טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMariya Zheleva
SubjectThe Role of T3SS and the Flagella of Salmonella Enterica
Serovar Typhimurium in its Survival on Plants
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Yaron Sima


Abstract

The emergence of large outbreaks linked to the consumption of contaminated plant products with Salmonella is likely one of the most important challenges the food industry and the consumers face. Still, the mechanisms Salmonella cells use to persist in the plant environment as part of their lifecycle, remain unclear. Here great emphasis was placed on understanding how the Type III Secretion Systems (T3SSs) and the flagella contribute to the survival of Salmonella Typhimurium on parsley.

During the infection in human, Salmonella uses flagella for attachment and motility, and utilizes T3SSs to translocate effectors into the host cell to promote colonization. Thus, our main goals were to investigate the importance of T3SSs and flagella in the attachment and survival of S. Typhimurium on/in parsley plants. The role of these virulence factors was determined by comparing the wild-type with its mutants, with deleted prgH, ssaK and flgE genes, essential for the construction and activity of T3SS-1, T3SS-2 and the flagella, respectively. Additionally, a double mutant, impaired in both T3SSs and a triple mutant impaired in both T3SSs and the flagella were used. For attachment experiment, we immersed harvested parsley in contaminated water containing Salmonella wild-type or mutants. S. Typhimurium was found to attach harvested parsley leaves with an average of 6.5 (±0.2) log CFU/g-1, with no detectable differences in the attachment between Salmonella wild-type and mutants. Moreover, ΔprgH, ΔflgE, ΔssaK, ΔssaKΔprgH, and ΔssaKΔprgHΔflgE did not affect enhanced proliferation of S. Typhimurium on harvested parsley after storage. This is indicative that, the successful attachment and proliferation of Salmonella on harvested parsley did not appear to be associated with T3SSs and the flagella. Confocal laser scanning microscopic images revealed that in absence of active T3SSs, S. Typhimurium uses various strategies in order to internalize parsley plant leaf. One of the key strategy is forming aggregates in the vein regions.

We carried out a comparison between epiphytic and endophytic survival of S. Typhimurium on and in growing parsley plants following spray irrigation/injection with contaminated water containing Salmonella wild-type or mutants. When mutants were sprayed, populations of ΔprgH decreased faster but all tested mutants could still be retrieved. Indicating that, functional T3SS-1 contributes to epiphytic survival of S. Typhimurium on the parsley phyllosphere. S. Typhimurium was injected to the leaves and its endophytic survival on parsley plants was investigated. During 7 days ΔprgH, ΔflgE, ΔssaK and ΔssaKΔprgH survived  as the wild-type. Moreover, the increased levels of the mutant defective in both secretion systems and the flagella, highlighted the possibility of S. Typhimurium to use additional mechanism to survive as an endophyte in growing plants. Mutants defective in both secretion systems and the flagella promoted forming of tissue damage, which indicates that plant colonization by S. Typhimurium is indeed an active process.

The outcome of our work demonstrated that, during Salmonella-plant interactions, T3SSs and flagella are not essential for the attachment, internalization and survival of the bacteria on/in parsley plants.