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

Abstract

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.

Keywords: 5-aminolevulinic acid; Propionibacterium acnes; acne; levulinic acid; porphyrin; strain; vitamin B12.

FIG 1

Acne-associated type IA-2 P. acnes strains produced significantly more porphyrins than health-associated type II strains. Each bar represents the porphyrins produced by a strain normalized to bacterial culture density. The means plus standard errors (error bars) of data obtained from at least three independent experiments with at least three replicates each are shown.

FIG 2

Vitamin B₁₂ supplementation significantly increased porphyrin production in acne-associated type IA-2 strains, but not in health-associated type II strains. P. acnes strains were cultured in medium with or without the addition of 10 µg/ml vitamin B₁₂. Each bar represents the porphyrins produced by a strain normalized to bacterial culture density. The means plus standard errors (error bars) of data obtained from at least three independent experiments with at least three replicates each are shown.

FIG 3

Vitamin B₁₂ supplementation repressed the expression of a vitamin B₁₂ biosynthesis gene, cbiL. The expression level of cbiL was quantified by qRT-PCR from P. acnes strains cultured with or without the addition of 10 µg/ml vitamin B₁₂. Strains of types IA-2 and II and an RT1 strain carrying deoR, HL025PA1, are shown. Each bar represents the fold change in gene expression of cbiL in cultures with vitamin B₁₂ supplementation compared to cultures without supplementation. The means plus standard deviations (error bars) of data obtained from at least two independent experiments with at least three replicates each are shown.

FIG 4

5-ALA increased porphyrin production, which was further enhanced by vitamin B₁₂ supplementation in acne-associated type IA-2 strains. P. acnes strains were cultured in medium with substrate 5-ALA (0.1 mg/ml) (grey bars) or without 5-ALA (white bars) or with both 5-ALA and vitamin B₁₂ (10 µg/ml) added (black bars). 5-ALA significantly increased porphyrin production in acne-associated type IA-2 strains (P < 0.0001) and in health-associated type II strains (P ≤ 0.06, except for HL001PA1). Vitamin B₁₂ supplementation further increased porphyrin production in the presence of 5-ALA in acne-associated type IA-2 strains, but not in health-associated type II strains. Each bar represents the porphyrins produced by each strain normalized to the bacterial culture density. The means plus standard errors (error bars) of data obtained from at least three independent experiments with at least three replicates each are shown.

FIG 5

Small-molecule inhibitor reduced porphyrin production in P. acnes, and its inhibition was counteracted by vitamin B₁₂ supplementation in acne-associated type IA-2 strains. P. acnes strains were cultured in medium with inhibitor LA (0.1 mg/ml) (grey bars) or without LA (white bars) or with both LA and vitamin B₁₂ (10 µg/ml) added (black bars). LA significantly reduced porphyrin biosynthesis in all strains except for HL001PA1 (P < 0.0001). Vitamin B₁₂ supplementation counteracted the inhibition of porphyrin biosynthesis by LA in acne-associated type IA-2 strains, but not in health-associated type II strains. Each bar represents the porphyrins produced by each strain normalized to the bacterial culture density. The means plus standard errors (error bars) of data obtained from at least three independent experiments with at least three replicates each are shown.

FIG 6

Health-associated strains carried and expressed deoR, a repressor gene in the porphyrin biosynthesis operon. deoR amplification from the cDNA and genomic DNA (gDNA) samples of multiple strains is shown in the gel image. 16S rRNA gene was used as a positive control. Strain HL045PA1, which is an acne-associated type IA-2 strain and does not carry deoR, is shown as a negative control.