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

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The microarray data showed that predominantly genes involved in amino acid metabolism are upregulated in the glnAP mutant. These genes, most of them belonging to the CodY regulon (9), are probably upregulated because the double mutant is starved for amino acids, glutamine in particular. Although glutamine was present in the medium used for the expression study, the gene regulatory network sensing and controlling general amino acid metabolism seemed perturbed. This is underscored by the differential expression of four transcriptional regulators, however, their function is yet unknown. This suggests that regulation of amino acid metabolism within the pneumococcal cell is quite complex, and has severe effects on fitness and virulence. The impact on virulence could also be explained by the twofold downregulation of the expression of htrA in the glnAP mutant, as it was shown previously that this gene is required for virulence in D39 (12). The gene gdhA, encoding glutamate dehydrogenase, does not appear to be required for virulence at any site in our infection models. No paralog of gdhA is present in the pneumococcal genome, which could complement its metabolic function. Regulation of gdhA by CodY has been described and might be the principle regulator in vivo, by repressing gdhA expression (9, 17). Possibly, gdhA expression is coordinated in such way that it is only activated in specific conditions (e.g., nitrogen limitation), and that these conditions are not encountered by the pneumococcus in our mouse models. We have described the behavior of mutants lacking genes of the GlnR-regulon, the regulatory system of glutamine and glutamate metabolism, during adherence to human pharyngeal epithelial cells in vitro and experimental virulence in mice. We have identified two genes that play a role in virulence, namely glnA and glnP. The gene glnA encodes glutamine synthetase, which is required for colonization, and glnP encodes a membrane glutamine / glutamate permease, which is required for survival in the lungs and, possibly, for the transition from the lungs to the blood circulation. This study provides novel insight into the nutritional requirements of S. pneumoniae within its host, more specifically the glutamine and glutamate requirements. The different phenotypes of mutants during in vitro adherence to human pharyngeal cells and colonization of the murine nasopharynx suggest that different host species (i.e., mice and humans) may have different adhesion properties and nutritional supply for the bacterium. GlnR-regulon and pneumococcal virulence Many studies have shown that expression of pneumococcal proteins during pathogenesis is required at different stages of infection (15, 21, 25). One study, performed by Oggioni and co-workers, presents evidence that the “transcriptional state” of the pneumococcal cells differs at particular niches within the host (24). The bacterium has to deal 175 175
Chapter 6 with the supply of nutrients that each site of the body offers. For this reason, co-regulation of site-specific metabolic and virulence factors by nutritional regulators is likely to occur. In this study, we report on the role of the glutamine / glutamate metabolism and its regulation during our experimental virulence models in mice. The site-specific requirements for GlnA and especially GlnP reported in this study might offer novel strategies to explore these molecules as future drug targets that specifically target invasive disease, while leaving colonization unaffected. 176 176 In conclusion, genes within the GlnR-regulon are of definite importance for bacterial survival within our experimental models of infection, with different subsets of the genes involved in glutamine / glutamate metabolism contributing to pneumococcal survival during different stages of infection.
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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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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
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
Page 144 and 145: (http://www.bd.com/ds/technicalCent
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 160 and 161: Introduction GlnR-regulon and pneum
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 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
Page 210 and 211: Summarizing discussion 21. Orihuela
Page 212 and 213: Samenvatting en discussie Curriculu
Page 214 and 215: Samenvatting en discussie kunnen ge
Page 216 and 217: Bevindingen die dit onderbouwen, we
Page 218 and 219: Curriculum Vitae Curriculum Vitae W
Page 220 and 221: Dankwoord Jeetje, dat het dan toch
Page 222: oekje en ik kijk uit naar de bijbeh
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