Single nucleotide resolution RNA-seq uncovers new regulatory mechanisms in the opportunistic pathogen Streptococcus agalactiae

Rosinski‐Chupin, Isabelle; Sauvage, Elisabeth; Sismeiro, Odile; Villain, Adrien; Cunha, Violette Da; Caliot, Marie-Elise; Dillies, Marie‐Agnès; Trieu-Cuot, Patrick; Bouloc, Philippe; Lartigue, Marie‐Frédérique; Glaser, Philippe

doi:10.1186/s12864-015-1583-4

Cited by 49 publications

(75 citation statements)

References 71 publications

(80 reference statements)

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“…Our operon prediction also yielded similar results to those reported for related human pathogen Streptococcus agalactiae , which encodes 891 operons and 484 monocistrons (Rosinski‐Chupin et al., ). Moreover, the distribution of 5’ UTR lengths in strain NZ131 is proportional to the distribution reported for S. agalactiae and the swine pathogen Streptococcus suis (Rosinski‐Chupin et al., ; Wu et al., ). To further validate our results, we compared our operon predictions with a computational prediction using ProOpDB.…”

Section: Discussionsupporting

confidence: 86%

Genome‐wide screening of potential RNase Y‐processed mRNAs in the M49 serotype Streptococcus pyogenes NZ131

Chen

Raghavan

et al. 2018

MicrobiologyOpen

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RNase Y is a major endoribonuclease in Group A streptococcus (GAS) and other Gram-positive bacteria. Our previous study showed that RNase Y was involved in mRNA degradation and processing in GAS. We hypothesized that mRNA processing regulated the expression of important GAS virulence factors via altering their mRNA stabilities and that RNase Y mediated at least some of the mRNA-processing events.The aims of this study were to (1) identify mRNAs that were processed by RNase Y and (2) confirm the mRNA-processing events. The transcriptomes of Streptococcus pyogenes NZ131 wild type and its RNase Y mutant (Δrny) were examined with RNAseq. The data were further analyzed to define GAS operons. The mRNA stabilities of the wild type and Δrny at subgene level were determined with tiling array analysis.Operons displaying segmental stability in the wild type but not in the Δrny were predicted to be RNase Y processed. Overall 865 operons were defined and their boundaries predicted. Further analysis narrowed down 15 mRNAs potentially processed by RNase Y. A selection of four candidates including folC1 (folylpolyglutamate synthetase), prtF (fibronectin-binding protein), speG (streptococcal exotoxin G), ropB (transcriptional regulator of speB), and ypaA (riboflavin transporter) mRNAs was examined with Northern blot analysis. However, only folC1 was confirmed to be processed, but it is unlikely that RNase Y is responsible. We conclude that GAS use RNase Y to selectively process mRNA, but the overall impact is confined to selected virulence factors. K E Y W O R D Sgenomics, RNA processing, RNase Y, Streptococcus pyogenes

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Section: Discussionsupporting

confidence: 86%

Genome‐wide screening of potential RNase Y‐processed mRNAs in the M49 serotype Streptococcus pyogenes NZ131

Chen

Raghavan

et al. 2018

MicrobiologyOpen

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“…Transcriptional Start Sites (TSS) were identified at the genome level in GBS strains A909 and BM110 using a differential RNA-seq approach as previously described for strain NEM316 [17]. A common TSS was identified 37 bp upstream of the start codon of the first gene ( orf ) of the PI-2b locus in both strains (Fig 4A).…”

Section: Resultsmentioning

confidence: 99%

“…Genome-wide mapping of transcription start sites in GBS strains A909 and BM110 was performed by using a differential RNA-sequencing protocol based on selective Tobacco Acid Pyrophosphatase (TAP) treatment and 5' adapter ligation as previously described [17, 28]. Briefly, for each strain, mixes of RNA prepared at mid-exponential and stationary growth phases in a rich culture medium (TH) and at the beginning of the stationary phase in a poor culture medium (RPMI supplemented with glucose 1% and pH-buffered with 50 mM Hepes) were enriched in mRNA with the MICROBExpress Kit (Ambion).…”

Section: Methodsmentioning

confidence: 99%

Regulation of PI-2b Pilus Expression in Hypervirulent Streptococcus agalactiae ST-17 BM110

et al. 2017

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The widely spread Streptococcus agalactiae (also known as Group B Streptococcus, GBS) “hypervirulent” ST17 clone is strongly associated with neonatal meningitis. The PI-2b locus is mainly found in ST17 strains but is also present in a few non ST17 human isolates such as the ST-7 prototype strain A909. Here, we analysed the expression of the PI-2b pilus in the ST17 strain BM110 as compared to the non ST17 A909. Comparative genome analyses revealed the presence of a 43-base pair (bp) hairpin-like structure in the upstream region of PI-2b operon in all 26 ST17 genomes, which was absent in the 8 non-ST17 strains carrying the PI-2b locus. Deletion of this 43-bp sequence in strain BM110 resulted in a 3- to 5-fold increased transcription of PI-2b. Characterization of PI-2b promoter region in A909 and BM110 strains was carried out by RNAseq, primer extension, qRT-PCR and transcriptional fusions with gfp as reporter gene. Our results indicate the presence of a single promoter (Ppi2b) with a transcriptional start site (TSS) mapped 37 bases upstream of the start codon of the first PI-2b gene. The large operon of 16 genes located upstream of PI-2b codes for the group B carbohydrate (also known as antigen B), a major constituent of the bacterial cell wall. We showed that the hairpin sequence located between antigen B and PI-2b operons is a transcriptional terminator. In A909, increased expression of PI-2b probably results from read-through transcription from antigen B operon. In addition, we showed that an extended 5’ promoter region is required for maximal transcription of gfp as a reporter gene in S. agalactiae from Ppi2b promoter. Gene reporter assays performed in Lactococcus lactis strain NZ9000, a related non-pathogenic Gram-positive species, revealed that GBS-specific regulatory factors are required to drive PI-2b transcription. PI-2b expression is up-regulated in the BM110ΔcovR mutant as compared to the parental BM110 strain, but this effect is probably indirect. Collectively, our results indicate that PI-2b expression is regulated in GBS ST17 strains, which may confer a selective advantage in the human host either by reducing host immune responses and/or increasing their dissemination potential.

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“…Each mutated gene was blast searched against the 300 sequenced GBS genomes in the NCBI database available in April 2015. On the basis of the identification of the transcription start sites mapped on the genome of GBS NEM316 (46), the relative position of each SNP located within noncoding regions was ascertained. Functional categories of the mutated genes were inferred on the basis of the SagaList annotation created from the GBS NEM316 sequencing project (47,48).…”

Section: Methodsmentioning

confidence: 99%

Whole-Genome Comparison Uncovers Genomic Mutations between Group B Streptococci Sampled from Infected Newborns and Their Mothers

Almeida¹,

Villain

Joubrel

et al. 2015

J Bacteriol

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Streptococcus agalactiae (group B Streptococcus or GBS), a commensal of the human gut and genitourinary tract, is a leading cause of neonatal infections, in which vertical transmission from mother to child remains the most frequent route of contamination. Here, we investigated whether the progression of GBS from carriage to disease is associated with genomic adaptation. Whole-genome comparison of 47 GBS samples from 19 mother-child pairs uncovered 21 single nucleotide polymorphisms (SNPs) and seven insertions/deletions. Of the SNPs detected, 16 appear to have been fixed in the population sampled whereas five mutations were found to be polymorphic. In the infant strains, 14 mutations were detected, including two independently fixed variants affecting the covRS locus, which is known to encode a major regulatory system of virulence. A one-nucleotide insertion was also identified in the promoter region of the highly immunogenic surface protein Rib gene. Gene expression analysis after incubation in human blood showed that these mutations influenced the expression of virulence-associated genes. Additional identification of three mutated strains in the mothers' milk raised the possibility of the newborns also being a source of contamination for their mothers. Overall, our work showed that GBS strains in carriage and disease scenarios might undergo adaptive changes following colonization. The types and locations of the mutations found, together with the experimental results showing their phenotypic impact, suggest that those in a context of infection were positively selected during the transition of GBS from commensal to pathogen, contributing to an increased capacity to cause disease. IMPORTANCEGroup B Streptococcus (GBS) is a major pathogen responsible for neonatal infections. Considering that its colonization of healthy adults is mostly asymptomatic, the mechanisms behind its switch from a commensal to an invasive state are largely unknown. In this work, we compared the genomic profile of GBS samples causing infections in newborns with that of the GBS colonizing their mothers. Multiple mutations were detected, namely, within key virulence factors, including the response regulator CovR and surface protein Rib, potentially affecting the pathogenesis of GBS. Their overall impact was supported by differences in the expression of virulence-associated genes in human blood. Our results suggest that during GBS's progression to disease, particular variants are positively selected, contributing to the ability of this bacterium to infect its host. Streptococcus agalactiae, or group B Streptococcus (GBS), is currently regarded as one of the leading causes of neonatal sepsis and meningitis worldwide (1-4). Known as a commensal of the digestive and genitourinary tracts of 10 to 30% of the human population, GBS is also a significant source of disease in immunocompromised adults and in the elderly (4-7). Apart from causing human infections, GBS is an etiological agent of bovine mastitis and invasive disease in fish (8-10). F...

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Single nucleotide resolution RNA-seq uncovers new regulatory mechanisms in the opportunistic pathogen Streptococcus agalactiae

Cited by 49 publications

References 71 publications

Genome‐wide screening of potential RNase Y‐processed mRNAs in the M49 serotype Streptococcus pyogenes NZ131

Genome‐wide screening of potential RNase Y‐processed mRNAs in the M49 serotype Streptococcus pyogenes NZ131

Regulation of PI-2b Pilus Expression in Hypervirulent Streptococcus agalactiae ST-17 BM110

Whole-Genome Comparison Uncovers Genomic Mutations between Group B Streptococci Sampled from Infected Newborns and Their Mothers

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