Porphyrin Production and Regulation in Cutaneous Propionibacteria.

Porphyrins are intermediate metabolites in the biosynthesis of vital molecules, including heme, cobalamin, and chlorophyll. Bacterial porphyrins are known to be proinflammatory, with high levels linked to inflammatory skin diseases. Propionibacterium species are dominant skin commensals and play essential roles in defending against pathogens and in triggering an inflammatory response. To better understand how the inflammatory potential of the skin microbiome may vary depending on its propionibacterial composition, we compared the production levels of porphyrins among Propionibacterium acnes, Propionibacterium granulosum, Propionibacterium avidum, and Propionibacterium humerusii strains. We found that porphyrin production varied among these species, with P. acnes type I strains producing significantly larger amounts of porphyrins than P. acnes type II and III strains and other Propionibacterium species. P. acnes strains that are highly associated with the common skin condition acne vulgaris responded to vitamin B12 supplementation with significantly higher porphyrin production. In contrast, vitamin B12 supplementation had no effect on the porphyrin production of health-associated P. acnes strains and other propionibacteria. We observed low-level porphyrin production in most Propionibacterium strains harboring the deoR repressor gene, with the exception of P. acnes strains belonging to type I clades IB-3 and IC. Our findings shed light on the proinflammatory potential of distinct phylogenetic lineages of P. acnes as well as other resident skin propionibacteria. We demonstrate that the overall species and strain composition is important in determining the metabolic output of the skin microbiome in health and disease.IMPORTANCE Porphyrins are a group of metabolites essential to the biosynthesis of heme, cobalamin, and chlorophyll in living organisms. Bacterial porphyrins can be proinflammatory, with high levels linked to human inflammatory diseases, including the common skin condition acne vulgaris. Propionibacteria are among the most abundant skin bacteria. Variations in propionibacteria composition on the skin may lead to different porphyrin levels and inflammatory potentials. This study characterized porphyrin production in all lineages of Propionibacterium acnes, the most dominant skin Propionibacterium, and other resident skin propionibacteria, including P. granulosum, P. avidum, and P. humerusii We revealed that P. acnes type I strains produced significantly more porphyrins than did type II and III strains and other Propionibacterium species. The findings from this study shed light on the proinflammatory potential of the skin microbiome and can be used to guide the development of effective acne treatments by modulating the skin microbiome and its metabolic activities.

Strain-Level Differences in Porphyrin Production and Regulation in Propionibacterium acnes Elucidate Disease Associations.

Propionibacterium acnes is an important skin commensal, but it is also considered a pathogenic factor in several diseases including acne vulgaris, the most common skin disease. While previous studies have revealed P. acnes strain-level differences in health and disease associations, the underlying molecular mechanisms remain unknown. Recently, we demonstrated that vitamin B12 supplementation increases P. acnes production of porphyrins, a group of proinflammatory metabolites important in acne development (D. Kang, B. Shi, M. C. Erfe, N. Craft, and H. Li, Sci. Transl. Med. 7:293ra103, 2015, doi:10.1126/scitranslmed.aab2009). In this study, we compared the porphyrin production and regulation of multiple P. acnes strains. We revealed that acne-associated type IA-2 strains inherently produced significantly higher levels of porphyrins, which were further enhanced by vitamin B12 supplementation. On the other hand, health-associated type II strains produced low levels of porphyrins and did not respond to vitamin B12. Using a small-molecule substrate and inhibitor, we demonstrated that porphyrin biosynthesis was modulated at the metabolic level. We identified a repressor gene (deoR) of porphyrin biosynthesis that was carried in all health-associated type II strains, but not in acne-associated type IA-2 strains. The expression of deoR suggests additional regulation of porphyrin production at the transcriptional level in health-associated strains. Our findings provide one potential molecular mechanism for the different contributions of P. acnes strains to skin health and disease and support the role of vitamin B12 in acne pathogenesis. Our study emphasizes the importance of understanding the role of the commensal microbial community in health and disease at the strain level and suggests potential utility of health-associated P. acnes strains in acne treatment. IMPORTANCE Propionibacterium acnes is a dominant bacterium residing on skin, and it has been thought to play a causal role in several diseases including acne, a common skin disease affecting more than 80% of people worldwide. While specific strains of P. acnes have been associated with either disease or healthy skin, the mechanisms remain unclear. Recently, we showed that vitamin B12 supplementation increased porphyrin production in P. acnes, leading to acne development (D. Kang, B. Shi, M. C. Erfe, N. Craft, and H. Li, Sci. Transl. Med. 7:293ra103, 2015, doi:10.1126/scitranslmed.aab2009). Here, we reveal that the levels of porphyrin production and vitamin B12 regulation are different between acne- and health-associated strains, suggesting a potential molecular mechanism for disease-associated strains in acne pathogenesis and for health-associated strains in skin health. This study highlights the importance of understanding the strain-level differences of the human microbiota in disease pathogenesis. Our findings also suggest the porphyrin biosynthesis pathway as a candidate drug target and use of health-associated strains as potential probiotics in novel acne therapeutics.

Conformational States and kinetics of the calcium binding domain of NADPH oxidase 5.

Superoxide generated by human NADPH oxidase 5 (NOX5) is of growing importance for various physiological and pathological processes. The activity of NOX5 appears to be regulated by a self-contained Ca(2+) binding domain (CaBD). Recently Bánfi et al. suggest that the conformational change of CaBD upon Ca(2+) binding is essential for domain-domain interaction and superoxide production. The authors studied its structural change using intrinsic Trp fluorescence and hydrophobic dye binding; however, their conformational study was not thorough and the kinetics of metal binding was not demonstrated. Here we generated the recombinant CaBD and an E99Q/E143Q mutant to characterize them using fluorescence spectroscopy. Ca(2+) binding to CaBD induces a conformational change that exposes hydrophobic patches and increases the quenching accessibilities of its Trp residues and AEDANS at Cys107. The circular dichroism spectra indicated no significant changes in the secondary structures of CaBD upon metal binding. Stopped-flow spectrometry revealed a fast Ca(2+) dissociation from the N-terminal half, followed by a slow Ca(2+) dissociation from the C-terminal half. Combined with a chemical stability study, we concluded that the C-terminal half of CaBD has a higher Ca(2+) binding affinity, a higher chemical stability, and a slow Ca(2+) dissociation. The Mg(2+)-bound CaBD was also investigated and the results indicate that its structure is similar to the apo form. The rate of Mg(2+) dissociation was close to that of Ca(2+) dissociation. Our data suggest that the N- and C-terminal halves of CaBD are not completely structurally independent.