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. 2024 Jul;20(7):1577-1596.
doi: 10.1080/15548627.2024.2330043. Epub 2024 Apr 21.

Streptococcus pneumoniae extracellular vesicles aggravate alveolar epithelial barrier disruption via autophagic degradation of OCLN (occludin)

Luqing Cui  1   2 , Ruicheng Yang  1   2   3 , Dong Huo  1   2 , Liang Li  1   2 , Xinyi Qu  1   2 , Jundan Wang  1   2 , Xinyi Wang  1   2 , Hulin Liu  1   2 , Huanchun Chen  1   2   3 , Xiangru Wang  1   2   3
Affiliations

Streptococcus pneumoniae extracellular vesicles aggravate alveolar epithelial barrier disruption via autophagic degradation of OCLN (occludin)

Luqing Cui et al. Autophagy. 2024 Jul.

Abstract

Streptococcus pneumoniae (S. pneumoniae) represents a major human bacterial pathogen leading to high morbidity and mortality in children and the elderly. Recent research emphasizes the role of extracellular vesicles (EVs) in bacterial pathogenicity. However, the contribution of S. pneumoniae EVs (pEVs) to host-microbe interactions has remained unclear. Here, we observed that S. pneumoniae infections in mice led to severe lung injuries and alveolar epithelial barrier (AEB) dysfunction. Infections of S. pneumoniae reduced the protein expression of tight junction protein OCLN (occludin) and activated macroautophagy/autophagy in lung tissues of mice and A549 cells. Mechanically, S. pneumoniae induced autophagosomal degradation of OCLN leading to AEB impairment in the A549 monolayer. S. pneumoniae released the pEVs that could be internalized by alveolar epithelial cells. Through proteomics, we profiled the cargo proteins inside pEVs and found that these pEVs contained many virulence factors, among which we identified a eukaryotic-like serine-threonine kinase protein StkP. The internalized StkP could induce the phosphorylation of BECN1 (beclin 1) at Ser93 and Ser96 sites, initiating autophagy and resulting in autophagy-dependent OCLN degradation and AEB dysfunction. Finally, the deletion of stkP in S. pneumoniae completely protected infected mice from death, significantly alleviated OCLN degradation in vivo, and largely abolished the AEB disruption caused by pEVs in vitro. Overall, our results suggested that pEVs played a crucial role in the spread of S. pneumoniae virulence factors. The cargo protein StkP in pEVs could communicate with host target proteins and even hijack the BECN1 autophagy initiation pathway, contributing to AEB disruption and bacterial pathogenicity.Abbreviations: AEB: alveolarepithelial barrier; AECs: alveolar epithelial cells; ATG16L1: autophagy related 16 like 1; ATP:adenosine 5'-triphosphate; BafA1: bafilomycin A1; BBB: blood-brain barrier; CFU: colony-forming unit; co-IP: co-immunoprecipitation; CQ:chloroquine; CTRL: control; DiO: 3,3'-dioctadecylox-acarbocyanineperchlorate; DOX: doxycycline; DTT: dithiothreitol; ECIS: electricalcell-substrate impedance sensing; eGFP: enhanced green fluorescentprotein; ermR: erythromycin-resistance expression cassette; Ery: erythromycin; eSTKs: eukaryotic-like serine-threoninekinases; EVs: extracellular vesicles; HA: hemagglutinin; H&E: hematoxylin and eosin; HsLC3B: human LC3B; hpi: hours post-infection; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LC/MS: liquid chromatography-mass spectrometry; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MVs: membranevesicles; NC:negative control; NETs:neutrophil extracellular traps; OD: optical density; OMVs: outer membrane vesicles; PBS: phosphate-buffered saline; pEVs: S.pneumoniaeextracellular vesicles; protK: proteinase K; Rapa: rapamycin; RNAi: RNA interference; S.aureus: Staphylococcusaureus; SNF:supernatant fluid; sgRNA: single guide RNA; S.pneumoniae: Streptococcuspneumoniae; S.suis: Streptococcussuis; TEER: trans-epithelium electrical resistance; moi: multiplicity ofinfection; TEM:transmission electron microscope; TJproteins: tight junction proteins; TJP1/ZO-1: tight junction protein1; TSA: tryptic soy agar; WB: western blot; WT: wild-type.

Keywords: Alveolar epithelial barrier; OCLN/occludin; S. pneumoniae; StkP; autophagy; pEVs.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
S. pneumoniae infections induced lung injuries possibly through the disruption of tight junctions and AEB integrity. (A-F) Balb/c mice were either control-treated or intranasal-infected by D39 at a CFU of 5 × 108 per mouse, and the tissues and whole-cell extracts were collected at 24 h after intranasal infection with bacteria. (A) bacterial burdens in the lungs, brains, and blood were determined respectively (n = 5). (B) Representative histological views of the lungs of mice by H&E staining. Arrows showed examples of disordered alveolar structures and neutrophil infiltration areas. Scale bars: 50 μm. (C and D) cytokine expressions of lung tissues were detected by cytokines array, and the fold changes compared to control were calculated according to gray levels. The indicated numbers from 1 to 10 in both C and D were corresponded to each other. (E and F) WB analysis of OCLN and LC3 levels of lung tissues at 24 hpi. (G) TEER changes of A549 cells in treating multiple dosages of D39 or PBS (mock) monitored by the electrical cell-substrate impedance sensing (ECIS) system. Data were collected and presented as mean ± SD from six replicated wells at each time point. (H) A549 cells were either control-treated or D39-infected at multiple moi (20, 50, and 100 moi), and whole-cell extracts were prepared at the respective time points (2, 4, and 8 hpi) for OCLN determination by WB. (I) Representative images of immunofluorescence staining of A549 cells infected with D39 for OCLN (red) co-stained with DAPI (blue). Scale bars: 20 μm.
Figure 2.
Figure 2.
S. pneumoniae infections induced autophagy in A549 cells. (A and B) WB analysis of the LC3 and SQSTM1 protein expressions in A549 cells in response to the infection of 50 moi of D39 at the respective time points (1, 2, 3, 4, 5, 6, 8, and 10 hpi). (C) A549 cells were infected with D39 (moi of 50 at 4 hpi) in the presence or absence of CQ (50 μM, 3 h) before LC3 and SQSTM1 WB analysis. (D-F) A549 cells were infected by 50 moi of D39 for 4 h, followed by fixation and imaging preparation. (D) the visualization of the ultrastructure of A549 cells via TEM. In the images, membrane-like vesicles in D39-infected cells were observed. Arrows indicated membrane-like autophagosomes and autolysosomes. Scale bars: 2 μm, 500 nm. (E) immunofluorescence microscopy visualization of LC3 puncta in D39-infected A549 cells. Confocal images of ptfLC3-transfected A549 cells were infected with D39 or treated for 12 h with 100 nM rapa. Scale bars: 10 μm. (F) images of mCherry-LC3 fluorescent dots (red), FITC-LAMP1 fluorescence (green), and the overlay with DAPI (blue), which shows colocalization of mCherry- and FITC-positive puncta. The rapa-treated group was set as the positive control. Scale bars: 10 μm.
Figure 3.
Figure 3.
S. pneumoniae infections induced autophagosomal degradation of TJ protein OCLN in A549 cells. (A) after the D39 infections, the total OCLN in the A549 cells was quantified by WB analyses. Blockage of autophagy by CQ caused an accumulation of OCLN in A549 cells. (B) after the knockdown of BECN1 in A549 cells, the autophagy activation and OCLN degradation were relieved under S. pneumoniae infections. (C) immunofluorescence microscopy visualization of the colocalization of Cy3-OCLN fluorescence (red) and the FITC-LAMP1 fluorescence (green) in D39-infected A549 cells. Scale bars: 10 μm. (D) schematic overview of autophagosome targeting and labeling using TurboID N-terminally fused to HsLC3B. A: autophagosome; N: nucleus. (E) A549 cells expressing HsLC3B-eGFP-MYC-TurboID were grown in the presence of DOX (12 h), BafA1 (2 h), D39 (4 h), biotin (1 h) and ATP (1 h) followed by fixation and immunolabeling with biotin-546, the colocalization of HsLC3B-eGFP-MYC-TurboID chimeras with biotinylated molecules were observed. Scale bars, 5 μm. (F) homogenates from HsLC3B-eGFP-MYC-TurboID chimera expressing A549 cells grown in the presence of DOX (12 h), BafA1 (2 h), D39 (4 h), biotin (1 h) and ATP (1 h) were left untreated or incubated with protK, Triton X-100, or both followed by immunoblotting. (G and H) lysates from the above protK-treated homogenates were immunoprecipitated with streptavidin magnetic beads, and the whole cell lysates (H: input) and beads-bound proteins from protK-protected proteins (G: IP) were respectively analyzed by immunoblotting. (I and J) A549 cells transiently expressing eGFP-SQSTM1, eGFP-LC3B, or eGFP-empty infected with D39 for 4 h were subjected to IP and assayed using an anti-eGFP antibody and protein A+G agarose. The total proteins (J: input) and bound proteins (I: IP) were analyzed by immunoblotting.
Figure 4.
Figure 4.
Isolation and characterization of pEvs released by S. pneumoniae cultured in THYE medium. (A) electron micrograph of vesicular structures budding from S. pneumoniae strain D39 (left) and the same vesicles in higher magnification (right). The red arrowheads pointed out the released pEvs from the cell wall. Scale bars: 200 nm. (B) schematic overview of the procedures for isolating, purifying, and identifying pEvs from S. pneumoniae strain D39. (C) pEvs were purified by density gradient ultracentrifugation (optiprep), and then, the separated fractions were visualized by silver-stained SDS-PAGE. (D and E) fractions 4–9 were pooled; OptiPrep was removed by ultracentrifugation, and the samples were determined and imaged by Zetasizer (D) and TEM (E) to observe the morphology and size distribution of pEvs from S. pneumoniae strain D39. pEvs were not visualized in fractions 10–12. Scale bars: 100 nm.
Figure 5.
Figure 5.
pEvs were internalized into A549 cells to activate autophagy and disrupt the integrity of AEB. (A) detection of intracellular pKH67-labeled (green) pEvs in A549 cells with zeiss LSM800 confocal laser scanning microscope at the indicated time points after exposure to 100 μg/mL pEvs. Scale bars: 20 μm. (B) fluorescence intensity analysis of intracellular DiO-labeled pEvs (excitation: 484 nm, emission: 501 nm); DiO-treated PBS was used in the mock group. (C) TEER changes of A549 monolayer in the treatment of multiple dosages of pEvs or PBS (mock) monitored by the ECIS system. All the data was collected and presented as mean ± SD from three replicated wells at each time point. (D and F) WB analysis of the LC3 and OCLN protein expressions in A549 cells in response to multiple dosages of pEvs. (E and G) A549 cells were incubated with pEvs (100 μg/mL) in the presence or absence of CQ (50 μM, 3 h) before WB analysis of LC3 (E) and OCLN (G).
Figure 6.
Figure 6.
pEvs-derived StkP activated autophagy by inducing BECN1 phosphorylation and resulted in autophagic degradation of epithelial OCLN in A549 cells. (A) purified pEvs from three independent biological replicates were analyzed by LC/MS. A total of 605 proteins were common to all three groups. The top 50 proteins ranked by average intensity (4D label-free) were listed. (B) lysates from A549 cells transiently expressing eGFP-tagged pEvs cargo proteins or eGFP-empty were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, eGFP, OCLN, or ACTB. (C) microscopy visualization of mCherry-LC3 fluorescent dots (red), LC3 puncta in eGFP-StkP- or eGFP-expressing A549 cells. Scale bar: 15 μm. (D) immunostaining of Cy3-OCLN (red) in A549 cells instantly expressing eGFP-StkP or eGFP alone. Scale bars: 20 μm. (E and F) lysates from A549 cells transiently expressing eGFP-StkP or eGFP and HA-BECN1 were performed IP with an anti-eGFP antibody and protein A+G agarose (E, upper two panels) or only an anti-HA magnetic beads (E, lower two panels). The total proteins (F: input) and respective bound proteins were analyzed by immunoblotting (E: IP). (G) WB detection of BECN1 phosphorylation (Ser93 and Ser96) level in A549 cells overexpressing eGFP or eGFP-stkP. (H) after overexpressing StkP in A549 cells, LC3 and OCLN were quantified by WB analyses. Blockage of autophagy by CQ caused increased expression of both LC3-II and OCLN. (I) WT BECN1 or BECN1S93,96A were overexpressed in BECN1 knockdown A549 cells (by shRNA), and western blotting analysis of OCLN expression was performed in WT BECN1 or BECN1S93,96A mutated A549 cells following introduction with eGFP or eGFP-StkP.
Figure 7.
Figure 7.
StkP-laden pEvs contributed to S. pneumoniae virulence and bacterial dissemination by disrupting AEB integrity. (A) Balb/c mice were intranasally challenged with D39-WT, D39-ΔstkP, and D39-C-ΔstkP at 5 × 108 CFU in 40 μL PBS; bacterial infected mice were continuously observed to obtain survival data (n = 10). (B) bacterial burdens in the lung tissues and blood were determined at 24 hpi (n = 5). (C) Representative histological views of the lung tissues of mice at 24 hpi by H&E staining. Scale bars: 100 μm. (D) WB analysis of the lung lysates at 24 hpi for indicated antibodies: BECN1, p-BECN1-Ser93,Ser96, LC3, and OCLN. (E) the expression and localization of LC3 and OCLN were observed in the mice’s lung tissues by immunofluorescent staining. Scale bars: 20 μm. Arrows indicated colocalization of increased LC3 and OCLN in lung tissues. (F) TEER changes of A549 monolayer incubated by 100 μg/mL of pEvs derived from D39-WT, D39-ΔstkP, and D39-C-ΔstkP respectively monitored by the ECIS system. All the data was collected and presented as mean ± SD from six replicated wells at each time point. (G) WB analysis of indicated protein in A549 cells treated by pEvs from D39-WT, D39-ΔstkP, and D39-C-ΔstkP, respectively. (H) OCLN integrity observation in different pEvs-treated A549 cells by confocal laser microscopy. Scale bars: 20 μm. (I) confocal imaging of LC3 puncta after incubation with 100 μg/mL of pEvs derived from D39-WT, D39-ΔstkP, or D39-C-ΔstkP, respectively. Scale bars: 10 μm.
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