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

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Discussion PsaR and pneumococcal virulence During colonization and infection of the human host, S. pneumoniae encounters fluctuating amounts of free Mn 2+ at the different niches where the bacterium resides. Since Mn 2+ is required for several cellular processes, proper regulation of manganese homeostasis is vital for pneumococcal physiology and virulence. The transcriptional repressor PsaR has been described to play an important role in this process, at least in the genetic background of strain EF3030 (serotype 19F) (28). However, we and others have previously shown that, even though transcriptional regulators themselves appear to be conserved between S. pneumoniae strains, they often have strain-specific impact on global transcription and virulence (4, 24, 40). To examine whether a similar strain-specificity holds true for PsaR, we identified its targets and its contribution to experimental virulence in two additional strains, TIGR4 and D39. We used a combination of transcriptional and proteome analyses for the identification of PsaR targets. Observed discrepancies between these two approaches could be the result of several factors, such as low levels of gene expression, protein instability, or post-translational regulation. The previously reported PsaR targets, the Psa operon, pcpA, and prtA (28, 32), were confirmed in TIGR4 and D39 by both transcriptomics and proteomics. In addition, we found sp1637, encoding a hypothetical protein, to be upregulated in both psaR mutants. Since the latter gene is located directly upstream of psaR (sp1638), we cannot entirely exclude the possibility that the derepression of sp1637 is caused by transcriptional read-through of the trimethoprim resistance cassette used to create the psaR knockout. We have previously shown that the common PsaR target pcpA is required for adherence to the human Detroit epithelial cell line and that the expression of this gene is also directly positively regulated by the nutritional regulator CodY (22). The inability of the codY-mutant to colonize the murine nasopharynx underscored, albeit indirectly, the involvement of PcpA in adherence and colonization. However, a recent study showed that PcpA is not involved in colonization but has a role in invasive disease (15). These contradictory results are possibly due to different experimental set-ups, however, the exact role of PcpA during pneumococcal pathogenesis remains unclear. In addition to the shared PsaR-targets (i.e., the overlapping genes between D39 and TIGR4), several genes were differentially expressed in D39 or TIGR4 only. This strainspecific differential expression might be due to direct regulation by PsaR, but is more likely to be either an indirect effect caused by an imbalance in Mn 2+ /Zn2 + -homeostasis due to the lack of PsaR or, possibly, downstream signaling of other regulators. Moreover, it seems that this 77 77
Chapter 3 effect was more severe for D39, since more genes and proteins were affected in their expression level in this strain. Downstream regulation or a disturbed balance in other cellular processes might be the cause of this differential expression, since manganese cations have been shown to be required for several bacterial cellular processes (26). For example, manganese cations have been shown to function as co-factors for enzymes in glycolysis, amongst others 6-phospho-β-glucosidase (50). The genes encoding 6-phospho-β-glucosidase and a phosphotransferase system (PTS) downstream of it (sp0303, sp0306, and sp0308), were upregulated in D39ΔpsaR, in line with indirect regulation. However, in D39 these genes are strongly downregulated in the presence of Zn 2+ , which is not in agreement with the opposite effect of Mn 2+ and Zn 2+ on PsaR-regulation (32). 78 78 In D39ΔpsaR, the gene ciaR, involved in competence development, was upregulated. It has been reported that Mn 2+ is required for genetic transformation (12), suggesting that downregulation of this gene might be a indirect effect of the psaR-mutation. Several other systems were downregulated in D39ΔpsaR alone, such as a putative bacteriocin system (sp0142-sp0146), the blp bacteriocin system, and the blp two component system (TCS13). The genes sp0141-sp0146 have also been shown to be regulated by the nutritional regulator CodY in D39 (22). Possibly, the downregulation of the blp bacteriocin genes is due to downregulation of the blp two component system (10, 11). Notably in TIGR4, a MerR family regulator (sp1856) and czcD (sp1857), encoding a Zn 2+ -efflux system, were upregulated in the psaR-mutant. These two genes have also been shown to be upregulated in the presence of Zn 2+ , underscoring the reported opposite effect of Mn 2+ and Zn 2+ on PsaR-regulation (32). The czcD gene has recently been shown to be regulated by SczA (sp1858) in reaction to increasing cellular zinc concentrations (34), but this efflux system might also be involved in Mn 2+ -homeostasis. This again indicates that these regulatory systems of cation homeostasis are intertwined (32). The expression of ply (encoding the pneumococcal toxin pneumolysin) was downregulated as a result of the psaR mutation during in vitro growth in TIGR4 only. If this downregulation also occurred during our infection experiments, it did not have a large effect on experimental virulence of the TIGR4 strain, as the ∆psaR mutant was as virulent as the wild-type in all our infection models. Only at 24h post-infection during experimental pneumonia, mice infected with the psaR-mutant had significantly lower bacterial loads in the lungs compared to those infected with wild-type. A role for PsaR in virulence has been reported in an EF3030 genetic background (28). During seven days of colonization no difference between the wild-type and isogenic psaR-
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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
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 44 and 45: correlated well with our in vitro m
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: Chapter 3 than the D39ΔpsaR-infect
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 100 and 101: The CodY regulon of S. pneumoniae w
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
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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
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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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