1. Bacteriophage inhibition of antibiotic-resistant pathogenic microbes and finding novel therapeutic strategies (PHARMS)
Antimicrobial resistance (AMR) is a major threat to global health, global economies, and humanity itself. Worldwide, AMR has killed more than 700,000 people, including over 25,000 cases in Europe every year. AMR is particularly deadly where malnutrition and co-infections are widespread, especially in developing countries. The rapid spread of AMR, and its devastating consequences for patients as well as healthy individuals, makes it one of the most important scientific challenges of our time. Research on novel AMR control strategies is clearly needed, despite the significant progress made in searching for new antibiotics. Viruses of bacteria, bacteriophages, are the nature’s most prevalent bacterial predators, which can be employed to fight AMR as a complement to antibiotic therapy. Coupling the bactericidal effects to host-recognition machineries, promising progress has been made in treating AMR bacteria using: i) lytic phages which can directly lyse hosts or a cocktail of different lytic phages to overcome bacterial resistance; ii) phage-encoded bactericidal peptides or enzymes, such as endolysins (lysins), which are peptidoglycan hydrolases involved in cell lysis during phage replication. To avoid adverse reactions caused by immune recognition of phages, phages with innate characteristics that are unlikely to elicit an immune response or mutant phages that are not recognized by the immune system are preferred for medical treatment. PHARMS will focus on three AMR bacteria: A. baumannii, H. influenzae, and H. pylori, all of which are major human pathogens in this year’s WHO guideline of twelve AMR bacterial families prioritized for R&D efforts. Three synergistic work packages are tailored for PHARMS: WP1 is designed to understand the infection strategies of phages in a high-throughput, cultivation-independent manner, and to provide information that guides targeted phage isolations; WP2 aims to targeted isolate representative divergent phages and understand the phage bactericide systematically in achieved divergent phage isolates, in order to engineer a new series of phage vectors with superior bactericidal potential by employing yeast-based engineering of phages; WP3 is designed to provide direct molecular confirmation for the function and mechanisms of phage-encoded bactericidal peptides and enzymes in selected phage isolates.
2. Functional roles of viruses in the natural environment and human-associated microenvironment
2.1 Impact of viruses in human(/animal) health and disease
The human microbiome is a unique polymicrobial community protecting the human host on the one hand from pathogenic assault by competing for sites of attachments and nutrients, as well as producing antimicrobial substances, and on the other hand altering the host’s metabolism by modulating lipid metabolism and glucose homeostasis, absorbing lipid-soluble vitamins, or activate respectively inactivate drugs. The composition of the microbiota thus affects metabolic, regulatory and morphogenetic networks. Members of the virome influence the phenotype of the host in a combinatorial manner by interacting with other members of the microbiome (such as other members of the virome itself, the bacterial microbiome or the mycobiome) and by interacting with individual variations in host genetics. Together these interactions may influence a range of phenotypes, which may be important for health and disease. We want to elucidate the functional roles of viruses especially with regard to disease progression. The method of choice for these questions is the application of metagenomic sequencing of a broad range of human samples deriving from healthy and diseased patients. In a further step, animal models are utilized to investigate the functional impact of exclusive enteral nutrition via microbiome and phageome changes in paediatric Crohn‘s disease.
2.2 Impact of viruses on microbial-driven contaminant biodegradation
Contamination with organic pollutants, such as petroleum hydrocarbons, is widespread in groundwater and a notorious threat to our water resources. Biodegradation is the single most important and sustainable process for contaminant breakdown. The biological activity that governed by numerous processes of which the current perspective is only evolving hydrology, biogeochemistry and environmental microbiology. One important piece of the puzzle, however, has been rarely touched: viruses. This project aims to elaborate a ground-breaking new perspective, the viral-driven degradation (Figure 1). Viruses are ubiquitous and abundant global players that impact microbial communities through mortality and horizontal gene transfer to modulate microbial metabolism. During the last years diverse phenomena critical to the biology of microbes have been described to be driven by viruses, especially with respect to rapid environmental changes. We hypothesize that degradation of contaminants is greatly impacted by viruses through (i) horizontally transfer host metabolic genes related to contaminant degradation, and (ii) specifically lysing key bacterial degraders. In a cutting-edge and interdisciplinary research endeavor, we want verify our hypotheses using model aerobic and anaerobic degraders for the group of benzene, toluene, ethylbenzene and xylenes (BTEX) and polycyclic aromatic hydrocarbons (PAHs). Pillaring on recent advances in methodology, the culture-independent, high-throughput approach “Viral-Tagging” which allows to link natural viruses to their hosts, and vice versa, carefully designed field surveys and microcosm experiments as well as metagenomics sequencing will be conducted.
3. Our Methods
3.1 Bioinformatic Toolbox
Despite growing interest in recent years, studying viromes remains a challenging endeavor due to several reasons, including the scarcity of viral genomic material (compared to microbial and human nucleic acid fraction) due to the small genome sizes of viruses and their low abundance in some cases. In a microbial community only 2–5% of the total DNA is generally of viral origin. Additionally, no conserved gene regions applicable for all viral types have been identified so far. Moreover, many viruses have not been characterized yet and are not included in viral databases. These facts impede straightforward contig assemblages as well as functional annotation of viral genomes and metagenomes. We built a human virome protein cluster (HVPC) database in order to improve and facilitate functional annotation and characterization of human viromes (Figure 2). This Human Viral Metagenomic Database for Diversity and Function Annotation constitutes of 12 terabases in total with more than 6 Million open reading frames (ORFs) and 927K function clusters. Further improvements such as using machine learning tools (random forest ad neural netwoks/deep learning) to identify host of viruses using signals including CRISPR, prophages, k-mer, etc. are currently on-going.
3.2 Viral Tagging (VT)
“Viral-Tagging” (VT) is a high-throughput, culture-independent means of experimentally linking wild viruses to a target host, and vice versa (Figure 3). The DNA of uncultivated viruses is labeled non-specifically with a fluorescent dye, then viruses are mixed with a ‘bait host’, and infected cells are collected by fluorescence-activated flow cytometric sorting. The infecting viral DNA is quantitatively amplified to produce viral-tagged metagenomes. VT enables researchers to broadly map how viruses change over space and time.
A. Viruses are fluorescently-labeled green, and then mixed with potential host bacteria that are flow-cytometrically green-negative, until “tagged” by fluorescently-labeled viruses. B. Flow cytometry data triggered on side scatter (SSC) for the fluorescently labeled virus and co-incubated host bacteria. Tagged cells infecting by viruses with extra fluorescence can be sorted out from non-infected cells. C. Representative micrographs of viral-tagged (green positive) and non-tagged (green negative) cells examined by a scanning flow cytometer to show the co-localization of the green VT signal to the red-autofluorescing cyanobacterial cells. Notably, >105 cells for each green positive and green negative populations were examined in a 10min experiment. D. Discrimination by PCR (40 cycles) of the host bacteria from the mixture of the virus, host and non-host bacteria in the viral tagged population (red in B) and a non-tagged population (black in B). The first experiment used strains in equal concentrations (Lane 1 & 3), while the second employed viruses and non-host at 100-fold higher density; lane 2 & 4).