Biodegradation of L-homocysteine by a newly isolated Niallia circulans YL01 from the gut microbiota of healthy humans

Technical Summary: Biodegradation of L-homocysteine by Niallia circulans YL01

Problem Statement
Hyperhomocysteinemia (HHcy), characterized by elevated serum L-homocysteine levels (>15 µmol/L), is a significant independent risk factor for cardiovascular and cerebrovascular diseases, including stroke and atherosclerosis. It is also linked to systemic disorders such as Alzheimer's disease, insulin resistance, and osteoporosis. Current management strategies rely on dietary interventions (reducing methionine intake) and pharmacological supplementation with B vitamins, folate, or betaine. However, these approaches often suffer from variable efficacy, potential side effects, and the risk of secondary deficiencies (e.g., vitamin B12 deficiency from prolonged protein avoidance). There is an urgent need for safer, more effective therapeutic strategies, particularly those leveraging the gut microbiota to modulate host metabolism. While some probiotic yeasts have shown L-homocysteine biodegrading capacity, there is a lack of diversity in bacterial strains capable of this function.

Methodology
The study employed a multi-faceted approach to isolate, characterize, and evaluate a novel bacterial strain:

• Isolation: A bacterial strain was isolated from the gut microbiota of healthy humans using a selective screening medium where L-homocysteine served as the sole carbon and energy source.
• Identification: The strain was identified via 16S rDNA gene sequencing and confirmed through whole-genome average nucleotide identity (ANI) analysis against reference strains, classifying it as Niallia circulans (designated strain YL01).
• Biodegradation Assays: The capacity of YL01 to degrade L-homocysteine was tested using both whole cells and cell-free extracts (CEs). High-Performance Liquid Chromatography (HPLC) with o-phthaldialdehyde (OPA) derivatization was used to quantify L-homocysteine concentrations.
• Genomic Analysis: Whole-genome sequencing (Illumina NovaSeq 6000) was performed to assemble the genome and annotate genes. Functional annotation utilized databases including COG, GO, KEGG, and CAZy. Specific attention was paid to genes encoding enzymes in the remethylation and transsulfuration pathways.
• Safety Evaluation: Preliminary safety was assessed through in silico analysis of virulence factors (VFDB) and antibiotic resistance genes (CARD), followed by in vitro assays for hemolytic activity, amino acid decarboxylase production, and gelatinase activity.

Key Results

• Strain Identification: The isolated strain, YL01, was confirmed as Niallia circulans with 100% ANI similarity to the type species. It is deposited in the CGMCC (Accession No. CGMCC 37025).
• Biodegradation Efficiency:
  • Whole Cells: YL01 cells removed 86% of an initial 5.5 g/L L-homocysteine concentration within 5 days.
  • Cell-Free Extracts (CEs): CEs containing 0.3 g/L protein removed 73% of an initial 2.0 g/L L-homocysteine concentration within 24 hours.
• Genomic Insights: The genome (4.27 Mbp, 35.5% GC) contains a complete metabolic pathway for L-homocysteine. Six key enzymes were identified, enabling biodegradation via two primary routes:
  1. Remethylation Pathway: Involving 5-methyltetrahydrofolate-homocysteine methyltransferase (metE), serine hydroxymethyltransferase (SHMT), methylenetetrahydrofolate reductase (metF), and S-adenosylmethionine synthetase (metK).
  2. Transsulfuration Pathway: Involving cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), converting L-homocysteine to L-cysteine.
• Safety Profile: In silico analysis indicated low pathogenic potential (only six genes with >75% similarity to virulence factors). Experimental assays confirmed the strain is non-hemolytic, non-gelatinolytic, and does not produce biogenic amines via amino acid decarboxylase activity.

Key Contributions

• Novel Strain Discovery: This study reports the first isolation of Niallia circulans YL01 from the human gut microbiota with demonstrated L-homocysteine biodegradation capabilities.
• Dual-Pathway Mechanism: Genomic analysis revealed that YL01 possesses a complete enzymatic toolkit for both the remethylation and transsulfuration pathways, suggesting a robust mechanism for L-homocysteine catabolism comparable to human metabolic systems.
• Safety Validation: The strain passed preliminary safety screens for hemolysis and toxin production, supporting its potential as a therapeutic candidate.
• Expansion of Microbial Diversity: The work addresses the limited diversity of known L-homocysteine-degrading bacteria, offering a new bacterial candidate distinct from previously studied probiotic yeasts.

Significance and Claims
The authors posit that Niallia circulans YL01 represents a promising candidate for a novel microbial therapeutic strategy to intervene in HHcy. The study claims that the strain's ability to efficiently degrade L-homocysteine via both whole cells and cell-free extracts, combined with its safety profile and complete metabolic pathway, provides a preliminary basis for its development as a functional food or therapeutic agent. The findings suggest that YL01 could offer a safer alternative or adjunct to current vitamin-based therapies. The authors note that while preliminary safety is established, further in vivo studies (acute oral toxicity and genotoxicity) are required to fully validate the strain's clinical potential.