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RNA-seq Analysis IDs Regulatory Mechanisms Involved in Pathogenesis of Opportunistic Bacteria

NEW YORK (GenomeWeb) – In an attempt to study in depth the regulatory mechanisms of pathogens that contribute to their physiology and toxicity, researchers at the Institut Pasteur in Paris used a combination of two transcriptome sequencing techniques to study Streptococcus agalactiae, a bacteria that frequently causes infection in newborns and is increasingly becoming an opportunistic pathogen in adults with underlying diseases.

The researchers used two strategies — differential RNA-seq and strand-specific RNA-seq — to identify transcription start sites, regulatory RNAs, and differential expression in an S. agalactiae strain grown under eight different environmental conditions and different genetic backgrounds.

They published their work in BMC Genomics in May.

S. agalactiae is present in the digestive tracts of 10 percent to 30 percent of the human population without causing infection. But it can switch from being commensal to pathogenic and is a major cause of infections in newborns, leading to pneumonia, septicemia, and meningitis. Increasingly, it is also causing infections in the elderly and adults with underlying diseases and has been associated with animal diseases, including massive epidemic outbreaks in fish farms and a bovine inflammatory disease called mastitis.

Thus far, the mechanisms that lead to the bacteria switching from benign to virulent are largely unknown. In order to identify those mechanisms, the researchers used transcriptome sequencing techniques to identify promoter regions, operon structure, small RNAs, and untranslated regions.

The team first performed differential RNA-seq, which distinguishes between primary transcripts and processed RNAs, on RNA extracted from an S. agalactiae strain grown under four different growth or stress conditions.

Sequencing predicted 1,210 transcription start sites (TSS), in which the team looked for reiterative transcription. Also known as transcript slippage, it is a process in which the RNA polymerase "stutters," leading to a transcript that differs at its 5' end from the DNA template by one or several bases. Reiterative transcription is a known regulatory mechanism in Escherichia coli and Bacillus subtilis.

The researchers found that 15 percent of the 1,210 TSS were associated with reiterative transcription and those regions were enriched for genes involved in nucleotide metabolism. The number of TSS associated with reiterative transcription was unexpectedly high, the researchers wrote, "suggesting new potential regulatory mechanisms." For instance, it might "serve as a mechanism to translate global information on the metabolic status of the cell into modifications in gene expression."

The researchers said that further analysis of reiterative transcriptions would "uncover the extent" of it in other bacterial species and "reveal new mechanisms of regulation."

Next, they combined the TSS analysis with strand-specific RNA-seq. Previous research on S. agalactiae has characterized 2,082 protein-coding genes, 36 pseudogenes, seven rRNA clusters, and 80 tRNA genes.

Under the different conditions in which they tested S. agalactiae they were able to identify gene expression from nearly 80 percent of the known, annotated genes. In addition, for 655 transcripts, they characterized the 3' end.

The researchers were especially interested in characterizing the RNA components involved in regulation, such as cis-regulatory regions in noncoding RNA that affect transcription and small RNAs.

They identified 39 cis-regulatory regions, 39 cis-antisense noncoding RNAs, and 47 small RNAs. There were 11 novel cis-regulatory regions and two responded to acidic conditions. Of those 47 sRNAs, 10 were differentially expressed in response to being grown under acidic conditions.

"Since adaptation to acidic conditions is important to colonize the vaginal niche and to survive in macrophage phagosome, these sRNAs might play a role in controlling the virulence of S. agalactiae," the authors wrote.

The researchers noted that although this was a first step toward understanding the mechanisms involved in causing S. agalactiae to switch from benign to pathogenic, the findings of novel regulatory regions that respond to different environments and the abundance of transcriptional start sites associated with reiterative transcription should "pave the way for further deciphering the regulatory networks that coordinate gene expression during the progression from commensalism to virulence."