The finding centers on CelF, a protein encoded by the C. burnetii gene CBU1825 and delivered into host cells through the bacterium’s Dot/Icm type IV secretion system. This secretion apparatus, which the pathogen uses to inject more than 140 candidate effector proteins into host cells, is essential for establishing the Coxiella-containing vacuole, or CCV — the modified phagolysosome in which the bacterium replicates. Unlike most intravacuolar pathogens that block fusion with lysosomes to avoid degradation, C. burnetii actively promotes it, thriving in the acidic, enzyme-rich environment that would kill most other microbes.

To understand how CelF contributes to this unusual lifestyle, the research team, led by Shan Zhang and Yufei Jiang at the State Key Laboratory of Pathogen and Biosecurity in Beijing, first confirmed that CelF is genuinely translocated into host cells. Using a β-lactamase-based translocation assay in THP-1 macrophage-like cells, they showed that CelF enters the host cytosol in a manner dependent on a functional Dot/Icm system. They then generated a knockdown strain using CRISPR interference, achieving approximately 90 percent reduction in celF transcription, and confirmed that the effect was specific by complementing the strain with a wild-type copy of the gene.

The consequences of losing CelF were striking. In THP-1 macrophages, HeLa cells, and BGMK cells, the CelF knockdown strain replicated poorly compared to wild-type bacteria. The characteristic large, LAMP1-positive vacuoles that wild-type C. burnetii forms were replaced by small, underdeveloped compartments. In a SCID mouse model, animals infected with the knockdown strain showed significantly reduced splenomegaly and lower bacterial loads in the spleen compared to those infected with wild-type or control strains. These results establish CelF as a genuine virulence factor required for the full pathogenic potential of C. burnetii.

Having confirmed that CelF matters for infection, the researchers turned to the question of what it actually does inside host cells. Their previous affinity purification–mass spectrometry screen had identified over 1,000 host proteins that physically interact with CelF, with significant enrichment in pathways related to the ubiquitin-proteasome system, autophagy, and membrane trafficking. Applying the MiST scoring algorithm to prioritize high-confidence candidates, they narrowed the list to 12 proteins, among them the proteasomal shuttle factor UBQLN1.

UBQLN1 is a fascinating molecule. It acts as a courier, using its C-terminal ubiquitin-associated domain to grab polyubiquitinated cargo and its N-terminal ubiquitin-like domain to dock at the 26S proteasome, specifically at the PSMD4 subunit, where it hands off its cargo for degradation. Co-immunoprecipitation experiments in HEK-293T cells confirmed that CelF binds UBQLN1, and an in vitro pull-down assay demonstrated that this interaction is direct. Truncation mapping revealed that CelF specifically engages the ubiquitin-like domain of UBQLN1 — the very same surface that normally contacts PSMD4.

This overlap set up a competitive scenario. The researchers used microscale thermophoresis to quantify the interactions. PSMD4 bound to UBQLN1 with a dissociation constant of approximately 5.12 micromolar. CelF bound UBQLN1 with a similar moderate affinity of about 15.82 micromolar. When UBQLN1 was pre-incubated with CelF before titration with PSMD4, the binding curve shifted dramatically to the right, with the apparent dissociation constant rising to 27.02 micromolar — clear evidence that CelF competes with PSMD4 for access to UBQLN1. Conversely, pre-incubating UBQLN1 with PSMD4 reduced the apparent affinity of the CelF-UBQLN1 interaction, confirming mutual exclusivity.

The functional consequence of this competition became apparent when the team examined the fate of SNAP29, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor, or SNARE, protein that participates in the STX17-SNAP29-VAMP8 complex responsible for fusing autophagosomes with lysosomes. Treatment with the proteasome inhibitor MG132 rescued SNAP29 levels in LPS-stimulated cells, while the lysosome inhibitor NH4Cl did not, establishing that SNAP29 is degraded through the ubiquitin-proteasome system. When CelF was expressed, SNAP29 protein levels rose, and cycloheximide chase experiments confirmed that the protein was genuinely more stable. Critically, ubiquitination assays showed that CelF did not alter the ubiquitination status of SNAP29 — the bacterium was not changing how the protein was tagged, only whether the tagged protein reached the proteasome.

With SNAP29 stabilized, the fusogenic SNARE complex assembles more efficiently. Co-immunoprecipitation revealed that CelF enhanced the interaction between STX17 and SNAP29 upon LPS stimulation, and that depleting CelF during infection impaired this assembly. Knocking down UBQLN1 with small interfering RNA increased SNARE complex formation, and reconstituting UBQLN1 restored levels to baseline, confirming that UBQLN1 mediates the regulatory effect. The downstream result was measurable: CelF-expressing cells showed increased numbers of red-only LC3 puncta in the tandem RFP-GFP-LC3 assay, indicating enhanced autophagosome-lysosome fusion, and this effect persisted even when lysosomal degradation was blocked with bafilomycin A1, placing CelF’s action upstream of the lysosomal step.

What makes this mechanism remarkable is its purely non-enzymatic nature. Most known pathogen-encoded modulators of the ubiquitin-proteasome system function as E3 ubiquitin ligases or deubiquitinases — enzymes that actively add or remove ubiquitin marks. Shigella flexneri’s IpaH9.8, for example, acts as an E3 ligase to degrade host guanylate-binding proteins. CelF possesses no recognizable enzymatic domain and leaves SNAP29’s ubiquitination untouched. Instead, it operates as a molecular decoy, occupying a binding surface on UBQLN1 that would otherwise engage the proteasome. This strategy allows selective modulation of a subset of UBQLN1 substrates without globally disrupting proteasomal activity, which would be toxic to the host cell. The researchers noted that CelF specifically elevated SNAP29 without causing broad accumulation of polyubiquitinated proteins, supporting the idea of a finely tuned intervention rather than a blunt shutdown.

The study also revealed that UBQLN1 knockdown alone promoted C. burnetii replication and partially rescued the growth defect of the CelF knockdown strain, providing genetic evidence that the UBQLN1-PSMD4 axis is a genuine regulatory checkpoint that the bacterium exploits. Furthermore, ectopic expression of SNAP29 in HeLa cells promoted wild-type bacterial growth and partially rescued the intracellular replication and vacuole maturation defects caused by CelF depletion, confirming that SNAP29 stabilization is functionally meaningful for the pathogen.

Beyond its implications for understanding Q fever pathogenesis, the identification of the UBQLN1-PSMD4-SNAP29 axis as a regulatory node for SNARE proteostasis has broader relevance. Dysregulation of SNAP29 and autophagosome-lysosome fusion has been implicated in neurodegenerative diseases, metabolic disorders, and cancer. The authors suggest that this axis could represent a therapeutic target for conditions characterized by impaired autophagic flux. The work also highlights a convergent evolutionary solution at the protein interaction interface, where a bacterial effector has evolved to mimic the proteasomal binding surface of a host shuttle factor — a strategy that differs fundamentally from the lysosome-avoidance tactics employed by pathogens such as Legionella pneumophila, and one that may inspire new approaches to targeting protein homeostasis in human disease.

Subject of Research: How the Coxiella burnetii effector CelF disrupts the UBQLN1-PSMD4 proteasomal axis to stabilize SNAP29 and promote lysosomal maturation of the Coxiella-containing vacuole

Article Title: Coxiella burnetii effector CelF reprograms host proteostasis via competitive disruption of the UBQLN1-PSMD4 axis to stabilize autophagic SNARE SNAP29 and modulate lysosomal metabolic maturation

Article References: Zhang, S., Jiang, Y., Lin, Y., Chen, X., Zhang, Y., Yu, Y., OuYang, X., Zhou, D., Song, Y., & Jiao, J. (2026). Coxiella burnetii effector CelF reprograms host proteostasis via competitive disruption of the UBQLN1-PSMD4 axis to stabilize autophagic SNARE SNAP29 and modulate lysosomal metabolic maturation. Frontiers in Physiology, 17, Article 1957980. https://doi.org/10.3389/fphys.2026.1957980

Image Credits: AI Generated

DOI: 10.3389/fphys.2026.1957980

Keywords: Coxiella burnetii, Q fever, CelF, UBQLN1, PSMD4, SNAP29, proteostasis, autophagy, lysosome, type IV secretion system, SNARE complex, molecular decoy