| AIMStaphylococcus aureus is a leading pathogen that may cause a variety of diseasesranging from moderate to severe skin and soft tissue infections to very serious diseasessuch as septic shock, toxic shock syndrome, or necrotizing pneumonia. As drug-resistantstrains emerge, especially methicillin-resistant S. aureus (MRSA)’s quickly epidemic,making the antibiotics which can treat SA infections much less. In addition, MRSA has anability of biofilm formation which also can resist antibiotics, this further enhance thedifficulties of anti-MRSA therapy. Constantly, the appearance of vancomycin-resistantStaphylococcus aureus (vancomycin-resistant MRSA strains, VRSA), or even thedetection of multidrug-resistant MRSA in recent years, forcing us to explore newstrategies to combat MRSA infections. Inhibition of bacterial Quorum sensing (QS)system can significantly weaken bacterial virulence, reduce the pressure to survive and noteasily induce drug-resistance, so it becomes one of the promising anti-MRSA strategies.The aim of this study is to supress expression of the key molecule agrA in SA’s agr quorum sensing system by antisense technique, to block agr signal pathway, consequentlydepress MRSA pathogenicity, and offering a potential drug target and a new thought tocombat MRSA infection.METHODS1. Design and synthesis of antisense oligonucleotides targeting agrA mRNA: Thesequence of agrA mRNA is obtained from Nucleotide database of NCBI and homologousof agrA among SA strains is performed by BLAST tool. Firstly, using Primer Premier5.0to forecast oligonucleotides(18~24bp) those can be combined to agrA mRNA; secondly, topredict the second structure of agrA mRNA and to calculate free energy of complementaryantisense oligonucleotides through RNA structure4.5;Lastly, combining the results ofPrimer Premier5.0and RNA structure4.5to select the excellent targets. Theseoligonucleotides are modified by LNA and covalently conjugated with the cell penetratingpeptide (KFF)3K(Cell penetrate peptide conjugated locked neucleic acid, PLNA).2. Influence of PLNAs on MRSA growth: CA-MRSA clone LAC(USA300) is usedas subject. The antibacterial effects of the selected PLNAs, PLNA34and PLNA522wereevaluated, by observing LAC’s growth. During test, the concentration of PLNAs is10μM,20μM and40μM respectively, PBS is treated as blank control.3. Decreased expression of MRSA virulent genes by PLNA34and PLNA522: MixPLNA34and PLNA522with LAC respectively, and incubate for5h, then collect LACpellet and supernatant. The transcription of agrA, effector RNAⅢ and downstreamvirulence genes, such as hla,pvl and psm, were analyzed by real-time quantitative PCR andthe expression of α-toxin was detected by western blot. The hemolytic activity ofsupernatant of different groups was analyzed by hemolysis assay. Analyzing influence ofsupernatant on chemotactic and lysis of human neutrophils can indirectly reflect PSMssecretion quantity. During test, the concentration of PLNAs is3.125μM,6.25μM and12.5μM respectively, PBS is treated as blank control.4. In vivo activity evaluation of PLNA34: For the subcutaneous infection model, C57BL/6Jmice were inoculated by subcutaneous injection with mixture of PLNA34and LACor PBS. Animals were examined for skin lesions at24-h intervals for a total of14days; the effect of PLNA34on elimination of LAC in infected skin was observed by measuringthe CFU; The protective effect of the PLNA34on skin was observed by HE staining.RESULTS1. Design and synthesis of antisense oligonucleotides targeting agrA mRNA: BLASTresults indicate that homologous of agrA in different SA strains, even including someSE(~80%), is very high, suggesting agrA could be a perfect, specific antisense target. Withthe help of Primer Premier5.0and RNA structure4.5, we finally select two potentialantisense oligonucleotides, through LNA modified and covalently conjugated with(KFF)3K, resulting in PLNA34and PLNA522.2. Influence of PLNAs on MRSA growth: The absorbance of different concentrationPLNA34groups and PLNA522groups are nearly same with PBS control group and thegrowth curve coincides almost completely, indicating that neither PLNA34nor PLNA522has antibacterial effect in vitro. This is same with the design concept.3. Decreased expression of MRSA virulent genes by PLNA34and PLNA522:Real-time PCR results show that both PLNA34and PLNA522can significantly restraintranscription of target gene agrA(p<0.001), the effector RNAⅢ(p<0.001), as well asdownstream virulence genes hla(p<0.001),pvl(p<0.001), psmα(p<0.001) and psmβ(PLNA34,p<0.001;PLNA522,p<0.01) under the concentration of12.5μM, whencomparing with PBS control group. Whereas, comparing with PLNA522, PLNA34has abetter inhibitory effect on these five genes (p<0.001), and this inhibition has concentrationdependence. Western blot results indicate that the level of α-toxin is greatly diminished byPLNA34in concentration dependent manner. However, PLNA522has little such effect.Hemolysis assay demonstrate that the PLNA522group and PBS control group have nosignificant difference, but PLNA34group has less hemolytic activity (vs PBS control,p<0.01; vs PLNA522, P<0.01). Human neutrophils chemotactic and lysis test also indicatethat the PLNA522group was almost siminar with the PBS control group, whereas,6.25μM and12.5μM of PLNA34groups can significantly decrease the degree ofneutrophils lysis (p<0.05). Besides, all PLNA34groups have less neutrophilschemotactic(3.135μM and6.25μM, p<0.01;12.5μM, p<0.001). In conclude, PLNA34has a better inhibitory effect on agr than PLNA522.4. In vivo activity evaluation of PLNA34: In subcutaneous infection model, PLNA34treated group have significantly less lesions, survival LAC and skin damage than modelgroup, and no abscess formation.CONCLUSION1. Both anti-agrA mRNA PLNA34and PLNA522selectively inhibit expression ofagrA, but PLNA34is better than PLNA522, it can effectively surpress LAC virulence andpromote immune system to eliminate LAC.2. AgrA might be a new target for anti-MRSA infection. Inhibiting AgrA’s expressionor making AgrA unfunctional what can depress agr quorum sensing system might offer anew strategy to combat with MRSA... |
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