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MS2-affinity purification coupled with RNA sequencing (MAPS) reveals S. aureus RsaC sRNA targetome

ABSTRACT: The human opportunistic pathogen Staphylococcus aureus produces numerous small regulatory RNAs (sRNAs) for which functions are still poorly understood. Here, we focused on an atypical and large sRNA called RsaC. Its length varies between different isolates due to the presence of repeated sequences at the 5’ end while its 3’ part is structurally independent and highly conserved. Using MS2-affinity purification coupled with RNA sequencing (MAPS) and quantitative differential proteomics, we identified sodA mRNA as a main target of RsaC sRNA. SodA is a Mn-dependent superoxide dismutase involved in oxidative stress response. We demonstrated that in presence of RsaC, S. aureus cells were less resistant to oxidative stress, in relation with lower activity of SodA enzyme. Remarkably, rsaC gene is co-transcribed with the major manganese ABC transporter MntABC and, consequently, RsaC is mainly produced in response to Mn starvation. This 3’UTR-derived sRNA is released from mntABC-RsaC precursor after cleavage by RNase III. By blocking the Mn-containing enzyme SodA synthesis, RsaC favors oxidative stress response mediated by SodM, an alternative SOD enzyme using either Mn or Fe as co-factor. Thus, RsaC may balance two interconnected defensive responses (i.e. against oxidative stress and manganese starvation) when S. aureus faces host immune cells.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE128741 | GEO | 2019/08/21

REPOSITORIES: GEO

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Project description:We assessed changes in gene expression in response to manganese availability for the human pathogen Corynebacterium diphtheriae. Total RNA was harvested from wild-type C. diphtheriae strain 1737 and an isogenic ΔmntR (dip0619) grown in semi-defined metal-limited media (mPGT) without or with 5 µM manganese chloride supplementation. Three biological replicates were prepared and five genes were assessed by real-time PCR to validate the array results: dip0124, dip0169, dip0615, dip1923, and dip2261. (Abstract) Corynebacterium diphtheriae is the causative agent of a severe respiratory disease in humans. The bacterial systems required for infection are poorly understood, but the acquisition of metals such as manganese (Mn) are likely critical for host colonization. MntR is a Mn-dependent transcriptional regulator in C. diphtheriae and was previously shown to repress the expression of the mntABCD genes, which encode an ABC metal transporter. However, other targets of Mn and MntR regulation in C. diphtheriae have not been identified. In this study, we use comparisons between the gene expression profiles of wild-type C. diphtheriae strain 1737 grown without or with Mn supplementation and comparisons of gene expression between wild-type and an mntR mutant to characterize the C. diphtheriae Mn and MntR regulon. MntR was observed to both repress and induce the expression of various target genes in a Mn-dependent manner. Genes induced by MntR include the Mn-superoxide dismutase, sodA, and the putative ABC transporter locus, iutABCD. DNA binding studies showed that MntR interacts at the promoter regions for several genes identified in the expression study, and a 17-bp consensus MntR DNA binding site was identified. We found that an mntR mutant displayed increased sensitivity to Mn and Cd that could be alleviated by the additional deletion of the mntA-D transport locus, providing evidence that the MntABCD transporter functions as a Mn uptake system in C. diphtheriae. The findings in this study further our understanding of metal uptake systems and global metal regulatory networks in this important human pathogen.

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MS2-affinity purification coupled with RNA sequencing (MAPS) reveals S. aureus RsaC sRNA targetome

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