טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentSinuani Fratty Ilana Aline
SubjectGlucanases in Salmonella Enterica and their Role in the
Plant-Bacteria Interactions
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Sima Yaron


Abstract

Food safety is a top public health priority, as unsafe food can cause pathogen infection in the general population at large and in particularly in higher risk populations. One of the major human pathogens causing high numbers of foodborne outbreaks associated with fruit and vegetables is Salmonella enterica. Irreversible attachment of S. enterica serovar Typhimurium to plants was demonstrated on lettuce, tomatoes, green peppers and parsley. Although many studies on the attachment, persistence and survival of Salmonella on plants have been reported, the mechanisms that enable the survival of Salmonella on and in plants have not been entirely resolved.

Previous studies showed that attachment and survival of Salmonella on plants are associated with the bacterial structure, biofilm formation and bacterial secretion systems. We suggest that Salmonella uses cell wall degrading enzymes (CWDE), similar to other phytopathogens, to enhance bacterial survival on/in plants. We screened the Salmonella Typhimurium genome for potential CWDE enzymes and from the list of suspected proteins chose BcsZ and BcsG for further analysis. BcsZ and BcsG demonstrated glucanase activity on carboxymethylcellulose and Avicel substrates. Expression studies demonstrated induction of BcsG by plant polysaccharides and parsley extracts. To examine the impact of BcsZ and BcsG on Salmonella’s survival on/in plants, we constructed S. Typhimurium overexpressing BcsZ and BcsG (pBcsZ and pBcsG) and knockout mutants ∆BcsZ and ∆BcsG, and examined the epiphytic and endophytic survival of these S. Typhimurium strains on parsley leaves. The epiphytic experiments were composed of plant overhead irrigation with contaminated solutions of the wild-type, pBcsZ, pBcsG, ∆BcsZ and ∆BcsG strains, and examination of bacterial survival 7 days post irrigation. We demonstrated that pBcsZ and pBcsG affected the survival of S. Typhimurium on parsley leaves by improving long-term bacterial survival compared to the wild-type and the knockout mutants. For endophytic survival, we infiltrated the same bacterial strains to parsley leaves, and observed long-term bacterial survival up to 14 days when infiltrated with pBcsZ.  

Interestingly, seven days post bacterial inoculation, the leaves infiltrated with pBcsZ developed necrotic lesions upon leaf surface, a phenomenon that has never been observed before with the wild-type or other Salmonella mutants. To understand the cause for the hypersensitive response, we infiltrated the bacterial lysate, and observed necrotic lesions in the leaves infiltrated with the pBcsZ lysate. To understand if the enzyme activity causes the necrotic lesions, we infiltrated separately the denaturized enzymes and the degraded polysaccharides into parsley leaves and observed necrotic lesions on the leaves infiltrated with the degradation products of the polysaccharides.

Since BcsZ is a periplasmic enzyme, we suggest that subpopulation of Salmonella undergoes cell lysis on the plant and that the released BcsZ enzyme degrades the plant cell wall. This enables further survival of Salmonella on parsley plants, but also trigger the plant immune system. Shortly, we suggest that BcsZ and BcsG act as CWDEs in Salmonella and contribute to the plant-Salmonella interactions.