Metal ions and protein folding: conformational and functional interplay

Dissertation presented to obtain a PhD degree in Biochemistry at Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

The role of the efflux mechanisms in multidrug resistance in Mycobacterium tuberculosis

Abstract The emergence of multi and extensively drug resistant tuberculosis (MDRTB and XDRTB) has increased the concern of public health authorities around the world. The World Health Organization has defined MDRTB as tuberculosis (TB) caused by organisms resistant to at least isoniazid and rifampicin, the main first-line drugs used in TB therapy, whereas XDRTB refers to TB resistant not only to isoniazid and rifampicin, but also to a fluoroquinolone and to at least one of the three injectable second-line drugs, kanamycin, amikacin and capreomycin. Resistance in Mycobacterium tuberculosis is mainly due to the occurrence of spontaneous mutations and followed by selection of mutants by subsequent treatment. However, some resistant clinical isolates do not present mutations in any genes associated with resistance to a given antibiotic, which suggests that other mechanism(s) are involved in the development of drug resistance, namely the presence of efflux pump systems that extrude the drug to the exterior of the cell, preventing access to its target. Increased efflux activity can occur in response to prolonged exposure to subinhibitory concentrations of anti-TB drugs, a situation that may result from inadequate TB therapy. The inhibition of efflux activity with a non-antibiotic inhibitor may restore activity of an antibiotic subject to efflux and thus provide a way to enhance the activity of current anti-TB drugs. The work described in this thesis foccus on the study of efflux mechanisms in the development of multidrug resistance in M. tuberculosis and how phenotypic resistance, mediated by efflux pumps, correlates with genetic resistance. In order to accomplish this goal, several experimental protocols were developed using biological models such as Escherichia coli, the fast growing mycobacteria Mycobacterium smegmatis, and Mycobacterium avium, before their application to M. tuberculosis. This approach allowed the study of the mechanisms that result in the physiological adaptation of E. coli to subinhibitory concentrations of tetracycline (Chapter II), the development of a fluorometric method that allows the detection and quantification of efflux of ethidium bromide (Chapter III), the characterization of the ethidium bromide transport in M. smegmatis (Chapter IV) and the contribution of efflux activity to macrolide resistance in Mycobacterium avium complex (Chapter V). Finally, the methods developed allowed the study of the role of efflux pumps in M. tuberculosis strains induced to isoniazid resistance (Chapter VI). By this manner, in Chapter II it was possible to observe that the physiological adaptation of E. coli to tetracycline results from an interplay between events at the genetic level and protein folding that decrease permeability of the cell envelope and increase efflux pump activity. Furthermore, Chapter III describes the development of a semi-automated fluorometric method that allowed the correlation of this efflux activity with the transport kinetics of ethidium bromide (a known efflux pump substrate) in E. coli and the identification of efflux inhibitors. Concerning M. smegmatis, we have compared the wild-type M. smegmatis mc2155 with knockout mutants for LfrA and MspA for their ability to transport ethidium bromide. The results presented in Chapter IV showed that MspA, the major porin in M. smegmatis, plays an important role in the entrance of ethidium bromide and antibiotics into the cell and that efflux via the LfrA pump is involved in low-level resistance to these compounds in M. smegmatis. Chapter V describes the study of the contribution of efflux pumps to macrolide resistance in clinical M. avium complex isolates. It was demonstrated that resistance to clarithromycin was significantly reduced in the presence of efflux inhibitors such as thioridazine, chlorpromazine and verapamil. These same inhibitors decreased efflux of ethidium bromide and increased the retention of [14C]-erythromycin in these isolates. Finaly, the methods developed with the experimental models mentioned above allowed the study of the role of efflux pumps on M. tuberculosis strains induced to isoniazid resistance. This is described in Chapter VI of this Thesis, where it is demonstrated that induced resistance to isoniazid does not involve mutations in any of the genes known to be associated with isoniazid resistance, but an efflux system that is sensitive to efflux inhibitors. These inhibitors decreased the efflux of ethidium bromide and also reduced the minimum inhibitory concentration of isoniazid in these strains. Moreover, expression analysis showed overexpression of genes that code for efflux pumps in the induced strains relatively to the non-induced parental strains. In conclusion, the work described in this thesis demonstrates that efflux pumps play an important role in the development of drug resistance, namely in mycobacteria. A strategy to overcome efflux-mediated resistance may consist on the use of compounds that inhibit efflux activity, restoring the activity of antimicrobials that are efflux pump substrates, a useful approach particularly in TB where the most effective treatment regimens are becoming uneffective due to the increase of MDRTB/XDRTB.

Metal ions modulate the folding and stability of the tumor suppressor protein S100A2

Febs Journal (2009)276:1776-1786

Design of bio-inspired ionic liquids for protein stabilisation

Ionic Liquids (ILs) consist in organic salts that are liquid at/or near room temperature. Since ILs are entirely composed of ions, the formation of ion pairs is expected to be one essential feature for describing solvation in ILs. In recent years, protein - ionic liquid (P-IL) interactions have been the subject of intensive studies mainly because of their capability to promote folding/unfolding of proteins. However, the ion pairs and their lifetimes in ILs in P-IL thematic is dismissed, since the action of ILs is therefore the result of a subtle equilibrium between anion-cation interaction, ion-solvent and ion-protein interaction. The work developed in this thesis innovates in this thematic, once the design of ILs for protein stabilisation was bio-inspired in the high concentration of organic charged metabolites found in cell milieu. Although this perception is overlooked, those combined concentrations have been estimated to be ~300 mM among the macromolecules at concentrations exceeding 300 g/L (macromolecular crowding) and transient ion-pair can naturally occur with a potential specific biological role. Hence the main objective of this work is to develop new bio-ILs with a detectable ion-pair and understand its effects on protein structure and stability, under crowding environment, using advanced NMR techniques and calorimetric techniques. The choline-glutamate ([Ch][Glu]) IL was synthesized and characterized. The ion-pair was detected in water solutions using mainly the selective NOE NMR technique. Through the same technique, it was possible to detect a similar ion-pair promotion under synthetic and natural crowding environments. Using NMR spectroscopy (protein diffusion, HSQC experiments, and hydrogen-deuterium exchange) and differential scanning calorimetry (DSC), the model protein GB1 (production and purification in isotopic enrichment media) it was studied in the presence of [Ch][Glu] under macromolecular crowding conditions (PEG, BSA, lysozyme). Under dilute condition, it is possible to assert that the [Ch][Glu] induces a preferential hydration by weak and non-specific interactions, which leads to a significant stabilisation. On the other hand, under crowding environment, the [Ch][Glu] ion pair is promoted, destabilising the protein by favourable weak hydrophobic interactions , which disrupt the hydration layer of the protein. However, this capability can mitigates the effect of protein crowders. Overall, this work explored the ion-pair existence and its consequences on proteins in conditions similar to cell milieu. In this way, the charged metabolites found in cell can be understood as key for protein stabilisation.