Cobamide sharing drives skin microbiome dynamics

The human skin microbiome is a key player in human health, with diverse functions ranging from defense against pathogens to education of the immune system. While recent studies have begun to shed light on the valuable role that skin microorganisms have in maintaining a healthy skin barrier, a detailed understanding of the complex interactions that shape healthy skin microbial communities is limited. Cobamides, the vitamin B12 class of cofactor, are essential for organisms across the tree of life. Because this vitamin is only produced by a limited fraction of prokaryotes, cobamide sharing has been shown to mediate community dynamics within microbial communities. Here, we provide the first large-scale unbiased metagenomic assessment of cobamide biosynthesis and utilization in the skin microbiome. We show that while numerous and diverse taxa across the major bacterial phyla on the skin are cobamide dependent, relatively few species encode for de novo cobamide biosynthesis. We find that cobamide sharing shapes the network structure in microbial communities across the different microenvironments of the skin and that changes in community structure and microbiome diversity are driven by the abundance of cobamide producers in the Corynebacterium genus, in both healthy and disease skin states. Lastly, we find that de novo cobamide biosynthesis is enriched only in host-associated Corynebacterium species, including those prevalent on human skin. We confirm that the cofactor is produced in excess through quantification of cobamide production by skin-associated species isolated in the laboratory. Taken together, our results support a role for cobamide sharing within skin microbial communities, which we predict stabilizes the microbiome and mediates host interactions.

representing 12 genes within the de novo cobamide biosynthesis pathway, cbiZ as a marker of 149 cobamide remodeling, and single-copy core gene rpoB as a marker of community structure, we 150 found that samples from sebaceous sites harbored the overall highest median number of hits to 151 cobamide biosynthesis genes, followed by dry, moist, and foot samples (Supplemental Figure  152 1A). 153 154 To assess the contribution of different taxa to cobamide biosynthesis, the metagenomic 155 sequence classifier pipeline Kraken and Bracken was used to classify the resulting gene hits. 156 The top taxa encoding for biosynthetic genes in descending order were determined to be 157 Propionibacteriaceae, Corynebacteriaceae, Veillonellaceae, Streptococcaceae, 158 Dermacoccaceae, and Pseudomonadaceae. Within individual metagenomes, the contribution of 159 each taxon to cobamide biosynthesis gene hits was calculated by dividing the number of 160 biosynthesis gene hits assigned to a given taxa by the total number of biosynthesis gene hits 161 within the sample. We found that Propionibacteriaceae was the dominant contributor to 162 cobamide biosynthesis, particularly in sebaceous sites (Figure 2A). Although few species within the skin microbiome synthesize cobamides de novo, we predict that 193 a larger proportion use cobamides. We determined the prevalence of the cobamide transport 194 protein btuB and 19 enzymes that carry out diverse cobamide dependent reactions. The median 195 number of cobamide-dependent gene hits across samples varied by microenvironment 196 (Supplemental Figure 1). Across the sebaceous, moist, and dry microenvironments, 197 Propionibacteriaceae was the dominant family encoding for the cobamide-dependent enzymes 198 D-ornithine aminomutase, methylmalonyl-CoA mutase, and ribonucleotide reductase class II 199 ( Figure 3). In contrast, across the remaining cobamide dependent enzymes, hits were assigned 200 to phylogenetically diverse taxa across the four major phyla on the skin (Actinobacteria, 201 Firmicutes, Proteobacteria, and Bacteroidetes) (Grice and Segre, 2011). Cobamide-dependent 202 enzymes involved in primary metabolism, including methionine synthase, epoxyqueosine 203 7 reductase, ribonucleotide reductase, and ethanolamine lyase, were the most common cobamide 204 dependent enzymes in the dataset (Supplemental Figure 3). Notably, only 1% of species 205 appreciably contribute to de novo cobamide biosynthesis (n=18 species), yet approximately 206 39% of species encode for cobamide dependent enzymes (n=638 species encoding at least 207 one cobamide-dependent enzyme) (Supplemental Figure 3). While the true number of de novo 208 cobamide producers may be underestimated due to filtering of rare and singleton hits prior to 209 analysis, these species likely represent the core cobamide producers found on the skin. Overall,210 these results support a model of cobamide sharing, where a much larger number of skin taxa 211 require cobamides than can produce the cofactor de novo. 212 213

Regulation of cobamide biosynthesis is species-specific 214
To further delineate cobamide usage within the skin microbiome, we identified cobalamin 215 riboswitches within the metagenomes. Cobalamin riboswitches are cobamide-binding elements 216 found in the untranslated region of bacterial mRNAs that regulate expression of genes or 217 transcripts involved in cobamide-dependent metabolism, biosynthesis, and cobamide transport 218 (Garst et al., 2011;Nahvi et al., 2004;Polaski et al., 2017). We show that phylogenetically 219 diverse skin taxa encode for cobalamin riboswitches, with Propionibacteriaceae being the 220 dominant taxa ( Figure 4A). At the species level, these hits were found predominantly within C. 221 acnes genomes. 222

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To identify the pathways regulated by cobamides in C. acnes, we mapped cobalamin riboswitch 224 sequence reads to the C. acnes KPA171202 reference genome. We find that riboswitches are 225 distributed across the genome in numerous regions, regulating pathways involved in ABC 226 transport, cobalt transport, cobamide biosynthesis, and cobamide-dependent and -independent 227 reactions ( Figure 4B). Three of the C. acnes cobalamin riboswitches (Regions 6, 7, and 8) are 228 located upstream of pseudogenes or genes of unknown function ( Figure 4B). Manual curation of 229 these sequences suggest that the small pseudogenes are hypothetical adhesin protein 230 fragments and the larger downstream sequences are thrombospondin type-3 repeat containing 231 proteins (Supplemental Material S4). The role of cobalamin riboswitches in regulation of these 232 genes is unknown, but suggests that cobalamin riboswitches are regulating diverse functions 233 yet to be discovered. 234

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We found fewer cobalamin riboswitches in the genomes of other species relative to C. acnes. 236 hypothesize that changes at the community level are associated with the presence of these 287 cobamide-producing species. To assess this, we first explored the relationship between 288 microbiome diversity and cobamide-producing Corynebacteria (CPC) abundance within healthy 289 skin metagenomes. NMDS ordination of Bray-Curtis dissimilarity indices revealed clustering that 290 follows increasing gradients of both alpha diversity and CPC abundance, where alpha diversity 291 increases as CPC abundance increases ( Figure 6A, Supplemental Table 2). This was most 292 striking for samples from sebaceous, moist, and foot sites. In contrast, this pattern of clustering 293 was not observed for Cutibacterium cobamide producers, but rather samples with the highest 294 Cutibacterium relative abundances often were the least diverse (Supplemental Figure 5). 295 Furthermore, communities with a low CPC abundance were usually dominated by Cutibacterium 296 acnes, whereas communities with high abundance showed an expansion of other skin taxa and 297 an overall more even species distribution within the community (Supplemental Figure 6). 298 Consistent with our analysis of riboswitch regulation, these results further support a model 299 where Corynebacterium species constitutively produce cobamides as a shared common good, 300 promoting microbiome diversity and structure. On the other hand, tightly regulated production by 301 Cutibacterium species permits niche expansion and lower diversity. 302 303 Cobamide production is depleted in atopic dermatitis 304 10 A decrease in microbiome diversity is associated with increased pathogen colonization in 305 dermatological disease such as atopic dermatitis (AD) (Paller et al., 2019;Williams, 2005). To 306 assess the potential role of CPC in the AD skin microbiome, we analyzed 417 metagenomes 307 from a cohort of 11 pediatric AD patients and 7 healthy controls. Microbiome structures 308 exhibited a higher level of variability compared to the adult cohorts, with weak clustering of 309 samples based on alpha diversity or CPC abundance. A subset of samples collected from moist 310 sites during a flare formed a distinct cluster exhibiting low CPC abundance and alpha diversity 311 (Supplemental Figure 7). AD skin symptoms often present in moist sites such as the antecubital 312 fossa (bend of the elbow) and popliteal fossa (bend of the knee), suggesting a relationship 313 between microbiome structure, diversity, and CPC abundance during AD flares. Consistent with 314 this hypothesis, we observed that CPC abundance is significantly reduced in AD patients at 315 baseline (p=0.0018) as well as during flares (p=0.0050) compared to healthy controls (Figure 316 6C). Overall, differential CPC abundance is detected between disease states, suggesting a 319 relationship between these members and microbial community structure in atopic dermatitis. 320 321

Cobamide biosynthesis is enriched in host-associated Corynebacterium species 322
Until recently, species of the Corynebacterium genus have been underappreciated as significant 323 members of skin microbial communities, predominantly due to the difficulty of growing these 324 species in the lab, which is a result of their nutritionally fastidious and slow-growing nature 325 (Grice and Segre, 2011  for all or nearly all of the genes required for de novo cobamide biosynthesis, and notably, 21 out 361 of 22 of these predicted cobamide producers are host-associated. Taken together, these results 362 demonstrate a range of cobamide biosynthetic capabilities by Corynebacteria, with de novo 363 producing species being almost exclusively host-associated, despite reduced genome size. 364 Thus, we hypothesize a role for cobamides in mediating host-microbe interactions. 365 366 Skin commensal Corynebacterium amycolatum produces high levels of cobamides. 367 From our metagenomic and comparative genomic analyses, we identified C. amycolatum as a 368 de novo cobamide producer. To test in vitro production of cobamides by this species, we 369 isolated a strain of C. amycolatum from healthy skin, cultured it in a minimal growth medium, 370 and prepared cell extracts from the intracellular metabolite content. We tested the cell extract in 371 a microbiological assay using the indicator strain E. coli ATCC 14169, whose growth is 372 proportional to cobamide concentration from 0.1 to 1.5 ng/mL (Supplemental Figure 10A). When 373 diluted 10,000-to 50,000-fold, C. amycolatum cell extracts yielded growth of E. coli within the 374 linear range (Supplemental Figure 10B), with an average cobamide amount of 1.51 ± 0.135 µg 375 per gram of wet cell weight and an average intracellular concentration of 11.3 ± 2.37 µM. 376 Physiological requirements of cobamides range from nanomolar to even picomolar 377 concentrations (Sokolovskaya et al., 2020), supporting our hypothesis that C. amycolatum 378 produces the cofactor in excess quantities to support cobamide sharing in the community. Corynebacteria are well-equipped for growth on the skin due to their "lipid-loving" and 427 halotolerant nature, allowing them to thrive in moist and sebaceous skin microenvironments 428 (Scharschmidt and Fischbach, 2013). However, many questions remain about the processes 429 that govern skin colonization by this relatively understudied skin taxa and how these processes 430 may impact or be impacted by microbe-microbe and microbe-host interactions on the skin. We 431 identified several Corynebacterium species to be de novo cobamide producers on the skin, and 432 further, that the abundance of these species impacts microbial community dynamics through 433 promotion of diversity. This suggests skin-associated Corynebacteria are a keystone species, 434 leading us to perform a comparative genomics study of the entire genus. As expected, host-435 associated species have significantly smaller genomes, but unexpectedly, they are enriched for 436 de novo cobamide biosynthesis as compared to environment-associated species. Retention of 437 the energetically costly 25-enzyme cobamide biosynthesis pathway within host-associated 438 14 species, even with reduced genome size, suggests that synthesis of this cofactor is 439 advantageous for host niche colonization. 440

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A key question that arises is why some Corynebacterium species have retained the de novo 442 cobamide biosynthesis pathway, while others have not. Our results showed that 443 Corynebacterium species encode for cobamide-dependent methionine synthase, 444 methylmalonyl-CoA mutase, and ethanolamine ammonia lyase, consistent with previous 445 findings by Shelton et al. (Shelton et al., 2019). Therefore, cobamides are likely produced by 446 Corynebacteria to fulfill metabolic requirements in methionine, propionate, and 447 glycerophospholipid metabolism. Alternative cobamide-independent pathways exist for these 448 functions, therefore cobamides may confer a distinct advantage for these species. Indeed, 449 metE, the cobamide-independent methionine synthase, is sensitive to oxidative stress and has 450 reduced turnover compared to metH (González et al., 1992 abundance of these cobamide-producing Corynebacteria is strongly associated with increased 472 microbiome diversity and disease state, supporting our hypothesis that cobamides are important 473 mediators of microbiome structure and skin health. We also show that within the 474 Corynebacterium genus, de novo cobamide biosynthesis is uniquely a host-associated function. 475 Future studies to interrogate the role of cobamides in microbe-microbe and microbe-host 476 interactions will provide insight into the key roles that microbially-derived metabolites play in 477 microbial community dynamics and host health. BioProject IDs PRJNA46333 and PRJNA266117, respectively. Metagenomic reads across 694 multiple SRA run accessions from the same biological sample were pooled, processed for 695 quality control, and assigned taxonomy using the methods outlined above. In all, the 696 metagenomic data represents the skin microbial communities across 21 distinct sites from 66 697 healthy individuals. Sample information is described in Supplemental Material S1 and S2. 698   The total normalized hits for cobamide-dependent enzymes, cobamide transport protein btuB, and SCG rpoB are shown (total hits normalized to profile HMM coverage and sequence depth), with the taxonomic abundance of the hits expanded as relative proportions above. Hits to distinct B12-dependent radical SAM proteins are grouped together as "B12-dep radical SAM".   A) The SPIEC-EASI method was used to identify microbial associations within each microenvironment of three independent skin microbiome datasets. Consensus networks are shown, representing associations identified in at least 2 of the 3 datasets. Species are represented by nodes and colored by phylum. Green and pink edges represent positive and negative associations, respectively. Node shape represents cobamide biosynthesis category and node size reflects mean species relative abundance within each microenvironment. Cobamide dependent species are outlined in black. In each final network, B) the number of species classified to each cobamide biosynthesis category, C) the number of species that are cobamide dependent or independent, D) the percentage of total edges that fall into each cobamide biosynthesis edge category, and E) the percentage of total edges that exist between cobamide producers and cobamide dependent species that are non-producers or precursor salvagers is shown. NP=Non-producer, P=Producer, S=Precursor salvager.