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

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CodY was found to be required for adherence and colonization (12 and chapter 4). Many transport systems involved in nitrogen uptake and metabolism were found to be regulated by CodY. A changed expression of one of these genes (or a combination of these) might be responsible for the lack-of-colonization phenotype and the lower adherence of the codY mutant. The pcpA gene was more highly expressed in the codY mutant, however, as described above, it seems not very likely that this gene is required for colonization and adherence. Summarizing discussion The transcriptional regulator GlnR was investigated in chapters 5 and 6. This glutamine/glutamate-dependent regulator itself was found not to be essential for pneumococcal virulence in mice, but its target genes were: glutamine synthetase GlnA was required for efficient colonization and the glutamine permease GlnP was required for survival in the lungs (14). A double mutant for glnA and glnP appeared to be avirulent in all our models. Interestingly, microarray analysis showed that in this double mutant many CodY targets were overexpressed, indicating that the double mutant was in severe nutritional stress. Our results clearly show that both the CodY and GlnR regulatory systems are important for pneumococcal virulence. Given the strain-specificity observed by us and others for other regulatory systems, it will be interesting to see if these two highly conserved regulatory proteins also have strain-specific features. Concluding remarks In this thesis four different regulatory systems have been investigated. In different ways, all were shown to be involved in virulence. A major finding in this thesis is that the target genes of several conserved regulators appear to be strain-specific (chapters 2 and 3), which is rather surprising considering that these regulatory systems are conserved among the sequenced strains and even among other streptococci. Strain-specificity complicates the discovery of new antigens that could serve as novel vaccine targets. Besides strain-specific regulation of gene expression, genes are also expressed site-specifically, e.g., expressed at the nasopharynx but not in the blood. One can argue that for vaccination purposes, the ideal situation would be expression of an antigen only during invasive disease and not during colonization; this will leave colonization unaffected and will prevent invasive disease. The benefit of leaving colonization unaffected is that it keeps other potentially pathogenic bacteria from colonizing due to competition between pneumococcus and other respiratory pathogens (4, 23). 205 205
Chapter 8 206 206 Another important finding described in this thesis is requirement of the two nutritional regulators CodY and GlnR for full pneumococcal virulence (chapter 4, 5, and 6). Obviously, nutrition is the most important process in (bacterial) survival; without food no living organism can survive. Under normal conditions, pneumococcus only inhabits the nasopharynx of humans. This highly specific niche probably has a highly specific pool of metabolizable nutrients for which pneumococcus has developed specific metabolic routes. This makes it very likely that expression (or repression) of genes involved in nutrient uptake/breakdown and colonization are linked and co-regulated. Chapters 4, 5, and 6 have contributed to a general understanding of this gene regulation. Future research should be focusing on elucidation of these regulatory pathways to get a better understanding of pneumococcal behavior. For instance, CodY is repressing its gene targets during colonization (12), but it remains to be investigated whether CodY-regulated genes are required for invasive disease. It will be interesting to see the virulence phenotype of a CodY-overexpressing pneumococcus during a pneumonia or bacteremia model of infection. In summary, this thesis highlights regulatory networks controlling the interplay between pneumococcal nutrition and virulence. Unraveling the gene transcriptional networks will help predict bacterial behavior and, consequently, will aid future disease prevention and treatment strategies.
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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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Nitrogen Metabolism in S. pneumonia
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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
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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 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 218 and 219: Curriculum Vitae Curriculum Vitae W
Page 220 and 221: Dankwoord Jeetje, dat het dan toch
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