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

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correlated well with our in vitro microarray and real-time PCR data, supporting the hypothesis that RR09 has a role in the regulation of this pathogenicity islet. Gene regulation by RR09 in S. pneumoniae Figure 6. Expression of sp0060 to sp0066 in wild-type and ∆rr09 mutant D39, TIGR4, and 0100993 as determined by real-time PCR. The dashed line indicates the twofold change microarray cutoff value for differential expression. wt, wild type. Strain-dependent regulation of sp0063 does not explain the observed difference in virulence While our analysis indicated that RR09 has a prominent role in regulation of sugar metabolism in D39, only three putative PTSs were found to be regulated by RR09 in TIGR4. It seems unlikely that there is a difference in the sugar diets of D39 and TIGR4, as both genomes basically contain the same putative PTSs (20 PTSs common to both strains and one PTS specific for TIGR4) (9, 28). A clear example of a PTS locus differentially regulated by RR09 in D39 and TIGR4 was the locus comprising sp0060 to sp0066, and therefore we also examined expression of this locus by real-time PCR in a third strain, 0100993. This and previous studies showed that RR09 mutants of the three strains used here all have different phenotypes in murine models of infection (2). While strong downregulation of sp0060 to 43 43
Chapter 2 sp0066 was confirmmed to occuur in D39∆rrr09, no RR R09-dependdent regulatiion was obs served in either TTIGR4 or 0100993 (Fiig. 6). The observed downregulat d tion of this locus in D39∆rr09 may result in redduced sugarr uptake annd thus a smaller s suppply of carrbohydrates for the polysaccharide cappsule, whichh in turn migght explain the aviruleent phenotyppe of D39∆r rr09. To test thiss hypothesiss, an sp00663 mutant wwas construc cted and tessted in a mmurine mode el, and it was commpared withh the D39 wwild-type strain and th he rr09 muutant. There was no sig gnificant differennce in the ssurvival rattes betweenn wild-type strain D399- and sp00063 mutant-infected mice; aall five wildd-type strainn-infected mmice died, as did fourr of the five ∆sp0063- -infected mice. Likewise, thee blood counnts at 24 h ppostinfectio on were simmilar (Fig. 7) ). In agreem ment with our prevvious work (3), the rr009 mutant wwas essentia ally aviruleent; all infeccted mice survived, s and the blood counnts at 24 h wwere beloww the detecti ion limit ( 83 CFU/ml). Indeed, the t mice showedd no overt clinical signs of infectioon. Thus, th he gene prooduct of sp00063 alone does d not significaantly contriibute to virrulence in DD39 in this s model annd hence dooes not exp plain the dramatic phenotype seen afterr deletion oof rr09 in D39. D The ∆rrr09 derivattives of TIG GR4 and 01009933 conferredd a less sevvere in vivoo phenotype e. In vivo eexpression oof sp0063 could c be demonsstrated for bboth D39 annd TIGR, annd the levels in TIGR44 were higheer (almost fourfold) f (Fig. 5aa). In TIGR44, sp0063 aappears to be regulated by RR09 inn vivo (Fig. 5b) but not t in vitro (Fig. 6) ), highlightiing the impportance of measuring gene expression in vvivo. Moreo over, this finding provides fu further evideence that thhe different tial regulation of this ooperon observed in vitro dooes not expllain the diffference betwween D39 and a TIGR4 in vivo. Allso, there ap ppears to be a reddundancy oof PTS in thhe pneumococcal geno ome, whichh could commplement th he lower level off expressionn of the PTTS. This is uunderscored d by our preeliminary ddata, which indicate that the rr09 and spp0063 mutants are ablee to ferment mannose (nnot shown). 44 44
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 14 and 15: histidine kinase is the sensor prot
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 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
Page 65 and 66: Chapter 3 between the wild-type and
Page 67 and 68: Chapter 3 S. pneumoniae TIGR4 by CS
Page 69 and 70: Chapter 3 performance liquid chroma
Page 71 and 72: Chapter 3 Results Transcriptional a
Page 73 and 74: Chapter 3 Table 3. Differentially e
Page 75 and 76: Chapter 3 Figure 2. Colonization mo
Page 77 and 78: Chapter 3 than the D39ΔpsaR-infect
Page 79 and 80: Chapter 3 effect was more severe fo
Page 81 and 82: Chapter 3 in TIGR4ΔpsaR. However,
Page 83 and 84: Chapter 3 References 1. Adrian, P.
Page 85 and 86: Chapter 3 21. Hemsley, C., E. Joyce
Page 87 and 88: Chapter 3 40. McCluskey, J., J. Hin
Page 90 and 91: CHAPTER 4 CodY of Streptococcus pne
Page 92 and 93: Introduction Bacteria encounter var
Page 94 and 95:
Materials and Methods Bacterial str
Page 96 and 97:
Table 2. Oligonucleotide primers us
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The CodY regulon of S. pneumoniae I
Page 100 and 101:
The CodY regulon of S. pneumoniae w
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Accession numbers The microarray da
Page 104 and 105:
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
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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
Page 184 and 185:
CHAPTER 7 Pneumococcal gene regulat
Page 186 and 187:
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
Page 196 and 197:
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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