Novel cytotoxic glycosyltransferase effectors of Legionella Abstract: Legionella pneumophila and Legionella longbeachae are Gram negative bacteria which cause Legionnaires disease a severe form of pneumonia. The pathogens replicate inside host cells. Therefore the bacteria produce a large array of effectors which are secreted by the Dot Icm (Defective in organelle trafficking Intracellular multiplication) type 4 secretion system (T4SS) into the cytosol of the target cell. Many of the injected effectors possess enzyme activities and thereby interfere with host cell functions to promote intracellular bacterial multiplication. Some L. pneumophila effectors (e.g. Lgt1 3 and SetA) harbor glycosyltransferase (GT) activities and share structural similarity with clostridial glucosylating toxins which are the prototypes of glucosylating virulence factors. Aim of the study was the identification of novel putative Legionella glycosyltransferases by genome sequence analysis and their characterization by biochemical molecular and cell biological methods. In the first part of the thesis the L. longbeachae protein Llo1578 was analyzed which shares amino acids sequence similarity with the Lgt glucosyltransferases from L. pneumophila. Llo1578 displayed in vitro glucohydrolase and autoglucosylating activities using UDP glucose as a sugar donor. Expression of Llo1578 in yeast exhibited cytotoxic effects which were dependent on the glucosylating activity of the protein. Thus the variants of Llo1578 with the mutations in the identified catalytic residues W23 D126 D142 N177 and W326 did not affect yeast growth. However in contrast to the Lgt glucosyltransferases Llo1578 did not affect in vitro translation and did not modify EF1A the substrate of the Lgt effectors. Therefore this study suggests that Llo1578 likely belongs to a new group of Legionella glucosyltransferases with an unknown acceptor specificity. The second part of the thesis was focused on the novel L. pneumophila effector LtpM (Legionella translocating protein M). The study revealed that LtpM is a two domain protein which consists of an N terminal glycosyltransferase domain and a C terminal phosphatidyl inositol 3 phosphate (PI3P) binding domain. My studies showed that the N terminal domain of LtpM is responsible for its glucohydrolase activity with UDP glucose as the preferred activated sugar. The affinity of LtpM for PI3P was determined by surface plasmon resonance (SPR) spectroscopy and revealed a Kd value of 591 ± 117 n ...