Directed polar secretion of protease from single cells of Vibrio cholerae via the type II secretion pathway

Abstract

Bacteria have long been thought of as little more than sacks of homogeneously distributed enzymes. However, recent cytological studies indicate that bacteria are compartmentalized with proteins involved in processes such as cell division, motility, chemotaxis, and development located at distinct sites. We have used the green fluorescent protein as a reporter to determine the cellular distribution of the extracellular protein secretion (eps)-encoded type II secretion complex responsible for extracellular secretion of cholera toxin and hemagglutinin/protease in Vibrio cholerae. Real-time monitoring of green fluorescent protein fused to EpsM in living cells indicated that, like the single polar flagellum, the Eps complex is located at the old pole after cell division. Eps-dependent protease secretion was also visualized in single cells by fluorescence microscopy by using intramolecularly quenched casein. This analysis demonstrated that active protease secretion is focused at the poles and colocalizes with the site of the polar Eps apparatus. These results suggest that the type II secretion complex is responsible for directed delivery of virulence factors during cholera pathogenesis.

Figure 1

Polar distribution of GFP fused to either EpsL or EpsM. The location of different GFP constructs was determined by fluorescent microscopy by using FITC filter. (A) GFP-EpsL expression in a V. cholerae epsL mutant. (B) GFP-EpsM expression in a V. cholerae epsM mutant. (C) GFP expression in the epsL mutant. (D) GFP-EpsL expression in E. coli MC1061. (E) GFP-EpsM expression in E. coli MC1061. (F) GFP-EpsL and EpsM coexpression in E. coli MC1061. The arrow indicates the brighter pole in a cell with bipolar fluorescence.

Figure 2

Polar localization of native Eps proteins. Cells of wild-type V. cholerae were fixed with paraformaldehyde, treated with lysozyme, and subjected to immunofluorescence by using anti-EpsG (A) or anti-EpsL (B) antibodies and detected with Alexa fluor 488-conjugated F(ab′)₂ goat anti-rabbit IgG. C shows result observed with secondary antibody only.

Figure 3

The Eps apparatus is localized to the “old” pole. Cells of V. cholerae epsM mutant that express GFP-EpsM were placed on a thin film of agarose-M9GM on a slide and subjected to time-lapse fluorescence and phase-contrast microscopy. The images were overlaid with the use of Adobe photoshop 5.0. The numbers indicate time in minutes. The white arrows indicate the appearance of new fluorescent poles. The black arrow shows the cell division site.

Figure 4

The Eps apparatus and flagellum assemble at the same pole. Fixed cells of V. cholerae epsM mutant that express GFP-EpsM were subjected to immunofluorescence by using antiflagellin antibodies and detected with Alexa fluor 546 goat anti-rabbit IgG. FITC and TRITC filters were used for visualization of GFP-EpsM and the single flagellum, respectively. The bacteria were visualized by phase-contrast microscopy. The images were overlaid with the use of Adobe photoshop 5.0.

Figure 5

Polar Eps-dependent HA/protease secretion in single cells. Cells of V. cholerae were embedded in agarose containing M9 salts, amino acids, BODIPY TR-X casein, and IPTG to induce HA/protease expression and grown overnight at 37°C. The slides were subjected to fluorescence microscopy by using a TRITC filter. (A) Wild-type V. cholerae TRH7000 expressing HA/protease from pHAP. (B) TRH7000. (C) epsM mutant PU3 expressing HA/protease from pHAP. (D) PU3. The corresponding phase-contrast images are shown next to the fluorescent images to indicate the presence of bacteria on each slide.

Figure 6

Colocalization of the Eps apparatus and the site of Eps-dependent protease secretion. Cells of wild-type V. cholerae that contained plasmids pHAP and pGFP-EPSM-ara were embedded in agarose containing M9 salts, amino acids, BODIPY TR-X casein, and IPTG and arabinose to induce HA/protease and GFP-EpsM expression, respectively. The slide was incubated at 37°C overnight, and fluorescent images were retrieved by using a TRITC filter to detect casein hydrolysis (A) or an FITC filter to detect GFP-EpsM (B). The images were imported into Adobe photoshop and layered for the purpose of colocalization, which is shown as yellow (C). A cell in which GFP-EpsM is detected at only one pole and this pole alone secretes HA/protease is indicated by an arrow.