Forming links in the forage : a (meta)genome-centric metaproteomic approach to connect rumen microbial functions

Abstract

The rumen microbiome operates in close symbiosis with its ruminant host, where the rumen functions as a fermentation chamber for the conversion of complex recalcitrant carbohydrates into nutrients that represent the primary source of energy for the animal. Dynamic interactions between bacterial, protozoal, fungal, viral and archaeal microorganisms in the rumen are associated with important host productivity traits, such as feed efficiency, animal health and greenhouse gas production (Dillard, 2019; FAO, 2020; Huws et al., 2018; Jami et al., 2014; Matthews et al., 2019; McCann et al., 2016). Therefore, understanding and unravelling the metabolic functions carried out by the complex rumen microbiome is of both industrial and scientific interest. Such knowledge can contribute to strategies to circumvent substantial challenges related to greenhouse gas mitigation from agriculture as well as advancing the livestock industry to securely meet the dietary requirements of a growing human population, without compromising animal health and wellbeing. Advances in modern molecular techniques have enabled the study and insight into genetic information and functional activity of complex microbiomes. The application of so-called “meta-omics” approaches enhances the recovery of the molecules that constitute a microbiome, and their interpretation facilitates improved predictions of the metabolic functions of intricate microbiomes in their natural ecosystems. Furthermore, untangling the dynamic interactions between microbial members in host associated microbiomes such as the rumen of herbivores can contribute to forming valuable links to host metabolism. In this thesis, the functions of the host associated rumen have been explored microbiome utilizing a combined metagenomic and metaproteomic approach. First, Paper I present key methods and considerations for meaningful metaproteomic analysis and consequent reconstruction of metabolically active populations of the rumen. Further the (meta)genome centric metaproteomic approach was applied to different rumen microbiome datasets to investigate the effects of dietary modulation and how they relate to key metabolism in connection to host metabolism in Paper II. Specifically, this enabled the exploration of the role of understudied rumen ciliate protozoal populations in the rumen microbiome of cattle and goats fed a starch-rich diet. Further, this led to the reconstruction of key protozoal metabolism related to carbohydrate and hydrogen metabolism, in addition to bacterial predation and volatile fatty acid production. Despite the starch degrading reputation of certain protozoal species, we observed a decrease in protozoal populations in low methane-emitting animals that were fed starch-rich feeds. In contrast, an increase in starch degrading bacterial populations producing succinate and propionate indicated a putative shift in hydrogen metabolism with broader implications for methane production. Lastly, Paper III investigated carbohydrate degradation of the red seaweed Mazzaella japonica through the rumen as well as the distal regions of the gastrointestinal tract in cattle. Contrary to initial assumptions, we observed higher activity of seaweed degradation in the lower gastrointestinal tract than in the rumen. We were further able to reconstruct an expressed co-regulated polysaccharide utilization locus with putative seaweed degrading properties from a Bacteroides- affiliated species, emphasising the influence of the lower gastrointestinal tract on degradation of complex carbohydrates and putative effects on host metabolism. Overall, the results presented in thesis show how the integration of metagenomics to metaproteomic dataset can provide added resolution to understand the functional contributions of understudied microbial populations or metabolic niches of the host-associated microbiomes in ruminants. Further, the results presented can contribute to the identification of microbial populations and changes in microbial structure are associated with the host, towards sustainable improvements of animal health and productivity and development of strategies to mitigate greenhouse gas emissions from agriculture.

Mikrobiomet i vomma fungerer i symbiose med sin drøvtyggende vert, der vomma fungerer som et fermenteringskammer for omdannelsen av komplekse karbohydrater til næringsstoffer, som representerer dyrets hovedkilde for energi. Dynamiske interaksjoner mellom mikroorganismer i vomma, som bakterier, protozoer, mikrobielle sopper, virus og arker, er assosierte med viktige produktivitetstrekk for verten, slik som fôrutnyttelse, dyrehelse og klimagassutslipp (Dillard, 2019; FAO, 2020; Huws et al., 2018; Jami et al., 2014; Matthews et al., 2019; McCann et al., 2016). Derfor er forståelsen av de metabolske funksjonene som utføres av den komplekse vommikrobiotaen både av industriell og vitenskapelig interesse. Denne kunnskapen kan bidra til å utvikle strategier for å imøtegå betydelige utfordringer knyttet til klimagassutslipp fra landbruket. Dette vil også kunne forbedre husdyrnæringen i møte med et økende behov for mat til en stadig voksende befolkning, uten å gå på bekostning av dyrehelse og dyrevelferd. Utvikling av moderne molekylære teknikker har muliggjort studier av og innsikt i genetisk informasjon og funksjonell aktivitet i komplekse mikrobiom. Utnyttelsen av såkalte ‘meta-omics’-tilnærminger gjør at vi kan øke gjenskaping av molekyler som utgjør et mikrobiom, og tolkningen av disse fasiliterer bedre prediksjoner av de metabolske funksjonene til intrikate mikrobiom i deres naturlige økosystem. Oppklaring av de dynamiske interaksjonene mellom mikrobielle medlemmer i vertsassosierte mikrobiom, slik som i vomma av planteetere, kan bidra til å danne verdifulle koblinger til vertsmetabolismen.