Common skin bacteria protect their host from oxidative stress through secreted antioxidant RoxP

Abstract

Cutibacterium acnes is an abundant skin commensal with several proposed mutualistic functions. A protein with strong antioxidant activity was recently identified from the C. acnes secretome. This protein, termed RoxP, facilitated aerobic bacterial growth in vitro and ex vivo. As reducing events naturally occurred outside of the bacterial cell, it was further hypothesized that RoxP could also serve to modulate redox status of human skin. The biological function of RoxP was here assessed in vitro and in vivo, through oxidatively stressed cell cultures and through protein quantification from skin affected by oxidative disease (actinic keratosis and basal cell carcinoma), respectively. 16S rDNA amplicon deep sequencing and single locus sequence typing was used to correlate bacterial prevalence to cutaneous RoxP abundances. We show that RoxP positively influence the viability of monocytes and keratinocytes exposed to oxidative stress, and that a congruent concentration decline of RoxP can be observed in skin affected by oxidative disease. Basal cell carcinoma was moreover associated with microbial dysbiosis, characterized by reduced C. acnes prevalence. C. acnes's secretion of RoxP, an exogenous but naturally occurring antioxidant on human skin, is likely to positively influence the human host. Results furthermore attest to its prospective usability as a biopharmaceutical.

Figure 1

C. acnes phylotype I express significantly more roxP than other phylotypes, particularly during stationary phase growth. RNA was extracted from exponential and/or stationary phase C. acnes cultures, representative of type I (n = 6), type II (n = 5) and type III (n = 2) strains. qPCR quantifications of roxP mRNA were run in biological and technological duplicates, normalized against gapdh transcripts and evaluated using double delta Cq analysis. Grouped with their respective phylotypes, statistical significance was subsequently determined through a student’s t-test. ***p < 0.001, ****p < 0.0001.

Figure 2

RoxP homologs display comparable activity. Two distinct RoxP variants, natively expressed from KPA171202 and AD24 C. acnes, were incubated with either (A) pre-formed ABTS⁺ radicals or (B) pre-formed ABTS⁺ radicals plus NaCl (150 mM). Reduction of substrate was measured as absorbance decrease at 734 nm.

Figure 3

RoxP antioxidant activity is highly resilient to adverse conditions. The ability of RoxP to reduce ABTS radical cations, denoted by a decrease in absorbance at 734 nm, was tested in multiple settings. Different concentrations of recombinant RoxP (rRoxP) (0.2–3.2 μM) in sodium phosphate buffer (20 mM, pH 7.4) (A), rRoxP (2.7 μM) incubated with salt solutions (B), pre-heated rRoxP (2.7 μM) incubated for 20 min in temperatures ranging from RT to 98 °C and allowed to cool down to ambient temperature prior to activity measurements (C), or rRoxP (2 μM) stored at RT for up to 2 weeks (D) were added to a solution of pre-formed ABTS⁺ radicals and the absorbance recorded after 30 sec.

Figure 4

RoxP can protect human cells from oxidative damage in vitro. Human monocyte (A) or keratinocyte (B) cell lines were exposed to oxidative stress through addition of paraquat, while adding RoxP momentarily afterwards. Cells were incubated for 24 h (keratinocytes) or 48 h (monocytes) before cell viability was analyzed through MTT assays. Representative microscopy visualizations of monocytes after paraquat (C) or paraquat + RoxP (D) are demonstrated. Statistics were calculated using a student’s t-test.

Figure 5

RoxP is less prevalent in skin regions with oxidative disease. Individuals with either healthy skin (control, n = 18), actinic keratosis (AK, n = 18) or basal cell carcinoma (BCC, n = 18) were swabbed at diseased (affected) and healthy (non-affected) regions. Absolute quantity of RoxP was determined by RoxP-MIP capacitive biosensor measurements. All individual data points were included in the analysis.

Figure 6

Dysbiosis of the skin microbiome in AK and BCC. Box plot of the relative abundances of the five most abundant genera and four most abundant species in the four groups (A). Whiskers shows the 5–95% percentile and median values are shown as vertical lines. Differentially abundant taxa based of LefSe analysis are marked with an*. (B) Determination of relative abundancies of C. acnes phylotypes in AK and BCC. C. acnes type II abundances increase in AK-affected skin sites compared to controls (increase from 15% to 39%), whereas type IA strains decline (from 70% to 45%).