William Mohn

Therapies targeting the microbiome hold great promise to improve the wellbeing of children. Their success will depend on understanding host-microbiome conversations. The gut microbiome differs between geographically diverse populations. Does this contribute to distinct immune phenotypes? Does the same perturbance impact these diverse populations in the same way? How does the composition of the fungal microbiome compare to bacterial? What tools are needed to listen to these conversations where it matters the most, in vulnerable populations living in low-resource settings? These questions can be ethically asked in humans.To find answers to these questions, I assessed the relationship between systemic immune responses and the gut microbiome and compared bacterial to fungal colonization in geographically diverse child cohorts, hypothesizing that population-specific microbiomes correlated to distinct systemic innate immune phenotypes. To test this, we profiled the bacterial microbiomes of 2-year-old children from Belgium, Canada, Ecuador, and South Africa and measured their cytokine responses to innate stimuli. Certain immune differences between the cohorts correlated with the abundance of select bacterial taxa. Splenocytes of germ-free mice inoculated with human stools responded to stimulation in a manner consistent with the corresponding human donors, indicating that microbiomes can direct systemic innate immunity.I hypothesized that immune responses of HIV-exposed uninfected (HEU) and HIV-unexposed uninfected (HUU) children within each site differed in population-specific ways and that these differences correlated to distinct microbomes. While HEU were distinguished from HUU children by immune responses and microbiome composition in population-specific ways, differences in the microbiome did not correlate with altered immune phenotypes. I compared Bacterial vs. fungal composition over the first 5 years of life in a rural African population and found that both changed in kingdom-specific ways, suggesting they were shaped by separate selective forces. To overcome the hurdle of investigating newborns, I designed and implemented an experimental protocol in a low-resource setting that allows extraction of ‘big data’ out of the very small samples. Taken together, these findings suggest that therapies targeting the microbiome must consider population differences, and that placing newborns in low-resource settings at the forefront of our research is not only warranted, but feasible.

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Asthma is the most common childhood medical condition and it accounts for nearly two million hospital visits and thousands of deaths per year. Despite its immense societal burden, there is no cure, there are very few treatment options, and no prevention strategies. In fact, the etiology of asthma remains elusive. There is an emerging understanding that an association exists between the gut microbiome and asthma. Herein, we provide evidence that the microbiome impacts early-life immune development with consequences for asthma via the production of short chain fatty acid (SCFA) metabolites. We found that mice treated with vancomycin have an altered microbiome and metabolite profile, exhibit exacerbated Th2 responses, and are more susceptible to allergic lung inflammation. Here we show that dietary supplementation of SCFAs ameliorates this enhanced asthma susceptibility by modulating the activity of T cells and dendritic cells. Informed by this animal model, we sought to determine whether alterations in microbiome carbohydrate fermentation pathways could also be identified in human infants prior to developing atopic disease using shotgun metagenomic sequencing of the gut microbiome. We found that the microbiome of infants that went on to develop asthma later in childhood lacked genes encoding key functional enzymes for carbohydrate breakdown and butyrate production. To better understand the imprint of SCFAs on immune development, we successfully transferred the phenotypes of both heightened and dampened Th2 skewing via bone marrow transplants to irradiated recipient mice. Consistent with the hypothesis that the transferred phenotype is encoded within the epigenome, we found unique regulatory states, as defined by DNA acetylation, within the genomes of purified hematopoietic stem and progenitor cells of recipient mice that received BM transplants from dysbiotic mice. Altogether, this research highlights the role of microbially-derived metabolites, SCFAs, in the development of asthma and atopy. We present a new understanding of the intricate relationship between the microbiome, microbial metabolites and asthma. Knowledge of this process will have potential practical applications in the prevention and treatment of disease.

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Forest industries are expected to bolster the renewable resource economy, but must contend with ecological challenges in maintaining the long-term fertility of forest plantation soils, and technological challenges in converting forest biomass into industrially relevant sources of carbon and energy. This thesis advances research related to both, first by describing the broad changes in soil microbial communities in the decades following timber harvesting, their implications for soil processes and the influence of biomass retention for mitigation (Chapter 3) and, second, by conducting the first comprehensive culture-independent survey of lignocellulolytic organisms in forest soils to expand knowledge of their diversity and catabolic capabilities (Chapter 4). Analysis of over 1,300 bacterial (16S rRNA gene) and fungal (ITS region) pyrotag libraries demonstrated consistent changes in microbial communities at harvested sites across North America, such as i) the increase of desiccation- and heat-tolerant organisms, ii) the general decline of ectomycorrhizal (EM) fungi with a rise of select EM genera (Suillus and Thelephora), iii) the moderation of population shifts by organic matter retention and iv) changes in the functional character of harvested soils, including reduced methanotrophic populations and cellulolytic activity. Biogeographical differences in community structure revealed the potential for variation in the impacts of harvesting. Overall, a number of taxonomic groups were identified that may be important indicators for assessing the long-term impact of timber harvesting. Stable isotope probing revealed the degradation of model hemicellulose, cellulose and lignin substrates by specialized taxa, active on a sole substrate, and groups capable of degrading all three plant polymers, such as members of Burkholderiales and Caulobacteraceae. Bacterial lignin-degraders were more active than fungi in soil microcosms, represented by taxa with characterized lignolytic capability (Sphingobacteriaceae and Sphingomonadaceae) and novel taxa, such as members of Elusimicrobia and Acidobacteria. Differences in lignocellulolytic populations were observed among ecozones and soil layers. Mineral soils harboured a greater proportion of poorly characterized functional taxa and represent reservoirs of unexplored catabolic diversity. Metagenome assembly was ~3 to 20-fold higher as a result of SIP, providing a trove of sequence data containing carbohydrate- and lignin-active enzymes from lignolytic and cellulolytic taxa for future characterization.

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Actinomycetes are an abundant bacterial group in soil, with a critical role in the decomposition of organic matter. Rhodococcus jostii strain RHA1 is of particular interest to the field of bioremediation because it can degrade a broad range of organic compounds, both natural and xenobiotic. Understanding the factors contributing to the desiccation resistance of RHA1 will enrich our basic knowledge of this common soil stress and may help advance bioremediation technologies for contaminated soils subject to droughts.Here I report the first transcriptomic analysis of a Gram-positive bacterium during desiccation. Filtered RHA1 cells incubated at either low relative humidity, as an air-drying treatment, or high relative humidity, as a control, were transcriptionally profiled over a comprehensive time series. Also, the morphology of RHA1 cells was characterized by cryofixation scanning electron microscopy during each treatment. Desiccation resulted in a transcriptional response of 819 differentially regulated genes, 8-times more than in the control. Included among the highly up-regulated desiccation-specific genes was dps1 (induced 33-fold), encoding an oxidative stress protection protein which has not previously been directly associated with desiccation, as well as sigF3 (induced 58-fold), encoding a sigma factor possibly involved in the regulatory response to desiccation.RHA1 mutants with dps1 or both of its dps homologs deleted were challenged with oxidative stressors under a variety of assay conditions. The mutants were also exposed to physiological stresses that generate reactive oxygen species intracellularly, including desiccation. In all cases, the dps− mutants did not have impaired oxidative stress resistance – a novel finding with respect to bacterial dps-null strains. Additionally, the RHA1 dps-null mutant did not have substantially lower survival compared to the wild type when challenged with metal toxicity or DNA-damaging agents or when they were cocultured through multiple cycles of starvation. Nevertheless, expression of RHA1 dps1 in an Escherichia coli dps– mutant restored its hydrogen peroxide resistance. Purified RHA1 Dps1 was shown to have ferroxidase activity and thereby to protect DNA from oxidative damage. The general insensitivity of the RHA1 dps-null mutant may be representative of a large group of Actinobacteria for which robust oxidative stress tolerance is an important adaptation.

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