Transcriptional response of Streptococcus pneumoniae to varying concentrations of carbohydrates and metal ions

S. pneumoniae is one of the most common human pathogen that resides in nasopharynx. It is responsible for millions of death every year all over the world, especially in young children. S. pneumoniae can spread from nasopharynx to different parts of the human body where it may encounter different nutrients including various concentration of metal ions and carbohydrates. It has an ability to survive on these nutrients by tuning it’s genome according. This thesis contributes to the understanding of sugar- and metal-ions dependent regulation of virulence genes/operons including fcs operon, PsaR regulon and AdcR regulon.
Chapter 2 deals with the optimization of conditions for bacterial transcriptome analysis from cell harvest to DNA microarray analysis. Taking into account the time, costs and accuracy of the experiments, this technology platform proves to be very useful and universally applicable for studying bacterial transcriptomes. Here, we perform DNA microarray analysis with Streptococcus pneumoniae as a case-study by comparing the transcriptional responses of S. pneumoniae grown in the presence of varying L-serine concentrations in the medium. Total RNA was isolated by using a Macaloid method using an RNA isolation kit and the quality of RNA was checked by using an RNA quality check kit. cDNA was prepared using reverse transcriptase and the cDNA samples were labelled using one of two amine-reactive fluorescent dyes, . Homemade DNA microarray slides were used for hybridization of the labelled cDNA samples and microarray data were analyzed by using a cDNA microarray data pre-processing framework (Microprep). Finally, Cyber-T was used to analyze the data generated using Microprep for the identification of statistically significant differentially expressed genes. Furthermore, in-house built software packages (PePPER, FIVA, DISCLOSE, PROSECUTOR, Genome2D) were used to analyze data.

In Chapter 3 explores the impact of fucose on the transcriptome of S. pneumoniae D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (fcs operon), was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the fcs operon, as a transcriptional activator of the fcs operon. We also predict a 19-bp putative FcsR regulatory site (5’-ATTTGAACATTATTCAAGT-3’) in the promoter region of the fcs operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the fcs operon.
Manganese (Mn2+)-, zinc (Zn2+)- and copper (Cu2+) play significant roles in transcriptional gene regulation, physiology and virulence of Streptococcus pneumoniae. So far, the effect of the important transition metal ion cobalt (Co2+) on gene expression of S. pneumoniae has not yet been explored. Chapter 4 reveals the impact of Co2+ stress on the transcriptome of S. pneumoniae strain D39. BLAST searches revealed that the genome of S. pneumoniae encodes a putative Co2+-transport operon (cbi operon), the expression of which we show here to be induced by a high Co2+ concentration. Furthermore, we found that Co2+, as has been shown previously for Zn2+, can cause derepression of the genes of the PsaR virulence regulon, encoding the Mn2+-uptake system PsaBCA, the choline binding protein PcpA and the cell-wall associated serine protease PrtA. Interestingly, although Mn2+ represses expression of the PsaR regulon and Co2+ leads to derepression, both metal ions stimulate interaction of PsaR with its target promoters. These data will be discussed in the light of previous studies on similar metal-responsive transcriptional regulators.
Chapter 5 examines the Ni2+-dependent regulation of PsaR regulon (pcpA, psaBCA and prtA). Here, we combine qRT-PCR analysis, metal accumulation assays, β-galactosidase assays and electrophoretic mobility shift assays to show that the elevated concentration of Ni2+ leads to the higher expression of PsaR regulon. Our ICP-MS data demonstrated that the induced expression of PsaR regulon is directly linked to high concentration of Ni2+, which reduces the cell-associated concentration of Mn2+. In vitro studies with purified PsaR protein showed that Ni2+ diminishes the Mn2+-dependent interaction of PsaR to the promoter regions of its target genes. Additionally, Ni2+-dependent role of transcriptional regulator PsaR in the regulation of PsaR regulon is studied by the DNA microarray analysis.
Chapter 6 explains the transcriptome analysis of S. pneumoniae D39 in presence of Ni2+. The regulation of virulence genes in the human pathogen Streptococcus pneumoniae is an important characteristic of transition metal ions. Metal ions such as Mn2+, Zn2+, Cu2+and Co2+ have been shown to regulate the expression of various virulence genes. The effect of Ni2+ on global gene expression of S. pneumoniae has not yet been investigated. Here, we identify a number of differentially expressed genes/ operons in the presence of a high extracellular concentration of Ni2+. The virulence genes belonging to the AdcR regulon (adcRCBA, phtA, phtB, phtD and phtE), the PsaR regulon (prtA, pcpA and psaBCA) and Zn2+/ Co2+ -efflux system czcD were highly upregulated in the presence of Ni2+. By means of DNA microarray analysis, electrophoretic mobility shift assays and transcriptional lacZ-reporter studies, we show that Ni2+ is directly involved in the derepression of adcRCBA, adcAII-phD, phtA, phtB and phtE via the Zn2+-dependent repressor AdcR. The opposite effect of Zn2+ and Ni2+ on the AdcR regulon is also explored in detail. In conclusion, the role of Ni2+ in the regulation of virulence genes belonging to the AdcR regulon has been further elucidated.