Analysis of a Spontaneous Non-Motile and Avirulent Mutant Shows That FliM Is Required for Full Endoflagella Assembly in Leptospira interrogans

Abstract

Pathogenic Leptospira strains are responsible for leptospirosis, a worldwide emerging zoonotic disease. These spirochetes are unique amongst bacteria because of their corkscrew-like cell morphology and their periplasmic flagella. Motility is reported as an important virulence determinant, probably favoring entry and dissemination of pathogenic Leptospira in the host. However, proteins constituting the periplasmic flagella and their role in cell shape, motility and virulence remain poorly described. In this study, we characterized a spontaneous L. interrogans mutant strain lacking motility, correlated with the loss of the characteristic hook-shaped ends, and virulence in the animal model. Whole genome sequencing allowed the identification of one nucleotide deletion in the fliM gene resulting in a premature stop codon, thereby preventing the production of flagellar motor switch protein FliM. Genetic complementation restored cell morphology, motility and virulence comparable to those of wild type cells. Analyses of purified periplasmic flagella revealed a defect in flagella assembly, resulting in shortened flagella compared to the wild type strain. This also correlated with a lower amount of major filament proteins FlaA and FlaB. Altogether, these findings demonstrate that FliM is required for full and correct assembly of the flagella which is essential for motility and virulence.

Fig 1. Morphology and motility of 702^mot-, WT and 702^compl strains.

(A) Observation under dark-field microscopy at ×20 or ×200 magnifications of cultures in liquid EMJH. (B) Spread of bacteria on soft 0.5% agar EMJH plates observed after 10 days of incubation. Identical results were obtained on 0.3% agar plates (S2 Fig). (C) Images taken every 0.1 s during 0.6 s under dark-field microscopy at ×200 magnification.

Fig 2. Mutation in fliM gene in 702^mot- strain.

(A) Genetic locus of fliM gene in L. interrogans. The arrow indicates the localization of the SNP in the 702^mot- strain. Gene nomenclature corresponds to the homologs in L. interrogans serovar Copenhageni strain Fiocruz L1-130. (B) fliM nucleotide and corresponding amino acid sequences in mutated and WT strains. The deletion at position 105 of the coding sequence in strain 702^mot- induces a stop codon. (C) Detection of FliM and LipL41 by immunoblots. Uncropped gels are presented in S4 Fig.

Fig 3. Virulence of 702^mot-, WT and 702^compl strains.

The hamsters were infected intraperitoneally with the three strains. One group of 5 hamsters received 10⁸ leptospires of 702^mot- strain, 2 groups of 5 hamsters received 10⁸ or 10⁶ leptospires of WT strain and 2 groups of 10 hamsters received 10⁸ or 10⁶ leptospires of 702^compl strain.

Fig 4. Flagella integrity in 702^mot-, WT and 702^compl strains.

(A) Immunofluorescence assay using FlaB antibody on methanol permeabilized cells detected by Cy3 coupled secondary antibodies (yellow) and DAPI counterstaining (blue). Merged images are presented. (B and C) Observation of flagella preparations by transmission electron microscopy after negative staining with uranyl acetate at ×4,800 magnification (B) and ×30,000 magnification (C). The arrows point flagellar motors.

Fig 5. Flagellar protein content in 702^mot-, 702^compl and WT strains.

(A) Purified flagella analyzed by Coomassie stained SDS-PAGE. (B and C) Detection of FlaA2 and FlaB by immunoblots in (B) purified flagella and (C) whole cell lysates. Uncropped gels are presented in S7 Fig.