Global Transcriptome Analysis of the Human Pathogens Chlamydia trachomatis and Chlamydia pneumoniae [Elektronische Ressource] / Marco Albrecht. Betreuer: Lauster Roland

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Global Transcriptome Analysis of the Human Pathogens Chlamydia trachomatis and Chlamydia pneumoniae vorgelegt von Diplom-Ingenieur Marco Albrecht aus Meerane Von der Fakultät III - Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktor der Ingenieurwissenschaften – Dr.-Ing. – genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr. Vera Meyer Berichter: Prof. Dr. Roland Lauster Berichter: Prof. Dr. Thomas Rudel Berichter: Prof. Dr. Ulf Stahl Tag der wissenschaftlichen Aussprache: 16.05.2011 Berlin 2011 D 83 TABLE OF CONTENT CONTENT 1 Summary .......................................................................................................................................... 3 1.1 Abstract ... 3 1.2 Zusammenfassung ................... 5 2 Introduction ...................................................................................................................................... 7 2.1 The Obligate Intracellular Bacterial Order Chlamydiales ....................................................... 7 2.1.1 The Chlamydial Life Cycle ......................................................... 8 2.1.2 Pathogenesis of Chlamydia trachomatis ................................................................... 10 2.1.3 Pathog Chlamydia pneumoniae ... 11 2.1.4 Chlamydia and Host Cell Apoptosis ......... 11 2.1.5 The Chlamydia Genomes .......................
Publié le : samedi 1 janvier 2011
Lecture(s) : 42
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Source : D-NB.INFO/1018072705/34
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Global Transcriptome Analysis of the Human Pathogens
Chlamydia trachomatisand Chlamydia pneumoniae
vorgelegt von Diplom-Ingenieur Marco Albrecht aus Meerane Von der Fakultät III - Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktor der Ingenieurwissenschaften Dr.-Ing.genehmigte Dissertation Promotionsausschuss:
Vorsitzender: Prof. Dr. Vera Meyer Berichter: Prof. Dr. Roland Lauster Berichter: Prof. Dr. Thomas Rudel Berichter: Prof. Dr. Ulf Stahl Tag der wissenschaftlichen Aussprache: 16.05.2011 Berlin 2011 D 83
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TABLE OFCONTENT
Summary .......................................................................................................................................... 31.1Abstract ................................................................................................................................... 31.25Zusammenfassung ................................................................................................................... Introduction ...................................................................................................................................... 72.1....................................................... 7The Obligate Intracellular Bacterial Order Chlamydiales 2.1.1The Chlamydial Life Cycle ......................................................................................... 82.1.2Pathogenesis ofChlamydia trachomatis................................................................... 102.1.3Pathogenesis ofChlamydia pneumoniae................................................................... 112.1.4Chlamydiaand Host Cell Apoptosis ......................................................................... 112.1.5TheChlamydiaGenomes .......................................................................................... 122.1.6Gene Regulation inChlamydia................................................................................. 132.1.7.......................................................................................... 15The Chlamydial Plasmids 2.2Transcriptome Analysis by Differential RNA-Sequencing (dRNA-seq) .............................. 162.3Bacterial Small Non-coding RNAs (sRNA).......................................................................... 172.3.1Classes of Bacterial sRNAs ....................................................................................... 172.3.2Mechanisms of RNA-Based Gene Regulation .......................................................... 182.3.3Identification of Bacterial sRNAs ............................................................................. 192.3.4Target Identification of RNA-Based Gene Regulation ............................................. 202.3.5Small RNAs inChlamydia........................................................................................ 20Aim of Study .................................................................................................................................. 22Material .......................................................................................................................................... 234.1Bacterial Strains .................................................................................................................... 234.2............................................................................................................ 23Eukaryotic Cell Lines 4.323Chemicals .............................................................................................................................. 4.4Kits ........................................................................................................................................ 244.5Antibodies ............................................................................................................................. 244.6Enzymes ................................................................................................................................ 244.7Buffers and Solutions ............................................................................................................ 244.8Oligonucleotides.................................................................................................................... 264.9.................................................................................................. 26Lab Ware and Consumables 4.10Equipment ......................................................................................................................... 264.11Software ............................................................................................................................ 27Methods.......................................................................................................................................... 285.1Cell Biology .......................................................................................................................... 285.1.1.................................................................... 28General Handling of Human Cell Lines 5.2Microbiology ......................................................................................................................... 285.2.1Propagation and Purification ofChlamydia trachomatis.......................................... 285.2.2Propagation and Purification ofChlamydia pneumoniae.......................................... 285.2.3Infection and Estimation of Bacteria Stock Titre ...................................................... 295.2.4Isolation of EB and RB ofChlamydia trachomatis................................................... 295.2.5Isolation of EB and RB ofChlamydia pneumoniae.................................................. 295.2.6Electron Microscopy of Purified EB and RB ............................................................ 305.3Biochemistry ......................................................................................................................... 305.3.1Preparation ofC. pneumoniaeLysates ...................................................................... 305.3.2Fractionation of Lysates on a Density Gradient ........................................................ 30
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5.3.3SDS-Polyacrylamide Gel Electrophoresis ................................................................. 315.3.4Western Blot Analysis of Proteins ............................................................................ 315.4Molecular biology ................................................................................................................. 325.4.1Isolation of RNA from Purified Chlamydia .............................................................. 325.4.2Polyacrylamide Gel Electrophoresis of RNA and Northern Blot.............................. 325.4.3Next-Generation Sequencing of theChlamydiaTranscriptomes .............................. 335.5Computational Biology ......................................................................................................... 345.5.1Processing of Sequencing Raw Data ......................................................................... 345.5.2Visualisation of Transcriptome Sequencing Data ..................................................... 345.5.3Analysis of Transcriptional Start Sites and Read Numbers per Gene ....................... 345.5.4..................................................................................... 35Promoter Sequence Analysis Results ............................................................................................................................................ 366.1Characterization of theChlamydia trachomatisTranscriptome by dRNA-seq..................... 366.1.1Purification ofChlamydia trachomatisEB and RB .................................................. 366.1.2Deep Sequencing of PurifiedChlamydia trachomatisEB and RB ........................... 366.1.3Annotation of Transcriptional Start Sites (TSS)........................................................ 396.1.4Discovery of Putative Small Non-Coding RNAs ofChlamydia trachomatis........... 426.1.5Differential Gene Expression in EB and RB ............................................................. 466.1.6The Chlamydial Cryptic Plasmid Encodes Abundant sRNAs................................... 486.2Characterization of theC. pneumoniaeTranscriptome by dRNA-seq .................................. 506.2.1Purification ofC. pneumoniaeEB and RB................................................................ 506.2.2Deep Sequencing of PurifiedC. pneumoniaeEB and RB ........................................ 516.2.3Annotation of Transcriptional Start Sites (TSS)........................................................ 536.2.4Discovery of Putative Small Non-Coding RNAs ofChlamydia pneumoniae75...........6.2.5Analysis of Polycistronic Transcripts........................................................................ 636.2.6Differentially Expressed Genes inC. pneumoniaeEB and RB................................. 656.2.7Analysis ofC. pneumoniaePromoters ...................................................................... 67Discussion ...................................................................................................................................... 717.1Transcriptome Analysis by dRNA-seq.................................................................................. 717.2Transcriptome Analysis ofChlamydia trachomatis.............................................................. 727.2.1Comparison with Tiling Array Study ........................................................................ 747.3Transcriptome Analysis ofChlamydia pneumoniae............................................................. 757.4Comparison betweenC. trachomatisandC. pneumoniae77Transcriptomes ........................... 7.5Future Perspectives................................................................................................................ 80References ...................................................................................................................................... 81Appendix ........................................................................................................................................ 919.1Detailed Lists of Transcriptional Start Sites (TSS) ............................................................... 919.1.1TSS ofC. trachomatis............................................................................................... 919.1.2TSS ofC. pneumoniae............................................................................................. 1179.2Abbreviations ...................................................................................................................... 1519.3List of Figures ..................................................................................................................... 1539.4List of Tables....................................................................................................................... 154
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SUMMARY
SUMMARY
1.1Abstract Chlamydiaobligate intracellular pathogenic bacteria that share a unique biphasic life are cycle.Chlamydia trachomatis is the most common bacterial cause of sexually transmitted infections and blinding trachoma.Chlamydia pneumoniae is responsible for a wide range of acute and chronic diseases, including respiratory infections and atherosclerosis.Chlamydiashare a unique bi-phasic life cycle that switches from infectious but metabolically inactive elementary bodies (EB) to non-infectious but metabolically active reticular bodies (RB).
Since genetic tools to manipulate the genome and methods to culture the bacteria outside the host cell are lacking, genome sequence analysis has been the main approach to get insight into the biology of allChlamydiales. Although the genomes of several strains have been sequenced yet, very little information is available on the gene structure of these bacteria. The genome sequences ofC. trachomatisandC. pneumoniaeare available since 1998 and 1999, respectively. Genome annotation and most information on transcript organisation are based on comparative computational approaches.
Massively parallel cDNA sequencing has revolutionized global transcriptome analysis in recent years. Here, a differential RNA-sequencing (dRNA-seq) approach is applied that uses enzymatic enrichment for primary transcripts to define the transcriptome of purified elementary bodies (EB) and reticulate bodies (RB) of the strainsC. trachomatis L2b andC. pneumoniaeCWL029, respectively. Using dRNA-seq technique, primary transcriptional start sites (TSS) of annotated genes and novel genes like small non-coding RNAs could be mapped in a thus far unprecedented resolution as a complement to the genome sequence.
By massive parallel sequencing a detailed map of the transcriptomes of twoChlamydia species was generated experimentally. ForC. trachomatis363 transcriptional start sites (TSS) of annotated genes could be determined. Semi-quantitative analysis of mapped cDNA reads revealed differences in the RNA levels of 84 genes isolated from EB and RB, respectively. Furthermore, 42 genome- and 1 plasmid-derived novel non-coding RNAs were discovered and in part validated by Northern blotting. The genome encoded non-coding RNA, ctrR0332, was found to be one of the most abundant and differentially expressed transcripts in EB and RB, implying an important role in the developmental cycle ofC. trachomatis.
565 transcriptional start sites and 246 polycistronic transcripts could be determined forC. pneumoniae.Furthermore, numerous transcripts in intergenic regions and antisense to annotated ORFs which encode putative non-coding RNAs were identified and in part verified by Northern hybridisation. Most of them arecis-ortrans-encoded non-coding RNAs. By experimental methods a distinct TSS or an affiliation to an operon could be identified for 861 out of 1074 genes (80%) ofC. pneumoniae. Based on the transcriptome sequencing data the annotation of several genes could be
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corrected. An analysis of differentially expressed genes identified more than 50 genes to be more abundant in EB, most of them with unknown function. Based on the experimentally verified TSS the promoter sequences of 531 genes were analysed for common motifs. This analysis revealed a standard sigma factor binding site whereas alternative sigma factor binding sites are not conserved among Chlamydia.
This detailed catalogue of TSS and operon structure will help to understand the organization, control and function of genes of these important pathogens. It is valuable information for further analysis of gene regulation. This is the first study that differentially analysed transcriptomes of purified EB and RB which will help to understand the regulation of the unique, yet unknown, conversion mechanisms between the two life cycle forms.
SUMMARY
1.2Zusammenfassung Chlamydien sind obligat intrazelluläre humanpathogene Bakterien, welche einen einzigartigen zweiphasigen Lebenszyklus besitzen.Chlamydia trachomatis ist die häufigste Ursache für sexuell übertragbare Infektionen und Bindehautinfektionen die zur Erblindung führen.Chlamydia pneumoniaeist für ein breites Spektrum an akuten und chronischen Erkrankungen wie Atemwegsinfektionen und Atherosklerose verantwortlich. Chlamydien besitzen einen einzigartigen zweiphasigen Entwicklungszyklus, welcher zwischen infektiösen aber metabolisch inaktiven Elementarkörpern (EB) und nichtinfektiösen aber metabolisch aktiven Retikularkörpern (RB) alterniert.
Es gibt bis heute keine Möglichkeit zur genetischen Manipulation von Chlamydien und keine Methode um Chlamydien außerhalb der Wirtszelle zu kultivieren. Deshalb stellt die Genomanalyse die Hauptmethode dar, um Einblicke in die Biologie derChlamydialeszu erhalten. Obwohl die Genome von mehreren Stämmen bereits sequenziert wurden, sind sehr wenige Informationen über die Genstruktur dieser Bakterien bekannt. Die Genomsequenzen vonC. trachomatisundpneumoniae C. sind seit 1998 beziehungsweise 1999 bekannt. Die Annotation des Genoms und der Großteil der Informationen über die Organisation des Transkriptoms basiert auf vergleichenden computergestützten Analysen und Vorhersagen.
Durch die Einführung von Hochdurchsatz-Sequenziermethoden wurde die globale Transkriptomanalyse revolutioniert. In dieser Arbeit wurde eine Methode zur differentiellen RNA-Sequenzierung (dRNA-seq) angewendet, welche eine enzymatische Anreicherung von Primärtranskripten verwendet, um das primäre Transkriptom von aufgereinigten RB sowie EB der beiden StämmeC. trachomatisL2b undC. pneumoniaezu bestimmen. Mit dieser dRNA- CWL029 seq-Technik ist es möglich, primäre Transkriptionsstartstellen (TSS) von annotierten Genen zu bestimmen, sowie neue Gene, wie kleine nicht-codierende RNAs, zu entdecken. Diese Kartierung der TSS ist mit einer Auflösung bis zu einem Nukleotid möglich.
Durch Hochdurchsatzsequenzierung wurden experimentell detaillierte Transkriptomkarten der beidenChlamydien-Spezies geschaffen. FürC. trachomatiskonnten 363 TSS von annotierten Genen bestimmt werden. Durch semiquantitative Analyse der kartierten cDNA Sequenzen wurden 84 in EB und RB differentiell exprimierte Gene bestimmt. Weiterhin konnten 42 genom- und eine plasmid-kodierte kleine nicht-kodierende RNA entdeckt und teilweise experimentell durch Northern Blotting verifiziert werden. Die genomkodierte nichtkodierende RNA Ctr0332 war eine der am stärksten exprimierten und differentiell exprimierten Transkripte, was auf eine Rolle im Entwicklungszyklus vonC. trachomatishindeutet.
Bei der Analyse vonC. pneumoniaewurden 565 TSS sowie 246 polycistronische Transkripte bestimmt. Außerdem wurde eine Vielzahl an Transkripten in intergenischen Regionen und antisense zu annotierten Genen entdeckt, die potentielle nichtkodierende RNAs darstellen. Diese wurden teilweise durch Northern Blotting verifiziert. Durch diesen experimentellen Ansatz der
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Hochdurchsatzsequenzierung wurde für insgesamt 861 der 1074 annotierten Gene (entspricht 80%) vonC. pneumoniaeentweder eine primäre TSS oder eine Zugehörigkeit zu einem Operon identifiziert. Basierend auf diesem Transkriptom-Datensatz konnte die Annotation für eine Reihe von Genen korrigiert werden. Eine Analyse von differentiell regulierten Genen zeigte, dass über 50 Gene in EB angereichert sind, die meisten mit völlig unbekannter Funktion. Durch die experimentelle Bestimmung der TSS war es möglich, die Promotorsequenzen von 531 proteinkodierenden Genen zu analysieren. Diese Analyse zeigte, dass die Mehrheit der Gene durch den Standardsigmafaktor erkannt wird und es bei den untersuchten Chlamydien keine stark konservierten Promotormotive für alternative Sigmafaktoren gibt.
Dieser detaillierte Datensatz der primären TSS sowie der Operonstrukturen leistet einen fundamentalen Beitrag zum Verständnis der Genorganisation und -funktion dieser wichtigen Humanpathoge. Er bildet eine wertvolle Grundlage für zukünftige Untersuchungen der Genkontrolle. Dies ist die erste Arbeit, welche die differentiellen Transkriptome von aufgereinigten EB und RB untersucht hat. Die Ergebnisse können helfen den einzigartigen und bisher größtenteils unbekannten Konversionsmechanismus zwischen den beiden Entwicklungszyklusformen zu verstehen.
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INTRODUCTION
INTRODUCTION
2.1The Obligate Intracellular Bacterial Order Chlamydiales The bacterial order Chlamydiales comprises obligate intracellular Gram-negative pathogens that share a unique developmental cycle in which multiplication only occurs within a eukaryotic host cell (Moulder 1982). During the last decades numerous new organisms have been identified from different animal and environmental sources which led to the division of the family Chlamydiaceae into the two generaChlamydiaandChlamydophila(Everettet al.1999) shown in figure 1. This taxonomy is controversial and therefore this work refers to the use of a single genus,Chlamydia, as suggested by Schachteret al.(2001).
Bacteria of the familyChlamydiaceaeare the etiological agents of numerous important human and animal diseases. The familyChlamydiaceae contains three human pathogenic species namely Chlamydia trachomatis (C.trachomatis),Chlamydia pneumoniae (C. pneumoniae),and Chlamydia psittaci.C. trachomatisis the most prevalent cause of genital tract infection in the western world and the ocular serovars result in blinding trachoma in developmental countries.C. pneumoniaeis the causing agent of community-acquired pneumonia, with millions of infections each year and chronic infections are associated with an enhanced risk of developing atherosclerosis (Kuo et al. 1993),
Figure 1: Taxonomy of the order Chlamydiales. Phylogenetic tree of the order Chlamydiales based on ribosomal RNA homology. Species that have human as their major host are marked in red. The division of the family Chlamydiaceae into the two generaChlamydiaand Chlamydophilais controversially discussed in the community. This work refers to a single genusChlamydia.Lengths of lines are not proportional to the real phylogenetical distances. Adapted from (Bush and Everett 2001).
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cerebrovascular disease (Balinal. et  1998) and asthma (Hahnal. et  1991).C. psittaci can cause psittacosis in humans which is a zoonotic infectious disease. Transmission usually originates from contact to birds, especially poultry (Beeckman and Vanrompay 2009).
Although the genomes of several species are available for research for some years already, the mechanisms of pathogenicity are far from being understood (Subtil and Dautry-Varsat 2004). The major challenge when working withChlamydiais the lack of an experimental genetic system and the unavailability of anin vitrosystem outside a host cell. To date there is no transformation culturing method available to maintain exogenous DNA insideChlamydia. The consequence is the lack of any in vivotranscription assay.
2.1.1The Chlamydial Life Cycle The major feature ofChlamydiais their unique biphasic developmental cycle which alternates between two morphological forms, the elementary body (EB) and the reticulate body (RB) (Moulder 1991). EB are small, spore-like bacterial particles with a diameter of approximately 0.3 µm. They are metabolically inert, extracellular, infectious particles, able to attach to and invade susceptible cells. Their biological function is to survive extracellularly until a suitable host cell is found for infection. The bacterial nucleoid is highly condensed by bacterial histone-like proteins HctA and HctB (Brickmanet al.1993). EB contain significant quantities of mRNA and ribosomes, despite there is no transcriptional activity. EB have a unique outer membrane with little or no peptidoglycan which is cross-linked by inter- and intramolecular cysteine bonds and contains large amounts of outer membrane proteins such as OmpA, OmcA and OmcB. The cell wall of EB seems to contain components of a type three secretion system (Fieldset al.2003).
Chlamydia can infect numerous cell types and have developed several, possibly redundant, mechanisms for host cell attachment and entry. The exact interaction of bacterial adhesion and host cell receptor is still unclear. Several chlamydial membrane proteins such as OmpA, OmcB and the polymorphic outer membrane protein (Pmp) family have been suggested to play a role (Grimwoodet al.The attachment of 2001). Chlamydia to the host cell seems to be a two-stage process. Initial attachment is reversible and occurs through electrostatic interactions between heparin sulphate containing glycosaminoglycan and OmpA (Stephenset al.2001). The second step is irreversible and the exact mechanism is still unknown (Carabeo and Hackstadt 2001).
After adhesion (see figure 2, stage 1), a type three system secreted protein called Tarp (Translocated actin-recruiting phosphoprotein) is injected into the host cell. The protein is rapidly phosphorylated at tyrosine residues followed by actin recruitment (Cliftonal. et  2004). The mechanism of internalization of the EB into the host is still unknown (Scidmore-Carlson and Hackstadt 2000). Inside the host cell the EB are located in a membrane bound vesicle called inclusion (Figure 2, stage 2). They become metabolically active, enlarge in size, and differentiate into RB that undergo binary fission (Figure 2, stages 3 and 5).
INTRODUCTION
The reticulate bodies are larger than EB (1 µm in diameter) located inside the inclusion and bound by an inner and outer membrane like other Gram-negative bacteria. RB are non-infective and larger than EB due to their diffuse nucleic acid structure. RB divide by binary fission every 2-3 hours during the logarithmic growth phase and after a number of divisions the developmental process becomes asynchronous (McClarty 1994; Moulder 1991). Some RB start to differentiate back into EB (Figure 2, stage 5) followed by the release of the EB, which is accompanied by host cell death after 48 hours (C. trachomatis) to 96 hours (C. pneumoniae)depending on the species and growth conditions. Then a new infection cycle starts with EB attaching neighbouring cells or infecting another organism (Figure 2, stage 7).
The regulation of RB-to-EB conversion is largely unknown. Attempts to identify regulatory elements that switch gene regulation have failed so far. However, two histone-like proteins Hc1 and Hc2 have been identified that are expressed late in RB and are thought to initiate the conversion by
Figure 2: The life cycle of Chlamydiales. (1) Elementary bodies (EB) attach to the host cell and invade the cell. (2) EB differentiate into reticulate bodies (RB) inside a vesicle called inclusion, followed by intracellular growth and division (3, 5). Persistence stimuli like cytokines or starvation of the host cell lead to the formation of persistence bodies (PB), which can enter the life cycle again after reactivation(5). Upon redifferentiation of the RB into EB, the lysis of the host cell is induced, and the EB escape from the host cell to start a new infection cycle. Figure adapted from Byrne and Ojcius (2004).
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