Functional prediction of proteins from the human gut archaeome

Abstract

The human gastrointestinal tract contains diverse microbial communities, including archaea. Among them, Methanobrevibacter smithii represents a highly active and clinically relevant methanogenic archaeon, being involved in gastrointestinal disorders, such as inflammatory bowel disease and obesity. Herein, we present an integrated approach using sequence and structure information to improve the annotation of M. smithii proteins using advanced protein structure prediction and annotation tools, such as AlphaFold2, trRosetta, ProFunc, and DeepFri. Of an initial set of 873 481 archaeal proteins, we found 707 754 proteins exclusively present in the human gut. Having analysed archaeal proteins together with 87 282 994 bacterial proteins, we identified unique archaeal proteins and archaeal-bacterial homologs. We then predicted and characterized functional domains and structures of 73 unique and homologous archaeal protein clusters linked the human gut and M. smithii. We refined annotations based on the predicted structures, extending existing sequence similarity-based annotations. We identified gut-specific archaeal proteins that may be involved in defense mechanisms, virulence, adhesion, and the degradation of toxic substances. Interestingly, we identified potential glycosyltransferases that could be associated with N-linked and O-glycosylation. Additionally, we found preliminary evidence for interdomain horizontal gene transfer between Clostridia species and M. smithii, which includes sporulation Stage V proteins AE and AD. Our study broadens the understanding of archaeal biology, particularly M. smithii, and highlights the importance of considering both sequence and structure for the prediction of protein function.

Keywords: archaea; gut microbiome; methanogens; protein structure.

Figure 1

(A) Flowchart demonstrating major steps of the analysis; the Venn diagram demonstrates the number of shared KOs assigned to archaeal and bacterial sourmash clusters; (B) funnels illustrating the protein count at each stage of protein selection; MM2, MMseqs2 clusters; SCs, sourmash clusters.

Figure 2

Relative metagenomic occurrence and average metatranscriptomic read coverage of proteins in the (A) unique and (B) homologous groups of clusters with archaeal proteins; MG, metagenomics; MT, metatranscriptomics.

Figure 3

Schematic proposal highlighting proteins specific to gut-associated archaea with described functions: u1, Type II restriction endonuclease BglII; u2, intimin/invasin-like protein with a Ig-like domain; u3, intimin/invasin-like protein; u4, Unr protein; u22, Type I restriction–modification EcoKI enzyme, specificity subunit; u24, polypeptide N-acetylgalactosaminyltransferase; h1, 4-amino-4-deoxy-l-arabinose transferase or related glycosyltransferases of PMT family; h2,3,4,6, dolichyl-phosphate-mannose–protein mannosyltransferase 1; h5, dolichyl-diphosphooligosaccharide–protein glycosyltransferase subunit STT3B; h7, Propanediol utilization protein pduA; h11, phosphoenolpyruvate-dependent PTS system, IIA component; h28, transthyretin-like protein; h31, 2-AEP aminotransferase.

Figure 4

Gene synteny for sporulation stage V genes AE and AD from their respective sourmash clusters (A) h9 and (B) h20; gene expression of target genes (spoVAE and spoVAD) as well as genes from flanking regions are demonstrated below each sequence and are colored correspondingly. Genes with key archaeal functions: (A) pyrimidine metabolism (K18678, phytol kinase), methane metabolism (K11781, 5-amino-6-(d-ribitylamino)uracil–l-tyrosine 4-hydroxyphenyl transferase), and thiamine metabolism (K00878, hydroxyethylthiazole kinase; K00788, thiamine-phosphate pyrophosphorylase); (B) pyrimidine metabolism (K22026, nucleoside kinase; K18678, phytol kinase) and methane metabolism (K11781, 5-amino-6-(d-ribitylamino)uracil–l-tyrosine 4-hydroxyphenyl transferase).

Figure 5

Genomic context of the archaeal flanking regions up- and downstream of the (A) spoVAE and (B) spoVAD gene clusters in the M. smithii strain DSM 861.