Leptospira transcriptome sequencing using long-read technology reveals unannotated transcripts and potential polyadenylation of mRNA molecules

Leptospirosis caused by the spirochete bacteria Leptospira is an emerging zoonosis that causes life-threatening disease in humans and animals. However, a detailed understanding of Leptospira’s RNA profiles is limited. In this study, we sequenced and analyzed the transcriptome of multiple Leptospira serovars using Oxford Nanopore Technologies’ direct cDNA and direct RNA sequencing methods. We identified several operon transcriptional units, novel RNA coding regions, and evidence of potential posttranscriptional polyadenylation in the Leptospira transcriptome. Some coding regions of multiple RNA molecules that have not been previously annotated could be potential sRNA or ncRNA molecules that support gene expression regulation purposes in Leptospira. Many of the relative positions of these unannotated RNA coding regions were consistent in their neighboring coding regions across the reference genomes of two pathogenic Leptospira analyzed in this study. Besides, the majority of the unannotated coding regions and operon transcriptional units were not detected in the nonpathogenic Leptospira, suggesting potential virulence-related functions for these RNA molecules coding regions. Overall, our study confirms the utility of ONT’s sequencing in studying prokaryotic transcriptome profiles and offers a tool to improve our understanding of the structural composition of RNA molecules and prokaryotic polyadenylation. However, the findings from our study also warrant that the presence of homopolymers of adenine bases in the transcripts may interfere with the interpretation of bacterial transcriptome profiles. Carefully designed experiments are needed to unravel the role of the features described in this study in Leptospira virulence and pathogenesis. Author Summary Leptospirosis caused by the spirochete bacteria, Leptospira, is recognized as one of the most widespread zoonotic diseases. Leptospirosis is a neglected disease and has been estimated to cause over one million annual human clinical cases and over 60,000 deaths. Leptospira are maintained in the renal tubules of asymptomatic animal reservoirs, and in contaminated environments, such as soil and water. Studying gene expression profiles is an important component of bacterial pathogenesis studies, and a number of methods are available to do it. Our study compared various transcriptome sequencing methods using one of the latest next generation sequencing tools (Oxford Nanopore Technologies) and evaluated the compositions of RNA in different Leptospira’s transcriptomes. We identified many previously undescribed and potentially significant features within the Leptospira transcriptome that may contribute to the virulence and pathogenesis of Leptospira sp. Our findings lead to new opportunities for researchers to sequence prokaryotic RNA molecules and to unravel many regulatory mechanisms.