A Novel Heme-Degrading Enzyme that Regulates Heme and Iron Homeostasis and Promotes Virulence in Enterococcus faecalis

Abstract

Enterococcus faecalis, a gut commensal, is a leading cause of opportunistic infections. Its virulence is linked to its ability to thrive in hostile environments, which includes host-imposed metal starvation. We recently showed that E. faecalis evades iron starvation using five dedicated transporters that collectively scavenge iron from host tissues and other iron-deprived conditions. Interestingly, heme, the most abundant source of iron in the human body, supported growth of a strain lacking all five iron transporters ({Delta}5Fe). To release iron from heme, many bacterial pathogens utilize heme oxygenase enzymes to degrade the porphyrin that coordinates the iron ion of heme. Although E. faecalis lacks these enzymes, bioinformatics revealed a potential ortholog of the anaerobic heme-degrading enzyme anaerobilin synthase, found in Escherichia coli and a few other Gram-negative bacteria. Here, we demonstrated that deletion of OG1RF_RS05575 in E. faecalis ({Delta}RS05575) or in the {Delta}5Fe background ({Delta}5Fe{Delta}RS05575) led to intracellular heme accumulation and hypersensitivity under anaerobic conditions, suggesting RS05575 encodes an anaerobilin synthase, the first of its kind described in Gram- positive bacteria. Additionally, deletion of RS05575, either alone or in the {Delta}5Fe background, impaired E. faecalis colonization in the mouse gastrointestinal tract and virulence in mouse peritonitis and rabbit infective endocarditis models. These results reveal that RS05575 is responsible for anaerobic degradation of heme and identify this relatively new enzyme class as a novel factor in bacterial pathogenesis. Findings from this study are likely to have broad implications, as homologues of RS05575 are found in other Gram-positive facultative anaerobes. IMPORTANCEHeme is an important nutrient for bacterial pathogens, mainly for its ability to serve as an iron source during infection. While bacteria are known to release iron from heme using enzymes called heme oxygenases, a new family of anaerobic heme-degrading enzymes has been described recently in Gram-negative bacteria. Here, we report the first description of anaerobic heme degradation by a Gram-positive bacterium, the opportunistic pathogen Enterococcus faecalis, and link activity of this enzyme to their ability to colonize and infect the host. We also show that homologues of this enzyme are found in many Gram-positive facultative anaerobes, implying that the ability to degrade heme under anaerobic conditions may be an overlooked fitness and virulence factor of bacterial pathogens.