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

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The CodY regulon of S. pneumoniae washed with 70% ethanol, air dried, and resuspended in 10 µl formamide loading buffer (containing bromophenol-blue and xylene-blue). These samples were heated for 1 min at 99ºC, and applied to a preheated 8% polyacrylamide denaturing sequencing gel. Gels were air-dried, and autoradiography was performed by exposing the gel to an X-ray film. In vitro pneumococcal adherence assay Adherence of pneumococci to epithelial cells was studied essentially as described previously (7, 27, 28). In short, monolayers of the human pharyngeal cell line Detroit 562 (ATCC CCL-138) were washed twice with 1 ml PBS. Aliquots of bacteria (grown to mid-log in Todd Hewitt Yeast-broth) stored at -80°C were thawed rapidly, harvested by centrifugation, and resuspended in RPMI 1640 medium without phenol red (Invitrogen) supplemented with 1% FCS to 1x10 7 CFU/ml. One ml of bacterial suspension was allowed to adhere for 2h, after which non-adherent bacteria were removed by three washes with 1 ml PBS. For quantification of adherence, epithelial cells were subsequently detached by treatment with 25% Trypsin, 1 mM EDTA in PBS and lysed by the addition of ice-cold 0.025% Triton X-100 in PBS. Serial 10-fold dilutions were plated on blood agar plates to count the number of adherent bacteria, and corrected mathematically to account for small differences in count in the initial inoculum. Wild-type and mutant strains grew comparably in RMPI medium without Detroit 562 cells. Experimental mouse models Nine-week old female outbred CD-1 mice (Harlan, Horst, Netherlands) were used for all infection models. Prior to infection, D39 wild-type and ΔcodY were passaged in mice to maintain virulence as described previously (25). Cultures of S. pneumoniae D39 or ΔcodY were grown to an OD600 of 0.3, and stored in aliquots at -80�C in 10% glycerol. Prior to infection, these aliquots were spun down and bacteria were resuspended in sterile PBS. Mice were lightly anesthetized with 2.5% (vol/vol) isoflurane / O2, and infected intranasally with 10 6 CFU of bacteria as described previously (24). At predetermined time points after infection, groups of mice were sacrificed by cervical dislocation and samples of various sites were taken to determine the bacterial load. In the colonization model, five mice per group were infected with 10 µl of PBS containing 10 6 CFU of either D39 wild-type or ΔcodY, a volume small enough to only infect the nose (nasopharynx) of the mice. Bacteria were recovered from the nasopharynx by flushing the nostrils with 2 ml of sterile PBS (26), and lungs were removed from the body and homogenized in 2 ml of sterile PBS using a hand held 99 99
Chapter 4 homogenizer (polytron PT 1200, Kinematica AG). Viable bacteria in nasal lavage fluid and homogenized lungs were counted by plating serial 10-fold dilutions on Colombia blood agar (Oxoid) supplemented with 5% (vol/vol) defibrinated sheep blood (Biotrading). Time points for sampling were 0, 24, 48, 96 and 192 h post-infection. For the pneumoniae model, five mice per group were infected with 50 µl of PBS containing 10 6 CFU of either D39 wild-type or ΔcodY. Viable bacteria were recovered and quantified from the different sites as described above. In addition, a blood sample was removed by a cardiac puncture using a 1-ml syringe. Time points for sampling were 0, 12, 24, and 36h post-infection. In the sepsis model, six mice per group were infected in a tail vein with 10 6 CFU resuspended in 100 µl of sterile PBS. Bacteria were recovered from the blood by a lateral tail vein puncture from the same mouse at 0, 12, 24, and 36h post-infection and quantified as described above. Bacteriology results are expressed as geometric mean ± standard errors of the mean (SEM). Comparison of bacterial loads was performed using Student’s t test. In all analyses, p
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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
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
Page 98 and 99: The CodY regulon of S. pneumoniae I
Page 102 and 103: Accession numbers The microarray da
Page 104 and 105: Table 3. Differentially expressed g
Page 106 and 107: Binding of CodY to target promoters
Page 108 and 109: The CodY regulon of S. pneumoniae E
Page 110 and 111: The CodY regulon of S. pneumoniae F
Page 112 and 113: Discussion CodY has been described
Page 114 and 115: levels of adherence in D39. Using a
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
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
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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
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CHAPTER 7 Pneumococcal gene regulat
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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
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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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oekje en ik kijk uit naar de bijbeh
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