CRISPR interference (CRISPRi)-mediated silencing of non-coding RNA (ncRNA) in saprophyte and pathogenic strains of Leptospira

Abstract

Leptospirosis is a worldwide zoonosis recently included in the list of Neglected Tropical Diseases. In urban centers, rodents play the major role as reservoirs of the disease by allowing colonization of leptospires in the proximal renal tubules and excreting them alive in the urine. The genus Leptospira encompasses saprophytic and pathogenic species, the latter being the etiological agent of the disease. The generation of Leptospira mutants is a key aspect in elucidating virulence factors and the basic biology of the bacterium; however, the recovery of mutants is hampered by the longer growth times of pathogenic strains in traditional culture media. The optimization and application of new media will promote studies in mutagenesis. Recent studies in Leptospira have shown differential expression of non-coding RNA (ncRNA) under conditions that mimic host infection. To date, little is known about the function of ncRNA in Leptospira spp. and their role in virulence. Silencing of these ncRNAs has never been done in Leptospira species. The CRISPR interference technique (CRISPRi) has been used in many organisms for specific gene silencing and, more recently, ncRNAs. This technique employs a catalytically inactive Cas9, dCas9, to cause a physical hindrance to RNA polymerase, then blocking transcription. We have satisfactorily applied the CRISPRi tool to saprophytic strains for target gene silencing, exploring new aspects of leptospiral basic biology. However, the tool has not yet been applied to pathogenic strains, given the longer growth times required for colony formation. With the present project, it is intended, through the optimization of the culture medium, to apply the CRISPRi tool to pathogenic Leptospira strains for ncRNA silencing. At first, the major ncRNA RNase P will be used for validation of silencing in saprophytic and pathogenic strains and then comparison of RNAseq data between distinct virulence profile strains available in the host laboratory will be used to define new differentially expressed ncRNA targets to elucidate their role in the virulence of leptospires, which is still poorly understood. The effect of ncRNA silencing on the expression of genes will be evaluated by RNAseq and proteomics. (AU)