Academic literature on the topic 'Rv0158'

ABSTRACT In the murine model of infection, a Mycobacterium tuberculosis mce1 operon mutant elicits an aberrant granulomatous response, resulting in uncontrolled replication and failure to enter a persistent state. In this study, we demonstrate that the mce1 genes can be transcribed as a 13-gene polycistronic message encompassing Rv0166 to Rv0178. Quantitative reverse transcriptase PCR and immunoblot analyses revealed that the mce1 genes and proteins are expressed during in vitro growth but are significantly down-regulated in intracellular bacilli isolated from murine macrophages. A homologue of the FadR subfamily of GntR transcriptional regulators, Rv0165c (designated Mce1R), lies upstream and is divergently transcribed from the operon. To investigate whether this gene plays a role in regulation of mce1 expression, we created an M. tuberculosis mce1R deletion mutant. There was no difference in expression of mce1 operon genes in Δmce1R compared to expression in the wild type during logarithmic growth in vitro. However, in bacilli isolated from murine macrophages, expression of mce1 genes was significantly higher in Δmce1R. In addition, overexpression of mce1R resulted in repression of the mce1 genes. These data demonstrate that Mce1R is a negative regulator that acts intracellularly to repress expression of the mce1 operon. We propose that Mce1R facilitates balanced temporal expression of the mce1 products required for organized granuloma formation, which is both protective to the host and necessary for the persistence of M. tuberculosis.

The metabolic pathway of phospholipids is one of the most important physiologic pathways in Mycobacterium tuberculosis , a typical intracellular bacterium. The hemolytic phospholipase lip gene (Rv0183) is one of 24 phospholipase genes that have been demonstrated to play critical roles in the metabolism of phospholipids in M. tuberculosis. Quantitative RT–PCR and flow cytometry were used to elucidate the immunological and pathogenic implications of the Rv0183 gene on the inflammatory response following persistent expression of Rv0183 in mouse alveolar macrophage RAW264.7 cells. Our results demonstrate that a time-course-dependent ectopic expression of Rv0183 significantly elevated the expression of IL-6, NF-κB, TLR-2, TLR-6, TNFα, and MyD88 in these alveolar macrophage cells. Furthermore, the persistent expression of Rv0183 induced RAW264.7 cell apoptosis in vitro. These findings demonstrate that the expression of Rv0183 induces an inflammatory response and cell apoptosis in the host cells, suggesting that Rv0183 may play an important role in the virulence and pathogenesis of M. tuberculosis infection.

ABSTRACT The pe and ppe genes are unique to mycobacteria and are widely speculated to play a role in tuberculosis pathogenesis. However, little is known about how expression of these genes is controlled. Elucidating the regulatory control of genes found exclusively in mycobacteria, such as the pe and ppe gene families, may be key to understanding the success of this pathogen. In this study, we used a transposon mutagenesis approach to elucidate pe and ppe regulation. This resulted in the identification of Rv0485, a previously uncharacterized transcriptional regulator. Microarray and quantitative real-time PCR analysis confirmed that disruption of Rv0485 reduced the expression of the pe13 and ppe18 gene pair (Rv1195 and Rv1196), defined the Rv0485 regulon, and emphasized the lack of global regulation of pe and ppe genes. The in vivo phenotype of the Rv0485 transposon mutant strain (Rv0485::Tn) was investigated in the mouse model, where it was demonstrated that the mutation has minimal effect on bacterial organ burden. Despite this, disruption of Rv0485 allowed mice to survive for significantly longer, with substantially reduced lung pathology in comparison with mice infected with wild-type Mycobacterium tuberculosis. Infection of immune-deficient SCID mice with the Rv0485::Tn strain also resulted in extended survival times, suggesting that Rv0485 plays a role in modulation of innate immune responses. This is further supported by the finding that disruption of Rv0485 resulted in reduced secretion of proinflammatory cytokines by infected murine macrophages. In summary, we have demonstrated that disruption of a previously uncharacterized transcriptional regulator, Rv0485, results in reduced expression of pe13 and ppe18 and attenuation of M. tuberculosis virulence.

The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units·mg−1 at 37 °C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.

ABSTRACT Latent tuberculosis represents a high-risk burden for one-third of the world population. Previous analysis of murine tuberculosis identified a novel transcriptional regulator encoded by Rv0348 that could control the establishment of persistent tuberculosis. Disruption of the Rv0348 gene from the genome of the virulent H37Rv strain of Mycobacterium tuberculosis revealed a global impact on the transcriptional profiles of 163 genes, including induction of the mammalian cell entry (mce1) operon and the repression of a significant number of genes involved in hypoxia and starvation responses. Nonetheless, gel shift assays did not reveal direct binding between Rv0348 and a set of regulated promoters, suggesting an indirect regulatory role. However, when expressed in Mycobacterium smegmatis, the Rv0348 transcripts were significantly responsive to different levels of hypoxia and the encoded protein was shown to regulate genes involved in hypoxia [e.g., Rv3130c (tgs1)] and intracellular survival (e.g., mce1), among other genes. Interestingly, the colonization level of the ΔmosR mutant strain was significantly lower than that of the wild-type strain of M. tuberculosis, suggesting its attenuation in the murine model of tuberculosis. Taken together, our analyses indicated that the Rv0348 gene encodes a novel transcriptional factor that regulates several operons involved in mycobacterial survival, especially during hypoxia; hence, we propose that Rv0348 be renamed mosR for regulator of mycobacterial operons of survival.

ABSTRACT We report on Mycobacterium tuberculosis Rv0241c and Rv3389c, representing two physiologically functional 3-hydroxyacyl-thioester dehydratases (Htd). These enzymes are potentially entrained in type 2 fatty acid synthase (FASII). Mycobacterial FASII is involved in the synthesis of mycolic acids, which are the major constituents of the protective layer around the pathogen, shielding it from noxious chemicals and the host's immune system. Mycolic acids are additionally associated with the virulence and resilience of M. tuberculosis. Here, Rv0241c and Rv3389c, which are distinct from the previously identified heterodimers Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC) but also the homodimer Rv0130 (HtdZ), were identified by expressing the corresponding candidate open reading frames in Saccharomyces cerevisiae htd2Δ cells lacking mitochondrial 3-hydroxyacyl-acyl carrier protein dehydratase activity, followed by scoring for phenotype rescue. The htd2Δ mutant fails to produce sufficient levels of lipoic acid and does not respire or grow on nonfermentable carbon sources. Soluble protein extracts made from mutant htd2Δ cells expressing mitochondrially targeted Rv0241c or Rv3389c contained 3-hydroxyacyl-thioester hydratase activity. Moreover, mutant yeast cells expressing Rv0241c or Rv3389c were able to recover their respiratory growth on glycerol medium and efficiently reduce 2,3,5-triphenyltetrazolium chloride. Additionally, expression of mitochondrial Rv0241c or Rv3389c in htd2Δ cells also restored de novo lipoic acid synthesis to 92 and 40% of the level in the wild-type strain, respectively. We propose naming Rv0241c and Rv3389c as HtdX and HtdY, respectively, and discuss the implications of our finding with reference to Rv0098, a candidate mycobacterial FabZ homologue with intrinsic thioesterase and hydratase activities that lacks the eukaryotic-like hydratase-2 motif.

Background Bedaquiline is a core drug for treatment of rifampicin-resistant tuberculosis. Few genomic variants have been statistically associated with bedaquiline resistance. Alternative approaches for determining the genotypic-phenotypic association are needed to guide clinical care. Methods Using published phenotype data for variants in Rv0678, atpE, pepQ and Rv1979c genes in 756 Mycobacterium tuberculosis isolates and survey data of the opinion of 33 experts, we applied Bayesian methods to estimate the posterior probability of bedaquiline resistance and corresponding 95% credible intervals. Results Experts agreed on the role of Rv0678, and atpE, were uncertain about the role of pepQ and Rv1979c variants and overestimated the probability of bedaquiline resistance for most variant types, resulting in lower posterior probabilities compared to prior estimates. The posterior median probability of bedaquiline resistance was low for synonymous mutations in atpE (0.1%) and Rv0678 (3.3%), high for missense mutations in atpE (60.8%) and nonsense mutations in Rv0678 (55.1%), relatively low for missense (31.5%) mutations and frameshift (30.0%) in Rv0678 and low for missense mutations in pepQ (2.6%) and Rv1979c (2.9%), but 95% credible intervals were wide. Conclusions Bayesian probability estimates of bedaquiline resistance given the presence of a specific mutation could be useful for clinical decision-making as it presents interpretable probabilities compared to standard odds ratios. For a newly emerging variant, the probability of resistance for the variant type and gene can still be used to guide clinical decision-making. Future studies should investigate the feasibility of using Bayesian probabilities for bedaquiline resistance in clinical practice.

Tuberculosis continues to be a major threat to the human population. Global efforts to eradicate the disease are ongoing but are hampered by the increasing occurrence of multidrug-resistant strains of Mycobacterium tuberculosis. Therefore, the development of new treatment, and the exploration of new druggable targets and treatment strategies, are of high importance. Rv0183/mtbMGL, is a monoacylglycerol lipase of M. tuberculosis and it is involved in providing fatty acids and glycerol as building blocks and as an energy source. Since the lipase is expressed during the dormant and active phase of an infection, Rv0183/mtbMGL is an interesting target for inhibition. In this work, we determined the crystal structures of a surface-entropy reduced variant K74A Rv0183/mtbMGL in its free form and in complex with a substrate mimicking inhibitor. The two structures reveal conformational changes in the cap region that forms a major part of the substrate/inhibitor binding region. We present a completely closed conformation in the free form and semi-closed conformation in the ligand-bound form. These conformations differ from the previously published, completely open conformation of Rv0183/mtbMGL. Thus, this work demonstrates the high conformational plasticity of the cap from open to closed conformations and provides useful insights into changes in the substrate-binding pocket, the target of potential small-molecule inhibitors.

This thesis presents the cloning, purification, crystallization, and structural studies of two unknown proteins from Mycobacterium tuberculosis, and of an aminotransferase from Mycobacterium smegmatis. Structural knowledge of these proteins is of highest interest for structure-based drug design, which is one of the approaches that can be used in order to fight tuberculosis (TB). The structure of the conserved hypothetical protein Rv0216 was refined to a resolution of 1.9 Å. The structure exhibits a so-called double hotdog-fold, similar to known hydratases. However, only parts of the hydratase active site are conserved in Rv0216, and no function could be assigned to the protein. Several Rv0216-like protein sequences were found in a variety of actino- and proteobacteria, suggesting that these proteins form a new protein family. Furthermore, other hotdog-folded proteins in M. tuberculosis were identified, of which a few are likely to be hydratases or dehydratases involved in the fatty acid metabolism. The structure of Rv0130 exhibits a single hotdog-fold and contains a highly conserved R-hydratase motif. Rv0130 was shown to hydrate fatty acid coenzyme A derivatives with a length of six to eight carbons. The Rv0130 active site is situated in a long tunnel, formed by a kink in the central hotdog-helix, which indicate that it can utilize long fatty acid chains as well. A number of previously predicted hotdog-folded proteins also feature a similar tunnel. The structure of branched chain aminotransferase (BCAT) of M. smegmatis was determined in the apo-form and in complex with an aminooxy inhibitor. Mycobacterial BCAT is very similar to the human BCAT, apart for one important difference in the active site. Gly243 is a threonine in the human BCAT, a difference that offers specificity in inhibition and substrate recognition of these proteins. The aminooxy compound and MES were found to inhibit the mycobacterial BCAT activities. The aminooxy compound inhibits by blocking the substrate-pocket. A second inhibitor-binding site was identified through the binding of a MES molecule. Therefore, both the MES-binding site and the substrate-pocket of M. smegmatis BCAT are suggested to be potential sites for the development of new inhibitors against tuberculosis.

Mycobacterium tuberculosis (Mtb) is evolutionarily equipped to resist exogenous reactive oxygen species but shows vulnerability to an increase in endogenous ROS (eROS). Since eROS is an unavoidable consequence of aerobic metabolism, understanding how eROS levels are controlled is essential yet remains uncharacterized. By combining the Mrx1-roGFP2 redox biosensor with transposon mutagenesis, we identified 368 genes (redoxosome) responsible for maintaining non-toxic levels of eROS in Mtb. Integrating redoxosome with a global network of protein-protein interactions and transcriptional regulators revealed a hypothetical protein (rv0158) as a top node managing eROS and redox homeostasis in Mtb. RNA sequencing, seahorse XF flux measurements, and lipid analysis indicate that rv0158 is required to balance the deployment of fatty acid substrates between lipid anabolism and oxidation. Disruption of rv0158 perturbed redox balance in a carbon-source-specific manner, promoted killing in response to anti-TB drugs, reduced survival in macrophages, and lowered persistence in mice. We describe a novel pathogen response to moxifloxacin. Mtb, unlike Escherichia coli, decreases respiration in response to moxifloxacin. Nevertheless, cells were killed, as ROS increased due to NADH-dependent reductive stress. Moxifloxacin lethality was mitigated by supplementing bacterial cultures with a ROS scavenger (thiourea), and an iron chelator (bipyridyl), indicating ROS is part and not a consequence of death processes. Treatment with N-acetyl cysteine (NAC) accelerated respiration and ROS production, increased moxifloxacin lethality, and lowered the mutant prevention concentration. Thus, redox and bioenergetic imbalance contribute to the moxifloxacin-mediated killing of Mtb. These results provide a way to make fluoroquinolones more effective anti-tuberculosis agents. We have previously reported that Mtb H37Rv sets up a gradient of mycothiol redox potential: EMSH-oxidized (-240 mV) to EMSH-reduced (-320 mV) inside macrophages, where the EMSH -reduced Mtb subpopulation are significantly more tolerant to anti-TB drugs. Therefore, one of the keys to subverting drug-tolerance is to impede the emergence of EMSH -reduced subpopulation by inducing overwhelming oxidative stress. In this study, we exposed THP1-macrophages infected with Mtb H37Rv expressing Mrx1-roGFP2-biosensor, to a library of FDA-approved drugs (Enzo Life Sciences; BML-2842) and scored for the oxidative shift in the Mtb- EMSH at 24 hours post infection. Based on their activity to trigger oxidative stress inside the bacterium, non-cytotoxicity to host, and inhibition of bacterial growth inside macrophages, C5 molecule emerged as the top hit. Pre-treatment with C5 potentiated killing of Mtb by all tested antibiotics (isoniazid, rifampicin, and moxifloxacin) and reduced drug-tolerance.
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Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world’s human population. Despite the multiplicity of antimicrobial mechanisms mounted by its host, M. tuberculosis shows a remarkable ability to survive either by evoking survival strategies or by interference with critical macrophage functions that are required to successfully respond to the infection. It has been postulated that the outcome of exposure to M. tuberculosis (in terms of disease symptoms) largely depends upon the selective gene expression of tuberculosis bacilli along with activation of specific signaling pathways in the infected host cells during different phases of infection. In this perspective, determination of the complete genome sequence of Mycobacterium tuberculosis has provided crucial information with respect to the physiology of this bacterium and the pathogenesis of tuberculosis. However, putative functional annotation to all hypothetical proteins coded by M. tuberculosis genome remains complex. One important outcome of the genome-sequencing project was the discovery of two new multigene families designated PE and PPE. About 10% of the M. tuberculosis coding capacity is devoted to the PE and PPE genes, named for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N terminus of their gene products. In addition to these motifs, proteins of PE family share N-terminal domains of approximately 100 amino acids, whereas the PPE proteins possess an N-terminal domain of about 180 amino acids. Many PE and PPE proteins are composed only of these N-terminal homologous domains. However, other members possess an additional C-terminal segment of variable length, often composed of multiple copies of polymorphic GC rich sequences (PGRS). The uniqueness of the PE genes is further illustrated by the fact that these genes are restricted to mycobacteria. However, despite their abundance in mycobacteria, very little is known regarding the expression or the functions of PE family genes. Although the PE and PPE families of mycobacterial proteins are the focus of intense research, no precise function has so far been unraveled for any member of these families. In perspective of above-mentioned observations, we have chosen Rv0754 as a representative PE family gene. Rv0754 was shown to be upregulated in tubercle bacilli upon infection of bone marrow derived macrophages as well as in M. tuberculosis isolated from alveolar macrophages of infected mice. In the current investigation, we demonstrate that Rv0754 is hypoxia responsive gene based on promoter or transcript expression analysis. Further, extensive bioinformatics analysis predicated that Rv0754 posses possible Phosphoglycerate Mutase domain, an enzyme known for its significant role not only in the glycolytic pathway of the carbohydrate metabolism, but also for the crucial cell fate decision during conditions like oxidative stress as well as infection. Experimental data clearly suggests that hypoxic environment dependent expression of Rv0754 imparts resistance to macrophages from oxidative stress. These findings could be attributed to the presence of catalytically active Phosphoglycerate Mutase domain of Rv0754. More often, sophisticated regulation/modulation of key signaling events regulate the critical cell fate decisions during oxidative stress. In this context, TLR2 dependent triggering of PI3K-ERK1/2- NF-κB signaling axis by Rv0754 may be operative in imparting resistance to oxidative stress. Further, Rv0754 triggers COX-2 expression by activating PI3K-ERK1/2-NF-κB cascade in mouse macrophages. These observations are of relevance as Rv0754 is associated with cell wall and is exposed outside the surface of the bacterium suggesting the possible access to intracellular compartments of the infected macrophages. Additionally, Rv0754 elicited humoral antibody reactivities in a panel of human sera or in cerebrospinal fluid samples obtained from different clinical categories of tuberculosis patients. DNA immunizations experiments in mice clearly suggested that Rv0754 is an immunodominant antigen demonstrating significant T cell and humoral reactivity. These observations clearly advocate that Rv0754 protein is expressed in vivo during active infection with M. tuberculosis and that the Rv0754 is immunogenic. Taken together, our findings suggest that Rv0754 is a novel PE_PGRS protein with unique features which could generate conditions that favor survival of the mycobacteria.

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world’s human population. Despite the multiplicity of antimicrobial mechanisms mounted by its host, M. tuberculosis shows a remarkable ability to survive either by evoking survival strategies or by interference with critical macrophage functions that are required to successfully respond to the infection. It has been postulated that the outcome of exposure to M. tuberculosis (in terms of disease symptoms) largely depends upon the selective gene expression of tuberculosis bacilli along with activation of specific signaling pathways in the infected host cells during different phases of infection. In this perspective, determination of the complete genome sequence of Mycobacterium tuberculosis has provided crucial information with respect to the physiology of this bacterium and the pathogenesis of tuberculosis. However, putative functional annotation to all hypothetical proteins coded by M. tuberculosis genome remains complex. One important outcome of the genome-sequencing project was the discovery of two new multigene families designated PE and PPE. About 10% of the M. tuberculosis coding capacity is devoted to the PE and PPE genes, named for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N terminus of their gene products. In addition to these motifs, proteins of PE family share N-terminal domains of approximately 100 amino acids, whereas the PPE proteins possess an N-terminal domain of about 180 amino acids. Many PE and PPE proteins are composed only of these N-terminal homologous domains. However, other members possess an additional C-terminal segment of variable length, often composed of multiple copies of polymorphic GC rich sequences (PGRS). The uniqueness of the PE genes is further illustrated by the fact that these genes are restricted to mycobacteria. However, despite their abundance in mycobacteria, very little is known regarding the expression or the functions of PE family genes. Although the PE and PPE families of mycobacterial proteins are the focus of intense research, no precise function has so far been unraveled for any member of these families. In perspective of above-mentioned observations, we have chosen Rv0754 as a representative PE family gene. Rv0754 was shown to be upregulated in tubercle bacilli upon infection of bone marrow derived macrophages as well as in M. tuberculosis isolated from alveolar macrophages of infected mice. In the current investigation, we demonstrate that Rv0754 is hypoxia responsive gene based on promoter or transcript expression analysis. Further, extensive bioinformatics analysis predicated that Rv0754 posses possible Phosphoglycerate Mutase domain, an enzyme known for its significant role not only in the glycolytic pathway of the carbohydrate metabolism, but also for the crucial cell fate decision during conditions like oxidative stress as well as infection. Experimental data clearly suggests that hypoxic environment dependent expression of Rv0754 imparts resistance to macrophages from oxidative stress. These findings could be attributed to the presence of catalytically active Phosphoglycerate Mutase domain of Rv0754. More often, sophisticated regulation/modulation of key signaling events regulate the critical cell fate decisions during oxidative stress. In this context, TLR2 dependent triggering of PI3K-ERK1/2- NF-κB signaling axis by Rv0754 may be operative in imparting resistance to oxidative stress. Further, Rv0754 triggers COX-2 expression by activating PI3K-ERK1/2-NF-κB cascade in mouse macrophages. These observations are of relevance as Rv0754 is associated with cell wall and is exposed outside the surface of the bacterium suggesting the possible access to intracellular compartments of the infected macrophages. Additionally, Rv0754 elicited humoral antibody reactivities in a panel of human sera or in cerebrospinal fluid samples obtained from different clinical categories of tuberculosis patients. DNA immunizations experiments in mice clearly suggested that Rv0754 is an immunodominant antigen demonstrating significant T cell and humoral reactivity. These observations clearly advocate that Rv0754 protein is expressed in vivo during active infection with M. tuberculosis and that the Rv0754 is immunogenic. Taken together, our findings suggest that Rv0754 is a novel PE_PGRS protein with unique features which could generate conditions that favor survival of the mycobacteria.

Yes
Objectives: Resistance-associated variants (RAVs) in Rv0678, a regulator of the MmpS5-MmpL5 efflux pump, have been shown to lead to increased MICs of bedaquiline (2- to 8- fold) and clofazimine (2- to 4-fold). The prevalence of these Rv0678 RAVs in clinical isolates and their impact on treatment outcomes are important factors to take into account in bedaquiline treatment guidelines. Methods: Baseline isolates from two bedaquiline MDR-TB clinical trials were sequenced for Rv0678 RAVs and corresponding bedaquiline MICs were determined on 7H11 agar. Rv0678 RAVs were also investigated in non-MDRTB sequences of a population-based cohort. Results: Rv0678 RAVs were identified in 23/347 (6.3%) of MDR-TB baseline isolates. Surprisingly, bedaquiline MICs for these isolates were high (>0.24 mg/L, n¼8), normal (0.03 0.24 mg/L, n¼11) or low(<0.03 mg/L, n¼4). A variant at position 11 in the intergenic region mmpS5–Rv0678 was identified in 39 isolates (11.3%) and appeared to increase the susceptibility to bedaquiline. In non-MDR-TB isolates, the frequency of Rv0678 RAVs was lower (6/ 852 or 0.7%). Competition experiments suggested that rifampicin was not the drug selecting for Rv0678 RAVs. Conclusions: RAVs in Rv0678 occur more frequently in MDR-TB patients than previously anticipated, are not associated with prior use of bedaquiline or clofazimine, and in the majority of cases do not lead to bedaquiline MICs above the provisional breakpoint (0.24mg/L). Their origin remains unknown. Given the variety of RAVs in Rv0678 and their variable effects on the MIC, only phenotypic drug-susceptibility methods can currently be used to assess bedaquiline susceptibility.
This work was supported by Janssen Pharmaceutica. N. C. was supported by a Baekeland PhD scholarship from the Flemish Institute for Scientific Technology (IWT 130308, Belgium). C. J. M., L. R. and B. d. J. were supported by a European Research Council Starting Grant INTERRUPTB (311725).

The emergence of drug resistant strains of Plasmodium has given a new face to the old disease, malaria. One of the approaches is to block metabolic pathways of the pathogen. The current thesis describes the X-ray crystallographic analysis of two enzymes of the fatty acid biosynthesis pathway of the malaria parasite Plasmodium falciparum. In order to understand the functional mechanism and mode of inhibitor binding, enzyme-inhibitor complexes were characterized, which could help in further improvement of the efficacy of the inhibitors and hence to fight against the disease. The introductory chapter of the thesis presents a discussion on malaria and different metabolic pathways of the pathogen which could be suitable targets for novel antimalarials. In continuation to that, the pathway of our choice the fatty acid biosynthesis and an overview of the structural features of the enzymes involved in the pathway that have been characterized from different organisms are also described. The second chapter includes the tools of X-ray crystallography that were used for structural studies of the present work. It also discusses the biochemical, biophysical and other computational methods used to further characterize the enzymes under study. Triclosan, a well known inhibitor of Enoyl Acyl Carrier Protein Reductase (FabI) from several pathogenic organisms, is a promising lead compound to design effective drugs. The X-ray crystal structures of Plasmodium falciparum FabI (PfFabI), in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations, were determined and compared with triclosan binding which form the basis of chapter 3. The structures revealed that 4 and 2’ substituted compounds have more interactions with the protein, cofactor and solvent molecules as compared to triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2’ position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water based inhibitor design. 2’ and 4’ unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensates for the lost interactions due to the unsubstitution at 2’ and 4’. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2’ and 4’ positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors. Certain residues in the substrate binding tunnel of PfFabI were mutated to identify the role of these residues in substrate binding and protein stability, which forms the 4th chapter of the thesis. The substrate binding site residue Ala372 of PfFabI has been mutated to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli FabI (EcFabI) which has a Methionine at the structurally similar position of Ala372 of PfFabI. Kinetic studies of the mutants of PfFabI and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a 3-fold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. A second set of mutants generated to check this hypothesis exhibited loss of function, except in one case where, the crystal structure showed that the substrate binding loop is stabilized by a water bridge network. The main focus of chapter 5 is β-Hydroxyacyl-acyl carrier protein dehydratase of Plasmoduim falciparum (PfFabZ) which catalyzes the third and important reaction of the fatty acid elongation cycle. The crystal structure of PfFabZ was available in its hexameric (active) and dimeric (inactive) forms. However, until now PfFabZ has not been crystallized with any bound inhibitors. We have designed a new condition to crystallize PfFabZ with its inhibitors bound in the active site, and determined the crystal structures of three of these complexes. This is the first report of the crystal structures of PfFabZ with competitive inhibitor complexes and the first such study on any FabZ enzyme with active site inhibitors. These inhibitors in the active site stabilize the substrate binding loop, revealing the substrate binding tunnel with an overall shape of “U”. In the crystal structure, the residue Phe169 located in the middle of the tunnel was found to be in two different conformations, open and closed, implying that it controls the length of the tunnel and makes it suitable for accommodating longer substrates merely by changing its side chain conformation. The hydrophobic nature of the substrate binding channel signifies the specificity for the hydrophobic tail of fatty acid substrates. The volume of the active site tunnel is determined by the sequence as well as by the conformation of the substrate binding site loop region and varies between organisms for accommodating fatty acids of different chain lengths. All PfFabZ inhibitors reported here bind to the active site through specific contacts like hydrogen bonds with catalytic residues and hydrophobic interactions. This report on the crystal structures of the complexes of PfFabZ provides the structural basis of the inhibitory mechanism of the enzyme, that could be used to improve the potency of inhibitors against an important component of fatty acid synthesis common to many infectious organisms. The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. The analysis discussed in chapter 6, led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects. Rv0098, predicted to be the FabZ of Mycobacterium tuberculosis, was cloned, expressed, purified, crystallized, and X-ray diffraction data were collected. Molecular replacement trials with all “hot dog” fold proteins failed to yield any significant solution due to the low sequence similarity (<20%) of Rv0098 compared to other FabZs. During the trials of structure solution by multiple isomorphous replacement method, structure of Rv0098 was published and it was shown to be a long-chain fatty acyl-CoA thioesterase (FcoT). The crystal structure of Rv0098 did not explain the molecular basis of substrate specificity of varying chain lengths. Molecular dynamics studies were carried out, which revealed that certain residues of the substrate binding tunnel are flexible and thus modulates the length of the tunnel. Flexibility of the loop at the base of the tunnel was also found to be important for determining the length of the tunnel for accommodating appropriate substrates. The structural basis of accommodating long chain substrates by Rv0098 is discussed in chapter 7, by combining the crystallographic and molecular dynamics studies. Part of the work presented in the thesis has been reported in the following publications. Karmodiya, K., Sajad, S., Sinha, S., Maity, K., Suguna, K. and Surolia, N. (2007) Conformational stability and thermodynamic characterization of homotetrameric Plasmodium falciparum beta-ketoacyl-ACP reductase. IUBMB Life 59, 441-9. Pidugu, L. S., Maity, K., Ramaswamy, K., Surolia, N. and Suguna, K. (2009) Analysis of proteins with the 'hot dog' fold: prediction of function and identification of catalytic residues of hypothetical proteins. BMC Struct Biol 9, 37. Kapoor, N., Banerjee, T., Babu, P., Maity, K., Surolia, N. and Surolia, A. (2009) Design, development, synthesis, and docking analysis of 2'-substituted triclosan analogs as inhibitors for Plasmodium falciparum enoyl-ACP reductase. IUBMB Life 61, 1083-91. Maity, K., Bhargav, S. P., Sankaran, B., Surolia, N., Surolia, A. and Suguna, K. (2010) X-ray crystallographic analysis of the complexes of enoyl acyl carrier protein reductase of Plasmodium falciparum with triclosan variants to elucidate the importance of different functional groups in enzyme inhibition. IUBMB Life 62, 467-76. Maity, K., Banerjee, T., Narayanappa, P., Surolia, N., Surolia, A. and Suguna, K. (2010) Effect of substrate binding loop mutations on the structure, kinetics and inhibition of Enoyl Acyl Carrier Protein Reductase from Plasmodium falciparum. (Communicated) Maity, K., Bharat, S. V., Kapoor, N., Surolia, N., Surolia, A. and Suguna, K. (2010) Insights into the functional and inhibitory mechanism of the β-Hydroxyacyl-Acyl Carrier Protein Dehydratase of Plasmodium falciparum from the crystal structures of its complexes with active site inhibitors. (Communicated)

The emergence of drug resistant strains of Plasmodium has given a new face to the old disease, malaria. One of the approaches is to block metabolic pathways of the pathogen. The current thesis describes the X-ray crystallographic analysis of two enzymes of the fatty acid biosynthesis pathway of the malaria parasite Plasmodium falciparum. In order to understand the functional mechanism and mode of inhibitor binding, enzyme-inhibitor complexes were characterized, which could help in further improvement of the efficacy of the inhibitors and hence to fight against the disease. The introductory chapter of the thesis presents a discussion on malaria and different metabolic pathways of the pathogen which could be suitable targets for novel antimalarials. In continuation to that, the pathway of our choice the fatty acid biosynthesis and an overview of the structural features of the enzymes involved in the pathway that have been characterized from different organisms are also described. The second chapter includes the tools of X-ray crystallography that were used for structural studies of the present work. It also discusses the biochemical, biophysical and other computational methods used to further characterize the enzymes under study. Triclosan, a well known inhibitor of Enoyl Acyl Carrier Protein Reductase (FabI) from several pathogenic organisms, is a promising lead compound to design effective drugs. The X-ray crystal structures of Plasmodium falciparum FabI (PfFabI), in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations, were determined and compared with triclosan binding which form the basis of chapter 3. The structures revealed that 4 and 2’ substituted compounds have more interactions with the protein, cofactor and solvent molecules as compared to triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2’ position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water based inhibitor design. 2’ and 4’ unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensates for the lost interactions due to the unsubstitution at 2’ and 4’. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2’ and 4’ positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors. Certain residues in the substrate binding tunnel of PfFabI were mutated to identify the role of these residues in substrate binding and protein stability, which forms the 4th chapter of the thesis. The substrate binding site residue Ala372 of PfFabI has been mutated to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli FabI (EcFabI) which has a Methionine at the structurally similar position of Ala372 of PfFabI. Kinetic studies of the mutants of PfFabI and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a 3-fold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. A second set of mutants generated to check this hypothesis exhibited loss of function, except in one case where, the crystal structure showed that the substrate binding loop is stabilized by a water bridge network. The main focus of chapter 5 is β-Hydroxyacyl-acyl carrier protein dehydratase of Plasmoduim falciparum (PfFabZ) which catalyzes the third and important reaction of the fatty acid elongation cycle. The crystal structure of PfFabZ was available in its hexameric (active) and dimeric (inactive) forms. However, until now PfFabZ has not been crystallized with any bound inhibitors. We have designed a new condition to crystallize PfFabZ with its inhibitors bound in the active site, and determined the crystal structures of three of these complexes. This is the first report of the crystal structures of PfFabZ with competitive inhibitor complexes and the first such study on any FabZ enzyme with active site inhibitors. These inhibitors in the active site stabilize the substrate binding loop, revealing the substrate binding tunnel with an overall shape of “U”. In the crystal structure, the residue Phe169 located in the middle of the tunnel was found to be in two different conformations, open and closed, implying that it controls the length of the tunnel and makes it suitable for accommodating longer substrates merely by changing its side chain conformation. The hydrophobic nature of the substrate binding channel signifies the specificity for the hydrophobic tail of fatty acid substrates. The volume of the active site tunnel is determined by the sequence as well as by the conformation of the substrate binding site loop region and varies between organisms for accommodating fatty acids of different chain lengths. All PfFabZ inhibitors reported here bind to the active site through specific contacts like hydrogen bonds with catalytic residues and hydrophobic interactions. This report on the crystal structures of the complexes of PfFabZ provides the structural basis of the inhibitory mechanism of the enzyme, that could be used to improve the potency of inhibitors against an important component of fatty acid synthesis common to many infectious organisms. The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. The analysis discussed in chapter 6, led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects. Rv0098, predicted to be the FabZ of Mycobacterium tuberculosis, was cloned, expressed, purified, crystallized, and X-ray diffraction data were collected. Molecular replacement trials with all “hot dog” fold proteins failed to yield any significant solution due to the low sequence similarity (<20%) of Rv0098 compared to other FabZs. During the trials of structure solution by multiple isomorphous replacement method, structure of Rv0098 was published and it was shown to be a long-chain fatty acyl-CoA thioesterase (FcoT). The crystal structure of Rv0098 did not explain the molecular basis of substrate specificity of varying chain lengths. Molecular dynamics studies were carried out, which revealed that certain residues of the substrate binding tunnel are flexible and thus modulates the length of the tunnel. Flexibility of the loop at the base of the tunnel was also found to be important for determining the length of the tunnel for accommodating appropriate substrates. The structural basis of accommodating long chain substrates by Rv0098 is discussed in chapter 7, by combining the crystallographic and molecular dynamics studies. Part of the work presented in the thesis has been reported in the following publications. Karmodiya, K., Sajad, S., Sinha, S., Maity, K., Suguna, K. and Surolia, N. (2007) Conformational stability and thermodynamic characterization of homotetrameric Plasmodium falciparum beta-ketoacyl-ACP reductase. IUBMB Life 59, 441-9. Pidugu, L. S., Maity, K., Ramaswamy, K., Surolia, N. and Suguna, K. (2009) Analysis of proteins with the 'hot dog' fold: prediction of function and identification of catalytic residues of hypothetical proteins. BMC Struct Biol 9, 37. Kapoor, N., Banerjee, T., Babu, P., Maity, K., Surolia, N. and Surolia, A. (2009) Design, development, synthesis, and docking analysis of 2'-substituted triclosan analogs as inhibitors for Plasmodium falciparum enoyl-ACP reductase. IUBMB Life 61, 1083-91. Maity, K., Bhargav, S. P., Sankaran, B., Surolia, N., Surolia, A. and Suguna, K. (2010) X-ray crystallographic analysis of the complexes of enoyl acyl carrier protein reductase of Plasmodium falciparum with triclosan variants to elucidate the importance of different functional groups in enzyme inhibition. IUBMB Life 62, 467-76. Maity, K., Banerjee, T., Narayanappa, P., Surolia, N., Surolia, A. and Suguna, K. (2010) Effect of substrate binding loop mutations on the structure, kinetics and inhibition of Enoyl Acyl Carrier Protein Reductase from Plasmodium falciparum. (Communicated) Maity, K., Bharat, S. V., Kapoor, N., Surolia, N., Surolia, A. and Suguna, K. (2010) Insights into the functional and inhibitory mechanism of the β-Hydroxyacyl-Acyl Carrier Protein Dehydratase of Plasmodium falciparum from the crystal structures of its complexes with active site inhibitors. (Communicated)