Inactivation of the fliY gene encoding a flagellar motor switch protein attenuates mobility and virulence of Leptospira interrogans strain Lai

Abstract

Background: Pathogenic Leptospira species cause leptospirosis, a zoonotic disease of global importance. The spirochete displays active rotative mobility which may contribute to invasion and diffusion of the pathogen in hosts. FliY is a flagellar motor switch protein that controls flagellar motor direction in other microbes, but its role in Leptospira, and paricularly in pathogenicity remains unknown.

Results: A suicide plasmid for the fliY gene of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai that was disrupted by inserting the ampicillin resistance gene (bla) was constructed, and the inactivation of fliY gene in a mutant (fliY-) was confirmed by PCR and Western Blot analysis. The inactivation resulted in the mRNA absence of fliP and fliQ genes which are located downstream of the fliY gene in the same operon. The mutant displayed visibly weakened rotative motion in liquid medium and its migration on semisolid medium was also markedly attenuated compared to the wild-type strain. Compared to the wild-type strain, the mutant showed much lower levels of adhesion to murine macrophages and apoptosis-inducing ability, and its lethality to guinea pigs was also significantly decreased.

Conclusion: Inactivation of fliY, by the method used in this paper, clearly had polar effects on downstream genes. The phentotypes observed, including lower pathogenicity, could be a consequence of fliY inactivation, but also a consequence of the polar effects.

Figure 1

Amplification and expression of the fliY gene and purification of the rFliY protein. Panel A, showing PCR analysis. Lane 1: DNA marker (TaKaRa, China); lane 2: the amplification segment of the entire fliY gene; lane 3: blank control. Panel B, showing SDS-PAGE analysis. Lane 1: protein marker (TaKaRa); lane 2: pET32a with no insertion of the fliY gene; lane 3: the expressed recombinant protein, rFliY; lane 4: the purified rFliY protein.

Figure 2

Confirmation for insertion mutantion of fliY gene in the fliY^- mutant. Panel A, showing PCR analysis. Lane 1: DNA marker (TaKaRa); lane 2: the amplification segment (2019 bp) of mutated fliY gene from the fliY^- mutant; lane 3: the amplification segment (1065 bp) of the fliY gene from the wild-type strain; lane 4: blank control for PCR. Panel B, showing Western Blot analysis. Lane 1: protein marker (TaKaRa); lane 2: the fliY^- mutant lacking the FliY protein; lane 3: the wild-type strain expressing the FliY protein; lane 4: blank control for Western Blot assay. rFliY antiserum was used as the primary antibody.

Figure 3

Genes present with the fliY gene within the same predicted operon. Annotation of the genes (gene name/product): fliY/flagellar motor switch protein; LA2612/flagellar protein required for flagellar formation; fliP/flagellar biosynthesis protein; fliQ and fliR/flagellar biosynthetic proteins and type III secretion apparatus proteins; flhB/flagellar protein; flhA/flagellar biosynthesis protein; flhF/flagellar GTP-binding protein; LA2605/ParA protein; and LA2604/hypothetical protein.

Figure 4

Colony sizes of the fliY^-mutant and wild-type strain on semisolid Korthof agar. The colonies with different sizes formed by the fliY^- mutant (A) and wild-type strain (B) on semisolid Korthof agar. The leptospires were cultured on 8% RS semisolid Korthof plate for three weeks. This experiment was repeated three times.

Figure 5

Adhesion of the fliY^- mutant and wild-type strain to J774A.1 cells. Adhesion of the wild-type strain (A) and fliY^- mutant (B). The arrow indicates the adhering leptospires on J774A.1 cells. This experiment was repeated three times. Magnification × 400.

Figure 6

Adhesion ratios of the fliY^- mutant and wild-type strain to J774A.1 cells after different incubation times. Adhesion was quantified as described in Methods. *: P < 0.05, wild-type strain compared with the mutant.

Figure 7

Apoptosis ratios of J774A.1 cells induced by the fliY^- mutant and wild-type strain. Panel A: lower left quadrants indicate unstained normal cells; lower right quadrants, the early apoptotic cells binding Annexin-V; upper left quadrants, the necrotic cells binding PI; and upper right quadrants, the late apoptotic/necrotic cells binding both Annexin-V and PI. Cell death was measured by flow cytometry as described in Methods. Panel B: proportion of early apoptotic cells (annexin-V⁺/PI^-) after infection for different times. The data are expressed as mean ± SD for three independent experiments. Panel C: proportion of late apoptotic/necrotic cells (annexin-V⁺/PI⁺) after infection for different times. The data are expressed as mean ± SD for three independent experiments. *:P < 0.05, wild-type strain compared with the mutant.

Figure 8

Strategy for preparing the fliY^- mutant using the suicide plasmid p2NIL^fliY-bla.