Integrative multi-omics analysis of autism spectrum disorder reveals unique microbial macromolecules interactions

• Reduced Microbial Diversity: Children with ASD displayed significantly less microbial diversity in the gut microbiota compared to the control group. • Microbial Community Shuffling: We observed a characteristic community shuffling pattern, highlighting potential microbial crosstalk in ASD. • Perturbed Macromolecules: Identified macromolecules (metaproteins/metabolites) and associated pathways may influence brain function and neurological traits related to ASD behavior. • Host Response Proteome: The host proteome response in ASD primarily involves proteins related to nervous system development and immune response. • Comprehensive Multiomics Integration: The integration of genomics, metaproteomics, and metabolomics provided a comprehensive portfolio of the gut microbiota, revealing potential macromolecule production linked to ASD. Gut microbiota alterations have been implicated in Autism Spectrum Disorder (ASD), yet the mechanisms linking these changes to ASD pathophysiology remain unclear. This study utilized a multi-omics approach to uncover mechanisms linking gut microbiota to ASD by examining microbial diversity, bacterial metaproteins, associated metabolic pathways and host proteome. The gut microbiota of 30 children with severe ASD and 30 healthy controls was analyzed. Microbial diversity was assessed using 16S rRNA V3 and V4 sequencing. A novel metaproteomics pipeline identified bacterial proteins, while untargeted metabolomics explored altered metabolic pathways. Finally, multi-omics integration was employed to connect macromolecular changes to neurodevelopmental deficits. Children with ASD exhibited significant alterations in gut microbiota, including lower diversity and richness compared to controls. Tyzzerella was uniquely associated with the ASD group. Microbial network analysis revealed rewiring and reduced stability in ASD. Major metaproteins identified were produced by Bifidobacterium and Klebsiella (e.g., xylose isomerase and NADH peroxidase). Metabolomics profiling identified neurotransmitters (e.g., glutamate, DOPAC), lipids, and amino acids capable of crossing the blood–brain barrier, potentially contributing to neurodevelopmental and immune dysregulation. Host proteome analysis revealed altered proteins, including kallikrein (KLK1) and transthyretin (TTR), involved in neuroinflammation and immune regulation. Finally, multi-omics integration supported single-omics findings and reinforced the hypothesis that gut microbiota and their macromolecular products may contribute to ASD-associated symptoms. The integration of multi-omics data provided critical evidence that alteration in gut microbiota and associated macromolecule production may play a role in ASD-related symptoms and co-morbidities. Key bacterial metaproteins and metabolites were identified as potential contributors to neurological and immune dysregulation in ASD, underscoring possible novel targets for therapeutic intervention.