BLOC1S1 Attenuates B. Melitensis 16M LPS-Triggered Autophagy by Spatial Confinement of TDP-43

Abstract

Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is considered to have anti-Brucella potential. However, the effect of BLOC1S1 on Brucella autophagy has not yet been studied. This study investigates the interplay between Brucella lipopolysaccharide (LPS) and BLOC1S1 in modulating autophagy within goat spermatogonial stem cells (mGSCs-I-SB). Using LPS from B. melitensis 16M, its capacity is demonstrated to induce AMPK-dependent autophagy, contrasting with Escherichia coli LPS, which shows no significant effect. Mechanistically, B. melitensis 16M LPS activates AMPK signaling, elevates LC3B-II/LC3B-I ratios, and upregulates lysosomal and pro-inflammatory genes. BLOC1S1 overexpression attenuates autophagy, reducing autolysosome formation (TEM) and LC3B-II/I ratio. RNA sequencing and proteomic analyses reveal BLOC1S1-mediated transcriptional reprogramming of lysosomal pathways and mitochondrial metabolism. Co-immunoprecipitation and subcellular localization studies reveal that TDP-43 is a key interacting partner and that BLOC1S1 sequesters TDP-43 in the cytoplasm, inhibiting its nuclear translocation-dependent ATG7 mRNA stability and enhancing autophagy. These findings delineate a dual regulatory mechanism: B. melitensis 16M LPS-driven, AMPK-dependent autophagy induction, and BLOC1S1-mediated autophagic suppression through spatial control of TDP-43. These results advance understanding of host-pathogen interactions in brucellosis and identify BLOC1S1 as a potential therapeutic target for bacterial persistence and TDP-43-related pathologies.

Keywords: Brucella spp; BLOC1S1; LPS; TDP‐43; autophagy.

Figure 1

Brucella infection downregulates BLOC1S1 expression in goat testes. A) Venn diagram demonstrating RIDD‐targeted genes with reduced expression following Brucella infection. B) Hematoxylin and eosin (H&E) staining of testicular sections from Brucella‐infected or not‐infected goats. Enlarged regions (top to bottom) highlight characteristic pathological changes: disrupted testicular architecture, fibrotic remodeling, and seminiferous tubule vacuolation. Scale bar: 125 and 300 µm. C) According to structural disorganization in seminiferous tubules, fibrosis and vacuolar degeneration, the sections as in (B) were evaluated and scored for lesion severity using the following scoring system, which is scored as the sum of the three lesion scores: 0 = no lesions; 1 = minimal with lesions involving <5% of tissue; 2 = moderate with focally extensive areas of lesions (5–25% of tissue); 3 = moderate to severe with focally extensive areas of lesions (>25% to 50% of tissue); 4 = severe with large confluent areas of lesions (>50% of tissue). D) Transmission electron micrographs showing increased lysosomal abundance in infected testicular cells, red arrows indicate lysosomes. Scale bar: 2 µm. E) Statistical analysis of the lysosomes amounts of goat testis infected or not infected by Brucella ssp., as in (D). F) Immunofluorescence analysis of BLOC1S1 expression (red) in testicular tissue, with Hoechst 33342 counterstaining for nuclei (blue). Scale bar: 125 µm. G) Fluorescence intensity quantification of goat testis infected or not infected by Brucella ssp., as in (F). H) Relative BLOC1S1 mRNA levels in goat testis infected or not infected by Brucella ssp. (n = 3). I) BLOC1S1 protein expression in o goat testis infected or not infected by Brucella ssp. J) Quantitative analysis of BLOC1S1 protein levels normalized to β‐actin (n = 3). ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

Figure 2

BLOC1S1 overexpression elevates lysosome‐associated gene expression in mGSCs‐I‐SB. A) Volcano plot displaying differentially expressed genes (DEGs) identified by RNA‐seq analysis between oeBLOC1S1 and CAG cells. B) RNA‐seq profiling of lysosome‐associated gene expression in oeBLOC1S1 versus CAG mGSCs‐I‐SB cells. C) Relative BLOC1S1, LAMP2, PLBD1, TLR2, CXCR2, RAB20, mTOR and THBS1 mRNA levels in oeBLOC1S1 versus CAG mGSCs‐I‐SB cells (n = 3). D) Top 20 enriched KEGG pathways ranked by significance (p‐value) for upregulated and downregulated DEGs in oeBLOC1S1 versus CAG cells. E) Top 20 enriched GO biological processes by significance (p‐value) for upregulated and downregulated DEGs in oeBLOC1S1 versus CAG cells. ^* p < 0.05, ^** p < 0.01.

Figure 3

Brucella 16M LPS induces AMPK‐dependent autophagy in mGSCs‐I‐SB. A) Relative mRNA Levels of NF‐κB, TLR4, TNF‐α, and IL‐1β in mGSCs‐I‐SB cells (n = 3), these cells were treated with 16M LPS (500 ng/mL) for 0, 1, 2, 4, 6, 12, and 24 h. B) Relative mRNA Levels of NF‐κB, TLR4, TNF‐α, and IL‐1β in mGSCs‐I‐SB cells (n = 3), these cells were treated with 0, 50, 100, 200, 500, and 1000 ng/mL 16M LPS for 24 h. C) Relative mRNA Levels of PYCARD, CXCL1, DNTBP1, IL‐18, IRF3, and LAMP2 in oeBLOC1S1 and CAG mGSCs‐I‐SB cells (n = 3), these cells were treated with 1000 ng/mL 16M LPS for 24 h. D) Relative mRNA Levels of PYCARD, CXCL1, DNTBP1, IL‐18, IRF3, and LAMP2 in oeBLOC1S1 and CAG mGSCs‐I‐SB cells (n = 3), these cells were treated with 1000 ng/mL E.coil LPS for 24 h. E) Flow cytometry analysis of oeBLOC1S1 and CAG mGSCs‐I‐SB cells following staining with apoptosis‐specific probes Annexin V‐FITC/PI, these cells were treated with 500, 1000 ng/mL 16M LPS or 1000 ng/mL E.coil LPS for 24 h. F) The protein amounts of β‐ACTIN, BLOC1S1, LC3B, DDX5, NF‐κB, phospho‐NF‐κB, IL‐6, and IL‐6 ST in mGSCs‐I‐SB cells, these cells were treated with 1000 ng/mL 16M LPS or 1000 ng/mL E.coil LPS for 24 h. G) Statistical analysis of the protein amounts of BLOC1S1, LC3B, DDX5, NF‐κB, phospho‐NF‐κB, IL‐6, and IL‐6 ST in mGSCs‐I‐SB cells treated as in (F). The relative amounts of these proteins were normalized to β‐ACTIN. H) The protein amounts of β‐ACTIN, BLOC1S1, LC3B, AMPK, phospho‐AMPK in mGSCs‐I‐SB cells, these cells were treated with or without 16M LPS (1000 ng/mL) for 24 h in the presence or absence of AMPK inhibitor Dorsomorphin (10 µm). I) Statistical analysis of the protein amounts of BLOC1S1, LC3B, AMPK, phospho‐AMPK in mGSCs‐I‐SB cells treated as in (H). The relative amounts of these proteins were normalized to β‐ACTIN. ns: not significant. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

Figure 4

BLOC1S1 overexpression attenuates 16M LPS‐induced autophagic activation. A) Transmission electron microscopy visualization of cell junctions in oeBLOC1S1 or CAG mGSCs‐I‐SB cells treated with or without 16M LPS for 24 h. The red arrows indicate autophagosome‐like vesicles (autophagosomes and autolysosomes). The red box are higher magnification images. Scale bar: 2 and 0.5 µm. B) Statistical analysis of the autophagosome‐like vesicles amounts of in oeBLOC1S1 or CAG mGSCs‐I‐SB cells treated as in (A). C) Relative mRNA Levels of LAMP1, LAMP2, LC3, BECN1, UVRAG, and PYCARD in oeBLOC1S1 or CAG mGSCs‐I‐SB cells (n = 3), these cells were treated as (A). D) The protein amounts of β‐ACTIN, BLOC1S1, LC3B, ULK1, AKT, phospho‐AKT, PI3K, phospho‐PI3K, AMPK, phospho‐AMPK, mTOR, DDX5, NF‐κB, phospho‐NF‐κB, IL‐6, and IL‐6 ST in oeBLOC1S1 or CAG mGSCs‐I‐SB cells, these cells were treated as (A). E) The protein amounts of LC3B in oeBLOC1S1 or CAG mGSCs‐I‐SB cells, these cells were treated with or without 16M LPS (1000 ng/mL) for 24 h in the presence or absence of lysosomes inhibitor BafA1 (100 nM). F) Analysis of O₂ consumption in oeBLOC1S1 or CAG mGSCs‐I‐SB cells treated with or without 16M LPS or E.coil LPS for 24 h. The rates of oxygen consumption (OCR) were first measured on 2×10⁴ cells of each groups under basal conditions and then with sequential additions of oligomycin (1.5 µm), FCCP (0.8 µm), rotenone (3.0 µm), and antimycin A (1.5 µm) at the indicated times to determine different parameters of mitochondrial functions. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

Figure 5

BLOC1S1 attenuates 16M LPS‐induced autophagy by restrictingTDP‐43 nuclear translocation. A) Total lysates were extracted from mGSCs‐I‐SB transfected by FLAG or FLAG‐BLOC1S1 fusion protein vectors for Co‐IP experiments. Equal number of proteins was immunoprecipitated separately with Anti‐FLAG M2 Magnetic Beads. Western blots show expression of BLOC1S1 with specific antibodies. B) Venn diagrams showing overlap of FLAG‐ and FLAG‐BLOC1S1‐associated proteins determined by mass spectrometry. C) Table lists FLAG‐BLOC1S1‐associated proteins. D) Two different fusion protein vectors, pBiFC‐VN173, pBiFC‐VC155, pBiFC‐VN173‐BLOC1S1, and pBiFC‐VC155‐TDP‐43 were transfected into HEK293T cells, and green fluorescence expression was observed after 48 h. Stronger green fluorescence indicates that the two proteins bind to each other. Cell nucleus were stained with DAPI (blue). Scale bar: 125 µm. E) Quantification of nuclear‐cytoplasmic ratio fluorescence signal for Figure 4D. F) Two different fusion protein vectors, pBiFC‐VN173‐BLOC1S1 and pBiFC‐VC155‐TDP‐43, were transfected into HEK293T cells, lysates were immunoprecipitated with anti‐FLAG or anti‐HA antibodies, and immunoprecipitated protein complex was examined. G) The protein amounts of β‐ACTIN, TDP‐43, ATG7 in oeBLOC1S1 or CAG mGSCs‐I‐SB cells, these cells were treated with or without 16M LPS for 24 h. H) The cytoplasmic and nuclear protein amounts of Histone H3, β‐ACTIN, BLOC1S1, TDP‐43, TFEB in oeBLOC1S1 or CAG mGSCs‐I‐SB cells, these cells were treated as (F). I) The 16M LPS induces elevated intracellular autophagy levels in mGSCs‐I‐SB cells through the AMPK pathway. BLOC1S1 interacts with TDP‐43, restricting its nuclear translocation, which subsequently reduces ATG7 expression and ultimately decreases autophagy levels (created by BioRender). ns: not significant, ^*** p < 0.001.