Scientific Achievements - Thomas F. Meyer Laboratory
Genetic basis of microbial behaviours and virulence mechanisms (1978 – 1998)
How genes function in the continual processes of microbial reproduction, evolution and interaction with other organisms, particularly the host, is of fundamental importance to our understanding of infections. From the beginning of his research career, Thomas Meyer was concerned with genetic aspects of infectious agents. Early studies included the understanding of gene replication mechanisms, the elucidation of the genetic principles underlying the antigenic and functional variation of microbial surface proteins involving intragenic recombination and slipped-strand mispairing, unravelling the principles of natural transformation events, as well as the development of sophisticated genetic tools for elucidating crucial virulence functions. Pioneering discoveries from this time also included the characterization a novel secretion mechanism of auto-transported proteins, the adhesin function of a pilus tip located protein and the heterologous expression of the meningococcal capsular gene complex.
- During his Ph.D. under the supervision of Klaus Geider and mentoring by Hartmut Hoffmann-Berling and Heinz Schaller, TFM purified enzymatic components and achieved replication of circular double-stranded phage DNA in vitro, revealing basic insights into DNA amplification mechanisms even prior to the invention of PCR (e.g. Meyer et al. 1979 Nature; Meyer & Geider 1982 Nature).
- Triggered by an initial report on the variable feature of the gonococcal pilin protein, TFM, supervised as a postdoctoral researcher by Maggie So, cloned the first Neisseria gene, pilin, which served as the starting point for broad investigations into the genetic principles of pilin variation in Neisseria gonorrhoeae. This work established the first paradigm of a complex genetic system for antigenic variation in bacteria, based on diverse recombination processes between silent and expressed gene copies (e.g. Meyer et al. 1982 Cell; and as an independent researcher: Haas & Meyer 1986 Cell; Haas et al. 1987 PNAS; Gibbs et al. 1989 Nature).
- After accepting an independent position in Heidelberg, TFM initiated a project to analyze the genetic basis of opacity variation of gonococcal colonies. This led to the discovery of a variable pentameric nucleotide repeat sequence that defined the translational reading frame of otherwise intact genes, encoding members of the family of opacity associated proteins (Opa) in the pathogenic Neisseria species. It is the first example of such a gene variation mechanism based on short sequence repeats amongst microbes and was later referred to as slipped strand mispairing (Stern et al 1984 Cell; Stern et al 1986 Cell; Stern et al 1987 Mol Microbiol).
- Cloning and analysis of the gene encoding the IgA protease of Neisseria gonorrhoeae revealed the first, and at its time best-understood, self-secreted protein of gram-negative bacterial species, thus constituting the paradigm of autotransporter secretion, later also referred to as type V secretion. Autotransporters were subsequently associated with hundreds of key virulence determinants and adhesins in other gram-negative bacterial species. Key articles: Halter et al 1984 EMBO J; Pohlner et al. 1987 Nature.
- The mechanistic assessment of the autotransporter system enabled the secretion of heterologous proteins from recombinant E. coli to either expose them to the bacterial surface or release them to the extracellular environment. These studies established a paradigm for many similar secretion systems explored by other groups. Systematic analyses in the TFM laboratory further gave insight into the critical role of periplasmic oxidoreductases in protein folding (DsbA) whereby mutant bacteria facilitated secretion and proper folding of functional antibody as well as other biotechnologically relevant proteins (Klauser et al 1990 EMBO J; Klauser et al 1992 EMBO J; Klauser 1993 J Mol Biol; Jose et al. 1995 Mol Microbiol).
- The successful cloning of the large capsule gene cluster of serogroup B meningococci for the first time allowed insight into the structural and functional organization of this crucial pathogenesis determinant as well as an understanding of its phase variable features (e.g. Frosch et al. 1989 PNAS).
- At a time when genetic transformation was recognized as a key driver of microbial diversity and genome evolution, one of the first demonstrations was the occurrence of a mosaic gene structure in the iga gene locus of Neisseria. This, together with the identification of key determinants of natural transformation competence, as well as natural transformation between co-cultured bacteria, provided evidence of the evolutionary importance of this process (Halter et al 1989 EMBO J; Frosch & Meyer 1992 FEMS Microbiol Lett; Facius et al 1993 Mol Microbiol; Fussenegger et al 1996 Mol Microbiol).
From insights into host cell mechanisms towards host-directed therapy (1991 – 2018)
With the knowledge on the function of virulence determinants, especially as obtained from the model organism Neisseria gonorrhoeae, the main research focus of the Meyer laboratory then turned towards the host cell and the host cell functions that comprise the target of bacterial virulence factors. Of particular interest was the characterization of the adhesin function of the major variable Neisseria surface proteins, resulting in the novel concept that antigenic variation is not only an immune escape mechanism but also a crucial instrument to enable functional diversity of microbes. Moreover, the expanding knowledge about host factors engaged in the infection process - including cell surface receptors, signaling molecules, transcription factors and small molecule messengers - nurtured recognition of the importance of host cells factors for their interactions with microbial pathogens. Thomas Meyer’s laboratory contributed substantially to this fascinating insight, and upon the discovery of RNA interference as a potent tool for knocking down human gene function, he was one of the first to recognize the value of this new technology for approaching the role of host factors during infection in a systematic way. Several genome-wide RNAi-based investigations have now led to comprehensive insights into the involvement of host factors in viral replication, bacterial survival and pathogen-induced signaling. Ultimately, the identification of critical host factors of infection led to the notion of using host factors as targets for host cell-directed therapy, e.g. on the basis of drug repositioning approaches.
- The identification and characterization of the variable Neisseria Opa proteins as cellular invasins marked the beginning of investigations into the involvement of host factors in the process of bacterial infections. These studies in the TFM laboratory showed that different members of the Opa protein family displayed different cell tropisms, reflected at the molecular level by two major receptor families, (a) the heparin sulfate proteoglycans in conjunction with serum factors, including vitronectin and fibronectin, and (b) the CEACAM receptors (Makino et al 1991 EMBO J; Kupsch et al 1993 EMBO J; Gray-Owen et al 1997 EMBO J; Billker et al 2002 EMBO J).
- The retractile type 4 pili produced by the pathogenic Neisseria species were found to exhibit two genetically and functionally separate binding features: One was broadly specific to erythrocytes and associated with the variable major pilin subunit PilE; the second one was highly specific for human epithelial cells and conferred by the minor pilus subunit PilC, functioning as a tip-located adhesion molecule and recognizing a proteinacious host cell receptor. Phase-variable PilC was thus identified as a key pilus component, displaying crucial functions in cell adherence, pilus assembly, and natural transformation (Rudel et al 1992 Mol Microbiol; Rudel et al 1995 Nature).
- Binding of the gonococcal pili to human epithelial cells elicited a so far unknown feature, characterized by the recruitment of host cell caveolin (Cav1) to the sites of attachment. Via a cascade of cellular signaling events, this led to the formation of cortical plaques whereby actin prevented the internalization of attached bacteria. This demonstrated for the first time the anti-phagocytic function of type 4 pili. Interestingly, this extracellular stabilization was overcome via an ON-switch of the Opa invasins, thus revealing antagonistic functions of the two variable Neisseria adhesin families (Boettcher et al. 2010 PLoS Biol).
- Strikingly, the seemingly inert neisserial PorB, the most abundant outer membrane protein of Neisseria, was recognized as a GTP / ATP modulated porin. Moreover, PorB was shown to translocate into host cells, where it associates with mitochondria and modulates the apoptosis of infected cells. These observations led to numerous subsequent studies on the infection strategies displayed by the pathogenic Neisseria species. Key publications: Rudel et al 1996 Cell; Müller et al 1999 EMBO J; Müller et al 2000 EMBO J; Müller et al 2002 EMBO J.
- Fascinating insight into the engagement of host cell functions upon infection was provided by looking at key signaling molecules known to regulate host cell function, e.g. via phosphorylation, nuclear translocation or small messenger molecules. Using pathogenic Neisseria species, several new pathways could be identified that were intimately involved in bacterial internalization (host cell invasion) and inflammatory responses (Grassmé et al 1997 Cell; Naumann et al 1997 J Exp Med; Hauck et al 1998 EMBO J;
- While investigating the features of the obligate intracellular bacterium Chlamydia trachomatis, particularly interesting observations were made upon the assessment of signaling routes associated with host cell survival and intracellular vesicular trafficking. During the process of pathogen propagation and maturation different cellular features appeared to be required, and these ranged from the modulation of signaling routes involved in the stress response to endocytic maturation and Golgi compartment function - providing a substantially extended view on the life style of an intracellular pathogen (Mäurer et al 2007 PLoS Pathogens; Heuer et al. 2009 Nature; Gurumurthy et al 2010 Science Signaling; Mehlitz et al 2010 J Cell Biology; Al-Younes et al 2011 Autophagy; Al-Zeer et al 2013 Autophagy).
- Large-scale analyses of host factors engaged in viral and intracellular bacterial pathogen propagation were initiated following the first successful applications of RNAi in human epithelial cells and, despite the various pitfalls of such applications and the demand for high-quality standards, resulted in a wide-ranging understanding of host cell factor function during the course of viral and bacterial infections. Comparative and meta-analytic studies led to the identification of central signaling nodes important for more than one pathogen. The essential requirement of host factors for infection also nurtured the idea that identified hits could potentially be used as targets for a novel type of anti-infective therapy. Validation of this promising host cell-directed approach was obtained in vivo for chikungunya virus and Chlamydia trachomatis (Karlas et al 2010 Nature; Tripathi 2015 Cell Host Microbe; Karlas 2016 Nat Commun; Rother 2018 Cell Host Microbe)
Impact of chronic bacterial infections on the emergence of human cancer (2000 – ongoing)
Motivated by the profound ways in which some pathogens interfere with normal signalling pathways of their hosts and the growing epidemiological indications that some bacterial infections are linked to cancer development, Thomas Meyer’s group again broke new ground by asking questions about the fate of infected host cells. Not much is known about whether infected cells (or stem cells) can survive and give rise to progeny. If they do – even if this is a rare occurrence - and if they are inherently damaged, this would represent a risk for cancer or other age-related diseases. Helicobacter pylori, as a paradigm of a cancer-inducing bacterium, was therefore chosen as a model to explore its long-term effect on host cells. In particular, the inflammatory features of the bacterium combined with its unique ability to escape the mucosal defense and thereby ensure life-long persistence, turned into the focus of interest and led to crucial discoveries. H. pylori and several other pathogens turned out to cause DNA damage and to alter DNA damage response pathways, enabling mutations. However, it became clear that a full understanding of the fate and impact of mutant cells in the infected tissue requires a comprehensive understanding of tissue homeostasis, in particular with regard to stem cell plasticity and tissue regeneration. Analyses of these processes in the presence and absence of infected cells have already provided unprecendented insight into the principles of how alterations in tissue homeostasis caused by persistent infection could shift the balance towards disease progression.
- With the recognition of H. pylori’s cagPAI type 4 secretion system (T4SS) as a significant virulence attribute, the laboratory was at the forefront of establishing a molecular basis for its function as an inflammatory and potentially transforming determinant. They revealed the translocation of the CagA effector protein into host cells and its phosphorylation by Src kinase and uncovered crucial signaling routes induced by CagA, including its function in downregulating innate epithelial defence factors. Importantly, they recognized that a factor other than CagA is responsible for the vast pro-inflammatory activity of the T4SS and identified this as a metabolite of bacterial LPS synthesis. According to their most recent data, T4SS co-secretes a β-ADP-heptose, which stimulates the early response transcription factor NF-κB via a novel innate response signaling route involving Alpk1 and the formation of TIFAsomes. This supports the notion that inflammation is a bystander effect of CagA translocation, which serves bacterial survival and persistence. Key publications: Backert et al 2000 Cell Microbiol; Selbach et al 2002 J Biol Chem; Selbach et al 2003 EMBO J; Bauer 2012 Cell Host Microbe; Koch et al 2012 PNAS; Belogolova 2013 Cellular Microbiology; Bielig 2014 PLoS Pathogen; Koeppel 2014 Cell Rep; Zimmermann 2017 Cell Rep; Pfannkuch 2019 FASEB J.
- How does H. pylori cope with the intense inflammation and the subsequent anti-microbial response it provokes? A compelling answer was provided by the elucidation of a mechanism that effectively prevents the mucosal immune effector function: The observation rests on the discovery of an enzyme, glucosyl-a-cholesterol transferase, that causes depletion of host cell cholesterol and thereby inactivation of lipid raft-dependent cytokine receptors, including those for IFN-g and IL-22. This mechanism establishes immune protected niches on the mucosal surface, which allow H. pylori to persist despite the intense inflammatory response in the vicinity of infected sites. The recent observation sheds light on the putative mechanism by which H. pylori induces a precancerous condition and explains the failure of previous efforts in vaccinating against this persistent pathogen (Lebrun 2006 J Biol Chem; Wunder et al 2006 Nature Medicine; Aebischer 2008 Gut; Morey et al 2017 Gastroenterology).
- For studying the mechanisms of infection and cell transformation in vitro it was necessary to establish a suitable epithelial model. Although early efforts of the laboratory provided initial progress in understanding gastric infections, a breakthrough was obtained upon the establishment of gastric organoids, and recently by the creation of epithelial mucosoids that combine quasi-indefinite growth with the reproduction of in-vivo-like infection conditions – technologies that have already revealed fascinating insights. (Boxberger 1993 Eur J Cell Biol; Boxberger 1994 Epithel Cell Biol; Schlaermann et al 2016 Gut; Boccellato et al 2018 Gut. )
- As a complementary approach for studying infections in the tissue context, novel mouse models have been established and have provided deep insight into the plasticity of gastric stem cells and the regeneration of the epithelium under normal and infected conditions. These studies, which involve state-of-the art genetic lineage tracing systems as well as single molecule in situ detection, are suited to more broadly understand the occurrence of tissue damage and emergence of gastrointestinal cancers. They provide a novel view on the concerted actions of inflammatory cytokines and morphogens, such as Wnt and R-spondin, in the preservation of the mucosal tissue (Sigal et al 2017 Nature; Sigal et al 2019 Nat Cell Biol; Harnack et al 2019 Nat Comm).
- While tackling H. pylori as a paradigm of a cancer-inducing bacterium, the observed features provided a basis for assessing similar processes in other bacterial species, such as Chlamydia trachomatis in the female genital tract. Here, one of the most striking observations was the DNA damage induced during infection. Despite this, host cells continued to proliferate and, instead, responded with the down-regulation of the guardian of the genome, the tumour suppressor p53, explaining their previous observations on the anti-apoptotic features of Chlamydia. Moreover, infected cells acquired a cancer-related metabolic state, known as the Warburg effect, thus providing deep insight into the cancer-promoting potential of certain bacterial pathogens (Chumduri 2013 Cell Host Microbe; Gonzalez 2014 Nature Comm; Chumduri 2016 Nat Rev Mol Cell Biol; Zadora et al 2019 Cell Rep).
- To better understand infections of the female genital tract and their long-term consequences, genuine tissue and organoid models have been established for the human fallopian tube epithelium. This tissue is widely considered to provide the precursors of ovarian cancer. These models thus facilitate current studies on the early stages of putative premalignant transformation events at the site of chronic infection induced by sexually transmitted pathogens, including Chlamydia (Kessler et al 2012 Am J Pathology; Kessler et al 2015 Nat Commun; Hoffmann et al 2020 EMBO).
Using advanced techniques and thorough bioinformatics analyses, we aim to identify genetic signatures that bacterial pathogens leave behind in human cancers, proving a causative role in carcinogenesis. Our first highlight of this ambitious research shows that E. Coli's genotoxin, colibactin, causes double-strand breaks, resulting in a specific pattern of mutations in human cells and colorectal cancer (Dziubańska-Kusibab et al. 2020 Nature Medicine).