Gene regulation in Streptococcus pneumoniae - RePub - Erasmus ...

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histidine kinase is the sensor protein that resides in the cell membrane. Upon sensing and binding of its ligand (e.g., sugars or peptides), the HK is activated and autophosphorylation of a conserved histidine residue occurs. Usually, HK proteins are found as homodimers and phosphorylation of the histidine residue on the first HK catalyzes phosphorylation of the histidine residue on the second HK of the dimer (14). The second component is a cytosolic protein called the response regulator (RR), which is a DNA-binding protein. The phosphoryl group of the HK is transferred to an aspartate residue on the RR, after which the RR undergoes a conformation change and becomes activated. The activated RR then acts as a transcriptional regulator, repressing or activating gene expression. Introduction Sequence analysis of the pneumococcal genome has revealed the presence of 13 TCSs plus one so-called orphan response regulator (RitR) (46, 60). Knock-out mutants of these genes were used to investigate their role in virulence of different serogroups in mice (30, 61). Following these two large screening studies, more detailed studies have led to further insight into the importance of pneumococcal TCSs in pathogenesis (for a complete overview see 46). The most studied TCS in S. pneumoniae is the ComDE system (TCS12), which is part of the competence machinery (24). Natural competence is the state in which a bacterium is able to take up DNA from its environment. Sequestered DNA can be inserted into the chromosome, and novel genetic properties and pathogenic features can be acquired. The histidine kinase ComD senses competence stimulating peptide (CSP), a small peptide of which the precursor is produced by comC and is transported and cleaved to its mature state by ComAB. When CSP accumulates to a critical concentration outside the cell, ComD will be phosphorylated, and transfer of the phosphoryl group will activate the RR ComE, which in turn activates the other components of the competence machinery. The transcriptional regulator ComX is also activated, which subsequently activates many other genes involved in DNA uptake and processing, bacteriocin production, and stress response (32, 49). The ComDE TCS was demonstrated to be important for virulence, more specifically ComD was shown to be involved in pneumonia and bacteremia in serotype 2 strain D39 (3), serotype 3 strain 0100993 (31) and serotype 4 strain TIGR4 (20). Using microarray-based transcriptional profiling, over 200 CSP-responsive genes were identified (12, 49). These induced or repressed genes included several known virulence factors such as autolysin lytA, a stress response protein htrA, and the choline binding protein cbpD. TCS04 was demonstrated to be involved in virulence during murine pneumonia (61), and later the involvement was shown to be strain-dependent (37). Transcriptome analysis of 13 13
Chapter 1 mutants for rr04 revealed variation between strains in RR04-regulated genes, potentially reflecting the need for expression of a specific set of genes during pneumococcal virulence, which varies between strains. Especially the psa-operon (encoding a manganese transporter) was strain-specifically regulated (37). 14 14 TCS02 (also known as vic, micAB, yycFG, and 492) was found to be the only essential TCS in pneumococcus, regulating cell wall and fatty acid metabolism and the expression of the virulence factor PspA (30, 39, 43, 61). One of the important gene targets of RR02 is the pcsB gene encoding a murein-hydrolase; overexpression of this gene could negate the essentiality of RR02, allowing deletion of rr02 (41, 42). TCS05 (or CiaRH) was also shown to be important for virulence, mediated by activating the major virulence factor HtrA (22). Mutants for the response regulator CiaR displayed the same attenuation as mutants for HtrA, in a murine intranasal infection model (22). In addition, this TCS plays a role in competence and antibiotic resistance (16, 17, 64). TCS09 was found to significantly contribute to virulence in murine models of infection (7). Mutants for the response regulator in two different strains displayed different virulence properties. D39∆rr09 was avirulent, whereas 0100993∆rr09 was fully virulent upon intraperitoneal and intravenous infection. In contrast, upon intranasal infection, this mutant was unable to survive in lung tissue and in the blood, suggesting a role in dissemination from lungs to blood (7). This study by Blue & Mitchell was one of the first to illustrate that gene regulatory systems may have different impacts on the virulence potential of different strains (7). Nutritional gene regulation All bacteria need nutrients to sustain and to replicate. Acquisition of these molecules or their precursors is required for both anabolic and catabolic activities of the cell. During in vitro growth pneumococci convert glucose into the acids acetate and/or lactate, thereby generating energy and producing molecules needed for other cellular processes. In addition, dedicated transporters exist to take up nutrients like sugars or amino acids from the environment. However, little is known about pneumococcal metabolism in vivo during pathogenesis, while such knowledge would be crucial to increase our understanding of pneumococcal virulence. Gene regulatory pathways play an essential role in controlling the expression of genes necessary for uptake and consumption of particular metabolites. Various publications have reported the co-regulation of metabolism and virulence factors, and proteins involved in
Page 1 and 2: Gene regulation in Streptococcus pn
Page 3 and 4: Cover image by Duve van Boggelen (w
Page 5 and 6: Promotiecommissie Promotoren: Prof.
Page 8 and 9: CHAPTER 1 Introduction 7
Page 10 and 11: Introduction Figure 1. Infections c
Page 12 and 13: Interestingly, autolysis induced by
Page 16 and 17: metabolism are often required for v
Page 18 and 19: egulatory systems, aiming to elucid
Page 20 and 21: 11. Cockeran, R., A. J. Theron, H.
Page 22 and 23: 33. Li, S., S. J. Kelly, E. Lamani,
Page 24 and 25: 53. Romero, P., R. Lopez, and E. Ga
Page 26 and 27: CHAPTER 2 Regulation of gene expres
Page 28 and 29: Introduction Streptococcus pneumoni
Page 30 and 31: Materiaals and Meethods Gene regula
Page 32 and 33: (Invitrogen), 0.2 mM aminoallyl dUT
Page 34 and 35: Isolation of pneumococcal RNA durin
Page 36 and 37: Resultss and Discuussion Gene regul
Page 38 and 39: Table 1. Genes regulated by RR09 in
Page 40 and 41: phosphofructokinase, and a fructose
Page 42 and 43: Figure 4. Expression of rlrA and rr
Page 44 and 45: correlated well with our in vitro m
Page 46 and 47: Figure 7. Bacterial loads in blood
Page 48 and 49: Acknowledgments This work was suppo
Page 50 and 51: 10. Ibrahim, Y. M., A. R. Kerr, J.
Page 52 and 53: genome sequence of a virulent isola
Page 54 and 55: Gene regulation by RR09 in S. pneum
Page 56 and 57: Gene regulation by RR09 in S. pneum
Page 58 and 59: Table S4. Genes up- and downregulat
Page 60: Table S6. Genes up- and downregulat
Page 63 and 64: Chapter 3 Abstract 62 62 Previous s
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Chapter 3 between the wild-type and
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Chapter 3 S. pneumoniae TIGR4 by CS
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Chapter 3 performance liquid chroma
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Chapter 3 Results Transcriptional a
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Chapter 3 Table 3. Differentially e
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Chapter 3 Figure 2. Colonization mo
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Chapter 3 than the D39ΔpsaR-infect
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Chapter 3 effect was more severe fo
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Chapter 3 in TIGR4ΔpsaR. However,
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Chapter 3 References 1. Adrian, P.
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Chapter 3 21. Hemsley, C., E. Joyce
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Chapter 3 40. McCluskey, J., J. Hin
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CHAPTER 4 CodY of Streptococcus pne
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Introduction Bacteria encounter var
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Materials and Methods Bacterial str
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Table 2. Oligonucleotide primers us
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The CodY regulon of S. pneumoniae I
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The CodY regulon of S. pneumoniae w
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Accession numbers The microarray da
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Table 3. Differentially expressed g
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Binding of CodY to target promoters
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The CodY regulon of S. pneumoniae E
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The CodY regulon of S. pneumoniae F
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Discussion CodY has been described
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levels of adherence in D39. Using a
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References The CodY regulon of S. p
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Glass. 2001. Genome of the bacteriu
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40. Shivers, R. P., S. S. Dineen, a
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CHAPTER 5 Regulation of glutamine a
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Introduction Regulation of nitrogen
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Materials and Methods Nitrogen Meta
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TK136 D39 Δcps; Km R capsule-less
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gene from pORI28, was fused to the
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Construction of lacZ fusions Chromo
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Enzyme assays Cell-free extracts, u
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Reverse-transcriptase PCR RNA isola
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Table 3. Summary of transcriptome c
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effect caused by altered intracellu
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examine whether zwf is only transcr
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(http://www.bd.com/ds/technicalCent
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H6-GlnR alone at the concentration
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Discussion In this study, we charac
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Acknowledgements TK, JB and HB are
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11. den Hengst, C. D., S. A. van Hi
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Nitrogen Metabolism in S. pneumonia
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52. Wray, L. V., Jr., J. M. Zalieck
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CHAPTER 6 Site-specific contributio
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Introduction GlnR-regulon and pneum
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Materials and Methods Bacterial str
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eferred to as t=0. Bacteriology res
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Results GlnR-regulon and pneumococc
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Figure 3. Colonization model. Bacte
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GlnR-regulon and pneumococcal virul
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Table 2. Differentially expressed g
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Discussion The ability to adequatel
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The microarray data showed that pre
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Acknowledgments WTH is supported by
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12. Ibrahim, Y. M., A. R. Kerr, J.
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33. Zalieckas, J. M., L. V. Wray, J
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CHAPTER 7 Pneumococcal gene regulat
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Introduction Regulation of gene exp
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Gene regulation and metabolism in S
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makes it possible for the pneumococ
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A CcpA homologue exists in the pneu
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double mutant, suggesting direct re
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References Gene regulation and meta
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23. Kerr, A. R., P. V. Adrian, S. E
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45. Sonenshein, A. L. 2005. CodY, a
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CHAPTER 8 Summarizing discussion 20
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that the observed virulence phenoty
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CodY was found to be required for a
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References Summarizing discussion 1
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Summarizing discussion 21. Orihuela
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Samenvatting en discussie Curriculu
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Samenvatting en discussie kunnen ge
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Bevindingen die dit onderbouwen, we
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Curriculum Vitae Curriculum Vitae W
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Dankwoord Jeetje, dat het dan toch
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oekje en ik kijk uit naar de bijbeh
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