Characterization of LE3 and LE4, the only lytic phages known to infect the spirochete Leptospira
Résumé
Leptospira is a phylogenetically unique group of bacteria, and includes the causative agents of leptospirosis, the most globally prevalent zoonosis. Bacteriophages in Leptospira are largely unexplored. To date, a genomic sequence is available for only one temperate leptophage called LE1. Here, we sequenced and analysed the first genomes of the lytic phages LE3 and LE4 that can infect the saprophyte Leptospira biflexa using the lipopolysaccharide O-antigen as receptor. Bioinformatics analysis showed that the 48-kb LE3 and LE4 genomes are similar and contain 62% genes whose function cannot be predicted. Mass spectrometry led to the identification of 21 and 23 phage proteins in LE3 and LE4, respectively. However we did not identify significant similarities with other phage genomes. A search for prophages close to LE4 in the Leptospira genomes allowed for the identification of a related plasmid in L. interrogans and a prophage-like region in the draft genome of a clinical isolate of L. mayottensis. Long-read whole genome sequencing of the L. mayottensis revealed that the genome contained a LE4 phage-like circular plasmid. Further isolation and genomic comparison of leptophages should reveal their role in the genetic evolution of Leptospira. Recently, there has been a renewed interest in bacteriophages for their potential use as alternatives to conventional antibiotics 1 , and also in understanding their contribution in evolution of bacteria 2,3. In addition, phages could be used to develop new genetic tools such as replicative vectors from different compatibility groups and phage-delivery systems. Unfortunately, little is known about the diversity of phages among the genus Leptospira. Leptospira are ubiquitous organisms that are found as free-living saprophytes in environmental water and soil, or as pathogens that can cause acute or chronic infections in animals. A third group that is composed of intermediate species (in regards to their pathogenesis) of Leptospira, is phylogenetically closely related to the pathogenic species and can cause mild infections 4. Leptospirosis is an emerging waterborne zoonosis which results in more than one million human cases a year with a fatality rate frequently exceeding 10% 5. To the best of our knowledge, the only phages that have been isolated, purified, and phenotypically characterized in the genus Leptospira are: vB_LbiM_LE1 (renamed 6 , and abbreviated LE1), vB_LbiM_LE3 (LE3), and vB_LbiM_LE4 (LE4) 7. These tailed phages have been isolated from urban sewage and infect the saprophyte L. biflexa. The LE1 temperate phage genome was previously sequenced 8 and an Escherichia coli-L. biflexa shuttle vector was generated by cloning the replication origin of LE1 9. Until now, the virulent phages LE3 and LE4 have not been further characterized at the genomic and proteomic levels. In addition to these three phages, phage-like particles were also observed following mitomycin C induction of a pathogenic strain carrying a circular plasmid with phage-related genes, but these phage-like particles were not purified 10. Recently, comparative analyses of genome sequences have suggested the existence of prophages and genomic islands within the genus Leptospira 11,12. Putative prophages are found in infectious Leptospira species, including pathogenic and intermediate species, and absent in saprophytic Leptospira species, suggesting that phages may have had a major role in the emergence of the pathogens and/or in the acquisition of virulence factors. Analysis of the complete genomes of 20 Leptospira species led to the description of several predicted prophage regions 11 , including LE1-like and Mu-like prophages. In addition, the existence of two other groups of prophages have been proposed: a 22-kb region which was initially described in L. interrogans serovar Lai but that is present in most
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