus that the adduct inhibits the enoyl-[acyl-carrier-protein]-reductase (InhA) [9] and thus inhibits synthesis of the mycolic acid component of the mycobacterial cell wall. There was an earlier controversy as to the nature of the inhibited enzyme and a more recent study has used computational methods to investigate the range of targets for the adduct [10]. Understanding of the molecular changes which lead to isoniazid resistance [11], have been important not only in understanding resistance but also in understanding the mechanism of action of this mainstay of anti-tubercular therapy and of identifying new possible treatments [6,7,12]. Whilst INH is still a front line treatment and is the drug of choice for latent TB, it is however a drug which has been associated with a wide range of adverse side effects. The most common of these is hepatotoxicity [13], followed by neuropathy [14], and also a condition resembling systemic lupus erythematosus (SLE) [15,16]. The mode of action of isoniazid oxidation results in the formation of a covalent bond with NAD+. The effectiveness of isoniazid in combating TB and its mode of action being is the “yin” to the “yang” in relation to its side effects. This review focuses on one particular side effect induced by isoniazid, namely systemic lupus erythematosus (SLE). The condition of drug-induced lupus is shared with a wide range of other drugs [17], and has also been reviewed recently in an excellent online article which sets out the facts [18]. The most common other drugs associated with SLE include the anti-hypertensive, hydralazine, the anti-arrythmic procainamide Central Bringing Excellence in Open Access   Sim et al. (2018) Email: J Drug Des Res 5(1): 1065 (2018) 2/5 studies in different populations [15,32]. The implication is that the difference in the clearance of the drug in slow NAT acetylators creates sufficient of a non-acetylated metabolite to be involved in the adverse reaction. The competition between NAT-acetylation and KatG-activation of INH in humans has also been described in mycobacterial cells themselves [44]. It was demonstrated that mycobacterial cells have an enzyme which N-acetylates INH [26,45], and this has also been demonstrated to contribute to sensitivity to INH in mycobacterial gene deletion and overexpression studies [44,45]. Genetic mutations in the nat gene in clinical isolates of Mycobacterium tuberculosis [11,24,25], have demonstrated that whilst the nat gene does show mutations it makes a minor contribution clinically to INH resistance with the mutations in InhA and KatG genes being of most importance [11,12]. Whilst genetic variation in the mechanisms for pumping INH from the mycobacterial cells has also been identified, but it has also been found to contribute only marginally to the overall INH resistance [11,12]. Structural studies on NAT enzymes from mycobacteria in which each NAT protein has a very similar amino acid sequence [46] have demonstrated INH in the binding pocket of the NAT enzyme from M. smegmatis [47]. In a separate study, hydralazine has been located in the binding site of the NAT enzyme from M. marinum [48], which has shed light on the reaction mechanism for N-acetylation. Interestingly the nat gene itself and the operon in which it is found is essential for mycobacterial survival inside cells [45] and has been explored as a target for antibacterial therapy [49-51]. MECHANISM OF THERAPEUTIC ACTION AND ADVERSE REACTION Isoniazid is activated inside macrophage and the enzyme KatG which catalyses the activation is essential for the action of isoniazid (Figure 1). Once it is activated, the moiety forms a covalent interaction with NAD+ and the adduct formed gives rise to a complex which stops InhA working in the formation of mycolic acids [9]. It has been argued that the adverse reaction in humans is caused by an oxidation reaction perhaps in activated macrophages [34]. It is clear that there is a sub population of individuals who are susceptible to drug-induced SLE. Not all individuals get the adverse reaction. The incidence of INH-induced SLE is low (much less than 5%) although in hydralazine-induced lupus the incidence is higher with up to 12% in the early days when higher doses were used [31,41]. In order to understand the contribution of genetics, studies have been carried out to investigate the Human Leukocyte Antigen (HLA) type of patients who experience SLE-like symptoms. These studies have identified that individuals who carry the HLA DR4 type are more prevalent in the adverse reactors [52], along with those who are slow acetylators for NAT. In addition to the HLA DR4 type, it has been observed that there is an increased incidence of side effects on individuals carrying the C4A-null type a class 3 HLA antigen [53]. It is well established that deletion of the genes for the early components of the classical pathway of complement are at increased risk of developing SLE and the C4A-null type is a particular risk feature [54]. These studies have been confirmed for hydralazine-induced SLE in (which is still used in the USA but only in special circumstances in the UK) and also the anti-arthritic drug penicillamine. Isoniazid and hydralazine are chemically similar, both being hydrazine compounds (Figure 2), and this review focuses on a the nature of SLE induced by isoniazid, using examples derived from isoniazid’s interaction with the immune system in comparison with hydralazine also. The emergence of HIV and concommitant increase in TB, including paediatric TB [19], has resulted in an increased interest in isoniazid toxicity and this has been particularly important in relation to understanding the presentation of instances where children have suffered adverse side effects [14]. ISONIAZID USE Isoniazid is still the main front line drug against tuberculosis, despite the growing problem of resistance. It is usually used in combination with other anti-tuberculars for latent TB and in ongoing drug regimens [20-22]. In addition, isoniazid is being used prophylactically in latent TB [23], and it has been studied in relation to treatment of children who are not receiving anti-viral agents for HIV and appears to have a positive effect in reducing deaths from TB. INH resistance in TB has been widely studied and the overwhelming evidence suggests that mutations in the InhA gene and the KatG gene account for the majority of the incidences of resistance in clinical isolates [11,12], but in addition mutations in the gene encoding for the mycobacterial pumps and in the arylamine N-acetyltransferase (nat) gene in mycobacteria have been implicated. The latter two appear to have a minor effect [11,24-26]. PATTERN OF SIDE EFFECTS SLE is one of the less common side effects of isoniazid therapy. The diagnosis relies on the appearance of a combination of a range of indicators such as rash, joint involvement, and is particularly linked with the appearance of autoantibodies [2729], which have been noted in a similar fashion to hydralazine and procainamide induced SLE [30-32]. One of the key features of the diagnosis of INH-induced SLE has been the recovery and reversal of symptoms on removal of the drug and predictive assays have been reported relating to the induction of autoantibodies [15,28], such as the antibodies identified aga

Linked ArticlesThis article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc

Background and PurposeWith the emergence of extensively drug‐resistant tuberculosis, there is a need for new anti‐tubercular drugs that work through novel mechanisms of action. The meta cleavage product hydrolase, HsaD, has been demonstrated to be critical for the survival of Mycobacterium tuberculosis in macrophages and is encoded in an operon involved in cholesterol catabolism, which is identical in M. tuberculosis and M. bovis BCG.Experimental ApproachWe generated a mutant strain of M. bovis BCG with a deletion of hsaD and tested its growth on cholesterol. Using a fragment based approach, over 1000 compounds were screened by a combination of differential scanning fluorimetry, NMR spectroscopy and enzymatic assay with pure recombinant HsaD to identify potential inhibitors. We used enzymological and structural studies to investigate derivatives of the inhibitors identified and to test their effects on growth of M. bovis BCG and M. tuberculosis.Key ResultsThe hsaD deleted strain was unable to grow on cholesterol as sole carbon source but did grow on glucose. Of seven chemically distinct ‘hits’ from the library, two chemical classes of fragments were found to bind in the vicinity of the active site of HsaD by X‐ray crystallography. The compounds also inhibited growth of M. tuberculosis on cholesterol. The most potent inhibitor of HsaD was also found to be the best inhibitor of mycobacterial growth on cholesterol‐supplemented minimal medium.Conclusions and ImplicationsWe propose that HsaD is a novel therapeutic target, which should be fully exploited in order to design and discover new anti‐tubercular drugs.Linked ArticlesThis article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro‐organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc

Linked ArticlesThis article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro‐organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc

The 7th International Workshop on N-Acetyltransferases (NAT), held from 18 to 20 June 2016, was hosted by Brunhilde Blömeke and her team at the Trier University (Germany). The workshop addressed important aspects and latest advancements in the fields of NAT enzymes, endogenous functions of NATs, NAT gene nomenclature, genetic polymorphisms, and their associations with diseases as well as their use in diagnosis. Representatives from the leading teams performing research on NATs presented their excellent work, discussed the latest results, and created new ideas in the field of N-acetyltransferase research.

Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, includingMycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with intracellular mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Results. Macrophages infected with intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. Conclusion. These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Results. Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. Conclusion. These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

Plant-pathogenic fungi and their hosts engage in chemical warfare, attacking each other with toxic products of secondary metabolism and defending themselves via an arsenal of xenobiotic metabolizing enzymes. One such enzyme is homologous to arylamine N-acetyltransferase (NAT) and has been identified in Fusarium infecting cereal plants as responsible for detoxification of host defence compound 2-benzoxazolinone. Here we investigate functional diversification of NAT enzymes in crop-compromising species of Fusarium and Aspergillus, identifying three groups of homologues: Isoenzymes of the first group are found in all species and catalyse reactions with acetyl-CoA or propionyl-CoA. The second group is restricted to the plant pathogens and is active with malonyl-CoA in Fusarium species infecting cereals. The third group generates minimal activity with acyl-CoA compounds that bind non-selectively to the proteins. We propose that fungal NAT isoenzymes may have evolved to perform diverse functions, potentially relevant to pathogen fitness, acetyl-CoA/propionyl-CoA intracellular balance and secondary metabolism.

Azoreductases are a family of diverse enzymes found in many pathogenic bacteria as well as distant homologues being present in eukarya. In addition to having azoreductase activity, these enzymes are also suggested to have NAD(P)H quinone oxidoreductase (NQO) activity which leads to a proposed role in plant pathogenesis. Azoreductases have also been suggested to play a role in the mammalian pathogenesis of Pseudomonas aeruginosa. In view of the importance of P. aeruginosa as a pathogen, we therefore characterized recombinant enzymes following expression of a group of putative azoreductase genes from P. aeruginosa expressed in Escherichia coli. The enzymes include members of the arsenic-resistance protein H (ArsH), tryptophan repressor-binding protein A (WrbA), modulator of drug activity B (MdaB) and YieF families. The ArsH, MdaB and YieF family members all show azoreductase and NQO activities. In contrast, WrbA is the first enzyme to show NQO activity but does not reduce any of the 11 azo compounds tested under a wide range of conditions. These studies will allow further investigation of the possible role of these enzymes in the pathogenesis of P. aeruginosa.

Diagnosing child TB is a challenge in 22 High Burden Countries (HBCs) including Bangladesh. WHO estimates the case detection rate should be 6-10% of total TB cases. Recent publication shows it can be 4-22%, with sporadic reports form HBCs up to 25%. In Bangladesh, case detection was 3.1% in 2011, 2.85% in 2012 and it was 2.74% in 2013. Moreover, case detection rate in the sub-district hospitals (Upazila Health Complex-UHC) were even lower than the national average. One of the important reasons is lack of clinical skills of doctors and awareness of health care workers on child TB at these community hospitals. To develop capacity among these groups of stakeholder, between 2012-13 Bangladesh has developed generic interactive training modules, training video, flip chart and other training aids on Child TB. By using these tools, capacity building program for doctors and health care workers was conducted between November 2013-July 2014. This was done with support from USAID under the guidance of National Tuberculosis Control Program (NTP) with Bangladesh Pediatric Association (BPA) as technical & implementing partner. Total 1181 doctors and 8345 health care workers have been trained on child TB in 17 districts and 122 sub-districts of Dhaka Division. Preand post-test analysis, of doctors trained, showed statistically significant (p<0.001) improvement of knowledge of the participant irrespective of age, qualification and portfolio. Andragogy modular training methodology was applied; and has been highly appreciated and enjoyed by the participant. Health Care worker also showed enthusiasm on the orientation. Case detection rate showed a raising trend in the sub-district hospitals of Dhaka division. The number of cases detected in the project area was 752 in 2013, while 992 cases were detected till 30 September 2014 (132% of 2013) sparing one quarter (October to December) of 2014. Year end data will provide further light on increment. Since no other intervention was undertaken during this period in Dhaka Division, it can be assumed that the increase in the case detection rate was due to this capacity development program of the doctors and health care workers. LINEAGE 7 MYCOBACTERIUM TUBERCULOSIS STRAINS ARE ASSOCIATED WITH LONGER PATIENT DELAY IN PULMONARY TB PATIENTS SA Yimer, G Norheim, A Namouchi, ED Zegeye, W Kinander, T Tønjum,S Bekele, T Mannsåker, G Bjune, A Aseffa, C Holm-Hansen Department of Microbiology, Unit for Genome Dynamics, Oslo University Hospital, PO Box 4950, Nydalen, NO0424 Oslo, Norway, Email: yimsolo@yahoo.com Background This study investigates the genetic diversity of Mycobacterium tuberculosis (Mtb) strains among pulmonary TB patients in Amhara Region, Ethiopia, and the association between specific Mtb linages, sociodemographic and clinical parameters. Methods DNA was isolated from Mtb-positive sputum specimens (n=240) and analyzed by PCR/24-locus MIRU-VNTR and spoligotyping. Bioinformatics was used to assign Mtb genotypes to global lineages. Associations between patient characteristics and genotype were evaluated using logistic regression analysis. Results Mtb strains (n=138) were assigned to seven sub-lineages, four of which were not represented in the MIRUVNTRplus database. The largest sub-lineages (n=60; 26.0%) belonged to Central Asian (CAS), the next (n=36; 15.6%) to lineage 7, and the third (n=35; 15.2%) to Haarlem. The four novel sub-lineages designated NW-ETH3, NW-ETH1, NW-ETH2, NW-ETH4 included 24 (10.4%), 18 (7.8%), 8 (3.5%) and 5 (2.2%) isolates, respectively. Patients infected with lineage 7 strains were highly likely to delay in seeking medical attention compared to patients infected with CAS strains (AOR=4.7, 95% CI 1.6, 13.5). Cases of Harlem infection (OR= 2.8 95% CI 1.2, 6.6) and NW-ETH3 (OR= 2.8 95% CI 1.0, 7.3) appeared in defined clusters. Conclusion The study revealed a high diversity of modern and pre-modern Mtb lineages of which approximately 25% were not previously reported. Infection with Mtb lineage 7 strains is associated with longer patient delay that suggests a possible increased duration of illness among these patients. Intensified active case finding and contact tracing activities in the study region are needed to expedite diagnosis and treatment of TB. RESISTANCE TO PYRAZINAMIDE IN RUSSIAN MYCOBACTERIUM TUBERCULOSIS ISOLATES D.A. Maslov, O.B. Bekker, T.G. Smirnova, Elena E. Larionova, Sofya N. Andreevskaya, L.N. Chernousova, Y. Zhang, V.N. Danilenko d Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia; Central TB Research Institute, Russian Academy of Medical Sciences, Moscow, Russia; Division of Global Health Equity, Brigham and Women's Hospital, Harvard University, Boston, MA 02115, USA; NPO SRC «BIOAN», Moscow, Russia. Corresponding author V.N. Danilenko: Vavilov Institute of General Genetics, Gubkin str. 3, 119991, Moscow, GSP-1, Russia; e-mail: valerid@vigg.ru. Tuberculosis (TB) is a major global health problem with an estimated of 9.0 million new TB cases and 1.5 million TB deaths in 2013. Pyrazinamide (PZA) is a key frontline drug in TB chemotherapy, which shortens the lengthy anti-TB therapy to 6 months due to its sterilizing ability against persisters. PZA is recommended for treatment of both drug-susceptible and drug-resistant TB cases. The phenotypic testing for PZA susceptibility can show both false-positive and false-negative results (Piersimoni et al., 2006). Up to 40% of false-positive resistance can be obtained on Bactec MGIT 960 (Chedore et al., 2010). Sequencing of pncA gene is also recommended (Simons et al., 2012). Mutations in gene pncA coding pyrazinamidase (PZase) are the major mechanism of PZA resistance in M. tuberculosis (Scorpio and Zhang, 1996; Scorpio et al., 1997; Cheng et al., 2000). Mutations in rpsA gene, encoding S1 ribosomal protein, a key element of trans-translation, were found in some PZA-resistant M. tuberculosis isolates lacking mutations in pncA (Shi et al., 2011). Another gene involved in PZA-resistance is panD, encoding an aspartate decarboxylase (Zhang et al. 2013; Shi et al., 2014). We analyzed 64 M. tuberculosis clinical isolates from patients from the European part of Russia. The isolates were classified in 3 groups: 20 isolates from patients with long and ineffective chemotherapy; 21 isolates from patients with long and effective chemotherapy; a control group of 23 drug-sensitive isolates from new-TB cases. The collection included 6 monoor poly-drug resistant isolates, 25 MDR and 10 XDR isolates. The genotyping by spoligotyping revealed 70% of the isolates to belong to Beijing lineage. The share of Beijing genotypes varied from 48% in the control group to 95% in the group with long and ineffective treatment. The isolates were tested for phenotypic PZA resistance in Bactec MGIT 960 system and for PZase activity using Wayne method. The genes controlling PZA resistance (pncA, rpsA and panD) from these isolates were sequenced. Twenty-four isolates were found to be PZA-resistant after the first Bactec MGIT 960 DST, twentythree of them carrying mutations in pncA and showing negative PZase activity; five more phenotypically sensitive to PZA isolates also harbored mutations in pncA and had negative PZase activity, three of them confirmed PZA resistance on the second DST. Mutations in rpsA and panD not leading to PZA resistance in clinical diagnostics concentration (100 mkg/ml) were found, which still may lead to low-level PZA resistance. We have found 1 PZA-resistant isolate with no mutations in known genes, which may harbor a new mechanism of

Water soluble quinones are a group of cytotoxic anti-bacterial compounds that are secreted by many species of plants, invertebrates, fungi and bacteria. Studies in a number of species have shown the importance of quinones in response to pathogenic bacteria of the genus Pseudomonas. Two electron reduction is an important mechanism of quinone detoxification as it generates the less toxic quinol. In most organisms this reaction is carried out by a group of flavoenzymes known as NAD(P)H quinone oxidoreductases. Azoreductases have previously been separate from this group, however using azoreductases from Pseudomonas aeruginosa we show that they can rapidly reduce quinones. Azoreductases from the same organism are also shown to have distinct substrate specificity profiles allowing them to reduce a wide range of quinones. The azoreductase family is also shown to be more extensive than originally thought, due to the large sequence divergence amongst its members. As both NAD(P)H quinone oxidoreductases and azoreductases have related reaction mechanisms it is proposed that they form an enzyme superfamily. The ubiquitous and diverse nature of azoreductases alongside their broad substrate specificity, indicates they play a wide role in cellular survival under adverse conditions.