Prakoso, D. | bioRxiv Science

bioRxiv2023

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 Leptospiras 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 ONTs 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 SummaryLeptospirosis 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 Leptospiras 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.

microbiology↗

bioRxiv2022

Leptospira enrichment culture followed by ONT Nanopore sequencing allows better detection of Leptospira presence and diversity in water and soil samples.

BackgroundLeptospirosis, a life-threatening disease in humans and animals, is one of the most widespread global zoonosis. Contaminated soil and water are the major transmission sources in humans and animals. Clusters of disease outbreaks are common during rainy seasons. Methodology/Principal FindingsIn this study, to detect the presence of Leptospira, we applied PCR, direct metagenomic sequencing, and enrichment culture followed by metagenomic sequencing on water and soil samples. Direct sequencing and enrichment cultures followed by PCR or sequencing effectively detected pathogenic and nonpathogenic Leptospira compared to direct PCR and 16S amplification-based metagenomic sequencing in soil or water samples. Among multiple culture media evaluated, Ellinghausen-McCullough-Johnson-Harris (EMJH) media containing antimicrobial agents was superior in recovering and detecting Leptospira from the environmental samples. Our results show that enrichment culture followed by PCR can be used to confirm the presence of pathogenic Leptospira in environmental samples. Metagenomic sequencing on enrichment cultures effectively detects the abundance and diversity of Leptospira spp from environmental samples. Conclusions/SignificanceThe selection of methodology is critical when testing environmental samples for the presence of Leptospira. Selective enrichment culture improves Leptospira detection efficacy by PCR or metagenomic sequencing and can be used successfully to understand the presence and diversity of pathogenic Leptospira during environmental surveillance. Author SummaryLeptospirosis, a life-threatening disease in humans and animals, is one of the most widespread global zoonosis. Contaminated soil and water are major sources of transmission in humans and animals. For this reason, clusters of disease outbreaks are common during the rainy season. In this study, Leptospira enrichment cultures followed by PCR and sequencing detected pathogenic and nonpathogenic Leptospira in soil and water samples. The pathogenic and intermediate groups of Leptospira were more prevalent in soil samples tested. Metagenomic sequencing on enrichment culture is effective in detecting the abundance and diversity of various Leptospira spp. in environmental samples. Soil samples in proximity to water may be an ideal niche for Leptospira growth and survival and may be an appropriate sample of choice for testing.

microbiology↗