Georgios Belibasakis

The advent of antibiotic therapy over the past century has brought along the global healthcare threat of antibiotic resistances. This biological complication can render pathogens unresponsive to treatment and permit the development of life-threatening infections. Oral infections are very common painful conditions, that impinge on the patient´s well-being. However, on some occasions they may spread beyond the neighboring tissue even to distant vital organs via the blood stream, where they can become potentially lethal for the patients. These infections may also cause airway obstruction or cause damage to vital structures in the head and neck area. Effective antibiotic treatment can prove to be lifesaving in such cases. On the contrary, the presence of resistances among the involved microorganisms (‘resistome’) may imperil the outcome. Important knowledge gaps exist in this healthcare domain, including lack of consensus on risk factors or standard treatment protocols, or insufficient estimation of antibiotic resistance levels within microbial communities and human populations. To contribute to bridging those gaps, this project aims to evaluate retrospectively the prevalence of oral infections, particularly of dental-origin, that required antibiotic treatment, while assessing the most common types of antibiotic resistances present. Patient registry data will be retrieved from Karolinska University Hospital, Huddinge in a complementary approach between clinical and laboratory hubs, as summarized in Workpackage-1. Workpackage-2 aims to prospectively evaluate antibiotic resistances in samples retrieved from oral infection referral patients at Karolinska University Laboratoty, by performing functional and molecular characterization of the isolates at the Department of Dental Medicine (ANA Futura Laboratories). Deeper molecular characterization of known and unknown resistance genes will be acquired by whole-genome sequencing (WGS) of the resistant bacterial isolates. This project will describe the current use of antibiotics in treating oral infections and initiate the mapping the ‘oral resistome’, while paving the way for establishing a cross-Departmental regional Reference Centre for Antibiotic Resistances. Over time, the dynamic data collection will deliver much-needed scientific evidence for strategies and guidelines for antibiotic stewardship programs for treatment of oral infections and beyond.

The Gram-positive anaerobic rod Filifactor alocis is an emerging oral pathogen associated with multiple oral infections. We discovered in  F. alocis a hitherto unknown RTX toxin, which we named FtxA. It is homologous to B. pertussis adenylate cyclase toxin (ACT) CyaA and to A. actinomycetemcomitans leukotoxin LtxA. FtxA is the 1st ACT and the 2nd RTX identified among the oral microbiome.The mechanisms by which it may act as a virulence factor of F. alocis to regulate the oral microbial ecology and immune response is an uncharted territory and the project´s main aim. In the first segment of the project, clinical isolates and genetically manupulated F. alocis strains will be used to decipher the fucntional roles of FtxA in F. alocis microbe-microbe ecological competition and host-microbe immuno-pathogenic interactions of F. alocis. This approach will employ biofilms, immune cells and organotypic tissue in vitro models and metaproteomics. In the second segment, the virulence contribution of FtxA will be evaluated in a murine model of F. alocis infection, using single-cell proteomics. Moreover, the expression levels of FtxA will be evaluated in a cohort of periodontitis patients, where the oral levels of F. alocis have already been defined before and after treatment. By deciphering the fucntional properties of FtxA , the project will lay the foundation for novel prevention and therapeutic strategies against infections caused by F. alocis or other RTX-producing species.

1) Project file. Modulation of dental plaque interactions with host cells by the periodontal pathogen Tannerella forsythia 2) Content of research project. The oral cavity, similarly to other human organs, is colonized by various microorganisms. Bacterial colonization is controlled by the host immune system and most oral bacteria are beneficial for humans. However, overgrow of some microorganisms might lead to the development of periodontitis, one of the most frequent infectious diseases of adults world-wide, which results in tooth loss, if untreated. In most cases, periodontitis is associated with increased numbers of the "red complex" bacteria - Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia - in the oral biofilm (dental plaque). These bacteria possess specific virulence factors, which facilitate invasion into the human body and cause tissue destruction. Studies in the last years provided evidence that the periodontal pathogen P. gingivalis is able to manipulate distinct components of the host immune system and, consequently, changes the interaction between the oral microbial community as a whole and the immune system. This ability of P. gingivalis is now considered as a key for the progression of periodontitis. 3) Hypothesis. It is currently unclear if other members of the red complex group possess a comparable ability to manipulate the host immune system. Based on own recent data we hypothesize that the periodontal pathogen T. forsythia is an emerging candidate for research into that direction. Thus, this project is designed to investigate T. forsythia for its host-manipulative ability. 4) Methods. To test our hypothesis, we have chosen a combined cell biology / microbiology approach. We will use a biofilm model consisting of five commensal oral microorganisms into which T. forsythia will be incorporated. To delineate effects caused by this pathogen, we will compare the reactions of different human cells involved in the immune response to the biofilm, once with and once without T. forsythia. We will set-up different biofilm-cell co-culture models and we will specifically investigate the biofilm-induced response in epithelial cells, neutrophils, macrophages, and gingival cells, which form the body`s first line of defense against bacterial infection. We will measure several functional parameters involved in the host response against infection and in the control of microbial growth. 5) Explanation indicating what is new/special about the project. The data obtained in this interdisciplinary project will unravel if and how T. forsythia is involved in manipulating the host immune response and disturbing the balance between the immune system and the oral microbial community. The results of this project might pinpoint novel therapeutic and prophylaxis approaches against periodontitis. Above that, learning about new facets of microbial-host interactions will contribute to increasing our general understanding of how bacteria thrive in our body.

The globally occurring gum disease periodontitis critically involves the pathogenic bacterium Tannerella forsythia acting as a late colonizer of a polymicrobial biofilm, commonly known as dental plaque. We found that this bacterium has a unique cell surface protein glycosylation, which underpins its pathogenicity by influencing the relationship with other biofilm bacteria and the host through dedicated glycobiological and glycoimmunological interactions

a specific sugar acid (nonulosonic acid) seems to be pivotal to these interactions. A comparison of all publicly available genome sequences of periodontitis-associated T. forsythia strains revealed the presence of a distinct gene cluster that underlays protein glycosylation. In contrast, the genome of the novel, putative periodontal health-associated Tannerella species BU063, which is the closest phylogenetic relative to T. forsythia, has a different glycosylation gene cluster lacking genes for sugar acid biosynthesis. We hypothesize that an altered glycome contributes to the difference of periodontal pathogenesis by Tannerella sp. BU063 and provides a new foundation to further understand the genome evolution and mechanisms of bacteria-host interaction in closely related oral microbes with different pathogenicity potential. Initial data indicate differences in surface-layer protein glycosylation and immunogenicity of Tannerella sp. BU063 compared to T. forsythia. Our novel procedures for cultivation and genetic manipulation of Tannerella sp. BU063 make detailed glycobiological investigations possible. We will investigate the glycoinfrastructure of the Tannerella sp. BU063 cell envelope and determine possible implications for the bacterium`s biofilm lifestyle and interaction with the host. Methods include anaerobic bacterial cultivation, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) for glycan structure elucidation, glycoproteomics, genetic manipulation, immune response profiling, biofilm techniques, and imaging. This project generates glycobiology information on a novel oral Tannerella species and aims at delineating glycobiology-mediated mechanisms that could support the association of this bacterium with periodontal health. In relation to the glycobiology knowledge of the pathogenic relative T. forsythia, this project might provide novel insight into the pathogenesis of periodontitis and pinpoint novel therapeutic and prophylaxis approaches against the disease. It also furthers our knowledge on the Bacteroidetes phylum, to which Tannerella species are affiliated, since this bacterial phylum is prominent not only in the oral but also in the gut microbiome. This project is a collaboration between Christina Schäffer (principal investigator

microbiology, glycobiology, biofilm, imaging), Friedrich Altmann (MS), Markus Blaukopf (NMR), all Universität für Bodenkultur, Vienna, Austria, Oleh Andrukhov (immunology

Medical University, Vienna, Austria), and Georgios N. Belibasakis (interaction studies with selected host cells

Karolinska Institutet, Huddinge, Sweden).