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"Mycobacterium tuberculosis causes tuberculosis, one of the most life-threatening diseases of all time. It infects the host macrophages and survives in its phagosome. The host phagosome is a very hostile environment where M. tuberculosis copes with high concentration of transition metals (Zn2+, Cu2+), low levels of others (Mn2+, Fe2+) and acidic pH. P-ATPases are membrane proteins that transport various ions against their electrochemical gradients utilizing the energy of ATP hydrolysis. Based on their primary sequences; seven of the twelve mycobacterial ATPases are classified as putative heavy metal transporters and a K+-ATPase, while the substrate of four (CtpE, CtpF, CtpH and CtpI) remains unknown. Consistent with their membrane topology and conserved amino acids, CtpE and CtpF are possibly P2 or P3-ATPases that transport alkali metals or protons. We examined the cellular roles of orthologous CtpE and CtpF in M. smegmatis, a non-pathogenic model organism. We hypothesized that these novel P- ATPases play an important role in transporting alkali metals and/or protons. We analyzed growth fitness of strains carrying mutations of the coding gens of these enzymes, in presence of various metals and different pHs, as well as the gene expression levels under different stress conditions. We observed that the M. smegmatis mutant strains, lacking of CtpF or CtpE, are sensitive to high concentrations (mM) of Mn2+. Furthermore, CtpE mutant is sensitive to alkali pH. Our results indicate that CtpE and CtpF might be an Mn2+ or H+-ATPase that are required for cell’s homeostasis sustainability."

Common mycobacterial diseases, including tuberculosis and leprosy, contribute to major mortality and morbidity worldwide. Despite evidence of an important role of host genetic factors in susceptibility to these infections, few compelling genetic associations have been identified with previous candidate gene and linkage approaches. This thesis investigates the genetic factors of human immunity to these mycobacterial diseases using a high-throughput approach of association testing. To assess genetic susceptibility to tuberculosis, I have conducted a genome-wide association study in the Gambian population as part of the Wellcome Trust Case Control Consortium (WTCCC). The study reveals the region flanking CADM1 as a potential susceptibility locus. Combining this study with a Ghanaian cohort further implicates two genetic loci at chromosome 18q11.2 (P = 9.2x10⁻⁹) and PARD3B (P = 1.4x10⁻⁶). For leprosy, I have performed a gene-centric association study in the New Delhi Indian population. Evidence of significant association was observed in the HLA-DRB1/DQA1 (P = 4.9x10⁻¹⁴) and TLR1 (P = 1.7x10⁻⁹) loci. These studies identify important genomic regions that may be involved in immunity to tuberculosis and leprosy. Further analysis revealed a significant immunogenetic overlap between tuberculosis and leprosy. This provides proof-of-principle for the subsequent aggregate analysis for mycobacterial susceptibility, which suggests that the steroid biosynthesis pathway may be important in anti-mycobacterial immunity. This thesis represents one of the largest studies to identify the genetic factors for human immunity against mycobacteria. These novel findings will further enhance vaccine and pharmaceutical efforts into prevention and treatment of these mycobacterial diseases.

Charles University, Faculty of Pharmacy, Hradec Králové Field of study: Specialist in laboratory methods Author: Bc. Tomáš Hampl Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma thesis: Mycobacterium tuberculosis as causative agent of grave illness The aim of this diploma thesis is to deal with tuberculosis, describe the pathogen, taxonomic classification, describe the origin, course and clinical manifestations of the disease, diagnosis, including laboratory diagnostics, epidemiology of tuberculosis, treatment and prevention. Tuberculosis (TBC) is one of the deadliest infectious diseases in the world caused by bacterium M. tuberculosis in humans. TBC is sometimes referred as a disease of the past, but the recent rise in cases of the disease, and especially the spread of forms of drug-resistant TBC, makes the disease very dangerous. Currently, a major problem is the alarming rise in resistant and multi-resistant tuberculosis, which is not counteracted by commonly used first-line drugs. Therefore, there is a constant emphasis around the world on discovering and testing new potential antituberculotics that could help patients with resistant and multidrug-resistant TBC. In the experimental part, the thesis deals with screening of antimycobacterial activity of potential antituberculotics...

Charles University Faculty of Pharmacy in Hradec Kralove Department of Biological and Medical Sciences Study program: Specialist on Laboratory Methods Author: Bc. Tereza Pchálková Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma thesis: In vitro screening of potential antimycobacterial compounds active against fast growing strains of Mycobacterium genus II Key words: Mycobacteria, Tuberculosis, Antituberculotics, Microdilution broth method, Minimum inhibition concentration Background: The aim of this diploma thesis has been screening of in vitro antimycobacterial activity of novel compounds against fast growing strains of the genus Mycobacterium (Mycobacterium smegmatis and Mycobacterium aurum). Another aim has been predicting the structure-activity relationships for tested compounds. Methods: The technique used for activity determination was microdilution broth method. The value of minimum inhibition concentration for each compound was determined. The evaluation was performed visually and spectrophotometrically using the Alamar blue indicator. Results: A total of 79 compounds were tested. 22 tested compounds showed significant activity against mycobacteria. The substances were sorted according to the similarity in chemical structure into 10 groups. From a chemical point of view, these...

Tuberculosis is one of the most prevalent infectious diseases globally due to the successful survival mechanisms displayed by Mycobacterium tuberculosis (Mtb). Mtb primarily infects alveolar macrophages (AMs) and is able to live intracellularly for extended periods of time due to a number of virulence factors which inhibit the antibacterial mechanisms of the AMs. This aspect of the Mtb life cycle means TB treatments suffer from poor bioavailability and efficacy. Additionally, the rise in resistant strains of Mtb means the use of higher doses and the use of alternative second and third line drugs which increase the risk of systemic toxicity. Drug encapsulation is a novel approach that can provide more favourable drug pharmacokinetics and pharmacodynamics. The aim of this project was to develop a liposomal drug delivery system to target Mtb infected alveolar macrophages. The system involved the encapsulation of two drugs; the antibiotic gatifloxacin (GFLX) and Mtb virulence factor inhibitor CV7. The hypothesis was that the two different antibacterial mechanisms would work in synergy and increase the efficacy of the treatment. AM targeting and receptor-mediated endocytic uptake was encouraged by the presence of a ligand attached to the surface of the liposome. Furthermore a pH-sensitive release mechanism was to be incorporated into the liposome to encourage the release of the encapsulated drugs in the vicinity of the intracellular bacteria. The intention was to produce a drug delivery system to enable a TB therapy regime of fewer, lower doses to increase compliance and reduce systemic toxicity by increasing efficacy through improved bioavailability. GFLX was successfully encapsulated using a weak base active loading method. To establish encapsulation efficiency, a homogeneous fluorescence assay able to quantify intra- and extra-liposomal gatifloxacin simultaneously was developed. pH-sensitive release of the payload could be achieved using a pH-sensitive peptide with a novel design based on chimeric structure, namely P3. CV7 was successfully encapsulated using a weak acid active loading method. CV7 liposomes were able to be functionalised by the incorporation of a mannose ligand on the surface of the liposome. An inhibition assay using the target enzyme of CV7, MptpB, was optimised to assess efficacy of liposomally encapsulated and released CV7. Flow cytometry and confocal microscopy studies confirmed that the liposomal formulations were internalised by the target macrophage cell line, J774a.1. Mannose liposomes conveyed superior uptake kinetics. Further confocal microscopy showed that after internalisation the liposomes entered the endolysosomal pathway and colocalised with BCG. A BCG-macrophage infection model was used to determine the intracellular efficacy of the liposomal formulations. Encapsulated CV7 displayed increased efficacy over free CV7, while encapsulation in functionalised liposomes showed better efficacy still. The encapsulation of GFLX did not increase the efficacy of GFLX and synergy between the two drugs was not achieved. In conclusion, the liposomal encapsulation of CV7 increased uptake of the drug by the target cell line and facilitated colocalisation of the drug with the target pathogen thereby increasing efficacy. Such a formulation could potentially increase bioavailability and efficacy in vivo for a more tolerable TB therapy.