Due to factors such as aging populations and environmental influences, PD has become one of the fastest-growing neurological disorders globally. Meanwhile, the role of the gut microbiota in contributing to the pathogenesis of various neurodegenerative diseases, including PD, is becoming increasingly recognized and underscored in recent scientific discourse20. The human gut is estimated to host a staggering quadrillion microbes, and the exploration of gut bacteria's role in health and disease has captured widespread attention. Some gut microbiome organisms like Bacteroides vulgatus, Parabacteroides distasonis, Lactobacillus salivarius and Clostridium bacteria may influence neuroinflammatory signaling and affect PD pathogenesis and brain function21, 22. In addition, several microbial metabolites impact mechanisms to explain the role of the microbiome in the etiology of PD have been proposed, including compromising gut and blood-brain barrier integrity, fostering inflammatory cascades, affecting endocrine signaling pathways, and influencing neuronal survival23. Yet, these factors do not comprehensively account for the etiology of PD, prompting researchers to persist in their quest for potential causes.
While the gut microbiota is intricately linked to PD and could be among its primary triggers, the role of enteroviruses is frequently underestimated. Recent research has demonstrated that viruses can exert a substantial influence on the physiology of their bacterial hosts, consequently altering the structure of bacterial communities. Thus, we conducted a targeted study on the virus group in PD. In our research, we examined the alterations in the gut virome across two cohorts, totaling 158 samples. Cohort 1 included 39 couples from Hubei in central China, and Cohort 2 consisted of 40 couples from Shanghai in eastern China. Our study enhances the previous metagenomic datasets based on the gut virome of PD by adding more information. Metagenome based virome sequencing improves the observed diversity of viruses, as it can capture more information about previously overlooked viruses. Compared with the HSs, the two groups PD patients in our study exhibited a significant increase in viral richness (number of observed vOTUs) and Shannon index. Delving into the diversity of viral families within PD patients sheds light on their potential roles in modulating the disease's pathogenesis. Specifically, comparison of the family-level composition revealed that the virome PD patients had an increased abundance of Circoviridae, Myoviridae, Siphoviridae, Podoviridae, Herelleviridae, p-crAss-like and Salasmaviridae but that HSs only showed a higher abundance of Quimbyviridae. Currently, the Caudovirales order is classified into nine distinct families, with four of them (Myoviridae, Podoviridae, Siphoviridae, Herelleviridae) being particularly widespread and polyphyletic. These families are recognized as both prevalent and among the most abundant phage groups in the human gut virome24, 25. Meanwhile, Mayneris et al. revealed that the lactic acid bacteria such as Streptococcus, Lactobacillus, Lactococcus, and Enterococcus from the Lactobacillales order positively correlate with certain Caudovirales levels, there exists an inverse association with Bacteroides species, indicating the complex interactions between bacteriophages and bacteria in the gut may impact the progression of PD9, 26. The p-crAss-like phages have been reported to be enriched in the gut of individuals with Ankylosing Spondylitis (AS)27, yet targeted research remains scarce, and their function within the gut is still unclear. The Salasmaviridae family with over 200 diverse vOTUs spanning more than 20 viral subfamilies, infecting a wide variety of gut-associated Firmicutes and Actinobacteria28. However, the Firmicutes and Actinobacteria phyla, encompassing numerous bacteria that produce short-chain fatty acids (SCFAs), are pivotal for maintaining intestinal health and fostering immune regulation, which plays a significant protective role in the progression of PD29. Thus, the enrichment of these seven viruses in PD suggests that they may play an important role in its pathogenesis. The Quimbyviridae family, distinguished by its widespread presence and high variability, primarily targets Bacteroides and is believed to undergo an obligate lytic lifecycle. This viral family, newly discovered, is recognized for its prominent and pervasive occurrence in the human GI tract30.
We used the combined probability values from independent tests in two cohorts to obtain 640 vOTUs with significant differences between PD patients and HSs. Within the intersection of both cohorts, 479 vOTUs were found to be enriched in PD patients, while 96 vOTUs were found to be enriched HSs. The PD-enriched vOTUs presented a higher proportion of Siphoviridae viruses and Myoviridae; the HS-enriched vOTUs contained more viruses of Quimbyviridae.
Turning our attention to the viral hosts, we discovered that most PD-enriched vOTUs (428; 74.43%) could be assigned to at least one prokaryotic host. The dominant hosts of PD-enriched vOTUs included members of Alistipes, Lawsonibacter, Oscillibacter, Faecalibacterium, Ruthenibacterium, Intestinimonas, Flavonifractor, Fournierella, and multiple unknown genera from Oscillospiraceae and other taxa. By contrast, the HS-enriched vOTUs were Bacteroides and Prevotella species. Alistipes, a relatively newly identified genus of bacteria, plays a significant role in dysbiosis and disease, being pathogenic in colorectal cancer and associated with mental indicators of depression31. There are few articles on the relationship between Lawsonibacter and human diseases, though one species of Lawsonibacter was identified as a butyrate-producing bacterium32. The Oscillibacter was shown to be involved in the integrity of the mouse intestinal barrier33. The reduction of Faecalibacterium may compromise gut-barrier function, increasing the enteric nervous system's (ENS) vulnerability to enteric pathogen infections and elevating the risk of α-synuclein formation, with its abundance inversely associated with PD duration, suggesting a connection to the progression and neuropathology of PD34, 35. Flavonifractor plautii is a flavonoid-degrading bacterium that was reported to affect antigen-induced T helper 2 cell (Th2) immune responses in mice, and it is also increased in young-onset colorectal cancer patients36, 37. Despite the scarcity of literature on the link between Ruthenibacterium, Fournierella and Intestinimonas and human diseases, these species emerge as the most enriched taxa within the PD gut microbiome, which may exist potential significance in PD pathology38, 39. Analogously, reduced Prevotella levels in PD may initiate a compensatory response, leading to elevated intestinal permeability and subsequent inflammation40. These studies suggest the close association between the host bacteria of these PD-enriched vOTUs and PD.
In addition to exploring the composition of virome differences, we also focused on the functions of viral signatures associated with PD, included thymidylate synthase (K00560), ATP-dependent Clp protease proteolytic subunit (K01358) and integrase (K14059). For example, the thymidylate synthase (K00560) is an important enzyme involved in one-carbon metabolism that has been linked to neurodegeneration41. The ClpP serves as an essential component of mitochondrial Clp proteases that play a role in the prevention of premature aging42, 43. Although ClpP presence in viruses has not been confirmed, we noticed 18 PD-associated vOTUs with high-confidence ClpP (>80% protein similarity; Supplementary figure 1). Similarly, integrases (K14059), prevalent in viruses, notably retroviruses like HIV, and in certain mobile genetic elements, play a pivotal role in integrating viral DNA into the host genome, a crucial step for establishing persistent infections. This suggests an enhanced viral invasiveness in PD patients44. The marked disparities in these genes between PD patients and the healthy population may serve as a non-invasive biomarker for diagnosing PD onset, though further research is required.
PD, which directly impairs the central nervous system's function, necessitates early diagnosis as it may become incurable if identified at a later stage. Nowadays, numerous efforts to identify biomarkers for early recognition of PD have been undertaken, yet despite these attempts, the significant role of viruses in screening PD biomarkers has frequently been overlooked. Based on the identified gene markers16 or six different taxa (Eubacterium, Capnocytophaga, Phascolarctobacterium, Akkermansia, and mOTUs classified only at the Firmicutes or broader Bacterial level)45, the models generally achieved the performance of AUCs between 0.80 to 0.84. Here, with only 575 gut vOTUs, we achieved high precision (73.8%). By further refinement, the higher AUC (average of 85.4%) was obtained when a subset of the top 22 ranked vOTUs was used. Additionally, the model demonstrated strong predictive performance on independent samples, further reinforcing the reproducibility of the virome as a reliable biomarker for diagnosing PD evidenced by AUC scores of 78.2% and 72.8% across two cohorts. These findings represent an encouraging advancement, indicating the substantial diagnostic potential of the gut virome in distinguishing PD, underscores the need for further in-depth research.
The impact of our study is limited by the relatively small number of participants and the homogeneity of the sample. Future research should include larger, more diverse cohorts to validate and expand upon these results.
Drawing on cross-cohort metagenome shotgun sequencing data, we have pinpointed viral signatures that distinguish significantly between Parkinson's Disease (PD) patients and healthy subjects (HS). These distinctive viral markers hold promise as novel targets for PD intervention, offering fresh avenues for the development of preventative and therapeutic strategies against PD. Moreover, the high efficacy and repeatability of our predictive model (AUC > 0.854), despite utilizing only a limited set of viruses, underscores its considerable potential for clinical implementation.