Mechanisms of Talaromyces marneffei induced CNS injury: Synergistic roles of tauopathy, pyroptosis, and microglial inflammation

Abstract

Talaromyces marneffei (T. marneffei) is an opportunistic, dimorphic fungus that has been clinically reported to involve the central nervous system (CNS). However, its neuroinvasive capacity and the underlying pathogenic mechanisms remain poorly understood. In this study, T. marneffei infection was found to impair learning, memory, and motor balance in mice, as demonstrated by Morris water maze and accelerating rotarod assays. Viable T. marneffei was subsequently isolated from hippocampus and cortex through fungal cultures, confirming its ability to invade the CNS. Histopathological analysis revealed CNS damage following infection, characterized by neuronal pyknosis, neurofibrillary tangles, and an increased presence of glial cells in the cortex. To investigate the underlying mechanisms, transcriptomic profiling showed significant enrichment of neurodegeneration pathways, with elevated Tau and GSDMD expression in hippocampal and cortical regions as well as N2a cells. Additionally, neuronal damage was exacerbated by indirect neurotoxicity mediated by microglial M1 polarization, accompanied by increased levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β). In conclusion, our findings uncover for the first time a dual-pathogenic mechanism by which T. marneffei induces CNS injury: direct neuronal damage via Tau pathology and GSDMD-dependent pyroptosis and indirect inflammation-mediated injury via M1-polarized microglia. These insights not only advance our understanding of fungal neuroinvasion but also provide a foundation for the development of targeted therapies.

Keywords: Talaromyces marneffei; central nervous system; microglia inflammation; neurodegeneration; pyroptosis.

Figure 1.

Effects of T. marneffei infection on learning, memory and motor balance in mice. A:T. marneffei infection prolongs escape latency in mice (left). Representative movement trajectories of two groups of mice in space exploration experiment (right). B: T. marneffei infection reduces the number of times mice swim across the escape platform. C: Effect of T. marneffei infection on motor balance in mice was compared based on the duration of time on the rod, distance rotated, and speed of rotation during falling. *P < 0.05, **P < 0.01.

Figure 2.

Hematoxylin-Eosin (H&E) staining and fungal culture analyses of T. marneffei-infected mouse brain tissues demonstrated fungal invasion and viability. A:H&E staining results of hippocampus CA1 and cerebral cortex in control group and T. marneffei-infected group. Control group (top-left): Hippocampal CA1 region exhibits well-organized neuronal architecture with intact structure and no observable neuronal pyknosis (arrowheads). T. marneffei-infected group (top-right): Notable reduction in pyramidal neurons, scattered neuronal pyknosis (arrowheads), and neurofilament entanglement in the CA1 region. Control group (bottom-left): The cortical region demonstrates well-organized neuronal alignment with intact cell membranes and homogeneous staining (arrowheads). T. marneffei-infected group (bottom-right): cortical neurons retain structural organization but exhibit increased glial cells with hyperchromatic nuclei (arrowheads), indicative of reactive gliosis. B: Fungal culture results of mouse brain tissue. No colony growth was observed in the PDA culture medium of the hippocampus (top-left) and cerebral cortex (bottom-left) of control group mice. Rose-red villous colonies were cultured from the hippocampus (top-middle) and cerebral cortex (bottom-middle) of T. marneffei-infected mice at 25°C. Gray-white cheese-like colonies were cultured from the cerebral cortex (top-right) and hippocampus (bottom-right) of T. marneffei-infected mice at 37 °C. C: LPCB smear of T. marneffei-infected mice brain tissue culture colonies at 25 °C (left) and 37 °C (right).

Figure 3.

T. marneffei causes N2a neuronal injury through direct invasion and indirect microglial-mediated mechanisms. A:The N2a cells co-cultured with GFP-labeled T. marneffei were observed using a fluorescence microscope. B: The viability of N2a cells was assessed using the CCK8 kit to detect changes after co-culturing with T. marneffei. C: Flow cytometry to detect the rise in the proportion of apoptosis after co-culturing with T. marneffei. D: JC-1 kit was used to detect mitochondrial membrane potential of N2a cells decreased after co-culturing with T. marneffei. E: Flow cytometry to detect the rise in the proportion of apoptosis after infection with T. marneffei. F: CCK8 kit was detect the cells viability of N2a, which co-cultured with T. marneffei -infected BV2 cells. *P < 0.05, ***P < 0.001.

Figure 4.

KEGG pathway analysis reveals upregulation of pathways of neurodegeneration in T. marneffei-infected mice cerebral cortex. A:Volcano plot showing differentially expressed genes (DEGs). DEGs were defined as genes with |fold-change| ≥ 1.5 and FDR < 0.05 (red: 435 up-regulated; blue: 409 down-regulated). Each biological replicate consists of 2 samples (n = 2). B: DEGs clustering heatmap, each row represents a gene, red represents a significantly up-regulated gene, blue represents a significantly down-regulated gene. C: KEGG enrichment analysis, the size of the dot represents the number of genes enriched in the pathway, the color of the dot represents the significance of the difference of the pathway.

Figure 5.

T. marneffei infection induces Tau pathology and pyroptosis in mice hippocampus and cortex. A:The protein expression level of pyroptosis and neurodegenerative lesion critical proteins from the hippocampus of mice infected with T. marneffei by western blot. B: the protein expression level of pyroptosis and neurodegenerative lesion critical proteins from the cortex of mice infected with T. marneffei by western blot. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

T. marneffei infection induces Tau pathology, pyroptosis and apoptosis in N2a cells. A-B: The protein expression levels of critical proteins involved in pyroptosis and neurodegenerative lesions were examined in N2a cells co-cultured with T. marneffei for 48 hours using western blot analysis. *P < 0.05, **P < 0.01.

Figure 7.

T. marneffei infection increases the levels of inflammatory factors, leading to M1 polarization of microglia. A:Immune infiltration analysis, the violin plot illustrates the distribution of cell type marker genes in each cluster using density curves, with the height of each plot representing the frequency of cells exhibiting relevant gene expression levels. B: qPCR detect the effect of T. marneffei invasion on the release of TNF-α, IL-1β, and IL-6 in mice cerebral cortex. C: Fluorescence microscope was used to observe BV2 cells invaded by T. marneffei stain with GFP fluorescent label. D-E: ELISA and qPCR detect the effect of T. marneffei invasion on the release of TNF-α, IL-1β, and IL-6 in BV2 cells. *P < 0.05, **P < 0.01, ns: No significant differences.