Redefining the Small Regulatory RNA Transcriptome in Streptococcus pneumoniae Serotype 2 Strain D39

Abstract

Streptococcus pneumoniae (pneumococcus) is a major human respiratory pathogen and a leading cause of bacterial pneumonia worldwide. Small regulatory RNAs (sRNAs), which often act by posttranscriptionally regulating gene expression, have been shown to be crucial for the virulence of S. pneumoniae and other bacterial pathogens. Over 170 putative sRNAs have been identified in the S. pneumoniae TIGR4 strain (serotype 4) through transcriptomic studies, and a subset of these sRNAs has been further implicated in regulating pneumococcal pathogenesis. However, there is little overlap in the sRNAs identified among these studies, which indicates that the approaches used for sRNA identification were not sufficiently sensitive and robust and that there are likely many more undiscovered sRNAs encoded in the S. pneumoniae genome. Here, we sought to comprehensively identify sRNAs in Avery’s virulent S. pneumoniae strain D39 using two independent RNA sequencing (RNA-seq)-based approaches. We developed an unbiased method for identifying novel sRNAs from bacterial RNA-seq data and have further tested the specificity of our analysis program toward identifying sRNAs encoded by both strains D39 and TIGR4. Interestingly, the genes for 15% of the putative sRNAs identified in strain TIGR4, including ones previously implicated in virulence, are not present in the strain D39 genome, suggesting that the differences in sRNA repertoires between these two serotypes may contribute to their strain-specific virulence properties. Finally, this study has identified 66 new sRNA candidates in strain D39, 30 of which have been further validated, raising the total number of sRNAs that have been identified in strain D39 to 112. IMPORTANCE Recent work has shown that sRNAs play crucial roles in S. pneumoniae pathogenesis, as inactivation of nearly one-third of the putative sRNA genes identified in one study led to reduced fitness or virulence in a murine model. Yet our understanding of sRNA-mediated gene regulation in S. pneumoniae has been hindered by limited knowledge about these regulatory RNAs, including which sRNAs are synthesized by different S. pneumoniae strains. We sought to address this problem by developing a sensitive sRNA detection technique to identify sRNAs in S. pneumoniae D39. A comparison of our data set reported here to those of other RNA-seq studies for S. pneumoniae strain D39 and TIGR4 has provided new insights into the S. pneumoniae sRNA transcriptome.

Grad‐seq in a Gram‐positive bacterium reveals exonucleolytic sRNA activation in competence control

“…Taken together, our Grad‐seq study adds a new level of information to the increasing number of high‐throughput studies of the pneumococcus. It will provide an important link between predictions of protein–protein interactions (Wuchty et al , ) and the wealth of recent transcriptomics‐based studies (Acebo et al , ; Mann et al , ; Aprianto et al , ; Slager et al , , ; Warrier et al , ; Sinha et al , ) in the quest to better understand the molecular underpinnings of pneumococcal physiology and virulence.…”

“…The pneumococcus is a leading human pathogen, causing diverse infectious diseases such as otitis media, sinusitis, sepsis, meningitis, and pneumonia, and is responsible for more than one million deaths annually (O'Brien et al , ; Henriques‐Normark & Tuomanen, ). Unsurprisingly, pneumococcal transcriptomes, including condition‐specific sRNA expression, have been extensively characterized (Acebo et al , ; Mann et al , ; Aprianto et al , ; Slager et al , , ; Warrier et al , ; Sinha et al , ) and potential binary protein–protein interactions have also been predicted (Wuchty et al , ). Although these studies yielded invaluable new insight into the molecular underpinnings of pneumococcal virulence, they did not cover functional RNA–protein interactions, especially for sRNAs with known roles in these processes.…”

Non-coding RNAs in pneumonia diagnosis

“…На модельных животных, зараженных S. pneumoniae, было зарегистрировано 42 некодирующих малых бактериальных РНК длиной более 100 нуклеотидов, уровень которых был увеличен более чем в 2 раза в образцах плевральной жидкости по сравнению с культуральной средой [58]. При сравнении разных штаммов S. pneumoniae было выявлено, что 15% малых РНК одного штамма не синтезируются в клетках другого штамма [59]. Разные штаммы S. aureus также отличались наборами малых РНК, профили экспрессии некоторых из них различались в сыворотке человека относительно культуральной среды.…”

Competence beyond Genes: Filling in the Details of the Pneumococcal Competence Transcriptome by a Systems Approach

“…In addition, paired-end and "cappable" RNA-seq approaches and predictions of factor-independent transcription terminators were compiled to indicate the precise boundaries of single-gene and multigene operons and noncoding small RNAs (sRNAs) throughout the D39 genome. These analyses revealed a wealth of genomic features, including new sRNAs, whose number has since been expanded using a different bioinformatic approach (22); the presence of regulatory riboswitches in the 5= untranslated regions (5=-UTRs) of numerous operons but the absence of 5=-UTRs upstream of others; a strong bias toward adenine (A) at transcription start sites; and the presence of likely regulatory motifs in most promoter regions. Importantly, this gold mine of information was assembled and made available to other researchers in an easy-to-use, intuitive website named PneumoBrowse (20).…”