Innovative approaches to explore host-microbiota interactions and to discover novel antibiotics – functional metamining of the human intestinal microbiome

Project Leader
Justus Reunanen, Ph.D., Doc.

Biocenter Oulu, University of Oulu; Cancer and Translational Medicine Research Unit, University of Oulu

Background and Significance

Human intestinal microbiota in health and in inflammatory bowel diseases. The human gastrointestinal tract (GI-tract) is heavily colonized by a multitude of microbes, collectively called the microbiota. The microbiota provide us with metabolic capabilities not encoded in the human genome, e.g. the ability to utilize energy stored in dietary polysaccharides otherwise undigestible to us. Our microbiota also protects us against pathogens by competing for nutrients and binding sites within the GI-tract. Furthermore, cell numbers of GI-tract microbiota outcompete the host by a factor of ten, and therefore it can be seen as an organ in organ, composed of hundreds of species. It has been shown that >90 % of intestinal bacteria belong to the divisions Bacteroidetes and Firmicutes, whereas the rest of the phylotypes fall mainly into the Proteobacteria, Actinobacteria, Fusobacteria, and Verrucomicrobia. The GI-tract microbiota has been a subject of intensive research during the last years, and its importance in health and disease is only starting to be realized because of recent breakthrough observations linking the human microbiota composition with major human diseases, such as diabetes and obesity. In healthy adults the composition of microbiota has been shown to be highly subject-specific and stable; indeed, only a limited number of phylotypes are shared by different individuals, forming the common core microbiota that is functionally redundant.

The incidence of chronic inflammatory bowel disease (IBD) has been steadily increasing since the World War II in the US and EU, and it has been estimated to reach the magnitude of a worldwide epidemic in the coming years. IBD is characterized by chronic inflammation of the intestinal epithelium by an unknown reason. IBD manifests itself with two distinct types, ulcerative colitis (UC) and Crohn’s disease (CD). However, dysbiosis of the intestinal ecosystem is a common feature in both clinical conditions, as revealed by decreased microbial species diversity and distinctive changes in relative proportions of major phyla and genera members. Well described examples are decrease of Firmicutes and Bacteroidetes and increase of Desulfovibrio and Fusobacterium species in UC; in CD patients reduction of Firmicutes complexity, disappearance of Faecalibacterium and Roseburia (in ileal CD), and rise of Enterobacteriaceae and Rumicoccus gnavus, are commonly observed. The exact causative origins leading to IBD pathogenesis are not currently known, but it has become clear that the disease development is linked to multiple environmental factors, such as diet, socioeconomic status, antibiotic use, and exposure to microbes, to name a few. In fact, genetic predisposition to disease progression has been approximated to account only for 23 % in CD and 16 % in UC incidences. However, it is currently widely accepted that the intestinal microbiota is a fundamental driving force in the development of intestinal inflammation and resulting mucosal lesions, and that some microbial strains enhance the inflammatory response, whereas others attenuate it.   

Human antimicrobial peptides in the intestine. Human antimicrobial peptides (AMPs) are proteinaceous antibiotics with wide variety of antimicrobial activities in terms of mechanism of activation, action, and antimicrobial spectrum. Despite the wealth of knowledge about different AMP structures, mode of actions and expression patterns, it is striking that there is no much data about the bactericidal activities of human AMPs against human intestinal commensals, since studies related to antimicrobial spectra of different AMPS have by far mostly dealt with human pathogens. Most of the AMPs are upregulated in IBD, indicating that IBD-related microbiota members should be fairly tolerant against the majority of human AMPs. This assumption is further evidenced by the fact, that in IBD an increased colonization of the inflamed gut epithelial surface is frequently observed. Taken into account that the concentrations of individual AMPs are on the range of dozens, or even hundreds of micrograms/ml mucus, the human intestinal mucosal epithelium represents a surface as hostile than that of an agar plate with multiple antibiotics, and therefore it is justified to assume that there must be many yet unexplored bacterial AMP-resistance mechanisms -both in healthy and IBD-related microbiota- to support microbial growth and survival on such a harsh ecological niche.

Multiresistant bacteria as a global threat. Another major line in our research is exploration of novel antibiotic molecules. Bacterial resistance to antibiotics is an increasing problem in public health and economy, since treatment of bacterial infections has become complicated by the ability of bacteria to develop resistance to antimicrobial agents. Over the past 80 years, antibiotics have enabled tremendous advances in modern medicine and revolutionized agricultural and industrial practice. However, the introduction of every new antimicrobial product has been closely followed by the emergence of resistance against it. Many decades of excessive antibiotic use has applied unprecedented selective pressure towards high-level antimicrobial resistance, rendering entire classes of antibiotics redundant and threatening to bring about the end of the ‘antibiotic era’.

Human intestinal microbiota as a reservoir of genes with novel functions. In humans, more than 100 trillion microorganisms, mostly bacteria, colonize the gastrointestinal tract (GI-tract). Cell numbers of GI-tract microbiota outcompete the host by a factor of ten, and therefore it can be seen as an organ in organ, composed of hundreds of species. In healthy adults the composition of microbiota is highly subject-specific and stable; indeed, only a limited number of phylotypes are shared by different individuals. The combined collection of bacterial genomes within each individual’s intestine is called the (intestinal) metagenome. Since each individual metagenome contains approximately ~5x106 genes (compare with “only” 2x104 genes in human genome), the combined catalogue of all genes in different metagenomes (the human intestinal microbiome) is enourmous.  

Bacteriocins as a reservoir of novel antimicrobial molecules. Bacteriocins are bacterially produced peptides that are active against other bacteria. It has been estimated that most bacteria produce at least one bacteriocin, and thus it is not surprising that bacteriocins have been suggested as the next ample source of novel antibiotics. It has been shown that bacteriocin producing bacteria colonize intestine more efficiently than their isogenic mutant strains devoid of bacteriocin production. It is important to note, that many bacteriocins are strain-specific, i.e. they are active against certain, but not all strains of a given species. Therefore bacteriocins offer a tempting alternative to antibiotics, as usage of bacteriocins to combat infections would be strain-specific, and therefore would not disturb the healthy microbiota, as currently used antibiotics do.

Bacteriocin-producers have traditionally been isolated from fermented food products, but there are also few examples of intestinal isolates. However, the search of bacteriocin-producers is not only laborious and time-consuming, but it is also inherently limited to discover bacteriocins produced only by cultivable microbes. As vast majority of the human intestinal microbiota members cannot be cultivated, this approach is severely limited in its power to mine the metagenomic reservoir of novel bacteriocins.

Recent Progress

Phage display is a powerful yet simple functional genomic tool for screening protein-ligand interactions. Phage display relies on the expression of foreign peptides or proteins on the surface of phages, which can be screened (“biopanned”) based on their ability to bind to immobilized molecules or cells. The power of phage display is based on the facts, that i) it allows biopanning of billions of unique peptides or proteins in a single reaction tube, ii) a physical link between displayed polypeptides and DNA encoding them is maintained and easily determined. We have developed a novel state-of-the-art phage display technique, metagenomic phage display, which allows functional study of all proteins encoded by any metagenomes – also of those encoded by uncultivable bacteria.

We have successfully used this system to identify several key genes utilized by normal intestinal microbiota to interact with the host.

We are currently collecting tissue and fecal samples from healthy people, IBD-patients, and from individuals who have recently been identified as intestinal carriers of multidrug resistant bacteria.

Future Goals

We aim at discovering i) the fundamental immunity mechanisms necessary for any given bacterium to adapt to the hostile environmental conditions of the human intestinal tract created by continuous secretion of a multitude of host defence molecules by the intestinal epithelium, ii) novel bacteriocins active against multidrug resistant bacteria. The knowledge gained in this study will open up multiple new avenues for rational design and development of novel antibiotics, bacterial or molecular replacement therapies, molecular nutrition, oral vaccines, and product development in food industry.  As such, it is easy to realize the many perspectives it offers for the future research dealing with microbes and man.     

Publications 2016-

Ottman N, Huuskonen L, Reunanen J, Boeren S, Klievink J, Smidt H, Belzer C, de Vos WM. Characterization of Outer Membrane Proteome of Akkermansia muciniphila Reveals Sets of Novel Proteins Exposed to the Human Intestine. Front Microbiol 7:1157. doi: 10.3389/fmicb.2016.01157, 2016.

Tytgat HL, Douillard FP, Reunanen J, Rasinkangas P, Hendrickx AP, Laine PK, Paulin L, Satokari R, de Vos WM. Lactobacillus rhamnosus GG Outcompetes Enterococcus faecium via Mucus-Binding Pili: Evidence for a Novel and Heterospecific Probiotic Mechanism. Appl Environ Microbiol 82(19):5756-62, 2016.

Tytgat HL, van Teijlingen NH, Sullan RM, Douillard FP, Rasinkangas P, Messing M, Reunanen J, Satokari R, Vanderleyden J, Dufrêne YF, Geijtenbeek TB, de Vos WM, Lebeer S. Probiotic Gut Microbiota Isolate Interacts with Dendritic Cells via Glycosylated Heterotrimeric Pili. PLoS One 11(3):e0151824  doi: 10.1371/journal.pone.0151824, 2016.

Research Group Members

Project Leader:
Justus Reunanen, Ph.D., Academy of Finland Research Fellow 2016-2021 (Academy of Finland)

Senior and Post-doctoral Investigators:
Johanna Korvala, Ph.D. (Academy of Finland)

Ph.D. Students:
Anna Sorjamaa, M.Sc., Ph.D. student (Academy of Finland)

Last updated: 10/5/2017