BIOLOGICAL processes are governed through specific interactions of macromolecules. The three-dimensional structural information of the macromolecules is necessary to understand the basis of molecular recognition. A large number of protein structures have been determined at a high resolution using various experimental techniques such as X-ray crystallography, NMR, electron microscopy and made publicly available through the Protein Data Bank. In the recent years, comprehending function by studying a large number of related proteins is proving to be very fruitful for understanding their biological role and gaining mechanistic insights into molecular recognition. Availability of large-scale structural data has indeed made this task of predicting the protein function from three-dimensional structure, feasible. Structural bioinformatics, a branch of bioinformatics, has evolved into a separate discipline to rationalize and classify the information present in three-dimensional structures and derive meaningful biological insights. This has provided a better understanding of biological processes at a higher resolution in several cases. Most of the structural bioinformatics approaches so far, have focused on fold-level analysis of proteins and their relationship to sequences. It has long been recognized that sequence-fold or fold-function relationships are highly complex. Information on one aspect cannot be readily extrapolated to the other. To a significant extent, this can be overcome by understanding similarities in proteins by comparing their binding site structures. In this thesis, the primary focus is on analyzing the small-molecule ligand binding sites in protein structures, as most of the biological processes ranging from enzyme catalysis to complex signaling cascades are mediated through protein-ligand interactions. Moreover, given that the precise geometry and the chemical properties of the residues at the ligand binding sites dictate the molecular recognition capabilities, focusing on these sites at the structural level, is likely to yield more direct insights on protein function. The study of binding sites at the structural level poses several problems mainly because the residues at the site may be sequentially discontinuous but spatially proximal. Further, the order of the binding site residues in primary sequence, in most of cases has no significance for ligand binding. Compounding these difficulties are additional factors such as, non-uniform contribution to binding from different residues, and size-variations in binding sites even across closely related proteins. As a result, methods available to study ligand-binding sites in proteins, especially on a large-scale are limited, warranting exploration of new approaches. In the present work, new methods and tools have been developed to address some of these challenges in binding site analysis. First, a novel tool for site-based function annotation of protein structures, called PocketAnnotate was developed ( http://proline.biochem.iisc.ernet. in/pocketannotate/). PocketAnnotate, detects the putative binding sites from a given protein structure and compares them to known binding sites in PDB to derive functional annotation in terms of ligand association. Since the tool derives functional annotation at the level of binding sites, it has an advantage over other methods that solely utilize fold or sequence information. This becomes even more important for cases where there is no detectable homology with entries in existing databases, as Pocket Annotate does not depend on evolutionary based information for annotation. Second, a web-accessible tool for in silico almandine scanning mutations of binding site residues called ABS-Scan has been developed ( http://proline.biochem.iisc.ernet.in/abscan/). This tool helps in assessing the contribution of the individual residues of binding sites in the protein towards ligand recognition. All residues, one at a time, in a binding site are mutated systematically to an alanine and the ability of the corresponding mutant to bind a given ligand is analyzed. The contribution of each residue towards ligand binding is calculated through a G value derived by comparing the binding affinity to the wild-type protein-ligand complex. Third, a database called Protein-Ligand Interaction Clusters (PLIC) has been developed to identify and analyze the information of similarity across binding sites in PDB, which has been provided in the form of a web-accessible database ( http://proline.biochem.iisc.ernet/ PLIC). Protein-ligand interactions are primarily explored using three different computational approaches - (i) binding site characteristics including pocket shape, nature of residues and interaction profiles with different kinds of chemical probes, (ii) atomic contacts between protein and ligands (iii) binding energetics involved in interactions derived from scoring functions developed for docking. The information on variations in these features derived from different computational tools is also included in the database for enabling the characterization of the binding sites. As a case study to demonstrate the usefulness of these tools, they have been applied to decipher the complexity of S-adenosyl methionine interactions with the protein. Around 1,213 binding sites of SAM or SAM-like compounds could be extracted from the PLIC database. The SAM or SAM-like compounds were observed to interact with ∼18 different protein-fold types. The variations in different protein-ligand contacts across fold types were analyzed. The fold-specific interaction properties and contribution of individual residues towards SAM binding are identified. The tools developed and example analyses using them are described in Chapter 2. Chapter 3 describes a large-scale pocketome analysis from structural complexes in PDB, in an effort to characterize the known pocket space of protein-ligand interactions. Tools devel-opted as described in Chapter 2 are used for this. A set of 84,846 binding sites compiled from PDB, have been comprehensively analyzed with an objective of obtaining (a) classification of binding sites, (b) sequence-fold-site relationships among proteins, (c) a minimal set of physicochemical attributes sufficient to explain ligand recognition specificity and (d) site-type specific signatures in terms of physicochemical features. A new method to describe binding sites was developed in the form of BScIds such that the structural fold information is well captured. Binding sites and similarities among them were abstracted in the form of networks where each node represents a binding site and an edge between two nodes represents significant similarity between the sites at the structural level. Pocketome networks were constructed from the large-scale information on protein-ligand interactions in the PLIC database. The large pocketome network was then studied to derive relationships between protein folds and chemical entities they interact with. A classification of the binding pockets was achieved by analyzing the pocketome network using graph theoretical approaches combined with clustering methods. 10,858 clusters were identified from the network, each indicating a site-type. Thus, it can be said that there are about 10,858 site-types. Classification of ligand associations into specific site-types helps greatly in resolving the complex relationships by yielding specific site-type ligand associations. The observed classification was further probed to understand the basis of ligand recognition by representing the pockets through feature vectors. These features capture a wide range of physicochemical properties that can be used to derive site-type specific signatures and explore the pocket-space of protein-ligand interactions. A principal component analysis of these features reveals that binding site feature space is continuous in the entire PDB and minor changes in specific features can give rise to significant differences in ligand specificity, consequently defining their distinct functional roles. The weights were also derived for these features through the use of different information theoretic approaches to explain the multiple-specificity of protein-ligand interactions. Analysis of binding sites arising from contribution of residues from different protein fold-types revealed increasing diversity of physicochemical properties at the site, supporting the hypothesis that combination of folds could give rise to new binding sites. Given that a finer appreciation of the molecular mechanisms within the cell is possible only with the structural information, the next objective was to explore if a structural view of an entire proteome can be obtained and if a pocketome could be constructed and analyzed. With this in mind, the causative agent of tuberculosis - Mycobacterium tuberculosis (Mtb) was chosen. Mtb is also being studied in the laboratory from a systems biology perspective, which enabled exploration of how systems and the structural perspectives could be combined and applied for drug discovery. Chapters 4 to 6 describe this effort. The genome sequence of Mycobacterium tuberculosis (Mtb) H37Rv, indicates the presence of ∼4,000 protein coding genes, of which experimentally determined structures are available for ∼300 proteins. Further, advances in homology modeling methods have made it feasible to obtain structural models for many more proteins in the proteome. Chapter 4 describes the efforts for obtaining the Mtb structural proteome, through which the three-dimensional struc-tures were derived for ∼70% of the proteins in the genome. Functional annotation of each protein was derived based on fold-based functional assignments, binding-site comparisons and consequent ligand associations. PocketAnnotate, a site-based function annotation pipeline was utilized for this purpose and is described in Chapter 2. Besides these, the annotation covers detection of various sequence and sub-structural motifs and quaternary structure predictions based on the corresponding templates. The study provides a unique opportunity to obtain a global perspective of the fold distribution in the genome. The annotation indicates that cellular metabolism can be achieved with only 219 unique folds. New insights about the folds that predominate in the genome, as well as the fold-combinations that make up multi-domain proteins are also obtained. 1,728 binding pockets have been associated with ligands through binding site identification and sub-structure similarity analyses, yielding a list of ligands that can participate in various biochemical events in the mycobacterial cell. A web-accessible database MtbStructuralproteome has been developed to make the data and the analyses available to the community, ( http://proline.physics.iisc.ernet.in/Tbstructuralannotation). The resource, being one of the first to be based on structure-based functional annotations at a genome scale, is expected to be useful for better understanding of tuberculosis and for application in drug discovery. The reported annotation pipeline is fairly generic and can be applied to other genomes as well. Chapter 5 describes the characterization of the Mtb pocketome. For the structural models of the Mtb proteome described in chapter 4, a genome-scale binding site prediction exercise was carried out using three different computational methods and subsequently obtaining consensus predictions. The three methods were independent and were based on considering geometry, inter-molecular energies with probes and sequence conservations in evolutionarily related proteins respectively. In all, 13,858 consensus binding pockets were predicted in 2,877 proteins. The pocket space within Mtb was then explored through systematic all-pair comparisons of binding sites. The number of site-types within Mtb was found to be 6,584, as compared to the ∼400 structural folds and 1,831 unique sequence families. This reveals that the pocket space is larger than the sequence or fold-space, suggesting that variations at the site-level contribute significantly to functional repertoire of the organism. By comparing the pockets with the PDB sites enclosing known ligands, around 6906 binding sites were observed to exhibit significant similarity in the entire pockets to some or the other known binding site in PDB. 1,213 metabolites could be mapped onto 665 enzymes covering most of the metabolic pathways. The identified ligands serve as a predicted metabolome for unit abundances of the proteins. A list of proteins containing unique pockets is also identified. The binding pockets, similarities they share within Mtb and the ligands mapped onto them are all made available in a web-accessible database at http://proline.biochem.iisc.ernet.in/mtbpocketome/. The availability of structural information of the pocketome at a genome-scale opens up several opportunities in drug discovery. They can be directly applied for understanding mechanism of drug action, predicting adverse effects and pharmacodynamics of a drug. Moreover, it enables exploration of new ideas in drug discovery. Polypharmacology is a new concept that aims at modulating multiple drug targets through a single chemical entity. Currently, there are no established approaches to either select appropriate target sets or design polypharmacological drugs. In this study, a structural-proteomics approach is explored to first characterize the pocketome and then utilize it to identify similar binding sites. The knowledge of similarity relationships between the binding sites within the genome can be used in identifying possible polypharmacological drug targets. A pocket similarity based clustering of binding site residues resulted in identification of binding site sets, each having a theoretical potential to interact with a common ligand. A polypharmacological index was formulated to rank targets by incorporating a measure of drug ability and similarity to other pockets within the proteome. By comparing with known drug binding sites from databases such as the Drug Bank, the study has yielded a ready shortlist that includes sets of promising drug targets with polypharmacological possibilities and at the same time has identified possible drug candidates either directly for repurposing or at the least as significant lead clues that can be used to design new drug molecules against the entire group of proteins in each set. This analysis presents a rational approach to identify targets with polypharmacological potential, clues about lead compounds and a list of candidates for drug repurposing. This thesis demonstrates the feasibility of utilizing the structural bioinformatics approaches at a genome-scale. The tools developed for analyzing large-scale data on protein-ligand inter-actions could be applied to characterize the pocket-space of protein-ligand interactions. The network theory approaches applied in this work, make large-scale data tractable and enable binding-site typing. The binding site analysis at a genome-scale for Mtb is first of its kind and has provided novel insights into the pocket space. The binding site analysis performed on a genome-scale for Mtb provided an opportunity to rationalize the polypharmacological target selection and explore drugs for repurposing in TB. In the larger context, structural modelling of a proteome, mapping the small-molecule binding space in it and understanding the determinants of small-molecule recognition forms a major step in defining a proteome at higher resolution. This in turn will serve as a valuable input towards the emerging field of structural-systems biology, which seeks to understand the biological models at a systems level without compromising on the resolution of the study.

A tuberculose é uma doença crônica granulomatosa caracterizada por um déficit de imunidade antígeno específica do hospedeiro, cuja resposta imune é ativamente regulada por citocinas. No Brasil há mais de 50 milhões de habitantes infectados pelo Mycobacterium tuberculosis. O objetivo foi avaliar a linfoproliferação e a produção de citocinas por células mononucleares do sangue periférico (PBMC) estimuladas por quatro diferentes antígenos do M. tuberculosis, um complexo, o antígeno sonicado, e três purificados, ESAT-6, antígeno 85B e antígeno HBHA, eventuais candidatos à vacina anti-tuberculose. Para avaliação da produção de IFN-g e IL-10 foram utilizados dois métodos: Elispot e Elisa à partir de sobrenadante de cultura de PBMC. Para essas avaliações, os pacientes com tuberculose ativa (TB-A) foram comparados a dois subgrupos de indivíduos controles. O primeiro subgrupo foi constituído por indivíduos saudáveis PPD+ e o segundo por indivíduos curados de um episódio de tuberculose (TB-C). Nossos resultados de linfoproliferação e de Elisa revelaram diminuição da resposta linfoproliferativa e da produção de IFN-g dos pacientes em comparação com os indivíduos PPD+, enquanto os indivíduos TB-C apresentaram em geral resultados intermediários. Observou-se também que as respostas à PHA não diferiam significativamente entre os grupos, ressaltando a natureza antígeno específica da hiporreatividade na tuberculose. Adicionalmente, verificamos maior reatividade ao antígeno complexo, sonicado, que aos antígenos purificados, e entre estes, a reatividade foi maior para ESAT-6 e 85B que para HBHA, A resposta ao HBHA pode ter sido eventualmente subestimada por razões técnicas, como utilização de dose sub-ótima ou perda da atividade biológica. Em relação ao Elispot para IFN-g, não pudemos observar diferenças entre os grupos, tanto quando se considerou o número total de spots, como quando se contou apenas spots com diâmetro > 65 mm, apresentando portanto uma sensibilidade aparentemente menor comparado aos outros 2 métodos. A comparação entre os métodos revelou pouca correlação entre seus resultados, que pode ser eventualmente explicado pela diferente contribuição das populações celulares (T CD4+ e T CD8+) para cada uma das provas munológicas. Finalmente, a análise da produção de IL-10 medida por Elisa no sobrenadante de cultura e por spots de IL10, também não revelou diferenças entre os grupos. Convém notar que o Elisa detectou baixas concentrações de IL-10 nos sobrenadantes, porém o Elispot demonstrou número elevado de spots e boa correlação entre as resposta aos antígenos. Em conclusão, nossos resultados sugerem que métodos \'clássicos\', e já estabelecidos, como linfoproliferação e Elisa, persistem válidos para se avaliar a imunidade celular, e que em nossas condições laboratoriais, a técnica de Elispot não representou, até o momento, uma melhora na qualidade da avaliação imunológica. / Tuberculosis is a chronic granulomatous disease characterized by a deficit of the antigen-specific immunity of the host, whose immune response is actively regulated by cytokines. In Brazil there are 50 million people infected with Mycobacterium tuberculosis. The objective of the present work was to evaluate the lymphoproliferative response e the IFN-g response by peripheral blood mononuclear cells (PBMC) indiced with 4 different antigens isolated from Mycobacterium tuberculosis: a complex, crude, the sonicate antigen, and 3 other, purified ones, Esat-6, 85B, and HBHA, the last 3 eventual candidates to the design of a vaccine against tuberculosis. We used 2 methods to evaluate the IFN-g and IL-10 productions, namely Elispot and Elisa of supernatant of PBMC cultures. We studied a group of active tuberculosis patients (TB-A), and compared them with controls individuals comprising 2 groups, one made of healthy PPD+ individuals and the second one of individuals who have been cured from an episode of tuberculosis in the past (TB-C). Our results of lymphoproliferation and Elisa revealed decrease in the lymphoproliferative and IFN-g responses by patients\' PBMC as compared to the PPD+ group, with the TB-C group in general presenting intermediate results. We also observed that the responses to the mitogen PHA were not statisically different among the groups, denoting the antigen-specific nature of the immune deficit in tuberculosis. In addition, we verified that stronger reactivity to the complex antigen than with the purified antigens, and, among the latter, the reactivity was stronger with Esat-6 and 85B as compared to HBHA, Reactivity to HBHA may have been understimated due to technical reasons, such as loss of .the biological activity of the molecule or use of a sub-optimal dose. By using the Elispot for IFN-g we were not able to detect differences among the groups, even when we counted all spots formed or spots with more than > 65 mm in diameter. Thus our Elispot for IFN-g apparently showed lower sensitivity than the other 2 methods. Furthermore, comparisons between the methods revealed low correlation between their results, a finding that may be explained by the differning contribution of different subpopulations (T CD4+ and T CD8+) to each of the results. Finally, analysis of the production of IL-10 as measured by Elisa in the culture supernatants as well as by Elispot revealed no differences among the groups. It is noteworthy that the levels of IL-10 detected by Elisa were low, but the Elispot revealed high number of spots and a good correlation between the antigen responses. In conclusion, we may say that our well standardized \'classical\' methods Elisa and lymphoproliferation persist useful to evaluate cellular immunity responses, and that the Elispot technique, up to now and in our laboratorial conditions, did not represent an improvement in the quality of the immunological evaluation.

Proteins are essential for the growth, survival and maintenance of the cell. Understanding the functional roles of proteins helps to decipher the working of macromolecular assemblies and cellular machinery of living organisms. A thorough investigation of the link between sequence, structure and function of proteins, helps in building a comprehensive understanding of the complex biological systems. Proteins have been observed to be composed of single and multiple domains. Analysis of proteins encoded in diverse genomes shows the ubiquitous nature of multi-domain proteins. Though the majority of eukaryotic proteins are multi-domain in nature, 3-D structures of only a small proportion of multi-domain proteins are known due to difficulties in crystallizing such proteins. While functions of individual domains are generally extensively studied, the complex interplay of functions of domains is not well understood for most multi-domain proteins. Paucity of structural and functional data, affects our understanding of the evolution of structure and function of multi-domain proteins. The broad objective of this thesis is to achieve an enhanced understanding of structure and function of protein domains by computational analysis of sequence and structural data. Special attention is paid in the first few chapters of this thesis on the multi-domain proteins. Classification of multi-domain proteins by implementation of an alignment-free sequence comparison method has been achieved in Chapters 2 and 3. Studies on organization, interactions and interdependence of domain-domain interactions in multi-domain proteins with respect to sequential separation between domains and N to C-terminal domain order have been described in Chapters 4 and 5. The functional and structural repertoire of organisms can be comprehensively studied and compared using functional and structural domain annotations. Chapter 6, 7 and 8 represent the proteome-wide structure and function comparisons of various pathogenic and non-pathogenic microorganisms. These comparisons help in identifying proteins implicated in virulence of the pathogen and thus predict putative targets for disease treatment and prevention. Chapter 1 forms an introduction to the main subject area of this thesis. Starting with describing protein structure and function, details of the four levels of hierarchical organization of protein structure have been provided, along with the databases that document protein sequences and structures. Classification of protein domains considered as the realm of function, structure and evolution has been described. The usefulness of classification of proteins at the domain level has been highlighted in terms of providing an enhanced understanding of protein structure and function and also their evolutionary relatedness. The details of structure, function and evolution of multi-domain proteins have also been outlined in chapter 1. ! Chapter 2 aims to achieve a biologically meaningful classification scheme for multi-domain protein sequences. The overall function of a multi-domain protein is determined by the functional and structural interplay of its constituent domains. Traditional sequence-based methods utilize only the domain-level information to classify proteins. This does not take into account the contributions of accessory domains and linker regions towards the overall function of a multi-domain protein. An alignment-free protein sequence comparison tool, CLAP (CLAssification of Proteins) previously developed in this laboratory, was assessed and improved when the author joined the group. CLAP was developed especially to handle multi-domain protein sequences without a requirement of defining domain boundaries and sequential order of domains (domain architecture). ! The working principle of CLAP involves comparison of all against all windows of 5-residue sequence patterns between two protein sequences. The sequences compared could be full-length comprising of all the domains in the two proteins. This compilation of comparison is represented as the Local Matching Scores (LMS) between protein sequences (nslab.iisc.ernet.in/clap/). It has been previously shown that the execution time of CLAP is ~7 times faster than other protein sequence comparison methods that employ alignment of sequences. In Chapter 2, CLAP-based classification has been carried out on two test datasets of proteins containing (i) Tyrosine phosphatase domain family and (ii) SH3-domain family. The former dataset comprises both single and multi-domain proteins that sometimes consist of domain repeats of the tyrosine phosphatase domain. The latter dataset consists only of multi-domain proteins with one copy of the SH3-domain. At the domain-level CLAP-based classification scheme resulted in a clustering similar to that obtained from an alignment-based method, ClustalW. CLAP-based clusters obtained for full-length datasets were shown to comprise of proteins with similar functions and domain architectures. Hence, a protein classification scheme is shown to work efficiently that is independent of domain definitions and requires only the full-length amino acid sequences as input.! Chapter 3 explores the limitations of CLAP in large-scale protein sequence comparisons. The potential advantages of full-length protein sequence classification, combined with the availability of the alignment-free sequence comparison tool, CLAP, motivated the conceptualization of full-length sequence classification of the entire protein repertoire. Before undertaking this mammoth task, working of CLAP was tested for a large dataset of 239,461 protein sequences. Chapter 3 discusses the technical details of computation, storage and retrieval of CLAP scores for a large dataset in a feasible timeframe. CLAP scores were examined for protein pairs of same domain architecture and ~22% of these showed 0 CLAP similarity scores. This led to investigation of the sensitivity of CLAP with respect to sequence divergence. Several test datasets of proteins belonging to the same SCOP fold were constructed and CLAP-based classification of these proteins was examined at inter and intra-SCOP family level. CLAP was successful in efficiently clustering evolutionary related proteins (defined as proteins within the same SCOP superfamily) if their sequence identity >35%. At lower sequence identities, CLAP fails to recognize any evolutionary relatedness. Another test dataset consisting of two-domain proteins with domain order swapped was constructed. Domain order swap refers to domain architectures of type AB and BA, consisting of domains A and B. A condition that the sequence identities of homologous domains were greater than 35% was imposed. CLAP could effectively cluster together proteins of the same domain architectures in this case. Thus, the sequence identity threshold of 35% at the domain-level improves the accuracy of CLAP. The analysis also showed that for highly divergent sequences, the expectation of 5-residue pattern match was likely a stringent criterion. Thus, a modification in the 5-residue identical pattern match criterion, by considering even similar residue and gaps within matched patterns may be required to effectuate CLAP-based clustering of remotely related protein sequences. Thus, this study highlights the limitations of CLAP with respect to large-scale analysis and its sensitivity to sequence divergence. ! Chapters 4 and 5 discuss the computational analysis of inter-domain interactions with respect to sequential distance and domain order. Knowledge of domain composition and 3-D structures of individual domains in a multi-domain protein may not be sufficient to predict the tertiary structure of the multi-domain protein. Substantial information about the nature of domain-domain interfaces helps in prediction of the tertiary as well as the quaternary structure of a protein. Therefore, chapter 4 explores the possible relationship between the sequential distance separating two domains in a multi-domain protein and the extent of their interaction. With increasing sequential separation between any two domains, the extent of inter-domain interactions showed a gradual decrease. The trend was more apparent when sequential separation between domains is measured in terms of number of intervening domains. Irrespective of the linker length, extensive interactions were seen more often between contiguous domains than between non-contiguous domains. Contiguous domains show a broader interface area and lower proportion of non-interacting domains (interface area: 0 Å2 to - 4400 Å2, 2.3% non-interacting domains) than non-contiguous domains (interface area: 0 Å2 to - 2000 Å2, 34.7% non-interacting domains). Additionally, as inter-protein interactions are mediated through constituent domains, rules of protein-protein interactions were applied to domain-domain interactions. Tight binding between domains is denoted as putative permanent domain-domain interactions and domains that may dissociate and associate with relatively weak interactions to regulate functional activity are denoted as putative transient domain-domain interactions. An interface area threshold of 600 Å2 was utilized as a binary classifier to distinguish between putative permanent and putative transient domain-domain interactions. Therefore, the state of interaction of a domain pair is defined as either putative permanent or putative transient interaction. Contiguous domains showed a predominance of putative permanent nature of inter-domain interface, whereas non-contiguous domains showed a prevalence of putative transient interfaces. The state of interaction of various SCOP superfamily pairs was studied across different proteins in the dataset. SCOP superfamily pairs mostly showed a conserved state of interaction, i.e. either putative permanent or putative transient in all their occurrences across different proteins. Thus, it is noted that contiguous domains interact extensively more often than non-contiguous domains and specific superfamily pairs tend to interact in a conserved manner. In conclusion, a combination of interface area and other inter-domain properties along with experimental validation will help strengthen the binary classification scheme of putative permanent and transient domain-domain interactions.! Chapter 5 provides structural analysis of domain pairs occurring in different sequential domain orders in mutli-domain proteins. The function and regulation of a multi-domain protein is predominantly determined by the domain-domain interactions. These in turn are influenced by the sequential order of domains in a protein. With domains defined using evolutionary and structural relatedness (SCOP superfamily), their conservation of structure and function was studied across domain order reversal. A domain order reversal indicates different sequential orders of the concerned domains, which may be identified in proteins of same or different domain compositions. Domain order reversals of domains A and B can be indicated in protein pair consisting of the domain architectures xAxBx and xBxAx, where x indicates 0 or more domains. A total of 161 pairs of domain order reversals were identified in 77 pairs of PDB entries. For most of the comparisons between proteins with different domain composition and architecture, large differences in the relative spatial orientation of domains were observed. Although preservation of state of interaction was observed for ~75% of the comparisons, none of the inter-domain interfaces of domains in different order displayed high interface similarity. These domain order reversals in multi-domain proteins are contributed by a limited number of 15 SCOP superfamilies. Majority of the superfamilies undergoing order reversal either function as transporters or regulatory domains and very few are enzymes. A higher proportion of domain order reversals were observed in domains separated by 0 or 1 domains than those separated by more than 1 domain. A thorough analysis of various structural features of domains undergoing order reversal indicates that only one order of domains is strongly preferred over all possible orders. This may be due to either evolutionary selection of one of the orders and its conservation throughout generations, or the fact that domain order reversals rarely conserve the interface between the domains. Further studies (Chapters 6 to 8) utilize the available computational techniques for structural and functional annotation of proteins encoded in a few bacterial genomes. Based on these annotations, proteome-wide structure and function comparisons were performed between two sets of pathogenic and non-pathogenic bacteria. The first study compares the pathogenic Mycobacterium tuberculosis to the closely related organism Mycobacterium smegmatis which is non-pathogenic. The second study primarily identified biologically feasible host-pathogen interactions between the human host and the pathogen Leptospira interrogans and also compared leptospiral-host interactions of the pathogenic Leptospira interrogans and of the saprophytic Leptospira biflexa with the human host. Chapter 6 describes the function and structure annotation of proteins encoded in the genome of M. smegmatis MC2-155. M. smegmatis is a widely used model organism for understanding the pathophysiology of M. tuberculosis, the primary causative agent of tuberculosis in humans. M. smegmatis and M. tuberculosis species of the mycobacterial genus share several features like a similar cell-wall architecture, the ability to oxidise carbon monoxide aerobically and share a huge number of homologues. These features render M. smegmatis particularly useful in identifying critical cellular pathways of M. tuberculosis to inhibit its growth in the human host. In spite of the similarities between M. smegmatis and M. tuberculosis, there are stark differences between the two due to their diverse niche and lifestyle. While there are innumerable studies reporting the structure, function and interaction properties of M. tuberculosis proteins, there is a lack of high quality annotation of M. smegmatis proteins. This makes the understanding of the biology of M. smegmatis extremely important for investigating its competence as a good model organism for M. tuberculosis. With the implementation of available sequence and structural profile-based search procedures, functional and structural characterization could be achieved for ~92% of the M. smegmatis proteome. Structural and functional domain definitions were obtained for a total of 5695 of 6717 proteins in M. smegmatis. Residue coverage >70% was achieved for 4567 proteins, which constitute ~68% of the proteome. Domain unassigned regions more than 30 residues were assessed for their potential to be associated to a domain. For 1022 proteins with no recognizable domains, putative structural and functional information was inferred for 328 proteins by the use of distance relationship detection and fold recognition methods. Although 916 sequences of 1022 proteins with no recognizable domains were found to be specific to M. smegmatis species, 98 of these are specific to its MC2-155 strain. Of the 1828 M. smegmatis proteins classified as conserved hypothetical proteins, 1038 proteins were successfully characterized. A total of 33 Domains of Unknown Function (DUFs) occurring in M. smegmatis could be associated to structural domains. A high representation of the tetR and GntR family of transcription regulators was noted in the functional repertoire of M. smegmatis proteome. As M. smegmatis is a soil-dwelling bacterium, transcriptional regulators are crucial for helping it to adapt and survive the environmental stress. Similarly, the ABC transporter and MFS domain families are highly represented in the M. smegmatis proteome. These are important in enabling the bacteria to uptake carbohydrate from diverse environmental sources. A lower number of virulent proteins were identified in M. smegmatis, which justifies its non-pathogenicity. Thus, a detailed functional and structural annotation of the M. smegmatis proteome was achieved in Chapter 6. Chapter 7 delineates the similarities and difference in the structure and function of proteins encoded in the genomes of the pathogenic M. tuberculosis and the non-pathogenic M. smegmatis. The protocol employed in Chapter 6 to achieve the proteome-wide structure and function annotation of M. smegmatis was also applied to M. tuberculosis proteome in Chapter 7. The number of proteins encoded by the genome of M. smegmatis strain MC2-155 (6717 proteins) is comparatively higher than that in M. tuberculosis strain H37Rv (4018 proteins). A total of 2720 high confidence orthologues sharing ≥30% sequence identity were identified in M. tuberculosis with respect to M. smegmatis. Based on the orthologue information, specific functional clusters, essential proteins, metabolic pathways, transporters and toxin-antitoxin systems of M. tuberculosis were inspected for conservation in M. smegmatis. Among the several categories analysed, 53 metabolic pathways, 44 membrane transporter proteins belonging to secondary transporters and ATP-dependent transporter classes, 73 toxin-antitoxin systems, 23 M. tuberculosis-specific targets, 10 broad-spectrum targets and 34 targets implicated in persistence of M. tuberculosis could not detect any orthologues in M. smegmatis. Several of the MFS superfamily transporters act as drug efflux pumps and are hence associated with drug resistance in M. tuberculosis. The relative abundances of MFS and ABC superfamily transporters are higher in M. smegmatis than in M. tuberculosis. As these transporters are involved in carbohydrate uptake, their higher representation in M. smegmatis than in M. tuberculosis highlights the lack of proficiency of M. tuberculosis to assimilate diverse carbon sources. In the case of porins, MspA-like and OmpA-like porins are selectively present in either M. smegmatis or M. tuberculosis. These differences help to elucidate protein clusters for which M. smegmatis may not be the best model organism to study M. tuberculosis proteins.! At the domain-level, ATP-binding domain of ABC transporters, tetracycline transcriptional regulator (tetR) domain family, major facilitator superfamily (MFS) domain family, AMP-binding domain family and enoyl-CoA hydrolase domain family are highly represented in both M. smegmatis and M. tuberculosis proteomes. These domains play an essential role in the carbohydrate uptake systems and drug-efflux pumps among other diverse functions in mycobacteria. There are several differentially represented domain families in M. tuberculosis and M. smegmatis. For example, the pentapeptide-repeat domain, PE, PPE and PIN domains although abundantly present in M. tuberculosis, are very rare in M. smegmatis. Therefore, such uniquely or differentially represented functional and structural domains in M. tuberculosis as compared to M. smegmatis may be linked to pathogenicity or adaptation of M. tuberculosis in the host. Hence, major differences between M. tuberculosis and M. smegmatis were identified, not only in terms of domain populations but also in terms of domain combinations. Thus, Chapter 7 highlights the similarities and differences between M. smegmatis and M. tuberculosis proteomes in terms of structure and function. These differences provide an understanding of selective utilization of M. smegmatis as a model organism to study M. tuberculosis. ! In Chapter 8, computational tools have been employed to predict biologically feasible host-pathogen interactions between the human host and the pathogenic, Leptospira interrogans. Sensitive profile-based search procedures were used to specifically identify practical drug targets in the genome of Leptospira interrogans, the causative agent of the globally widespread zoonotic disease, Leptospirosis. Traditionally, the genus Leptospira is classified into two species complex- the pathogenic L. interrogans and the non-pathogenic saprophyte L. biflexa. The pathogen gains entry into the human host through direct or indirect contact with fluids of infected animals. Several ambiguities exist in the understanding of L. interrogans pathogenesis. An integration of multiple computational approaches guided by experimentally derived protein-protein interactions, was utilized for recognition of host-pathogen protein-protein interactions. The initial step involved the identification of similarities of host and L. interrogans proteins with crystal structures of experimentally known transient protein-protein complexes. Further, conservation of interfacial nature was used to obtain high confidence predictions for putative host-pathogen protein-protein interactions. These predictions were subjected to further selection based on subcellular localization of proteins of the human host and L. interrogans, and tissue-specific expression profiles of the host proteins. A total of 49 protein-protein interactions mediated by 24 L. interrogans proteins and 17 host proteins were identified and these may be subjected to further experimental investigations to assess their in vivo relevance. The functional relevance of similarities and differences between the pathogenic and non-pathogenic leptospires in terms of interactions with the host has also been explored. For this, protein-protein interactions across human host and the non-pathogenic saprophyte L. biflexa were also predicted. Nearly 39 leptospiral-host interactions were recognized to be similar across both the pathogen and saprophyte in the context of processes that influence the host. The overlapping leptospiral-host interactions of L. interrogans and L. biflexa proteins with the human host proteins are primarily associated with establishment of its entry into the human host. These include adhesion of the leptospiral proteins to host cells, survival in host environment such as iron acquisition and binding to components of extracellular matrix and plasma. The disjoint sets of leptospiral-host interactions are species-specific interactions, more importantly indicative of the establishment of infection by L. interrogans in the human host and immune clearance of L. biflexa by the human host. With respect to L. interrogans, these specific interactions include interference with blood coagulation cascade and dissemination to target organs by means of disruption of cell junction assembly. On the other hand, species-specific interactions of L. biflexa proteins include those with components of host immune system. ! In spite of the limited availability of experimental evidence, these help in identifying functionally relevant interactions between host and pathogen by integrating multiple lines of evidence. Thus, inferences from computational prediction of host-pathogen interactions act as guidelines for experimental studies investigating the in vivo relevance of these predicted protein-protein interactions. This will further help in developing effective measures for treatment and disease prevention. In summary, Chapters 2 and 3 describe the implementation, advantages and limitations of the alignment-free full-length sequence comparison method, CLAP. Chapter 4 and 5 are dedicated to understand the domain-domain interactions in multi-domain protein sequences and structures. In Chapters 6, 7 and 8 the computational analyses of the mycobacterial species and leptospiral species helped in an enhanced understanding of the functional repertoire of these bacteria. These studies were undertaken by utilizing the biological sequence data available in public databases and implementation of powerful homology-detection techniques. The supplemental data associated with the chapters is provided in a compact disc attached with this thesis.!

Made available in DSpace on 2014-07-29T15:30:34Z (GMT). No. of bitstreams: 1 Dissertacao Loanda Carvalho S Limongi.pdf: 1004813 bytes, checksum: 09f423fef50cc45223e734df788a0c39 (MD5) Previous issue date: 2009-12-18 / Pleural tuberculosis (TBP) has often spontaneous resolution, even without treatment. However, the lack of proper diagnosis and treatment can lead to the occurrence of pulmonary and/or extra-pulmonary tuberculosis in 65% of the cases, in the five years subsequent. The objective of this study was to evaluate the effectiveness of the recombinant Hsp-X antigen (rHsp-X) in an enzyme immunoassay for determining the presence of TBP in patients with pleural effusion. For this study we used 132 samples of serum and pleural fluid (PF) from patients with pleural effusion. Of these, 97 samples were from TBP patients and 35 patients with diseases other than tuberculosis (NTBP) (28 patients with metastatic cancer, 1 patient with congestive heart failure, 2 patients whit liver failure, 1 patient with systemic lupus erythematosus and 3 patients with parapneumonic effusion). The IgM levels of PF were higher in patients with PTB than NTBP, the sensitivity and specificity of the ELISA for the PF was 42% and 83%, respectively. Testing for IgG from the serum and the PF against the recombinant antigen Hsp-X were not effective in discriminating TB patients from other diseases, the sensitivity was found to be 13% and 16% using serum and LP, respectively, with the same specificity of 83%. TBP patients presented higher levels of IgA specific for the rHSP-X than NTBP, the IgA ELISA using PF presented better sensitivity (65%) than using serum (39%), while the IgA ELISA for both types of samples presented the same specificity (83%). In conclusion, the recognition of Mycobacterium tuberculosis rHsp-X by IgA antibodies from the PF of patients with TBP reveals its possible role in the development of a diagnostic test for additional cases of TBP. Further studies must be conducted to confirm this hypothesis. / A tuberculose pleural (TBP) frequentemente tem resolução espontânea, mesmo sem tratamento. No entanto, a ausência de diagnóstico e tratamento pode levar, nos cinco anos subseqüentes, à ocorrência de tuberculose pulmonar e/ ou extrapulmonar em 65% dos casos. O objetivo deste estudo foi avaliar a eficácia da utilização do antígeno Hsp-X em um ensaio imunoenzimático para determinação da presença de TBP em indivíduos com derrame pleural. Para o estudo foram utilizadas 132 amostras de soro e líquido pleural (LP) de pacientes com derrame pleural. Destas, 97 amostras eram de pacientes TBP e 35 de pacientes com outras doenças não tuberculosas (NTBP) (28 pacientes por câncer metastático, 1 pacientes por insuficiência cardíaca congestiva, 2 paciente por insuficiência hepática, 1 paciente por lúpus eritematoso sistêmico e 3 pacientes por derrames parapneumônicos). Os níveis de IgM do LP foram mais elevados nos pacientes com TBP do que os NTBP, a sensibilidade do ELISA para do LP de 42%, para a especificidade de 83%. Os ensaios para pesquisa de IgG, do soro e do LP, contra o antígeno Hsp-X não foram eficazes em discriminar pacientes com TBP de outras doenças, a sensibilidade encontrada foi de 13% e 16%, utilizando o soro e o LP, respectivamente, para a mesma especificidade de 83%. A densidade óptica dos ensaios para pesquisa de IgA, específicos para o antígeno rHsp-X, foi mais elevada nos pacientes TBP do que os NTBP, o ELISA com a IgA do LP foi melhor que o ELISA do soro, a sensibilidade foi de 65% e 39%, respectivamente, para a mesma especificidade de 83%. Concluímos que o reconhecimento de Hsp-X recombinante de Mycobacterium tuberculosis pelos anticorpos IgA do LP de pacientes com TBP indicam que esse antígeno poderá ser utilizado para o desenvolvimento de um teste de diagnóstico complementar para casos de TBP. Estudos adicionais precisam ser realizados para confirmar tal hipótese.

The ability to fine-tune gene-expression in the adverse conditions during pre and post infectious stages has contributed in no small measure to the success of Mycobacterium tuberculosis as the deadly pathogen. Multiple sigma factors, transcription regulators, and diverse two component systemshave facilitated tailoring the metabolic pathways to meet the challenges faced by the pathogen. Over the last decade, studies have been initiated to understand the various facets of transcription in mycobacteria. Although not as extensive as the work in other model systems, such as Escherichia coli and eukaryotes, it is evident from these initial studies that the machinery is conserved,yetmany aspects of transcription and its regulation seem to be different in mycobacteria.The work presented in the thesis deals with some of the steps in the process, primarily initiation in the context of the distinct physiology of M. tuberculosis. The detailed kinetic and equilibrium study of a few selected promoters of M. tuberculosis viz.PgyrB1, PgyrR, PrrnPCL1 and PmetU is described in Chapter 2.Different stages of transcription initiation that have been analyzed include promoter specific binding of RNAP, isomerization, abortive initiation and promoter clearance.The equilibrium binding and kinetic studies of various steps reveal distinct rate limiting events for each of the promoter, which also differed markedly in their characteristics from the respective promoters of Mycobacterium smegmatis. In addition, a novel aspect of the transcription initiation at the gyr promoter was unraveled. The marked differences in the transcription initiation pathway seen with rrn and gyr promoters of M. smegmatis and M. tuberculosis suggest that such species specific differences in the regulation of expression of the crucial housekeeping genes could be one of the key determinants contributing to the differences in growth rate and lifestyle of the two organisms. In Chapter 3, the mechanism of growth phase dependent control (GPDC) at a few of the M. tuberculosis promoters has been investigated. The experiments described in the chapter are carried out to demonstrate a different pattern of interaction between the promoters and sigma A (SigA) of M. tuberculosis to facilitate the iNTPs and pppGpp mediated regulation. Instead of cytosine and methionine, thymine at three nucleotides downstream to -10 element and leucine232 in SigA are found to be essential for iNTPs and pppGpp mediated response at the rrn and gyr promoters of the organism. The specificity of the interaction is substantiated by mutational replacements, either in the discriminator or in SigA, which abolish the nucleotide mediated regulation in vitro or in vivo. In chapter 4, the long standing hypothesis that deals with interdependence of the transcription elongation kinetics and the growth rates has been addressed. Previous studies suggest that the rate of synthesis of the key molecules in cells affects the growth kinetics. In order to validate, the kinetics of elongation of RNAPs from M. tuberculosis, M. smegmatis and E. coli whose growth rates vary from very slow to fast is measured. Surface Plasmon Resonance (SPR) is used to monitor the transcription in real time and kinetic equations are applied to calculate the elongation rates. Further, the effects of the composition of the template DNA on the elongation rates of RNAP from E. coli and M. smegmatis, whose genomes show difference in the GC content are explored. The results obtained from the analysis support the hypothesis and also reveal the effect of template composition on elongation rates of RNAP.

Structural investigations on two mycobacterial enzymes were carried out. Tuberculosis still remains a major threat to mankind even though drugs against it have been in use for many decades. The emergence of drug resistant strains of the bacteria calls for the identification of new targets based on which new drugs can be developed to combat the disease. A thorough understanding of the functioning of the target molecules is essential for this approach. We have taken up the structural studies on two such molecules, aspartic proteinases and adenylyl cyclases, of Mycobacterium tuberculosis with a view to obtain insights into their mechanisms of action at the atomic level. The work presented in the thesis includes (i) the identification, cloning, expression, purification and structure determination of a putative aspartic proteinase domain of M. tuberculosis and (ii) the crystal structure of an adenylyl cyclase of M. tuberculosis and its mutant; and also of an adenylyl cyclase from M. avium. Chapter 1 presents an overview of aspartic proteinases and nucleotide cyclases with an emphasis on their structural features. The methods employed during the course of the present work are described in Chapter 2. Work on the putative aspartic proteinase domain identified in M. tuberculosis is presented in Chapter 3. The structure of the aspartic proteinase domain is the first structural report of such domain from any bacteria. A search in the genome of M. tuberculosis showed a weak similarity to the HIV aspartic proteinase sequence. This region corresponds to the C-terminal domain of a PE family protein in M. tuberculosis. The presence of two signature motifs, DTG and DSG, of aspartic proteinases in the full sequence of this domain encouraged us to take up further studies on this domain. Previous reports identifying the protein as a surface antigen and our findings on the occurrence of similar domains in two other PE proteins of M. tuberculosis and also in other pathological strains of Mycobacteria indicated that these domains probably play an important role in infecting the host. The crystal structure of one of the domains showed that it has a pepsin-like fold and the catalytic site architecture of known aspartic proteinases. However, no proteolytic activity was detected. The size of the molecule is intermediate to eukaryotic pepsins and the homodimeric retroviral pepsins. A close examination of the binding site revealed subtle differences when compared to the active enzyme structures. Appropriate mutations of some of the residues in this region to convert it to an active enzyme did not make it active. Once the in vivo function of these putative aspartic proteinase domains is established, their potential to act as drug targets can be probed as the PE proteins are present exclusively in pathogenic Mycobacteria. As part of an ongoing project on adenylyl cyclases of Mycobacteria, we have taken up the structure analysis of the catalytic domains of two adenylyl cyclases; Rv1625c from M. tuberculosis and Ma1120 from M. avium. This work is described in Chapter 4. The wild-type of Rv1625c crystallized as a domain swapped head to head inactive dimer even though it is an active dimer in solution and expected to have a head to tail arrangement as in the previously reported structures of the active forms of the enzyme. Mutation of a phenylalanine residue presumed to occur at the subunit interface of the active dimeric structure of the enzyme to an arginine residue, a conserved residue of guanylyl cyclases, resulted in reduced adenylyl cyclase activity. This mutant crystallized as a monomer though it was expected to be an active dimer. Similarly, Ma1120 also has a monomeric structure in the crystal in spite of showing activity in solution. Though our aim was to capture the active dimers in the crystalline state we did not succeed in this effort in any of the three cases. The catalytic reaction probably takes place very rapidly with the formation of a transient active form of the dimer which cannot be easily crystallized. However, the analysis revealed new structures which are likely to represent the stable states of the enzyme when it is required to stay inactive in certain conditions. We have also established that the N-terminal segments of the enzyme, a loop at the dimeric interface and external factors like pH are involved in determining the oligomeric status of the enzyme thereby regulating its function. Publications 1 Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16; Deivanayaga V. Barathy and K. Suguna; FEBS Open Bio (In Press) 2 New structural forms of mycobacterial adenylyl cyclases (in preparation)

Living organisms employ different kinds of mechanisms, to regulate the functions of genes or their products, which may help in maintaining homeostasis inside the cell or may help in fighting hostile environment in the case of pathogenic organisms. These mechanisms act at the transcriptional, post-transcriptional, translational, and post-translational levels. In order to understand the physiology of an organism, it is essential to obtain an in-depth knowledge of such mechanisms, in which several proteins participate in interlinked pathways. In this regard, the present study focuses on two such proteins: (i). the newly identified Fic (Filamentation induced by cAMP) protein; and (ii). NDK (Nucleoside Diphosphate Kinase), which had been studied for decades. Fic protein and NDK share several common features: (i). both use nucleoside triphosphate (NTPs) or nucleoside diphosphate (NDPs) or their derivatives as one of their substrates; (ii). they have been found to be involved in diverse cellular pathways, involving different types of substrates that form the second substrate of these proteins; (iii). both are ubiquitously present in all the living organisms - from bacteria to humans to plants. However, there is very little information on these proteins from mycobacterial systems, which include some major human pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, which are the causative agents of Tuberculosis and Leprosy, respectively. In view of these reasons, in the present study, the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis, were analysed, as it might be of medical significance for effectively combating the pathogen. The Chapter 1 of the thesis contains the Introduction to the research work and Chapter 2 is on the overall Materials and Methods. The remaining chapters pertain to the data obtained on the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis. Chapter 3. Cloning, Expression and Purification of Mycobacterium tuberculosis Fic The role of FIC (Filamentation induced by cAMP) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylylation), in microorganisms, higher eukaryotes, and plants is emerging. In order to understand the biological role of FIC domain containing proteins in mycobacteria, the gene for the FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, MtuFic, was cloned, overexpressed, purified to homogeneity, and biochemically characterised. Neither the His-tagged nor the GST-tagged MtuFic protein, overexpressed in Escherichia coli, nor expression of Mtufic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtuFic (MBP-MtuFic) could be obtained partly in the soluble fraction. Denatured-refolded protein was used for the antibody generation in mice and rabbit. The cellular localisation and secretion of MtuFic were characterised using the antibody. Chapter 4. Biochemical Characterisation of Mycobacterium tuberculosis Fic Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. MtuFic, possesses the critical His144 residue, in the characteristic FIC Motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The activity of MtuFic was consistent with the biochemical activities hitherto reported for a variety of bacterial FIC domain containing proteins. Studies were also carried out on NMPylylation in the presence of eukaryotic proteins and eukaryotic and mycobacterial cell lysates. Although formation of NMPs from NTPs mediated by MBP-MtuFic could be detected, we could not identify any protein as the target substrate either in the human macrophage (THP1) cells or in the M. tuberculosis cells. VopSΔ30 (kind gift from Dr. Kim Orth), along with human G proteins as targets, were used as the positive controls. Various possibilities for the inability to detect a protein target substrate are discussed. Chapter 5. Transcriptional Analysis of Mycobacterium tuberculosis fic Gene (Mtufic) In parallel, in order to understand the transcriptional regulation of Mtufic, primer extension analysis was carried out. The Transcription Start Site (TSS; +1 site) of Mtufic were mapped under different growth/stress conditions, which tubercle bacilli encounter in human host. Mtufic got expressed mainly through two transcripts, T1 and T2, arising from two different transcription start sites (TSS). Putative promoter regions were cloned in a promoter probe vector, which expresses a GFP protein of very high intensity, in order to qualitatively detect the activity of the promoters. The half-life of the gfp mRNA was determined to be 4 min and therefore justifiably quantitated the Mtufic promoter activity by determining the gfp mRNA levels. The levels of Mtufic mRNA were two-fold higher under nutrient-depleted stationary phase of growth, as compared to the levels at mid-log phase. The activity of P1 and P2, as quantitated real-time using the short half-life gfpm2+ mRNA levels in Mycobacterium smegmatis transformants, showed that the activity of P2 was upregulated two-fold under nutrient-depleted stationary phase of growth, while that of P1 remained unaltered while of P1 and P2 were low under hypoxia. Co-transcription of Mtufic, with the immediate upstream gene, Rv3642c, of unknown function, was observed. Taken together, the data strongly indicated that the expression of Mtufic gets altered under nutrient-depleted and hypoxic conditions, which are the stress conditions experienced by tubercle bacilli in granuloma in tuberculosis patients. Chapter 6. Functional Characterisation of Mycobacterial FtsZ-NDK Interaction During the past few decades, our laboratory has been carrying out extensive molecular and functional studies on the cytokinetic protein, FtsZ, of different mycobacterial species, and of a variety of other mycobacterial proteins that are believed to be interacting with the cell division machinery. In this regard, in parallel to the work on MtuFic, we carried out work on the identification and characterisation of the proteins that interact with mycobacterial FtsZ. In this context, we found for the first time that the nucleoside diphosphate kinase (NDK), which can generate NTPs from ATP/GTP and NDPs, interacts with FtsZ and that the interaction was conserved across several mycobacterial species. Therefore, the FtsZ-NDK interaction was extensively characterised in vitro, using the recombinant, purified FtsZ and NDK proteins from different mycobacterial species. This novel finding on the interaction of NDK with FtsZ adds another role to NDK, namely in bacterial cell division.

The DNA binding proteins under starvation (Dps) from M. smegmatis are cage-like structures which internalize iron and bind DNA. They provide resistance to the cells from free radical damage, and physically protect the DNA from the harmful effects of reactive oxygen species by DNA compaction. The work compiled in this thesis has been an effort to study oligomerization and dynamics of iron metabolism by these nano-protein compartments. Chapter 1 gives a general introduction on stress, especially oxidative stress, and the ways bacteria fight back the host resistance systems. This has been elaborated from the point of view of the Dps proteins which is the focus of our work. Also, the competition for iron among the host and pathogens, and the modes of iron trafficking of the pathogens from host organisms has been summarized. Finally, the structural aspects of ferritin family proteins to which Dps belongs, has been discussed. Chapter 2 elaborates on the oligomerization pathways of the first M. smegmatis Dps MsDps1, which exists in vitro as two oligomeric forms. The GFP-tagging has been used to locate the Dps1 proteins by live cell imaging and the over-expression of these proteins during nutrient limiting conditions has been studied. The crystal structure of a point mutant F47E in the background of MsDps1, which shows no dodecamerization in vitro, has been solved. The possible ways of dodecamerization of MsDps1 has been concluded by analyzing the intermediates via glutaraldehyde cross-linking and native electrospray mass spectrometry. Chapter 3 documents the gating machinery of iron in MsDps2 protein, the second M. smegmatis Dps protein. Through graph theoretical approaches, a tight histidine-aspartate cluster was identified at the ferritin-like trimeric pore which harbors the channel for the entry and exit of iron. Sitespecific variants of MsDps2 were generated to disrupt this ionic knot, and the mutants were further assayed for ferroxidation, iron uptake and iron release properties. Our studies in MsDps2 show the importance of counter-acting positive and negatively charged residues for efficient assimilation and dispersion of iron. Chapter 4 describes crystallization studies of MsDps2 pore variants, done in an attempt to connect the changes in functional properties described in chapter 3, with structural alterations of the point mutants. We show here that the gating mechanism happens by alterations in side chain configuration at the pore and does not alter the over-all stability of the proteins. Chapter 5 is the final section where we have employed site specific mutations and cocrystallization studies to elucidate the behaviour of MsDps2 proteins upon the addition of iron. By studying the effect of substitutions at conserved sites near ferroxidation center, we attempt to arrive at a pathway which iron atoms take to reach the ferroxidation site. Also, by crystallization of proteins loaded with varying amounts of iron we tried to map the changes in the protein structure in the presence of its ligand. Chapter 6 concludes briefly the work that has been documented in this thesis. Appendix I relates the role of N-terminal tail for DNA binding in MsDp2. Appendix II gives the technical details of a modified protein preparation and oligomerization process for his-tagged MsDps1 protein. Appendix III gives the maps of the plasmids used in this study.

L’émergence de la résistance à une multitude d’agents antimicrobiens chez des bactéries pathogènes est considérée comme une menace majeure pour la santé publique (2). Ces souches sont reconnues comme des organismes multirésistants aux médicaments ou MDR (multidrug-resistant) (4). Les recherches progressent chez les bactéries, à Gram positif, à Gram négatif et acido-alcoolo-résistantes au vu de l’ampleur de la menace pour la santé publique, ces bactéries multirésistantes sont devenues les cibles potentielles à cette fin de recherche. De ce fait, les objectifs de la présente étude ont consisté en la caractérisation structurale et fonctionnelle de différents transporteurs transmembranaires de la famille des RND (Resistance-Nodulation-Division) encore énigmatiques, à savoir: le MmpL3 chez Mycobacterium tuberculosis (Mtb) via l’étude de son orthologue CmpL1 chez Corynebacterium glutamicum (Cgl) et le TriAxBC chez Pseudomonas aeruginosa (P. aeruginosa). Ainsi, comme première démarche présentée dans le chapitre 2, la structure du transporteur MmpL3 Mtb (un transporteur d'acides mycoliques – sous forme de tréhalose de monomycolates (ou TMM) - essentiel pour la viabilité de Mtb) (5) et celle de son orthologue CmpL1 Cgl ont été prédites via le serveur I-TASSER (6-8). Ces structures ont été validées par la suite en comparant à la carte électronique générée pour CmpL1 (18 Å) par des analyses de microscopie électronique en transmission à coloration négative (TEM). La caractérisation du transporteur CmpL1 purifié par chromatographie à exclusion stérique a confirmé le complexe trimérique de taille avoisinant les 315 KDa (incluant la couronne du détergent) en accord avec des analyses par gel SDS-PAGE. Des études génétiques et biochimiques en collaboration ont d’autre part identifié des résidus engagés dans le transport du TMM chez MmpL3 ainsi que d’autres impliqués dans la résistance à des inhibiteurs ciblant ce transporteur. L’ensemble de ces données a mis en évidence la localisation des résidus essentiels au transport et à la résistance au niveau du canal central du modèle trimérique de MmpL3. La région de MmpL3 activant le transport par force protomotrice a été localisée au niveau d’une cavité centrale qui est une caractéristique intrinsèque de la famille des RND. Les cartes électroniques de faible résolution déjà obtenues pour la protéine CmpL1 font de ce projet une des directions futures du laboratoire. Dans le chapitre 3, nous illustrons le deuxième aspect du présent projet qui repose sur l’extension de l’étude du potentiel thérapeutique du ciblage du transporteur transmembranaire MmpL3 chez les différentes souches de Mycobacterium. Nos collaborateurs ont effectué une analyse biochimique de l’effet thérapeutique des inhibiteurs les plus prometteurs du transporteur MmpL3 Mtb sur certaines souches mycobactériennes non-tuberculeuses (NTB) multi-résistantes. Basés sur nos modélisations structurales comparatives obtenues par I-TASSER (6-8), nous avons pu complémenter les informations biochimiques en soulignant les similitudes et les différences de structure entre les souches TB et NTB ainsi que leurs impacts fonctionnels. Ce chapitre met en évidence l’intérêt du ciblage thérapeutique de MmpL3 chez les espèces NTB. En effet, l’efficacité de certains inhibiteurs de MmpL3 Mtb sélectionnés sur le traitement des infections pulmonaires NTB promet de pouvoir généraliser cette nouvelle voie de traitement pour d’autres souches multi-résistantes NTB voire à contribuer à remédier à la problématique de la résistance aux antibiotiques et décomplexifier le traitement actuel. D’autres études en collaboration entreprenant les mêmes approches d’études structurales ont été réalisées pour les transporteurs tripartites TriAxBC (P. aeruginosa), des pompes à efflux appartenant à la famille des RND. Le but du chapitre 4 était de générer une structure du complexe et de déchiffrer son mode d’assemblage et d’expulsion des antibiotiques vers le milieu externe. Un modèle à structure quaternaire de TriAxBC a été prédit par I-TASSER (6-8) et validé contre sa carte électronique à 4.3 Å générée en Cryo-EM. Le complexe TriAxBC a été également caractérisé par filtration sur gel confirmant une taille approximative de 620 KDa et sa composition en trimère par visualisation sur gel SDS-PAGE. En conclusion, nous avons pu à travers cette étude combiner différentes approches biochimiques, génétiques et structurales soutenant la nécessité d’une approche multidisciplinaire pour l’approfondissement de la compréhension de la structure et du mode de fonctionnement des transporteurs RND. Ces derniers demeurent toujours énigmatiques; toutefois, nos avancées et d’autres à venir permettront la génération de nouveaux médicaments spécifiques traitant les bactéries multirésistantes. / The emergence of resistance to a multitude of antimicrobial agents in pathogenic bacteria is considered a major threat to public health (2). These strains are recognized as multidrug resistant organisms (MDR) (4). Research is progressing in Gram positive, Gram positive high GC and Gram negative bacteria, and given the scale of the public health threat, these MDR have become potential targets for this research. The objectives of the present study consist of the structural and functional characterization of various transmembrane transporters of the still enigmatic RND (Resistance-Nodulation-Division) family, namely: MmpL3 in Mycobacterium tuberculosis (Mtb) via the study of its ortholog CmpL1 in Corynebacterium glutamicum (Cgl) and TriAxBC in Pseudomonas aeruginosa (P. aeruginosa). The first component of this project, presented in Chapter 2, studies the structure of the transporter MmpL3 Mtb (a TMM mycolic acid transporter essential for the viability of Mtb (5) and that of its CmpL1 Cgl orthologue, which have been predicted via the I- Tasser Pack (6-8). These structures were subsequently validated by comparing to the electronic map generated for CmpL1 (18 Å) by negative staining transmission electron microscopy (TEM). Characterization of the purified CmpL1 transporter by size exclusion chromatography confirmed the trimeric complex size around 315 KDa (including the detergent crown) corroborated by SDS-PAGE gel analyses. Collaborative genetic and biochemical studies have also identified residues involved in the transport of TMM in MmpL3 as well as those residues conferring antibiotic resistance. This data highlighted the location of the essential residues of transport and resistance in the central channel of the trimeric Mmpl3 model. The MmpL3 region activating proto-motor transport has been located at a central cavity, which is an intrinsic feature of the RND family. The low-resolution electronic maps obtained for the protein CmpL1 may serve as the foundation of future studies. In Chapter 3 we explore the therapeutic potential of the targeting of the transmembrane transporter MmpL3 in different Mycobacterium strains. Our collaborators studied the therapeutic effect of the most promising inhibitors of the MmpL3 Mtb transporter on certain multi-resistant mycobacterial non-tuberculous (NTB) strains. Based on our comparative structural modeling obtained by I-TASSER (6-8), we supplemented the biochemical data by highlighting the structural similarities and differences between the TB and NTB strains as well as their functional impacts. This chapter highlights the interest of direct or indirect targeting of MmpL3 in NTB species. Indeed, the efficacy of certain selected MmpL3 Mtb inhibitors on the treatment of NTB pulmonary infection have potential as generalizable treatment options for other NTB multi-resistant strains, or even to help address the problem of resistance to antibiotics and simplify current combination approaches. Other collaborative studies undertaking the same structural approaches were carried out for TriAxBC tripartite carriers (P. aeruginosa), efflux pumps belonging to the RND family. The purpose of Chapter 4 was to generate a structure of the complex and decipher its mode of assembly and expulsion of antibiotics from the intracellular environment. A quaternary structure model of TriAxBC was predicted by I-TASSER (6-8) and validated against its 4.3 Å electronic map generated by Cryo-EM. The TriAxBC complex was also characterized by gel filtration confirming an approximate size of 620 KDa and its trimer composition by SDS-PAGE. In conclusion, this study is combining different biochemical, genetic and structural approaches to highlight the need for a multidisciplinary approach to characterizing the structure function of RND transporters. The latter remain enigmatic; however, our contribution and the progress of others will allow the generation of new specific drugs targeting multiresistant strains.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Setting This study was conducted in the Tygerberg district, Cape Town, in the Western Cape, South Africa Background The evaluation of early tuberculosis (TB) treatment response is based on month 2 sputum culture status. This method of evaluation has a number of limitations: the test requires relatively advanced laboratory infrastructure and procedures, it takes several weeks to obtain results and is a relatively a poor marker at predicting treatment response. The discovery of potential host markers which reflect the efficacy of early treatment would be of great importance for clinical management of individual patients. The treatment failure would be detectable earlier than at week 8 of treatment. The duration of clinical trials of new anti-tuberculosis drugs may also be substantially reduced by such markers if these would be measurable earlier than at week 8 of therapy. Objectives 1) To evaluate diluted, 7-day whole blood cultures stimulated with live Mycobacterium tuberculosis (M.tb) for the presence of host markers of early TB treatment response 2) To evaluate an overnight, undiluted, M.tb antigen stimulated whole blood culture Quantiferon Gold In Tube (QFT-GIT) supernatants for host markers of early TB treatment response The study designs were as follows: In study one, baseline samples and samples from week 1, week 2 and week 4 of treatment from 30 cured TB patients were selected from a larger biomarker study, in which whole blood was stimulated with live M.tb or left unstimulated. Fifty seven host markers were measured in supernatants by multiplex cytokine arrays. In study two, baseline samples and samples from week 2 and week 8 of treatment from 19 cured TB patients were randomly selected from the placebo group in a micronutrient supplement study. QFT-GIT supernatants from these participants were assessed through multiplex cytokine arrays for levels of fifty seven host markers. All of the participants in both studies were Human Immunodeficiency Virus (HIV) negative. Changes in marker expression over time and between fast and slow responders to treatment were evaluated. Comparability between the two culture methods was assessed for markers that were evaluated in both studies. Results In study one, the majority of host markers showed significant changes over time in the unstimulated supernatants. Only GRO and IL-1beta changed significantly in an antigen-specific manner (background levels subtracted). No significant changes were observed between fast and slow responders. In study two, the majority of host markers showed significant changes over time in the unstimulated supernatants whereas only MDC and IL-4 changed during the observation period in antigen stimulated levels. Significant differences were observed between fast and slow responders at pre-treatment for IL-13 Ag-Nil and IL-1betaAg-Nil . Conclusion This study revealed, antigen-specific responses showed only limited potential for early TB treatment response monitoring, but may have potential in differentiating between treatment outcomes. Future investigations may have to include later time points during treatment as these were not included in the present assessment. The QFT-GIT samples do not appear to be equivalent to live M.tb stimulated 7-day whole blood assays. / AFRIKAANSE OPSOMMING: Instelling Die studie is uitgevoer in die Tygerbergdistrik, Kaapstad, Wes-Kaap, Suid-Afrika. Agtergrond Die evaluering van die respons op vroeë tuberkulose (TB) behandeling word gebaseer op die status van maand 2 sputum kulture. Hierdie evalueringsmetode het ‘n paar beperkinge: die toets benodig relatief gevorderde laboratorium infrastruktuur en prosedures, die toetsuitslae is eers na ‘n paar weke beskikbaar en dit is n relatiewe swak merker om repons op behandeling te voorspel. Die ontdekking van potensiële selfmerkers wat die doeltreffendheid van vroeë behandeling weerspieël sal van groot belang wees vir die kliniese bestuur van individuele pasiënte. Mislukking van die behandeling sal sodoende voor week 8 van behandeling waargeneem kan word. Die tydsduur van kliniese proewe van nuwe anti-tuberkulose medikasie mag ook baie verkort word met sulke merkers as dit voor week 8 van behandeling gemeet kan word. Doelwitte 1) Om verdunde, 7-dae oue volbloedkulture, met lewende Mikobakterium tuberkulosis (M.tb) gestimuleer, te evalueer vir die teenwoordigheid van vroeë TB behandeling respons selfmerkers. 2) Om die supernatant van oornag, onverdunde, M.tb antigeen gestimuleerde volbloedkulture Quantiferon Gold In Tube (QFT-GIT) vir vroeë behandeling respons selfmerkers te evalueer. Die studie-ontwerpe was soos volg: Met studie een is basislynmonsters en monsters verkry na week 1, week 2 en week 4 van behandeling van 30 geneesde TB-pasiënte geselekteer uit ‘n groter biomerkerstudie waarin die volbloed met lewende M.tb gestimuleer is of ongestimuleer gelaat is. Sewe-en-vyftig selfmerkers is in die supernatante gemeet deur middel van multipleks sitokine arrays. Met studie twee is basislynmonsters en monsters verkry na week 2 en week 8 van behandeling van 19 geneesde TB-pasiënte lukraak uit die plasebo-groep in ‘n mikrovoedingstowwe-aanvullingstudie geselekteer. Vlakke van 57 selfmerkers is in die QFT-GIT supernatante van hierdie deelnemers, deur middel van die multipleks sitokine arrays, bepaal. Al die deelnemers van beide studies was HIV negatief. Veranderinge in merker-uitdrukking oor tyd, asook tussen vinnige en stadige respons tot behandeling, is ge-evalueer. Die vergelykbaarheid van die twee kultuurmetodes is geassesseer ten opsigte van die ge-evalueerde merkers in albei studies. Resultate Met studie een het die meerderheid van die selfmerkers in die ongestimuleerde supernatante kenmerkende verandering oor tyd gewys. Slegs GRO en IL-1beta het aansienlik verander in die antigeenspesifieke wyse (agtergrond vlakke afgetrek). Geen kenmerkende veranderinge was waargeneem tussen die vinnige en stadige respons pasiënte nie. Met studie twee het die meerderheid van die selfmerkers aansienlike veranderinge oor tyd in die ongestimuleerde supernatante gewys, in vergelyking waar net die MDC en IL-4 veranderinge gedurende die observasie periode in antigeen gestimuleerde vlakke getoon het. Kenmerkende verskille is tussen die vinnige en stadige respons pasiënte in voorbehandeling vir IL-13 Ag-Nil en IL-1betaAg-Nil waargeneem. Gevolgtrekking Die studie bewys dat antigeenspesifieke response slegs beperkte potensiaal vir vroeë TB behandeling respons monitering het, maar mag die potensiaall vir onderskeidende behandeling uitkomste hê. Toekomstige ondersoeke sal dalk latere tydpunte gedurende die behandeling moet insluit aangesien dit nie in hierdie evaluasie ingesluit is nie. Die QFT-IT monsters verskyn nie as gelykwaardig met die lewendig M.tb gestimuleerde 7-dae volbloed toetse nie.