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

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Discussion The ability to adequately adapt to changes in the availability of nutrients is a prerequisite for bacterial survival. Several studies have suggested that nitrogen metabolism, and especially glutamine metabolism, is important for virulence of pathogens (18, 29, 31). The transcriptional repressor GlnR regulates, together with glutamine synthetase GlnA, genes involved in glutamine / glutamate uptake and conversion in S. pneumoniae, L. lactis, and B. subtilis (7, 17, 19). DNA-binding assays have demonstrated that GlnR-repression is dependent on GlnA, but the exact mechanism remains unknown (17). As a result, GlnR- targets are derepressed in a glnA-mutant. In this study, we investigated the contribution of the transcriptional regulator GlnR and its target genes to pneumococcal virulence, in particular its contribution during colonization, pneumonia, and bacteremia in mice. Absence of GlnR had no significant effect on bacterial virulence in any of our three infection models used. It could well be that levels of available glutamine at the nasopharynx and in the lungs are considerably low and, hence, no repression by GlnR is triggered. Alternatively, all genes of the GlnR-regulon might be expressed in the ΔglnR strain, and redundant expression of these genes might not influence the ability to colonize or cause disease. The conversion of glutamate to glutamine appears to be required during colonization, as the mutant lacking GlnA was found to be attenuated in colonization of the murine nasopharynx. Alternatively, the lack of GlnA might affect regulation by GlnR through derepression of its gene targets, although this appears less likely given the lack-of- colonization phenotype of the glnR-mutant. Attenuation of the glnA-mutant was not observed in lungs and in the blood stream during the pneumonia and bacteremia models of infection, indicating that GlnA is not required for bacterial survival in lungs and the transition from the lungs to the blood in our infection models. GlnR-regulon and pneumococcal virulence GlnP, which is part of the main glutamine / glutamate ABC transporter GlnPQ, is not required for colonization of the murine nasopharynx. Interestingly, GlnP was found to be involved in adherence to Detroit 562 cells, suggesting a role in colonization in humans. It cannot be excluded that the difference between in vitro adherence and the murine colonization model reflects the differences between the in vitro and in vivo setting. For instance, expression of glnP might be required in RPMI medium, but not in the murine (or human) nasopharynx. In group B streptococci it has been proposed that glnQ, also part of the glutamine / glutamate transporter, is involved in adherence to fibronectin and virulence in rats, possibly by modulating cytoplasmic glutamine levels (29). GlnP was also identified as a 173 173
Chapter 6 candidate for fibronectin binding using phage display library analysis (1). However, binding of fibronectin by GlnP of streptococci has never been confirmed, suggesting an indirect involvement (1, 29). In the pneumonia model, mice infected with the glnP-mutant showed a lower bacterial load in the lungs at 36h than wild-type infected mice. Strikingly, while the number of wild-type bacteria increased with time, fewer glnP-mutants reached the blood stream from 24h post-infection onwards. This suggests a role for GlnP in the transition from the lungs to the blood stream. Uptake of glutamine could be of importance at this stage of infection, i.e., entering the blood stream. The impaired glutamine uptake in the glnP-mutant might therefore create a growth disadvantage leading to a smaller number of bacteria in the blood circulation. However, we cannot rule out the possibility that the ΔglnP phenotype, i.e., impaired transition from lungs to blood, is caused by multiple factors. Fewer bacteria cause less tissue damage in the lungs and this will consequently lead to less spill-over into the blood. One such example might be lower pneumolysin concentrations due to lower bacterial loads in the lungs (11). 174 174 The glnA-glnP double mutant displayed an attenuated phenotype similar to the glnA- mutant in the colonization model, most likely caused by the glnA mutation only, given the lack-of-colonization phenotype of the glnP-mutant. Moreover, since GlnP is part of the main glutamine and glutamate transporter (17), our data suggest that pneumococcus, while colonizing the murine nasopharynx, is able to acquire glutamine and glutamate through other processes such as peptide uptake and degradation. Different results were obtained in the lungs; here the glnA-mutation alone did not result in attenuation whereas mutation of both glnA and glnP did, suggesting that pneumococcus cannot easily use other sources for acquisition of glutamine at this particular site. The double mutant is not able to convert glutamate to glutamine (GlnA), and is not able to take up free glutamate or glutamine (GlnP). Consequently, the double mutant has to rely on another system for acquiring these amino acids. If so, such a system is apparently not able to complement the lack of GlnA and GlnP sufficiently when pneumococci reach the lungs or blood circulation, in contrast to the nasopharynx, where only glutamine synthesis (GlnA) contributes to survival. The glnAP mutant was able to grow in defibrinated blood in vitro, but this growth was slower and to a lower cell density than the wild-type. Addition of glutamine rescued the phenotype of ΔglnAP to wild-type growth (data not shown). This suggests that, when added in high concentrations, glutamine can enter the cell, possibly by other uptake systems with very low affinity for glutamine or by passive diffusion.
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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
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
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 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
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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