Genetic Determinants of Hydrogen Sulfide Biosynthesis in Fusobacterium nucleatum Are Required for Bacterial Fitness, Antibiotic Sensitivity, and Virulence

The Gram-negative anaerobe Fusobacterium nucleatum is a major producer of hydrogen sulfide (H(2)S), a volatile sulfur compound that causes halitosis. Here, we dissected the genetic determinants of H(2)S production and its role in bacterial fitness and virulence in this important member of the oral microbiome. F. nucleatum possesses four enzymes, CysK1, CysK2, Hly, and MegL, that presumably metabolize l-cysteine to H(2)S, and CysK1 was previously shown to account for most H(2)S production in vitro, based on correlations of enzymatic activities with gene expression at mid-log phase. Our molecular studies showed that cysK1 and megL were highly expressed at the late exponential growth phase, concomitant with high-level H(2)S production, while the expression levels of the other genes remained substantially lower during all growth phases. Although the genetic deletion of cysK1 without supplementation with a CysK1-catalyzed product, lanthionine, caused cell death, the conditional ΔcysK1 mutant and a mutant lacking hly were highly proficient in H(2)S production. In contrast, a mutant devoid of megL showed drastically reduced H(2)S production, and a cysK2 mutant showed only minor deficiencies. Intriguingly, the exposure of these mutants to various antibiotics revealed that only the megL mutant displayed altered susceptibility compared to the parental strain: partial sensitivity to nalidixic acid and resistance to kanamycin. Most significantly, the megL mutant was attenuated in virulence in a mouse model of preterm birth, with considerable defects in the spread to amniotic fluid and the colonization of the placenta and fetus. Evidently, the l-methionine γ-lyase MegL is a major H(2)S-producing enzyme in fusobacterial cells that significantly contributes to fusobacterial virulence and antibiotic susceptibility.