טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKatsman Marina
SubjectIdentification of New Effector Proteins from Salmonella
Enterica and Study of their Role in the Bacteria-
Host Interactions
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Sima Yaron


Abstract

     Salmonella enterica uses two type III secretion systems (TTSSs) to inject more than 40 effector proteins into host cells. These translocated effector proteins manipulate various host cell processes, but it is hypothesized that more functional unknown effectors contribute to the host-bacteria interaction.  The identification of new effectors is difficult due to their very diverse sequences and activities. The aims of this study were to identify new effectors, translocated by these two systems, and to investigate their influence on the Salmonella - host cells interactions, using different host models.

     We applied two cycles of a machine-learning classification approach to identify putative effectors in four S. enterica serovars. From the obtained list of suspected effectors, nine top-ranked proteins were investigated for secretion to the medium and translocation to host cells. Cultures of HeLa cells and macrophages were used as host models. Each gene was fused to a TEM-1 domain that served as reporter for translocation, and was expressed in S. Typhimurium wild-type and mutants of TTSS-1 and TTSS-2. Translocation of the investigated proteins was analyzed by microscopy and flow cytometry.

     In total, out of nine suspected proteins, seven proteins were positive for translocation into host cells. STM4158, STM3155, STM1055 were translocated by both TTSSs, SEN1975 was positive for TTSS-1 and the STM2138, a Tir chaperone, was translocated by other, yet-unknown, mechanisms. STM1089 and STM4316 were translocated not only in TTSSs dependent manner but also by a mechanism that involves the flagellar apparatus. In addition, we show that SEN1386 and SEN0532 were negative for translocation to host cells.

     Two new effectors STM1089 and STM4158 were chosen for further analysis of their function. Results demonstrated that they have an effect on the invasion, survival and virulence of Salmonella in the host cells. STM4158 inhibited ROS response in the host cells while STM1089 promoted it. In addition, STM4158 promoted host cells death 24hr post infection, while STM1089 reduced it.

    In this study, the combination of advanced machine learning and experimental methods proved to be a powerful approach for new effectors discovery in Salmonella. This study, which led to identification of seven new translocated proteins, deepens our understanding of the virulence and host evasion mechanisms adopted by Salmonella, and might help to elucidate the molecular underpinning of the differences in degree of virulence among serovars.

       In the second part of the research, we developed a new model of plant host (basil cell culture) and showed that TTSSs have an effect on the attachment, survival, hosts' immune response and virulence of Salmonella on basil cells. Our results demonstrated, for the first time, that (1) some of the effectors are delivered directly into the plant cell (2) three effector proteins: AvrA, STM4158 and STM1089 function in a similar manner in both, mammalian and plant hosts. This can imply on the possibility that Salmonella employs similar effector arsenal to exploit plants and animals as hosts. The findings from this part of the research will improve our knowledge about the mechanisms used by Salmonella in its interactions with plants.