טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKatsman Marina
SubjectIdentification and Characterization of New Type III
Secretion System (T3SS) Effector Proteins in
Salmonella Entrica Serovars
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Sima Yaron


Abstract

Gram-negative pathogens use type III secretion systems (T3SSs) to inject effector proteins into host cells. The translocated effector proteins manipulate host cell processes. The genes encoding the structural components of the T3SSs are conserved among bacterial species and can be identified by sequence homology. In contrast, the sequences of secreted effector proteins are less conserved and are therefore difficult to identify. Salmonella enterica harbors two T3SSs. The aim of this project was to identify and characterize new effectors translocated by these two systems in the foodborne pathogen Salmonella enterica. Identification was conducted using a novel machine learning approach. We received a list of suspicious genes form different Salmonella serotypes and investigated whether they are indeed translocated to the host cells, by using microbiological and molecular biological methods. Each gene was fused to TEM-1 domain that served as reporter for translocation. We constructed plasmids expressing candidate effector-TEM1 fusions, which were introduced into the WT S. Typhimurium strain. HeLa cells and macrophages J774 were infected with the bacteria, and translocation of the effector was analyzed by microscopy and FACS. In order to confirm that the newly identified proteins are secreted by one of the two Salmonella T3SSs, the experiments were repeated with three mutants defective in one of these systems or both. Results revealed that five suspicious proteins are positive for translocation, three of them are translocated by both T3SS-1 and T3SS-2, one seems to be positive for T3SS-1, and Tir chaperone is translocated independently from both T3SS-1 and T3SS-2.

The new effectors were included in the second phase screening of the machine learning algorithms. Indeed phase 2 screening increased the prediction score of few unknown genes. In addition, we showed that two of the new protein effectors have an effect on the viability of HeLa cells and J774A cells and they increased the activity of caspase-3 (apoptosis) in HeLa cells compared with negative controls. Discovering new effectors and their function will deepen our understanding of the virulence and host evasion mechanisms adopted by Salmonella and may provide novel approaches for treating the infections. Moreover, it will provide tools to distinguish between serotypes with different degree of virulence.