Genome-Wide Transcriptional Start Site Mapping and sRNA Identification in the Pathogen Leptospira interrogans

Abstract

Leptospira are emerging zoonotic pathogens transmitted from animals to humans typically through contaminated environmental sources of water and soil. Regulatory pathways of pathogenic Leptospira spp. underlying the adaptive response to different hosts and environmental conditions remains elusive. In this study, we provide the first global Transcriptional Start Site (TSS) map of a Leptospira species. RNA was obtained from the pathogen Leptospira interrogans grown at 30°C (optimal in vitro temperature) and 37°C (host temperature) and selectively enriched for 5′ ends of native transcripts. A total of 2865 and 2866 primary TSS (pTSS) were predicted in the genome of L. interrogans at 30 and 37°C, respectively. The majority of the pTSSs were located between 0 and 10 nucleotides from the translational start site, suggesting that leaderless transcripts are a common feature of the leptospiral translational landscape. Comparative differential RNA-sequencing (dRNA-seq) analysis revealed conservation of most pTSS at 30 and 37°C. Promoter prediction algorithms allow the identification of the binding sites of the alternative sigma factor sigma 54. However, other motifs were not identified indicating that Leptospira consensus promoter sequences are inherently different from the Escherichia coli model. RNA sequencing also identified 277 and 226 putative small regulatory RNAs (sRNAs) at 30 and 37°C, respectively, including eight validated sRNAs by Northern blots. These results provide the first global view of TSS and the repertoire of sRNAs in L. interrogans. These data will establish a foundation for future experimental work on gene regulation under various environmental conditions including those in the host.

Structure and function of the Leptospira interrogans peroxide stress regulator (PerR), an atypical PerR devoid of a structural metal-binding site

“…6B). These motifs are located between 38 and 152 nucleotides upstream of the transcription start site of the perR ORF, which appears to be identical to the first nucleotide of the translation start codon (28).…”

Identification of a cis-acting determinant limiting expression of sphingomyelinase genesph2inLeptospira interroganswith agfpreporter plasmid

“…interrogans will aid selection of the correct junction for promoter fusions to gfp (37). A 309 transcription start site was not detected for sph2 in the study, consistent with the limited 310 expression of sph2 in non-Pomona strains under standard culture conditions.…”

Pathogenic <i>Leptospira</i>: Advances in understanding the molecular pathogenesis and virulence

“…Various previous studies have documented the L. interrogans transcript (Lo et al, 2010;Pinne et al, 2012;Iraola et al, 2016). Recent investigations generated the Transcriptional Start Site (TSS) map of Leptospira species (Zhukova et al, 2017), results of which showed that a larger proportion (2,866) of the primary TSS (pTSS) were predicted in the genome of L. interrogans. Interestingly, significant numbers of the pTSSs were located within the first 10 nucleotides from the start point of translation, observations that signify translational regions of Leptospiral species that display characteristic leaderless transcripts (Zhukova et al, 2017).…”

“…Recent investigations generated the Transcriptional Start Site (TSS) map of Leptospira species (Zhukova et al, 2017), results of which showed that a larger proportion (2,866) of the primary TSS (pTSS) were predicted in the genome of L. interrogans. Interestingly, significant numbers of the pTSSs were located within the first 10 nucleotides from the start point of translation, observations that signify translational regions of Leptospiral species that display characteristic leaderless transcripts (Zhukova et al, 2017). Additional studies on the L. interrogans genome sequence identified two genes (LIC11122 and LIC12287) that code for two novel proteins that were characterized and labeled as a sigma factor and a potential functional protein, respectively (Figueredo et al, 2017).…”

“…Recent studies (Xue et al, 2013;Iraola et al, 2016;Schons-Fonseca et al, 2016) and reviews (Murray, 2015;Picardeau, 2015) have described the application of transcriptomic technologies to decipher mechanisms of Leptospira pathogenesis (Zhukova et al, 2017). Reports from the RNA-sequence technology that enabled the generation of the first transcriptome (Iraola et al, 2016) facilitated the investigation of changes in the transcript turnover that were associated with the biofilm growth and description of the functional Leptospira pathways (Ko et al, 2009).…”