الفهرس 
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Abstract
Staphylococcus aureus is a Gram-positive opportunistic pathogen of a global concern that colonize in the nares and on the skin of approximately 30% of population. Colonization acts as a predisposing factor contributing to the increased rates of infections. S. aureus is highly dangerous worldwide bacteria that can cause variety of diseases throughout the human body, which ranges in severity from soft tissue infections to highly life-threatening diseases such as necrotizing fasciitis, hemorrhagic pneumonias, osteomyelitis, endocarditis, toxic shock syndrome, bloodstream infections. S. aureus compromises multiple cell-wall associated elements function to facilitate the S. aureus adhesion to the extracellular matrix proteins, fibrin and platelets.
Intriguingly, S. aureus experiences varied range of environmental temperatures during both its survival and /or virulence activity to colonize and/or infect the body host, respectively.
A long term studies on regulation of bacterial genes have revealed that RNA molecules introduce a promising target for novel therapeutic. In particular, the molecules that could potentially stabilize the inhibitory structure of RNAT within a virulence-associated gene would abolish or diminish the ability of S. aureus to cause diseases. RNATs are cis-acting ribo-regulators proven to mediate target gene expression that substantially implicated in multiple key bacterial processes such as, stress-response, nutrient acquisition and virulence, in response to ambient temperature fluctuation. This study is the first of its kind to identify and characterize RNATs in S. aureus that expanded the foundational understanding of RNATs via direct investigation the role of this increasingly appreciated class of regulators in monitoring gene regulation in S. aureus.
During this study the following was done:
• Preliminary studies were accomplished by in silico analysis and compared with the RNAseq data to predict RNATs within the chromosome of S. aureus in CA-MRSA strain USA300.
• Prioritized list of 10 putative virulence-associated RNATs was generated.
1. The oligonucleotides, containing the nucleic acid sequence composing each putative S. aureus RNAT with compatible overhang, were ligated between the constitutive promoter and ATG-less reporter gfp gene of the plasmid pXG-10.
2. The annealed oligos were cloned into plasmid pXG-10 previously restriction digested with NsiI and NheI.
3. Each reporter plasmid was then introduced into E. coli DH5α and then the resulted strain was cultured to the mid-logarithmic phase of growth at different temperatures; 25°C and/or 30°C, 37°C and 40°C.
• The total RNA and total protein were isolated form each culture and the relative amount of the gfp transcript and Gfp protein were then measured using quantitative real-time PCR and western blot analyses to determine the functionality of putative S. aureus RNATs and the ability of the putative RNATs to confer temperature-dependent post-transcriptional regulation was tested.
• cidA RNAT was selected to continue the study.
1. The 5’- rapid amplification of cDNA ends (5’-RACE) was performed to identify the transcriptional start site of cidA 5’UTR.
2. RT-reverse transcriptase was performed to confirm the RNAseq data that revealed two transcripts of cidA 5’UTR; cidA (36 nt) and long cidA (82 nt).
3. Mutational analysis of the long cidA RNATs that have shown thermal-dependent post-transcriptional regulation were performed by creating stabilized and destabilized reporters to investigate the contribution of the predicted RBS-sequestering inhibitory structure within each putative RNAT in mediating temperature-dependent post-transcriptional regulation.
4. The 5’UTR of long cidA RNAT was cloned in the S. aureus under regulation of the native promoter.
5. The cidA-gfp-promoter insert was generated via SOEing PCR reaction and then the resulted construct cidA-gfp-promoter insert was ligated into plasmid pZero-Blunt TOPO.
6. The reporter was then inserted into integration vector pJC1112, both were previously digested with EcoR1enzyme.
7. The pJC1112-prom-cidA-gfp was expressed into S. aureus RN9011 (RN4220 containing the integrase plasmid pRN7023 by electroporation and then was sequence for the correct insert. The plasmid was then integrated onto the S. aureus RN9011 chromosome within S. aureus pathogenicity island 1 (SaPI1) to create the strain SAPI1::cidA-gfp.
The obtained findings could be summarized in the following:
1. Ten putative RNATs were successfully generated to be tested for the functionality and if they will show post-transcriptional dependent thermoregulation.
2. While five of the ten putative RNATs named sspB (the long and the short), ssaA, SAUSA300_1056, SAUSA300_1973, SAUSA300_0223 did not show any temperature-dependent regulation, five putative RNATs named cidA, SAUSA300_1052, SAUSA300_0651, clfB and aur were able to confer varied post-transcriptional temperature-dependent regulation.
3. Obviously, cidA and aur RNATs were the ones that show the most significant regulation distinct form the other functional RNATs SAUSA300_1052, SAUSA300_0651 and clfB.
4. cidA RNAT was selected for further investigations in order to confirm its functionality and it is role in S. aureus bacteria.
5. Two transcriptional start sites were successfully identified for the cidA and long cidA RNAT.
6. Two transcripts of cidA 5’UTR; cidA (36 nt) and long cidA (82 nt) that was initially predicted by RNAseq data was confirmed using RT-PCR.
7. Both mutant constructs; stabilized and destabilized long cidA RNAT were successfully generated and have demonstrated the expected results by which means reduction and increasing in the amount of Gfp protein, respectively, but had no impact on the gfp transcript.
8. The long cidA RNAT integrated onto the S. aureus RN9011 chromosome, representing the native host, has successfully conferred the same thermoregulation that was shown in E. coli-based study system
9. The relative abundance of total cidA transcript and long cidA transcript were measured by the quantitative real-time analysis which showed that long cidA transcript represent 3% of the total cidA transcript pool in wild-type S. aureu