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Unraveling the molecular mechanism of nitrosative stress resistance in tuberculosis

Final Report Summary - NOSTRESS (Unraveling the molecular mechanism of nitrosative stress resistance in tuberculosis)

Executive summary:

Tuberculosis is today amongst the major worldwide health threats. Treatment failure is unfortunately becoming more usual, especially in countries lacking the long and costly treatment adapted to patients. Thus, tuberculosis causes two million deaths every year and latently persists in over one billion individuals worldwide. Current treatments are challenged by multidrug resistant strains, drug side effects, and co-infections. Therefore, identification of potent, safety antimycobacterial agents is mandatory. However, the success of this strategy is largely determined by the detailed knowledge of their mechanism of action, which in turn depends on the validation of suitable biological targets.

The NOSTRESS project (please see http://www.ub.edu/cbdd/Nostress online) pursues the definition of new, complementary therapeutic approaches by identifying the molecular basis of the nitrosative stress resistance of M. tuberculosis. Our working hypothesis is that a decrease in the Nitric oxide (NO) resistance of the microorganism should reduce significantly the capability to rest in latency, thus contributing to increase the efficacy of the therapeutic treatment. In this context, understanding of the NO detoxification activity played by M. tuberculosis trHbN is essential.

The outcome of the project should provide a firm basis to assess the viability of trHbN as a therapeutic target, and set up the background to exploit this knowledge in the design of innovative therapeutic strategies to fight the disease.

Project context and objectives:

Tuberculosis is today amongst the major worldwide health threats. Besides the spread of the disease, strains of Mycobacterium tuberculosis, i.e. the causative agent of tuberculosis, have slowly emerged. As a consequence, treatment failure is unfortunately becoming more usual, especially in countries lacking the long and costly treatment adapted to patients. Thus, because of lack of treatment or lack of adapted treatment, M. tuberculosis infection causes two million deaths every year and latently persists in over one billion individuals worldwide.

The chemotherapy of tuberculosis started with the introduction of streptomycin in 1946, and nearly a decade later a therapeutic strategy based on the combination of streptomycin, p-aminosalicylic acid and isoniazid was adopted as a standard treatment by the western world. In the last two decades, research on M. tuberculosis has undergone much progress. At the laboratory level, the genome of the microorganism has been unravelled. Renewed screening for antimycobacterial substances has been made, leading to novel compounds with growth inhibitory properties and the identification of several mechanisms of action. Thus, antimycobacterial agents are known to inhibit the biosynthesis of fatty acids (isoniazid, pyrazinamide), peptidoglycans (ethambutol) or proteins (streptomycin, linezolide), and Deoxyribonucleic acid (DNA)-based processes (rifampin, fluoroquinolones). In turn, this has facilitated the definition of different regimes based on the combination of different drugs, even though the success is complicated by the occurrence of multidrug resistant strains, drug side effects, and co-infections with Human immunodeficiency virus (HIV). Many antituberculosis drugs have been used for decades with little or no knowledge about the molecular basis of their mechanism of action. In fact, the determination of the biochemical processes targeted by antituberculosis drugs is still ongoing, and should permit us in future to validate already known biological targets and to identify new potential ones.

Despite effective control of M. tuberculosis growth in healthy individuals, the microorganism is rarely completely eliminated from the body. The pathogen resides primarily in macrophages, a cell type that normally controls bacterial growth. Although numerous aspects of the immune system are responsible for slowing the growth of M. tuberculosis, increasing data suggest that nitric oxide (NO) is essential for this process. NO has antimicrobial activity and is produced by the inducible nitric oxide synthase in macrophages. Recent studies have suggested that the truncated hemoglobin N (trHbN) from M. tuberculosis might play a crucial role as a defence mechanism against nitric oxide (NO) produced by macrophages during the initial growth infection stage, thus contributing to restrict the bacteria in latency. NO detoxification mechanisms have evolved in several pathogenic microorganisms to reduce the potential damage produced by the nitrosative stress. In the particular case of M. tuberculosis, trHbN is postulated to act as a NO dioxygenase converting NO into the harmless nitrate anion.

The truncated hemoglobin N from M. tuberculosis

Truncated hemoglobins comprise a family clearly distinct from all other globins. They have primary structures that are 20-40 residues shorter than mammalian Hbs. Phylogenetic analysis of trHb sequences showed that they can be classified into three groups, designated I, II and III. The members are orthologous within each group and paralogous across the groups, forming a diverse family, as indicated by pairwise identity values that are occasionally lower than 20 %.

The trHbN protein from M. tuberculosis pertains to group I. The typical tertiary structure is based on a two-over-two helical sandwich compared with the three-overthree helical sandwich of the classical Hb fold. The proximal HisF8 heme-linked residue is conserved in both Hb and trHb families, and a distal tyrosine at position B10 is found in almost all the trHb family members sequenced to date. Despite their small size compared to vertebrate globins, several trHbs host a protein matrix apolar tunnel system that connects the heme pocket with the protein surface. In trHbN from M. tuberculosis the tunnel system is built by two perpendicular branches, of about 8 and 20 Å length, respectively, that have been proposed to control diatomic ligand diffusion to/from the heme as the rate-limiting step in NO conversion to nitrate.

Several lines of evidence support the protective role played by trHbN in M. tuberculosis. against NO stress. First, NO conversion to nitrate by oxy-trHbN occurs at a rate faster than O2 binding to the deoxy protein. Second, the most direct evidence for supporting the molecular mechanism of action comes from comparison of wild type protein and mutants.

Aims of the project

The basic assumption of this project is that the nitrosative stress resistance of M. tuberculosis might be largely diminished by affecting the activity of the trHbN, thus contributing to decreasing the capability of the microorganism to rest in latency in the infected organism. The hypothetical NO detoxification role makes trHbN a potential therapeutic target, as the design of effectors that might affect the efficiency of the NO detoxification should be valuable to weaken the resistance of the microorganism in the framework of a multitarget therapeutic strategy.

On the basis of the preceding considerations, this project pursues the following objectives:
i) to examine the hypothetical molecular mechanism underlying the NO dioxygenase activity of trHbN in M. tuberculosis,
ii) to establish the differences in structure-function relationships in trHbN from M. tuberculosis and related trHbs, and
iii) to identify the redox protein system that helps trHbN to restore the ferrous staterequired to initiate the NO detoxification cycle.

The outcome of the project should provide a firm basis to assess the viability of trHbN as a therapeutic target, and set up the background to exploit this knowledge in the design of innovative therapeutic strategies to fight the disease.

Project results:

The NO detoxification role makes trHbN (and putative proteins that might participate in interaction networks, such as reductases) a potential therapeutic target. Accordingly, we can hypothesise that the design of effectors that might affect the efficiency of the NO detoxification activity should be valuable to weaken the resistance of the microorganism in the framework of a multitarget therapeutic strategy. To achieve this goal, however, it is necessary to identify the molecular mechanism that underlies the NO detoxification activity of trHbN, which in turn makes it necessary to unravel the relationship between structure, dynamics and functional role of the protein.

To this end, the project aims to gain deeper knowledge into the molecular basis of NO resistance by defining the following research lines:
(i) In a direct approach, the viability of the molecular mechanism of trHbN will be corroborated by analysing mutants corresponding to selected mutations of those residues that are expected to play a crucial role in the mechanism of ligand migration.
(ii) In an indirect approach, we will examine the structure-function relationships by examining the linkage existing between changes in sequence composition and structure with differences in biological function for selected trHbs.
(iii) Finally, we will identify the molecular system responsible for the final reduction step leading from ferric to ferrous state of trHbN, which enables the protein to start a new NO detoxification cycle.

Potential impact:

The aim of this research project is to contribute to the definition of new, complementary therapeutic approaches by identifying the molecular basis of the nitrosative resistance of M. tuberculosis.

In this context, our studies have lead to a deeper understanding at the microscopic level of the molecular mechanism associated with the NO detoxification activity of truncated hemoglobin N. This mechanism unrabvels the relationship between structure and dynamics, which are imbricated in order to facilitate efficient mechanisms to ensure NO detoxification by the pxygenated protein. On the other hand, the elucidation of a potential type II flavohemoglobin opens new avenues to expand our knowledge about the resistence mechanisms implicated in protecting the bacillus against nitrosative stress.

From a therapeutical point of view, it is reasonable to expect that the peculiar features found from preliminary studies for the flavohemoglobin provide a basis for exploring the potential druggability of this protein as putative target in future studies.