Bacterial pathogens are constantly regulating the expression of their genes in response to changing environmental conditions and signals from the host. Timely and adequate levels of gene expression are essential for obtaining nutrients and evading the host immune system. The aim of this thesis was to study regulatory mechanisms of virulence-related genes in the bacterial pathogens Francicella novicida and Streptococcus pyogenes.
The focus of chapter one is on the regulation of the important virulence factor streptolysin S (SLS), which is responsible for the hemolytic phenotype of the human pathogen S. pyogenes. First, we investigated the role of the ribonuclease (RNase) Y in the transcriptional and post-transcriptional regulation of sagA, which codes for the precursor of SLS. We found that RNase Y promotes the production of a small RNA (sRNA) from the sagA transcript. However, no role of RNase Y in the regulation of the sagA transcript at the post-transcriptional level was observed. Yet, RNase Y promotes sagA transcription indirectly, affecting the hemolytic activity in a growth phasedependent manner. Next, we studied the function of sagA 5′ untranslated region (UTR) as a putative cis-acting regulatory RNA. We show that the sagA 5′ UTR contains a secondary structure that may affect the accessibility to the ribosomal binding site (RBS) and that this structure is possibly modulated by direct binding to a ligand. Moreover, our results indicate that removing fragments of the 5′ UTR has a negative effect on sagA expression, possibly by stabilizing the RBS-blocking structure. While investigating the identity of the putative ligand that affects the sagA 5′ UTR structure, we developed a method for testing the activity of riboswitches. Using this method, we validated three predicted riboswitches in S. pyogenes.
In chapter two, we characterized the mechanism by which F. novicida CRISPRCas9 (FnoCas9) represses the expression of bacterial lipoproteins (BLPs), allowing evasion of the host immune system. We show that FnoCas9 is a dual-function protein that, in addition to its canonical DNA nuclease activity, evolved the ability to regulate transcription. In this newly-described mechanism, the non-canonical RNA duplex tracrRNA:scaRNA guides FnoCas9 to the DNA target located downstream of the promoter of the BLP-coding genes (FTN_1103 and FTN_1101), causing transcriptional interference. The endogenous targets contain a protospacer-adjacent motif (PAM) and a sequence that is complementary to scaRNA, promoting FnoCas9 binding. While the mechanism is reminiscent of DNA targeting in the canonical immunity function of CRISPR-Cas9, with scaRNA fulfilling a similar function than crRNA, reduced complementarity between scaRNA and the DNA promotes binding but does not allow cleavage. This system can also be engineered to repress other genes, expanding the toolbox of CRISPR applications.
|Advisor:||Turgay , Kürşad , Landthaler , Markus , Charpentier , Emmanuelle|
|School:||Humboldt Universitaet zu Berlin (Germany)|
|Source:||DAI-C 81/8(E), Dissertation Abstracts International|
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