Metagenomic And Transcriptomic Insights Into The Skin Microbiome And Host Response

Culture-independent, high-throughput sequencing techniques have enabled us to characterize the complex communities of resident microorgansims living in and on our skin. These microbes contribute to cutaneous health, by providing colonization resistance and regulating immunity, however disruption of skin microbiota has been linked to diseases, like acne and atopic dermatitis. While the microbiome is a promising therapeutic target, we do not fully understand the mechanisms underlying the microbial contributions to disease pathogenesis. It is therefore crucial to develop comprehensive knowledge of both the structure of the skin microbiome and the host functions it stimulates. Here, we present a critical analysis of the composition and metabolic potential of healthy human skin microbiota and how these communities modulate the gene expression of their hosts. In the first section, we optimize methodologies for skin microbiome studies and utilize the best approaches to characterize the bacteria, viruses, and fungi colonizing healthy skin. We applied amplification and sequencing of two hyper variable regions of the 16S rRNA gene, in addition to whole metagenomic shotgun sequencing, to cutaneous swab samples from a healthy cohort. We demonstrate that shotgun sequencing yields the most accurate profiles of the skin microbiome, but that sequencing of the V1-V3 region of the 16S rRNA gene is a suitable, cost-effective alternative. We also reveal significant taxonomic, functional, and temporal diversity of skin microbial communities and highlight potential multi-kingdom interactions between skin phages and their bacterial hosts. The second section focuses on the response of the cutaneous transcriptome to colonization by resident microflora. We used RNA sequencing to compare gene expression in skin collected from sterile, germ-free mice, and mice conventionally raised in the presence of microbiota. We find that the skin microbiome primes the cutaneous immune system, through increases in both frequency of innate immune cell populations and expression of immune response genes. We also reveal that the skin microbiome transcriptionally regulates epidermal development and differentiation, suggesting a novel role for microorganisms in skin barrier structure and function. Together, the work presented in this thesis highlights the complex dynamics of skin microbial communities and their impact on the host

Three transmission events of Vibrio cholerae O1 into Lusaka, Zambia

Cholera has been present and recurring in Zambia since 1977. However, there is a paucity of data on genetic relatedness and diversity of the Vibrio cholerae isolates responsible for these outbreaks. Understanding whether the outbreaks are seeded from existing local isolates or if the outbreaks represent separate transmission events can inform public health decisions. Seventy-two V. cholerae isolates from outbreaks in 2009/2010, 2016, and 2017/2018 in Zambia were characterized using multilocus variable number tandem repeat analysis (MLVA) and whole genome sequencing (WGS). The isolates had eight distinct MLVA genotypes that clustered into three MLVA clonal complexes (CCs). Each CC contained isolates from only one outbreak. The results from WGS revealed both clustered and dispersed single nucleotide variants. The genetic relatedness of isolates based on WGS was consistent with the MLVA, each CC was a distinct genetic lineage and had nearest neighbors from other East African countries. In Lusaka, isolates from the same outbreak were more closely related to themselves and isolates from other countries than to isolates from other outbreaks in other years. Our observations are consistent with i) the presence of random mutation and alternative mechanisms of nucleotide variation, and ii) three separate transmission events of V. cholerae into Lusaka, Zambia. We suggest that locally, case-area targeted invention strategies and regionally, well-coordinated plans be in place to effectively control future cholera outbreaks.https://doi.org/10.1186/s12879-021-06259-

HmmUFOtu: An HMM and phylogenetic placement based ultra-fast taxonomic assignment and OTU picking tool for microbiome amplicon sequencing studies

Abstract Culture-independent analysis of microbial communities frequently relies on amplification and sequencing of the prokaryotic 16S ribosomal RNA gene. Typical analysis pipelines group sequences into operational taxonomic units (OTUs) to infer taxonomic and phylogenetic relationships. Here, we present HmmUFOtu, a novel tool for processing microbiome amplicon sequencing data, which performs rapid per-read phylogenetic placement, followed by phylogenetically informed clustering into OTUs and taxonomy assignment. Compared to standard pipelines, HmmUFOtu more accurately and reliably recapitulates microbial community diversity and composition in simulated and real datasets without relying on heuristics or sacrificing speed or accuracy

Evolutionary and functional implications of hypervariable loci within the skin virome

Localized genomic variability is crucial for the ongoing conflicts between infectious microbes and their hosts. An understanding of evolutionary and adaptive patterns associated with genomic variability will help guide development of vaccines and antimicrobial agents. While most analyses of the human microbiome have focused on taxonomic classification and gene annotation, we investigated genomic variation of skin-associated viral communities. We evaluated patterns of viral genomic variation across 16 healthy human volunteers. Human papillomavirus (HPV) and Staphylococcus phages contained 106 and 465 regions of diversification, or hypervariable loci, respectively. Propionibacterium phage genomes were minimally divergent and contained no hypervariable loci. Genes containing hypervariable loci were involved in functions including host tropism and immune evasion. HPV and Staphylococcus phage hypervariable loci were associated with purifying selection. Amino acid substitution patterns were virus dependent, as were predictions of their phenotypic effects. We identified diversity generating retroelements as one likely mechanism driving hypervariability. We validated these findings in an independently collected skin metagenomic sequence dataset, suggesting that these features of skin virome genomic variability are widespread. Our results highlight the genomic variation landscape of the skin virome and provide a foundation for better understanding community viral evolution and the functional implications of genomic diversification of skin viruses

Commensal microbiota modulate gene expression in the skin

Abstract Background The skin harbors complex communities of resident microorganisms, yet little is known of their physiological roles and the molecular mechanisms that mediate cutaneous host-microbe interactions. Here, we profiled skin transcriptomes of mice reared in the presence and absence of microbiota to elucidate the range of pathways and functions modulated in the skin by the microbiota. Results A total of 2820 genes were differentially regulated in response to microbial colonization and were enriched in gene ontology (GO) terms related to the host-immune response and epidermal differentiation. Innate immune response genes and genes involved in cytokine activity were generally upregulated in response to microbiota and included genes encoding toll-like receptors, antimicrobial peptides, the complement cascade, and genes involved in IL-1 family cytokine signaling and homing of T cells. Our results also reveal a role for the microbiota in modulating epidermal differentiation and development, with differential expression of genes in the epidermal differentiation complex (EDC). Genes with correlated co-expression patterns were enriched in binding sites for the transcription factors Klf4, AP-1, and SP-1, all implicated as regulators of epidermal differentiation. Finally, we identified transcriptional signatures of microbial regulation common to both the skin and the gastrointestinal tract. Conclusions With this foundational approach, we establish a critical resource for understanding the genome-wide implications of microbially mediated gene expression in the skin and emphasize prospective ways in which the microbiome contributes to skin health and disease

Current progress and future opportunities in applications of bioinformatics for biodefense and pathogen detection: report from the Winter Mid-Atlantic Microbiome Meet-up, College Park, MD, January 10, 2018

Abstract The Mid-Atlantic Microbiome Meet-up (M3) organization brings together academic, government, and industry groups to share ideas and develop best practices for microbiome research. In January of 2018, M3 held its fourth meeting, which focused on recent advances in biodefense, specifically those relating to infectious disease, and the use of metagenomic methods for pathogen detection. Presentations highlighted the utility of next-generation sequencing technologies for identifying and tracking microbial community members across space and time. However, they also stressed the current limitations of genomic approaches for biodefense, including insufficient sensitivity to detect low-abundance pathogens and the inability to quantify viable organisms. Participants discussed ways in which the community can improve software usability and shared new computational tools for metagenomic processing, assembly, annotation, and visualization. Looking to the future, they identified the need for better bioinformatics toolkits for longitudinal analyses, improved sample processing approaches for characterizing viruses and fungi, and more consistent maintenance of database resources. Finally, they addressed the necessity of improving data standards to incentivize data sharing. Here, we summarize the presentations and discussions from the meeting, identifying the areas where microbiome analyses have improved our ability to detect and manage biological threats and infectious disease, as well as gaps of knowledge in the field that require future funding and focus