Role of FlgT in anchoring the flagellum of Vibrio cholerae

Abstract

Flagellar motility has long been regarded as an important virulence factor. In Vibrio cholerae, the single polar flagellum is essential for motility as well as for proper attachment and colonization. In this study, we demonstrate that the novel flagellar protein FlgT is involved in anchoring the flagellum to the V. cholerae cell. A screen for novel colonization factors by use of TnphoA mutagenesis identified flgT. An in-frame deletion of flgT established that FlgT is required for attachment, colonization, and motility. Transmission electron microscopy revealed that while the flgT mutant is capable of assembling a phenotypically normal flagellum, the flgT population is mostly aflagellate compared to the wild-type population. Further analyses indicated that the flagellum of the flgT mutant is released into the culture supernatant from the cell upon completion of assembly. Additionally, hook basal body complexes appear to be released along with the filament. These results indicate that FlgT functions to stabilize the flagellar apparatus at the pole of the cell.

FIG. 1.

The ΔflgT mutant is defective in attachment and colonization. (A) V. cholerae strain O395 (WT) and ΔflaA and ΔflgT mutants were competed against an O395 ΔlacZ strain (KSK258) for in vitro attachment to HT-29 epithelial cells. Statistical analyses (unpaired Student's t test) were performed between each mutant and the WT, as indicated by asterisks. ***, P < 0.0001. (B) V. cholerae strain O395 (WT) and ΔflaA and ΔflgT mutants were competed against an O395 ΔlacZ strain (KSK258) for in vivo colonization of infant mice, using four to seven mice per group. Statistical analyses (unpaired Student's t test) were performed between each mutant and the WT, as indicated by asterisks. ***, P < 0.0001.

FIG. 2.

FlgT is essential for motility and flagellar stability. (A) Zones of motility of V. cholerae WT, ΔflaA, ΔflgT, and ΔflgT/pFlgT-His₆ strains in motility agar. (B) Transmission electron micrographs showing the polar flagella of the V. cholerae WT (i) and the ΔflgT strains. The two ΔflgT mutant cell types are shown in panels ii (flagellate) and iii (aflagellate). Grids were prepared at an OD₆₀₀ of ∼2.0. Bar, 500 nm. (C) Graphical representation of percent flagellated cells of the V. cholerae WT, ΔflaA, and ΔflgT strains during mid-log (OD₆₀₀ = 0.5), late-log (OD₆₀₀ = 2.0), and stationary (OD₆₀₀ = 4.0) phases. A total of 100 cells per strain were examined via transmission electron microscopy. Brackets indicate statistical comparisons (unpaired Student's t test) between the indicated data sets. ***, P < 0.0001; *, P < 0.05.

FIG. 3.

The flagellum produced by the ΔflgT mutant does not remain associated with the bacterial cell. (A) Anti-flagellin antiserum was used to probe whole-cell extracts prepared from the V. cholerae WT, ΔflaA, and ΔflgT strains at an OD₆₀₀ of 4.0. (B) Anti-flagellin antiserum was used to probe the supernatants from the cultures that were used to prepare the whole-cell extracts shown in panel A. Results were similar at an OD₆₀₀ of 2.0; however, the overall levels of flagellin were reduced (data not shown).

FIG. 4.

Flagella with the HBB complex are released into the culture supernatant of the flgT mutant. (i to iii) Transmission electron micrographs of flagella released into the culture supernatant by the flgT mutant. (iv) Transmission electron micrograph of the polar flagellum of an osmotically shocked WT cell. Arrows point to the base of the flagellum. Grids were prepared at an OD₆₀₀ of ∼2.0. Bar, 100 nm.

FIG. 5.

FlgT is involved in anchoring the flagellum. (i and ii) Transmission electron micrographs of flagella being released from the flgT mutant. (iii) Transmission electron micrograph of the flagellar pole of the WT. Arrows indicate the flagellar apparatus. Grids were prepared at an OD₆₀₀ of ∼2.0. Bar, 100 nm.

FIG. 6.

Cellular localization of FlgT. (A) Anti-FlgT immunoblot of periplasmic fractions isolated from V. cholerae WT, ΔflgT, ΔflgT/pFlgT-His₆, and ΔflgT/vector strains. The asterisk indicates a cross-reactive band. The apparent molecular weights of the protein ladder are listed to the left of the gel. (B) Subcellular fractionation was carried out on the ΔflgT/pFlgT-His₆ strain. The cytoplasm (lane C), periplasm (lane P), inner membrane (lane IM), and outer membrane (lane OM) fractions were isolated and separated by SDS-PAGE, followed by anti-His and anti-FlgT immunoblotting. Anti-VieA, anti-DsbA, anti-ToxR, and anti-OmpW immunoblots were performed to confirm fraction purity.

FIG. 7.

Role of FlgT in anchoring the flagellum to the V. cholerae cell body. For WT cells, the flagellum is securely anchored to the cell, which is also true for the motX mutant. Although the motX mutant lacks the stator components that make up the motor, flagellar stability is maintained, presumably via FlgT. In the case of the flgT mutant, the flagellum is similar to that of the motX mutant in that it does not assemble the T ring. However, unlike the case in the motX mutant, once flagellar assembly is complete, the flagellum is released from the cell of the flgT mutant, suggesting that FlgT plays a crucial role in anchoring the flagellum to the cell. It is hypothesized that FlgT could provide additional support to the preexisting anchoring components, such as the P and L rings. Another possibility is that FlgT stabilizes the membrane surrounding the flagellar apparatus. It also seems likely that FlgT functions as an indirect anchoring protein, such that FlgT forms a complex with another protein(s) in order to anchor the flagellar apparatus to the cell.