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

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Introduction GlnR-regulon and pneumococcal virulence Streptococcus pneumoniae is often carried asymptomatically in the human nasopharynx. However, this Gram-positive bacterium can cause disease upon dissemination to other sites of the body, such as otitis media, pneumonia, septicemia, and meningitis (3). To survive at various sites in the host S. pneumoniae needs to metabolize available nutrients. In line with this, several systems involved in nutrition and metabolism have been suggested to be important for virulence. For example, PsaA is part of a Mn(II) uptake system and contributes to colonization and invasive disease (2, 13), while the Ami-AliA/AliB oligopeptide uptake system contributes to colonization only (15). Nitrogen metabolism is of utmost importance for bacterial survival, and is therefore strictly regulated. Glutamine and glutamate serve as main sources of nitrogen in the cell. We have recently shown that in pneumococcus, expression of genes involved in glutamine metabolism is regulated by the transcriptional regulator GlnR, which has a similar function in Bacillus subtilis and Lactococcus lactis (5, 17, 19) (Fig.1). In the pneumococcus, the GlnRregulon consists of two operons, glnRA and glnPQ-zwf, and the gdhA gene. The gene glnA encodes glutamine synthetase GlnA, which forms glutamine out of glutamate and ammonium while hydrolyzing ATP. The glnPQ genes encode the main glutamine / glutamate transporter, and zwf encodes glucose-6-phosphate 1-dehydrogenase, an enzyme involved in pentose metabolism (17). The gene gdhA encodes glutamate dehydrogenase, which converts 2oxoglutarate and ammonium to glutamate, thereby hydrolyzing NAD(P)H. We have demonstrated that GlnR binds to a conserved operator sequence present in the promoter regions of its target genes (17). Regulation by GlnR is dependent on GlnA, as the GlnR targets are derepressed in a glnA mutant and GlnA stimulates binding of GlnR to its operator sequence (17). In addition, GlnR-regulation is responsive to glutamine and ammonium (17, 19). Importantly, expression of gdhA has been shown to be under control of a second regulatory protein, CodY (9, 17). This transcriptional repressor has been studied extensively in B. subtilis and L. lactis (6, 23, 28, 30), and recently in pneumococcus (9). Targets of CodY have been shown to consist mainly of genes involved in amino acid biosynthesis and uptake. It can be expected that GdhA plays an important role in central amino acid metabolism as the expression of its gene is tightly regulated by GlnR and CodY. In B. subtilis, a GlnR-ortholog is present, TnrA, which regulates various genes, including genes involved in nitrogen metabolism (30, 33). TnrA activates several genes during low nitrogen availability and its DNA recognition site is similar to that of GlnR (5, 30, 33). In pneumococcus no TnrA 159 159
Chapter 6 homologue is present, indicating differences in regulation of nitrogen metabolism with B. subtilis. Figure 1. The pneumococcal GlnR-regulon. GlnR regulates the expression of the glnRA and glnPQ-zwf operons, and the gdhA gene. Repression of gene expression by GlnR is responsive to high concentrations glutamine, glutamate, and ammonium. Moreover, regulation by GlnR requires cellular GlnA for full repression of target genes. Signature-tagged mutagenesis screens have suggested that several pneumococcal genes involved in glutamine metabolism are required for full virulence (8, 20, 27). In group B streptococci, a mutant deficient in glnQ, the gene encoding a glutamate transporter, is less capable to adhere to and invade A549 respiratory epithelial cells in vitro. Furthermore, this mutant showed a decreased virulence in a rat model of infection (29). In Mycobacterium tuberculosis, glutamine synthetase GlnA1 is essential for growth in macrophages (31). In another intracellular pathogen, Salmonella typhimurium, glnA is regulated by the Ntr-system (18). This gene shares an operon with genes encoding the two component system NtrB/C. Mutation in either glnA, ntrB, or ntrC resulted in a marked reduction of virulence and a reduced ability to survive within host cells (18). In this study, we assessed the importance of glutamine and glutamate metabolism in S. pneumoniae by evaluating the contribution of the glutamine / glutamate regulator GlnR and its target genes, glnP, glnA, and gdhA, to pneumococcal virulence. To this end, we investigated the ability of mutants for these genes to adhere to a human pharyngeal cell line in vitro. Furthermore, we tested the individual contribution of these genes in three murine infection models, representing the three major phases of pneumococcal disease: colonization, pneumonia, and bacteremia. In addition, we used microarray analysis to examine the global gene expression of the glnA-glnP mutant, which was severely affected in virulence. 160 160
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Gene regulation in Streptococcus pn
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Cover image by Duve van Boggelen (w
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Promotiecommissie Promotoren: Prof.
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CHAPTER 1 Introduction 7
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Introduction Figure 1. Infections c
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Interestingly, autolysis induced by
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histidine kinase is the sensor prot
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metabolism are often required for v
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egulatory systems, aiming to elucid
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11. Cockeran, R., A. J. Theron, H.
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33. Li, S., S. J. Kelly, E. Lamani,
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53. Romero, P., R. Lopez, and E. Ga
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CHAPTER 2 Regulation of gene expres
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Introduction Streptococcus pneumoni
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Materiaals and Meethods Gene regula
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(Invitrogen), 0.2 mM aminoallyl dUT
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Isolation of pneumococcal RNA durin
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Resultss and Discuussion Gene regul
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Table 1. Genes regulated by RR09 in
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phosphofructokinase, and a fructose
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Figure 4. Expression of rlrA and rr
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correlated well with our in vitro m
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Figure 7. Bacterial loads in blood
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Acknowledgments This work was suppo
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10. Ibrahim, Y. M., A. R. Kerr, J.
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genome sequence of a virulent isola
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Gene regulation by RR09 in S. pneum
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Gene regulation by RR09 in S. pneum
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Table S4. Genes up- and downregulat
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Table S6. Genes up- and downregulat
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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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Page 112 and 113: Discussion CodY has been described
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Page 116 and 117: References The CodY regulon of S. p
Page 118 and 119: Glass. 2001. Genome of the bacteriu
Page 120 and 121: 40. Shivers, R. P., S. S. Dineen, a
Page 122 and 123: CHAPTER 5 Regulation of glutamine a
Page 124 and 125: Introduction Regulation of nitrogen
Page 126 and 127: Materials and Methods Nitrogen Meta
Page 128 and 129: TK136 D39 Δcps; Km R capsule-less
Page 130 and 131: gene from pORI28, was fused to the
Page 132 and 133: Construction of lacZ fusions Chromo
Page 134 and 135: Enzyme assays Cell-free extracts, u
Page 136 and 137: Reverse-transcriptase PCR RNA isola
Page 138 and 139: Table 3. Summary of transcriptome c
Page 140 and 141: effect caused by altered intracellu
Page 142 and 143: examine whether zwf is only transcr
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Page 146 and 147: H6-GlnR alone at the concentration
Page 148 and 149: Discussion In this study, we charac
Page 150 and 151: Acknowledgements TK, JB and HB are
Page 152 and 153: 11. den Hengst, C. D., S. A. van Hi
Page 154 and 155: Nitrogen Metabolism in S. pneumonia
Page 156 and 157: 52. Wray, L. V., Jr., J. M. Zalieck
Page 158 and 159: CHAPTER 6 Site-specific contributio
Page 162 and 163: Materials and Methods Bacterial str
Page 164 and 165: eferred to as t=0. Bacteriology res
Page 166 and 167: Results GlnR-regulon and pneumococc
Page 168 and 169: Figure 3. Colonization model. Bacte
Page 170 and 171: GlnR-regulon and pneumococcal virul
Page 172 and 173: Table 2. Differentially expressed g
Page 174 and 175: Discussion The ability to adequatel
Page 176 and 177: The microarray data showed that pre
Page 178 and 179: Acknowledgments WTH is supported by
Page 180 and 181: 12. Ibrahim, Y. M., A. R. Kerr, J.
Page 182 and 183: 33. Zalieckas, J. M., L. V. Wray, J
Page 184 and 185: CHAPTER 7 Pneumococcal gene regulat
Page 186 and 187: Introduction Regulation of gene exp
Page 188 and 189: Gene regulation and metabolism in S
Page 190 and 191: makes it possible for the pneumococ
Page 192 and 193: A CcpA homologue exists in the pneu
Page 194 and 195: double mutant, suggesting direct re
Page 196 and 197: References Gene regulation and meta
Page 198 and 199: 23. Kerr, A. R., P. V. Adrian, S. E
Page 200 and 201: 45. Sonenshein, A. L. 2005. CodY, a
Page 202 and 203: CHAPTER 8 Summarizing discussion 20
Page 204 and 205: that the observed virulence phenoty
Page 206 and 207: CodY was found to be required for a
Page 208 and 209: 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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