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, 75 (19), 6076-86

Internalization of Salmonella Enterica in Leaves Is Induced by Light and Involves Chemotaxis and Penetration Through Open Stomata

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Internalization of Salmonella Enterica in Leaves Is Induced by Light and Involves Chemotaxis and Penetration Through Open Stomata

Yulia Kroupitski et al. Appl Environ Microbiol.

Abstract

Outbreaks of salmonellosis related to consumption of fresh produce have raised interest in Salmonella-plant interactions leading to plant colonization. Incubation of gfp-tagged Salmonella enterica with iceberg lettuce leaves in the light resulted in aggregation of bacteria near open stomata and invasion into the inner leaf tissue. In contrast, incubation in the dark resulted in a scattered attachment pattern and very poor stomatal internalization. Forcing stomatal opening in the dark by fusicoccin had no significant effect on Salmonella internalization. These results imply that the pathogen is attracted to nutrients produced de novo by photosynthetically active cells. Indeed, mutations affecting Salmonella motility and chemotaxis significantly inhibited bacterial internalization. These findings suggest a mechanistic account for entry of Salmonella into the plant's apoplast and imply that either Salmonella antigens are not well recognized by the stoma-based innate immunity or that this pathogen has evolved means to evade it. Internalization of leaves may provide a partial explanation for the failure of sanitizers to efficiently eradicate food-borne pathogens in leafy greens.

Figures

FIG. 1.
FIG. 1.
Interactions of S. enterica serovar Typhimurium with lettuce leaves. (A) S. enterica serovar Typhimurium was incubated for 2 h with a 3- by 3-cm piece of lettuce leaf as described in Materials and Methods and shown. A small (ca. 1- by 0.5-cm) centrally located piece of leaf (not containing large veins) was mounted on a microscopic slide and examined by confocal microscopy. (B and C) Microscopic images of GFP-tagged Salmonella (green) showing both diffuse and stomatal-associated attachment (B) and a higher magnification of a single stoma harboring Salmonella cells are presented (C). Red fluorescence indicates autofluorescence of the chlorophyll of guard cells. The fluorescent images were overlaid with the transmitted light image obtained using Nomarski differential interference contrast. (D) SEM image showing the complex topography of a single stomatal region and multiple bacteria (potentially Salmonella) residing within the stomatal space. Bars, 50 μm (B), 5 μm (C), and 10 μm (D).
FIG. 2.
FIG. 2.
Photomicrographs showing the distribution (depth) of Salmonella cells in lettuce leaf tissues following exposure to light (A) and dark (B) for 2 h at room temperature. Representative photomicrographs showing fluorescent images along a z section overlaid with DIC images are shown. (A) Under illumination, numerous green fluorescent bacteria were observed beneath stomata (indicated by white arrows) and in the intercellular space in the underlying parenchyma cells. (B) Following incubation in the dark, GFP-labeled bacteria were localized on the leaf surface and no bacterium was observed in inner tissues. Red fluorescence indicates autofluorescence of chlorophyll within chloroplasts. Since the epidermis is devoid of chloroplasts (besides the guard cells), the presence of chloroplast (red) and nearby Salmonella (green) in the same focal plane confirms the localization of Salmonella cells within the parenchymal tissue. (C) A three-dimensional reconstruction of confocal microscopy images taken of the same leaf section shown in panel A further demonstrate the existence of S. enterica cells (green) inside a stoma (rectangle outlined by a white broken line) as well as deeper within the parenchymal tissue, characterized by the presence of chloroplasts (red autofluorescence). The yellow color corresponds to the localization of bacteria (green) and chloroplast (red) close together. The distance in microns (0 to 200 μm) for the images is indicated.
FIG. 2.
FIG. 2.
Photomicrographs showing the distribution (depth) of Salmonella cells in lettuce leaf tissues following exposure to light (A) and dark (B) for 2 h at room temperature. Representative photomicrographs showing fluorescent images along a z section overlaid with DIC images are shown. (A) Under illumination, numerous green fluorescent bacteria were observed beneath stomata (indicated by white arrows) and in the intercellular space in the underlying parenchyma cells. (B) Following incubation in the dark, GFP-labeled bacteria were localized on the leaf surface and no bacterium was observed in inner tissues. Red fluorescence indicates autofluorescence of chlorophyll within chloroplasts. Since the epidermis is devoid of chloroplasts (besides the guard cells), the presence of chloroplast (red) and nearby Salmonella (green) in the same focal plane confirms the localization of Salmonella cells within the parenchymal tissue. (C) A three-dimensional reconstruction of confocal microscopy images taken of the same leaf section shown in panel A further demonstrate the existence of S. enterica cells (green) inside a stoma (rectangle outlined by a white broken line) as well as deeper within the parenchymal tissue, characterized by the presence of chloroplasts (red autofluorescence). The yellow color corresponds to the localization of bacteria (green) and chloroplast (red) close together. The distance in microns (0 to 200 μm) for the images is indicated.
FIG. 3.
FIG. 3.
Microscopic photomicrographs showing stomatal guard cells (white arrows) following 20 min of preconditioning at the following light intensities: 100 (A), 3 (B), and 0 (C) μE m−2 s−1. Bars, 100 μm.
FIG. 4.
FIG. 4.
Effect of light on the localization of Salmonella in leaf tissue. (A) Incidence of S. enterica serovar Typhimurium (STm) on leaf surface and in internal tissue. Preexposure refers to experiments in which the whole lettuce head was preexposed to light (100 μE m−2 s−1) for 30 min before the leaves were cut and taken for internalization assay performed in the dark. Each experiment was performed in triplicate and repeated at least twice at different days. Different letters indicate significant difference (P < 0.05) between the means of surface (capital letters) and internal (lowercase letters) fields harboring bacteria by analysis of variance by the Tukey-Kramer multiple-comparison test. (B) Confocal microscopy images showing GFP-tagged bacteria residing on the surface of the leaf and in internal leaf tissues following 2-h internalization assay. Internal leaf tissue images are composed of a stack of fluorescent images taken every 1.2 μm to a depth of 100 μm along a z section of the same field. All images were overlaid with DIC images. Note Salmonella aggregation near and within stomata (indicated by white arrows) under illumination, but not in the dark. Bars, 50 μm.
FIG. 5.
FIG. 5.
S. enterica serovar Typhimurium does not trigger stomatal closure. (A) Confocal microscopic images of lettuce leaf exposed to saline (control), S. enterica serovar Typhimurium (STm), and P. syringae pv. tomato (Pst) DC3000 for 2 h in light. White arrows indicate stomata. (B) Stomatal aperture in lettuce leaf exposed to saline (control), S. enterica serovar Typhimurium, and P. syringae pv. tomato DC3000. Results are shown as means plus standard deviations (error bars) (n = 60). Different letters indicate significant differences (P < 0.05) between the means by analysis of variance by the Tukey-Kramer multiple-comparison test.
FIG. 6.
FIG. 6.
Effect of temperature on the incidence of Salmonella enterica serovar Typhimurium (STm) in leaf tissue. Internalization experiments were performed in light (3.0 μE m−2 s−1). The data represent the mean plus standard deviations (error bars) for two independent experiments, each performed in triplicate. Different letters indicate significant differences (P < 0.05) between the means of surface (capital letters) and internal (lowercase letters) fields containing bacteria by analysis of variance by the Tukey-Kramer multiple-comparison test.
FIG. 7.
FIG. 7.
Effects of fliGHI and cheY mutations (A and B) and exogenous sugars (C) on Salmonella localization in leaf tissue. Internalization experiments were conducted in light (100 μE m−2 s−1) for 2 h. (A and C) Incidence of S. enterica serovar Typhimurium (STm) on the leaf surface and in internal tissues. (B) Confocal microscopy image stacks show bacterial distribution in the various leaf locations. Sucrose, glucose, and fructose were added to the bacterial suspension at a concentration of 100 mM. Leaf extract was prepared from lettuce leaves adapted to light for 1 h. Control denotes bacterial suspension in saline only. The data represent the means plus standard deviations (error bars) from at least two independent experiments, each performed in triplicate. Different letters indicate significant differences (P < 0.05) between the means of surface (capital letters) and internal (lowercase letters) fields harboring bacteria by analysis of variance by the Tukey-Kramer multiple-comparison test. WT, wild type.
FIG. 8.
FIG. 8.
A model summarizing our current understanding regarding Salmonella internalization through stomata. Red circles denote putative chemoattractant nutrients produced by stomatal guard cells and by parenchyma cells during photosynthesis.

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