Abstract:
One of modern medicine’s most innovative and emerging fields in response to complex, multifactorial diseases is to unravel molecular dependencies from a systems point of view. One key factor driving this research is the growing recognition of the critical role in human health and disease played by the microbiome, an aspect that was previously little appreciated and understood. In addition, the overuse of antibiotics in our society is leading to a growing number of antibiotic-resistance-associated infections and, consequently, deaths. Further, antibiotic treatment is linked with dysbiosis, resulting in diseases. Often, those drugs are not pathogen-specific and also collaterally damage commensal bacteria. One approach to face these challenges, which is gaining more attention, is modulation of the microbiome to specifically eradicate pathogens and restore health in the host. Several strategies, such as probiotics, prebiotics, and fecal microbiota transplantation (FMT) are well-recognized already in supporting the host’s health. However, while it is fully acknowledged that microbiome-modulation holds much more potential to counteract many diseases, applications are still in an early stage. Currently, the investigation of the complex interplay of the microbiome and the host attracts scientific attention and expands the understanding of the microbiome’s far-reaching influence on the body. Its impact extends beyond colonized body regions, creating connections between different organs as well as linking the host’s environment to internal physiological processes. A deeper understanding of these intricate networks, including the key metabolites involved and the microbes that influence them, could enable us to precisely modulate relevant microbial populations. This knowledge holds the potential to prevent or treat diseases in a targeted manner while minimizing secondary unintended effects on the host. To harness this potential, we must understand how to precisely regulate microbes – either by modulating their abundance or by selectively activating or inhibiting metabolic pathways – without disrupting the homeostasis of the microbiome and the host. This requires insight into both microbe-host interactions and the complex network of microbial interactions within a community. Screenings of patient samples from healthy and diseased donors are valuable for finding correlations and identifying key species for disorders and diseases but cannot elucidate the interactions between microbes. For that, microbial communities need to be investigated in more detail.
This dissertation investigates the interactions between human gut commensals and the pathobiont Clostridium perfringens. One part focuses on the development of tools to study C. perfringens and members of a synthetic community in co-cultures or communities. In this section, I focused on developing strain-specific primers for quantifying strain abundance using quantitative polymerase chain reaction (qPCR), worked on the development of a real-time fluorescence reporter system, and established a multiple bioreactor system (MBS) that was used to study microbes under controlled pH conditions and continuous nutrient supply. I show the suitability of this MBS for microbial community studies by using it to investigate the effects of a proton-pump inhibitor (PPI) and pH changes on communities and how this affects colonization resistance against Clostridioides difficile.
In another part of this dissertation, I combined the developed tools with proteome and metabolome analysis to elucidate the molecular response of C. perfringens to the presence of each member of our synthetic community and vice versa. To further investigate these molecular responses and, consequently, the nature of these inter-species interactions, a novel approach was pursued to genetically modify C. perfringens. A newly developed CRISPR-Cas9 application for C. perfringens demonstrated its functionality through the successful deletion of two genes. This culminated in the insight into interspecies level interaction on physiological, metabolic, and proteomic levels, relevant for C. perfringens growth and virulence, providing leads for future therapeutic applications.
This study demonstrates the relevance and functionality of the developed tools for investigating microbial interactions. While C. perfringens is used here as a model microbe, the approaches presented can be applied to other communities and strains.
inter-species interactions