Expanding the genetic toolbox for Leptospira species by generation of fluorescent bacteria

Abstract

Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence of Leptospira by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the gfp gene had no significant effects on growth in vivo and pathogenicity in L. interrogans. We constructed an expression vector for L. biflexa that contains the lacI repressor, an inducible lac promoter, and gfp as the reporter, demonstrating that the lac system is functional in Leptospira. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) in L. biflexa transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of Leptospira spp.

FIG. 1.

Schematic diagram of plasmid constructs used to express constitutively GFP and mRFP1. The determinants for replication in L. biflexa (parAB and rep), as well as spectinomycin (SpcR) and kanamycin (KmR) resistance cassettes, are indicated.

FIG. 2.

Micrographs of GFP-expressing Leptospira strains visualized on slides by fluorescence microscopy. (A) GFPmut-expressing L. biflexa; (B) GFPmut-expressing L. interrogans strain Lai; (C) GFPmut-expressing L. interrogans strain Fiocruz. Bar, 5 μm.

FIG. 3.

Spectrofluorometric analysis of fluorescence from Leptospira transformants. Data are the means of results of two independent experiments, with bars indicating the ranges. The fluorescence intensity (y axis) was quantified in 100-μl aliquots of cell suspension containing 5 × 10⁸ bacteria.

FIG. 4.

Induction of fluorescence in a lac-inducible expression system. Cultures of Patoc SLHG lacI were untreated (0 mM IPTG) or induced with 1 mM IPTG. Samples were collected at the times indicated (h), and fluorescence assays were performed. GFP fluorescence (OD) from triplicate samples of each culture was standardized according to a cell density of 5 × 10⁸ spirochetes; results are presented as the mean OD/1 × 10⁸ bacteria ± standard deviation. (A) Schematic representation of the relevant regions and restriction sites of the L. biflexa shuttle plasmid used in this study. (B) Western blot analysis of GFP using anti-6× His monoclonal antibodies. Cultures of L. biflexa and Patoc SLHG lacI were untreated or induced with 1 mM IPTG. Cells were collected 1 week postinduction. Total protein extracted from 1 × 10⁸ spirochetes was loaded in each gel lane. Molecular masses (kDa) are indicated on the left. (C) Kinetics of green fluorescence production from the lac repressor/operator expression construct.

FIG. 5.

Heat shock stimulates transcription of hsp20 and groES in L. biflexa. Cells were grown at 30°C, and half the culture was transferred to 37°C for 4 h before the cells were harvested for fluorescence measurement and RNA isolation. The results are from one representative experiment and are the means of technical duplicates. The error bars represent the standard deviations. (A) Measured fluorescence relative to the OD₄₂₀ of the culture, in arbitrary fluorescence units (AU), at 30°C and 37°C. The white bars represent the fluorescence at 30°C, whereas the black bars represent the fluorescence at 37°C. P_hsp20-gfp, Patoc HspG; P_groES-gfp, Patoc GroG. (B) Levels of induction of the hsp20 and groES promoters after heat shock in strain Patoc HspG (light gray bars) and Patoc GroG (dark gray bars), respectively. The target transcript for real-time RT-PCR is indicated below each bar. The transcript levels were normalized to the amount of rpoB transcripts in the cells. The dashed line indicates a ratio of one.