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Physiological functions of mycobacterial outer membrane channel proteins [Elektronische Ressource] / vorgelegt von Frank Wolschendorf

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62 pages
Als Dissertation genehmigt Physiological functions of mycobacterial von der Naturwissenschaftlichen Fakultät outer membrane channel proteins der Universität Erlangen-Nürnberg Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Tag der mündlichen Prüfung: 12. Dezember 2008 Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch vorgelegt von Erstbericherstatter: Prof. Dr. Wolfgang Hillen Frank Wolschendorf Zweitberichterstatter: Prof. Michael Niederweis aus Grobengereuth / Thüringen und: Prof. Dr. Mary Jackson (geb. in Pößneck) Danke / Thanks Mein besonderer Dank gilt Prof. Dr. Michael Niederweis für die Vergabe dieses Projektes, die ausgezeichnete Betreuung, seinen wissenschaftlichen Rat, seine ständige Diskussionsbereitschaft und die hervorragenden Arbeitsbedingungen. Insbesondere die meinen Eltern Martin und Beate, Etablierung des „mycolabs“ an der „University of Alabama at Birmingham“ war eine meinen Großeltern Paul und Annerose, großartige und bereichernde Erfahrung und ich danke ihm für sein Vertrauen. sowie meinem Großvater Günther Wolschendorf Ich bedanke mich auch recht herzlich bei Herrn Prof. Dr.
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   Physiological functions of mycobacterial outer membrane channel proteins         Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgradesvorgelegt von Frank Wolschendorf aus Grobengereuth / Thüringen (geb. in Pößneck)
Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 12. Dezember 2008 Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch Erstbericherstatter: Prof. Dr. Wolfgang Hillen Zweitberichterstatter: Prof. Dr. Michael Niederweis und: Prof. Dr. Mary Jackson
meinen Eltern Martin und Beate, meinen Großeltern Paul und Annerose, sowie meinem Großvater Günther Wolschendorf
I Schwierigkeiten nmitten der liegt die Möglichkeit. (Albert Einstein)
Danke / Thanks Mein besonderer Dank gilt Prof. Dr. Michael Niederweis für die Vergabe dieses Projektes, die ausgezeichnete Betreuung, seinen wissenschaftlichen Rat, seine ständige Diskussionsbereitschaft und die hervorragenden Arbeitsbedingungen. Insbesondere die Etablierung des mycolabs an der University of Alabama at Birmingham war eine großartige und bereichernde Erfahrung und ich danke ihm für sein Vertrauen. Ich bedanke mich auch recht herzlich bei Herrn Prof. Dr. Wolfgang Hillen, der meine Promotion an der Naturwissenschaftlichen Fakultät der Friedrich-Alexander Universität Erlangen-Nürnberg ermöglichte und unterstützte. Ferner danke ich den Professoren Dr. W. Hillen, Dr. M. Niederweis, Dr. M. Jackson, Dr. C. Koch und Dr. G. Kreimer entsprechend für die Erstellung der Gutachten und die Übernahme der Prüfungspflichten. I thank my former roommates and colleagues Hoffi, Jenni, Kristin, Elli and Didi for an unforgetable experience in Sweet Home Alabama. I am very thankful to Tobi for being so reliable in taking care of all the dirty things in the labratory. I have to thank Jonathan, Yoitsna, Veneet, Shveta and Jennifer for the excellent technical support by preparing media, buffers and countless other things. I also thank iron-Chris,attB-Jason, nitrate-Rachel, in-your-a..(peep)-Axel, robot-Ryan, nano-Mikhail, drug-Olga, Houhui the Great and oracle-guru Ying for making the mycolab the most awesome, brisky and fittest tubercle-lab in the world.Von ganzem Herzen bedanke ich mich bei meinen Eltern Martin & Beate sowie meinen Großeltern Paul & Annerose, die mich all die Jahre so großartig unterstützten und auf die ich mich immer verlassen konnte. Mein Dank geht auch an meine Großmutter Hermine und meinen Großvater Günther, an den ich mich gern zurückerinnere. Der ganzen Familie danke ich für die vielen schönen Stunden am Telefon und all die Karten und Hilfs-Packete, über die ich mich immer sehr gefreut habe. Überraschungseier sind der Hit!
Index v
1. Zusammenfassung .........................................................................................1 1. Summary .........................................................................................................2 2. Introduction.....................................................................................................3 2.1.ThegenusMycobacterium.....................................................................................3 2.1.1. Taxonomy.................................................................................................................................... 3 2.1.2. Evolutionary pathway of the tubercle bacilli ................................................................................. 3 2.1.3. Medical relevance of mycobacteria ............................................................................................. 4 2.2. The mycobacterial outer membrane and its proteins ............................................. 5 2.2.1. Transport processes across mycobacterial outer membranes .................................................... 6 2.2.2. Porin mediated diffusion of hydrophilic solutes inM. smegmatis................................................. 7 2.2.3. The role ofM. tuberculosisOmpA in outer membrane permeability ............................................ 8 2.2.4. A proteome-wide screen for outer membrane proteins ofM. tuberculosis................................... 9 2.2.5. Rv1698 ofM. tuberculosis ..................................10is a channel-forming outer membrane protein 2.3. Uptake of phosphates by mycobacteria ............................................................... 11 2.4. Copper metabolism in mycobacteria.................................................................... 12 2.4.1. Metabolic requirements for copper .............................................................................................12 2.4.2. Cytochromecoxidase ofM. tuberculosis.................................................................................1..2 2.4.3. Mycobacterial superoxide dismutase..........................................................................................13 2.4.4. Copper toxicity............................................................................................................................14 2.5. Copper transport mechanisms in bacteria ........................................................... 15 2.5.1. Gram-positive bacteria................................................................................................................15 2.5.2. Gram-negative bacteria ..............................................................................................................15 2.5.3. Mycobacteria ..............................................................................................................................16 2.6. Gene deletions by allelic exchange in mycobacteria ........................................... 17 3. Aims of this thesis ........................................................................................19 Part 1: The role of MspA in uptake of inorganic ions .................................................. 19 Part 2: The physiological role of the outer membrane channel protein Rv1698 in M. tuberculosis..................................................................19.......................................... 4. Materials and Methods .................................................................................20 4.1.Material................................................................................................................20 4.1.1. Bacterial strains ..........................................................................................................................20 
Index vi
4.1.2. Plasmids .....................................................................................................................................20 4.1.3. Oligonucleotides .........................................................................................................................22 4.1.4. Antibodies...................................................................................................................................22 4.1.5. Peptide library.............................................................................................................................23 4.2.Methods...............................................................................................................24 4.2.1. Standard protocols .....................................................................................................................24 4.2.2. Growth conditions .......................................................................................................................25 4.2.3. Cloning of deletion vectors .........................................................................................................26 4.2.4. Protocol for gene deletions in mycobacteria ...............................................................................28 4.2.5. Induction of gene expression by acetamide inM. smegmatis9...2.................................................. 5. Results...........................................................................................................31 5.1. Phosphates can diffuse through the MspA channel............................................. 31 5.2. Porin-mediated membrane permeability of phosphates inM. smegmatis........... 32 5.3. A new generation of gene deletion systems for mycobacteria ............................. 41 5.3.1. Construction of a new suicide deletion vector forM. smegmatis. ...............................................43 5.3.2. A new general gene deletion system for mycobacteria ..............................................................43 5.3.3. The introduction of reporter genes into conditionally-replicating deletion vectors.......................44 5.4. Rv1698 homologs in mycobacteria ...................................................................... 45 5.5. Deletion of putative outer membrane proteins in mycobacteria ........................... 46 5.5.1. Gene deletion protocol for mycobacteria ....................................................................................46 5.5.2. Deletion strategy forrv1698/msm_3747..36................................................................................. 5.5.3. Deletion ofmsm 3747inM. smegmatis5.6.................................................................................... _ 5.5.4. Deletion ofrv1698inM. tuberculosis........76.................................................................................. 5.6 The role of MctB in mycobacteria.......................................................................... 69 5.7. Surface accessibility of Rv1698 (MctB) by proteinase K...................................... 85 5.8. MctB is exported in a folded conformation ........................................................... 86 5.9. MctB in whole cells is not accessible to polyclonal antibodies ............................. 87 5.10. Monoclonal antibodies against MctB.................................................................. 88 5.10.1. Specificity of monoclonal antibodies .........................................................................................89 5.10.2. Epitope mapping.......................................................................................................................90 5.10.3. Cross-reactivity of hybridoma clone 5D1.23 .............................................................................91 
 
Index vii 6. Discussion.....................................................................................................93 6.1. The physiological function of Rv1698 and its homologs. ..................................... 93 6.2. The response ofM. tuberculosisto elevated copper levels ................................. 94 6.3. Copper as a defense mechanism againstM. tuberculosisin macrophages. ....... 96 6.4. Copper homeostasis inM. tuberculosis............................................................... 97 7. Conclusions................................................................................................100 8. Authors contribution .................................................................................101 9. Outlook ........................................................................................................102 9.1. Virulence ofM. tuberculosis 102mutants deficient in copper homeostasis.............. 9.2. Interaction of MctB with other proteins............................................................... 102 9.3.TheroleofMctA.................................................................................................103 10. References.................................................................................................104 11. Abbreviations ............................................................................................115              
Zusammenfassung 1 1. Zusammenfassung Mykobakterien sind von großer Bedeutung, da Tuberkulose die weltweit verbreitetste Infektionskrankheit mit 1.7 Millionen Todesopfern darstellt. Die außergewöhnlich niedrige Durchlässigkeit der mykobakteriellen äußeren Membran gilt als Hauptursache für deren Widerstandsfähigkeit gegenüber vielen Antibiotika. Äußere Membranproteine in Gram-negativen Bakterien sind wichtig für die Nahrungsaufnahme, Sekretion und deren Infektionsvermögen.Escherichia coli hat mehr als 60 verschiedene äußere Membranproteine. Keines hat Ähnlichkeit mit mykobakteriellen Proteinen. Bisher sind das Porin MspA vonMycobacterium smegmatisund das Kanal-bildende Protein OmpA vonMycobacterium tuberculosis einzigen bekannten mykobakteriellen die Membranproteine. Rv1698 vonM. tuberculosis wurde von uns als Kanalprotein der äußeren Membran mit unbekannter Funktion entdeckt. Zellen der entsprechenden Mutante reicherten 100-mal mehr Kupfer an als der Wildtyp. EineM. smegmatisMutante der das Homolog Msm_3747 fehlt, zeigte einen 11-mal höheren Kupfergehalt. Das Aufnahmevermögen für Glukose blieb jedoch unverändert. Diese Ergebnisse zeigen, dass diese Kanalproteine eine wichtige Rolle für die Ausscheidung von Kupferionen über die mykobakterielle äußere Membran spielen. Damit wurde die Ausscheidung von Kupfer als ein wichtiger Mechanismus identifiziert, mit dem Mykobakterien der Ansammlung von giftigen Kupferionen in der Zelle entgegenwirken. Rv1698 ist somit das erste mykobakterielle äußere Membranprotein mit nachgewiesener Ausscheidungs-funktion. Rv1698 hat kein Kupferbindemotiv. Ebenso fehlen der äußeren Membran die Energiequellen für den Transport entgegen dem Konzentrationsgefälle. Darum ist es wahrscheinlich, dass Rv1698 mit Kupfer-spezifischen Transportproteinen der inneren Membran interagiert. Diese sind in der Regel substratspezifisch und stellen die Energie für den Transport bereit. Darum ist zu vermuten, dass die mykobakteriellen Transportsysteme prinzipiell denen in Gram-negativen Bakterien ähneln. Kupfer über einer Konzentration von 25 µM hemmt das Wachstum vonM. tuberculosis auf künstlichem Nährmedium. Ähnliche Konzentrationen wurden in Phagosomen von Interferon- Makrophagen gemessen. Dies lässt vermuten, dass stimulierten Makrophagen das Wachstum vonM. tuberculosis Hilfe von Kupfer eindämmen. mit Aufnahmemechanismen für Kupferionen sind unbekannt. Wir haben gezeigt, dass Porinmutanten vonM. smegmatisim Gegensatz zum Wildtyp auf nahezu Kupfer-freiem Medium kaum wachsen und eine Toleranz bei erhöhtem Kupfergehalt zeigen. Kupferaufnahme erfolgt somit über Porine. Kanäle in der mykobakteriellen äußeren Membran sind daher unverzichtbar für die Aufnahme und Ausscheidung überschüssiger Kupferionen. Diese Ergebnisse erweitern dramatisch unser Verständnis über mykobakterielle Transportprozesse.
Summary 2
1. Summary Mycobacterium tuberculosis isof deaths resulting from a single the leading cause infectious disease with 1.7 million victims annually. The exceptionally low permeability of the outer membrane contributes to the intrinsic resistance of mycobacteria to many antibiotics. Despite the well-documented importance of outer membrane proteins for nutrient uptake, secretion, and host-pathogen interactions in Gram-negative bacteria, only the porin MspA ofM. smegmatis the channel-forming protein OmpA of and M. tuberculosis have been characterized as mycobacterial integral outer membrane proteins. By contrast,E. coli more than 60 proteins to functionalize its outer uses membrane, none of which has significant sequence similarity to anyM. tuberculosisprotein. Rv1698 ofM. tuberculosiswas discovered by us as an outer membrane channel protein with unknown function. Intracellular copper in anM. tuberculosismutant lacking Rv1698 was 100-fold increased. AnM. smegmatis lacking the close homolog mutant _ re copper than the wild-type, while uptake o g Msm 3747 accumulated 11-fold mo f lucose remained unchanged. These results demonstrated that Rv1698-like channel proteins are required for copper efflux across the mycobacterial outer membrane and that secretion of Cu+ a mechanism by which isM. tuberculosis maintains copper homeostasis to prevent copper toxicity. Rv1698 is the first identified mycobacterial channel protein that is involved in efflux across the outer membrane. In addition, Rv1698 lacks a predicted copper binding motive and there is no energy source in the outer membrane that would support efflux trough the Rv1698 channels against the concentration gradient. Thus, Rv1698 is likely recruited by Cu+ specific inner membrane translocases that determine substrate specificity and provide energy for the transport. These findings indicate that mycobacteria possess multicomponent efflux systems that are functionally similar to those of Gram-negative bacteria. We also found thatM. tuberculosis not grow at did Cu2+ µM. The amount of copper in phagosomes of concentrations above 25 macrophages stimulated with interferon- increases to similar concentrations after infection withM. tuberculosis. Thus, macrophages appear to utilize copper to control intracellular growth ofM. tuberculosis. Uptake pathways for the essential micronutrient copper are unknown in mycobacteria. However, anM. smegmatis porin mutant did not grow with trace amounts of copper (<1 µM), but was more resistant than wild-type, demonstrating that channel proteins are required for copper uptake across the outer membrane. These outer membrane channels are essential components of a considerably revised model of copper homeostasis inM. tuberculosis. The implications of these findings for our understanding of transport mechanisms and, in particular efflux systems, in mycobacteria are profound.
Introduction
3
2. Introduction 2.1. The genus Mycobacterium 2.1.1. Taxonomy Mycobacteria are Gram-positive aerophilic bacteria with a high G+C content and show a rough morphology with uneven formed branched cells. Taxonomically, mycobacteria belong to the genusMycobacterium is the single genus within the family of which Mycobacteriaceaeorder Actinomycetales. This order includes various the  in microorganisms, but mycobacteria and allied taxa are easily distinguished by their ability to synthesize mycolic acids (Rastogi et al., 2001). Mycobacteria possess the longest mycolic acids consisting of up to 90 carbon atoms (Barryet al.,1998) which confer acid-fastness to these bacilli. The genus is divided in slow- and fast-growing mycobacteria, which corresponds to phylogenetic data derived from 16S rRNA sequences (Rogall et al., Fast-growing species with generation times of less than 5 hours are mostly 1990). non-pathogenic, saprophytic soil bacteria such asMycobacterium smegmatis,M. phleiandM. chelonae. Slow-growing species have generation times of 20 hours and longer and are often pathogenic such asM. tuberculosis, the causative agent of tuberculosis (TB) andM. leprae, the pathogen causing leprosy. 2.1.2. Evolutionary pathway of the tubercle bacilli Speciation of recent members of theM. tuberculosis is estimated to have complex occurred during the last 15,000 to 20,000 years (Kapur et al., The complex 1994). consists ofM. tuberculosis,M. canettii,M. africanum,M. microtiandM. bovis(including M. bovisBCG). All members have identical rRNA sequences (Boddinghauset al.,1990; Broschet al.,2001) and exceptional little sequence variation resulting in 99.9% identity of their genomes at the nucleotide level (Sreevatsan al., et Musser 1997; al., et 2000). The subspecies can only be distinguished by a few phenotypic and genotypic characteristics but show great variety in terms of host range and pathogenicity (Brosch et al., 2001; Brosch al., et 2002).Before genome sequences were available it was believed thatM. tuberculosisevolved fromM. bovisby adaptation of an animal pathogen to the human host (Gonzalez-Flecha and Demple 1995). However, analysis of the genomes revealed thatM. bovis undergone numerous deletions relative to hasM. tuberculosisto be part of a separate lineage represented by therefore seems  andM. africanum,M. microtiandM. bovis. This group is defined by successive loss of DNA in
Introduction 4 relation toM. tuberculosis in decreasing genome sizes (Garnier resulting et al., 2003). There are 14 regions of difference (RD1-14) that are absent inM. bovisBCG relative to M. tuberculosis 10 of those regions have been used as evolutionary markers to and propose the evolutionary pathway of the tubercle bacilli within theM. tuberculosiscomplex (Fig. 2.1) (Broschet al.,2002).
Figure 2.1:Scheme of the proposed evolutionary pathway of the tubercle bacilli. The scheme is based on the presence or absence of conserved deleted regions and on sequence polymorphisms in five selected genes. Blue arrows indicate that strains are characterized bykatG463. (Leu), CTGgyrA95 ACC (Thr), typic9a5tecadiinsworraneerG.snismorgap1grouofrlA.ThThr) that strains belong to group 2 characterized bykatG463CGG (Arg),gyrA ( e CC red arrow indicates that strains belong to group 3, characterized bykatG463 (Arg), CGGgyrA95 (Ser), as defined by Sreevatsan AGC al., et 1997. The figure was taken from Broschet al.,2002. 2.1.3. Medical relevance of mycobacteria Mycobacteria are of great importance becauseM. tuberculosis the leading cause of is deaths resulting from a single infectious disease with 9.2 million new cases and 1.7 million deaths in 2006 (world health organization report 2008). The world health organization estimates that one third of the worlds population is infected and about 5 to 10% of infected people will become sick or infectious during their lifetime (WHO report, Factsheet No 104, revised 2007). Infection withM. tuberculosis not lead does
Introduction 5 unavoidably to disease, since the immune system can control the bacilli in check forcing them to adapt to prolonged periods of dormancy in tissues (Wayne 1994). It is suggested that the ability to shift down into non-replicating stages is crucial for the ability of tubercle bacilli to be dormant in the host for years or decades (Wayne and Hayes 1996). Immunocompetent individuals harboring latentM. tuberculosis carry % a 2-23 lifetime risk of reactivating tuberculosis, while patients with HIV reactivate tuberculosis at a much higher rate (Parrish et al., 1998). A problem is the rise of multi-drug resistant (MDR) strains (Bleed et al., 2001) which are resistant against at least rifampicin and isoniazid. Nearly 5% of all new infections are caused by MDR strains causing 500,000 tuberculosis cases per year. In addition, an extensively drug-resistance (XDR) form of tuberculosis has been reported in 45 countries. XDR tuberculosis is virtually untreatable and seriously threatens control efforts. Treatment of tuberculosis is difficult since only a few antibiotics are effective against fully susceptibleM. tuberculosis strains. Chemotherapy takes up to 6 month and must be extended for up to 2 years for MDR tuberculosis. The fact that many antibiotics do not affectM. tuberculosis can mostly be attributed to the unique mycobacterial cell wall which shields the mycobacterial cell from antibiotics and other toxic molecules due to its very low permeability (Brennan and Nikaido 1995).  2.2. The mycobacterial outer membrane and its proteins Due to its paramount importance as a pathogen, the growth and nutritional requirements ofM. tuberculosishave been intensively studied since its discovery more than a century ago (Koch 1882). However, nutrient transport inM. tuberculosisis still poorly understood despite a wealth of genomic data (Niederweis 2008). This is true in particular for transport processes across the outer membrane. The outer membrane ofM. bovisBCG and ofM. smegmatiswas visualized by cryo-electron microscopy (Fig. 2.2) and showed that mycolic acids are an essential component of this unusual supported lipid bilayer (Hoffmann et al., Mycolic acids are believed to form the inner leaflet of an 2008). asymmetrical bilayer while lipids that are extractable by organic solvents are assumed to form the outer leaflet (Minnikin 1982). X-ray diffraction of isolated mycobacterial cell walls showed that the mycolic acids are oriented parallel to each other and perpendicular to the plane of the cell envelope (Nikaido 1993). Its unique architecture and composition raise the question of how the mycobacterial outer membrane is
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