Development of Transcriptional Fusions to Assess Leptospira interrogans Promoter Activity

Abstract

BackgroundLeptospirosis is a zoonotic infectious disease that affects both humans and animals. The existing genetic tools for Leptospira spp. have improved our understanding of the biology of this spirochete as well as the interaction of pathogenic leptospires with the mammalian host. However, new tools are necessary to provide novel and useful information to the field.Methodology and Principal FindingsA series of promoter-probe vectors carrying a reporter gene encoding green fluorescent protein (GFP) were constructed for use in L. biflexa. They were tested by constructing transcriptional fusions between the lipL41, Leptospiral Immunoglobulin-like A (ligA) and Sphingomielynase 2 (sph2) promoters from L. interrogans and the reporter gene. ligA and sph2 promoters were the most active, in comparison to the lipL41 promoter and the non-induced controls. The results obtained are in agreement with LigA expression from the L. interrogans Fiocruz L1-130 strain.ConclusionsThe novel vectors facilitated the in vitro evaluation of L. interrogans promoter activity under defined growth conditions which simulate the mammalian host environment. The fluorescence and rt-PCR data obtained closely reflected transcriptional regulation of the promoters, thus demonstrating the suitability of these vectors for assessing promoter activity in L. biflexa.

Positive Regulation of Leptospira interrogans kdp Expression by KdpE as Demonstrated with a Novel β-Galactosidase Reporter in Leptospira biflexa

“…The chromosomal bgaL reporter has several advantages over the plasmid-based green fluorescent protein reporter described in earlier studies (3,8). First, since only one plasmid capable of replicating stably in L. biflexa is available, placing the fusion on the chromosome allows genes encoding candidate regulatory factors to be cloned easily into the plasmid to test whether they can control fusion expression in trans.…”

“…4D), permitting the study of weakly expressed genes. In contrast, the relatively high background fluorescence of L. biflexa may obscure changes in the expression of weak promoters fused to gfp (8). Third, the copy number of the bgaL fusion remains stable, since the fusion is located on the chromosome while the copy number of plasmid-borne fusions may be affected by culture conditions.…”

“…In a recent study, expression of the L. interrogans adhesins LigA and LigB in L. biflexa enabled L. biflexa to serve as a surrogate host for studies of leptospiral binding to fibronectin and fibrinogen (9). Recently, a plasmid-based green fluorescent protein (GFP) reporter was developed to measure promoter activities of leptospiral genes in L. biflexa (3,8). When the L. biflexa hsp20 and groES heat shock promoters were fused to gfp, GFP levels increased with temperature (3).…”

“…When the L. biflexa hsp20 and groES heat shock promoters were fused to gfp, GFP levels increased with temperature (3). The gfp reporter was also used to assess the promoter activities of the L. interrogans genes lig, sph2, and lipL41 in L. biflexa grown under different culture conditions (8).…”

Leptospira and Leptospirosis

“…The Photorhabdus luminescens luxCDABE cassette and gfp and mRFP1 alleles have been transferred into Leptospira strains to produce luminescent and fluorescent leptospires, respectively (Aviat et al 2010;Murray et al 2010a). While the bacteria expressing the fluorescent and luminescent alleles were not sufficiently bright to be clearly visible from outside the infected animal or recorded with conventional microscopes, the lux construct could be used as a viability reporter for cell populations and the gfp construct can be used as a reporter gene system (Aviat et al 2010;Cerqueira et al 2011). A reporter gene system that enables translational fusion of leptospiral genes directly to the leptospiral chromosomal gene of bgal (β-galactosidase) has also been developed (Matsunaga and Coutinho 2012).…”