Antisense RNA Enhanced Sensitivity of Methicillin-resistant Staphylococcus Aureus to Hydrogen Peroxide


 Background: Staphylococcus aureus (S. aureus) is the leading cause of various infective diseases including topical soft tissue infections. Due to an increasing prevalence of the methicillin-resistant Staphylococcus aureus (MRSA), S. aureus infection become more challenging to being treated over recent years. The goals of this study were to investigate the roles of antisense yycF/G RNAs in the regulation of the biofilm formation and pathogenicity. Methods: MRSA ASyycF/G overexpression mutants were constructed. The biofilm biomass was determined by crystal violet microtiter assay and scanning electron mi-ceroscopy (SEM). Quantitative RT-PCR and western blotting analysis were used to detect the transcripts and translations of biofilm-related genes. The effects of the antisense RNAs (ASyycF/G) strategy on the susceptibility of biofilm-producing S. aureus to hydrogen peroxide were also investigated. Result: The ASyycF/G transcript leaded to reduction in the biofilm formation. Overexpression of ASyycF/G inhibited the transcripts and translations of biofilm-related genes. The sensibility to vancomycin was improved in ASyycF/G overexpression MRSA. Conclusions: The biofilm biomass and the transcripts of the pathogenicity associated genes decreased in the ASyycF/G overexpression mutant. Thus, the current evidence may provide a supplementary strategy for managing MRSA infections.


Introduction
Staphylococcus aureus (S. aureus), as Gram-positive opportunistic pathogen, is the leading cause of various infective diseases including topical soft tissue infections, osteomyelitis, and even high mortality endocarditis [1]. Due to an increasing prevalence of multi-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), S. aureus infection become more challenging to being treated over recent years [2]. Bio lm formation of MRSA is responsible for persistent infections and di cult to being eradicated as bio lm conditions which is much more resistant to environmental stimulus. Hydrogen peroxide (H 2 O 2 ), as a common biocide, is used for cleaning and debriding chronic wound infections.
However, the activity of H 2 O 2 is signi cant decreased in against bio lm with drug resistant bacteria.
Thus, targeting bio lm has become an alternative strategy to attenuate the pathogenicity of bacterium.
In bacteria, two-component signal transduction systems (TCSs) play an essential activity on adaptation to environment changes [3]. In S. aureus, 16 TCSs including the only essential YycFG are encoded contribute to the pathogenicity and virulence [4][5]. Commonly YycFG the only essential TCS also known VicRK/WalRK TCS was consisting of a sensor histidine protein kinase YycG and its cognate response regulator YycF [6][7]. Our previous study showed YycFG could modulate extracellular polysaccharide (EPS) synthesis via ica operon, which is associated with bio lm construction [7]. Hence, YycFG TCS represents a promising target to modulate S. aureus bio lm.
An antisense oligonucleotide can hybridize to the target mRNA by base-pairing recognition [9]. It can inhibit translation of the corresponding gene by targeting sequences within pre-mRNA as a kind of antimicrobial oligonucleotides to co-administered with biocides [9][10]. The yycF gene, as the rst gene of yycFG operon [11], encodes YycF response regulator. From this point we designed an antisense yycF (ASyycF) according to yycF sequence to inhibit the expression of yycF, which has a positive association with ica expression [7]. We hypothesis this ASyycF could enhance H 2 O 2 bactericide effect on methicillinresistant S. aureus by depression the construction of ica-dependent bio lm as a novel antimicrobial agent for infection elimination.

Methods And Materials
Bacterial strains and bio lms growth conditions Methicillin-resistant S. aureus strain ATCC43300 provided by the Department of Laboratory Medicine (West China Hospital, Sichuan University, Chengdu, China) was cultured in tryptic soy broth (TSB). After overnight incubation at (37ºC, 5% CO 2 ). Five hundred micro lters of S. aureus overnight suspension was inoculated into 10 mL fresh TSB medium to mid-logarithmic phase with OD 600 value at 0.5. For the formation of bio lm, the sterilized glass disks (diameter in 14 mm) were applied for 24 hours bio lms for further analyses according to our previous study [12].

Construction of S. aureus antisense yycF/yycG overexpression strains
Recombination of plasmid pDL278 containing antisense yycF or yycG was conducted by inserting antisense yycF (ASyycF) or yycG (ASyycG) sequence into restriction sites between BamHI and EcoRI by Sangon Biotech (Shanghai, China). According to our previous study, an ASyycF overexpression methicillin-resistant S. aureus (ASyycG mutants) or an ASyycG overexpression methicillin-resistant S. aureus (ASyycF mutants) was constructed by adding recombinant pDL278 ASyycG or ASyycF plasmid. The empty pDL278 plasmid did not exert any effects on the viability of S. aureus [12].
Analysis of gene expression using quantitative real-time PCR (qRT-PCR) MRSA cDNA Synthesis Kit (Thermo Scienti c). The quantitative real-time PCR assays were applied with LightCycler 480 system (Roche, Basel, Switzerland) with the primers listed in Table 1 with the 16Sr RNA gene as an internal control. Each sample was analyzed in triplicate, and the threshold cycle values (CT) were quanti ed [7]. Table 1 Sequences of primers in this study

Western Blotting
For protein extraction, the bacterial cells including MRSA + H 2 O 2 _group, ASyycG + H 2 O 2 group, and ASyycF + H 2 O 2 group were mechanically disrupted and collected by centrifugation as previously described [12]. For western blot analysis, the collected protein was probed with puri ed YycG-and YycFspeci c antibodies (1:1000, HuaBio Biotechnology, Hangzhou, China). A BioRad GS-700 Imaging Densitometer was used to determine the signal density of Western blot bands for comparation.
Bio lms exposure to hydrogen peroxide The microtiter dish assay and epi uorescence staining for bio lm biomass The microtiter dish assay was applied to evaluate the biomass of treated bio lm after cultured with another 24 hours with crystal violet (CV) following previous protocol [12]. The dye bound to the bio lms was transferred into a new plate and the absorbance was measured with a microplate reader (ELX800, Gene) under OD 600 nm.
For epi uorescence staining, the bio lms were labeled with SYTO9 (LIVE/DEAD Bacterial Viability Kit reagent; BacLight, Invitrogen, Grand Island, NY, USA); live cells were stained green, while dead cells appeared red. The cells were visualized using epi uorescence microscopy (Nikon Eclipse TE-2000S, Melville, NY) at 40×magni cation. Notably, three random elds in each specimen were selected.
Characterizing bio lm morphologies

Data analysis
All statistical data were analyzed in SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The Shapiro-Wilk test was used to analyze the distribution of data, and the Bartlett test was used to determine the homogeneity of variances. For parametric testing, we adopted a one-way ANOVA analysis to assess the statistical signi cance of variables followed by the Tukey test. The signi cant differences of data were set at P < 0.05.

Results
Antisense yycF regulated the bio lm associated genes and protein in MRSA Quantitative RT-PCR analyses demonstrated that the expression levels of the yycG and yycF genes in were signi cantly reduced, which may be attributable to the overexpression of ASyycF in the S. aureus treated with H 2 O 2 . Furthermore, the expression levels of icaA related to the oxide stress reaction and bio lm-associated ica genes were more signi cantly reduced by ASyycF overexpression in the ASyycG strains treated with H 2 O 2 than in the MRSA parent strains (P < 0.05; Fig. 1A). Consistently, the YycF protein expression was signi cantly downregulated in the ASyycF group treated with H 2 O 2 compared with the MRSA parent strains (Fig. 1B). These results indicated that antisense yycF inhibited the production of the YycF, which may have contributed to reductions in downstream icaA gene expression.

Antisense yycF sensitized MRSA to H 2 O 2 intervention and vancomycin antibiotic
The SEM results similarly showed the bio lm construction in ASyycF + H 2 O 2 group most sparse than other groups ( Fig. 2A). Vancomycin is the primary option for MRSA infections. By E-test, the sensitivity of MRSA to vancomycin decreased from 5 to 3 mg/L after ASyycG overexpression and 5 to 1 mg/L after ASyycF overexpression (Fig. 2B). These results also showed that antisense yycF was much more effectively than antisense yycG on enhance the susceptibility of MRSA to H 2 O 2 .
The antibacterial effect of H 2 O 2 was signi cantly enhanced by ASyycF and ASyycG compared with MRSA group even after 24hrs intervention with H 2 O 2 and culture in TSB (Fig. 3A). Quantitively, we evaluated the ability of the MRSA strains to form bio lms after 24hrs intervention with H 2 O 2 and culture in TSB. Immuno uorescence density for the dead/live ratios exhibited ASyycF + H 2 O 2 group was at 0.3 around (Fig. 3B). The biomass was quanti ed via a microtiter dish assay, and ASyycF + H 2 O 2 group exhibited more reduced bio lm formation compared with MRSA group from 1.4 to 0.8. Similarly, AsyycG + H 2 O 2 group reduced bio lm formation compared with MRSA group, but less effective than ASyycF + H 2 O 2 group (Fig. 3C).

Discussion
Staphylococcus aureus is a major human pathogen, which is responsible for a wide range of infective diseases. With the development of antibiotic resistant strains such as MRSA, S. aureus-based infections are becoming more di cult to being treated [13]. The propensity of bacteria to produce bio lms was one of most essential factors, which contributed to the pathogenesis and resistance [14]. In this study, antisense yycF strategy was applied to MRSA strains to signi cantly down-regulate bio lm production associated YycFG pathway, which could as a promising strategy to overcome this bio lm developed infections.
Two-component systems (TCSs) were ubiquitously found in bacteria [15]. The highly conserved YycFG (also known as WalRK) TCSs reveal a major role in controlling bio lm formation in low-G + C grampositive bacteria including S. aureus [7]. Antisense is a kind of complementary sequences hybridized to their target mRNA and inhibits the transcriptions of the corresponding genes [16]. In our study, the expression of YycFG TCS and bio lm production associated ica genes were accordingly down-regulated by antisense yycF or yycG. In S. epidermidis, a classical staphylococci bacterium, bio lm formation could also be inhibited via the blockade of extracellular domain of the YycG protein [17][18]. From this point, YycFG TCS may be a potential candidate for the eradication of staphylococci infections. YycG proteins act as a sensor sensing environmental signals and YycF could directly regulate different sets of vital functional genes by binding to the promoter regions [19]. Our results also showed antisense yycF not only depresses the sensitive of MRSA strains to environmental clues but also disrupted cytoplasmic adaptive activities by downregulation both YycF and YycG expressions.
In S. aureus, the only polysaccharide intercellular adhesin (PIA) is associated with ica locus [20]. PIA strongly contributes to bio lm formation leading resistance to antimicrobial agent. According to our previous study, ica expression was controlled by YycFG [7]. In our study, the antisense could cause a signi cant bio lm reduction by inhibiting YycFG as well as ica expression. Two-component signal transduction systems were essential in response to environmental stimuli, involving adaptation to oxidative stress [21]. Therefore, there will be an adaptive regulation when occurred with H 2 O 2 treatment.
In present study, YycFG expression can be signi cantly decreased by ASyycF, even under oxidative stress intervention with H 2 O 2 . From this point, ASyycF has a potential as a kind of enhancer to improve the antibacterial e ciency of H 2 O 2 by suppression resistance and adaption to oxidative stress. S. aureus is the leading cause of osteomyelitis with bio lm formation on the destructed bone matrix or necrotic bone, 1,000 times more tolerant to antibiotic and greatly contributions to cause of treatment recalcitrance were identi ed [22]. Without the shelter of bio lm, the susceptibility of pathogen was reversible [23]. The vancomycin was a gold treatment for clinical MRSA infections [24]. With the interruption of antisense, MRSA bio lms were signi cantly reduced without the shelter of bio lm, which enhanced the sensitivity of vancomycin. In addition, the resistance of hydrogen peroxide, which was a common biocide used for cleaning and debriding infections was destructed synergically by the antisense strategies [25].
Antisense oligonucleotides provide a therapeutic potential for treating infection disease [26]. However, nucleases are capable to degrading the antisense oligonucleotides rapidly, which limit their e cacy [27]. Hydrogel scaffolds such as alginate-based hydrogel has been designed for infection inducing bone defects [28]. They could be employed as gene carrier's protection from the in ammation-mediated degradation and control release [29]. Our previous study showed nano sized platform such as graphene oxide as a kind of nano knife could signi cantly improving the transformation effectiveness of antisense oligonucleotides [30]. Therefore, further investigations on composition nano material functioned hydrogel will widen the possibility of antisense strategy for clinical infection control.

Conclusions
In the current study, overexpression of ASyycF/G signi cantly downregulate bio lm formation and the transcripts of the pathogenicity associated genes. Also, the sensibility to vancomycin was improved in ASyycF/G overexpression MRSA. Furthermore, the antisense RNAs, as post-transcriptional regulators, reveal a potential supplementary strategy for managing MRSA infections. Consent for publication: All authors understand that the text and any pictures or videos published in the article, and give our consent for information to be published in JOSR.  ASyycF/G modulated the bio lm organization and antibiotics sensitivity A. SEM images of MRSA strains after ASyycF or ASyycG overexpression; B. E-test for the sensitivity of MRSA to vancomycin.