Effects and mechanisms of simulated microgravity on fusobacterium nucleatum and multiomic analysis

With the continuous advancement of deep-space exploration missions, the potential threat of microorganisms in the closed environment of space stations to astronauts' health has attracted increasing attention. Fusobacterium nucleatum, as an opportunistic pathogen colonizing the oral cavity and mucosal sites, is closely associated with various diseases including periodontal disease and colorectal cancer. Under extreme space conditions such as microgravity and radiation, the growth, virulence, drug resistance, and metabolic characteristics of microorganisms may undergo significant alterations, thereby increasing the risk of infection for astronauts. However, current research on the effects of microgravity on anaerobic bacteria remains extremely limited, and the phenotypic changes and molecular regulatory mechanisms of F. nucleatum under simulated microgravity conditions have not yet been elucidated, making it difficult to provide a scientific basis for the prevention and control of anaerobic bacterial contamination and infection risk assessment in space stations.

In a recent study published in Space: Science & Technology, researchers from the Air Force Medical Center systematically revealed the effects of simulated microgravity on Fusobacterium nucleatum and its molecular mechanisms through multi-omics integrative analysis. The study cultured F. nucleatum in a clinostat at 25 rpm and 37°C under anaerobic conditions for 3 days to simulate microgravity, with static culture and horizontal shaking culture groups serving as controls. Phenotypic experiments including scanning electron microscopy, growth curve determination, carbon source utilization and drug susceptibility analysis, and biofilm quantification were comprehensively conducted, integrated with transcriptome sequencing, TMT-labeled proteomics, and untargeted metabolomics to construct a transcriptome–proteome–metabolome multi-omics correlation network. The results demonstrated that under simulated microgravity, the growth rate of F. nucleatum was significantly accelerated, biofilm thickness increased from approximately 22 μm in the control group to 44 μm, and the capacity for utilizing various carbon sources was enhanced. The expression levels of molecular chaperone proteins (ClpB, GroL) and energy metabolism-related proteins were upregulated, and the differential metabolites were mainly enriched in ABC transporters, D-amino acid metabolism, and arginine biosynthesis pathways. The multi-omics network revealed that trehalose exhibited strong positive correlations with ClpB and GroL, while diaminopimelic acid showed negative correlations with metabolic regulators, indicating that the strain underwent metabolic reprogramming under microgravity. This study is the first to systematically reveal the multi-level regulatory mechanisms of simulated microgravity on the anaerobic bacterium Fusobacterium nucleatum, providing important scientific evidence for the prevention and control of anaerobic bacterial contamination and infection risk assessment in space stations.

First, this study focuses on the phenotypic effects of simulated microgravity on Fusobacterium nucleatum and, through rigorous experimental design, reveals significant alterations in the growth, carbon source utilization, and biofilm formation of this bacterium. Fusobacterium nucleatum is an opportunistic pathogen colonizing the oral cavity and mucosal sites, closely associated with various oral and systemic diseases; however, research on its behavior under microgravity conditions has remained unexplored. As shown in Fig. 1, the study divided F. nucleatum into three groups: the original strain (FNO, static culture), the anaerobic normal gravity group (FNN, horizontal shaking culture), and the anaerobic simulated microgravity group (FNS, clinostat rotation at a constant speed of 25 rpm), all cultured at 37°C under anaerobic conditions for 3 days. Scanning electron microscopy revealed that the surface of FNS cells exhibited extensive accumulation of flocculent material, whereas the surfaces of FNO and FNN remained smooth. Growth curve measurements demonstrated that the proliferation rate of FNS was significantly higher than that of FNN and FNO between 30 and 72 hours, with no significant difference observed between FNN and FNO. Carbon source utilization experiments showed that FNS exhibited enhanced capacity to utilize carbon sources such as 3-methylglucose, fucose, rhamnose, and sorbitol, along with increased sensitivity to fusidic acid and tetrazolium salts, and decreased sensitivity to sodium butyrate. More importantly, biofilm formation assays (Figs. 2 and 3) indicated that the biofilm thickness of FNS reached 44.16 μm, significantly higher than that of FNN (22.50 μm) and FNO (21.96 μm), with viable bacteria predominating in the biofilm. These phenotypic changes suggest that simulated microgravity significantly enhances the survival and adaptive capacity of Fusobacterium nucleatum.

Second, through transcriptomic and proteomic analyses, this study systematically reveals the molecular regulatory network underlying the phenotypic changes of Fusobacterium nucleatum driven by simulated microgravity. Transcriptome sequencing identified 109 differentially expressed genes between FNS and FNN, of which 32 were upregulated and 77 downregulated. As shown in Fig. 4, genes associated with protein folding and molecular chaperone functions—including clpB, groL, and grpE—were significantly upregulated in FNS, and energy metabolism-related genes such as pflB and nhaC also exhibited upregulated trends. Clusters of Orthologous Groups (COG) functional classification showed that the differentially expressed genes were mainly enriched in categories such as energy production and conversion, amino acid transport and metabolism, and nucleotide transport and metabolism. Gene Ontology (GO) enrichment analysis further indicated that upregulated genes were primarily involved in biological processes including protein folding, protein refolding, and ATP-dependent protein folding. Proteomic analysis detected 337 differentially expressed proteins (158 upregulated and 179 downregulated), which were mainly involved in catalytic activity, binding functions, and transporter activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the differentially expressed proteins were significantly enriched in pathways such as carbon metabolism, amino acid biosynthesis, glycolysis/gluconeogenesis, butyrate metabolism, and ABC transporters. Integration of transcriptomic and proteomic data revealed that 20 genes exhibited consistent differential expression trends across both omics levels. Among these, proteins related to ATP production, nucleotide metabolism, and carbohydrate metabolism were significantly upregulated in FNS, providing a molecular basis for accelerated growth; meanwhile, the upregulation of genes involved in carbon metabolism and amino acid biosynthesis—such as groL, galE, and pflB—provided potential support for enhanced biofilm formation.

Finally, through metabolomics and multi-omics integrative network analysis, this study further reveals the metabolic reprogramming characteristics and synergistic regulatory mechanisms of Fusobacterium nucleatum under simulated microgravity. Untargeted metabolomics was employed to detect metabolite differences in both cell pellets and culture supernatants. Partial least squares discriminant analysis (PLS-DA) showed that the metabolic profiles of FNS and FNN were clearly distinguishable. In the supernatants, 463 differential metabolite ions were identified (208 upregulated and 255 downregulated), while in the pellets, 483 differential metabolite ions were identified (82 upregulated and 401 downregulated). KEGG pathway enrichment analysis indicated that differential metabolites in the supernatants were mainly enriched in pathways such as tryptophan metabolism, D-amino acid metabolism, arginine biosynthesis, and ABC transporters; those in the pellets were mainly enriched in arginine and proline metabolism, D-amino acid metabolism, folate biosynthesis, and ABC transporters. The commonly enriched pathways across both compartments were ABC transporters, D-amino acid metabolism, and arginine biosynthesis, suggesting that these pathways serve as core metabolic hubs in the response to simulated microgravity. As shown in Fig. 5, the multi-omics integrative network revealed complex regulatory relationships among genes, proteins, and metabolites: trehalose—a known osmoprotectant and stress-responsive metabolite—exhibited strong positive correlations with the molecular chaperone proteins ClpB and GroL; inosine and ornithine showed positive correlations with transport- and amino acid metabolism-related proteins; and the peptidoglycan precursor diaminopimelic acid exhibited negative correlations with nifJ (a pyruvate metabolism gene) and porin family proteins, suggesting that the strain may undergo suppression of cell wall synthesis pathways or adaptive reprogramming of metabolic flux under microgravity. This multi-omics network reveals the complex molecular mechanisms by which Fusobacterium nucleatum achieves adaptive survival under simulated microgravity through the coordinated regulation of multi-level pathways including stress responses, substance transport, and energy metabolism, providing important scientific evidence for the prevention and control of anaerobic bacterial contamination and infection risk assessment in space stations.