Transcriptome Sequencing Reveals Wide Expression Reprogramming of Basal and Unknown Genes in Leptospira biflexa Biofilms

In this work, we describe the first transcriptome based on RNA-seq technology focused on studying transcriptional changes associated with biofilm growth in a member of the genus Leptospira. As many pathogenic species of this genus can survive inside the host but also persist in environmental water, mostly forming biofilms, identifying the molecular basis of this capacity can impact the understanding of how leptospires are able to fulfill a complete life cycle that alternates between adaptation to the host and adaptation to hostile external environmental conditions. We identified several genes and regulatory networks that can be the kickoff for deepening understanding of the molecular mechanisms involving bacterial persistence via biofilm formation; understanding this is important for the future development of tools for controlling leptospirosis. ABSTRACT The genus Leptospira is composed of pathogenic and saprophytic spirochetes. Pathogenic Leptospira is the etiological agent of leptospirosis, a globally spread neglected disease. A key ecological feature of some pathogenic species is their ability to survive both within and outside the host. For most leptospires, the ability to persist outside the host is associated with biofilm formation, a most important bacterial strategy to face and overcome hostile environmental conditions. The architecture and biochemistry of leptospiral biofilms are rather well understood; however, the genetic program underpinning biofilm formation remains mostly unknown. In this work, we used the saprophyte Leptospira biflexa as a model organism to assess over- and underrepresented transcripts during the biofilm state, using transcriptome sequencing (RNA-seq) technology. Our results showed that some basal biological processes like DNA replication and cell division are downregulated in the mature biofilm. Additionally, we identified significant expression reprogramming for genes involved in motility, sugar/lipid metabolism, and iron scavenging, as well as for outer membrane-encoding genes. A careful manual annotation process allowed us to assign molecular functions to many previously uncharacterized genes that are probably involved in biofilm metabolism. We also provided evidence for the presence of small regulatory RNAs in this species. Finally, coexpression networks were reconstructed to pinpoint functionally related gene clusters that may explain how biofilm maintenance is regulated. Beyond elucidating some genetic aspects of biofilm formation, this work reveals a number of pathways whose functional dissection may impact our understanding of leptospiral biology, in particular how these organisms adapt to environmental changes. IMPORTANCE In this work, we describe the first transcriptome based on RNA-seq technology focused on studying transcriptional changes associated with biofilm growth in a member of the genus Leptospira. As many pathogenic species of this genus can survive inside the host but also persist in environmental water, mostly forming biofilms, identifying the molecular basis of this capacity can impact the understanding of how leptospires are able to fulfill a complete life cycle that alternates between adaptation to the host and adaptation to hostile external environmental conditions. We identified several genes and regulatory networks that can be the kickoff for deepening understanding of the molecular mechanisms involving bacterial persistence via biofilm formation; understanding this is important for the future development of tools for controlling leptospirosis.