Avian pathogenic Escherichia coli virulence protein Hcp2a induces incomplete autophagy in chicken HD11 cells

Abstract

The hemolysin co-regulator protein (Hcp) is a core virulence protein of the Type VI Secretion System (T6SS) in avian pathogenic Escherichia coli (APEC) and plays a critical role in host-pathogen interactions. While several bacterial effectors are known to subvert host immunity by modulating autophagy, whether and how the APEC Hcp2a protein influences autophagy in chicken macrophages (HD11) remains unclear. Here, we demonstrate that Hcp2a is efficiently internalized into HD11 cells and induces significant cytotoxicity. Notably, Hcp2a treatment elicited an autophagic response, as evidenced by elevated LC3-II levels and increased formation of autophagic vesicles. However, this was accompanied by p62/SQSTM1 aggregation and impaired autophagic flux, indicating a blockade in autophagosome-lysosome degradation. Quantitative proteomic analysis revealed significant down-regulation of the lysosomal pathway, particularly of key components involved in acidification and proteolysis. Functional assays confirmed that Hcp2a causes lysosomal dysfunction, characterized by the reduction in acidic lysosomal compartments and decreased levels of mature cathepsin D (CTSD). These defects ultimately impair the degradative capacity of lysosomes following autophagosome fusion, culminating in the block of autophagic flux. Our findings uncover a mechanism by which APEC Hcp2a interrupts host autophagy through lysosomal impairment, providing novel insights into APEC pathogenesis and bacterial immune evasion strategies.

Introduction

Avian pathogenic Escherichia coli (APEC) is a major poultry pathogen, often causing systemic infections, septicemia, and organ damage, and leading to substantial economic losses in the poultry industry (Guerra et al., 2018; Li et al., 2020; Nawaz et al., 2024). The pathogenicity of APEC is driven by a repertoire of virulence factors, including adhesins, invasins, iron acquisition systems, secretion systems, and toxins (Ahmed et al., 2013; Awad et al., 2020; Hu et al., 2022). Among these, the type VI secretion system (T6SS) is a key virulence determinant in APEC. Hemolysin co-regulator protein 2a (Hcp2a)—a core structural and effector component of T6SS—not only constitutes part of the secretion apparatus but is also closely associated with APEC pathogenicity and survival (Ma et al., 2017; Wu et al., 2023).

Autophagy is a protective mechanism that maintains cellular homeostasis and acts as a key component of innate immune responses, capable of degrading damaged organelles, misfolded proteins, and invading pathogens (Kundu et al., 2025; Mizushima and Komatsu, 2011). Recent studies have demonstrated that APEC can induce host autophagy through the secretion of outer membrane vesicles (OMVs) (Cui et al., 2026). Furthermore, accumulating evidence has revealed that bacterial type VI secretion system (T6SS) effectors can induce host autophagy (Jiang et al., 2016; Trunk et al., 2018; Wang et al., 2023; Yu et al., 2015). For instance, TplE, a T6SS effector from Pseudomonas aeruginosa, activates the unfolded protein response (UPR) via the IRE1α-XBP1 pathway, leading to enhanced autophagic flux (Jiang et al., 2016). Similarly, in Vibrio parahaemolyticus, the T6SS2 effector VgrG2 has been demonstrated to induce autophagy in macrophages (Yu et al., 2015).

During long-term co-evolution with their hosts, pathogenic bacteria have developed sophisticated strategies to subvert the autophagic process, thereby promoting intracellular survival (Chowdhury et al., 2026; Liang et al., 2021; Meng et al., 2025; Winchell et al., 2016). For example, Shigella virulence factor VirA blocks autophagosome formation by interfering with Rab1 (Mattock and Blocker, 2017). Lysosomes, as the terminal degradative compartments of autophagy, are also frequently targeted by pathogens to evade host immunity (Rijal and Gomer, 2026; Sreelatha et al., 2013). Acinetobacter baumannii, for instance, activates transcription factor EB to induce autophagy while simultaneously impairing lysosomal acidification, thereby blocking autophagic flux (Parra-Millán et al., 2018). Mycobacterium tuberculosis (Mtb) effector protein Mce3C directly targets and inhibits the lysosomal core protease cathepsin B (CTSB), thereby disrupting lysosomal degradation function (Lu et al., 2025b).

Despite the established role of T6SS effectors in modulating host autophagy, whether and how the APEC effector Hcp2a regulates autophagy in avian host cells remains poorly understood. In this study, using the chicken macrophage cell line HD11 as a model, we reveal that Hcp2a induces incomplete autophagy by impairing lysosomal function, providing new insights into APEC pathogenesis and bacterial immune evasion strategies.

Materials and methods

Cell culture

The chicken macrophage cell line HD11 was obtained from the Key Laboratory of Veterinary Pathobiology and Disease Prevention and Control of Anhui Province. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FSP500, ExCell Bio, Suzhou, China) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin; C0222, Beyotime, Shanghai, China) at 37 °C in a humidified 5% CO₂ incubator.

Purification of Hcp2a protein

The Hcp2a protein was extracted and purified as previously described (Chen et al., 2023; Lu et al., 2025a). Briefly, Escherichia coli BL21 strains carrying the pET28a‑hcp2a plasmid were revived and grown in LB medium containing kanamycin. When the optical density at 600 nm (OD₆₀₀) reached 0.6–0.8, protein expression was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) (A600168-0025, Sangon Biotech, Shanghai, China). Cells were harvested by centrifugation, washed with phosphate‑buffered saline (PBS), and lysed by ultrasonication. After centrifugation, the supernatant was subjected to affinity purification using His‑tag magnetic beads (20561ES, Yeasen, Shanghai, China) according to the manufacturer’s protocol. The bound proteins were washed to remove nonspecific contaminants and eluted. Eluates were immediately analyzed by SDS‑PAGE to assess purity. The target protein was then concentrated and buffer‑exchanged into PBS using Amicon® Ultra centrifugal filters (3 kDa cutoff; UFC801096, Merck, Germany). Endotoxin was removed with the ToxinEraser™ Endotoxin Removal Kit (L00338, GenScript, Nanjing, China), and endotoxin levels were measured. Protein concentration was determined using a BCA Protein Assay Kit (EC0001, SparkJade, Shandong, China). Finally, the purified protein was sterilized by filtration through a 0.22 μm membrane, aliquoted, and stored at −80 °C until use.

Immunofluorescence

HD11 cells seeded in 24-well plates were treated with 50 μg/mL Hcp2a protein for 12 h. Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with PBS containing 0.1% Tween-20 for 15 min, and blocked with 5% skim milk for 2 hours. Subsequently, cells were incubated with the following primary antibodies diluted in 5% skim milk/Tris-buffered saline containing Tween-20 (TBST) for 1 h: His‑tag mouse monoclonal antibody (30405ES10, Yeasen, Shanghai, China); anti-P62 (18420-1-AP, Proteintech, Wuhan, China); anti-LC3 (14600-1-AP, Proteintech, Wuhan, China); anti-LAMP1(222466, Zenbio, Chengdu, China). After washing with TBST, cells were incubated with the following secondary antibodies for 1 h in the dark: FITC‑affiniPure goat anti‑mouse IgG(H+L) (33207ES60, Yeasen, Shanghai, China), ABflo® 488-conjugated Goat anti-Rabbit IgG (H+L) (ABclonal, Wuhan, China; AS053) and ABflo® 594-conjugated Goat anti-mouse IgG (H+L) (ABclonal, Wuhan, China; AS054). Nuclei were stained with DAPI for 10 min. Samples were observed under laser confocal microscope (SP8 LIGHTNING, Leica, Germany), and images were acquired.

Cell viability assay

Cytotoxicity of Hcp2a protein toward HD11 cells was assessed using the Cell Counting Kit-8 (CCK-8) assay kit (CT0001-C, SparkJade, Shandong, China). Briefly, cells seeded in a 96-well plate were treated with different concentrations (50 or 100 μg/mL) of Hcp2a for 6h, 12 h or 18 h. Subsequently, 100 μL of DMEM medium containing 10% CCK-8 was added to each well, and the plates were incubated at 37 °C for 1 hour. Absorbance at 450 nm was measured using a microplate reader, and relative cell viability values were calculated.

Western blot

After treatment, cells were lysed on ice with Radio-Immunoprecipitation Assay (RIPA) lysis solution (Epizyme, Shanghai, China; PC101). Protein samples were separated by SDS‑PAGE and transferred to PVDF membranes. The membranes were blocked with TBST containing 5% skimmed milk for 2 h and then incubated overnight at 4 °C with the following primary antibodies diluted in 5% skim milk/TBST: anti-LC3 (1:3000; 14600-1-AP, Proteintech, Wuhan, China), anti-GAPDH (1:50,000; 10494-1-AP, Proteintech, Wuhan, China), anti-P62 (1:40,000; 18420-1-AP, Proteintech, Wuhan, China), anti-CTSD (1:5000; 21327-1-AP, Proteintech, Wuhan, China), anti-β-actin (1:10000; bs-0061R, Bioss, Beijing, China). After washing with TBST, membranes were incubated at 37 °C for 1 h with the following secondary antibodies: HRP-labeled goat anti-rabbit IgG (1:10,000; SA00001-2, Proteintech, Wuhan, China) and HRP-labeled goat anti-mouse IgG (1:10,000; SA00001-1, Proteintech, Wuhan, China), followed by further washing. Signals were detected using ECL reagent and captured with a gel imaging system. Band intensities were quantified using ImageJ software.

Cellular transfection

GFP‑LC3 and mCherry‑GFP‑LC3 plasmids were extracted from E. coli cultures using a plasmid extraction kit (TD429-100, Jianshibio, Beijing, China). HD11 cells were seeded in 24-well plates, and transfected at approximately 60% confluency using Lipo8000 transfection reagent (C0533, Beyotime, Shanghai, China) according to the instructions. Briefly, the medium was replaced with fresh medium before transfection. For each well, a transfection mixture containing DMEM, plasmid DNA, and transfection reagent was prepared and added dropwise to the cells. After 18 h of transfection, cells were treated with Hcp2a protein for 12 h. Subsequently, cells were washed with PBS, fixed with pre‑cooled 4% paraformaldehyde at 4 °C for 30 min, washed with PBST, and mounted with an anti‑fade mounting medium (EE0015, Sparkjade, Shandong, China) on glass slides. Finally, images were observed and captured under laser confocal microscope (SP8 LIGHTNING, Leica, Germany).

Transmission electron microscopy (TEM)

HD11 cells were seeded into 10 cm culture dishes and cultured to approximately 70% confluence. Cells were then treated with 100 µg/mL Hcp2a protein for 12 h at 37 °C, trypsinized, and collected by centrifugation. Cell pellets were resuspended in electron‑microscopy fixative (pre‑warmed to room temperature; P1126, Solarbio, Beijing, China) and fixed in the dark at room temperature for 30 min. Control samples were processed in parallel. Fixed samples were stored at 4 °C until further processing. TEM observation and image acquisition were performed by Wuhan Servicebio Technology Company to visualize autophagosome‑like vesicles.

Proteomics analysis

HD11 cells were seeded into 10 cm culture dishes and cultured to approximately 70% confluence. Cells were treated with 100 μg/mL Hcp2a protein for 12 h at 37 °C; a parallel untreated group served as the control. After treatment, cells were trypsinized, collected by centrifugation, washed twice with PBS, and pelleted. Cell pellets were sent to Novogene Technology Company (Beijing, China) for quantitative proteomic analysis using data‑independent acquisition (DIA) on an Orbitrap Astral high‑resolution mass spectrometer. Key steps included protein extraction, trypsin digestion, peptide desalting, DIA‑MS analysis, and database searching.

LysoSensor™ green DND-189 fluorescent probe assay

Cells were seeded on coverslips in 24‑well plates and grown to approximately 70% confluence. Cells were treated with 100 μg/mL Hcp2a protein for 12 h at 37 °C; cells treated with 10 μM chloroquine (CQ; MCE, New Jersey, USA; HY-17589A) served as a positive control. After incubation, cells were washed three times with PBS and incubated with 1 μM LysoSensor™ Green DND‑189 (40767ES, Yeasen, Shanghai, China) in pre‑warmed medium for 40 min at 37 °C. Following three additional PBS washes, coverslips were mounted on slides with DAPI‑containing mounting medium (EE0015, Sparkjade, Shandong, China) and imaged under laser confocal microscope.

Statistical analysis

Fluorescence puncta were quantified and Western blot band intensities were measured using ImageJ software. Data were processed and analyzed in GraphPad Prism 8. All measurement data in this study were expressed as mean ± standard deviation (mean ± SD). Statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons; for comparisons involving only two groups, two-tailed Student’s t-tests were used. Differences were considered significant at p < 0.05 and highly significant at p < 0.01.

Results

Hcp2a enters HD11 cells and reduces cell viability

First, we expressed and purified recombinant Hcp2a protein using established protocols and verified its purity by SDS-PAGE, which showed that the protein was obtained at high purity, without detectable contaminating bands, and was therefore suitable for subsequent experiments (Fig. 1A). To determine whether Hcp2a can enter cells to exert biological activity, we performed immunofluorescence using FITC‑labeled proteins. The results showed that Hcp2a was localized to intracellular or peri‑membrane regions of HD11 cells (Fig. 1B). Western blot analysis further detected full-length Hcp2a within the cells (Fig. 1C). Together, these data demonstrate that Hcp2a can enter HD11 cells.

Fig. 1.

Hcp2a protein enters HD11 cells and exerts cytotoxic effects. (A) SDS-PAGE analysis (Coomassie Brilliant Blue staining) showing the purity of recombinant Hcp2a protein during purification. Lanes: M, protein marker; L, flow-through; W1-W3, washing buffers; E1-E5, elution buffers. (B) HD11 cells were treated with 100 µg/mL Hcp2a for 12 h. FITC-labeled His-Hcp2a emitted green fluorescence, visualized by laser confocal microscopy (scale bar: 100 µm). (C) HD11 cells were treated with 100 µg/mL Hcp2a for 6 h or 12 h. After trypsin digestion to remove extracellular proteins, the protein levels of His-Hcp2a in cell lysates were detected by Western Blot. GAPDH was used as a loading control. (D) HD11 cells were treated with the indicated concentrations (50 or 100 µg/mL) of Hcp2a for 6 h, 12 h, or 18 h, and cell viability was assessed using the CCK‑8 assay. Data are presented as mean ± SD. Statistical significance is indicated by *P < 0.05 and **P < 0.01 compared with the control group.

We next evaluated the effect of Hcp2a on cell viability using the CCK‑8 assay. The results showed that treatment with 100 μg/mL Hcp2a for 12 h significantly reduced cell viability (P<0.01) (Fig. 1D). These results demonstrate that the Hcp2a protein enters HD11 cells and exert cytotoxic effects.

Hcp2a protein induces autophagy in HD11 cells

Accumulating evidence has demonstrated that bacterial effectors are capable of inducing autophagy in host cells (McEwan, 2017; Miller and Celli, 2016). We investigated whether Hcp2a triggers this process in HD11 cells. Conversion of LC3-I (cytoplasmic form) to LC3-II (autophagosomal membrane form) is a recognized marker of autophagy (Runwal et al., 2019). Therefore, we detected LC3 protein levels of cells after Hcp2a treatment by Western Blot. The results showed that LC3-II protein levels increased significantly at 12 h and 18 h of Hcp2a treatment (P<0.01) (Fig. 2A and B).

Fig. 2.

Hcp2a protein induces autophagy in HD11 cells. (A, B) HD11 cells were treated with the indicated concentrations (50 or 100 µg/mL) of Hcp2a for 6, 12, or 18 h. Protein levels of LC3-I/II and GAPDH (loading control) were assessed by western blot. Band intensities were quantified using ImageJ software. Data are presented as mean ± SD. *P < 0.05, **P < 0.01 versus the control group. (C) HD11 cells transfected with a GFP-LC3 plasmid were treated with 100 µg/mL Hcp2a for 6 or 12 h. GFP-LC3 puncta (green) were visualized by confocal microscopy (scale bar: 11.6 µm). Magnification, × 60. (D) HD11 cells were incubated with 100 µg/mL Hcp2a for 12 h and analyzed by transmission electron microscopy. Red arrows indicate autophagosome-like vesicles (scale bar: 2.0 µm).

To further examine LC3 localization, we transfected HD11 cells with a GFP‑LC3 plasmid and treated them with Hcp2a for 6 h or 12 h. Confocal microscopy revealed bright punctate GFP‑LC3 fluorescence in Hcp2a‑treated cells, whereas fluorescence in control cells remained diffuse (Fig. 2C), indicating that Hcp2a promotes LC3 aggregation.

Finally, we used transmission electron microscopy (TEM)—considered the gold standard for visualizing autophagy (Mizushima et al., 2010)—to directly observe autophagosome formation. TEM images showed more double‑membrane autophagosomes structures in Hcp2a‑treated cells compared with controls (Fig. 2D). In summary, these findings confirm that Hcp2a protein induces autophagy in chicken macrophages.

Hcp2a protein induces incomplete autophagy in HD11 cells

Accumulation of p62 is widely used as an indicator of impaired autophagic degradation (Frankel et al., 2011). To evaluate whether autophagic clearance was affected, we first measured p62 protein levels. The results showed that intracellular p62 protein level was significantly elevated (P<0.01) with the prolongation of treatment time compared with the control (Fig. 3A and B). Immunofluorescence staining further revealed bright p62 aggregates in Hcp2a‑treated cells that became more prominent over time (Fig. 3C). Although p62 accumulation is consistent with blocked autophagic flux, we cannot completely exclude alternative mechanisms such as NRF2 activation; nevertheless, the mCherry-GFP-LC3 data directly support flux blockade (Choi et al., 2024).

Fig. 3.

Hcp2a protein induces incomplete autophagy in HD11 cells. (A, B) HD11 cells were treated with the indicated concentrations (50 or 100 µg/mL) of Hcp2a for 6, 12, or 18 h. Protein levels of P62 and GAPDH (loading control) were assessed by Western blot. Band intensities were quantified using ImageJ software. Data are presented as mean ± SD. Statistical significance is indicated by *P < 0.05, **P < 0.01 versus the control group. (C) HD11 cells were treated with 100 µg/mL Hcp2a for 6 h or 12 h. The immunofluorescence analysis was performed using an anti-p62 antibody (green) and DAPI (blue), and the samples were visualized by confocal microscopy (scale bar: 5 µm). Magnification, × 60. (D) HD11 cells were transfected with the mCherry-GFP-LC3 plasmid and then treated with 100 µg/mL Hcp2a for 12 h or 18 h. Yellow puncta (mCherry+/GFP+, autophagosomes) are indicative of blocked autophagic flux (scale bars: 11.6 µm). Magnification, × 60.

To further monitor the autophagy flux, we utilized the mCherry-GFP-LC3 tandem reporter. In this system, GFP fluorescence is quenched in acidic lysosomes, whereas mCherry remains stable. Thus, the persistence of yellow puncta (mCherry+/GFP+) indicates autophagosomes that have not undergone successful lysosomal dissolution. As shown in Fig. 3D and E, Hcp2a‑treated cells exhibited a significant increase in yellow LC3 puncta (P<0.01), while control cells exhibited predominantly diffuse fluorescence. This pattern demonstrates a blockade in the autophagic degradation process, providing direct evidence that Hcp2a disrupts the completion of autophagy.

Taken together, these results demonstrate that Hcp2a triggers autophagosome formation but impairs lysosomal degradation, leading to incomplete autophagy and a blockade in autophagic flux.

Down-regulation of lysosome-associated pathways in HD11 cells treated with Hcp2a protein

To further investigate the mechanism underlying the observed incomplete autophagy, we performed quantitative proteomic sequencing. Compared with the control, there were 233 up-regulated proteins and 307 down-regulated proteins in the Hcp2a-treated group (Group C) (Fig. 4A). Clustering heat map results showed significant inter-group differences and high intra-group consistency, indicating that the data quality was reliable and suitable for subsequent in-depth analysis (Fig. 4B).

Fig. 4.

Proteomic analysis of HD11 cells treated with Hcp2a protein. HD11 cells were treated with 100 µg/mL Hcp2a for 12 h and subjected to data‑independent acquisition (DIA)‑based quantitative proteomics. Groups: A, control; C, Hcp2a‑treated. (A) Volcano plots of differentially expressed proteins. Red dots denote significantly upregulated proteins (fold change > 1.5, P < 0.05); green dots denote significantly downregulated proteins (fold change < 0.66, P < 0.05). (B) Hierarchical‑clustering heatmap of differentially expressed proteins (red, high expression; blue, low expression). (C) Gene Ontology (GO) enrichment analysis of differentially expressed proteins. (D) KEGG pathway enrichment analysis. Bubble size indicates the number of enriched proteins; color represents the –log₁₀(P‑value). In panel D, the left panel shows upregulated pathways, the right panel shows downregulated pathways, with the lysosome pathway highlighted in red.

Gene Ontology (GO) enrichment analysis indicated that differentially expressed proteins in Hcp2a‑treated cells were significantly enriched in biological processes such as immune response, DNA metabolism, protein dephosphorylation, and DNA replication. For cellular components, Hcp2a‑treated cells showed enrichment in extracellular region and cytoskeleton. In molecular function, Hcp2a‑treated cells were enriched in hydrolase activity, ion binding, DNA binding, cytokine receptor binding, and phosphorylated protein phosphatase activity (Fig. 4C).

KEGG pathway analysis revealed that up‑regulated proteins in Hcp2a‑treated cells were enriched in 17 pathways, including cytokine‑cytokine receptor interaction, RIG‑I‑like receptor signaling, Toll‑like receptor signaling, NOD‑like receptor signaling, cellular senescence, and MAPK signaling. Notably, the lysosome pathway was significantly downregulated upon Hcp2a treatment. In addition, pathways such as adrenergic signaling in cardiomyocytes, DNA replication, cell cycle, unsaturated fatty acid biosynthesis, and purine metabolism were also significantly downregulated (Fig. 4D).

Collectively, these proteomic results indicate that Hcp2a treatment activates cellular stress‑immune and signal‑transduction pathways, while simultaneously impairing critical cellular functions through suppression of lysosomal degradation, disruption of energy supply, interference with cell cycle progression, inhibition of basal metabolism, and exacerbation of oxidative stress.

Lysosomal dysfunction mediates incomplete autophagy in Hcp2a-treated HD11 cell

Autophagy relies on functional lysosomes, and lysosomal impairment disrupts the autophagic cascade. KEGG analysis revealed significant down‑regulation of the lysosomal pathway. The major down-regulated proteins include lysosomal acid hydrolase and V-type proton ATPase protein lipid subunit, which are essential for maintaining lysosomal acidity and degradative capacity (Table 1).

Table 1.

Downregulated proteins in the lysosomal pathway (Hcp2a.vs. Control).

Protein ID	Description	Gene name	Ratio of difference	p-value
A0A1D5PM49	N-acetylgalactosamine-6-sulfatase	GALNS	0.544	0.026
A0A8V0X637	Sphingomyelin phosphodiesterase 1	SMPD1	0.485	0.024
A0A8V0X8Z3	Solute carrier family 17member 5	SLC17A5	0.596	0.021
A0A8V0XVC0	Insulin like growth factor 2 receptor	IGF2R	0.254	0.006
A0A8V0YC98	Alpha-galactosidase	NAGA	0.580	0.023
A0A8V0YVB6	cathepsin X	CTSZ	0.515	0.007
A0A8V0Z0Z3	V-type proton ATPase proteolipid subunit	ATP6V0C	0.396	0.020
A0A8V0ZAV4	Galactocerebrosidase	GALC	0.507	0.014
A0A8V0ZD93	Beta-hexosaminidase	HEXA	0.626	0.031
A0A8V1AFH0	Cathepsin S	CTSS	0.485	0.008
P43233	Cathepsin B	CTSB	0.647	0.037
Q05744	Cathepsin D	CTSD	0.635	0.003
Q5ZJS7	NPC intracellular cholesterol transporter 2	NPC2	0.560	0.009
Q90744	Alpha-N-acetylgalactosaminidase	NAGA	0.631	0.009
A0A1D5PM49	N-acetylgalactosamine-6-sulfatase	GALNS	0.544	0.026
A0A8V0X637	Sphingomyelin phosphodiesterase 1	SMPD1	0.485	0.024
A0A8V0X8Z3	Solute carrier family 17 member 5	SLC17A5	0.596	0.021
A0A8V0XVC0	Insulin like growth factor 2 receptor	IGF2R	0.254	0.006

To examine the effect of Hcp2a on lysosomal acidification, we used the pH‑sensitive probe LysoSensor™ Green DND‑189, which accumulates in acidic compartments and emits bright green fluorescence. Chloroquine (CQ), which raises lysosomal pH and blocks autophagosome‑lysosome fusion, served as a positive control (Mauthe et al., 2018). Fluorescence microscopy showed that the green fluorescent aggregates in the Hcp2a protein-treated group were significantly reduced compared with the control, similar to the CQ-treated group, which indicated that the acidic lysosomal compartments was reduced and the lysosomal acidic environment was disrupted by Hcp2a treatment (Fig. 5A).

Fig. 5.

Hcp2a treatment impairs lysosomal function in HD11 cells. (A) HD11 cells were treated with 100 µg/mL Hcp2a for 12 h. Chloroquine (CQ) was used as a positive control at a concentration of 10 μM for 6 h before staining. Lysosomal acidification was detected using LysoSensor™ Green DND-189. Acidic compartments appeared as green fluorescence under confocal microscopy (scale bar: 11.6 µm). Magnification, × 60. (B, C) Cells were treated with 100 µg/mL Hcp2a for 3, 6, or 12 h. The protein expression level of intracellular cathepsin D (CTSD) was assessed by western blot. GAPDH was used as an internal control. The bar graph shows the mature CTSD/pro-CTSD ratio (mean ± SD; **P < 0.01 vs. control group). (D) Co-localization of LC3 (green) and LAMP1 (red) in cells treated with 100 µg/mL Hcp2a for 12 h. Nuclei were stained with DAPI (blue) (scale bar: 5 µm). Magnification, × 60.

Cathepsin D (CTSD), a major lysosomal aspartic protease, is required for degradation within autolysosomes, and its deficiency leads to impaired autophagic flow (Di et al., 2020; Gallwitz et al., 2024; Suzuki et al., 2022). Western blot analysis of CTSD showed that the ratio of intracellular mature somatic CTSD to precursor CTSD was significantly decreased in the Hcp2a-treated cells (P<0.01) (Fig. 5B and C).

In addition, in order to investigate whether autophagosomes and lysosomes can fuse normally, we labeled the LC3 (autophagosome marker) and LAMP1 (lysosome marker) with immunofluorescence staining. Hcp2a‑treated cells displayed increased yellow puncta (Fig. 5D). The increased co-localization of LC3 and LAMP1 suggests that fusion may still occur, but degradation within autolysosomes is likely arrested.

Together, these results demonstrate that Hcp2a‑induced incomplete autophagy is primarily due to lysosomal deacidification and impaired degradative function.

Discussion

Avian pathogenic Escherichia coli (APEC) is a significant pathogen in poultry, and its Type VI Secretion System (T6SS) effector Hcp2a is a key virulence determinant (de Pace et al., 2010). In this study, we demonstrated that the APEC T6SS effector Hcp2a induces incomplete autophagy in chicken HD11 cells through lysosomal dysfunction.

Autophagy is a defense mechanism that removes intracellular pathogens, misfolded proteins and damaged organelles. However, bacterial effectors can interfere with autophagy in different ways to enhance bacterial survival (Jiao and Sun, 2019). LC3-II is a protein present in the autophagosome, whose expression and processing increase during autophagy and therefore serves as a marker of autophagy levels (Barth et al., 2010; Kabeya et al., 2000). In contrast, p62 (SQSTM1) binds directly to LC3 and promotes the degradation of ubiquitinated proteins and the autophagic process; its accumulation sometimes reflects impaired flux (Pankiv et al., 2007). The mCherry‑GFP‑LC3 tandem reporter is also widely used to monitor autophagic flux (Wang et al., 2022). In our experiments, the presence of persistent yellow puncta indicated that the acid‑sensitive GFP signal was not quenched, directly demonstrating a blockade at the late stage of autophagy. However, it should be noted that p62 accumulation may also arise partially from transcriptional upregulation, for instance via the reactive oxygen species (ROS)-responsive NRF2 pathway (Choi et al., 2024). Although our mCherry-GFP-LC3 data clearly support autophagic flux blockade, the current results cannot completely exclude the contribution of NRF2 activation to the elevated p62 levels. Future experiments using NRF2-specific inhibitors will help dissect the relative contributions of these two mechanisms to Hcp2a-induced p62 accumulation.

Autophagy can be categorized as complete or incomplete. In complete autophagy, autophagosomes fuse with lysosomes for content degradation, generally supporting cell survival. Incomplete autophagy, by contrast, often causes cellular damage or can be exploited by pathogens for replication (Xiong et al., 2019).

Lysosomes are highly acidic organelles that are mainly used to maintain cellular homeostasis and pathogen clearance (Gros and Muller, 2023). They receive substrates via fusion with endosomes or autophagosomes, or through chaperone-mediated and microautophagy pathways (Yim and Mizushima, 2020). Because autophagy is a major host cell defense mechanism, pathogens frequently target lysosomal function to evade immunity. For example, the effector VopQ of Vibrio parahaemolyticus binds the conserved Vo structural domain of the vesicular H(+)-ATPase, rapidly de‑acidifying lysosomes and blocking autophagic flux (Peng et al., 2020; Sreelatha et al., 2013); VacA, an effector protein secreted by Helicobacter pylori, induces autophagy but inhibits autophagosome‑lysosome fusion and reduces cathepsin D levels, impairing lysosomal catalytic activity (Raju et al., 2012; Wang et al., 2009). Our data place Hcp2a within this class of effectors: it down‑regulates V-ATPase components and acid hydrolases, leading to lysosomal de-acidification and reduced mature cathepsin D (CTSD) activity. V-ATPase, a multi-subunit complex that plays a key role in lysosomal acidification, mainly pumps protons into the lysosomal lumen to maintain an optimal pH (≈4.5-6.0) for over 60 acid hydrolases (Mindell, 2012). Its dysfunction leads to a substantial reduction in lysosomal degradation rates (Kosmidis et al., 2022; Ramirez et al., 2019; Vasanthakumar and Rubinstein, 2020).

In addition, lysosomal dysfunction leads to blocked autophagic flow and accumulation of undegraded autophagosomes, which can exacerbate cellular oxidative stress and damage (Stepien et al., 2020). The cellular immune stress-related signaling pathway revealed by proteome sequencing may be associated with impaired lysosomal degradation. Then, immunofluorescence co‑localization of LC3 and LAMP1 indicates that autophagosome‑lysosome fusion per se was not completely blocked; rather, the impairment appears to occur at the lysosomal degradation stage. The complete autophagic process involves autophagosome formation, fusion with lysosomes, and autolysosomes degradation. When lysosomal function is compromised, the cell cannot clear pathogens or damaged organelles, suggesting that Hcp2a‑induced blockade of autophagic flux may represent an immune‑evasion strategy.

In summary, our findings establish that the APEC T6SS effector Hcp2a induces incomplete autophagy in chicken HD11 cells through lysosomal dysfunction. This work provides a basis for understanding how APEC evades host immune responses. Future studies identifying the direct host targets of Hcp2a and investigating its effects in other cell types will further clarify its mechanism of action. A better understanding of this pathway may offer insights for developing strategies against APEC infections.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.

Declaration of AI and AI-assisted technologies in the writing process

Not applicable.

Funding

This work was supported by grants from the National Natural Science Foundation of China (Grant No. 32272959), Anhui Provincial University Excellent Young Talents Support Program (YQYB2023001), Research Funds of Joint Research Center for Food Nutrition and Health of IHM (2024SJY04) and Anhui Provincial Natural Science Foundation (2408085QC076).

CRediT authorship contribution statement

Yaqin Tian: Writing – original draft, Visualization, Validation, Methodology, Data curation. Yuqi Zhang: Visualization, Validation, Methodology, Data curation. Liting Lu: Visualization, Validation, Methodology, Investigation, Data curation. Shuhui Wang: Writing – review & editing, Visualization, Software, Formal analysis. Liyang Dai: Writing – review & editing, Visualization, Formal analysis. Haiyang Wang: Writing – review & editing, Visualization, Formal analysis. Saqib Nawaz: Writing – review & editing, Visualization, Formal analysis. Jiumeng Sun: Writing – review & editing, Visualization, Formal analysis. Ying Shao: Writing – review & editing, Visualization, Formal analysis. Zhengyu Wang: Writing – review & editing, Visualization. Jian Tu: Writing – review & editing, Visualization. Xiangjun Song: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Data curation.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.