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. 2023 Dec 19;14(6):e0197923.
doi: 10.1128/mbio.01979-23. Epub 2023 Dec 1.

Enteropathogenic E. coli infection co-elicits lysosomal exocytosis and lytic host cell death

Raisa Shtuhin-Rahav  1   2 , Aaron Olender  2   3 , Efrat Zlotkin-Rivkin  1   2 , Etan Amse Bouman  1   2 , Tsafi Danieli  4 , Yael Nir-Keren  4 , Aryeh M Weiss  5 , Ipsita Nandi  1   2 , Benjamin Aroeti  1   2
Affiliations

Enteropathogenic E. coli infection co-elicits lysosomal exocytosis and lytic host cell death

Raisa Shtuhin-Rahav et al. mBio. .

Abstract

Enteropathogenic Escherichia coli (EPEC) infection is a significant cause of gastroenteritis, mainly in children. Therefore, studying the mechanisms of EPEC infection is an important research theme. EPEC modulates its host cell life by injecting via a type III secretion machinery cell death modulating effector proteins. For instance, while EspF and Map promote mitochondrial cell death, EspZ antagonizes cell death. We show that these effectors also control lysosomal exocytosis, i.e., the trafficking of lysosomes to the host cell plasma membrane. Interestingly, the capacity of these effectors to induce or protect against cell death correlates completely with their ability to induce LE, suggesting that the two processes are interconnected. Modulating host cell death is critical for establishing bacterial attachment to the host and subsequent dissemination. Therefore, exploring the modes of LE involvement in host cell death is crucial for elucidating the mechanisms underlying EPEC infection and disease.

Keywords: EspF; EspZ; Map; cell death; enteropathogenic E. coli; host-pathogen interactions; lysosomal exocytosis; membrane repair; type III secreted effectors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
EPEC infection stimulates the secretion of lysosomal enzymes into the extracellular medium. (A) Secretion of CTHD from HeLa cells. HeLa cells were infected with EPEC-wt and EPEC-escV or remained uninfected. CTHD was detected in the extracellular media and cell lysates by immunoblotting (IB) using anti-CTHD antibodies. Probing with anti-α-tubulin antibodies was used to evaluate cell protein loading. A representative gel out of three independent experiments is shown. (B) Secretion of CTHD from polarized Caco-2BBe cells. Polarized Caco-2BBe cell monolayers were infected with the indicated EPEC strains or left uninfected. Media bathing the apical and basolateral surface of the cells were collected, and cells on filters were lysed, as described in Materials and Methods. CTHD and α-tubulin were detected in media and cell lysates by Western blotting. A representative gel out of three independent experiments is shown. (C) β-Hexosaminidase secretion. HeLa, Caco-2BBe, or MDCK cells were infected with the indicated EPEC strains or left uninfected. The extracellular media and cells were subjected to the β-hexosaminidase secretion assay described in Materials and Methods. Results are mean ± SE from four to six independent experiments.
Fig 2
Fig 2
Identifying type III secreted effectors that exert β-hexosaminidase secretion. (A) Screening for LEE-encoded effectors. MDCK cells were infected with the indicated EPEC strains, and the β-hexosaminidase secretion assay was applied, as described in Materials and Methods. Results are mean ± SE from seven independent experiments. (B) Effects of EspF and Map. MDCK or HeLa cells were infected with the indicated EPEC strains, and the β-hexosaminidase release assay was performed. Results are mean ± SE from 10 (MDCK) and 4 (HeLa) independent experiments. (C) The relative contribution of EspF and Map. HeLa cells were infected for 60 min at 37°C with the indicated EPEC strains. The indicated isopropyl-β-D-thiogalactopyranoside (IPTG) concentrations have been used to induce the effector expression, and the cells were subjected to the β-hexosaminidase release assay. Results are mean ± SE of three independent experiments. Asterisks indicate statistical significance compared to ∆map, espF.
Fig 3
Fig 3
Effects of EspF and Map domains on lysosomal enzyme secretion. (A) Effects of EspF domains. Upper panel: Schematic of the C-terminally FLAG-tagged EspFwt and EspF mutations used to inactivate each EspF domain. (i) The names of each EspFwt domain (MTS [26]; NTS [85]; SNX9 [86]; N-WASP [82]) are shown. The three redundant PRR domains of EspF are also indicated. The amino acids are numbered starting with 1 in the N-terminus. (ii) L16 is a leucine positioned in the N-terminal mitochondrial targeting sequence (marked with an asterisk). Its mutation to glutamate (L16E) abrogates mitochondrial targeting of EspF. (iii) The arginine-to-aspartic acid (R-D) mutations in each PRR inactivate SNX9 binding. (iv) The leucine-to-alanine (L-A) mutation in each PRR abolishes N-WASP binding. Lower panel: HeLa cells were infected with the indicated EPEC strains, and the β-hexosaminidase secretion assay was performed as described. Results are mean ± SE from five independent experiments. (B) Effects of Map domains. Upper panel: Schematic of the HA-tagged Mapwt effector protein and the mutations used to inactivate each Map domain. (i) The MTS (14, 17), the WxxxE RhoGEF (11), the mitochondrial toxicity region (MTR) (14), and the C-terminal TRL PDZ type I binding motif (87) are shown. The amino acids are numbered in sequence, starting with the N-terminus of the protein. (ii–v) The mutations used to inactivate each domain are indicated in red. (ii) The glutamate on the WxxxE GEF domain is mutated to alanine; (iii) the N-terminal MTS is replaced with the EspH N-terminal 25 amino acid; (iv) amino acids 101–152 of the MTR motif are removed; (v) the C-terminal TRL amino acids, which constitute the PDZ-type I binding motif, are substituted to alanine. All mutations have been described in reference (17). (A) Lower panel: HeLa cells were infected with the indicated EPEC strains, and the β-hexosaminidase secretion assay was applied. Results are mean ± SE from five independent experiments.
Fig 4
Fig 4
Effects of EPEC infection on PI uptake and Lamp-1 surface appearance in single cells. (A) Effects of EPEC-wt and EPEC-escV infection on HeLa cells. HeLa cells were infected with pre-activated EPEC-escV, or EPEC-wt, or remained uninfected. Cells were exposed to PI, and their surface was immunostained with anti-Lamp-1 antibodies recognizing the protein’s extracellular domain. Cells were then fixed, permeabilized, stained with DAPI and phalloidin-CF647, and processed for fluorescence microscopy. Representative images from three independent experiments are shown (upper panel). The mean PI vs Lamp-1 intensities per cell were determined and plotted on scatter and strip plots (lower panels). The scatter plots combine data from four different optical fields of view taken from two independent experiments, encompassing 1,000–1,500 cells in each condition (Control, escV, or wt). For each condition, Lamp-1 and PI fluorescence mean intensities were also plotted in a strip plot (lower right panels), whereby the black lines in the strip plots represent the mean intensity. A two-sided Welch’s t-test and Bonferroni’s correction were used to determine statistical significance. ****P = 1e−4, ns = not-significant. (B) Effects of EPEC-wt and EPEC-escV infection on Caco-2BBe cells. Cells were seeded on collagenated coverslips, infected with pre-activated EPEC-escV or EPEC-wt, exposed to PI uptake, immunostained with anti-Lamp-1 antibodies under conditions that allowed surface labeling, stained with DAPI and phalloidin-CF647, and processed for fluorescence microscopy, as in panel A. Representative images are shown (upper panel). The mean PI vs Lamp-1 intensities per cell were determined and plotted on scatter and strip plots (lower panels). A two-sided Welch’s t-test and Bonferroni’s correction were used to determine statistical significance. ****P = 1e−4, ns = not-significant. (C). Effects of EspF and Map. HeLa cells were infected with the indicated EPEC strains, and the PI and surface Lamp-1 fluorescence levels were analyzed, as described above and in Materials and Methods. A two-sided Welch’s t-test was applied to determine statistical significance. ****P = 1e−4, ns = not-significant.
Fig 5
Fig 5
Effects of EspZ on β-hexosaminidase release, cell permeability to PI, and Lamp-1 surface expression. (A) Effects on β-hexosaminidase release. MDCK, HeLa, or Caco-2BBe cell monolayers were infected with EPEC-escV, EPEC-wt, EPEC-ΔespZ*, and EPEC-ΔespZ*/pEspZ, and the β-hexosaminidase release was applied, as before. Results are mean ± SE from four independent experiments. (B) Effects on PI uptake and Lamp-1 surface expression in single cells. HeLa cells were infected with the indicated EPEC strains, and the PI and surface Lamp-1 fluorescence levels were analyzed as in Fig. 4. A representative fluorescence microscopy image is shown (left panel), and the mean fluorescence intensities of PI and Lamp-1 are shown as scatter and strip plots (right panels). A two-sided Welch’s t-test was performed to determine the statistical significance. ****P = 1e−4, ns = not-significant.
Fig 6
Fig 6
EPEC1 and EPEC1 expressing EspZ (EPEC1/pEspZ and EPEC2) effects on surface Lamp-1 clustering at infection sites and lysosomal enzyme secretion. (A) Impacts on surface Lamp-1 clustering. HeLa cells were infected with the indicated EPEC strains. Cells were subjected to PI uptake and Lamp-1 surface immunostaining, fixed, permeabilized, and stained with DAPI (to visualize host cell nuclei and bacterial microcolonies) and TR-phalloidin (to visualize F-actin). Cells were then imaged with confocal microscopy. Representative images of three independent experiments are shown (A, left). Arrows point toward infection sites. In the case of EPEC1 infection, white arrows indicate infecting microcolonies in which surface Lamp-1 has been visualized at the injection site, and yellow arrows point to a microcolony in which surface Lamp-1 clustering has not been detected. The degree of Lamp-1 clustering has been quantified, as previously described (19) (A, right). (B) Effects on lysosomal enzyme secretion. MDCK, HeLa, and Caco-2BBe cells were infected with the indicated EPEC strains, and β-hexosaminidase secretion levels were determined, as described in Fig. 1C and Materials and Methods. Results are mean ± SE from n = 4 to 6 independent experiments.
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