Molecular mechanisms of resistance to drugs and copper

der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades

vorgelegt von Alexander Speer aus Witten (geb. in Osnabrück)

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Als Dissertation genehmigt

von der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 12. Juli 2013

Vorsitzender des Promotionsorgans: Prof. Dr. Johannes Barth

Gutachter: Prof. Dr. A. Burkovski

Prof. Dr. M. Niederweis

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Danke / Thanks

Bedanken möchte ich mich zu allererst bei Prof. Dr. Michael Niederweis für die intensive Betreuung dieser Arbeit und seinen wissenschaftlichen Rat. Seine Aufgeschlossenheit und Enthusiasmus zu neuen Projekten und Ideen sorgten immer fuer eine spannende und abwechslungsreiche Arbeit.

Ich möchte mich auch bei Prof. Dr. Andreas Burkovski bedanken dessen Hilfsbereitschaft meine Zusammenarbeit mit Dr. Niederweis und die Erstellung dieser Arbeit ermöglichte. Zusätzlich danke ich Ihm und seinem Labor für aussergewöhnlich erfolgreiche Zusammenarbeit an einigen Teilprojekten dieser Studie.

Des Weiteren bedanke ich mich bei den Professoren Dr. A. Burkovski, Dr. G. Kreimer, Dr. M. Niederweis und Dr. T. Winkler entsprechend für die Erstellung der Gutachten und die Übernahme der Prüfungspflichten.

I would also like to thank all members of the Wolschendorf Lab for their excellent and fruitful collaboration during this work. I am grateful to Dr. Frank Wolschendorf for having always good advice and for his confidence in my work.

I would like to thank all current and former members of the Mycolab for creating a wonderful working environment. Special thanks go to Axel Siroy for sharing generously his endless knowledge about proteins with me. I want to thank Jason Huff for his dedicated BSL3 training and expert advice in genomic integration. For reading and adapting countless abstracts I would like to thank Jennifer Rowland. Jim Sun, I would like to thank for introducing me to the work with eukaryotic cultures. I am thanking Mikhail Pavlenok for his hospitality during my first year in the US and his technical help during emergencies inside and outside the lab. I would like to thank Olga Danilchanka for her constructive and honest criticism which improved and motivated my work. In addition I am thanking Olga for countless scientific discussions and advice that accelerated my work. I thank Ryan Wells, Virginia Meikle and Doreen William for their willingness to help at all times. Importantly, I thank Ying Wang for keeping the lab stocked.

I would like to thank the laboratory of Dr. Sue Michalek for providing us peritoneal . I am greatful to the laboratory of Dr. Stefan Bossmann for synthesizing the compounds ATSM and GTSM in huge amounts.

Von ganzem Herzen bedanke ich mich bei meiner Mutter Elisabeth Speer, die mich all die Jahre so großzügig unterstützt hat und bei meiner Schwester Friederike Speer auf die ich mich immer verlassen konnte.

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Table of Contents 1 Zusammenfassung...... 1 1 Summary ...... 3 2 Introduction ...... 5 2.1 Clinical relevance of M. tuberculosis ...... 5 2.2 The mycobacterial cell envelope ...... 6 2.2.1 Structure of the cell envelope of mycobacteria ...... 6 2.2.2 biosynthesis ...... 8 2.2.3 Mycobacterial lipids and lipoglycans ...... 9 2.2.4 Proteins of the mycobacterial outer membrane ...... 9 2.3 Pathogenesis ...... 10 2.3.1 Innate during bacterial infection ...... 10 2.3.2 M. tuberculosis adaptation to an intracellular lifestyle ...... 11 2.4 Dual role of copper in ...... 12 2.5 Role of copper during M. tuberculosis infection...... 12 2.6 Tuberculosis drug development ...... 15 2.7 Goal of this thesis ...... 16 3 Results ...... 17 3.1 Characterization of the rv1697-rv1698 operon in M. tuberculosis ...... 17 3.1.1 Rv1697 and rv1698 are encoded in one operon ...... 17 3.1.2 Subcellular localization of Rv1697 and Rv1698 ...... 18 3.1.2.1 Ms3748 and Ms3747 are membrane associated proteins ...... 18 3.1.2.2 Ms3748 and Ms3747 interact in vivo ...... 19 3.1.2.3 Ms3748 is translocated into the ...... 20 3.1.3 Characterization of rv1697 and rv1698 mutants and their homologs ...... 22 3.1.3.1 Construction of rv1697Δcth, ms3747Δcth and ΔcgR_1476 mutant strains ...... 22 3.1.3.2 Growth and morphological characterization of rv1697Δcth and ms3747Δcth ...... 25 3.1.3.3 Susceptibility of the ms3748Δcth mutant strains to SDS, copper and Malachite green ...... 27 3.1.3.4 Accumulation of Congo red, chenodeoxycholate and ethidium bromide ...... 29 3.1.3.5 sensitivity of rv1697Δcth and ms3748Δcth strains ...... 30 3.1.4 Cell wall analysis of ms3748Δcth and ΔcgR_1476 deletion strains ...... 32 3.1.4.1 Whole cell lipid analysis ...... 32

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3.1.4.2 Membrane vesicles from culture filtrate ...... 33 3.1.4.3 Differences in cell wall sugar content ...... 35 3.2 Screening for copper sensitivity enhancing compounds ...... 36 3.2.1 Assay development and optimization ...... 36 3.2.1.1 Media optimization ...... 36 3.2.1.2 Determination of the optimal copper concentration ...... 37 3.2.2 Assay validation and establishment of key assay parameters ...... 38 3.2.2.1 Effect of neocuproine on M. tuberculosis ...... 38 3.2.2.2. Determination of Z’-factors ...... 39 3.2.3 Limited pilot screen and analysis of hits ...... 40 3.2.3.1 Synergy between copper ions and Bis-dithiosemicarbazones ...... 40 3.2.3.2 Activity of diacetylbis(N(4)-methyl-3-thiosemicarbazone and glyoxalbis(N(4)- methyl-3-thiosemicarbazone against non-growing M. tuberculosis ...... 40 3.2.3.3 Therapeutic index of diacetylbis(N(4)-methyl-3-thiosemicarbazone and glyoxalbis(N(4)-methyl-3-thiosemicarbazone ...... 42 4 Discussion ...... 44 4.1 Rv1697 is an essential core protein of M. tuberculosis required for cell wall integrity ...... 44 4.1.1 Rv1697 of M. tuberculosis and its homologs in M. smegmatis and C. glutamicum have the same function ...... 44 4.1.2 Ms3748 and Ms3747 contribute to cell wall integrity ...... 44 4.1.3 Effects of ms3748Δcth on the mycobacterial cell wall ...... 45 4.1.4 The functions of Rv1697 and Rv1698 are connected ...... 46 4.1.5 Rv1697 is a potential drug target for TB chemotherapy ...... 47 4.1.6 Putative alternative function of Rv1697 ...... 48 4.2 Copper-boosting compounds: a novel concept for anti-mycobacterial drug discovery .....49 4.2.1 Assay development for new anti-TB drugs ...... 49 4.2.2 The therapeutic potential of diacetylbis(N(4)-methyl-3-thiosemicarbazone and glyoxalbis(N(4)-methyl-3-thiosemicarbazone ...... 51 4.2.3 Activity of diacetylbis(N(4)-methyl-3-thiosemicarbazone and glyoxalbis(N(4)-methyl-3- thiosemicarbazone against non-growing M. tuberculosis ...... 52 5 Material and Methods ...... 53 5.1 Material ...... 53 5.1.1 Chemicals and Enzymes ...... 53 5.1.2 Bacterial strains, media and growth conditions ...... 53

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5.2 Media and standard methods ...... 54 5.3 Protein analysis by SDS-PAGE and Western Blot ...... 57 5.4 Methods for nucleic acid isolation, recombination and detection ...... 58 5.4.1 Construction of plasmids ...... 58 5.4.2 Isolation of chromosomal DNA from M. tuberculosis ...... 64 5.4.3 Isolation of chromosomal DNA from M. smegmatis ...... 64 5.4.4 Southern blot analysis ...... 64 5.4.5 Preparation of RNA from M. bovis BCG, M. tuberculosis and RT-PCR Experiments .65 5.5 Construction of deletion mutants ...... 66 5.5.1 Construction of ms3748cth deletion mutant in M. smegmatis SMR5 ...... 66 5.5.2 Construction of rv1697cth deletion mutant M. tuberculosis mc26206 ...... 67 5.5.3 Construction of a cgR_1476 deletion mutant in C. glutamicum...... 68 5.6 Methods to determine susceptibility against drugs and biocides ...... 68 5.6.2 Drug sensitivity assays using microplate Alamar Blue assays ...... 68 5.6.3 Drug screening conditions and multi-dose response curves ...... 69 5.6.4 Activity of compounds against non-growing M. tuberculosis ...... 70 5.6.5 toxicity assays ...... 70 5.6.6 Drop assays and BlaTEM1 reporter assay ...... 70 5.7 Uptake and accumulation assays ...... 71 5.7.1 Uptake of 14C-chenodeoxycholate ...... 71 5.7.2 Uptake of ethidium bromide ...... 71 5.7.3 Accumulation of Congo red ...... 72 5.8 Isolation of subcellular protein fractions, lipids, mAGP and MVs ...... 72 5.8.1 Lipid extraction and analysis ...... 72 5.8.2 Isolation of mAGP ...... 73 5.8.3 Analysis of mAGP by GC-MS ...... 73 5.8.4 Subcellular fractionation of M. smegmatis ...... 74 5.8.5 Isolation, quantification and purification of membrane vesicles ...... 74 5.8.6 Preparation of SUVs ...... 75 5.9 Cross-linking and purification of Ms3748 and Ms3747 ...... 75 5.10 Light microscopy ...... 76 5.11 Transmission electron microscopy (TEM) ...... 76 6 References ...... 77

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7.1 Supplementary results ...... 90 7.2 List of authors that contributed to this work: ...... 92 7.3 Abbreviations ...... 93

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1 Zusammenfassung Es besteht ein dringender Bedarf für neue Anti-Tuberkulose-Medikamente aufgrund der Entstehung von multiresistenten Mycobacterium tuberculosis Stämmen. Die einzigartige Zellwand von M. tuberculosis bietet eine effiziente Permeabilitätsbarriere, die ein essentieller Teil der mycobakteriellen Zelle ist und einen entscheidenden Faktor zu der intrinsischen Antibiotikaresistenz von M. tuberculosis liefert. Daher sind Proteine, die zur Synthese der mykobakteriellen Zellwand benötigt werden wertvolle Drug-Tragets. Das Genprodukt des M. tuberculosis genes rv1697 ist ein Beispiel eines solchen Proteins; es ist annotiert als ein essentielles Protein in M. tuberculosis, das keine Homologe außerhalb der Gattung Corynebacterineae hat. Um die Funktion von Rv1697 zu untersuchen konstruierten wir eine partielle Deletionsmutante sowohl in M. tuberculosis als auch in M. smegmatis. Diese Mutanten produzierten eine verkürzte Version des Proteins ohne der C-terminalen hydrophoben Helix, die wahrscheinlich für eine korrekte subzelluläre Lokalisierung von Rv1697 oder Ms3748 erforderlich ist. In M. tuberculosis und M. smegmatis verursachte die Deletion des C-terminalen hydrophoben Helix einen drastischen Wachstumsdefekt und eine veränderte Koloniemorphologie. Des Weiteren wurde die Permeabilität der M. smegmatis ms3748Δcth Mutante zu Kongorot, Chenodesoxycholat und Ethidiumbromid erhöht. Zusätzlich war die M. tuberculosis Mutante von rv1697Δcth 16-fach anfälliger gegenüber den Antibiotika Ampicillin und Fusidinsäure und acht-fach anfälliger gegenüber . Wir fanden, dass M. smegmatis ms3748Δcth Membranvesikel in den Kulturüberstand freisetzte und Trehalose Mono-mycolate (TMM), die Vorstufe eines Lipids der äußeren Membran, in der Zelle akkumulierten. Die Deletion des rv1697 Homolog in C. glutamicum zeigte, dass der Verlust dieses Proteins zu einer Reduktion des Arabinose/Galactose-Verhältnis relativ zum Wildtyp geführt hat. Diese Phänotypen sind oft mit Zellwanddefekten verbunden. Zusammengenommen weisen diese Ergebnisse darauf hin, dass rv1697 und ms3748 Gene sind die an der Zellwand- Biosynthese von Mykobakterien beteiligt sind und stellen ein neuartiges Drug-Target dar, dessen Inhibierung synergistische Effekte mit den Antibiotika Ampicillin, Fusidinsäure und Rifampicin haben könnte. Wir zeigten, dass rv1697 und rv1698 in einem Operon kodiert sind und dass die Proteine in vivo interagieren. Rv1698 ist benoetigt für Kupferresistenz und Virulenz von M. tuberculosis, was möglicherweise auf Zellwanddefekte zurückzuführen ist. Dies führte zu der Hypothese, dass Kupferresistenzmechanismen bisher unerkannte Drug-Targets von M. tuberculosis darstellen. Daher entwickelten wir ein High-Throughput-Drug-Screening-Assay um Verbindungen zu identifizieren, die gezielt die Kupferresistenz von M. tuberculosis angreifen würden. Während eines anfänglichen Drug-Screenings identifizierten wir mehrere Beispiele von

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Kupfer komplexierenden Verbindungen, die M. tuberculosis in Gegenwart von Kupferionen in physiologischen Konzentrationen wirksam abtöten. Kupferionen verbesserten die anti- mykobakterielle Aktivität von Diacetylbis(N4-methyl-3-thiosemicarbazon) und Glyoxalbis(N4- methyl-3-thiosemicarbazon), während nur leichte toxische Wirkungen in Makrophagen auftraten. Zusätzlich zeigte Glyoxalbis(N4-methyl-3-thiosemicarbazon) Aktivität gegen nicht- replizierende M. tuberculosis Zellen. Diese Verbindungen bieten Beleg, dass Kupferresistenz ein vielversprechendes Drug-Target für M. tuberculosis-Chemotherapie ist.

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1 Summary There is an urgent need for new anti-Tuberculosis drugs due to the emergence of multidrug- resistant Mycobacterium tuberculosis strains. The unique cell wall of M. tuberculosis provides an efficient permeability barrier which is an essential part of the mycobacterial cell and a major determinant of M. tuberculosis’s intrinsic drug resistance. Hence, proteins required for the synthesis of the mycobacterial cell wall are likely to be valuable drug targets. The gene product of M. tuberculosis rv1697 is an example of such protein; it is annotated as an essential protein in M. tuberculosis and does not have homologs outside of the genus Corynebacterineae. To examine the function of Rv1697 we constructed a partial deletion mutant in both M. tuberculosis and M. smegmatis. These mutant strains produce a truncated version of the protein that lacks the C-terminal hydrophobic helix (cth), which is likely required for correct subcellular localization of Rv1697 or Ms3748. In both M. tuberculosis and M. smegmatis, the deletion of the C-terminal helix caused a drastic growth defect and distinct colony morphology. Further, the permeability of the M. smegmatis ms3748Δcth mutant to Congo red, chenodeoxycholate and ethidium bromide was increased. Additionally, the rv1697Δcth mutant of M. tuberculosis was 16-fold more susceptible to the ampicillin and fusidic acid and 8-fold more susceptible to rifampicin. Importantly, we found that M. smegmatis ms3748Δcth released membrane vesicles into the culture supernatant and accumulated trehalose mono-mycolates (TMM), a precursor of an outer membrane lipid. The deletion of the rv1697 homolog in C. glutamicum showed that the loss of this protein resulted in a reduction of arabinose/galactose ratio relative to wild type. These phenotypes are often associated with cell wall defects. Taken together these results suggest that the rv1697 and ms3748 genes are involved in the cell wall biosynthesis of mycobacteria and represent a novel drug target whose inhibition might have synergistic effects with the antibiotics ampicillin, fusidic acid and rifampicin. We demonstrated that rv1697 is encoded with rv1698 in an operon and that the proteins interact in vivo. Rv1698 is required for copper resistance and virulence of M. tuberculosis which might be due to cell wall defects, leading us to hypothesize that copper resistance mechanisms represent previously unrecognized drug targets of M. tuberculosis. Consequently, we developed a high-throughput drug screening assay to identify compounds that would target copper resistance in M. tuberculosis. During an initial drug screening effort, we identified several examples of copper complexing compounds that effectively kill M. tuberculosis in the presence of copper ions at physiological concentrations. Copper ions enhanced the anti-mycobacterial activity of diacetylbis(N(4)-methyl-3-thiosemicarbazone and glyoxalbis(N(4)-methyl-3-thiosemicarbazone while having limited toxic effects in macrophages. Additionally, glyoxalbis(N(4)-methyl-3-

3 thiosemicarbazone showed activity against non-replicating M. tuberculosis cells. These compounds provide proof-of-principle that copper resistance is a promising new drug target for M. tuberculosis chemotherapy.

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2 Introduction

2.1 Clinical relevance of M. tuberculosis

Robert Koch identified Mycobacterium tuberculosis (M. tuberculosis) as the etiological agent of the disease Tuberculosis (TB) in 1882 (Koch 1882). More than one century after its discovery, M. tuberculosis constitutes one of the single largest threat to human health (WorldHealthOrganization 2010). M. tuberculosis infection kills more people than any other bacterial pathogen. In 2010, there were more than 9 million Tuberculosis cases accounting for over 1.4 million deaths (WorldHealthOrganization 2010). Treatment of this disease is protracted and complicated because of the increased appearance of multi-drug resistant Tuberculosis (MDR-TB) during recent years. Delays in TB diagnosis and long and tedious TB treatment protocols with antibiotic drugs (at least 6 months with a combination of 4 drugs) result in low compliance rates, a major contributor to multi-drug resistance (Kant et al. 2010). The TB crisis is further worsened by the rise of extensively-drug resistant (XDR) M. tuberculosis strains, which, in some areas, already approach 30 % of all new TB cases reported (Skrahina et al. 2013). MDR-TB is resistant to two first-line drugs and rifampicin, the two most potent anti-TB drugs. XDR-TB is additionally resistant to any fluoroquinolone and at least one of three injectable second-line drugs (Pontali et al. 2013). Recently, resistant strains were reported that are totally drug resistant (TDR-TB) which raises concerns about future treatment options (Udwadia et al. 2012) (Velayati et al. 2009). New classes of anti-tuberculosis drugs with novel modes of action are urgently needed to prevent further spread of MDR/XDR M. tuberculosis strains. Infection with M. tuberculosis does not lead unavoidably to an outbreak of TB, as the introduced bacilli are capable of persisting within humans for long periods. In this clinically latent state, TB causes no obvious symptoms which makes the diagnosis of this disease difficult (Wayne 1994). Worldwide, there are 2 billion people infected with M. tuberculosis, mostly with the latent, asymptomatic form which can potentially convert into the life threatening acute form of M. tuberculosis infection (Stewart et al. 2003). Additionally, the mortality among HIV-1/M. tuberculosis co-infected individuals is extremely high. The HIV-1 and TB syndemics are driving the global expansion of TB and drug resistant strains (Koenig 2008). The World Health Organization (WHO) estimates that approximately 350,000 TB related deaths are associated with HIV-1 worldwide (WorldHealthOrganization 2010).

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2.2 The mycobacterial cell envelope

2.2.1 Structure of the cell envelope of mycobacteria

Mycobacteria are classified as Gram-positive bacteria based on 16S ribosomal RNA sequence comparison, but are more closely related to Gram-negative bacteria when comparing the whole genome (Fu et al. 2002). The cell wall of Gram-positive bacteria is composed of an inner membrane followed by a thick layer of and a capsule as the outer most layer. The discovery of an outer membrane in mycobacteria revealed that the cell wall organization is more reminiscent of Gram-negative bacteria whose thinner peptidoglycan layer is followed by an outer membrane composed of an asymmetric lipid bilayer (Minnikin 1982) (Hoffmann et al. 2008) (Zuber et al. 2008). However, the structure in detail contains many unique features only found in Corynebacterineae. The mycobacterial envelope consists of two major components: a typical plasma membrane surrounded by a cell wall core which includes the inner leaflet of the outer membrane. The cell wall core is composed of peptidoglycan covalently linked to an polymer that is again covalently linked to mycolic acids (Fig. 2.1). This macromolecule is also referred to as mycolated arabinogalactan peptidoglycan polymer (mAGP). The peptidoglycan in mycobacteria follows the structure of the A1γ class, which is the most common type of peptidoglycan in bacteria. The glycan chains of group A1γ are cross-linked by a tetra peptide consisting of L-alanyl-D-isoglutaminyl-meso-diaminopimelyl-d-alanine (Schleifer et al. 1972). In this peptide linker two meso- units are either cross-linked by an alanine or directly attached to each other by a peptide bond. The glycan chain consists of alternating subunits of N-acetylglucosamine (GlcNAc) and a modified muramic acid whose N- acetyl is further oxidized to N-glycolyl (Wietzerbin et al. 1974). The peptidoglycan is covalently linked to the C-6 atom of the muramic acid residues via a rhamnose linker and a phosphodiester (α-L-Rhap-(13)-D-GlcNAc-(1P)) to the galactan chain of the arabinogalactan polymer (McNeil et al. 1990). The galactan chain is made up of 30 Galf units

linked as a repeating disaccharide units [6-D-Galfβ15-D-Galfβ]15 (Daffé et al. 1990). At positions 8, 10 and 12 of the galactan chain three arabinan subunits are attached to the O-5 of the Galf residue and each unit contains 31 Araf. Most of the arabinan chain is linked by 5-linked α-D-Araf with a 3.5-α-D-Araf branching points at residues 14 and 18. Residues 17 and 18 are

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Figure 2.1: The cell envelope structure of M. tuberculosis. In this model mycolic acids span the entire outer membrane (OM). Extractable lipids are shown in both leaflets and include trehalose mono- and di- mycolates next to PIMs and acyltrehaloses. Lipids of the plasma membrane (PM) consist of phospholipids and PIMs. PG, peptidoglycan; AG, arabinogalactan. The Galf and Araf residues of AG are represented in blue and red, respectively. The succinyl and galactosamine residues of AG are in green. Manp residues in PIM, LM and LAM are in red; Araf residues are purple. Reproduced with permission (Kaur et al. 2009).

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part of the terminal Ara6 motif (Arafβ12Araf α 15(Arafβ12Araf α 13)-Araf α 15Araf α 1) leading to eight Araf units with exposed non-reducing termini (Daffé et al. 1990). Two-thirds of the Ara6 units are covalently linked over an ester bond at position C-5 of the last and the penultimate Araf unit to mycolic acids (McNeil et al. 1991). The mycolic acids are oriented parallel to each other and orthogonal to the plane of the envelope and build the inner leaflet of the asymmetric mycobacterial outer membrane. The outer leaflet is composed of extractable OM lipids that intercalate into the mycolic acid layer (Fig. 2.1).

2.2.2 Cell wall biosynthesis

The synthesis of mAGP is spatially separated into two compartments of the cell. It starts in the cytosol with the formation of the linker unit between the peptidoglycan and arabinogalactan. The petidoglycan building block GlcNAc-1-phosphate is transferred from UDP-GlcNAc to the carrier lipid decaprenyl diphosphate. Decaprenyl diphosphate (Dec-P) is synthesized from dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (Crick et al. 2001). A rhamnose from dTDP- Rha is added to the Dec-P-GlcNAc unit to build the linker unit Dec-P-P-GlcNAc-Rha. Subsequent residues of galactose in the furanose conformation (Galf) are added to this linker unit by cytosolic galactosyltransferases, whereby UDP-Galf units serve as the Galf donor (Kaur et al. 2009). In the periplasm, the arabinan is attached to the galactan. Furanosyl arabinose (Araf), which originates from the pentose phosphate pathway, gets linked to Dec-P to form the donor molecule decaprenylphosphoryl-Araf (DPA) (Crick et al. 2001). The carrier lipid Dec-P attached to DPA and Dec-P-P-GlcNAc-Rha-Galf allows the attachment of these units to the inner membrane and they are flipped over into the periplasmic space. A small multidrug resistance- like protein in M. tuberculosis was shown to facilitate this translocation of DPA (Larrouy- Maumus et al. 2012). In the periplasmic space several transmembrane arabinosyltranferases (AraTs) are required to build the complex structure of arabinan, each responsible for the polymerization of another linkage type (Alderwick et al. 2006) (Seidel et al. 2007) (Birch et al. 2008) (Skovierova et al. 2009). Mycolic acids are covalently linked to the non-reducing termini of the arabinan. The fatty acids chains of mycolates are synthesized by the synthase I&II complex in the cytosol and condensed by the Pks13 (Takayama et al. 2005). One mycolate molecule is transferred to trehalose to build trehalose-mono-mycolate (TMM). TMM is then translocated by the inner membrane transporter MmpL3 into the periplasmic space. In the

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periplasm, the multi enzyme complex Ag85 cleaves mycolate from TMM and transfers it to the non-reducing end of arabinose molecules (Grzegorzewicz et al. 2012) (Tahlan et al. 2012) (Belisle et al. 1997). Ag85 complex, formed by the proteins FbpA, FbpB and FbpC, is also able to condense two TMMs to one trehalose di-mycolate (TDM) molecule by cleavage of one trehalose molecule. The free trehalose is transported back into the cytosol by the inner membrane sugar transporter LpqY (Kalscheuer et al. 2010).

2.2.3 Mycobacterial lipids and lipoglycans

The mAGP is an insoluble macromolecule of the cell envelope which is interspersed with a variety of unique lipids and lipoglycans that contribute to cell wall integrity, pathogenesis and immune response (Crick et al. 2001). Trehalose di-mycolate (TDM) is an abundant OM lipid in mycobacteria and intercalates into the inner leaflet (Fig. 2.1). Mycolic acids have a remarkable length, up to 90 (M. tuberculosis) and 100 (Segniliparus) carbon atoms, and are the longest lipids known in nature (Hong et al. 2012) (Takayama et al. 2005). These lipids include modifications such as cyclopropane rings, , methoxy groups and double bonds (Hong et al. 2012) (Takayama et al. 2005). TDMs of M. tuberculosis have been shown in the murine model to be lethal if injected, and to arrest phagosome maturation in macrophages (Brennan 2003) (Indrigo et al. 2003). In addition, it has been proposed that macrophages have the ability to recognize M. tuberculosis infection by binding TDM via the C-type lectin receptor Mincle (Ishikawa et al. 2009). Phthiocerol dimycocerosates (PDIMs) are very hydrophobic branched chain lipids that are dispensable for in vitro growth but are important for virulence and cell wall permeability (Astarie-Dequeker et al. 2009) (Camacho et al. 2001). Another class of extractable lipids in mycobacteria are the lipoglycans. The cell envelope of mycobacterial species contains mannosylated glycans that are anchored to the inner and outer membrane by the phospholipid phosphatidylinositol. The most abundant lipoglycans are mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM) (Kaur et al. 2009). ManLAM modulates key signaling molecules in macrophages and contributes to virulence of M. tuberculosis (Rojas et al. 2000). LAM of M. tuberculosis was shown to bind toll receptors of macrophages which subsequentially blocks bactericidal responses.

2.2.4 Proteins of the mycobacterial outer membrane

The outer membrane of M. tuberculosis provides an efficient permeability barrier that protects the bacterium from antibiotics and bactericidal molecules encountered within the macrophage

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during the course of infection (Brennan et al. 1995) (Jarlier et al. 1990). At the same time this barrier has to enable uptake of small, hydrophilic nutrients such as iron, sulfate, phosphate and carbon sources. While hydrophobic compounds are taken up by passive diffusion through the lipid bilayer, the uptake of small, soluble nutrient molecules is accomplished by transmembrane water filled channels called porins (Wolschendorf et al. 2007) (Song et al. 2012) (Stephan et al. 2005) (Jones et al. 2010). The existence of channel-forming proteins in cell wall extracts of M. tuberculosis (Kartmann et al. 1999) and M. bovis BCG (Lichtinger et al. 1999) has been demonstrated, but these studies did not identify the proteins that are responsible for the channel activity. The first porin of mycobacteria (MspA) was found in the fast- growing non-virulent strain M. smegmatis (Niederweis et al. 1999). The crystal structure of this outer membrane protein revealed that MspA differs significantly from porins found in Gram-negative bacteria. Instead of an oligomer with each monomer providing one channel, in MspA eight monomers build one central channel (Faller et al. 2004). The loss of MspA and its paralogs MspC and MspD lead to increased resistance to ampicillin and cephaloridine (Danilchanka et al. 2008). Conversely, overexpression of mspA increased drug susceptibility of M. bovis BCG (Mailaender et al. 2004). However, an outer membrane channel protein in slow growing mycobacteria with a role in uptake has yet to be identified.

2.3 Pathogenesis

2.3.1 Innate immune system during bacterial infection

After inhalation into the lung, a bacterium gets engulfed (phagocytosed) by alveolar macrophages. The bacterium is trapped in a vacuole (phagosome) that consists of the macrophage plasma membrane. The phagosome matures over many steps to a hostile compartment in order to kill and degrade the bacterium, and present bacterial antigens on the surface of the macrophage (Flannagan et al. 2009); a key element of the adaptive immune response. In the early phagosome, the pathogen encounters highly reactive oxygen (ROS) and nitrogen species (NOS), generated by the NADH oxidase complex and nitric oxide synthase, respectively, at the phagosomal membrane (El-Benna et al. 2009). Furthermore, the macrophage functionalizes the phagosomal membrane by insertion of transporters that actively remove nutrients from the phagosomal lumen such as the divalent Fe2+ and Mn2+ ions (Vidal et al. 1993) (Forbes et al. 2001). Other ATPases localized to the phagosomal membrane are meant to increase the concentration of H+, Cu2+ and Zn2+ to convert the phagosome into an antibacterial environment (Lukacs et al. 1990). V-type ATPases decrease the pH of the

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phagosome from 7.0 to 4.5 which not only inhibits bacterial growth, but at the same time is the optimal pH for protease and lipase activity, which compromise bacterial membrane integrity (Flannagan et al. 2009). The heavy metals Cu2+ and Zn2+ transported into the phagosome have antimicrobial activity (see 2.4.2) and, among other mechanisms, generate ROS inside the phagosome and the bacteria following a Fenton-like reaction (White et al. 2009) (Botella et al. 2011) (Koppenol 2001). These processes are part of the maturation of the phagosome into the phagolysosome and results in the clearance of the engulfed bacterium.

2.3.2 M. tuberculosis adaptation to an intracellular lifestyle

M. tuberculosis belongs to the group of intracellular pathogens that possess the ability to survive and replicate within macrophages. M. tuberculosis employs an array of effector molecules to arrest phagosomal maturation. The secreted phosphatase PtpA was shown to interfere with the fusion event of phagosomes with vacuoles that contain H+ V-type ATPases. This prevents the presence of such ATPases on the surface of M. tuberculosis-containing phagosomes and therefore the pH of the phagosome remains near pH 6.5 (Wong et al. 2011). Instead of effectively killing a phagocytosed organism, an infected macrophage can induce a controlled cell death (apoptosis) that leaves the macrophage’s membranes intact (Hilbi et al. 1997). As a result, the M. tuberculosis surrounded by apoptotic bodies are engulfed by dendritic cells that deliver M. tuberculosis into the phagolysosome where both organisms get hydrolyzed and antigens of M. tuberculosis are then presented on the dendritic cell surface. M. tuberculosis inhibits apoptosis of the macrophage by, among other mechanisms, release of ManLAM into the extracellular space which prevents the influx of Ca2+ that is required for M. tuberculosis-induced apoptosis (Rojas et al. 2000). However, the infection of macrophages with M. tuberculosis causes the release of the cytokine IL-12 that stimulates T-cells to produce the protein interferon gamma (IFN-γ) that again, when taken up by macrophages, can override the inhibitory effects of M. tuberculosis infection, leading to clearance of M. tuberculosis (Cooper et al. 1995) (Flynn et al. 1993). Another mechanism M. tuberculosis uses to circumvent neutralization by the immune system has recently been proposed. M. tuberculosis was shown to possess the ability to escape the phagosome and enter the cytosol of the macrophage, from which M. tuberculosis escapes via necrosis (van der Wel et al. 2007) (Simeone et al. 2012).

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2.4 Dual role of copper in bacteria

Copper plays a dual function in the pathogenesis of M. tuberculosis. On the one hand copper is an essential trace element required for survival by all organisms from bacteria to humans. The wide range of tunable redox potentials of coordinated copper makes it an essential cofactor in enzymes used for electron transfer reactions in the presence of oxygen (Pena et al. 1999). On the other hand, copper ions are highly toxic and show antimicrobial properties employing several mechanisms. The ability of copper to change its redox state between the Cu+ and Cu2+ catalyzes the production of hydroxyl radicals via the Fenton and Haber-Weiss reactions under aerobic conditions (Koppenol 2001). The generated hydroxyl radicals can react with DNA, lipids and proteins resulting in irreparable damage (Halliwell et al. 1984) (Lee et al. 2002) (Zhu et al. 2002). However, it was shown in E. coli that under anaerobic conditions the toxic nature of copper is mainly due to reduction of Fe-S clusters in proteins (Macomber et al. 2007). Copper overload can also lead to the replacement of other metal ions with copper in metalloenzymes which results in non-functional proteins (Rowland et al. 2012).

2.5 Role of copper during M. tuberculosis infection

The investigation of copper’s role during the course of M. tuberculosis infections received more attention after it was shown that macrophages employ the antimicrobial properties of copper to fight off M. tuberculosis (Fig 2.2) (Wolschendorf et al. 2011). Using X-ray fluorescence microscopy, it was shown that M. tuberculosis containing phagosomes of macrophages show an IFN-γ dependent increased in copper (Wagner et al. 2005). Later it was shown that granulomatous lung tissue of M. tuberculosis infected guinea pigs have elevated amounts of copper compared to uninfected tissue samples (Wolschendorf et al. 2011). Using E. coli as a model it was demonstrated that a eukaryotic copper-transporting ATPase (ATP7A) translocated to the phagosome and to increase the phagosomal copper concentration of macrophages as response to an infection (Fig. 2.2) (White et al. 2009). Loss of ATP7A by RNAi- mediated knockdown resulted in decreased killing of phagocytosed E. coli (White et al. 2009). In M. tuberculosis transcriptional analysis identified two copper-responsive regulators: CsoR and RicR (Liu et al. 2007) (Festa et al. 2011). Expression of the gene ctpV, encoding an inner membrane transporter, is regulated by CsoR and was thought to be involved in copper homeostasis by transporting excess copper from the cytosol into the periplasmic space (Ward et al. 2010). CsoR is encoded in the same operon as ctpV, along with two additional genes of unknown function (Liu et al. 2007). The gene rv0846c is part of the RicR regulon and shows

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homology to the multicopperoxidase CueO of E. coli, which was shown to be critical for copper resistance by converting the Cu+ into the less toxic Cu2+ ions in the periplasm (Rowland et al. 2012). The gene rv1698 is not organized in a copper responsive regulon but the deletion of rv1698 caused a copper sensitive phenotype and decreased the bacterial burden in guinea pig infection experiments (Wolschendorf et al. 2011). Additionally, the loss of Rv1698 resulted in accumulation of copper in M. tuberculosis. However, the localization and the mechanisms how Rv1698 contributes to copper resistance remains unknown (Rowland et al. 2012). Nevertheless, the virulence defect of copper-sensitive M. tuberculosis mutants has illustrated the importance of copper during the course of infections and that copper is exploited as a natural defense mechanism of macrophages to control M. tuberculosis (Fig. 2.2).

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Figure 2.2: Copper homeostasis in macrophages. A. In resting macrophages CTR1 imports Cu+ which is bound by the chaperone ATOX1. ATP7A is localized to the Golgi Network to deliver Cu to copper- requiring proteins. Under copper stress ATP7A is trafficked to the plasma membrane to export excess copper into the extracellular space. B. After M. tuberculosis is taken up by phagocytosis into the macrophage, M. tuberculosis arrests the maturation of the phagosome. C. The phagosomal arrest is abolished upon activation of the infected macrophage with IFN-γ and the pH is reduced to pH 4.5. The expression of CTR1 and ATP7A increases and ATP7A is translocated to the phagolysosome. Reproduced with permission (Rowland et al. 2012).

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2.6 Tuberculosis drug development

The long treatment times of TB are one of the factors for the increasing occurrence of MDR strains. In addition 11-13 % of the worldwide TB patients are co-infected with HIV, which complicates treatments due to drug-drug interactions or overlapping toxic side effects which reduces the number of drugs that can be administrated (Koul et al. 2011). The increased daily pill burden of combined HIV/TB treatment reduces patient compliance and therefore builds an ideal environment for development of MDR-TB strains. The treatment of MDR-TB cases requires chemotherapy with eight to ten antibiotics over a period of 18-24 months (Koul et al. 2011). Drug resistance in mycobacteria is created and passed on by single nucleotide polymorphisms (SNP) in the chromosomal genome. There are no plasmid- or transposon-based horizontally transferred resistances to antibiotics described (Zhang et al. 2009). Some of these SNPs reduce bacterial fitness and are only maintained in a bacterial population as long as the selective pressure of the antibiotic maintains. Other resistances caused by SNPs, such as the mutations in the rpsL gene that confer high resistance to streptomycin, have no impact on bacterial fitness and remain stable in the genome even though the selective pressure has been absent for several decades (Bottger et al. 2008). Another mechanism of drug resistance is induced by hypoxia and starvation of M. tuberculosis during the course of infection. Under these conditions M. tuberculosis has the ability to change from an actively proliferating state into a persistent state, a condition characterized by low metabolic activity that causes resistance towards many antibiotics that target active processes (Franzblau et al. 2012). antibiotics were developed in the 1960s and approved by the FDA for TB treatment. However, no new TB drug was discovered in the 50 years after. Only recently, bedaquillin received FDA approval for treatment of MDR-TB cases (Cohen 2013). However, the FDA ordered a black box warning due to serious side effects such as irregular heart rhythms that can result in cardiac arrest. In fact, clinical trials demonstrated a 4.5-fold higher mortality in the bedaquillin treated group compared to the control group (Cohen 2013). In addition, the question whether bedaquillin can be combined with current HIV medications has yet to be addressed. This example and the fact that only 10 % of all drug candidates pass clinical trials with success illustrates the urgent need for new TB drugs (Robert Giffin 2009). Classical antimicrobial drugs target essential proteins whose inhibition leads to death or growth inhibition of the bacterium. Ideally, these targets are absent or have very low similarity in

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humans and other bacteria to ensure specific inhibition. In light of this, a pivotal study has identified essential genes in M. tuberculosis by the inability to generate transposon mutants of those genes (Sassetti et al. 2003). The bacterial cell wall is targeted by many antibiotics due to its indispensable nature to virtually all microorganisms. The targets range from inhibition of proteins in the cytosol (Makarov et al. 2009) or periplasm involved in lipid synthesis (Quemard et al. 1991) and lipid localization (Tahlan et al. 2012) or assembly of cell wall carbohydrates (Noguchi et al. 1978). The mycobacterial cell wall is the target of several TB drugs: the first line drugs INH (Banerjee et al. 1994) and EMB (Takayama et al. 1989), and several compounds in the TB clinical pipeline (Koul et al. 2011) (BTZ043, SQ109, AU1235, PA-824) (Makarov et al. 2009) (Tahlan et al. 2012) (Grzegorzewicz et al. 2012) (Stover et al. 2000). Ideally, new TB drugs should shorten treatment times, show no cross-resistance to MDR strains, allow compatibility with HIV treatment and target persistent M. tuberculosis.

2.7 Goal of this thesis

This work focused on two main goals. The first goal was to characterize the function of the gene rv1697 and its homologs in mycobacteria and corynebacteria. Furthermore, the potential for Rv1697 as a novel drug target was elucidated (chapter 3.1). The proteins Rv1697 and Rv1698 are encoded in an operon and interact in vivo. Previous studies have demonstrated that the deletion of rv1698 causes copper sensitivity and reduces the bacterial burden of M. tuberculosis during infection. The finding that copper resistance mechanisms are important for virulence of M. tuberculosis lead to the hypothesis that the susceptibility of M. tuberculosis to copper could be exploited by drugs. Hence, the second goal was the development of a drug screen to find copper sensitivity inducing drugs in M. tuberculosis (chapter 3.2).

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3 Results

3.1 Characterization of the rv1697-rv1698 operon in M. tuberculosis

3.1.1 Rv1697 and rv1698 are encoded in one operon

Rv1698 is required for copper resistance in vitro and virulence of M. tuberculosis in guinea pigs (Wolschendorf et al. 2011). To further elucidate the physiological functions of Rv1698, we investigated its conservation and genetic context among mycobacteria. The gene rv1697 is located directly upstream of rv1698 and these genes are predicted to be in an operon (Caspi et al. 2012). These proteins and the organization of the corresponding Figure 3.1: Reverse transcriptase PCR: The mRNA of M.bovis BCG and M. tuberculosis were genes are highly conserved among mycolic acid isolated and converted into cDNA where indicated containing bacteria (Table 3.1). To validate the (+RT). Primers 888 and 1075 binding in rv1697 and rv1698 were used for subsequential PCR operon prediction, mRNA of M. tuberculosis and M. amplification. Genomic DNA of M. tuberculosis was used as a positive control for the PCR. bovis BCG was isolated, reverse transcribed into cDNA and amplified by PCR with primers specific for the amplification of the intergenic region between rv1697 and rv1698. No DNA was amplified in the absence of reverse transcriptase confirming the absence of genomic DNA in the mRNA preparation. The amplification of a single DNA fragment from cDNA spanning rv1697 and rv1698 confirmed the organization of these genes in a transcriptional unit (Fig. 3.1).

Figure 3.2: Gene annotations and chromosomal organization of the rv1697-rv1698 operon in M. tuberculosis and its homologs in M. smegmatis and C. glutamicum. Homologous genes are highlighted with the same color. Sequence data and annotations were obtained from www.tigr.org. Genes are drawn to scale.

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Table 3.1: Sequence homology comparison of Rv1697 and Rv1698 in Corynebacterineae.

organism strain homologs of Rv1697 homologs of Rv1698 similarity identity similarity identity M.tuberculosis H37Rv 100 100 100 100 M. bovis BCG Pasteur 1173P2 100 100 100 100 M.marinum ATCC BAA-535 / 97.5 94.4 88 80.4 M M. leprae TN 94.9 90.6 83.6 77 M. avium strain 104 94.4 89.8 86.1 75.7 M.smegmatis ATCC 700084 / 88.4 77.9 75.5 62.1 mc(2)155 R. equi 103S 80.5 67.3 64.8 46.9 C. glutamicum R 60.1 41.2 47.5 30.1 Protein sequences were taken from www.uniprot.org, homology was calculated by pairwise sequence alignment using the EMBOSS Stretcher algorithm (EMBL-EBI). Similarity and identity is given in percent.

3.1.2 Subcellular localization of Rv1697 and Rv1698

3.1.2.1 Ms3748 and Ms3747 are membrane associated proteins

The protein Rv1697 and its homologs are predicted to contain a hydrophobic transmembrane C- terminal helix from residue I344 to V363 (Sonnhammer et al. 1998). In addition to this

Figure 3.3: (A) Prediction of transmembrane helices of Rv1697: Hydrophobic residues are black, polar residues are blue (-) and red (+). Model was generated by the SOSUI system (Hirokawa et al. 1998). (B) Subcellular localization of Ms3747 and Ms3748. WT M. smegmatis (SMR5) was lysed and water soluble and insoluble proteins were separated by high speed centrifugation. Proteins from each fraction were separated by SDS-PAGE and analyzed by Western blot. Ms3748 was detected by probing with antiserum against Rv1697. Ms3747 was detected by probing with monoclonal antibody against Rv1698. MspA and RNApol served as marker protein of the membrane and soluble proteins, respectively. 18

hydrophobic helix, a second hydrophilic transmembrane helix is predicted from residue G371 to S393. The interaction of these two helices is likely stabilized by hydrophilic interaction of polar residues in the center of the helices, calculated by a secondary structure prediction system

(SOSUI) (Fig. 3.3 A) (Hirokawa et al. 1998). The occurrence of the C-terminal double helices causes the N-terminus and the C-terminus of Rv1697 to be located on the same side of the membrane and build a C-terminal double helix (cth). In order examine whether Rv1697 is indeed associated with membranes, we performed subcellular fractionation. M. smegmatis cells were lysed by sonication and the water insoluble membrane particles were separated by centrifugation at 100,000 x g for 1 h (5.8.3). As a control for sufficient separation, the fractions

were probed for RNA polymerase, as a marker of soluble proteins, and for two markers for insoluble proteins, MspA (Niederweis et al. 1999) and Rv1698 (Wolschendorf et al. 2011) (Fig 3.3 B).Cross contamination of either fraction was very low. Western blot analysis using antibodies raised against Rv1697 detected Ms3748 in the insoluble fraction of the cell lysate, demonstrating that the protein Ms3748 is membrane associated.

3.1.2.2 Ms3748 and Ms3747 interact in vivo

The organization of rv1697 and rv1698 in an operon raised the question whether the encoded proteins are interaction partners. To address this question a pull down assay in combination with in vivo cross-linking experiments were performed. A 6xHis-tag was fused in frame C- terminal to Ms3748 and cloned with ms3747 into an operon. The proteins Ms3748His and Ms3747 were expressed episomally in the ms3748Δcth mutant (ML199, 3.2.2.1). Complementation assays showed that the His-tagged Ms3748 does not interfere with the function of the protein (Fig 3.4 A). Cells were lysed and the protein Ms3748His was purified by nickel-affinity chromatography. Western blot analysis demonstrated that Ms3747 was co-purified with Ms3748His suggesting the proteins physically interact. To ensure that the proteins interact also in vivo the Ms3748-Ms3747 protein complex was stabilized in whole cells by in vivo cross- linking with formaldehyde (5.9). Formaldehyde is known to penetrate the rapidly and to generate non-specific covalent bonds between residues of proteins which are in close proximity, approximately 2 Å (Zeng et al. 2006). The cells were lysed after cross-linking and the protein complexes were solubilized and isolated by affinity chromatography via the His-tagged Ms3748. To visualize generated protein complexes the samples were incubated with loading buffer at 37 °C to retain the formed cross-links or boiled to cleave the introduced bonds before separating the purified proteins by SDS-PAGE. Specific proteins were detected by immunoblotting with antibodies against Rv1697 and Rv1698. Ms3748 separated by SDS-PAGE

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Figure 3.4: (A) SDS susceptibility drop assay: A transcriptional fusion of ms3748 and ms3747 (pML2754) or ms3748His and ms3747 (pML2755) was expressed in the M. smegmatis strain ML199 (ms3748Δcth). SDS susceptibility was compared to ML199 containing an empty vector (pMS2) or WT (SMR5 pMS2). (B) Interaction of Ms3748 and Ms3747: Where indicated whole cells were cross-linked with 1% formaldehyde (CH2O). Ms3748His was purified under denaturing conditions by HPLC Ni2+-affinity chromatography. The fractions containing the majority of Ms3748His were boiled at 95 °C or incubated at 37 °C in loading buffer and separated by SDS-PAGE. Western blot analysis detected Ms3748His with antiserum against Rv1697 and Ms3747 was detected by probing with monoclonal Rv1698 antibody. has an electrophoretic mobility that correlates to its monomeric size of 42 kDa. A band of approximately 100 kDa can be detected that likely presents a dimer of Ms3748. After treating the M. smegmatis cells with formaldehyde the monomeric form of Ms3748 was reduced due to the formation of cross-linked Ms3748. The newly formed Ms3748 protein complexes show an electrophoretic shift towards the high molecular weight section of the blot. After cleaving the cross-links by heat the high molecular weight bands of Ms3748 disappeared and the amount of monomeric Ms3748 and Ms3747 increased. These data show that the physically interaction between Ms3748 and Ms3747 was stabilized during the purification process. This resulted in a higher amount of co-purified Ms3747 after cleavage of the formaldehyde induced cross-links (Fig 3.4 B). Furthermore, the cross-links between Ms3748 and Ms3747 were formed after treating intact cells with formaldehyde showing that this interaction takes place in vivo.

3.1.2.3 Ms3748 is translocated into the periplasm

The sequences of Rv1698 (M. tuberculosis) and its homologs contain a predicted Sec signal sequence. Hence, the proteins are transported across the inner membrane (Siroy et al., manuscript in preparation). By contrast, the sequences of Rv1697 or Ms3748 and its homologs do not contain any recognizable signal sequence. The interaction between Ms3748 and Ms3747 give rise to the question if Ms3748 is also translocated across the inner membrane. To

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determine whether Ms3748 is localized to the periplasm, a beta- lactamase reporter assay was performed (McCann et al. 2007). The M. smegmatis strain PM759 (Flores et al. 2005) lacks the native beta-lactamase gene

(blaS1), resulting in a strain

Figure 3.5: Beta-lactamase export assay: BlaTEM1 and fusion sensitive to beta-lactams. In this proteins of Ms3747 and Ms3748 with ‘BlaTEM1 were expressed in the M. assay the natural resistance smegmatis strains PM759 (ΔblaS1) and grown on 7H10 plates containing no and 75 μg/mL carbenicillin. As a negative control the plasmid towards beta-lactams can only be pML2167 expresses BlaTEM1 without signal sequence (‘blaTEM1). Full restored if the beta-lactamase length blaTEM1 is expressed from plasmid pML2165. The plasmid pML2755 encodes an N-terminal translational fusion of ‘BlaTEM1 with BlaTEM1 is translocated to the Ms3748 transcriptionally fused with ms3747 (’blaTEM1-ms3748, ms3747). Plasmid pML2741 encodes Ms3748 fused on the C-terminus to periplasm. As controls the strain ‘BlaTEM1 (ms3748-’blaTEM1). A C-terminal fusion of Ms3748 with PM759 was transformed with ‘BlaTEM1 transcriptionally fused with ms3747 is encoded on plasmid pML2757 (ms3748-’blaTEM1, ms3747). Ms3747 is fused on the C- gene expression constructs terminus to ‘BlaTEM1, encoded in pML1948 (ms3747-’blaTEM1). encoding blaTEM1 with (blaTEM1) and without (‘blaTEM1) signal sequence. Changes in beta-lactam resistance of M. smegmatis PM759 strains were determined by resistance to carbenicillin on agar plates. In order to examine whether a protein is translocated into the periplasm leaderless BlaTEM1 was translationally fused to Ms3748 or Ms3747. An Ms3747-BlaTEM1 fusion protein fully restored resistance to carbenicillin (Fig 3.5). The protein Ms3748 was fused C-terminally or N-terminally in frame to the leaderless sequence of the beta-lactamase BlaTEM1 (Tab 5.6) and expressed in the beta-lactam sensitive M. smegmatis strain PM759. The C-terminal fusion of Ms3748 to BlaTEM1 did confer low resistance towards carbenicillin. However, the expression of the same fusion protein in an operon with ms3747 increased resistance and allowed for growth on beta- lactam containing plates. These results show that Ms3748 is translocated into the periplasmic compartment. Interestingly, an N-terminal fusion of Ms3748 with BlaTEM1 encoded in an operon with ms3748 did not allow translocation of Ms3748 into the periplasm. These data suggests that the signal for the translocation of Ms3748 is located at the N-terminus. Taking into account the C-terminal double helix of Rv1697 and its homologs we suggest that both the C- terminus and the N-terminus are located together with the majority of the protein in the periplasm.

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3.1.3 Characterization of rv1697 and rv1698 mutants and their homologs

3.1.3.1 Construction of rv1697Δcth, ms3747Δcth and ΔcgR_1476 mutant strains

In order to investigate the function of Rv1697 and its homologs in mycobacteria we constructed a deletion mutant of ms3748 in M. smegmatis. We used M. smegmatis because of its relatively short doubling time (3 h) compared to species of the M. tuberculosis complex (24 h), allowing testing of several deletion strategies and characterization of the mutant strains in a relatively short time. Several attempts to delete large parts of ms3748 (residues G37-V367) in frame or the ms3748-ms3747 operon were unsuccessful and did not result in a double-crossover event. Hence, to examine the function of ms3748 we utilized a different strategy. Only the part of the gene encoding the C-terminal membrane helix was deleted. The primary hydrophobic helix was removed out of frame so that there is a stop codon after residue I344 resulting in a truncated version of ms3748. This deletion strategy yielded a double-crossover in M. smegmatis (designated ML199). Using the same approach as for the deletion of ms3748Δcth in M. smegmatis, an rv1697Δcth mutant was constructed in M. tuberculosis avirulent strain mc26206 (designated ML910). The genotypes in both mutants were confirmed by southern blot analysis (Fig 3.6 B, 3.7 D). We characterized expression of rv1697Δcth or ms3748Δcth and rv1698 or ms3747 in the deletion mutants. Use of the same gene deletion strategy in both M. tuberculosis and M. smegmatis resulted in differing effects on the expression levels of the truncated rv1697Δcth or ms3748Δcth gene and the downstream gene rv1698 or ms3747. Western blot analysis with antibodies raised against Rv1697 confirmed the presence of the truncated Rv1697Δcth protein in M. tuberculosis. The expression level of Rv1698, which is the operonic partner of rv1697 and encoded in the gene immediate downstream of rv1697, was not affected by this (Fig 3.6 C). However, Western blot analysis of the M. smegmatis mutant showed a much reduced expression level of the truncated protein Ms3748Δcth compared to wild type (Fig 3.7 B, C) and the Ms3747 protein was not detected by Western blot. Deletion of the C-terminal domain of Ms3748 resulted in a phenotypic ms3748Δcth / Δms3747 double mutant of M. smegmatis (designated ML199). After replacing the ms3748cth with a loxP site, polar effects might prevent transcription of the downstream gene ms3747. The reason why this phenomenon does not occur in the M. tuberculosis rv1697Δcth deletion strain is unclear. Nevertheless, the generation of a phenotypic ms3748Δcth / Δms3747 double mutant (ML199) allows for the opportunity to investigate both proteins in the same background by complementation with either ms3748 (pML951) or ms3747 (pML451) resulting in the

22 corresponding phenotypic single mutant or was fully complemented by expression of the entire ms3748-ms3747 operon (pML2654) (Fig 3.7 B). In contrast to mycobacteria, it was possible to delete the complete gene of the rv1697 homolog in C. glutamicum (cgR_1476). The deletion was carried out in frame so that six nucleotides of the gene remained at the 5’ end and 12 at the 3’ end and complemented with the rv1697-rv1698 operon under control of the native C. glutamicum promoter region (pML3111) (Fig. 3.8 A).

Figure 3.6: Construction of rv1697Δcth mutant. (A) Schematic representation of the chromosomal rv1697- rv1698 region of M. tuberculosis. The 69 bp encoding for the c-terminal hydrophobic helix of rv1697 were replaced by the 45 bp loxP site that introduces a stop codon into rv1697. Genes are drawn to scale. The probe used for southern blot analysis is indicated. A double-cross-over was obtained after counter selection (ML910) on sucrose, introducing a gfpm2+-hygromycin cassette flanked by two loxP sites. The loxP flanked gfpm2+-hygromycin cassette of ML910 was removed using the thermosensitive vector pML2714 expressing Cre-recombinase to yield the unmarked mutant ML911. (B) Southern blot analysis. Chromosomal DNA of M. tuberculosis WT (mc26206) and rv1697Δcth mutant (ML910) were digested with PacI and XmaI. After separating the DNA by gel electrophoresis the DNA was transferred to a membrane and a labeled DNA probe as indicated in A was used for detection of the complementary DNA. (C) Expression of Rv1697 and Rv1698 in M. tuberculosis: M. tuberculosis mc26206 (WT), ML910 (rv1697Δcth) and ML912 (rv1697Δcth + rv1697) were grown in 7H9, OADC, Tyloxapol and lysed by sonication. The whole cell lysate was separated by SDS-PAGE and presence of Rv1697 and Rv1698 was detected by Western blot analysis using polyclonal antibodies raised against Rv1697 and monoclonal antibodies generated against Rv1698. Detection of RNA polymerase served as loading control.

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Figure 3.7: Construction of ms3747Δcth mutant (ML199). (A) Schematic representation of the chromosomal ms3748-ms3747 region of M. smegmatis. A 96 bp long 3’ region of ms3748 encoding for a hydrophobic helix was replaced by homologues recombination with the 45 bp loxP site that introduces a stop codon (ML199). Homologues regions used are indicated as upstream and downstream. Genes are drawn to scale. (B) Expression of Ms3748 and Ms3747 in M. smegmatis. The mutant strain ms3748Δcth (ML199) was complemented singly with either ms3747 (pML451) and ms3748 (pML951) or as transcriptional fusion of ms3748-ms3747 (pML2654). All strains were grown in 7H9tc, hyg, tyloxapol prior to lysis by sonication. Whole cell lysate of M. smegmatis of all strains were separated by SDS-PAGE and presence of Ms3748 and Ms3747 was detected by Western blot analysis. Detection of RNA polymerase served as loading control. (C) Expression of ms3748Δcth in ML199. Small section of Western blot from Fig. 3.7 B was exposed two times longer using high sensitivity substrate (Table 5.2) to detect the truncated Ms3748Δcth protein. (D) Southern blot analysis. Chromosomal DNA of M. smegmatis SMR5 (WT) and mutant ML199 (ms3748Δcth) were digested with XmaI and DraI and analyzed by southern blotting using the probe as indicated in A.

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Figure 3.8: Construction of ΔcgR_1476 mutant. (A) Schematic representation of the chromosomal cgR_1476 region of C. glutamicum. The gene cgR_1476 was deleted in frame so that six nucleotides of the 5’ end and 12 nucleotides of 3’ end remain in the genome. (B) SDS susceptibility drop assay. The bacterial cells were filtered through 5 μm filter to avoid clumps and ten-fold serial dilutions were dropped on BHI agar plates containing no or 0.006% (w/v) SDS. The ΔcgR_1476 mutant (ML920) was complemented by transformation with pML3111 (rv1697- -3 rv1698). The colonies show the dilution of OD600 1x10 after three days of growth at 30 °C.

3.1.3.2 Growth and morphological characterization of rv1697Δcth and ms3747Δcth

The deletion of the C-terminal helix of Rv1697 caused a drastic growth defect of the M. tuberculosis mutant. The rv1697Δcth mutant required 16 weeks to grow on solid medium to a size that wild type M. tuberculosis reached after three weeks (Fig. 3.9 A). In M. smegmatis this deletion caused a less severe attenuation of growth from 3 days for wild type to five days for the mutant strain. In addition to the growth rate defect, both strains showed a drastic change in colony morphology (Fig. 3.9 A, B). Mycobacterial cell suspensions are prone to form aggregates which can be reduced by addition of detergents into the growth medium. The deletion strains showed a high tendency to aggregate in liquid culture regardless of the presence of detergents (Fig 7.1). In addition to the morphological changes of colonies on agar plates, light microscopy revealed that single cells of the rv1697Δcth deletion strain were elongated in size. The average cell length of the rv1697Δcth mutant increased significantly from 3.1 μm (WT) to 5.0 μm (rv1697Δcth) (p value < 0.05, n = 30, SD WT = 0.5, SD rv1697Δcth = 1.2) (Fig 3.10). Growth defect and morphological abnormalities were abrogated upon expression of rv1697 or the ms3748-ms3747 operon in M. tuberculosis or M. smegmatis, respectively.

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Figure 3.9: Growth defect and colony morphology of rv1697Δcth and ms3748Δcth mutants. (A) Single colonies of M. tuberculosis strains mc26206 (WT), ML910 (rv1697Δcth) and ML912 (rv1697Δcth + rv1697) were grown on 7H10, OADC, Hyg. (B) Colonies of M. smegmatis WT strain (SMR5) and mutant strain ms3748Δcth (ML199) transformed with the empty vector pMS2. The mutant strain ML199 was complemented singly with either ms3747 (pML451) or ms3748 (pML951) or as transcriptional fusion of ms3748-ms3747 (pML2654) and grown on 7H10tc, hyg, tyloxapol plates. M. tuberculosis and M. smegmatis cultures were filtered and serial diluted before plating. Pictures of colonies were taken from plates with less than ten colonies per plate.

Figure 3.10: Single cell morphology of rv1697Δcth mutants. Pictures of single M. tuberculosis cells; mc26206 (WT), ML910 (rv1697Δcth) or ML911 pML962 (rv1697Δcth + rv1697). Pictures were taken with a light microscope at 100 x magnification. The white bar represents 2 μm.

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3.1.3.3 Susceptibility of the ms3748Δcth mutant strains to SDS, copper and Malachite green

The genes rv1698 and ms3747 in M. tuberculosis and M. smegmatis, respectively, were shown to be required for copper resistance (Wolschendorf et al. 2011). To elucidate the role of Ms3748 in resistance to toxic compounds we used the phenotypic ms3748Δcth / Δms3747 double mutant. Using the drop assay we tested the sensitivity of the phenotypic ms3748Δcth / Δms3747 double mutant and its single complements towards copper. Unexpectedly, only the ms3748 complemented mutant ML199, missing only Ms3747, was sensitive to copper. The phenotypic ms3748Δcth / Δms3747 double mutant ML199 showed no impaired growth on copper containing plates (Fig 3.11 C). The morphological changes and the high tendency of the ms3748Δcth strain to aggregate is a phenotype often associated with general cell wall defects (Pawelczyk et al. 2011) (Bhatt et al. 2007) (Eckstein et al. 2000). Mutants with such defects are often sensitive to the detergent SDS, which causes cell wall stress (McDonough et al. 2005) (Chao et al. 2013) (Damveld et al. 2008). To test if the ms3748Δcth mutant shows sensitivity in the presence of SDS we compared the abilities of the M. smegmatis and C. glutamicum mutants to grow on SDS containing plates by using drop assays (Fig 3.11 A, Fig 3.8 B). The M. smegmatis phenotypic ms3748Δcth / Δms3747 double mutant ML199, the single complemented strains and the fully complemented strain expressing the ms3748-ms3747 operon were tested for their ability to grow on SDS containing agar plates. The M. smegmatis ΔtatC mutant served as an SDS sensitive control (McDonough et al. 2005). All M. smegmatis deletion strains showed sensitivity towards SDS and growth of the ms3748Δcth mutant was impaired in the presence of SDS similar to the ΔtatC mutant. The phenotypic Δms3747 strain, however, was more resistant to SDS than the ms3748Δcth mutant. Growth of the C. glutamicum ΔcgR_1476 mutant (ML920) on SDS containing plates was also impaired and could be rescued by the expression of rv1697-1698 operon (Fig 3.8 B). It should be noted that the sensitivity of ML920 was not as severe as for M. smegmatis mutants. These results suggest that alterations in the rv1697-1698 operon or its homologs in other mycobacteria or corynebacteria lead to a cell wall defect. A similar susceptibility pattern was observed when the M. smegmatis mutant ML199 and the single complemented strains were tested for their ability to grow in the presence of the compound Malachite green (Fig 3.11 B). Malachite green is a component of several mycobacterial growth media because of its broad-spectrum antibacterial activity. The Malachite green concentration in Middelbrook 7H10 is < 2 μM and allows growth of the M. smegmatis mutant ML199. However, in order to exclude cumulative susceptibility effects all experiments

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Figure 3.11: Susceptibility drop assays of ms3748Δcth deletion strains. The assays show M. smegmatis strains SMR5 (WT) and ML199 (ms3748Δcth). The mutant strain ML199 was complemented singly with either ms3748 (pML951), ms3747 (pML451) or with a transcriptional fusion encoding both genes (pML2654). The bacterial cells were filtered through 5 μm filter to remove clumps and 3 μL of ten-fold serial diluted cultures were dropped on 7H10tc/hygromycin plates containing (A) SDS, (B) Malachite green oxalate or (C) copper sulfate with stated concentrations. The first drop in each column contained cultures diluted to OD600 0.01. As an additional positive control for SDS susceptibility the JM567 (ΔtatC) strain was used (A).

were done in self made 7H10 (7H10tc) with no Malachite green (Table 5.3). Malachite green inhibits the growth of the phenotypic ms3748Δcth mutant and the phenotypic ms3748Δcth / Δms3747 double mutant at a concentration of 2 μM on agar plates. Increasing the concentration

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of Malachite green to 6 μM also inhibited the growth of the phenotypic Δms3747 mutant while the WT and the phenotypic ms3748Δcth / Δms3747 double mutant complemented with ms3748- ms3747 maintained the ability to grow.

3.1.3.4 Accumulation of Congo red, chenodeoxycholate and ethidium bromide

In order to directly measure the outer membrane permeability we performed ethidium bromide uptake assays. These assays have the advantage that the dye has to cross the outer and inner membrane in order to be taken up into the cytosol and only then produce fluorescence by intercalating into DNA (Freifelder 1971). Therefore we used ethidium bromide uptake to compare the cell wall permeability between M. smegmatis wild type and ms3748Δcth mutant strains. The phenotypic ms3748Δcth / Δms3747 double mutant and the phenotypic ms3748Δcth single mutant showed increased uptake of ethidium bromide compared to the wild type strain. The loss of only Ms3747 did not show any acceleration of ethidium bromide uptake (Fig 3.12 A). The high tendency of the mutant strains to form aggregates and the changed colony morphology might have been the result of altered surface properties. To address this question, the accumulation of the hydrophobic compounds Congo red on agar plates and the uptake of radio labeled chenodeoxycholate were investigated. The deletion of ms3748cth caused an approximately two-fold higher accumulation of the dye Congo red in M. smegmatis compared to the wild type strain. The strain ML199 complemented with ms3748, missing only Ms3747 showed no increased accumulation of Congo red compared to wild type (Fig. 3.12 B). Additionally, the strain ML199, missing both Ms3748Δcth and Ms3747 showed a two-fold increase of chenodeoxycholate uptake (Fig 7.2) indicating an increased outer membrane permeability in ML199. All uptake and accumulation experiments were complemented to wild type levels by expression of the ms3748-ms3747 operon. Together, these results reveal a role for Ms3748 in maintenance of the cell wall permeability barrier.

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Figure 3.12: Accumulation assays of ms3748cth deletion strains. The assays show M. smegmatis strains SMR5 (WT) and ML199 (ms3748Δcth). The mutant strain ML199 was complemented singly with either ms3748 (pML951), ms3747 (pML451) or with a transcriptional fusion of both genes (pML2654). (A) Uptake of ethidium bromide. Accumulation of ethidium bromide by M. smegmatis strains at a concentration of 1 μg/mL. Fluorescence was measured as relative fluorescence units (RFU) at an excitation wavelength of 530 nm and an emission wavelength of 590 nm every 2 min for 1 h. (B) Accumulation of Congo red. After filtration the single colonies were grown on 7H10tc, hyg 100 μg/mL Congo red. The dye was extracted from dried cells with DMSO. The amount of accumulated Congo red was quantified colorimetrically at 585 nm.

3.1.3.5 Antibiotic sensitivity of rv1697Δcth and ms3748Δcth strains

Next, we tested the susceptibility of the M. smegmatis and M. tuberculosis mutants towards a variety of antibiotics using the microplate Alamar Blue assay (MABA). The phenotypic M. smegmatis ms3748Δcth / ms3747 double mutant was eight-fold and four-fold more susceptible towards rifampicin and erythromycin, respectively (Table. 3.3). It is apparent that the double mutant was more susceptible to hydrophobic antibiotics. In contrast, lack of only Ms3747 had little effect on the drug susceptibility (Table 3.3). The deletion of the C-terminal helix of Rv1697 in M. tuberculosis caused an increased susceptibility towards a wide range of antibiotics (Table 3.2). Compared to wild type the rv1697Δcth strain was 16-fold more susceptible to fusidic acid and ampicillin. In addition the rv1697Δcth strain showed an eight-fold higher susceptibility towards the antibiotic rifampicin. The increased drug sensitivity was reversed by expressing either rv1697 or ms3748 in the rv1697Δcth M. tuberculosis strain (Table 3.2).

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Table 3.2: Drug susceptibility of M. tuberculosis rv1697Δcth deletion strain.

MIC90(μg/mL) WT rv1697Δcth rv1697Δcth + rv1697 rv1697Δcth + ms3748 Susceptibility factor WT/ rv1697Δcth Ampicillin 128 8 128 n.d 16 Fusidic acid 16 1 32 32 16 Rifampicin 0.06 0.0075 0.12 0.12 8 Novobiocin >25 8.3 n.d n.d >3 Ethambutol 3 1.5 3 n.d 2 Isoniazid 0.03 0.03 0.03 n.d 1 Kanamycin 2.5 2.5 2.5 n.d 1 Moxifloxacin 0.2 0.2 n.d n.d 1 Tetracyclin 12 12 12 n.d 1

The MICs were determined using the fluorometric Alamar Blue assay. The M. tuberculosis rv1697Δcth (ML910) mutant strain was complemented by episomal expression of rv1697 (pML961) or ms3748 (pML951) in the unmarked rv1697Δcth mutant ML911. WT: M. tuberculosis (mc26206), n.d.: not determined.

Table 3.3: Drug susceptibility of M. smegmatis ms3748Δcth deletion strain.

WT ms3748Δcth Partition empty empty +ms3747- Susceptibility coefficient Antibiotic +ms3747 +ms3748 vector vector ms3748 factor (Pow) 0.002-0.2 Ampicillin 250 125 125 125 250 2 (pH 7.2) 0.04-0.07 Tetracycline 2.4 1.2 nd. nd. nd. 2 (pH 7.0) 3.16 Clarithromycin 0.25 0.125 0.125 0.25 0.25 2

4.6-18.2 Erythromycin 2.5 0.625 0.625 1.25 2.5 4 (pH 7.4) 20.9 Rifampicin 16 2 2 8 16 8 (pH 7.5)

The assays show the M. smegmatis strains SMR5 (WT) and ML199 (ms3748Δcth) transformed with the empty vector pMS2. The mutant strain ML199 was complemented singly with either ms3748 (pML951), ms3747 (pML451) or with a transcriptional fusion encoding both genes (pML2654). The MICs were determined using the fluorometric Alamar Blue assay. The susceptibility factor was calculated by dividing the MIC of ML199 with the MIC of SMR5. The hydrophobicities of the drugs were determined experimentally and are given as apparent n-octanol–water partition coefficients (Pow) (Hansch et al. 1995) (Kim et al. 2009).

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3.1.4 Cell wall analysis of ms3748Δcth and ΔcgR_1476 deletion strains

3.1.4.1 Whole cell lipid analysis

The increased susceptibility to a wide variety of mostly hydrophobic compounds and the increased permeability to ethidium bromide suggest a general cell wall defect. Such defects have been observed in lipids biosynthesis mutants (Philalay et al. 2004) (Liu et al. 1999). To examine whether the ms3748-ms3747 operon plays a role in lipid metabolism or transport we performed lipid analysis. The M. smegmatis wild type and mutant strains were grown as surface pellicles before extractable lipids were removed from whole cells by chloroform/methanol extraction. The lipid extracts were analyzed by thin layer chromatography (TLC). Then, using the delipidated cells, the covalently bound lipids were cleaved from the cell wall by basic ester hydrolysis and analyzed by TLC. All main lipids were found to be synthesized by the mutant strains. However, we found the amounts of the lipid TDM and its precursor lipid TMM to be approximately two-fold higher in the M. smegmatis strains lacking the full length Ms3748 protein (Fig 3.13, 7.3).

Figure 3.13: Lipid analysis of whole cells and lipid vesicles. The thin layer chromatograms (TLCs) show lipid profiles of M. smegmatis strains WT (SMR5) and ML199 (ms3748Δcth). The mutant strain ML199 was complemented with pML2754 (ML199 cp) which encodes a transcriptional fusion of ms3748 and ms3747. For whole cell lipid analysis the bacteria were grown as surface pellicles. Lipid vesicles were purified from culture filtrate using high speed centrifugation and size exclusion chromatography. Lipids of whole cells and purified membrane vesicles were extracted with chloroform, methanol and water. TLCs were performed according to Methods (a) and (b) were resolved using solvent system E and lipids were visualized by copper sulfate in phosphoric acid. As a control, the first lane shows 4 mg of trehalose-di-mycolates (Sigma). Solvent system B was used to resolve TLCs (c) and (d). Anthrone was used to visualize sugar-containing lipids. The TLCs (e) and (f) were resolved with solvent system A and lipids were visualized by phosphomolybdic acid. GPLs, glycopeptidolipids; TAGs, triacylglycerols; TMM, trehalose monomycolate; TDM, trehalose dimycolate.

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3.1.4.2 Membrane vesicles from culture filtrate

We were wondering if the accumulation of TDM is accompanied by the release of membrane vesicles into the culture medium as it was described for deletion mutants that are impaired in arabinogalactan synthesis (Bou Raad et al. 2010). The deletion of the arabinofuranosyltransferase AftB of C. glutamicum was shown to cause outer membrane vesicles to be shed into the culture medium (Bou Raad et al. 2010). Thus, the C. glutamicum strain ΔaftB served in these experiments as a positive control. The culture filtrates of M. smegmatis wild type, mutant and complemented strain were spun down by high speed centrifugation. The obtained pellet of the mutant strain ML199 (ms3748Δcth) yielded a bigger pellet compared to those harvested from wild type or fully complemented mutant (Fig 3.15 A). To investigate whether the resulting pellet consisted of

Figure 3.14: TEM pictures of membrane vesicles. membrane vesicles, the pellets were Membrane vesicles released into the culture filtrate by M. smegmatis WT (SMR5) and ms3748Δcth (ML199) were resuspended and examined by obtained by centrifugation and applied to TEM after staining transmission electron microscopy (TEM) with uranyl acetate. The C. glutamicum WT and the aftB deletion strain were treated the same way as a positive using a uranyl acetate staining. As an control. Small unilamellar vesicles (SUVs) consisting of DPhPC and cholesterol (10:1) served as an additional additional positive control we generated positive control. Scale is indicated by white bar. small unilamellar vesicles (SUVs). The pellets of wild type and mutant strains of C. glutamicum and M. smegmatis contained vesicles (Fig 3.14). The irregular shape of the vesicles might be an artifact caused by the drying process during the staining procedure. In order to quantify the released membrane vesicles we used analytical size exclusion chromatography. The analysis revealed that the molecular weight of

33 vesicles produced by ML199 is about 2 MDa, much like the vesicles shed off by C. glutamicum ΔaftB (Fig 3.15 B, D). The phenotypic ms3748Δcth / Δms3747 double mutant released six times more membrane vesicles into the culture medium than the wild type and complemented strain, but eight times less than the C. glutamicum ΔaftB strain. The lipids of the fractions containing the major peak were extracted with chloroform/methanol and analyzed by TLC. The vesicles produced by the wild type and complemented strain contained triacylglycerols (TAGs), the outer membrane lipids glycopeptidolipids (GPLs) and TDM. Surprisingly, these outer membrane lipids were absent in the vesicles released by the phenotypic ms3748Δcth / Δms3747 double mutant and an increased amount of TAGs were detected (Fig 3.13, 7.3).

Figure 3.15: Purification and analysis of membrane vesicles. (A) Pellets of M. smegmatis culture filtrate in centrifugation tubes. The culture supernatant of WT (SMR5), ML199 (ms3748Δcth) and ML199 complemented with pML2654 (ms3748-ms3747) was filtered (pore size 0.22 μm) and a pellet was obtained after centrifugation (75,000 x g, 1 h, 4 °C). (B) and (C) show chromatograms of analytical size-exclusion chromatography read at OD260. (B) WT (C. glutamicum ATCC13032) and ΔaftB mutant (Seidel et al. 2007). (C) Shows analysis of vesicles produced by WT, ML199 and the complemented strain. Chromatography was performed using a TOSOH G3000SWXL column (Tosoh) on a Bio-Rad Duoflow HPLC chromatography system. The column was calibrated with blue dextran (~2 MDa), apoferritin (443 kDa), bovine serumalbumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome C (12.4 kDa) molecular mass standards.

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3.1.4.3 Differences in cell wall sugar content

It was possible to delete the M. tuberculosis rv1697 homolog cgR_1476 completely in C. glutamicum. To detect changes of the sugar composition in the cell wall polymer arabinogalactan the cell wall of the C. glutamicum strains were isolated and subjected to GC- MS analysis after complete acid hydrolysis (Tab. 3.4). The ΔcgR_1476 strain showed a reduced arabinose/galactose ratio compared to wild type, from 5.3 to 3.9. The phenotype could be complemented by expression of the rv1697-1698 operon. The reduced arabinose/galactose ratio was also observed in C. glutamicum mutants of arabinosyltransferases (Seidel et al. 2007). In order to examine whether cgR_1476 is involved in arabinogalactan polymerization we performed an arabinogalactan linkage analysis. Although there are differences in the ratio between the differently linked arabinogalactan sugar units, the fact that all different kinds of linked sugar units could be detected shows that cgR_1476 is not directly involved in the arabinogalactan assembly.

Table 3.4: Quantitative analysis of arabinogalactan composition. WT ΔcgR_1476 ΔcgR_1476 + rv1697-rv1698 Ara/Gal ratio 5.28 3.93 5.80 in mAGP

Table 3.5: Quantitative analysis of arabinogalactan linkage ΔcgR_1476 WT ΔcgR_1476 + rv1697- rv1698 t-Ara 11.9 4.4 5.8 2-Ara 6.8 7.8 6.5 5-Ara 36.8 41.3 29.4 3,5-Ara 12.6 9.7 7.2 2,5-Ara 8.7 17.5 9.6 t-Gal 1.0 1.0 1,0 5-Gal 12.6 11.7 7.7 6-Gal 3.5 2.9 1.7 5,6-Gal 3.9 3.9 2.4

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3.2 Screening for copper sensitivity enhancing compounds

The in here described development of a drug screen and data have been published in:

Speer, A., Shrestha, T.B., Harber, G.J., Michalek, S.M., Basaraba, R.J., Bossmann, S.H., Niederweis, M., Kutsch, O., Wolschendorf, F. Copper-boosting compounds: a novel concept for anti-mycobacterial drug development. Antimicrob. Agents. Chemother. 2013. 57(2):1089-91

3.2.1 Assay development and optimization

The discovery that copper resistance is a virulence factor for M. tuberculosis (Wolschendorf et al. 2011) lead us to consider a drug screening system to identify compounds that potentiate the anti-mycobacterial properties of copper ions and thereby target copper homeostasis of M. tuberculosis. To maintain the ability to screen under BSL2 conditions, we utilized a previously published avirulent variant of M. tuberculosis H37Rv designated as M. tuberculosis mc26230 (ΔRD1/ ΔpanCD) (Sambandamurthy et al. 2006). To our knowledge, none of the deleted genes affect copper-homeostasis or resistance to antibiotics. The screen was developed in a 96-well plate format. Microplate Alamar Blue assays were used to quantitatively determine cell viability (Primm et al. 2007).

3.2.1.1 Media optimization

As a first step we determined a suitable medium for the assay. According to our evaluation, the absence of copper ions in commonly used M. tuberculosis growth media, e.g. Loewenstein- Jensen, Brain Heart Infusion, Muller-Hinton, Sauton’s, Glycerol-alanine-salts medium or the presence of media constituents that sequester copper ions (e.g. albumin in Middlebrook 7H9 or 7H10 medium) would not have revealed drug induced copper sensitivity phenotypes of M. tuberculosis in previous screens. We found that the media supplement albumin is able to rescue the growth defect of the highly copper sensitive Δms3747 mutant of M. smegmatis and Δrv1698 mutant of M. tuberculosis (Wolschendorf et al. 2011). As our screening concept is designed to discover drugs that induce a Δrv1698-like copper phenotype in wild type strains we concluded that known copper sequestering media constituents such as albumin (Halliwell et al. 1984), certain amino acids (Neumann et al. 1967; Iqbal et al. 1990) and other protein based ingredients had to be omitted as they are likely to interfere with the performance of this assay. Based on

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these considerations, the majority of standard M. tuberculosis media were eliminated. Hartmans-deBont (HdB) minimal medium (Hartmans et al. 1992) was found to be the best fit for our purpose. This medium provides copper at 0.8 µM, which does not interfere with the assays described herein, but satisfy the metabolic requirements of M. tuberculosis for copper ions. HdB medium supports the growth of M. tuberculosis in the absence of albumin or protein derived supplements and copper can be added up to a concentration of 500 µM before precipitation occurs (data not shown).

3.2.1.2 Determination of the optimal copper concentration

The most effective way to distinguish between copper dependent and independent activities of test compounds is to conduct a parallel screen by which anti-mycobacterial properties of test compounds are being compared in the presence or absence of copper. It was therefore necessary to determine a copper concentration that by itself does not inhibit the growth of M. tuberculosis as the data would then be difficult to interpret. For that reason, we generated a copper susceptibility profile for the growth of M. tuberculosis in HdB medium at different copper concentrations using the well-established Alamar Blue assay. Optimal growth was observed until a copper concentration of 15 µM (Fig. 3.16). The rapid viability drop between 15 and 17.5 µM prompted us to avoid screening at 15 µM as compounds that insignificantly alter copper susceptibility would be detected as hit compounds by the assay. This would dramatically increase the number of false positive hits leading to a decrease in assay specificity. Hence, we chose to perform the parallel screen in the presence and absence of 10 µM copper.

Figure 3.17: Copper susceptibility profile of M. tuberculosis in HdB medium as determined by MABA. Reproduced with permission (Speer et al. 2013).

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3.2.2 Assay validation and establishment of key assay parameters

3.2.2.1 Effect of neocuproine on M. tuberculosis

Based on the recent characterizations of copper resistance pathways of M. tuberculosis, we distinguish two categories of copper-boosting compounds: (i) copper delivering agents, which complex copper and carry it across the outer membrane of mycobacteria and (ii) copper resistance pathway inhibitors, which inhibit activities of proteins that mediate copper resistance. Unfortunately, compounds that specifically inhibit copper resistance proteins of M. tuberculosis are still unknown. To ascertain that the here developed assay system is capable of identifying copper sensitivity inducing compounds we investigated if the copper complexing agent neocuproine could be used for the purpose of assay validation. Neocuproine is a well-studied membrane permeable copper-chelating agent (Chen et al. 2008) and could therefore act as a potential copper delivering agent. Its activity against M. tuberculosis was tested using MABA (Fig. 3.18 A). Under copper depleted conditions, neocuproine has only a mild inhibitory effect on the growth of M. tuberculosis with an IC50 of ~5

µM. However, in the presence of 10 µM copper the IC50 dropped by ~160-fold to ~30 nM (Fig. 3.18 A). These results revealed that the anti-mycobacterial properties of neocuproine are copper dependent rendering the assay suitable for the identification of copper boosting

compounds. However, neocuproine is toxic towards eukaryotic cells (IC50 = 0.2 μM; AID 504683). Bathocuproine, a membrane impermeable derivative of neocuproine (Chen et al. 2008) had no inhibitory activity in the presence or absence of copper (Fig. 3.18 B) despite its ability to complex copper ions. Because of their well-known copper complexing and contrasting membrane penetrating properties, neocuproine and bathocuproine provided useful probes for the assay optimization.

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Figure 3.18: Copper dependent activity of (A) neocuproine and (B) bathocuproine against M. tuberculosis in copper depleted (grey line) or copper supplemented (10 µM; black line) HdB medium. Molecular structures of the compounds are depicted within the graphs. These figures were taken from (Speer et al. 2013) with permission.

3.2.2.2. Determination of Z’-factors

To test the reliability of the assay, we performed a Z’-test (Iversen et al. 2004). The Z’-factor is a statistic value designed to reflect the dynamic range of the assay, as well as the variation associated with the signal measurements. This includes drift and edge effects associated with the 96-well plate format. As the Z’-factor is dimensionless it can be used for assay optimization purposes. A Z’-factor of 1.0 would

Figure 3.19: Drift and edge effects on a 96 plate were assessed on represent an ideal assay. To be “Max” (no treatment, black diamonds, or in the presence of 10 µM considered for high-throughput copper, grey triangles), “Min” (3 µM neocuproine + 10 µM copper, black squares) and “Mid” signals (3 µM neocuproine) organized in quadrants of screening, an assay should be a 96-well plate. Shaking and standing assay conditions are compared. Z’ characterized by a Z’-factor >0.5 factors were calculated based on “Max” and “Min” signals following recommendations from (Iversen et al. 2004). (Iversen et al. 2004). Using

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neocuproine as cell growth inhibitor, we optimized the assay conditions with the Z’-factor as a reference. We found that continuous shaking was not required during the 7 day incubation period. In fact, static culture conditions enhanced assay performance by minimizing evaporation from edge wells. This adjustment significantly increased the Z’-factor from 0.64 (agitated culture) to 0.89 (static culture) (Fig. 3.19).

3.2.3 Limited pilot screen and analysis of hits

To test the high-throughput capability of our assay, we performed a limited pilot screen using a small but random selection of compounds from our in-house small molecule library (Chembridge, Inc.) spiked with several antibiotics and known copper chelators which might function as potential copper delivery drugs. Several important insights were gained from this screening effort in regards to the implementation of the technology for HTS purposes and the feasibility of copper-boosting compounds as anti-TB drugs.

3.2.3.1 Synergy between copper ions and Bis-dithiosemicarbazones

Diacetylbis(N(4)-methyl-3- thiosemicarbazone) or ATSM (Fig. 3.20 A) emerged as a positive hit from the limited pilot screen. The lipophilic Cu2+(ATSM) complex has been originally developed for tumor imaging and diagnostics and is currently in several clinical trials (Xiao et al. 2008; Hung et al. 2012). Cu2+(ATSM) activity was confirmed in detailed dose-response experiments using infectious M. tuberculosis H37Rv (Fig. 3.20 B). In the absence of copper, ATSM at concentrations of up to 10 μM had no apparent inhibitory effect on M. tuberculosis. However, in

the presence of copper ATSM had an IC50 of ~0.6 μM and an IC90 of ~2.5 μM (Fig. 3.20 B). This finding encouraged us to test whether the structurally related compound glyoxalbis(N(4)- methyl-3-thiosemicarbazone) or GTSM (Fig. 3.20 A) would show an anti-mycobacterial activity. Under copper depleted conditions GTSM exhibited a potent inhibitory activity against M. tuberculosis, which was further enhanced in the presence of copper. The IC50 for GTSM with copper was ~60 nM and its IC90 was determined to be ~300 nM (Fig. 3.20 C). ATSM and GTSM used in this study were obtained from the Bossmann laboratory (Kansas State University, USA).

3.2.3.2 Activity of diacetylbis(N(4)-methyl-3-thiosemicarbazone) and glyoxalbis(N(4)- methyl-3-thiosemicarbazone) against non-growing M. tuberculosis

While treatment of active M. tuberculosis infection has been complicated by the development of drug resistant strains, the intrinsic drug-resistance phenotype of persistent M. tuberculosis

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infection poses a huge challenge to TB drug development. During latency, M. tuberculosis persists in a nutrient deprived environment and consequently rarely undergoes cell division, which contributes to the increased tolerance of latent M. tuberculosis to current anti-TB drugs (Esmail et al. 2011). Thus, drugs with the ability to sterilize M. tuberculosis lesions and kill latent M. tuberculosis would be highly desirable (Lienhardt et al. 2012). Therefore, we evaluated whether ATSM or GTSM would kill non-growing M. tuberculosis using a previously published and characterized starvation model of M. tuberculosis, which mimics the drug resistant phenotype of dormant M. tuberculosis (Byrne et al. 2007). M. tuberculosis mc26230 was cultured for 6 weeks in PBS. Then ATSM or GTSM were titrated on the cells in the presence and absence of copper. After eight days of exposure bacterial survival in liquid culture was determined after transferring an aliquot onto solid medium. Rifampicin was included as a control. In these experiments, we found that GTSM at concentrations of 2.5 µM was able to kill non-growing M. tuberculosis in the presence of copper (10 µM) (Fig. 3.20 D) while ATSM was inactive up to 10 µM.

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Figure 3.20: Activities of novel copper-boosting drug candidates. The bis(thiosemicarbazones (A) ATSM and GTSM were identified as compounds with potential copper-boosting activity in a limited pilot screen. The activity of (B) ATSM and (C) GTSM was detailed in multi-dose response curve experiments on mid-logarithmic phase growing M. tuberculosis and (D) nutrient starved non-growing persistent M. tuberculosis. These figures were taken with permission from (Speer et al. 2013).

3.2.3.3 Therapeutic index of diacetylbis(N(4)-methyl-3-thiosemicarbazone and glyoxalbis(N(4)-methyl-3-thiosemicarbazone

Tolerance of ATSM and GTSM in eukaryotes is implied by their use in various clinical settings and animal experiments. However, to ensure acceptable toxicity levels in in vitro models, we determined the therapeutic index of ATSM and GTSM on THP-1 cells (Fig. 3.21 A), a monocytic cell line, and mouse-derived peritoneal macrophages (Fig. 3.21 B). Both are important cellular model systems for M. tuberculosis infection. No toxic effects on these macrophages where

42 observed by MABA (Fig. Fig. 3.21 A) or microscopy (Fig. 3.21 B) within the solubility range of both compounds (10 μM). These results confirm the tolerance of ATSM and GTSM by eukaryotic cells. Based on the IC50 values for ATSM (Fig. 3.21 C) and GTSM (Fig. 3.21 D) on M. tuberculosis and THP-1 cells we calculated a therapeutic index of >16 and >20, respectively. The promising therapeutic indices justify further consideration of ATSM and GTSM as potential lead compounds for the development of new therapeutic agents against M. tuberculosis.

Figure 3.18: Cytotoxicity of ATSM and GTSM. Mouse peritoneal macrophages were exposed to 10 µM of ATSM or GTSM in 96-well plates. Viability was accessed 24 h post treatment by measuring the fluorescence of the cell health indicator AlamarBlue (A) according to the recommendations of the manufacturer (AbD Serotec) and by light microscopy (B). One representative dataset out of three independent evaluations, all with comparable outcomes, is shown. Magnification 40x. Viability of THP-1 cells and M. tuberculosis exposed to different concentrations of ATSM (C) or GTSM (D) was used to determine the therapeutic index. Figure was taken with permission from (Speer et al. 2013) and is shown with minor modifications.

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4 Discussion

4.1 Rv1697 is an essential core protein of M. tuberculosis required for cell wall integrity

4.1.1 Rv1697 of M. tuberculosis and its homologs in M. smegmatis and C. glutamicum have the same function

The genome of M. tuberculosis encodes 39 essential mycobacterial core genes that show no homology to other proteins outside the order Actinomycetales (Sassetti et al. 2003) (Marmiesse et al. 2004). Among these genes we found rv1697, which is organized in an operon with rv1698. This is of particular interest since rv1698 has been demonstrated to be required for full virulence of M. tuberculosis during infection studies, due to its contribution to copper resistance (Wolschendorf et al. 2011). Rv1697 is highly conserved among mycolic acid-containing bacteria, which suggests that the homologs of Rv1697 have the same function in these organisms. We confirmed this hypothesis by complementing the phenotypes of the rv1697Δcth mutation in M. tuberculosis by expression of ms3748, the homolog of rv1697 in M. smegmatis. Furthermore, the deletion of cgR_1476, the homolog of rv1697 in C. glutamicum, was complemented by expression of the M. tuberculosis rv1697, confirming the same function for rv1697 in both organisms.

4.1.2 Ms3748 and Ms3747 contribute to cell wall integrity

Cell wall defects often result in permeability defects. Interestingly, the ms3748Δcth mutant and the phenotypic Δms3747 mutant are characterized by increased susceptibility to Malachite green and SDS, and accumulate ethidium bromide much faster than wild type. These phenotypes indicate a general cell wall defect that enables these compounds to cross the membrane barrier more efficiently. Mycobacteria are intrinsically resistant to Malachite green possibly due to reducing enzymes in the cell wall (Tarnok et al. 1959). Thus, increased uptake of Malachite green due to a general permeability defect could overwhelm the reducing capacity of these enzymes. A cell wall defect in the ms3748Δcth mutant is further supported by the increased uptake of ethidium bromide, because this compound has to be taken up into the cytosol and must bind to DNA in order to generate fluorescence (Freifelder 1971). In addition, the is mainly responsible for resistance towards anionic detergents such as SDS. It is therefore not surprising that SDS sensitivity is routinely used as a standard experiment for determining outer membrane integrity loss (McDonough et al. 2005) (Chao et al.

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2013) (Damveld et al. 2008). The complete deletion of the rv1697 homolog in C. glutamicum also increases sensitivity to SDS indicating that the loss of cell wall integrity in M. tuberculosis and M. smegmatis is not an artifact of improper localization of the mycobacterial Rv1697Δcth and Ms3748Δcth proteins, but rather an effect of impaired function.

4.1.3 Effects of ms3748Δcth mutations on the mycobacterial cell wall

The high content of lipids in the mycobacterial cell envelope that are extractable or covalently bound to the cell wall are a major contributing factor to the high permeability barrier of M. tuberculosis (Rajni et al. 2011). Lipid analysis of M. smegmatis revealed that the lipids TMM and TDM accumulate in the strains expressing the truncated Ms3748Δcth protein. TMMs are precursor lipids that have to be transported from the cytosol into the periplasm before they are processed to TDM or mycolic acids, which are covalently attached to the cell wall (Kalscheuer et al. 2010). The presence of TDM in whole cell extracts implicates that TMM are transported into the periplasm (Grzegorzewicz et al. 2012). Our analysis showed no difference of cell wall attached mycolic acids between the ms3748Δcth and wild type strain, suggesting that TDM accumulation in the ms3748Δcth strain is not due to an impaired ability to transfer mycolic acids to arabinan. Instead, the accumulation of TMM and TDM might be a response to increased outer membrane permeability rather than direct involvement in mycolic acid synthesis or trafficking. The accumulating amount of TDM in the ms3748Δcth strain compared to wild type might also explain the changed surface properties of the ms3748Δcth strain, skewing towards a higher surface hydrophobicity that allows for increased uptake of the hydrophobic compounds chenodeoxycholate and Congo red. Accumulation of mycolic acids was also found after treatment of mycobacteria with the antibiotic ethambutol which targets Emb-arabinosyltransferases (Mikusova et al. 1995). Inhibition of these enzymes alters arabinogalactan assembly. As a consequence mycolic acids can no longer be attached to arabinogalactan and TMM’s and TDM’s accumulate (Mikusova et al. 1995). Furthermore, the deletion of the arabinosyltransferase aftB in C. glutamicum causes parts of the outer membrane to be shed into the culture supernatant, forming vesicles in the aqueous environment (Bou Raad et al. 2010). Interestingly, deletion of ms3748Δcth generated similar phenotypes: accumulation of TMM and TDM, and an increased amount of membrane vesicles found in the culture filtrate compared to wild type. The release of membrane vesicles is well studied in Gram-negative bacteria (Kuehn et al. 2005) and has also been described for mycobacteria (Prados-Rosales et al. 2011). These membrane

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vesicles are mostly made of lipids found in the outer membrane. In E. coli, formation of outer membrane vesicles (OMV) can have multiple causes independent of membrane stability (McBroom et al. 2006). However, increased production of OMV in C. glutamicum is due to incorrect arabinogalactan assembly (Bou Raad et al. 2010). In our case, lipids released in vesicles by ms3748Δcth mutant did not contain any outer membrane lipids. Instead, these OMVs contained high amounts of the carbon storage lipid TAG (Garton et al. 2002). This is in contrast to lipids found in vesicles released by the C. glutamicum arabinosyltransferase (aftB) deficient strain which contains mainly the outer membrane lipid TDM. A possible explanation for the compositional differences among OMVs from different sources might be that the alterations of the cell envelope caused by the ms3748Δcth mutation prevent lipids from being properly processed and from reaching their intended location within membrane structures of the cell envelope. Hence, the outer membrane lipids TMM and TDM accumulate within the cells of the ms3748Δcth mutant while other accumulating lipids such as TAG are being released as vesicles into the culture medium. The accumulation of outer membrane lipids and the production of membrane vesicles are phenotypes that the ms3748Δcth mutant and arabinosyltransferase mutants of s have in common. Deletion of the ms3748 homologue cgR_1476 in C. glutamicum reduced the arabinose/galactose ratio. A similar effect has also been described in arabinosyltransferase mutants (Seidel et al. 2007). However, a sugar linkage analysis of ms3748Δcth did not reveal a deficiency in arabinan linkage, in contrast to arabinosyltransferase deficient strains. It is clear that Rv1697 and its homologs play a critical role in maintaining cell wall integrity, but is unclear how these proteins accomplish that function.

4.1.4 The functions of Rv1697 and Rv1698 are connected

Pull down assays and in vivo cross-linking experiments demonstrated that Ms3748 and Ms3747 proteins interact with each other. Furthermore, we have demonstrated that both Ms3748 and Ms3747 are translocated into the periplasmic space, despite the absence of a detectable signal sequence in Ms3748 and its homologs. The beta-lactamase reporter assay showed that the resistance level mediated by Ms3748-beta-lactamase fusion proteins was further increased upon expression of ms3747, when the two genes were transcriptionally fused in an operon. One explanation could be that Ms3748 utilizes Ms3747 for co-translocation, as has been described previously for other bacterial protein complexes (Rodrigue et al. 1999). However, in the case of specific Ms3747- mediated translocation of Ms3748, deletion of Ms3747 should cause a

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Δms3748-like phenotype, which was not observed. Increased uptake of ethidium bromide and the accumulation of TMM and TDM are phenotypes only found in the ms3748Δcth mutant and not caused by the loss of Ms3747. Conversely, only the loss of Ms3747 caused sensitivity towards copper. The phenotypic ms3748∆cth single and phenotypic ms3748∆cth / Δms3747 double mutant did not show an increased copper sensitivity. The toxic effects of high copper concentration (25 μM) attenuate the growth of the wild type strain. The ms3748∆cth mutant has a general growth defect which is seemingly exacerbated in the presence of copper, and could mask copper-dependent growth inhibition. In addition, the ms3748Δcth mutation is associated with increased hydrophobicity that might make it more difficult for the hydrophilic copper ions to cross the cell wall, despite the severe cell wall integrity loss. Taking these observations into account, it is possible that the sensitivity of Rv1698 or Ms3747 deficient strains towards copper is due to a general cell wall defect and the consequential increase in copper influx may lead to intracellular accumulation of copper.

4.1.5 Rv1697 is a potential drug target for TB chemotherapy

Although elucidation of the function of Rv1697 would be of great importance, it is not necessary in order to employ Rv1697 as a drug target. Rv1697 is highly conserved in Corynebacterineae, and has no sequence similarity to any protein in eukaryotes. This lowers the risk of undesirable inhibition of host proteins. Reducing the drug treatment time for TB, currently a minimum of six months, is of critical importance. Current drugs are toxic to human cells, and the long treatment time puts great stress on the normal functions of the liver (Ichai et al. 2010). Additionally, specific inhibition of mycobacterial proteins is advantageous, since the patient gut microbiome may be spared, a disadvantage of broad spectrum antibiotics (Cotter et al. 2012). The realization that Rv1697 is involved in cell wall biosynthesis make this protein an interesting drug target for several reasons. First, inhibition of cell wall biosynthesis is thought to have bactericidal rather than bacteriostatic effects (Yount et al. 2013). Bactericidal agents allow not only the prevention of bacterial growth but actively support the immune system by neutralizing bacterial cells. Because Rv1697 is essential, we anticipate drugs targeting this protein will be bactericidal rather than bacteriostatic. Secondly, by targeting cell wall integrity, M. tuberculosis will become more sensitive to other drugs. The rv1697Δcth mutant demonstrated drastically increased sensitivity towards the first line drug rifampicin but also to ampicillin and fusidic acid. The latter two are unsuitable drugs for TB treatment due to their marginal activity. In vivo assays showed

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the concentration of ampicillin in pleural fluid to be higher than the MIC of ampicillin determined for rv1697Δcth mutant (Giachetto et al. 2004). These results are very encouraging when one takes into account that they were obtained from an impaired Rv1697 mutant rather than a complete deletion. This consideration indicates that even partial inhibition of Rv1697 would be favorable. A drug that shows low inhibition of Rv1697 and interferes with its function would not only cause a drastic growth defect, as seen in rv1697Δcth, but would also add the well-studied drugs ampicillin and fusidic acid to our limited arsenal of anti-TB drugs. Rv1697 is essential in mycobacteria and a classical drug screening approach measuring the ability of M. tuberculosis to proliferate in the presence of the tested compound could reveal an inhibitor. Drug screening campaigns against M. tuberculosis grown under standard culturing conditions identified compounds whose target has not been identified yet (Ananthan et al. 2009) (Maddry et al. 2009). It is possible that among those hits are compounds that inhibit Rv1697. One approach to find such compounds would be to retest hits from previous screens in a search for phenotypes affiliated with Rv1697 inhibition. For example, such an inhibitor should have the ability to synergize with ampicillin or fusidic acid at sublethal concentrations.

4.1.6 Putative alternative function of Rv1697

This work revealed that Rv1697 is involved in cell wall biosynthesis or assembly and is critical to maintaining the cell wall permeability barrier. So far, the function and specific mechanism by which Rv1697 contributes to cell wall integrity remains unknown. The predicted annotation of Rv1697 is thiamine pyrophosphokinase (TPK) (O33198_MYCTU), which are proteins required in other organisms during the scavenging process of thiamine from the extracellular space (Baker et al. 2001). Thiamine that is taken up from extracellular space gets phosphorylated by TPKs to yield the physiologically relevant form. However, the genome of M. tuberculosis encodes for all genes required to synthesize thiamine pyrophosphate de novo which leaves the question for Rv1697’s essentiality unanswered. Furthermore, the homology of rv1697 to TPKs is very low (7 out of 27 conserved residues required for thiamine binding) and M. smegmatis is incapable of taking up thiamine (data not shown), which renders thiamine recycling unnecessary. Although the substrate prediction could be wrong, a kinase activity might still be a possible function of this protein. Global metabolic studies are an alternative to examine kinase activity of Rv1697 and find the potential substrate (G. Larrouy-Maumusa 2012).

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4.2 Copper-boosting compounds: a novel concept for anti-mycobacterial drug discovery

Antibacterial properties of copper ions are well described (Faundez et al. 2004; Gyawali et al. 2011; Botella et al. 2012). However, repeated failure to translate this knowledge into effective antimicrobial treatment strategies accounts for the general perception that the activities of metal chelators would lack therapeutic specificity (Ananthan et al. 2009). Eukaryotic cells have distinct and highly efficient cellular and systemic mechanisms that protect them from copper overload. Chronic copper poisoning in humans is very rare and usually the result of genetic disorders that cause Menkes or Wilson disease (de Bie et al. 2007). The regulation of intestinal copper absorption protects humans from systemic copper overload (Uauy et al. 2008). At the cellular level, copper homeostasis is strictly regulated by copper import proteins (e.g. CTR1). Copper chaperones, but also glutathione and metallothioneins, control the reactivity of copper ions inside the cells and deliver them to cellular target enzymes (Xiao et al. 2008). Microbes lack this extra layer of compartmentalization and are more sensitive to copper overload, a weakness which is exploited by the human immune system (White et al. 2009). Phagocytes are able to increase the copper content in M. tuberculosis containing phagosomes (Wagner et al. 2005). If mycobacterial copper sequestration and efflux systems are overwhelmed, M. tuberculosis is no longer able to maintain crucial cellular functions and dies from multiplicative effects of copper intoxication (Ward et al. 2010). In a similar manner, copper resistance mechanisms contribute to the virulence of Pseudomonas aeroginosa, Streptococcus pneumonia and Salmonella enterica (Schwan et al. 2005; Achard et al. 2011; Shafeeq et al. 2011). In a recent study we found that genetic disruption of rv1698 in M. tuberculosis resulted in copper sensitivity and a strong decrease in virulence. These data suggest that M. tuberculosis is exposed to potentially toxic copper levels during the course of infection (Wolschendorf et al. 2011). Hence, the identification of pharmacologically relevant compounds that block copper resistance mechanisms of M. tuberculosis creates a novel opportunity for therapeutic interference. The ability to kill M. tuberculosis by copper overload is a new approach. Since resistance mechanisms are unlikely to have developed yet, our strategy may be of particular benefit for the control of MDR-TB. In addition, since copper toxicity has several cellular targets, potential resistances mechanisms may be more difficult to evolve (Rowland et al. 2012).

4.2.1 Assay development for new anti-TB drugs

During the development of the high-throughput assay for copper-boosting compounds described in this work, we considered M. smegmatis as a surrogate for M. tuberculosis due to its fast

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growing nature, but at the end rejected it. We were mainly concerned that the M. smegmatis screen would be biased for compounds that are active in M. smegmatis, but lack activity in M. tuberculosis. This concern arose upon the realization that some functions of the copper responsive RicR regulon are unique to pathogenic mycobacteria (Festa et al. 2011). By using M. smegmatis as a surrogate for M. tuberculosis, we might miss inhibitors that modulate these functions. Hence, we used the avirulent M. tuberculosis strain mc26230 as our surrogate for screen development, to minimize the risk of false positives hits and to maintain the ability to screen under BSL2 conditions. Comparisons to the virulent M. tuberculosis strain H37Rv showed that the deletions in mc26230 have no impact on copper resistance. The capability to screen under BSL2 conditions significantly simplified work and reduced costs. An additional consideration is the routine use of albumin in growth media of M. tuberculosis, which is capable of binding a wide variety of compounds, including metals and antibiotics (de Wolf et al. 2000). We have previously found that albumin is able to rescue the growth defect of the copper sensitive Δms3747 mutant of M. smegmatis on standard Middlebrook 7H10 medium (Wolschendorf et al. 2011) by binding up excess copper in the media. Many HTS campaigns to find anti-mycobacterial drugs were performed in albumin containing media, lowering the chance to find copper boosting drugs. We found HdB medium, which lacks albumin, to be most suited for this drug screen approach. The only possible class of compounds that we can predict to overcome natural copper resistance are copper complexing compounds, which can increase intracellular copper concentrations by serving as ionophores that transport copper ions across the mycobacterial outer membrane. The compounds neocuproine and bathocuproine are two such compounds and provided very useful chemical probes as internal controls. These compounds have similar and well-defined copper-complexing properties, but distinctly different characteristics regarding membrane permeability. While neocuproine is hydrophobic and capable of crossing the mycobacterial outer membrane, bathocuproine has charged groups and is membrane impermeable. As expected, bathocuproine was not effective against M. tuberculosis, independent of copper. Neocuproine provided excellent antibacterial properties in the presence of copper, but due to its cytotoxicity to eukaryotic cells, further characterization of that compound was not pursued (Peters et al. 2007). However, neocuproine and bathocuproine were suitable test compounds to demonstrate that the high throughput screening assay is capable of identifying potential new anti-tubercular compounds that act in a copper dependent manner.

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4.2.2 The therapeutic potential of diacetylbis(N(4)-methyl-3-thiosemicarbazone and glyoxalbis(N(4)-methyl-3-thiosemicarbazone

Compounds that act in a copper dependent manner could potentially boost the anti- mycobacterial properties of copper ions that reportedly accumulate at the sites of M. tuberculosis infection (Wolschendorf et al. 2011). In this study, we introduce ATSM and GTSM as proof-of-concept compounds for the idea that copper-boosting drugs can be developed for targeting mycobacterial infections. We hypothesize that in a therapeutic setting, copper homeostasis of M. tuberculosis could be targeted by (i) specific copper resistance pathway inhibitors, which would sensitize M. tuberculosis to the antibacterial effects of copper ions or (ii) by compounds that facilitate the transport of copper ions across the cell envelope of M. tuberculosis (copper delivery drugs). As both mechanisms would promote intracellular copper accumulation we referred to them as copper boosting drugs. While our preliminary pilot screen did not identify any potential copper resistance pathway inhibitors, we succeeded to identify potential copper delivery drugs that may reach lead compound status. ATSM and GTSM had potent copper-dependent activities against M. tuberculosis. The facts that ATSM can be orally administered (Hung et al. 2012) and is currently in several clinical trials for other applications, and that GTSM has been extensively tested in animals, nominates these compounds for further consideration as novel anti- mycobacterial compounds (Fujibayashi et al. 1997; Lewis et al. 2001; Soon et al. 2011; Hung et al. 2012). We found that the anti-mycobacterial activity of ATSM was entirely dependent on higher copper concentrations. In contrast, GTSM had anti-mycobacterial activity already in the absence of copper which suggests the presence of additional copper independent targets in M. tuberculosis. However, the activity of GTSM was further boosted in the presence of physiologically relevant copper concentrations. The combination of copper dependent and independent anti-mycobacterial functions could be advantageous for the in vivo activity of GTSM, as it promises activity in a greater variety of tissues and cell-specific environments, and thus may target a greater pool of phenotypically different M. tuberculosis cells. The copper- boost could then provide additional activity at the site of infection and within macrophages where copper ions are available (Wolschendorf et al. 2011) (White et al. 2009) (Wagner et al. 2005). 2+ 2+ Cu (ATSM) was less potent (MIC90 ~ 2.5μM) compared to Cu (GTSM) (MIC90 ~ 0.3 μM). However, it is reported that Cu2+ (ATSM) accumulates in hypoxic tissues (Fujibayashi et al. 1997) what is very interesting with respect to mycobacterial infections. Hypoxia is a key feature

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of M. tuberculosis-containing macrophages in lung granulomas (Daniel et al. 2011) where copper ions are abundant (Wolschendorf et al. 2011). In vivo studies on animals will reveal if the hypoxia-selectivity feature of ATSM may be exploitable for treatment of M. tuberculosis infections.

4.2.3 Activity of diacetylbis(N(4)-methyl-3-thiosemicarbazone) and glyoxalbis(N(4)- methyl-3-thiosemicarbazone) against non-growing M. tuberculosis

Another critical feature of M. tuberculosis pathogenicity is its ability to enter a latent state during infection. During latency, M. tuberculosis persists in a nutrient deprived environment and consequently rarely undergoes cell division, which contributes to the increased tolerance of latent M. tuberculosis to current anti-TB drugs (Esmail et al. 2012). Thus, drugs with the ability to sterilize M. tuberculosis lesions and kill latent M. tuberculosis would be highly desirable (Lienhardt et al. 2012). While treatment of active M. tuberculosis infection has been complicated by the development of drug-resistant strains, the intrinsic drug-resistance phenotype of persistent M. tuberculosis infection poses a huge challenge to TB drug development (WorldHealthOrganization 2010). GTSM, but not ATSM, had copper-dependent anti- mycobacterial activity against non-growing M. tuberculosis, providing some promise that copper-boosting drugs could eventually also act against latent M. tuberculosis infections in vivo. Taken together, our data suggest that copper delivery drugs could establish a novel class of anti-mycobacterial compounds, with ATSM and GTSM serving as first-in-class candidates. ATSM and GTSM provide experimental evidence that both replicating and non-replicating M. tuberculosis can be targeted by such compounds.

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5 Material and Methods

5.1 Material

5.1.1 Chemicals and Enzymes

Hygromycin B was purchased from Calbiochem. ATSM and GTSM were synthesized by the Bossmann laboratory (Kansas State University, KS, USA) as previously published (Dearling et al. 2002). The chemical library was purchased from Chembridge, Inc (San Diego, CA, USA). All other chemicals were purchased from Merck (Whitehouse Station, NJ, USA) or Sigma (St. Louis, MO, USA) at the highest purity available. Enzymes for DNA restriction and modification were purchased from New England Biolabs, Inc. (Ipswich, MA, USA) and Invitrogen (Grand Island, NY, USA). Oligonucleotides were obtained from Integrated DNA Technologies, Inc. (Coralville, IA, USA).

5.1.2 Bacterial strains, media and growth conditions

E. coli DH5α was used for cloning experiments and was routinely grown in Luria-Bertani broth at 37 °C. C. glutamicum was grown in brain heart infusion (BHI) medium (Becton, Dickinson) at 30 °C. M. smegmatis strains were grown in Middlebrook 7H9 medium (Difco) supplemented with 0.2 % glycerol and 0.02 % tyloxapol or on 7H10 Middlebrook medium (Difco) supplemented with 0.5 % glycerol and 0.02 % tyloxapol. M. tuberculosis H37Rv and M. bovis BCG were grown in Middlebrook 7H9 medium supplemented with 10 % OADC (Difico), 0.2 % glycerol and 0.02 % tyloxapol or on 7H10 Middlebrook medium supplemented with 10 % OADC, 0.5 % glycerol and 0.02 % tyloxapol. Middlebrook 7H10 medium for the M. tuberculosis strain mc26230 was additional supplemented with pantothenic acid 24 μg/mL and 0.2 % casamino acids and the M. tuberculosis strain mc26206 with pantothenic acid 24 μg/mL, leucin 50 μg/mL and 0.2 % casamino acids. Where indicated M. smegmatis or M. tuberculosis strains were grown in self- made 7H9 or on 7H10 without addition of copper or Malachite green (7H9/10tc - trace copper) or in Hartmanns de Bont (HdB) medium (Smeulders et al. 1999) supplemented with 0.5 % glucose, 0.02 % tyloxapol (HdBGT) and panthothenic acid, if applicable. When required antibiotics where used at the following concentrations: hygromycin (200 μg/ml for E. coli, 50 μg/ml for mycobacteria), kanamycin (30 μg/ml) and chloramphenicol (25 μg/ml for E.coli, 6 μg/ml for corynebacteria).

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Table 5.1: Bacterial strains and macrophages

Bacterial strains Parent vector, relevant genotype Source or reference E.coli DH5α recA1; endA1; gyrA96; thi; relA1; hsdR17(rK- (Sambrook et al. 1989) ;mK+); supE44; Ø80ΔlacZΔM15; ΔlacZYA-argF; UE169 M. smegmatis SMR5 mc2155 derivative, SmR (rpsL*) (Sander et al. 1996) M. smegmatis ML198 SMR5 derivative, ms3748cth::loxP-mycgfp2+- this study hyg-loxP, HygR M. smegmatis ML199 ML198 derivative, ms3748cth::loxP this study M. smegmatis JM567 mc2155 derivative, ΔtatC (McDonough et al. 2005) M. smegmatis PM759 mc2155 derivative, ΔblaS1, ΔlysA4, ΔrpsL6 (Flores et al. 2005) M. tuberculosis mc26230 H37Rv derivative, ΔRD1, ΔpanCD M. tuberculosis mc26206 H37Rv derivative, ΔpanCD, ΔleuCD Ranganathan et al., 2012 M. tuberculosis ML910 mc26206 derivative, rv1697cth::loxP-mycgfp2+- this study hyg-loxP, HygR M. tuberculosis ML911 ML910 derivative, rv1697cth::loxP this study M. tuberculosis ML912 ML910 derivative, Ms6::pML2688 this study M. tuberculosis H37Rv wild type ATCC25618 M. bovis BCG wild type, strain Institute Pasteur ATCC 27291 C. glutamicum wild type ATCC13032 C. glutamicum ML920 wild type derivative, ΔcgR_1476 this study C. glutamicum ΔaftB wild type derivative, ΔaftB (NCgl2780) (Bou Raad et al. 2010) Macrophages Description Source or reference

THP-1 Human monocytic cell line ATCC TIB-202

Peritoneal macrophages Isolated from peritoneal cavity of C57BL/6 Michalek Laboratory, female mice UAB, Birmingham, AL, USA) (Zhang et al. 2005)

5.2 Media and standard methods

Table 5.2: Standard methods

Standard methods Enzyme / Kit Source / Protocol PCR iProof High Fidelity Master Mix Bio Rad (see manufacturer manual), www.biorad.com Ligation T4 DNA-ligase New England Biolabs (see manufacturer manual), www.neb.com Digestion of DNA Restriction endonucleases New England Biolabs (see manufacturer manual), www.neb.com Blunt-ending of DNA T4 DNA-Polymerase New England Biolabs (see manufacturer manual), www.neb.com Small scale plasmid preparation FastPlasmid 5prime (see manufacturer manual), from E. coli Mini Kit www. 5prime.com

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Medium scale plasmid Plasmid Midi Kit Quiagen (see manufacturer preparation from E. coli manual), www.quiagen.com Recovery of DNA from gels and QIAquick gel extraction Kit Quiagen (see manufacturer solutes manual), www.quiagen.com DNA gelelectrophoresis - (Sambrook et al. 1989) Transformation of mycobacteria - (Parish et al. 2001) Preparation of E. coli calcium - (Sambrook et al. 1989) chloride competent cells Transformation of E. coli - (Sambrook et al. 1989) Western Blot detection ECL Western Blotting Pierce (see manufacturer manual), Substrate www.pierce.com Super Signal West Femto Max Thermo (see manufacturer manual), Sensitivity Substrate http://www.thermoscientificbio.com Denaturing polyacrylamide gel - (Schägger et al. 1987) electrophoresis (PAGE) Protein concentration BCA Protein Assay Kit Pierce (see manufacturer manual), www.pierce.com Isolation of peritoneal - Michalek laboratory, UAB, macrophages Birmingham, AL, USA Southern blot analysis DIG High Prime DNA Labeling Roche (see manufacturer manual), and Detection Starter Kit II www.roche-applied-science.com

Table 5.3: Media

Standard Media and Composition per liter Manufacturer/Uses Supplements LB broth 10 g NaCl E. coli 5 g Yeast extract 10 g Tryptone LB agar same as LB broth E. coli 15 g agar 7H9 broth 4.7 g Middlebrook 7H9 broth base 7H9 broth base is commercially 0.2% Glycerol available through (BD Difco, Franklin 0.05% Tyloxapol Lakes, NJ, USA) M. smegmatis M. tuberculosis (OADC required) 7H9 broth (tc) 0.5 g (NH4)2SO4 M. smegmatis 1.0 g KH2PO4 M. tuberculosis (OADC required) 2.5 g Na2HPO4 0.1 g Sodium citrate 50 mg MgSO4 0.5 mg CaCl2 1 mg ZnSO4 0.5 mg L-Glutamic acid 40 mg Ammonium iron (III)-citrate 1.0 mg Pyridoxine hydrochloride 0.5 mg Biotin 0.2% Glycerol 0.05% Tyloxapol 7H10 agar 9.5 g Middlebrook 7H10 agar base 7H10 agar base is commercially 0.2% Glycerol available through (BD Difco, Franklin 0.05% Tyloxapol Lakes, NJ, USA) M. smegmatis M. tuberculosis (OADC required, see Table 5.4) 7H10 agar (tc) 0.5 g (NH4)2SO4 M. smegmatis 1.5 g KH2PO4 M. tuberculosis (OADC required, see

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1.5 g Na2HPO4 Table 5.4) 0.4 g Sodium citrate 25 mg MgSO4 0.5 mg CaCl2 1 mg ZnSO4 0.5 mg L-Glutamic acid 40 mg Ammonium iron (III)-citrate 1.0 mg Pyridoxine hydrochloride 0.5 mg Biotin 0.2% Glycerol 0.05% Tyloxapol 12.6 g Agar Noble Hartman De Bond (Smeulders et al. 2.0 g (NH4)2SO4 M. smegmatis 1999) 1.45 g K2HPO4 · 3H2O M. tuberculosis 0.85 g NaH2PO4 · H2O 0.01 g EDTA · 2H2O 0.1 g MgCl2 · 6H2O 1.0 mg CaCl2 · 2H2O 2.0 mg NaMoO4 · 2H2O 0.4 mg CoCl2 · 6H2O 1.0 mg MnCl2 · 4H2O 2.0 mg ZnSO4 · 7H2O 5.0 mg FeSO4 · 7H2O 0.5 % Glucose 12.6 g Agar Noble (for solid media only) Sauton’s 0.56 g KH2PO4 M. smegmatis 0.56 g MgSO4 4.4 g L-Asparagine 56 mg Ferric Ammonium Citrate 67 mL Glycerol 2.2 g Citric acid 0.11 mL of 1% ZnSO4 pH 7.4 Brain Heart infusion broth 37 g BHI broth base BHI broth base is commercially available through BD Bacto, Franklin Lakes, NJ, USA) C. glutamicum BHI agar 37 g BHI broth base BHI broth base is commercially 15 g agar available through BD Bacto, Franklin Lakes, NJ, USA) C. glutamicum RPMI 1640 Ready to use liquid medium, THP-1 macrophage, includes amino acids, vitamins, Peritoneal macrophages inorganic salts, glucose and other Requires supplements: Heat components, formulation is available inactivated FBS (10%) and penicillin- from Invitrogen.com streptomycin-glutamate solution (1%) (see Table 5.4)

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Table 5.4: Media supplements

Supplements (stock Composition per liter Notes / Final concentrations concentration) OADC (Oleic acid-albumine- 0.5 g oleic acid required at 10% (v/v) in Middlebrook dextrose-catalase additive for 20 g dextrose 7H9 and 7H10 media for growth of Middlebrook media) 8.5 g NaCl M. tuberculosis (Difco) 40 mg catalase 50 g BSA fraction V Hygromycin (200 mg/mL) - 50 μg/ml for mycobacteria 200 μg/ml for E. coli Kanamycin (30 mg/mL) - 30 μg/ml for E. coli 30 μg/ml for mycobacteria Carbenicillin (100 mg/mL) - 100 μg/ml for E. coli at various concentrations for mycobacteria Chloramphenicol (25 mg/mL) - 6 μg/mL for C. glutamicum 25 μg/mL for E. coli Congo red (10 mg/mL) - 100ug/mL Sucrose (50 %, w/v) - 2% (w/v) for M. tuberculosis 10% (w/v) for M. smegmatis Copper sulfate (100 mM) - Used at various concentrations for mycobacteria Malachite green oxalate (3 mM) - Used at various concentrations for mycobacteria SDS (10 %) - Used at various concentrations for M. smegmatis and C. glutamicum heat inactivated FBS - 10 % (v/v) in RPMI 1640 Penicillin-Streptomycin-Glutamine 10,000 units/ml penicillin 1% (v/v) in RPMI 1640 solution (100x) 10 mg/ml of streptomycin 29.2 mg/ml of L-glutamine 10 mM citrate buffer (pH 6.0) 1.4 mg/ml NaCl

5.3 Protein analysis by SDS-PAGE and Western Blot

All protein samples were mixed with protein loading buffer (160 mM Tris-HCl pH 7.0, 12 % SDS, 32 % glycerol, 0.4 % Bromophenol blue) and boiled when required for 10 min or incubated at 37 °C for 1 h before loading on a 10 % SDS-PAGE gel. The PAGE was carried out using an anode buffer (0.1 M Tris-HCl pH 8.9) and a cathode buffer (0.1 M Tris-HCl, 0.1 M Tricine, 0.1 % SDS, pH 8.25) (Sambrook et al. 1989). The polyacrylamide gel was electroblotted overnight at 50 mA in transfer buffer (25 mM Tris-base, 192 mM Glycine, 0.1 % SDS, 20 % methanol) onto a polyvinylidene difluoride (PVDF) membrane. Subsequently, the membrane was blocked with 5 % fat free milk powder (Difco) in TBST (1.5 M NaCl, 0.5 % Tween 20, 0.1 M Tris-HCl, pH 8). Afterwards the membrane was probed with primary antibodies and horse radish conjugated secondary antibodies diluted in TBST. After each incubation step with an antibody containing solution the membrane was washed three times in TBST for 5 min. Immunoblots were developed using ECL Western blotting substrate according to the manufacturer’s recommendations.

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5.4 Methods for nucleic acid isolation, recombination and detection

5.4.1 Construction of plasmids

All plasmids utilized in this work and their features are listed in table 5.6. Plasmid construction is schemed in figure 5.1; oligonucleotides used throughout this study are listed in table 5.5. The temperature-sensitive vectors pML926 and pML2685 were used for the deletion of the C- terminal hydrophobic helix of ms3748 in M. smegmatis and rv1697 in M. tuberculosis, respectively. Deletion of rv1697cth in M. tuberculosis. The plasmid pML2685 is a derivative of the empty gene deletion vector pML2424 (Ofer et al. 2012). The approximately 1000 bp long homologues regions upstream and downstream of the rv1697cth were stepwise introduced. The upstream region was amplified by PCR from M. tuberculosis chromosomal DNA using primer pairs 2329/2328 and introduced into pML2424 after digestion with NsiI and PacI to yield the plasmid pML2684. The downstream region was amplified using the primer pair 2330/2331 and introduced into pML2684 using the restriction site SwaI and SpeI such that up- and downstream regions flanked the loxP-mycgfp2+-hyg-loxP fragment to yield the resulting plasmid pML2685. Deletion of ms3748cth in M. smegmatis. The knockout vector pML926 was constructed by amplification of the upstream region of ms3748cth with primers 1129 and 1128, and downstream region with 1131 and 1130. The upstream region was digested with BfrBI and PacI and cloned into similarly digested pML522 resulting in plasmid pML925. The downstream region was phosphorylated with T4 polynucleotide kinase after amplification and ligated into with SwaI linearized plasmid pML925 to yield the plasmid pML926. Deletion of cgR_1476 in C. glutamicum. In order to delete the gene cgR_1476 in C. glutamicum the deletion vector pK19-ΔmctA was constructed by using the empty pK19mobsacB C.glutamicum deletion vector. Two approximately 800 bp long fragments upstream and downstream of cgR_1476 were amplified from C. glutamicum genomic DNA using primer pairs mctA-Del-up-s2 / mctADel-up-rev and mctADel-down-for / mctADel-down-as2, respectively. The primers mctADel-up-rev and mctADel-down-for share a complement region that allowed in a second PCR the fusion of these DNA fragments using the primer set mctA-Del-up-s2 / mctADel- down-as2 and the two 800 bp fragments as templates. The resulting 1600 bp long DNA fragment and the vector pK19mobsacB were digested with XbaI and ligated to yield the plasmid pK19-ΔmctA. Vectors for complementation of deletion mutants and beta-lactamase repoter assay. For complementation of M. smegmatis mutants the shuttle vector pMN016 containing the strong

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mycobacterial promoter psmyc was chosen as a parent vector to construct various mycobacterial gene expression vectors for complementation of mutants and to express BlaTEM1 fusion proteins. The M. smegmatis gene ms3748 was amplified from genomic DNA using the primer pair 1168/1165. To obtain a 6xHis tagged version of Ms3748 the primer pair 1168/1166 was used. The obtained PCR fragments were digested with PacI and SwaI and ligated into pMN016 digested with the same enzymes resulting in pML950 (ms3748His) and pML951 (ms3748). To obtain complementation vector pML961 expressing rv1697, PCR amplification was performed using the primers 1255 and 1261, followed by digestion with PacI and HindIII and ligation into similarly digested pMN016. In order to construct an integrative rv1697 expression vector the promoter pUV15tetO was cloned from vector pSE100 into the empty Ms6 integration vector pML1222 using the restriction enzymes SpeI and HindIII yielding the plasmid pML2687. Afterwards the gene rv1697 was cloned in front of the pUV15tetO promoter from pML961 using the enzymes HindIII and PacI resulting in vector pML2688. In order to express the entire ms3748-ms3747 operon the gene ms3747 was amplified using the ms3747 expression vector pML451 as a template and the primer pair 1823/1822. The resulting PCR fragment was digested with HindIII and SwaI and ligated into the vectors pML950 and pML951 digested with the same restriction enzymes to yield the resulting vectors pML2655 and pML2654, respectively. The rv1697-rv1698 operon was amplified from M. tuberculosis genomic DNA using the primer pair 2879/2877 and fused by two step PCR with the 500 bp long promoter region upstream of cgR_1476 amplified using the primer pair 2880/2878. The resulting PCR fragment and the C. glutamicum shuttle vector pCGL487 were digested with PstI and BamHI and ligated to yield the plasmid pML3111. The BlaTEM1 expression vectors were constructed using the expression vector pMN016 as parent. The gene blaTEM1 was amplified from E.coli genomic DNA as full length and as leader less (‘blaTEM1) construct using the primer pairs 1734/3053 and 2196/3053, respectively. The resulting PCR fragments were digested with HindIII and PacI and ligated into pMN016 digested with same restriction enzymes to yield the vectors pML2165 (blaTEM1) and 2167 (‘blaTEM1). A translational fusion of Ms3748 and ‘BlaTEM1 was generated by fusing their genes using two step PCR and the primer pairs 682/3055 and 3053/3054 and as template the vectors pML951 and pML2167, respectively. During the second PCR amplification the two PCR fragments were fused using the primer pair 682/3054 to yield the ‘blaTEM1 fragment fused to the 3’ end of ms3748. Both, the PCR fragment and pMN016 were digested with HindIII and PacI and resulted in the plasmid pML2741 after ligation. In order to construct an ms3748-‘blaTEM1 fusion in an operon with ms3747 the primer pair 2885/2884 was used to amplify ms3747 by PCR from

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vector pML451, digested with HindIII and ClaI and ligated with pML2741, digested with the same restriction enzymes. An N-terminal translational fusion of Ms3748 with ‘BlaTEM1 was generated by two step PCR using the primer pairs 2619/2875 and 2874/894 to amplify the genes ‘blaTEM1 and ms3748, respectively. In the second PCR the primer pair 2619/894 was used to fuse the genes. Subsequential, the obtained PCR fragment and the vector pML2654 were digested with SphI and MluI and ligated to give pML2755.

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Figure 5.1: Cloning strategy for plasmids. Plasmid designations are given in gray boxes. The oligonucleotide pairs used for PCR amplification and utilized restriction enzymes for cloning are listed in white boxes. If chromosomal DNA was used as template for PCR the originating organism is indicated. In cases where a DNA fragment was obtained by digestion of a plasmid, the length of the utilized fragment is indicated. Two step PCR is indicated by four oligonucleotides and two templates in one white box. The oligonucleotide pairs used during the first reaction are separated by forward slashes and the used template DNA is noted below each primer pair. During the second PCR the outermost primers were employed.

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Table 5.5: Oligonucleotides,

Oligonucleotide Sequence (5’3’) 682 GCCTCGCCTTCCATCTCG 843 GCCAGCGCCAGGAAGACCGC 879 GCGGACCCGATGACCTGAAG 888 CGATTGCTGCGTAGGGTCTG 892 GCGACATCGACCGACTGCTC 894 GTAGACGCCGCCGTTGTGC 1075 GCTTTCACTGCGGATGAACTCAATT 1128 AGTACTGAAGATGTCAGCGCTGCTAAC 1129 AAATACGGCTGCGGTAGAGCGTG 1129 AAATACGGCTGCGGTAGAGCGTG 1130 CGTTAATTAATCGCGCGCCGACACCGCCGTG 1131 AACAACATGCATGACCGTCACCGAAGACGCAC 1165 AAATTAGTGGTGGTGGTGGTGGTGTCCACCCAGTGAGCCGATGATG 1166 AAATCATCCACCCAGTGAGCCGATGATG 1168 GCATGCTTAATTAAGCAGAAAGGAGGTTAATCTATGAAGATGTCAGCGCTGCTAAC 1255 CAAAGCTTCTAGGAGACCAAGTGCTGCAC 1261 CCGCATGCTTAATTAAGCAGAAAGGAGGTTAATCTATGAGGATGTCAGCGCTTCTGTCCCGTAAC 1734 CGTTAATTAAAGAAAGGAGGTTAATATGAGTATTCAACATTTCCG 1822 TAGTAAGCTTCTACTGCGGGACCGTCACCGAAG 1823 ATGCAATTTAAATATGATAACGCTACGGGCGCA 2196 CGTTAATTAAAGAAAGGAGGTTAATATGGCTCACCCAGAAACGCTGG 2328 ATATACTTAATTAAGTGGTTGCGGTAGAGCGTGG 2329 AATTGATGCATCATGAGGATGTCAGCGCTTC 2330 ATGATTTAAATTCCCGCACCGACGGCGTGGT 2331 ATTACACTAGTGACATCGCCCCGTGACCGGT 2619 CCGCCCGAAATGAGCACGATCCG 2874 CTCACCACCACCACCACCACTCTATGAAGATGTCAGCGCT 2875 GTGGTGGTGGTGGTGGTGAG 2877 CAGTGGATCCTACTGGGAAACCGTGACTG 2878 TGTGGAACCAAAGTCTCAACCTTTG 2879 CAAAGGTTGAGACTTTGGTTCCACAATGAGGATGTCAGCGCTTCT 2880 ATCGCTGCAGTGAGGTTGCTTTGACCTCCATTG 2884 TCATATCGATCTACTGCGGGACCGTCACCG 2885 TGATAAGCTTTATGATAACGCTACGGGCGCA TAAAGCTTTAGTGGTGGTGGTGGTGGTGAGTACTGGCGTAGTCCGGCACGTCGTACGGGTAGAT 3053 ATCCCAATGCTTAATCAGTGAGG 3054 GGAGCGGCCGCTGCTCACCCAGAAACGCTGGT 3055 AGCAGCGGCCGCTCCACCCAGTGAGCCGATGATGC GCGCGCTCTAGACGGAAAGCCCAAATCCGCCTA mctA-Del-up-s2 mctADel-up-rev CCTATTTGAACCCGCCCATTGTGGAACCAAAGTCTC mctADel-down-for CAATGGGCGGGTTCAAATAGGAAGGCAACATGG mctADel-down-as2 GCGCGCTCTAGATCAGGGTTGGCACGAAGCCGT The introduced restriction sites are underlined.

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Plasmid Parent vector, relevant genotype and properties Source or reference pCGL482 derivative wild type plasmid pBLI, CmR, 6270 bp (Peyret et al. 1993) pCreSacB pgroEL Cre, oriE, oriM, sacR, sacB, aph; 7891 bp gift from Dr. A.J.C. Steyn pK19mobsacB pMB1 origin, onT (RP4), lacZ, sacB, kanR, 5660bp (Schafer et al. 1994) pK19-ΔmctA pK19mobsacB, up-cgR_1476, dwn-cgR_1476 this study pML1222 ColE1 origin, Ms6 attP, loxP-hyg-int-tdTomato-loxP, 5037 bp this study pML1948 pMS2, psmyc ms3747-ΔSSblaTEM1 HA-tag HIS-tag, 7287bp Siroy et al., manuscript in preparation pML2165 pMS2, psmyc blaTEM1 HA-tag HIS-tag, 6426 bp this study pML2167 pMS2, psmyc ΔSSblaTEM1 HA-tag HIS-tag, 6363 bp this study pML2424 pUC origin, pAL5000ts, sacR, sacB, pwmyc tdTomato, loxP-psmyc (Ofer et al. 2012) mycgfp2+-hygR-loxP, 9527 bp pML2654 pMS2, psmyc ms3748, ms3747, 7655 bp this study pML2655 pMS2, psmyc ms3748 HIS-tag, ms3747, 7673 bp this study pML2684 pML2424, up-rv1697cth, 10548 bp this study pML2685 pML2684, down-rv1697cth, 11509 bp this study pML2687 pML1222, loxP-hyg-int-tdTomato- puv15tetO –loxP, 5466 bp this study pML2688 loxP-hyg-int-tdTomato- puv15tetO–rv1697-loxP, 6623 bp this study pML2714 pgroEL Cre, pUC origin, pAL5000ts origin, pimyc pamCherrym1, aph; (Ofer et al. 2012) 7613 bp pML2741 pMS2, psmyc ms3748 - ΔSSblaTEM1 HA-tag HIS-tag, 7563 bp this study pML2755 pMS2, psmyc ΔSSblaTEM1 HA-tag HIS-tag- ms3748, ms3747, 8506 this study bp pML2757 pMS2, psmyc ms3748 - ΔSSblaTEM1 HA-tag HIS-tag, ms3747, this study 8463 bp pML3111 pCGL482, pnative cgR_1476 rv1697-rv1698 this study pML451 pMS2, psmyc ms3747, 6469 bp (Wolschendorf et al. 2011) pML522 pUC origin, pAL5000ts origin, sacR, sacB, xylE, loxP-psmyc- (Wolschendorf et mycgfp2+-hyg-loxP, 9850 bp al. 2011) pML925 pML522, down-ms3748cth, 10872 bp this study pML926 pML925, up-ms3748cth, 11912 bp this study pML950 pMS2, psmyc ms3747 HIS-tag, 6735 bp this study pML951 pMS2, psmyc ms3747, 6717 bp this study pML961 pMS2, psmyc rv1697, 6695 bp this study pMN016 pMS2, psmyc mspA, 6164 bp (Stephan et al. 2005) pMS2 ColE1 origin, pAL5000 origin, hygR, 5229 bp (Kaps et al. 2001) pSE100 pMS2, puv15tetO ,5538 bp (Klotzsche et al. 2009) Table 5.6: Plasmids

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5.4.2 Isolation of chromosomal DNA from M. tuberculosis

Cells of a 10 mL stationary phase culture were harvested by centrifugation. The pellet was resuspended in 0.5 mL chloroform/methanol (3:1, v/v) and 0.5 mL Tris-buffered (pH 6.7) phenol/chloroform/isoamyl alcohol (25:24:1) were added. Freshly before usage β- mercaptoethanol was added to a final concentration of 1 % (v/v) to 0.75 mL GTC solution (4 M guanidium thiocyanate, 100 mM Tris-HCl, pH 7.5, 0.5 % Sarcosyl (w/v)) and added to the cell suspension. After careful resuspension with a pipette the solution was centrifuged (10,000 x g, 20 min, 4 °C). The upper layer was transferred into a new tube and the DNA was precipitated by addition of an equal amount of isopropanol. The precipitated DNA was washed twice with 70 % ethanol and allowed to dry at 37 °C before the pellet was resuspended in 100 μL water (Larsen et al. 2007).

5.4.3 Isolation of chromosomal DNA from M. smegmatis

The cell pellet of a 100 mL culture in stationary phase was resuspended in 3 ml TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA), 300 µl lysozyme (10 mg/ml), and 30 µl RNase (10 mg/ml) and incubated at 37 °C with shaking for 90 min. Afterwards, 210 µl of 20 % SDS and 60 µl Proteinase K (10 mg/ml) were added and the suspension was incubate at 65 °C for 45 min. After the addition of 600 µl of 5 M NaCl and 480 µl of 10% CTAB (cetyltrimethylammonium- bromide) solution incubation at 65 °C continued for another 30 min. To this suspension 4.5 ml of Tris-buffered (pH 6.7) phenol/chloroform/isoamyl alcohol (25:24:1) were added and carefully mixed with a pipette. After centrifugation (10,000 x g, 20 min, 4 °C) the upper layer was transferred into a new tube (Larsen et al. 2007). Precipitation of chromosomal DNA was carried out as described in 5.4.2.

5.4.4 Southern blot analysis

After isolation of chromosomal DNA from WT and mutant strains, 5 μg were digested with Dra/Xma or Pac/Xma for analysis of the M. smegmatis and M. tuberculosis genomic regions, respectively. After separation of the digested genomic DNA on a 1 % agarose gel, the gel was sequential incubated in depurination solution (0.25 M HCl), denaturation solution (1.5 M NaCl, 0.5 M NaOH) and neutralization buffer (0.5 M Tris-HCl, 1.5 M NaCl, pH 7.5) before the DNA was transferred to a positively charged nylon membrane by vacuum blotting using 10 x SSC (1.5 M NaCl, 0.15 M sodium citrate). The DNA was cross-linked to the membrane using a UV-

64 crosslinker (240,000 µJ) and prehybridized for 3 h at 42 °C in Dig-Easy hybridization solution (Roche). For analysis of the M. smegmatis ms3748Δcth and the M. tuberculosis rv1697Δcth genomic regions probes were generated by PCR from WT genomic DNA using the primer pairs 892/1129 and 888/1075, respectively. 3 ug of DNA-probe was randomly labeled with digoxigenin-dUDP overnight at 37 °C. Hybridization was carried out in the presence of 250 ng of digoxigenin-labeled PCR fragment at 57 °C and 48 °C overnight for the M. smegmatis and M. tuberculosis probe, respectively. The membrane was washed twice for 5 min at room temperature with 2 x SSC, 0.1 % SDS, and twice for 15 min at 68 °C with 0.1 x SSC, 0.1 % SDS. The hybridized digoxigenin-labeled probe was immune detected with an HRP-conjugated anti- digoxigenin antibody following the recommendations of the manufacturer (Roche, Tab 5.2).

5.4.5 Preparation of RNA from M. bovis BCG, M. tuberculosis and RT-PCR Experiments

Total RNA of M. tuberculosis H37Rv and M. bovis BCG were isolated by the TRIzol method as recommended by manufacture. Briefly, the cultures were grown in 100 ml of 7H9 medium supplemented with 10 % OADC. After addition of 35 ml of GTC buffer (5 M guanidium thiocyanate, 0.5 % sarcosyl, 0.5 % Tween 80, 1% β-mercaptoethanol) the cultures were centrifuged (10,000 x g, 10 min, 4 °C). The pellet was resuspended in 1.5 ml of TRIzol and lysed by agitation with glass beads (FastRNA Tubes-Blue) in a FastPrep© FP120 bead beater apparatus (Bio-101) for 3 × 45 s at level 6.5. Subsequential, 500 μl of chloroform was added to the suspensions, and the lysates were centrifuged (14,000 × g, 5 min, 4 °C). The upper phase was transferred to a new tube and an equal volume of isopropanol was added. The tubes were incubated for 20 min at –80 °C and centrifuged (14,000 × g, 20 min, 4 °C). The resulting pellet was washed with 70 % ethanol, dried, and resuspended in 100 μl of diethylpyrocarbonate- treated water (Ambion). Further purification of samples was performed using Nucleospin→RNAII kit (Macherey-Nagel) following the instructions of the manufacturer. RNA was sonicated to render DNA accessibility to DNase degradation (2 × 20 s at 20 % power) (Stephan et al. 2004). 5–10 μg of sonicated DNA was used for Turbo DNase treatment, which was done according to the manufacturer protocol (Ambion). The cDNA synthesis was performed using SuperScript III first strand synthesis system for RT-PCR (Invitrogen) according to the manufacturer protocol using random primers. AccuPrime Pfx SuperMix (Invitrogen) was employed for the PCR using primers #879 and #843. Thirty five cycles (30 s at 95 °C, 30 s at 58 °C, 30 s at 68 °C) were run to amplify the cDNA. The PCR products were analyzed using 1% agarose gels, which were stained with ethidium bromide.

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5.5 Construction of deletion mutants

The strategy for gene deletion in M. smegmatis and M. tuberculosis are based on thermosensitive deletion vectors that replicate only at lower temperatures (Song et al. 2009). If mycobacterial cells that carry such a thermosensitive deletion vector are switched from growth at temperatures less than 37 °C to growth at higher temperatures (>40°C), the vector cannot be passed on to progeny. Hence, only cells where the vector integrated into the genome will maintain the ability to replicate at these temperatures in the presence of the positive selection marker hygromycin and will form colonies on plates. Furthermore, deletion of the target genes requires two consecutive DNA crossover events between the homologues upstream and then between the homologues downstream regions (or vice versa) that are on the deletion vector and in the chromosome. This double crossover leads to the replacement of the chromosomal deletion sequence by a vector encoded gene deletion reporter cassette. This reporter cassette is flanked by loxP sites and is therefore excisable from the chromosome via the activity of Cre recombinase, if necessary. During the double crossover event, the backbone of the deletion vector is being removed. A positive deletion mutant candidate is therefore characterized by the absence of backbone markers (sucA/B and tdTomato or xylE) and the presence of markers which are part of the gene deletion reporter cassette (hyg and mycgfp2+) (Ofer et al. 2012) (Song et al. 2009).

5.5.1 Construction of ms3748cth deletion mutant in M. smegmatis SMR5

A hydrophobic helix of Ms3748 was predicted spanning from amino acid V345 to amino acid V367 (TMHMM server v. 2.0). Therefore, the ~ 1 kb fragments upstream and downstream of this hydrophobic helix were cloned into the deletion vector pML522 which carries the reporter genes mycgfp2+ and xylE to yield the ms3748cth deletion vector pML926. This thermosensitive deletion vector was transformed into M. smegmatis SMR5; colonies were selected on 7H10/Hyg

plates at 32 °C and then transferred into 7H9 liquid medium and grown at 32 °C until an OD600 of ~1.0 was reached. Of that culture, ten-fold serial dilutions were plated on Middlebrook 7H10 supplemented with 10 % sucrose and hygromycin and incubated at 40 °C to select for a double crossover mutant. A double crossover that causes the excision of the plasmid backbone was identified in by the loss of the reporter gene xylE and did not turn yellow in the presence of 1 % catechol. The double crossover of the M. smegmatis mutant ML198 was selected for six days before a colony was transferred into liquid medium. The remaining loxP-mycgfp2+-hyg-loxP cassette was removed using the vector pCreSacB1. Transformed colonies were selected

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7H10/kanamycin plates and subsequently grown in Middlebrook 7H9/kanamycin. Appropriate dilutions were plated on Middlebrook 7H10 medium supplemented with 10 % sucrose to counter select for pCreSacB, and incubated at 37 °C. After four days four mycgfp2+ negative colonies

were picked and grown in Middlebrook 7H9 broth. After the culture reached an OD600 of approximately 1.0, 5 μl of the cultures were dropped on Middlebrook 7H10 plates containing either kanamycin, hygromycin or no antibiotics. All colonies were sensitive to both antibiotics indicating that the hyg cassette had been efficiently excised and pCreSacB1 was removed. The correct genotype of the unmarked mutant M. smegmatis ms3748Δcth was confirmed by southern blot analysis and received the designation ML199.

5.5.2 Construction of rv1697cth deletion mutant M. tuberculosis mc26206

Similar to the M. smegmatis deletion strategy (sections 5.5, 5.5.1) we constructed a thermosensitive deletion vector to create an rv1697Δcth mutant of M. tuberculosis mc26206. However, the xylE reporter gene in the vector backbone was replaced by the gene of the red fluorescence protein TdTomato for practical reasons. The approximately 1 kb fragments of upstream and downstream region flanking the predicted (TMHMM server v. 2.0) C-terminal helix were cloned into the deletion vector pML2424 which carries the backbone reporter gene dt- tomato and the loxP-mycgfp2+-hyg-loxP KO reporter cassette, to yield the rv1697cth deletion vector pML2685. The vector pML2685 was transformed into M. tuberculosis mc26206 and selected on 7H10/hyg plates. After 28 days at 37 °C a colony was transferred in 10 mL of

properly supplemented 7H9/hyg medium. After the OD600 exceeded 1.0, ten-fold serial dilutions were plated on Middlebrook 7H10 supplemented with 2 % sucrose and hygromycin and incubated at 40 °C to select for a double crossover mutant that carries the loxP-mycgfp2+-hyg- loxP gene deletion reporter cassette in place of the C-terminal helix within the chromosomal rv1697 gene. After 16 weeks of growth, colonies of mutant candidates lacking red fluorescence, which were indicative for the absence of the tdTomato backbone reporter gene, were transferred into properly supplemented 7H9 medium. The culture was grown to stationary phase and then plated on Middlebrook 7H10/hyg after filtration through a 5 μm filter to remove contaminating wild type and single crossover mutant cells. Southern blot analysis confirmed the occurrence of the double crossover. The confirmed rv1697Δcth mutant of M. tuberculosis mc26206 received the designation ML910. To remove the loxP-mycgfp2+-hyg-loxP KO reporter cassette, competent cells were transformed with the thermosensitively replicating Cre- recombinase expression vector pML2714. Colonies were selected on 7H10/kan medium at 37

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°C. A gfp negative colony was picked and grown in Middlebrook 7H9 at 40 °C to prevent the plasmid pML2714 from replicating and to generate pML2714 free progeny. Ten-fold serial dilutions of that culture were then plated on Middlebrook 7H10 and grown at 40 °C until colonies appeared. One colony was picked and grown in Middlebrook 7H9 to yield the unmarked rv1697Δcth mutant ML911. Loss of hygromycin and kanamycin resistance was confirmed by the inability to grow in the presence of either antibiotic what confirmed the removal of the gene deletion reporter cassette from the genome and the loss of pML2714.

5.5.3 Construction of a cgR_1476 deletion mutant in C. glutamicum

The homolog gene of M. tuberculosis rv1697 in C. glutamicum is cgR_1476. Unlike in M. tuberculosis and M. smegmatis, where we were only able to delete the region of the C-terminal hydrophobic helix, we succeeded to generate a complete cgR_1476 deletion in C. glutamicum. The deletion vector was cloned as follows: The upstream and downstream region of cgR_1476 were fused by two step PCR and the resulting ~1600 bp fragment was cloned into the C. glutamicum deletion vector pK19mobsacB (Schafer et al. 1994) to give pK19-ΔmctA. We then followed a published procedure for obtaining the unmarked ΔcgR_1476 mutant of C. glutamicum. Briefly, transformation of wild type C. glutamicum resulted in the genomic integration of pK19-ΔmctA into the chromosomal cgR_1476 gene via homologues recombination (first crossover). This single crossover mutant was then grown on plates containing sucrose to select for mutants in which a second crossover event had led to the removal of the plasmid backbone from the chromosome. Colonies were then screened for loss of resistance to kanamycin as indication for loss of the vector backbone. The deletion of cgR_1476 was then confirmed by PCR.

5.6 Methods to determine susceptibility against drugs and biocides

5.6.2 Drug sensitivity assays using microplate Alamar Blue assays

The MICs of antibiotics for M. smegmatis, M. tuberculosis strains can be determined by microplate Alamar Blue assay (MABA) (Franzblau et al. 1998). Briefly, M. smegmatis strains were scraped from 7H10tc, tyloxapol 0.02 %, hygromycin 50 μg/mL plates and resuspended in

5 mL 7H9tc medium. After filtration through a 5 μm filter the OD600 was adjusted to 0.1 and the cells were grown for 8 h at 37 °C. M. tuberculosis strains were grown to mid-log phase in 7H9/OADC/tyloxapol. To break flocculated cells all cultures were sonicated for 3 min in a

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sonication bath (Fischer, FS60H) before filtration. For the initial drug solutions the compounds were solubilized in various solvents. Ampicillin (10 mg/mL), cycloserin (15 mg/mL), ethambutol (10 mg/mL) and kanamycin (30 mg/mL) were solubilized in water. Moxifloxacin was solubilized in 20 mM sodium hydroxid to a concentration of 10 mg/mL. Stock solutions of erythromycin (5 mg/mL), rifampicin (1 mg/mL) and tetracyclin (15 mg/mL) were prepared in dimethyl-sufloxide. Isoniazid (10 mg/mL) was solubilized in methanol. Aqueous stock solutions were sterilized by microfiltration (pore size 0.02 μm) prior use. Subsequent two-fold dilutions were prepared in 0.1 mL of water within the wells of black 96-well plates with clear-bottom. The outer perimeter wells of a 96-well plate were filled with 200 μL of sterile water to minimize evaporation. The filtered

bacterial cultures were diluted in two-fold medium to an OD600 of 0.04. For the assay, 0.1 mL of cells in two-fold medium were added to the wells containing 0.1 mL of drug dilutions. Each microplate included no drug and no cells controls. Microplates were incubated on a microplate shaker at 37◦°C and 350 rpm. To develop the assay, 40 μL of Alamar Blue solution (equal amounts of Alamar Blue and 10% Tween 80) was added and incubation continued until the no drug control produced a visible color change (blue to pink). Plates were analyzed using a microplate reader (SynergyHT, Biotek) in bottom-reading mode at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Percent viability was defined as (test well FU/mean FU of triplicate drug-free wells) x 100%. The lowest drug concentration inhibiting viability by ≥ 90% was considered the MIC. The MIC of ethambutol and isoniazid was determined by visual inspection (MABAvis).

5.6.3 Drug screening conditions and multi-dose response curves

To screen for drugs that induce a copper sensitive phenotype, we compared the activity of compounds against M. smegmatis and M. tuberculosis in the presence or absence of copper. M. tuberculosis was taken from frozen seed cultures and grown in HdB medium not exceeding 2 an OD600 of 2.0. Cell aggregates of M. smegmatis and M. tuberculosis mc 6230 in primary cultures were removed by filtration through a 5 μm filter. The filtrate was then diluted in 2-fold

HdB medium to an OD600 of 0.04. In 96-well plates, 100 μl of the bacterial cell suspension and 100 μl of the adjusted copper/compound mixture or the compound without extra copper were combined. Plates were sealed with parafilm, placed in plastic bags and incubated for about seven generation times (M. smegmatis: 18 h; M. tuberculosis: 7 days) at 37 °C. Alamar Blue was added as described under 5.6.3 and fluorescence was read on a SynergyHT plate reader instrument (BioTek).

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5.6.4 Activity of compounds against non-growing M. tuberculosis

To induce starvation, M. tuberculosis mc26230 was grown to stationary phase and washed three times with PBS/tyloxapol. Afterwards the cell pellet was resuspended in PBS/tyloxapol and cultured for six weeks. Then the drugs were titrated on the cells in the presence and absence of copper as described under 5.6.3. After eight days of exposure, a 5 μl sample aliquot was transferred onto Middlebrook 7H10 solid medium to evaluate bacterial survival. Growth was permitted for 16 days at 37 ºC. Rifampicin and isoniazid were included as controls.

5.6.5 Macrophage toxicity assays

Monocytic THP-1 cells were grown in RPMI medium supplemented with 10 % heat inactivated FBS and 1 % penicillin-streptomycin-glutamate solution. Peritoneal macrophages were isolated and cultured as previously described (Zhang et al. 2009). For toxicity assessment, 200,000 cells were seeded per well of a 96-well plate and compounds were added as indicated. After 24 h, Alamar Blue dye was added and metabolic activity was determined before dye conversion was completed in the untreated control wells according to the recommendations of the manufacturer (AbD Serotec).

5.6.6 Drop assays and BlaTEM1 reporter assay

Bacterial cells were grown on 7H10tc/tyloxapol/hygromycin plates. Afterwards, cells were scraped from the plate with a wooden stick and resuspended in a glass tube containing 5 mL PBS/tyloxapol using a mini vortexer. The cells were filtered through a 5 μm filter to break clumps

and yield a single cell suspension. After normalization of the OD600 to 1.0, 10-fold serial dilutions were prepared in PBS/tyloxapol. The 7H10tc agar was supplemented after cooling with the test compounds before pouring the plates. 3 μL of each dilution was deposited on the plates using a multi-channel pipette. The plates were incubated at 37 °C until single colonies of lowest dilutions reached a visible size.

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5.7 Uptake and accumulation assays

5.7.1 Uptake of 14C-chenodeoxycholate

The bacterial strains were grown on 7H10tc/tyloxapol plates and resuspended with a wooden stick into 7H9tc/tyloxapol. After filtration of the cell suspensions through a 5 μm filter a 50 mL

culture of 7H9tc/tyloxapol was inoculated at OD600 0.1 and grown at 37 °C, 200 rpm for 12 h. The cell cultures were harvested (3,270 x g, 10 min, 4 °C) and washed twice in ice cold uptake

buffer (50 mM Tris pH 6.9, 15 mM KCl, 10 mM NH4SO4, 1 mM MgSO4, 0.02 % tyloxapol).

Afterwards the cell pellet was resuspended in 12 mL uptake buffer and the OD600 was adjusted to 2.5. For dry weight determinations 4 ml of the cell suspension was centrifuged (3,200 x g, 10 min), resuspended in 0.6 mL uptake buffer and applied onto a pre-weight 0.45 μm membrane filter (Spin-X). After centrifugation (16,000 x g, 2 min) of the filter tube the flow through was discarded and the weight of the filter was determined after drying at 60 °C in a roto concentrator. 2 mL of the in uptake buffer resuspended cell suspension were preheated to 37 ˚C for 15 min (450 rpm) to equilibrate the cells. 10 μl of 51.3 mCi/mmol 14C-chenodeoxycholate (ARC Inc.) were added to each 2 ml sample and the tube was inverted twice to ensure mixing. At each time point 200 μL aliquots were transferred into a membrane filter tube that already contained 400 μL of killing buffer (3 % formalin, 30 mM LiCl) and centrifuged immediately (16,000 x g, 2 min). After two additional washes with killing buffer the flow through was discarded and the filter tube was transferred into a scintillation tube. The radioactivity was measured in a liquid scintillation counter (Beckman).

5.7.2 Uptake of ethidium bromide

The cells were scraped from 7H10tc/tyloxapol plates, resuspended in 7H9tc/tyloxapol and filtered through a 5 μm pore size filter to break clumps. Afterwards the cells were normalized to ◦ an OD600 of 0.1 and grown for 8 h at 37 °C. Before harvesting the cells, the OD600 was measured and the cells were resuspended to an OD600 of 0.5 in uptake buffer (50 mM sodium

phosphate pH 7.6, 5 mM MgSO4, 0.02 % tyloxapol). Subsequential, 100 μL of the cell suspension was aliquoted into a 96-well plate and the plate was transferred into a micro plate reader. After reading the OD600 ethidium bromide was added to each well to a final concentration of 1 μg/mL. To reduce background the fluorescence was measured in black, clear-bottomed plates in bottom-reading mode at an excitation wavelength of 530 nm and an emission wavelength of 590 nm.

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5.7.3 Accumulation of Congo red

Colonies of M. smegmatis were grown on 7H10tc plates containing Congo red (100 ug/mL) until the colonies reached a diameter of approximately 0.5 cm. The cells were carefully scraped from the plate and transferred into tubes. After drying the cells in a vaccum concentrator (Eppendorf, vacufuge) for 30 min at 60 °C the dry weight was determined. 3 mg of dried cells were extracted with 0.5 mL DMSO for 2 h in a heated sonication bath (Fisher, FS60H). After centrifugation (8,000 x g, 5 min) the supernatant was removed and the absorbance at 585 nm was determined in a 96-well plate using a microplate reader. For quantification of the extracted Congo red dye a standard curve was generated.

5.8 Isolation of subcellular protein fractions, lipids, mAGP and MVs

5.8.1 Lipid extraction and analysis

The M. smegmatis strains were grown as surface pellicles in a static culture at 37 °C in 200 mL Sauton’s medium for six days. After harvesting the cells by pouring off the medium the cells were transferred into a glass bottle and resuspended in 50 mL chloroform/methanol (1:2). The cell suspension was extracted for 24 h under vigorous stirring with a magnetic stirring bar. After separation of insoluble material by centrifugation (3,000 x g, 15 min) the supernatant was removed and transferred into a glass bottle. The pellet was resuspended in 10 mL of chloroform/methanol (2:1) and extracted for further 24 h using a magnetic stirring bar. The suspension was centrifuged (3,000 x g, 15 min) to remove the lipid containing supernatant from delipidated cells. The weight of the delipidated cells was determined after drying under vaccum at 30 °C. The supernatant from both extractions were combined and washed twice with equal amounts of 0.3 % aqueous sodium chloride. The lower organic phase was dried using a rotary evaporator (Eppendorf, vacufuge) and resuspended in chloroform according to the dry weight of the delipidated cells. This fraction contains the extractable lipids of M. smegmatis. In order to analyze the mycolic acids that are covalently linked to the mycobacterial cell envelope 80 mg of dried delipidated cells were resuspended in 3.5 mL of 15 % tetrabutylammonium hydroxide and refluxed at 100 °C overnight. After cooling to room temperature 3.5 mL water, 5 mL dichlormethane and 0.5 mL iodomethane were added to the reaction. The mixture was shaken on a platform for 4 h. After centrifugation the upper layer was discarded, the lower layer was washed three times with 10 mL water. The organic layer was dried under vacuum, extracted with 7 mL diethylether in a sonication bath (Fisher, FS60H) for 5 min. The insoluble parts were

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removed by centrifugation and the supernatant was dried and resuspended in 0.4 mL chloroform (Phetsuksiri et al. 1999). All lipid fractions were applied to 5 x 10 cm thin layer chromatography (TLC) glass plates (Macherey-Nagel, 0.25 mm silica gel 60, UV254) for TLC analysis and developed using several solvent systems. Sugar-containing compounds were visualized by spraying 0.2 % anthrone in concentrated sulfuric acid and charring at 110◦°C. The Dittmer-Lester reagent (Sigma) was used for revealing phosphorus-containing lipids. To visualize mycolic-acids, the TLC plates were sprayed with a 10 % copper sulfate in 8 % phosphoric acid solution and subsequently heated to 200◦°C. Where indicated an ethanolic solution of 20 % phosphomolybdic acid was used to visualize lipids on TLC plates after heating.

Lipids were identified either by their Rf value published previously (Deshayes et al. 2010) (Gil et al. 2010) or by running a standard on the same TLC plate. The following solvent systems were used. System A: petroleum ether/diethyl ether (7:3, vol/vol); B: chloroform/methanol (90:10, vol/vol); C: chloroform/methanol/water (60:16:2, vol/vol); D: chloroform/methanol/water (60:35:8, vol/vol); E: chloroform/methanol/water (20:4:0.5, vol/vol); F: three times n-hexane/ethylacetate (95:5, vol/vol).

5.8.2 Isolation of mAGP

Bacterial cell culture of C. glutamicum were grown to stationary phase and harvested by centrifugation (5,000 x g, 15 min, 4 °C). The cell pellet was resuspended in breaking buffer (2 % w/v Triton X-100 in PBS) at a concentration of 0.5 g (wet weight)/ mL. Afterwards the cell suspension was sonicated on ice (10 cycles, 60 s on, 90 s off, output 18 W). Cell walls were always separated from soluble material by centrifugation (27,000 x g, 15 min, 4 °C). After centrifugation the pellet was further extracted with breaking buffer overnight at 4 °C. The insoluble cell wall material was removed by centrifugation and the resulting pellet was extracted three times with 2 % SDS in PBS at 95 °C for 1 h. After washing the pellet three times with water, two times with 80 % aceton and one time with aceton the cell wall material was lyophilized using a vacuum concentrator.

5.8.3 Analysis of mAGP by GC-MS

The purified mAGP was analyzed by gas chromatography coupled mass spectrometry (GC-MS) as described previously (Seidel et al. 2007). Briefly, for the sugar composition analysis the mAGP was hydrolyzed using 2 M trifluoroacetic acid and reduced with sodium borohydride. The resulting alditols were per-O-acetylated and analyzed by gas chromatography. For the linkage

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analysis of the arabinogalactan the mAGP was per-O-methylated before hydrolysis using 2 M trifluoroacetic acid. After reduction with sodium borohydride the sugar derivatives were per-O- acetylated and examined by GC-MS.

5.8.4 Subcellular fractionation of M. smegmatis

50 mL of an M. smegmatis SMR5 culture was grown in 7H9tc/tyloxapol until OD600 reached 2.6. The cells were harvested by centrifugation and washed twice in PBS (140 mM NaCl, 2 mM KCl,

10 mM Na2HPO4/NaH2PO4 pH 7.4). Afterwards, the cells were resuspended in PBS (0.25 g/mL) containing 1 mM phenylmethanesulfonylfluoride and lysed by sonication (450 cycles, 1 s on, 1 s off, output 12 W) (MISONIX sonicator 3000). The crude lysate was incubated with 50 U Benzonase (Novagen) and 1 mg/ml lysozyme at 37 °C for 1 h while shaking at 200 rpm. Cell debris and unbroken cells were removed by centrifugation (3,000 x g, 10 min, 4 °C) and the supernatant was diluted 5-fold with PBS (WCL). To separate soluble from insoluble proteins the lysate was centrifuged at high speed (135,000 x g, 1 h, 4 °C). The soluble protein containing supernatant (SN1) was removed and the pellet (P1) was resuspended in PBS to the original volume. Both fractions were ultra centrifuged a second time. The supernatant of SN1 was transferred into a new tube and contains soluble proteins (SN2). The insoluble proteins containing pellet of P1 was separated from the supernatant and resuspended in PBS to the original volume (P2). The fractions were analyzed by SDS-PAGE following Western blot analysis.

5.8.5 Isolation, quantification and purification of membrane vesicles

M. smegmatis and C. glutamicum cells were grown in 300 mL 7H9 medium supplemented with tyloxapol or in BHI medium, respectively, until the cells reached stationary phase. After harvesting the cells by centrifugation (5,000 x g, 4 °C, 15 min) the culture supernatant was filtered (0.22 μm, milipore) to remove remaining cells. The cell pellets were dried (Eppendorf, vacufuge) and the weight was determined. In order to collect into the culture medium released membrane vesicles the culture filtrates were centrifuged at high speed (75,000 x g, 4 °C ,1 h) and the obtained membrane vesicle pellets were resuspended in PBS according to the weight of the dried cell pellets. For further purification and analysis the membrane vesicles were subjected to analytical size exclusion chromatography. The chromatography was performed

using a TOSOH G3000SWXL column (Tosoh) on a Bio-Rad Duoflow HPLC chromatography system fit with a 250 µL injection loop, a flow rate of 0.5 mL/min. The column was calibrated

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with blue dextran (~2 MDa), apoferritin (443 kDa), bovine serumalbumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome C (12.4 kDa) molecular mass standards. PBS was used as a solvent for all chromatographic runs and the elution of proteins and vesicles was detected by

changes in OD280. Fractions containing the major peak were pooled. The lipids of these fractions were extracted using chloroform and methanol and analyzed by TLC.

5.8.6 Preparation of SUVs

Giant unilamellar vesicles (GUVs) were prepared by electroswelling using the Vesicle Prep Pro apparatus (Nanion, Munich) (Kreir et al. 2008). 300 µL of a GUV suspension in 1 M sorbitol (Aldrich) were prepared from 20 µL of a solution containing 10 mM 1,2-diphytanoyl-sn-glycero- 3-phosphocholine (DPhPC) and 1 mM cholesterol (Avanti Polar Lipids, Sigma) in chloroform. Electroswelling of the lipid film was performed between two indium tin oxide (ITO) coated glass slides at 36 °C under a voltage of 3 V and a frequency of 5 Hz for 2 h (Kreir et al. 2008). The resulting GUVs were used to produce mono-dispersed small unilamellar vesicles (SUVs) of 100 nm diameter by extrusion (10 passes) through a polycarbonate membrane (100 nm pore size) using a mini-extruder (Avanti Polar Lipids) at 25 °C. The resulting suspension of SUVs was stored at 4 °C.

5.9 Cross-linking and purification of Ms3748 and Ms3747

Ms3748xHis and Ms3747 were co-expressed from plasmid pML2655 in the phenotypic ms3748Δcth / Δms3747 deletion strain ML199. Cells were grown in 7H9tc/tyloxapol and harvested at an OD600 of 2.6. The cells were washed twice in PBS/tyloxapol and resuspended to 2 g / mL in the same buffer. Subsequential, the cells were cross-linked for 4 h at 4◦°C by adding formaldehyde to a final concentration of 1 %. At the same time a control sample was carried through all steps with no formaldehyde added. To ensure a homogenous suspension the cells were stirred using a magnetic micro stirrer. The reaction was quenched by adding glycine to a final concentration of 150 mM and additionally washed twice in PBS. Afterwards, the cross- linked cells were lysed by ultra sonication (450 cycles, 1 s on, 1 s off, 12 W output) and treated with DNase (75 U Benzonase) and lysozyme 1 mg/mL for 1 h at 37 °C. The lysate was fractionated by high-speed centrifugation (100.000 x g, 1 h, 4◦°C) into water soluble and insoluble proteins. The proteins of the water insoluble fraction were resuspended in PBS containing 1% SDS and extracted under constant stirring at 4◦°C overnight. After allowing the extract to warm up to 20 °C the protein extract were separated from insoluble parts by

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centrifugation (16,000 x g, 10 min). The Ms3748His cross-links and untreated sample were purified under denaturing conditions by nickel affinity chromatography using an HPLC (Bio-Rad

Duoflow). The protein extract was diluted 10-fold with binding buffer (50 mM Na2HPO4 pH 7.6, 100 mM NaCl, 8 M Urea, 5 mM Imidazole) and bound to the column. Afterwards, the column was washed with 50 column volumes of binding buffer. The bound protein was eluted over a gradient of elution buffer (500 mM imidzole, 8 M Urea). The major fraction containing untreated and cross-linked Ms3748His were pooled and analyzed by SDS-PAGE Western blotting. The cross-links were cleaved by boiling the purified protein samples at 95◦°C for 40 min. Extracts showing uncleaved cross-links of Ms3748His were incubated at 37◦°C for 1 h to ensure sufficient binding of SDS before running a PAGE.

5.10 Light microscopy

To determine the length of single M. tuberculosis cells, bacteria that reached stationary phase in Middlebrook 7H9 medium were subjected to light microscopy using a axiovert 200 microscope (Zeiss) equipped with an axiocam MRC camera. Pictures of individual cells were made at a magnification of 1,000 fold and their actual size was determined using Axiovision 4.5 software (Zeiss).

5.11 Transmission electron microscopy (TEM)

Membrane vesicles isolated from culture filtrate by centrifugation and SUVs were stained with uranyl acetate as described previously (Hans W. Ackermann 2010). Briefly, 2 % uranyl acetate (Sigma) was dissolved in water, filtered (pore size 0.22 μm) and mixed with the membrane vesicles in a ratio of 1:1 for 30 min using a stirring bar. A drop of the membrane vesicles and uranyl acetate containing solution was placed on an electron microscopic grid and excess liquid was removed using a filter paper. The grid was dried overnight and stored in a desiccator before microscopy was performed using transmission electron microscope (FEI Tecnai F20 FEG).

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7 Appendix

7.1 Supplementary results

Figure 7.1: Cells of the ms3748Δcth mutant of M. smegmatis aggregates in clumps. WT (SMR5), ML199 (ms3748Δcth) and the with pML2654 (ms3748-ms3747) complemented strain were inoculated with an OD600 of 0.05 in 7H9tc/tyloxapol and grown for 24 h at 200 rpm, 37 °C.

Figure 7.2: [14C] chenodesoxycholate uptake experiments for M.smegmatis. The uptake rate is expressed as pmol of chendeoxycholate per milligram of dried cells. The uptake experiment was done in triplicate and is shown with standard deviations. Graph shows WT (SMR5), ML199 (ms3748Δcth) and ML199 complemented with pML2654 (ms3748-ms3747). Experiment was carried out three independent times with similar result.

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Figure 7.3: Lipid analysis of whole cells and membrane vesicles of M. smegmatis. Labels: Lipids from WT (SMR5) are in lanes labeled 1. Lipids from ML199 (ms3748Δcth) are analyzed in lanes 2. ML199 single complemented with ms3747 (pML451) is analyzed in lanes labeled with 3. The single complemented strain ML199 with ms3748 (pML951) is analyzed in lanes labeled with 4. Lipids of the double complemented strain (ML199 pML2654) were analyzed in lanes labeled with 5. Fractions: TLCs a-g show lipid profiles extracted from whole cells. TLCs h-l contain lipid extracts from membrane vesicles and a WT lipid extract from whole cells as control (WC). Covalently linked mycolic acids were cleaved from delipidated cells and analyzed in TLC m. Solvents Systems: TLC a and h were resolved using solvent system A. For TLCs b, i, f the solvent system B was used. To resolve the lipids on TLCs c,j,d,k the solvent system D was used. Solvents system C was used to resolve the lipids on TLC g. TLC e and l were resolved using solvent system E, while TLC m was resolved using three times solvent system F. Visualization: Sugar containing lipids of TLCs b,I,d,k and g were visualized using anthrone. Mycolic acid containing lipids of TLCs e, l and m were visualized using copper sulfate in phosphoric acid. Phospholipids of TLCs c and j were visualized with Dittmer-Lester reagent. TLC f was visualized by spraying water. As a control, the first lane in TLC e and l show 4 mg of Mtb TDM (Sigma). GMM, glycerol monomycolate; GPL, glycopeptidolipids, TAG, triacylglycerols; TDM, trehalose dimycolate; TMM, trehalose monomycolate, PE, phosphatidyl ethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIM, phosphoinositolmannosides; Tre, Trehalose. The labels α, α’ and epoxy refer to the three types of mycolic acids that are produced by M. smegmatis.

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7.2 List of authors that contributed to this work:

Olga Danilchanka1: Isolation of mRNA and RT-PCR. Gregory J. Harber1 and Suzanne M. Michalek1: Isolation of peritoneal macrophages. Nadine Rehm2 and Andreas Burkovski2: Construction of plasmid pK19-ΔmctA and the ΔcgR_1476 strain. Tej B. Shrestha4 and Stefan H. Bossmann4: Synthesis of ATSM and GTSM. Axel Siroy1: Analysis of membrane vesicles by HPLC and construction of plasmid pML1948. Frank Wolschendorf1: Construction of plasmids pML925, pML926, pML950, pML951 pML961. Sophie Zuberogoitia3 and Jérôme Nigou3: Analysis of arabinogalactan by GC-MS.

Affiliations:

1) Department of Microbiology, University of Alabama at Birmingham, Birmingham, USA

2) Department of Microbiology, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany

3) Department “Molecular Mechanisms of Mycobacterial Infections” Institute of Pharmacology and Structural Biology University of Toulouse Toulouse, France

4) Department of Chemistry, Kansas State University, Manhattan, USA

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7.3 Abbreviations

AraT Arabinosyltranferases AG Arabino-galactan Araf Arabinofuranose ATP Adenosine triphosphate ATP7A Copper-transporting ATPase ATSM Diacetylbis(N-(4)-methyl-3- thiosemicarbazone) BCG Calmette–Guérin BSL Biosafty-level Dec-P Decaprenyl diphosphate DMAPP Dimethylallyl diphosphate DNA Deoxyribonucleic acid DPA Decaprenylphosphoryl-Araf EMB Ethambutol FDA US food and drug administration FU Fluorescent unit Galf Galactofuranose GFP Green fluoresent protein GlcNAc N-Acetylglucosamine GMM Glycerol monomycolate GPL Glycopeptidolipids GTSM Glyoxalbis(N (4)-methyl-3-thiosemicarbazone) HIV Human immunodeficiency virus HPLC High perfromance liquif chromatography HRP Horse redish peroxidase HTS High troughput screen IC Inhibitory concentration IL Interleukine INH Isoniazid KO Knock out LAM Lipoarabinomannan LM Lipomannan loxP Recognitioin site for Cre-recombinase MABA Microplate alamar blue assay mAGP Mycolated arabinogalactan peptidoglycan polymer MDR Multi drug resistant MIC Minimal inhibitory concentration MOM Mycobacterial outer membrane NADH Nicotinamide adenine dinucleotide NFN-γ Interferon gamma NOS Nitrogen species OADC Oleic acid, albumin, dextrose, catalase ODx Optical density measured at λ= x nm OM Outer membrane OMV Outer membrane vesicle P Pellet PAGE Poly acrylamide gel electrophoresis PBS Phosphate Buffered Saline PCR Polymerase chain reaction PDIM Phthiocerol dimycocerosate PE Phosphatidyl ethanolamine; PG Peptido-glycan PG Phosphatidylglycerol; PI Phosphatidylinositol PIM Phosphoinositolmannosides PVDF Polyvinylidene fluoride RD Region of difference

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Rha Rhamnose RNA Ribonucleic acid ROS Reactive oxygen SDS Sodium docecylsulfate SN Supernatant SNP Single nucleotide polymorphisms TAG Triacylglycerols TB Tuberculosis TBST Tris-buffered saline with tween TDM Trehalose di-mycolate TDR Totally drug resistant TLC Thin layer chromatography TMM Trehalose mono-mycolate TPK Thiamine pyrophospho kinase Tris Tris-(hydroxymethyl-)aminomethan UDP Uracil-diphosphate WCL Whole cell lysate WHO World health organization WT Wild type

XDR Extensively drug resistant

Units Nucleotides U units A Adenine W watt C Cytosine ◦C degree Celsius G Guanine Ci Curie T Thymine Da Dalton g gram h hour L liter m meter M molar min minute s seconds

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