Lipoarabinomannan (LAM) and the mycolyl-arabinogalactan (mAG) complex are two major entities found in the cell wall of Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans. Given their important roles in the viability and virulence of the pathogen, enzymes involved in these pathways represent a rich source of potential therapeutic drug targets. As fundamental understanding of substrateenzyme interactions is often essential in the drug discovery process, the purpose of this study was to investigate the substrate specificities of an -(16)-mannosyltransferase (ManT) and a -(15,6)-galactofuranosyltransferase (GlfT2), two key enzymes involved in the biosynthesis of LAM and mAG, respectively. Although the ManT activity had been detected using an established radioactive assay, its substrate specificity remained poorly defined. The current study focused on the design, synthesis and evaluation of acceptor substrate analogs of ManT. Among those analogs prepared were those containing methoxy-, hydrogen-, and amino-substituted carbohydrate residues as well as epimeric derivatives. A homologous series of oxygen- and sulfur-linked mannosides were also prepared. Evaluation of these analogs revealed the steric requirements and hydrogen bonding interactions of the enzyme, and the effect of acceptor length on mannosyltransferase activity. Also, these results provided additional insight into the role of ManTs and allowed the current proposed pathway of LAM to be further revised. Another objective of the current study was to understand how GlfT2 catalyzes the alternating -(15) and -(16)-galactofuranosyl transfers in a single active site. A panel of mono- and dideoxy trisaccharide derivatives was synthesized, in which hydroxyl groups at either or both C-5 and C-6 positions on the sugar residues at the reducing ends were selectively removed. Biological evaluation of these analogs using a spectrophotometric assay, and structural analysis of some of the enzymatic products, showed that the removal of the hydroxyl group(s) in the acceptors appeared to have no dramatic effect on either GlfT2 activity or the regioselectivity of its galactosylation. These results suggest that groups other than the C-5 and C-6 hydroxyl groups of the acceptors are more critical for the enzyme catalysis. The identification of these key elements would be the further objective of this project. The results from these fundamental studies provide important information about how these enzymes interact with their substrates at the molecular level. More importantly, this work will serve as the basis for the further design of potential inhibitors, which are potential lead compounds for novel therapeutic agents that are active against tuberculosis.

No description available.

No description available.

All organisms must sense and respond to their environment in order to survive. The processes that allow a living cell to sense changes in its environment, and respond appropriately are collectively referred to as ‘signal transduction’. Cyclic AMP is a ubiquitously used second messenger molecule that plays diverse roles from hormone signalling in mammalian cells to catabolite repression in enteric bacteria. In several bacterial pathogens such as Pseudomonas aeruginosa, cAMP has also been found to mediate pathogenesis, usually by regulating the production of several virulence factors aiding in colonisation of the host. Cyclic AMP signalling has been suggested to regulate the virulence of the obligate intracellular Mycobacterium tuberculosis. Mycobacteria, including M. tuberculosis, code for a large number of adenylyl cyclases, enzymes that synthesise cAMP. Of the 16 putative adenylyl cyclases encoded by M. tuberculosis H37Rv, 10 have received extensive biochemical attention. A knockout of one of these cyclases, Rv0386, resulted in compromised virulence of M. tuberculosis. Ten proteins predicted to bind cAMP and mediate its cellular roles have also been identified in M. tuberculosis. Among these are the cAMP-regulated transcription factor, CRPMt, and cAMP-regulated protein acetyl transferase, KATmt. Comparatively little information is available, however, regarding the roles of cAMP-degrading machinery in mycobacteria. Two phosphodiesterases, with modest activity against cAMP in vitro, have been identified from M. tuberculosis, and are encoded by the Rv0805 and Rv2795c loci. Of these, Rv2795c has orthologs in all sequenced mycobacterial genomes. However, Rv0805-like proteins are coded only by slow growing mycobacteria such as the M. tuberculosis-complex, M. marinum and M. leprae, several of which are human or animal pathogens. Rv0805 belongs to the metallophosphoesterase superfamily of proteins, consisting of metal-dependent phosphoesterases with substrates ranging from large polymers like nucleic acids to small molecules like cAMP and glycerophospholipids. Like other metallophosphoesterases, Rv0805 displays promiscuous substrate utilisation and efficient hydrolysis of 2’3’-cAMP in vitro. Rv0805 also hydrolyses 3’5’-cAMP in vitro. Overexpression of Rv0805 is reported to lead to reduction in intracellular cAMP levels in M. smegmatis and M. tuberculosis, suggesting that it is capable of hydrolysing cAMP in the bacterial cell as well. The structure of Rv0805 revealed a sandwich-like α/β fold, typical of metallophosphoesterases, along with a relatively flexible C-terminal domain of unknown function. Despite extensive biochemical and structural information on Rv0805 however, its roles in mycobacteria remain unknown. In this study, the cellular roles of Rv0805 are explored and using information from biochemical and structural analyses, novel activities and interactions of Rv0805 have been identified. Rv0805, when expressed in M. smegmatis, led to a reduction in intracellular cAMP, as previously reported. However, the extent of reduction was modest (~30 %) and limited to the exponential phase of growth when both Rv0805 and intracellular cAMP are at their highest levels. Overexpression of Rv0805 also resulted in hypersensitivity to cell wall perturbants like crystal violet and sodium dodecyl sulphate (SDS) indicative of a change in the properties of the cell envelope of M. smegmatis. Importantly, these effects were independent of cAMP-hydrolysis by Rv0805, as overexpression of catalytically inactive Rv0805N97A also elicited similar changes. Unexpectedly, Rv0805 was localised to the cell envelope, both in M. tuberculosis as well as in M. smegmatis. The ability of Rv0805 to localise to the cell envelope was dependent on it C-terminus, as truncation of Rv0805 in this region (Rv0805Δ10, Rv0805Δ20 and Rv0805Δ40) resulted in progressively greater enrichment in the cytosol of M. smegmatis. Overexpression of Rv0805Δ40, which was localised almost completely to the cytosol, did not result in hypersensitivity to SDS, suggesting that cell envelope localisation, rather than cAMP-hydrolysis, was crucial for the cell envelope modifying roles of Rv0805. A possible mechanism behind the cell envelope-related effects of Rv0805 overexpression was the ability of the protein to interact with the cell wall of mycobacteria in a C-terminus-dependent manner. Purified Rv0805, but not Rv0805Δ40, could associate with crude mycobacterial cell wall as well as purified cell wall core polymer (mycolyl-arabinogalactan-peptidoglycan complex) in vitro. In addition to the C-terminus, the architecture of the active site was also crucial for this interaction as mutations in the active site that compromised metal-binding also resulted in poor interaction with the cell wall. Most significant among these residues was His207, which when mutated to Ala almost completely abrogated interaction with the cell wall in vitro. Further, Rv0805H207A was unable to localise to the cell envelope when expressed in M. smegmatis, even in the presence of the C-terminus, highlighting the importance of this residue in maintaining the structural integrity of Rv0805, and demonstrating that the structure of the C-terminus, rather than its sequence alone, played a role in cell envelope localisation and interaction. In order to verify that the observed sensitivity of Rv0805-overexpressing M. smegmatis to cell wall perturbants was due to a change in cell envelope properties atomic force microscopy was employed. Two distinct modes of operation were used to analyse surface and bulk properties of the mycobacterial cell envelope. These were tapping mode phase imaging, and contact mode force spectroscopy. Using tapping mode phase imaging, it was found that the cell surface of M. smegmatis was inherently heterogeneous in its mechanical properties. Further, contact mode force-spectroscopy revealed that the cell envelope of M. smegmatis in cross-section had at least three layers of varying stiffness. Typically, a middle layer of high stiffness was observed, sandwiched between two lower stiffness layers. This organisation is reminiscent of the current model of the mycobacterial cell envelope, possessing a central polysaccharide rich layer and outer and inner lipid rich layers. Treatment of wild type M. smegmatis with cell wall-perturbing antibiotics isoniazid and ethambutol resulted in markedly altered phase images, as well as significantly lower stiffness of the bacterial cell envelopes, validating that the methodology employed could indeed be used to assess cell wall perturbation in mycobacteria. Further, M. smegmatis harbouring deletions in cell envelope biosynthesis related genes, MSMEG_4722 and aftC, showed significantly lower cell wall stiffness than wild type M. smegmatis, providing evidence that genetic perturbation of the cell wall of mycobacteria could also be studied using atomic force microscopy. While phase imaging revealed similar surface properties of Rv0805-overexpressing and control M. smegmatis, force spectroscopy revealed significantly lower cell envelope stiffness, particularly of the middle layer, of the former. Cell envelope stiffness was, however, unaffected by expression of Rv0805Δ40 in M. smegmatis, providing direct evidence for C-terminus-dependent cell envelope perturbation upon Rv0805 overexpression. Additionally, overexpression of Rv0805N97A, but not Rv0805H207A led to reduced stiffness of the cell envelope of M. smegmatis, demonstrating that the cell wall remodelling activity of Rv0805 was independent of cAMP-hydrolysis, but dependent on cellular localisation and cell wall interaction. Like in M. smegmatis, overexpression of Rv0805 also led to lower cAMP levels in M. tuberculosis. Using a microarray-based transcriptomics approach, pathways affected by Rv0805 overexpression were identified. Rv0805 overexpression elicited a transcriptional response, leading to the down-regulation of a number of virulence associated genes such as whiB7, eis, prpC and prpD. Importantly, Rv0805-overexpression associated gene expression changes did not include genes regulated by CRPMt, the primary cAMP-regulated transcription factor in M. tuberculosis. Further, Rv0805N97A overexpression in M. tuberculosis led to similar changes in gene expression as overexpression of the wild type protein. These observations reiterated that, at least upon overexpression, the effects of Rv0805 were largely independent of cAMP-hydrolysis. Using overexpression in M. smegmatis and M. tuberculosis, cAMP-hydrolysis independent roles of Rv0805 in mycobacteria were identified. To further validate these observations, a knockout strain of the Rv0805 gene was generated in M. bovis BCG, a well-established model to study M. tuberculosis. Curiously, deletion of Rv0805 did not lead to a change in intracellular cAMP levels, demonstrating that cAMP-hydrolysis by Rv0805 may not contribute to the modulation of mycobacterial cAMP levels under standard laboratory growth conditions. Rv0805 deletion led to altered colony morphology and possible reduction in cell wall thickness, reaffirming the roles of this phosphodiesterase in regulating cell envelope physiology of mycobacteria. Additionally, Rv0805 deletion also resulted in compromised growth of M. bovis BCG in fatty acid-deficient media, implicating Rv0805 as a possible regulator of carbon metabolism. In summary, this thesis explores novel links between Rv0805 and the mycobacterial cell wall and elucidates the critical importance of the C-terminus domain of this metallophosphodiesterase in modulating its cellular localisation to, and interaction with, the mycobacterial cell envelope. En route to understanding the effects of Rv0805 overexpression on the cell wall of M. smegmatis, an atomic force microscopy-based methodology to assess perturbation of the cell envelope of mycobacteria was also developed. Finally, using a combination of biochemical and genetic analyses, cellular roles of Rv0805, independent of cAMP-hydrolysis, were identified in slow-growing mycobacteria. This study therefore provides direct evidence against the sole role of this mycobacterial phosphodiesterase as a regulator of intracellular cAMP levels, and opens up new avenues to understanding the cellular functions of Rv0805 and indeed other members of the metallophosphoesterase superfamily.

No description available.

Pathogenesis originating from mycobacterial invasion on host cells is prevalent and is a major challenge in efforts towards overcoming the burden of mycobacterial diseases. Complex architecture of mycobacterium cell wall includes an assortment of glycolipids, phospholipids, glycopeptidolipids (GPLs), peptidoglycans, arabinogalactans, lipoarabinomannans and mycolic acid. Aided by thick cell wall envelope, mycobacteria are known to survive in hostile environment. As most antibiotics target the log phase of the bacteria, bacterial survival is also largely dependent on its stationary phase. Mycobacteria have evolved colonization by means of biofilm formation in the stationary phase, so as to survive under stress and hostile conditions. Biofilms are the specialized form of phenotype which makes bacteria several fold resistant to antibiotics. Development of inhibitors against biofilms remains a challenge due to the poor permeability of molecules and coordination among cells. The first part of Chapter 1 of the thesis describes the details of formation of biofilm in the stationary phase of bacteria and understanding the molecular level details for making the strategies to overcome antidrug resistance of mycobacteria. Among the cyclic hosts, cyclodextrins are prominent. Due to their unique structural and physical properties, cyclodextrins can form inclusion complexes with a wide range of guest molecules. Although synthetic modifications of cyclodextrins through hydroxy groups are very common, modifications at backbone continue to be a challenge. Backbone modified cyclodextrins using different organic moieties were developed and their altered cavity properties were explored in many instances. Chemical synthesis of cyclic oligosaccharides is, in general, involved (i) a cyclo-oligomerization of linear oligosaccharide precursor and (ii) an one-pot polycondensation of appropriately designed monomer under suitable reaction conditions. The second part of Chapter 1 deals with a literature survey of skeletal modification of cyclodextrins, their synthesis and binding abilities with different guest molecules. In my research programme, synthesis and studies of oligosaccharide glycolipids relevant to mycobacterial cell wall were undertaken. Arabinofuranoside trisaccharide glycolipids, containing β-anomeric linkages at the non-reducing ends and double hexadecyloxy lipid moieties, interconnected to the sugar moiety through a glycerol core, were synthesized (Figure 1). Arabinan trisaccharides 1 with lipidic chain and 3 without lipidic chain comprise β-(1→2), β-(1→3) anomeric linkages at the non-reducing end, whereas in the case of arabinan trisaccharides 2 and 4, β-(1→2), β-(1→5) linkages are present between the furanoside units. In the scheme of synthesis of trisaccharide glycolipids, monosaccharide derivative and lipidic portions were individually prepared first and were assembled subsequently to secure the target glycolipids. Incorporation of β-arabinofuranoside linkages in trisaccharide arabinofuranosides 1-4 was achieved by low temperature activation of silyl group protected conformationally locked thioglycoside donor 5 (Figure 1), in the presence of N-iodosuccinimide (NIS) and silver trifluoromethanesulfonate (AgOTf). Figure 1. Molecular structures of trisaccharides 3, 4 and glycolipids 1, 2 with β-arabinofuranoside linkages at the non-reducing end and glycosyl donor 5. Following the synthesis, the efficacies of synthetic glycolipids to interact with surfactant protein A (SP-A) were assessed by using surface plasmon resonance (SPR) technique, from which association-dissociation rate constants and equilibrium binding constants were derived. SP-A, a lung innate immune system component, is known to bind with glycolipids present in the cell surface of a mycobacterial pathogen. From the analysis of SPR studies with glycolipids 1, 2 and SP-A, the association rate constants (ka) were found to be in the range of 0.3 to 0.85 M−1 s−1, whereas the dissociation rate constants (kd) were varied between 2.21 and 3.2×10−3 s−1. The equilibrium constants (Ka) values were in the range of 93 and 274 M−1. Trisaccharides 3 and 4, without lipidic chains, were also assessed for their efficacies to interact with SP-A. The association constants for 3 were found to be in the range of 2,470 to 9,430 M−1, whereas for the derivative 4, Ka values varied between 25,600 and 76,900 M−1. The association and equilibrium binding constants for 3 and 4 were found to be significantly higher when compared to glycolipids 1 and 2. In conjunction with our previous report, the present study shows that arabinofuranoside glycolipids, with β-anomeric linkages bind to SP-A with lesser extent as compared to α-anomers. Further, the studies of trisaccharides and glycolipids in mycobacterial growth and sliding motility assays were performed with model organism M. smegmatis and it was found that the synthetic compounds affected both growth and motility and the extent was lesser than that of α-anomeric glycosides and glycolipids. Chapter 2 of the thesis describes the details of synthesis, biophysical and biological studies of arabinan trisaccharide glycolipids, with β-anomeric linkages at the non-reducing end. Continuing the synthesis and studies of arabinan oligosaccharides, a linear arabinomannan pentasaccharide and heptasaccharide glycolipids 6 and 10, containing α-(1→2) and α-(1→3) linkages between core arabinofuranoside units, as well as, a branched arabinomannan pentasaccharide and heptasaccharide glycolipids 7 and 11, with α-(1→2) and α-(1→5) linkages between core arabinofuranoside units, were synthesized (Figures 2 and 3). Figure 2. Molecular structures of arabinomannan glycolipids 6 and 7 and the corresponding oligosaccharides 8 and 9. In addition to glycolipids, arabinomannan pentasaccharides without lipidic chain 8 and 9 and arabinomannan heptasaccharides without lipidic chain 12 and 13, were also synthesized. Synthesis was performed using trichloroacetimidate and thioglycosides as glycosyl donors. A block condensation methodology was adopted by which disaccharide donor and monosaccharide acceptor were chosen to assemble the pentasaccharide, by a two-fold glycosylation. Monosaccharide acceptors with and without lipidic chain were used in the glycosylations for the synthesis of glycolipids and pentasaccharides, respectively. Similarly, a trisaccharide thioglycoside donor and monosaccharide acceptors were chosen for the double glycosylation to synthesize heptasaccharides in the presence of NIS and AgOTf. Figure 3. Molecular structures of arabinomannan heptasaccharide glycolipids 10, 11 and corresponding heptasaccharides 12 and 13. Subsequent to synthesis, activities of pentasaccharide glycolipids were assayed on M. smegmatis bacterial growth, sliding motilities and also the effects on mycobacterial biofilms. Profound effects were observed with the synthetic compounds, to reduce the mycobacterial growth, sliding motilities and biofilm structures. Whereas reduction up to ~50% occurred on mycobacterial growth, as much as, 70% reduction in the motilities of the bacteria was observed in the presence of the synthetic glycolipids, at 100 µg mL-1 concentration. At the same concentration, 80–85% reduction in the biofilm was observed. These effects were more pronounced with branched glycolipids than linear analogues. Chapter 3 of the thesis presents the synthesis of linear and branched arabinomannan penta- and heptasaccharide glycolipids and biological studies of arabinomannan pentasaccharide glycolipids with M. smegmatis. Cyclodextrins, the most abundant naturally-occurring cyclic oligosaccharides, are valuable synthetic hosts, primarily as a result of their properties to form inclusion complexes with guest molecules. In spite of voluminous literature on the application of cyclodextrins, through modifications of hydroxy groups, modifications at the backbone continue to be a challenge. Skeletal modifications using aromatic, triazole, diyne, thioether and disulfide moieties were developed, that helped to alter the cavity properties of cyclodextrins. A programme was undertaken to synthesize backbone modified cyclic oligosaccharide, which was achieved using a monomer wherein a one carbon insertion is conducted at C4 of a pyranose, such that the hydroxy moiety at C4 is replaced with a hydroxymethyl moiety. In an approach, a linear trisaccharide monomer was anticipated to provide cyclic oligosaccharides in multiples of such a monomer. In the event, a trisaccharide linear monomer 14 was found to afford a cyclic trisaccharide macrocycle 15, as the major cyclo-oligomer (Scheme 1). Subsequent solid state structural studies show that the molecule confers a perfect trigonal symmetry in the P3 space group, in a narrow cone shape and a brick-wall type arrangement of molecules, such a geometry is hither-to unknown to a cyclic oligosaccharide (Figure 4). Furthermore, binding abilities of cyclic trisaccharide with few organic bases, such as 1-aminoadamantane and hexamethylenetetramine, was evaluated by the means of isothermal titration calorimetry and it was found that such a cyclic trisaccharide exhibits strong binding affinities towards 1-aminoadamantane in aqueous solutions, as compared to the same with naturally-occurring β-cyclodextrin. Scheme 1 Apart from cyclic trisaccharide, synthesis of cyclic tetrasaccharide 17, containing alternative anomeric α-(1→4) and β-(1→4) linkages was also undertaken by one-pot cyclo-oligomerization in the suitable reaction condition, from an activated disaccharide thioglycoside monomer 16, having β-(1→4) linkage at the non-reducing end (Scheme 2). Chapter 4 describes the synthesis of cyclic oligosaccharides 15 and 17, as well as, the details of solid state structure and binding studies of cyclic trisaccharide 15. Scheme 2 Figure 4. (a) Stick model of the crystal structure of 15, as viewed along the crystallographic c-axis; (b) trigonal view from crystal packing; (c) packing diagram crystal lattice, as viewed along the crystallographic b-axis, and without solvent inclusion and (d) packing diagram included with methanol (grey) and water (red) solvents, as viewed along the crystallographic c-axis. Hydrogen atoms are omitted for clarity in (c and d). In summary, the thesis presents (i) synthesis, biophysical and biological studies of synthetic arabinan and arabinomannan glycolipids, and (ii) synthesis, solid-state structural analysis and binding studies of glycosidic bond expanded cyclic oligosaccharides. Synthetic trisaccharide arabinofuranoside glycolipids containing β-anomeric linkages at the non-reducing end showed binding affinity towards pulmonary surfactant protein A, as assessed by surface plasmon resonance technique, with comparatively lower extent as compared to synthetic glycolipids having α-anomeric linkages. Linear and branched arabinomannan penta- and heptasaccharide glycolipids, having α-anomeric linkages were synthesized and biological studies with non-pathogenic strain M. smegmatis were conducted with pentasaccharide glycolipids. It was found that arabinomannan glycolipids inhibited the growth and sliding motility of mycobacteria. Importantly, disruption of biofilm and significant reduction in biofilm formation was observed in the presence of the synthetic glycolipids. Glycosidic bond expanded cyclic trisaccharide with anomeric α-(1→4) linkages and cyclic tetrasaccharide with alternative anomeric α-(1→4) and β-(1→4) linkages were prepared from suitably designed trisaccharide and disaccharide monomer, respectively, by cyclo-oligomerization. Solid-state structural analysis and binding studies of cyclic trisaccharide in solution by isothermal titration calorimetry were also conducted. Cyclic trisaccharide possessed a bowl shape and brick-wall type of arrangement in the solid-state structure, whereas it exhibited stronger binding affinity towards 1-aminoadamantane as compared to β-cyclodextrin in aqueous solution. Overall, the results presented in the thesis provide a possibility to develop new types of synthetic glycolipids that can act as inhibitors of biofilm formation of mycobacteria, as well as, to develop newer types of cyclic oligosaccharide synthetic hosts that can modify binding abilities towards various guest compounds.