טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentTal Ofek
SubjectPost Transcription Regulation of AvrA from Salmonella
Enteritidis by the Regulator CsrA
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Yaron Sima


Abstract

Salmonella enterica serotypes cause diverse clinical syndromes, which might be linked to varieties in virulence factors (effector proteins). These effectors are transported from the bacterial cytoplasm into the host cell and interact with the host's defense mechanisms. One of the effectors secreted by S. enterica is AvrA, which belongs to a family of ubiquitin-like acetyltransferases/ cysteine proteases expressed by animal and plant pathogens. Unlike other effectors that induce the immune response of the host, AvrA is anti-inflammatory. Approximately 80% of S. enterica serotypes contain the avrA gene, but only several serotypes express the AvrA protein under standard conditions. In a previous study in our lab, it was found that AvrA translation is regulated post-transcriptionally by the regulatory protein CsrA. In-vitro experiments showed that CsrA directly binds avrA mRNA, presumably by binding two or three putative binding sites (BS1-3), located in the avrA 5'-UTR: BS2 and BS3 overlap the ribosome binding site and BS1 is located upstream.

This work aimed to provide an evidence that the three putative CsrA binding sites are relevant for AvrA expression and that CsrA regulates AvrA expression via these binding sites in-vivo.

We constructed a set of plasmids containing gfp reporter gene under the native promoter of avrA or under mutated sequences with alternation of one, two or three CsrA binding sites. GFP expression and qRT-PCR analysis of S. Enteritidis PT11 99-847 ΔavrA carrying those plasmids, demonstrated that all three binding sites have a role in AvrA-GFP expression, that this role is dependent on the presence of the other sites, and that the regulation might be in RNA stability level. We suggest that binding to BS1 positively regulates AvrA-GFP expression only in the presence of at least one of BS2 or BS3, however BS1 alone negatively regulates AvrA-GFP expression. A comparison between GFP levels of S. Enteritidis PT11 99-847 ΔavrAΔcsrA and ΔavrA mutants reinforced the importance of BS1, as all the mutants lacking BS1 (single, double and triple) showed significantly higher fluorescence levels when CsrA was absent. In overexpression of CsrA, we also demonstrated that although BS1 is necessary for regulation by CsrA, its influence is dependent on the presence of another site. We also hypothesize that BS2 and/or BS3 are essential in intermediate levels of CsrA and might have a role in high CsrA levels.

We suggest an alternative mechanism of action: when binding BS1 alone, CsrA destabilizes avrA-gfp mRNA and there is a very poor production of AvrA-GFP. In effective levels, CsrA binds to BS1 and bridges towards BS2 or BS3, maybe causes a conformational change, and positively regulates AvrA-GFP production. In higher concentrations, the bridge is destabilized and the structure changes (CsrA maybe binds to additional sites), and AvrA-GFP production is lowered. In high concentrations, there might be also an indirect effect.

This study presents an evidence for the involvement of CsrA in the regulation of AvrA expression in-vivo and for the role of three CsrA binding sites in this regulation. This study also reveals the importance of CsrA in avrA mRNA stability.