bioRxiv2023
Corynebacterium is a predominant genus in the skin microbiome, yet its genetic diversity on skin is incompletely characterized and lacks a comprehensive set of reference genomes. Our work aims to investigate the distribution of Corynebacterium species on the skin, as well as to expand the existing genome reference catalog to enable more complete characterization of skin metagenomes. We used V1-V3 16S rRNA gene sequencing data from 14 body sites of 23 healthy volunteers to characterize Corynebacterium diversity and distribution across healthy human skin. Corynebacterium tuberculostearicum is the predominant species found on human skin and we identified two distinct C. tuberculostearicum ribotypes (A & B) that can be distinguished by variation in the 16S rRNA V1-V3 sequence. One is distributed across all body sites and the other found primarily on the feet. We performed whole genome sequencing of 40 C. tuberculostearicum isolates cultured from the skin of five healthy individuals across seven skin sites. We generated five closed genomes of diverse C. tuberculostearicum which revealed that C. tuberculostearicum isolates are largely syntenic and carry a diversity of methylation patterns, plasmids and CRISPR/Cas systems. The pangenome of C. tuberculostearicum is open with a core genome size of 1806 genes and a pangenome size of 5451 total genes. This expanded pangenome enabled the mapping of 24% more C. tuberculostearicum reads from shotgun metagenomic datasets derived from skin body sites. Finally, while the genomes from this study all fall within a C. tuberculostearicum species complex, the ribotype B isolates may constitute a new species. IMPORTANCEAmplicon sequencing data combined with isolate whole genome sequencing has expanded our understanding of Corynebacterium on the skin. Human skin is characterized by a diverse collection of Corynebacterium species but C. tuberculostearicum predominates many sites. Our work supports the emerging idea that C. tuberculostearicum is a species complex encompassing several distinct species. We produced a collection of genomes that help define this complex including a potentially new species which we are calling C. hallux based on a preference for sites on the feet, whole-genome average nucleotide identity, pangenomics and growth in skin-like media. This isolate collection and high-quality genome resource sets the stage for developing engineered strains for both basic and translational clinical studies. Microbiomes are shaped by taxa that are both characteristic to those sites and functionally important to that community. The genus Corynebacterium is one such taxa for the human skin and nares. Foundational studies using 16S rRNA gene sequencing and shotgun metagenomics by our lab (1, 2) and others (3) have established Corynebacterium as common members of the skin microbiome. While Corynebacterium have been positively correlated with the resolution of dysbiosis associated with eczema flares (4), the importance of the Corynebacterium spp. is less defined for skin disease severity in primary immune deficient patients (5, 6). Corynebacterium spp. are predominant members of the human aerodigestive tract microbiome (nares, oral cavity and respiratory tract) (3) and participate in microbe-microbe interactions with members of nasal microbiome (7, 8). Corynebacterium have been shown to engage with the host immune system, specifically C. accolens-promoted IL23-dependent inflammation in mice on a high-fat diet (9). C. bovis and C. mastiditis have been shown to predominate the microbiome of a ADAM10-deficient mouse model (10) as well as an ADAM17-deficient mouse model of eczema (11). Finally, C. tuberculostearicum has been shown to induce inflammation in human epidermal keratinocyte cell cultures (12). These studies establish Corynebacterium spp. as key members of the skin microbiome capable of both microbe-microbe and microbe-host interactions. A critical resource for understanding the biology of Corynebacterium on the skin is a robust collection of complete reference genomes, including isolates collected from a variety of individuals and body sites. Previously published genome collections from skin- or nares-resident species include Staphylococcus epidermidis (13), Cutibacterium acnes (14) and the recent comparative analysis of Dolosigranulum pigrum (15). Of note, while emerging bioinformatic methods and pipelines are now being employed to extract nearly-complete genomes (MAGs) from metagenomic assemblies of skin samples (16), MAGs are not yet a substitute for genomes from cultured isolates to understand strain level or pangenomic diversity. In addition to functional prediction, comparative genomics is increasingly being used to augment conventional microbiological methods to define or redefine taxonomic boundaries (17, 18), as well as describe the full extent of diversity within these boundaries (19). A pangenome, which encompasses the complete set of genes present within a set of genome sequences, enables the characterization of gene-level heterogeneity within a taxonomic group. The pangenome is commonly subdivided into the core genome, referring to genes present in all strains, and the accessory or dispensable genome, referring to those present in only one or some isolates. (The accessory pangenome can be further subdivided to reflect a wider range of gene uniqueness, e.g. singletons.) Thorough characterization of taxa is limited by the availability of representative and high-quality genome assemblies. Unfortunately, with the exceptions of clinically relevant Corynebacterium spp. (e.g., C. diphtheriae, C. striatum and C. pseudotuberculosis), the genus is inadequately sequenced, with 75% of species having fewer than six genomes. This includes common skin-associated species like C. tuberculostearicum with just five unique isolate genomes, only two of which are from skin. This work seeks first to characterize the distribution of Corynebacterium across 14 skin sites from 23 healthy volunteers. The second goal of this work focuses on what we identify as the predominant skin Corynebacterium species, C. tuberculostearicum. We have sequenced 23 distinct C. tuberculostearicum strains (n=40 genomes before dereplication), a five-fold increase in the number of publicly available, unique genomes (n=5). In addition to short-read assemblies, we generated five complete genomes which, along with the type strain (DSM44922), demonstrate that C. tuberculostearicum genomes are largely syntenic and carry a number of methylation systems as well as a CRISPR/Cas system. Genes from the C. tuberculostearicum genomes in our collection fall into 5451 gene clusters comprising the species pangenome. This expanded pangenome, as compared to existing public references, improved the mapping of C. tuberculostearicum metagenomic reads from unrelated healthy volunteers. In addition, we have identified a distinct C. tuberculostearicum clade that is highly enriched on the feet that may represent a new species, tentatively designated Corynebacterium hallux.