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

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that the observed virulence phenotype of the rr09 mutant was not caused by the downregulation of this gene. Summarizing discussion Another interesting finding was the RR09-dependent regulation of the rlrA pathogenicity islet (encoding a regulator, a structural pilus, and 3 sortases) (2). In TIGR4∆rr09, expression of this islet was downregulated, indicating direct or indirect regulation by RR09. In a recent paper, TCS08 has been shown to regulate the expression of the rlrA pathogenicity islet as well (31). In this paper, Song and co-workers found that in TIGR∆rr08 expression of the rlrA pathogenicity islet was increased in late stationary phase, which is in agreement with our findings (15). In another recent paper, Rosch and co-workers showed that the regulation of the rlrA pathogenicity islet is even more complex, with two other TCSs, and the transcriptional regulators MerR (or CzcD) and PsaR controlling the expression of the rlrA pathogenicity islet as well (27). As described in chapter 3, we confirmed regulation of expression of the rlrA pathogenicity islet in TIGR4 (13). In addition, MerR (sp1856) was also differentially expressed in the TIGR4 psaR mutant (13). Interestingly, it seems that multiple signals, e.g., manganese concentrations for PsaR and unknown for RR09, control the expression of this islet. Rosch and co-workers conclude that this pathogenicity islet (acquired by genetic recombination) integrated into the preexisting regulatory networks of pneumococcal strains, and that this integration might have occurred differently in the various strains (or serotypes). It will be interesting to see how TCSs and especially TCS09, regulate gene expression in other strains than the ones tested in chapter 2. In addition, it is as yet unknown whether the identified target genes are directly or indirectly regulated by RR09, or to which external signal the histidine kinase sensor protein responds. Sequencing of several different strains (or serotypes) and testing global gene expression might give some insight into the plasticity of pneumococcal two-component systems. The role of metallic cations during infection is a well-studied area of microbial pathogenesis, as is the competition between host and pathogen for these ions (29). During colonization and infection of humans, pneumococcus is exposed to fluctuating concentrations of cations, making strict regulation of their uptake of utmost importance. For example, manganese (Mn 2+ ) is essential to all bacteria, because it serves as a cofactor for several metalloenzymes (16). Fluctuation of Mn 2+ concentrations might serve as a trigger for sitespecific expression of virulence factors, since the concentration of Mn 2+ is much higher in saliva than in blood plasma (6). Like Mn 2+ , Fe 2+ , Fe 3+ , Zn 2+ and Cu 2+ have been described to be important to bacterial virulence (7, 9, 10, 30, 34). The pneumococcal Mn 2+ -regulator PsaR (17, 19) was investigated in chapter 3 (13). Previous studies showed that PsaR is important 203 203
Chapter 8 for virulence in a serotype 3 strain (11), and we extended these studies using two other strains, D39 (serotype 2) and TIGR4 (serotype 4). Mutants lacking psaR displayed strain-specific phenotypes, evident mainly from differences in its contribution to development of bacteremia in mice. In addition, we also observed genes to be affected in expression in the psaR mutant in only one of the tested strain backgrounds. Among these strain-specific regulated genes was the rlrA pathogenicity islet (see above) (27). The psa operon, prtA, and pcpA were confirmed as PsaR-targets in both D39 and TIGR4 in our study (17, 19), One of these common PsaR targets, the choline-binding protein PcpA, was demonstrated to be required for adherence to a human pharyngeal cell line (12, 28). Moreover, regulation of expression of this adherence factor was shown to be directly positively affected by the nutritional regulator CodY (12 and chapter 4), while PsaR negatively regulates the expression of pcpA. CodY was shown to bind to the pcpA promoter regions thereby activating transcription of pcpA (12). This suggests a direct mechanism for adherence of pneumococcal cells to pharyngeal cells as a reaction on nutrient availability (chapter 7). These results are underscored by several studies showing higher expression of psaA and pcpA in in vivo situations (e.g., during contact with nasopharynx epithelial cells, and in the murine nose, lungs and blood) (20, 21). Contradictory results were also described: pcpA is not involved in colonization, but in invasive disease (8, Cron et al., personal communication). In addition, Bootsma et al. found that expression of pcpA was lower in nasopharyngeal lavage fluid than in culture medium (Bootsma et al., personal communication, see also chapter 7). Thus, the exact role of PcpA during pneumococcal disease remains unclear. The data described above imply that strain-specificity of pneumococcus may complicate the examination of the cellular processes involved in virulence. Moreover, it suggests that expression of potential pneumococcal antigens may occur in a strain-specific manner, similar to the variety of capsular serotypes. Nitrogen is a vital element for virtually all biological molecules. Therefore, bacteria have to ensure that they can utilize nitrogen from their continuously changing environment. Bacteria have developed sophisticated uptake and metabolic systems for the acquisition of nitrogen-containing molecules. In several Gram-positive bacteria, these systems have been well studied, but not much was known about the importance of nitrogen regulatory systems during the pneumococcal life cycle. In chapters 4, 5, and 6 we investigated the two main regulatory systems controlling nitrogen uptake, metabolism and inter-conversion in S. pneumonia, CodY and GlnR (12, 14, 18). Since we already extensively described these regulatory systems in Chapter 7, they will only be discussed briefly here. 204 204
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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
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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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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
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Page 170 and 171: GlnR-regulon and pneumococcal virul
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Page 174 and 175: Discussion The ability to adequatel
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Page 178 and 179: Acknowledgments WTH is supported by
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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 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
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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
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