Inducible prophages and dietary compounds: Toward an integrated understanding of intestinal microbiota modulators

Abstract

The gastrointestinal tract of humans is densely colonised by a variety of different microorganisms referred to as the microbiota. The vast majority of these microorganisms are bacteria, and bacteria-infecting viruses termed bacteriophages (phages). The most important functions of the microbiota are shaping the host immune system, providing essential nutrients and protecting the host from pathogen colonisation, and these functions are thought to depend on a stable and diverse microbiota. However, the microbiota is constantly interacting with extrinsic and intrinsic factors that may alter its composition. For many factors, it is unknown if and to what extent they affect microbiota composition. Knowing which factors modulate the microbiota and how this occurs is critical because numerous studies have associated microbiota alterations with human health and disease. In this work, we assessed the impact of food as an extrinsic factor and developed an analytical framework to investigate the role of phages inserted in their bacterial host genome (prophages) as an intrinsic factor that shapes the gut microbial community. Food is a major factor that influences the intestinal microbiota. However, the composition of food is complex, and our understanding of how individual food components or orally administered substrates impact the composition of the gut microbiota is limited. Understanding the impact of different oral substrates could allow for targeted modulation of the gut microbiota, for example, by promoting the growth of some, but not other, community members. This could lead to new strategies using extrinsic factors to prevent disease and/or promote health. Chapter II describes my contribution to a study that tested whether a single dose of lactulose, which is solely fermented by certain gut bacteria, is sufficient to alter the microbiota composition in healthy human individuals. Moreover, we investigated whether hydrogen, a side product of fermentation, would promote the growth of enteropathogens and commensals, such as Escherichia coli. The results showed that a single high dose of lactulose was not sufficient to modulate the microbiota composition and thus is unlikely to alter colonisation resistance to pathogens. Although short-term intervention with lactulose was not effective in changing microbiota composition, another study in mice I contributed revealed that short-term intervention with dietary fat resulted in a microbial community shift and increased susceptibility to Enterobacteriaceae infections. Prophages are found in many intestinal bacteria, and the induction of a prophage influences its own host population and indirectly affects the entire niche-specific microbiota community. Prophage induction leads primarily to host cell lysis and the release of macromolecules and virions, which affect the environmental biochemical cycle and kill susceptible niche competitors. However, methodological challenges still limit our ability to detect inducible prophages and to understand how prophages could be used as intrinsic factors to shape the microbial community. For example, by identifying inducible prophages, we could potentially start to identify unique prophage-specific inducers. These could allow the implementation of prophages as an intrinsic factor to eliminate unfavourable, susceptible gut bacteria. Chapter III presents the establishment and experimental validation of mVIRs, an analytical tool to discover inducible prophages de novo and to locate them precisely within their reference genomes, which provides the basis for accurate, relative quantification of prophage induction. The sensitivity of mVIRs led to the discovery of prophages in strains with relative abundances < 1% in a low-complexity gut microbial community. This finding indicates the applicability of mVIRs as a tool to locate prophages not only in reference genomes and simple communities but also in more complex communities. Phage analysis of murine faecal samples demonstrated that dietary fat is an extrinsic factor that impacts bacterial community composition but also triggers a bacteria-independent shift in the phage community. This alteration was accompanied by an increase in the abundance of the integrase gene, indicating that a high-fat diet may act as a prophage inducer. Thus, we used mVIRs to investigate the effect of dietary fat as an extrinsic factor on prophages, which function as an intrinsic modulator within the intestinal microbiota community (Chapter IV). To this end, samples from Wotzka et al. (2019) were sequenced by metagenomics, and prophage detection by mVIRs was adapted for complex community samples. The phage-to-host analysis revealed increased prophage induction in a Lachnospiraceae species 24 hours post-administration of oleic acid. This showed that mVIRs can identify prophages and allows for quantifying phage activity in natural communities, which allows for the integration of extrinsic and intrinsic microbiota modulators. Overall, this work has shown that dietary compounds as extrinsic factors affect the bacterial and most probably the prophage communities and that the nature of the compound is essential. The development of a tool to identify and accurately locate inducible prophages provides the basis for reliable quantification of phage induction levels. This will enable researchers to study prophage induction and link prophages to variations in the gut microbial composition. Collectively, these novel insights will help develop future non-invasive strategies to monitor gut microbiota and promote healthy gut microbiota compositions.