Yajun Song

The aspA gene of Yersinia pestis exhibits a pronounced polymorphism at codon 363, with the dominant TTG allele encoding an inactive aspartate ammonia-lyase (AspA) while the GTG allele encoding an active enzyme. We previously demonstrated that this polymorphism drives an environment-dependent fitness trade-off, yet the underlying mechanism remains unclear. To address this, we first characterized the phenotypic differences between the two strains and revealed a clear growth-stress survival trade-off: the GTG (AspA-active) strain grew faster and produced more ATP, whereas the TTG (AspA-inactive) strain, despite slower growth, exhibited superior survival under nutrient limitation with oxidative, anaerobic, or hyperosmotic stress. Exogenous aspartate reversed the GTG strain’s disadvantages under anaerobic or cold stress. To dissect the metabolic basis, we performed ¹³ C-labeled metabolic flux analysis on these two strains. The GTG strain rapidly converted aspartate to fumarate via AspA, fueling the TCA cycle and boosting ATP production. In contrast, the TTG strain displayed stalled aspartate utilization and accumulated amino acids and lipids. Supplementation with TCA cycle intermediates (fumarate, succinate, or malate) partially rescued TTG growth, confirming that reduced TCA flux limits its proliferation. Collectively, AspA activity orchestrates a growth-stress survival trade-off through metabolic reprogramming, underlying the antagonistic pleiotropy of Y. pestis in aspartate-available or stressful environments. The “metabolic congestion” strategy of the TTG strain enhances survival under multiple stresses relevant to the flea gut, and it is plausible that this metabolic reprogramming may contribute to transmission and provide a selective advantage under flea-borne conditions. IMPORTANCE: The plague bacterium Yersinia pestis encounters oxidative, anaerobic, and hyperosmotic stresses—conditions relevant to its flea gut environment during transmission. This study reveals that a single, naturally occurring polymorphism in the aspA gene shifts the bacterium’s metabolism from a growth-focused mode to a “metabolic congestion” state that enhances survival under these stresses in vitro . Our work provides a mechanistic framework for understanding how metabolic reprogramming could influence adaptation to vector-like conditions, offering a plausible basis for the environment-dependent fitness trade-offs that may shape transmission dynamics.

Introduction: The ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway constitute the core proteolytic metabolic networks that maintain cellular protein homeostasis (proteostasis) and organelle metabolic identity. Intracellular pathogens frequently reprogram host metabolic pathways to establish replicative niches, yet how they manipulate proteostatic metabolism to remodel organelle maturation remains incompletely understood. Methods: CelF secretion was confirmed by TEM translocation assay. CRISPR-dCas9 was used to generate CelF knockdown and complementation strains, with celF transcription verified by RT-qPCR. Cell infection and SCID mouse models were used to assess intracellular survival, Coxiella-containing vacuole (CCV) maturation, and virulence of Coxiella burnetii. The effect of CelF on late autophagy was examined by dual-fluorescence LC3 and Western blotting. Then, UBQLN1 was identified as a CelF-interacting host protein by co-IP and pull-down, followed by truncation mapping. Finally, co-IP, UBQLN1 knockdown, Western blotting and MST assays were used to analyze the effect of CelF on SNARE assembly, SNAP29 degradation and ubiquitination, and competitive binding of CelF and PSMD4 to UBQLN1. Results: CelF depletion severely impaired the metabolic maturation of CCV, suppressed bacterial proliferation in macrophages, and attenuated virulence in vivo. Mechanistically, CelF acted as a molecular decoy that directly bound the ubiquitin-like (UBL) domain of the proteasomal shuttle factor UBQLN1, competitively displacing the proteasome subunit PSMD4. This competitive interference blocked UBQLN1-mediated proteasomal delivery of SNAP29 without altering its ubiquitination status, thereby enhancing SNAP29 protein stability and promoting assembly of the STX17-SNAP29-VAMP8 fusogenic complex. The augmented SNARE activity drove autophagosome-lysosome fusion, sustained autophagic flux, and facilitated lysosomal metabolic remodeling to support CCV biogenesis. Discussion: Unlike canonical pathogen-encoded UPS modulators that function as E3 ligases or deubiquitinases, CelF operates purely through competitive protein-protein interaction, representing a previously unrecognized paradigm for microbial manipulation of host proteostatic metabolism. Our findings reveal that intracellular pathogens can actively orchestrate organelle metabolic identity by rewiring host proteostasis, and identify the UBQLN1-PSMD4-SNAP29 axis as a novel regulatory checkpoint for SNARE proteostasis and lysosomal metabolic maturation.

Live imaging is essential for elucidating dynamic infection processes. Here, we developed a near-infrared fluorescent protein (NIR-FP)-based labeling strategy that enables integrated visualization of bacterial behavior in both cellular and animal infection models. Using Yersinia pestis as a model pathogen, we constructed stable fluorescent strains by either chromosomal insertion of the gene encoding NIR-FP miRFP670nano3 under the control of a strong endogenous promoter to obtain Yp-nano3, or by introducing a plasmid expressing the same reporter to obtain Yp-p-nano3. Both labeled strains exhibited a strong correlation between fluorescence intensity and bacterial counts, with Yp-nano3 exhibiting significantly higher brightness at the same bacterial count. Importantly, the labeled strains showed no measurable reduction in virulence under the tested experimental conditions, supporting their use in physiologically relevant infection studies. In cellular infection assays, sustained and robust fluorescence from the labeled bacteria enables continuous monitoring of pathogen–host cell interactions for up to 12 h. In a murine bubonic plague model, fluorescence signals were detectable in the inguinal lymph nodes (ILN) as early as 1 day post-infection with Yp-nano3 and increased progressively until the mice died. In contrast, mice infected with a highly attenuated Δpla mutant carrying the same NIR-FP label showed no detectable fluorescence in ILN throughout the infection. These results show that NIR-FP labeling, combined with in vivo imaging, provides a powerful approach for real-time tracking of the dissemination of Y. pestis in lymph nodes and for assessing bacterial virulence. Together, this work establishes an NIR-FP labeling platform suitable for real-time analysis of bacterial infection dynamics and host-pathogen interactions. IMPORTANCE: Live imaging techniques, such as bioluminescence imaging and fluorescence imaging, have been widely used to study infection dynamics. However, bioluminescence imaging is strongly limited by its dependence on exogenous substrate administration and its inability to explore direct interactions between pathogen and host cells. To overcome these limitations, we established an NIR-FP labeling platform that enables visualization of pathogen–host interactions at both cellular and animal levels, using Y. pestis as a model pathogen. This labeling method offers high stability and a strong linear relationship between fluorescence intensity and bacterial count, allowing real-time tracking of bacterial dissemination to lymph nodes and subsequent proliferation in vivo . Together, these attributes offer a novel and robust tool for investigating infection dynamics and underlying pathogenic mechanisms.

SlyA, a key transcriptional regulator in Yersinia pestis , is involved in stress adaptation and virulence, yet its regulatory mechanisms remain poorly understood. Here, we investigate the role of the conserved hypothetical protein YhcN and its regulation by SlyA in Y. pestis . We demonstrate that deletion of yhcN impairs bacterial viability under low-temperature and anaerobic conditions and growth. Intriguingly, both deletion and overexpression of slyA lead to upregulation of yhcN transcription, suggesting a complex regulatory interplay. Bioinformatics identified two SlyA-binding sites upstream of the predicted -35 box of the yhcN promoter. We propose that SlyA may mediate a complex regulation of yhcN , wherein basal SlyA level could repress the transcription of the yhcN , while elevated SlyA could activate it. This regulatory switch may enable Y. pestis to fine-tune YhcN expression under fluctuating environmental pressures. Taken together, our findings propose a novel and complex regulatory circuit involving SlyA and YhcN, advancing the understanding of transcriptional networks that govern stress adaptation in Y. pestis .

Chlamydia psittaci is an obligate intracellular pathogen that poses a significant threat to both human and animal health. The current therapeutic strategies are limited by the emergence of potential drug resistance, underscoring the urgent need for novel anti-chlamydial agents. In this study, we evaluated the anti-chlamydial activity of kaempferol, a natural occurring flavonoid. Potential targets of kaempferol and C. psittaci infection-related targets were identified through in silico screening. In cellular assays using both HeLa and THP-1 cells, kaempferol treatment significantly inhibits the intracellular growth of C. psittaci in a dose-dependent manner, while showing no host cell cytotoxicity at effective concentrations. Based on prior in silico virtual screening and our group’s systematic evaluation of the anti-chlamydial activity of flavonoids, kaempferol (40 μM) was selected as the target compound for this study due to its potent antimicrobial effects and its potential to modulate key host–pathogen interaction pathways. Mechanistic investigations revealed that kaempferol interferes with the trafficking of host-derived lipid to the bacterial inclusion, thereby depriving the pathogen of essential nutrients. Moreover, kaempferol treatment severely disrupted the normal differentiation of reticulate bodies (RBs) into infectious elementary bodies (EBs), resulting in an aberrant developmental cycle and a reduction in the production of new infectious progeny. This study demonstrates that kaempferol exerts anti-chlamydial activity by targeting two key processes of C. psittaci infection: lipid trafficking to inclusions and RB-EB conversion. These results highlight kaempferol as a promising lead compound for developing new therapeutic approaches against C. psittaci infections.

Introduction: The direct zoonotic risks associated with the growing online trade of live pets remain in adequately understood. Methods: We investigate a human case of severe psittacotic pneumonia in Zhejiang Province, China, in which the patient’s only avian exposure was through parrots recently purchased online. Using targeted probe-capture sequencing—a method designed for uncultivable, low-biomass pathogens—we recovered 13 high-quality Chlamydia psittaci genomes directly from the patient, the asymptomatic parrots, and their shared home environment. Results: Comparative genomic analysis revealed >99.99% average nucleotide identity across all samples, providing definitive evidence of parrot-to-human transmission. The causative strain was identified as a novel sequence type (ST388) within the known virulent genotype A. Conclusion: This investigation provides the first whole-genome confirmation of psittacosis transmission via online pet commerce. It establishes a practical genomic framework for investigating similar sporadic zoonoses and underscores the urgent need for targeted surveillance of this emerging and risk communication in this growing digital marketplace.

Toxin-antitoxin (TA) modules enable bacteria to persist under stressful environments. However, they are typically absent from host-associated prokaryotes due to their potential host toxicity. Here, the obligate intracellular bacterium spotted fever group (SFG) rickettsiae, which causes mild to severe human illness, was shown to harbor two vapBC TA modules. One of the vapBC modules (vapBC1) is crucial for Rickettsia to withstand accumulated host reactive oxidative species (ROS), via induction of bacterial dormancy through cleavage on the anti-codon loop of tRNA^fMet, thereby facilitating intracellular survival and infection in a mouse model. Another vapBC module (vapBC2) was found to be activated and toxin exposed to host cytoplasm, contributing to Rickettsia’s virulence and adaptability in its human host by non-specifically degrading host rRNAs rather than regulating rickettsial growth. Recognition of these rickettsial effectors contributes to understanding the intracellular adaptability and pathogenicity of all host-associated pathogens that harbor TA modules.

Yersinia pestis, the etiologic agent of plague, is a genetically monomorphic pathogen. By screening 1085 published Y. pestis genomes, we identified an unusual mutation hotspot within codon 363 of the aspartate ammonia-lyase gene aspA with at least six alleles in the population. Our investigation delves into the significance of the polymorphism at the aspA codon 363 and its impact on the fitness of Y. pestis strains. Notably, we found that the dominating TTG allele (L363), resulting in AspA inactivation, confers a fitness advantage to Y. pestis when competing with other bacteria inhabiting a similar niche, due to elevated expression of pesticin. However, the TTG strain exhibits fitness deficits under various stress conditions compared to the GTG strain (V363), which expresses an active AspA. Drawing from these observations, we propose an evolutionary hypothesis for Y. pestis at the aspA codon 363 locus. It is inferred that GTG represented the ancestral state at this locus. However, the TTG strain swiftly dominated populations in the early evolutionary stage of Y. pestis, likely owing to the V363L substitution that conferred a competitive advantage to Y. pestis over other bacteria. Subsequently, spontaneous mutations emerged, reinstating AspA activity, thereby offering fitness advantages across diverse environments and get fixed within the population. Our findings portraited a scenario of inactivating-restoring mutations relay in Y. pestis microevolution, shedding new lights on the evolutional dynamics of pathogens.

Chlamydia are Gram-negative, obligate intracellular bacterial pathogens that infect eukaryotic cells and reside within a host-derived vacuole known as the inclusion. To facilitate intracellular replication, these bacteria must engage in host-pathogen interactions to obtain nutrients and membranes required for the growth of the inclusion, thereby sustaining prolonged bacterial colonization. Autophagy is a highly conserved process that delivers cytoplasmic substrates to the lysosome for degradation. Pathogens have developed strategies to manipulate and/or exploit autophagy to promote their replication and persistence. This review delineates recent advances in elucidating the interplay between Chlamydia trachomatis infection and autophagy in recent years, emphasizing the intricate strategies employed by both the Chlamydia pathogens and host cells. Gaining a deeper understanding of these interactions could unveil novel strategies for the prevention and treatment of Chlamydia infection.