Characterization of virulence of Corynebacterium ulcerans (Charakterisierung der Virulenzeigenschaften von Corynebacterium ulcerans) Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Elena Hacker aus Erlangen Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 07. März 2016 Vorsitzender des Promotionsorgans: Prof. Dr. Jörn Wilms Gutachter: Prof. Dr. Andreas Burkovski apl. Prof. Dr. Andreas Tauch Table of contents Table of contents 1 Summary/Zusammenfassung ..................................................................................... 1 1.1 Summary ................................................................................................................. 1 1.2 Zusammenfassung .................................................................................................. 3 2 Introduction .................................................................................................................. 6 2.1 Corynebacteria ........................................................................................................ 6 2.2 Pathogenicity of Corynebacterium ulcerans............................................................. 8 2.2.1 Epidemiology, occurrence and disease symptoms ........................................... 8 2.2.2 Next generation sequencing of C. ulcerans genomes ......................................11 2.2.3 Virulence factors .............................................................................................12 2.2.3.1 Diphtheria toxin and lysogenic corynephages ..........................................12 2.2.3.2 Shiga-like toxin .........................................................................................15 2.2.3.3 Phospholipase D (PLD) ............................................................................16 2.2.3.4 Other pathogenicity determinants of C. ulcerans ......................................17 2.2.3.5 Identification and characterization of virulence factors ..............................22 2.3 Interaction of C. ulcerans with host cells and cell components ...............................24 2.4 Caenorhabditis elegans as infection model system for pathogenic corynebacteria .26 3 Aim of the work ...........................................................................................................29 4 Publications ................................................................................................................30 4.1 Colonization of human epithelial cell lines by Corynebacterium ulcerans from human and animal sources (Hacker et al., 2015a) .............................................................30 4.2 The killing of macrophages by Corynebacterium ulcerans (Hacker et al., 2015b) ...41 4.3 Characterization of DIP0733, a multi-functional virulence factor of Corynebacterium diphtheriae (Antunes et al., 2015a) .........................................................................69 4.4 Caenorhabditis elegans star formation and negative chemotaxis induced by infection with corynebacteria (Antunes et al., 2015b) ..............................................79 5 References ..................................................................................................................90 6 Curriculum vitae .......................................................................................................100 7 Acknowledgment/Danksagung ................................................................................102 Hacker, E. Summary/Zusammenfassung 1 Summary/Zusammenfassung 1.1 Summary The emerging infectious agent Corynebacterium ulcerans has been underestimated as human pathogen for a long time. C. ulcerans was mainly associated with mastitis in cattle, non-human primates, and other animals and is also known to be commensal in various domestic and wild animals. While infections in humans were formerly rarely reported and mainly associated with direct contact to infected cattle, these cases appear to be increasing in different industrialized countries during the last decade. Most often, these infections can be ascribed to zoonotic transmission through pet dogs and cats, although transmission of the bacterium from human to human cannot be excluded. Besides causing a respiratory diphtheria-like illness, C. ulcerans can also cause extrapharyngeal infections in humans, including severe pulmonary infections, skin ulcers, and severe necrotizing fasciitis (Mattos-Guaraldi et al., 2008; Meinel et al., 2014, 2015). Meanwhile, respiratory diphtheria-like illnesses caused by toxigenic C. ulcerans are reported more often in Western Europe than infections caused by the classical causative agent of diphtheria, Corynebacterium diphtheriae. Nevertheless, to date, only little is known about factors besides the diphtheria toxin that contribute to virulence of this pathogen and data elucidating mechanisms it uses for host colonization are missing. In this study, first investigations were made to analyze the interaction of C. ulcerans with host cells, and the contribution of different putative virulence factors in these processes. For this purpose, the two C. ulcerans strains 809, isolated from an 80-year-old woman with fatal pulmonary infection, and BR-AD22, isolated from an asymptomatic dog, were subjected to different cell-based assays. Two genes that have a putative function in virulence of C. ulcerans could be inactivated by insertion mutagenesis in strain BR-AD22. The pld gene codes for phospholipase D (PLD), the major virulence factor of Corynebacterium pseudotuberculosis. In this closely related species, PLD plays a crucial role in dissemination of the bacteria within the host. The resulting mutant in C. ulcerans BR-AD22 was designated ELHA1. In the second mutant strain ELHA3, CULC22_00609, a homolog of C. diphtheriae DIP0733 was inactivated. DIP0733 was found recently to be involved in adhesion to and invasion of epithelial cells and host cell death (Sabbadini et al., 2012; Antunes et al., 2015a). Adhesion and invasion assays with the two epithelial cell lines HeLa (cervix carcinoma cells) and Detroit562 (pharynx carcinoma cells) revealed that C. ulcerans is able to bind to epithelial cells in high amounts and multiply during in vitro infection. Moreover, the bacteria were able to invade the cytoplasm of epithelial cells. Mutant strains of the putative virulence factors phospholipase D and DIP0733 homolog CULC22_00609 showed no influence on colonization under the experimental conditions tested. The observed internalization into Hacker, E. 1 Summary/Zusammenfassung epithelial cells might be an effective mechanism for immune evasion of C. ulcerans supporting the establishment and progress of infections. Scanning electron microscopy of cells infected with C. ulcerans suggested a clustered and localized adhesion pattern, indicating the presence of a limited number of specific cell surface receptor sites. Typical V-shaped bacteria due to snapping division seen in fluorescence microscopy were a sign for growth both on the surface and inside the epithelial cells. Furthermore, as was shown by measurement of the transepithelial resistance of infected Detroit562 cell monolayers, C. ulcerans has a detrimental effect on eukaryotic cells (Hacker et al., 2015a). Once a pathogen has entered its host, it is immediately confronted with the innate immune system, helping the host to eliminate the foreign material again. To investigate the interaction of C. ulcerans with components of this defense system, the human monocytic cell line THP-1, which can be differentiated into macrophage-like cells, was infected with C. ulcerans wild type strains and corresponding mutants. The survival and intracellular replication of the bacteria in this phagocytic cell line was analyzed by counting intracellular CFU after different points in time. In addition, the reaction of the cells to the bacterial infection was studied by determination of cytokine levels, activation of NF-κB, and release of LDH as a sign of cell death. Through FACS analysis, the influence of C. ulcerans on cell death was investigated. We showed that this bacterium is not immediately inactivated, but rather was able to survive inside THP-1 macrophages for several hours and even replicate during the first hours of infection. Therefore, macrophage function is obviously impaired through C. ulcerans infection. This was confirmed by Lysotracker staining and fluorescence microscopy, which exhibited a delay in phagolysosome fusion. Upon infection, THP-1 cells produced high amounts of the cytokines IL-6 and G-CSF and the NF-κB signaling cascade was activated. Furthermore, LDH activity measured in the supernatants of infected cells indicated cell death. This idea was supported by detachment of adherent cells after infection with C. ulcerans. By the use of 7- AAD staining and FACS analysis, it was verified that this species causes a form of cell death in THP-1 cells resembling necrosis (Hacker et al., 2015b). Moreover, the nematode Caenorhabditis elegans was used as model system to study virulence properties of C. ulcerans in comparison with the closely related pathogen C. diphtheriae and the non-pathogenic species C. glutamicum (Antunes et al., 2015b). All these coryneform bacteria could induce star formation in C. elegans, a symptom which was previously described to occur upon infection with natural C. elegans pathogens.