Mycoplasma fermentans infection induces human necrotic neuronal cell death via IFITM3-mediated amyloid-β (1–42) deposition

Mycoplasma fermentans is a proposed risk factor of several neurological diseases that has been detected in necrotic brain lesions of acquired immunodeficiency syndrome patients, implying brain invasiveness. However, the pathogenic roles of M. fermentans in neuronal cells have not been investigated. In this study, we found that M. fermentans can infect and replicate in human neuronal cells, inducing necrotic cell death. Necrotic neuronal cell death was accompanied by intracellular amyloid-β (1–42) deposition, and targeted depletion of amyloid precursor protein by a short hairpin RNA (shRNA) abolished necrotic neuronal cell death. Differential gene expression analysis by RNA sequencing (RNA-seq) showed that interferon-induced transmembrane protein 3 (IFITM3) was dramatically upregulated by M. fermentans infection, and knockdown of IFITM3 abolished both amyloid-β (1–42) deposition and necrotic cell death. A toll-like receptor 4 antagonist inhibited M. fermentans infection-mediated IFITM3 upregulation. M. fermentans infection also induced necrotic neuronal cell death in the brain organoid. Thus, neuronal cell infection by M. fermentans directly induces necrotic cell death through IFITM3-mediated amyloid-β deposition. Our results suggest that M. fermentans is involved in neurological disease development and progression through necrotic neuronal cell death.


Necrotic neuronal cell death caused by M. fermentans is mediated by intracellular Aβ 1−42 deposition.
Infectious neuro-pathogens can drive amyloidosis and thereby play a protective role in innate immunity in brain 25 . Therefore, we tested Aβ deposition caused by M. fermentans, and phosphorylated tau (p-tau) that is induced by Aβ deposition, in human neuronal cells 26 . To measure deposition of p-tau and Aβ 1−42 , we analyzed these proteins in differentiated SH-SY5Y cells at 7, 12, and 19 dpi using western blotting. The results showed that p-tau (Ser202, Thr205) was significantly increased at 19 dpi, but t-tau was not increased significantly ( Fig. 2a-c). We also checked the quality of p-tau bands in the western blot; p-tau bands were confirmed by treating with calf intestinal phosphatase (CIP; Fig. 2d). In addition, intracellular Aβ 1−42 levels were increased at 19 dpi (Fig. 2e, f). Increased intracellular Aβ 1−42 was also confirmed using flow cytometry analysis (Fig. 2g, h). Immunocytochemical analysis also showed that both intracellular Aβ 1−42 and p-tau were increased in differentiated SH-SY5Y cells at 19 dpi, but t-tau was not increased (Fig. 2i, j).
To validate necrotic cell death caused by Aβ 1-42 , we generated stable human amyloid precursor protein (hAPP) knockdown and control knockdown SH-SY5Y cell lines using short hairpin RNA (shRNA) lentivirus, and we observed a decrease in hAPP mRNA level in hAPP knockdown SH-SY5Y cells compared with control cells (Fig. 2k). Necrotic neuronal cell death was dramatically inhibited in hAPP knockdown cells at 19 dpi (Fig. 2l,  m). These results indicate that M. fermentans induced intracellular Aβ 1-42 deposition, and this caused necrotic cell death.

M. fermentans infection induces Aβ 1-42 accumulation and necrotic neuronal cell death in brain organoids.
To evaluate the potential pathogenic impact of M. fermentans in a more physiologically relevant system, we utilized human brain organoids instead of a mouse infection model, since previous results showed that mouse neuronal cells are not susceptible to M. fermentans-induced necrotic cell death. We added M. fer-Scientific Reports | (2023) 13:6864 | https://doi.org/10.1038/s41598-023-34105-y www.nature.com/scientificreports/ mentans to human brain organoids at 40 days (Fig. 3a). At 26 dpi, infected brain organoids were decreased in size compared with controls (Fig. 3b, c). To confirm the infection and replication of M. fermentans in brain organoids, intracellular and secreted M. fermentans DNA levels were measured by qPCR. We detected intracellular and secreted M. fermentans DNA only in M. fermentans-infected brain organoids at 26 dpi (Supplementary Fig. S3a-d). We conducted immunohistochemical analysis to confirm necrotic cell death and Aβ 1-42 deposition in brain organoids at 26 dpi. After 67 days brain organoids exhibited a ventricular-like structure containing packed SOX2-positive neuronal progenitors, with a beta-tubulin III (TUJ1)-positive neuronal layer at the outer border ( Supplementary Fig. S3e) 27 . Additionally, the infected brain organoids contained more Aβ 1-42 in the disrupted TUJ1 and region around the outer border of brain organoids (Fig. 3d), indicating that M. fermentans can induce necrotic cell death through Aβ 1-42 deposition in mature neurons of brain organoids. We also confirmed increased p-tau and phosphorylated mixed lineage kinase domain-like (pMLKL) protein in infected brain organoids (Fig. 3e). Collectively, these results suggest that M. fermentans induces necrotic neuronal cell death by inducing Aβ 1-42 deposition in brain organoids as well as human neuronal cell lines. Our results also suggest that

Differential gene expression induced by M. fermentans infection.
We sought to identify the pathways involved in intracellular Aβ 1-42 deposition and necrotic cell death in M. fermentans-infected differentiated SH-SY5Y cells using RNA sequencing (RNA-seq) analysis. To identify genes implicated in necrotic cell death resulting from M. fermentans infection, we compared gene expression profiles between M. fermentans-infected samples collected at 19 dpi, when necrotic cell death was observed, and mock and 1 dpi samples, which did not display necrotic cell death. Analysis of 1dpi samples was conducted to distinguish genes associated with M. fermentans infection from those not related to necrotic cell death. Therefore, overlapping regions in the Venn diagram indicate genes that are specifically upregulated or downregulated in response to M. fermentans infection at 19 dpi and associated with necrotic cell death (Fig. 4a). We identified 1,144 upregulated genes and 543 downregulated genes after induction of necrotic cell death by M. fermentans infection (Fig. 4a), as well as the top five biological process categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with either upregulated or downregulated genes (Fig. 4b). The top enriched pathways linked to downregulated genes included cell cycle, cell division, and DNA replication (Fig. 4b), while the top enriched pathways associated with upregulated genes included signal transduction, inflammatory response, immune response, cytokine-cytokine receptor interaction, and tumor necrosis factor (TNF) signaling (Fig. 4b). Notably, inflammatory responses and TNF signaling are central pathways of cognitive decline and Aβ 1-42 deposition 28 . Interestingly, we found that growth differentiation factor 15 (GDF15), a blood marker for myalgic encephalomyelitis (ME)/CFS 29 and mitochondria dysfunction 30 , was upregulated in M. fermentans-infected cells (Fig. 4c). We also identified upregulated genes known to be associated with AD pathogenesis, namely TNF-α, apolipoprotein E (APOE), prion protein (PRNP), IFITM3, and interleukin-1β (IL-1β) (Fig. 4c). These upregulated genes were confirmed by measuring mRNA levels in M. fermentans-infected SH-SY5Y cells by qPCR at 19 dpi (Fig. 4d). TNF and IL-1β are key proinflammatory cytokines in neuroinflammation and degenerative conditions 28,31 , and APOE interacts with Aβ and promotes the aggregation and pathology of Aβ 32 . PRNP encodes PrP, a receptor for internalization of oligomeric Aβ, to induce tau hyperphosphorylation and necrotic cell death 26,33 . IFITM3 in neurons and astrocytes www.nature.com/scientificreports/ binds to γ-secretase and upregulates its activity, thereby increasing the production of Aβ 1-42 34 . IFITM3 is known to play a role in restricting infection by some viruses and bacteria, such as SARS-CoV-2 and Mycobacterium tuberculosis 35,36 . To investigate the potential positioning of genes (Fig. 4d) within the signaling pathway involved in M. fermentans-induced Aβ 1-42 deposition, we performed experiments to assess the effect of hAPP knockdown on their expression. The results revealed that knockdown of hAPP inhibited hIL-1β and hPRNP expression in M. fermentans-infected SH-SY5Y cells, while hAPOE expression was upregulated ( Supplementary Fig. S4). These findings provide further insights into the potential roles of these genes in the signaling pathway, and suggest their potential as upstream or downstream components.
M. fermentans induces IFITM3-mediated Aβ 1−42 accumulation, resulting in necrotic neuronal cell death. To confirm the function of IFITM3 in Aβ 1-42 deposition, we generated stable IFITM3 knockdown SH-SY5Y cells using shRNA lentivirus and infected these with M. fermentans. To evaluate the impact of IFITM3 on mycoplasma infection, we conducted experiments to measure M. fermentans growth in cells with IFITM3 knockdown. The results revealed a significantly higher infection level in cells with IFITM3 knockdown than in control knockdown cells (Supplementary Fig. S5). Moreover, at 12 dpi, M. fermentans-infected IFITM3 knockdown cells showed lower necrotic cell death than the control cells, although M. fermentans levels were higher in IFITM3 knockdown cells (Fig. 5a, b). ELISA demonstrated that M. fermentans increased Aβ 1-42 secretion, and that this was reduced by IFITM3 knockdown (Fig. 5c). Furthermore, using western blotting, we confirmed that IFITM3 and intracellular Aβ 1-42 protein levels were increased by M. fermentans infection, and that the levels of intracellular Aβ 1-42 proteins were reduced in IFITM3 knockdown cells (Fig. 5d).
A previous study showed that TLR2 and TLR4 ligands, and Mycobacterium tuberculosis infection, trigger IFITM3 to restrict mycobacterial growth 35 . RNA-seq data showed that TLR2 and TLR4 gene expression levels were increased in SH-SY5Y cells by M. fermentans at 19 dpi (Fig. 4c). Because proinflammatory cytokines can be associated with IFITM3 expression 35 , we explored the roles of IL-1β and TNF-α in IFITM3 upregulation during M. fermentans infection. However, neutralization of these cytokines did not affect IFITM3 upregulation, while the TLR4 antagonist inhibited necrotic cell death (Fig. 5e, f) and IFITM3 upregulation (Fig. 5g). A TLR2 antagonist had no inhibitory effect on necrotic neuronal cell death caused by M. fermentans infection (Fig. 5h, i). Therefore, we further investigated the effect of M. fermentans infection and found a significant upregulation of hTLR4 mRNA levels (Fig. 5j). Interestingly, we did not observe any significant change in the hAPP mRNA expression level, as confirmed by both RNA-seq (Fig. 4c) and qPCR (Fig. 5k). These results suggest that the observed increase in Aβ 1-42 production was not due to substrate upregulation, but rather to increased γ-secretase activity. Thus, M. fermentans may induce necrotic cell death via TLR4 signaling to upregulate IFITM3 for Aβ 1-42 deposition. In contrast to human neuronal cells, murine IFITM3 gene expression was not upregulated by M. fermentans (Supplementary Fig. S6a). This could be due to sequence differences in the ligand binding sites (560 of 843 amino acids, 66%) between murine and human TLR4 (Supplementary Fig. S6b). These results suggest that M. fermentans infection in human neuronal cells induces TLR4 signaling to increase Aβ 1−42 accumulation via an IFITM3-mediated pathway, resulting in necrotic cell death. To determine whether the observed effects are specific to M. fermentans infection or are a general response to TLR4-activating ligands, we performed additional experiments. We added CRX-527, a CD14-independent TLR4 agonist 37 , and α-enolase, a protein expressed on the cell surface of M. fermentans that can activate the CD14-dependent TLR4 signaling pathway [38][39][40] , to SH-SY5Y cells. The results showed that neither CRX-527 nor α-enolase alone induced cell death, upregulated hTLR4, hIL-6, hTNF-α, hIL-1β, or hIFITM3 mRNA levels, or increased intracellular Aβ 1-42 in SH-SY5Y cells ( Supplementary  Fig. S7a-e). These results are in line with those of previous research showing no impact of lipopolysaccharide (LPS) on SH-SY5Y cells [41][42][43][44][45] . Thus, the consequences of M. fermentans infection, such as Aβ 1-42 deposition and cell death, would appear to be specifically linked to TLR4 upregulation. We also found no increase in cell death of SH-SY5Y cells after adding the α-enolase to M. fermentans-infected cells ( Supplementary Fig. S7f) while an TLR4 antagonist blocked IFITM3 upregulation and necrotic cell death. These findings suggest that M. fermentans is sufficient for the activation of TLR-4 because α-enolase did not increase necrotic cell death.

Discussion
M. fermentans infection is associated with various neurological diseases such as CFS, GWS, ALS, and ASD [5][6][7][20][21][22][23][24] . This suggests that M. fermentans infection could have pathological relevance for neurological changes associated with shared symptoms in CFS, and GWS, ALS, and ASD, such as cognitive and neurological problems 46 . Indeed, mycoplasmas have been isolated from the brains of various types of animals, suggesting they can directly invade the CNS 8,9,11,15,16,[47][48][49] . Additionally, M. fermentans have been detected in numerous tissues including the brains of AIDS patients 9,10 , and was also detected in a case study of non-AIDS patients with acute fatal disease 13,14 . Thus, M. fermentans may be an underestimated pathogen in neurological diseases. We therefore investigated pathological changes in neuronal cells infected with M. fermentans. Surprisingly, M. fermentans could infect human neuronal cells, in which it replicated and induced necrotic cell death, but it could not induce necrotic cell death in mouse neuronal cells.
IFITM3, a member of the interferon-induced transmembrane protein family, is a restriction factor that prevents enveloped viral particles from entering host cells by blocking membrane fusion during endocytosis 50,51 . It also blocks mycobacterial growth 35 , plays a critical role in intrinsic antiviral immunity of human cells, and restricts cell entry of diverse viruses including influenza virus, West Nile virus, dengue virus, Ebola virus, and coronavirus 52 . IFITMs are potent antiviral effectors known for their ability to inhibit fusion between viral and cellular membranes, and they are widely studied for their potential as antiviral therapies 51,52 . Interestingly, a study revealed that IFITM3 in neurons can bind to γ-secretase to upregulate its activity, thereby increasing the production of Aβ 1-42 34 . In our current study, we also found that M. fermentans could upregulate IFITM3,    [38][39][40] . However, we observed that neither α-enolase nor the TLR4 agonist CRX-527 alone induced cell death or activated the TLR4 signaling pathway in SH-SY5Y cells. This finding is consistent with previous studies showing that direct application of LPS, a TLR4 agonist, has no effect on SH-SY5Y cell death [43][44][45] . Unlike other cells, SH-SY5Y cells do not exhibit cytokine transcription or release in response to LPS, possibly due to their low levels of TLR4 expression 41,42 . Thus, our results suggest that M. fermentans infection-mediated additional processes are required for necrotic cell death.
In addition, addition of α-enolase did not enhance the cell death effect beyond that observed with M. fermentans infection alone, whereas the TLR4 antagonist blocked cell death. This also suggests that M. fermentans infection is sufficient for TLR4 activation and induction of cell death.
In our experiments, we tested two mouse neuronal cell lines, neuro2a and HT-22, but neither of these cell lines exhibited IFITM3 upregulation or necrotic cell death after M. fermentans infection. However, we observed necrotic cell death of the human SH-SY5Y neuronal cell line. In addition, this effect by M. fermentans infection was observed in organoid cultures, minimizing the concern about extrapolating from a pure cell line approach. Thus, our results may indicate that M. fermentans-induced immunological responses to pathogen infection can differ depending on the species, as shown in a previous study 53 . Overall, our findings suggest that intracellular Aβ 1-42 deposition by TLR4-mediated IFITM3 upregulation is the main inducer of necrotic cell death.
The amphipathic helix of IFITM3 is essential for its antiviral activity by inducing membrane deformation in virus-infected cells 54 . However, a previous study revealed that IFITM3 also plays a modulatory role in γ-secretase activity by binding to PS1 near the active site, resulting in an increase in γ-secretase activity and Aβ production 34 . Thus, it is essential to consider the potential antiviral effects of any therapeutic targeting of IFITM3 since the determinants involved in its antiviral activity may also play a role in its interaction with γ-secretase and regulation of Aβ production. It is worth noting that oligomerization of IFITM3 is necessary for its antiviral activity through the induction of membrane stiffening 55 , whereas IFITM3 binds to PS1-NTF as a monomer or dimer 34 . This suggests that the antiviral mechanism of IFITM3 and its role in modulating γ-secretase activity may involve distinct determinants. Therefore, further investigations are necessary to fully comprehend the determinants of IFITM3 involved in this complex and their impact on γ-secretase activity, which could have significant implications for the development of targeted therapies that balance its antiviral and modulatory effects.
In our study, we observed a significant downregulation of IL-1β and PRNP gene expression in M. fermentansinfected SH-SY5Y cells following hAPP knockdown. This suggests that these genes may function downstream of the signaling pathway leading to Aβ 1-42 deposition in our experimental system, which is consistent with previous studies implicating IL-1β and PRNP in Aβ-induced neuroinflammation and neurotoxicity 26,28,31,33 . Interestingly, we also observed upregulation of APOE expression following hAPP knockdown in M. fermentans-infected SH-SY5Y cells. APOE has been implicated in AD pathology, where it plays a complex role in the clearance and aggregation of Aβ in an isoform-specific manner 32 . Therefore, this finding may reflect a compensatory mechanism that counteracts Aβ-induced neurotoxicity. However, the exact role of the Aβ pathway in human PRNP or APOE remains unclear. Therefore, it is important to note that the observed effects of hAPP knockdown on gene expression may be indirect, and further studies are needed to confirm their precise positioning within the signaling pathway leading to Aβ deposition. Nonetheless, our findings provide important insights into the potential roles of IL-1β, PRNP, and APOE in this process, and suggest new avenues for future research.
M. fermentans has been found in various tissues of AIDS patients, leading to its designation as an opportunistic pathogen 4 . Interestingly, several studies showed that M. fermentans is present in saliva in about half of the population (110 of 201, 54.7%; 49 of 110, 44%), suggesting that the organism colonizes the human mouth and is transmitted easily to others 56,57 . However, blood infection did not occur frequently in healthy individuals [5][6][7][20][21][22][23][24] . It is possible that the organism invades blood and various tissues including the brain in immunocompromised individuals, altering physiological conditions and causing M. fermentans-related neurological diseases. Thus, our results suggest the possibility that opportunistic CNS infection by M. fermentans may occur in immune-altered individuals, resulting in other types of neurological diseases.
In conclusion, in vitro and ex vivo analyses revealed that M. fermentans can function in a pathogenic capacity in human neuronal cells. Cellular and molecular analyses of M. fermentans pathogenicity revealed that M. fermentans directly induces necrotic neuronal cell death by IFITM3-mediataed Aβ 1-42 deposition. Thus, our results suggest that M. fermentans is involved in the pathogenicity of neurological diseases. Furthermore, our findings could help expand our understanding of neurological diseases caused by mycoplasmal infections.

Differentiation of SH-SY5Y cells and preparation of conditioned medium.
To induce differentiated SH-SY5Y cells, they were seeded into 3 ml of medium at a density of 2 × 10 5 cells per a well in a 6-well cell culture plate. After 24 h, cells were treated with 80 mM PMA (Sigma-Aldrich, Cat. No. P1585-1MG) for 7 days. Infection was performed at an MOI of 0.01, and medium was replaced with fresh medium after 24 h. At 7, 12, and 19 dpi, cells were used for analysis. At 19 dpi, the cell culture medium was replaced with fresh medium, and after 2 days of incubation, the conditioned medium was harvested and centrifuged at 900 × g at 4 °C for 30 min, and supernatants were passed through a 0.45 μm pore size membrane filter. The conditioned medium was then diluted 1:3 in fresh culture medium and used to treat fresh differentiated SH-SY5Y cells.  Statistical analysis. The significance of differences between two groups was analyzed using unpaired Student's t-tests using Origin2021 software (OriginLab Corporation, Northampton, MA, USA). Quantification of protein expression levels in western blotting was performed using ImageJ software, GAPDH served as an internal control for normalization, and Hoechst was used for normalization of immunocytochemical data. All results are expressed as the mean ± standard deviation (SD). For all statistical tests, p-values ≤ 0.05 were considered significant.

Data availability
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author. All RNA-seq data generated in this study are available through the NCBI Sequence Read Archive (SRA) through accession numbers SRR22252649-SRR22252651 under BioProject PRJNA900032.