IMPACT OF THE GLOBAL REGULATOR SARA ON TOXINS PRODUCTION IN STAPHYLOCOCCUS AUREUS

UAMS-1 wild type clinical strain, its sarA mutant strain and a revertant strain. Genes encoding for proteases (including sspA, aur, scpA and sspB ) , genes encoding for hyaluronidases (including hysA 1 and hysA 2) and gene encoding for staphylokinase ( sak ) were upregulated in response to the sarA mutation. On contrary, genes encoding for lipases (including lip1 and lip2 ) were downregulated. Moreover, the quantitative production of protease, hyaluronidase and staphylokinase was significantly increased due to the sarA mutation. On the other hand, the quantitative production of lipase was significantly altered. Overall, our findings indicate that SarA is an effective global regulator of toxins production that can suggest new prospective therapeutics for the treatment of S. aureus infections.


INTRODUCTION
In the clinical context, Staphylococcus aureus (S. aureus) is considered the most pathogenic organism in the Gram-positive Staphylococcus genus (Bitrus et al., 2018).It is a commensal bacterium, which colonizes the majority of the human population (Tong et al., 2015).S. aureus becomes opportunistic when it gains access to open wounds, or when it colonizes populations suffering from lowered immunity including immunocompromised patients, diabetics, elderly people and children (Tong et al., 2015;Bitrus et al., 2018).This opportunistic pathogen can lead to high morbidity and mortality.S. aureus can cause a vast variety of diseases ranging from mild and moderate skin infections to more severe and lifethreatening infections such as endocarditis, food poisoning, osteomyelitis and toxic shock syndrome (Becker, 2018;Hamzah et al., 2019).Disease severity and the multiple pathogenic implications of S. aureus are attributed to the diversity of virulence factors that act in a complicated harmonious manner such as the production of biofilm, surface proteins, exfoliative toxins, exoenzymes and exotoxins (Connolly et al., 2017).Staphylococcal ability to produce these virulence factors enables its attachment to the host tissues and evading the host immune system to subsequently cause its cellular and systemic toxic effect (Ionescu et al., 2015).Indeed, S. aureus produces many potent exotoxins, co-factors and exoenzymes, among which, proteases, lipases, staphylokinase and hyaluronidases are especially important  .Furthermore, staphylokinase is a 136 amino acid long co-factor found associated with the cell surface and in the environment of cell culture.It can bind to host antibacterial peptides including α defensins and revoke their antibacterial action.It does not have proteolytic ability on its own, but its activity arises after the formation of 1:1 complex with plasmin to form Sak-plasmin active complex that converts plasminogen (PLG) into plasmin with broad proteolytic range (Pietrocola et al., 2017; Tam & Torres, 2019).Besides, the ability of S. aureus to go through tissues rich in hyaluronic acid (HA) is based on its secreted hyaluronidase enzyme or hyaluronate lyase that was previously known as the spreading factor.Hyaluronidase is encoded by hysA1, however, in some S. aureus strains, another type of hyaluronidase enzyme was found that is about 75.9% identical to hyaluronate lyase 1 and is encoded by hysA2 (Tam & Torres, 2019).
In the current study we depicted effect of the global regulator SarA on protease, lipase, staphylokinase and hyaluronidase production in S. aureus.This was fulfilled by comparing the gene expression as well as quantitative production of these toxins between wild type, sarA mutant and revertant S. aureus strains.

Bacterial strains and culture conditions
S. aureus strains utilized in this study are described in Table 1 (Beenken et al., 2003;Morrison et al., 2012).Tryptic soy broth (TSB) was used for routine cultivation of S. aureus strains.Moreover, extraction of toxins and their quantitative assay were carried out in TSB.
Staphylococcus aureus (S. aureus) is a Gram-positive bacterium that is responsible for many infectious diseases in human, some of which may be fatal.The regulation of toxins production and virulence factors in this pathogen is controlled by many global regulators including the staphylococcal accessory regulator, SarA.In this study, we outlined the influence of SarA on toxins production in S. aureus.The expression of genes encoding toxins was measured using real-time PCR and toxins production was quantitatively assayed.Gene expression and quantitative toxins production were compared in three staphylococcal strains; UAMS-1 wild type clinical strain, its sarA mutant strain and a revertant strain.Genes encoding for proteases (including sspA, aur, scpA and sspB), genes encoding for hyaluronidases (including hysA1 and hysA2) and gene encoding for staphylokinase (sak) were upregulated in response to the sarA mutation.On contrary, genes encoding for lipases (including lip1 and lip2) were downregulated.Moreover, the quantitative production of protease, hyaluronidase and staphylokinase was significantly increased due to the sarA mutation.On the other hand, the quantitative production of lipase was significantly altered.Overall, our findings indicate that SarA is an effective global regulator of toxins production that can suggest new prospective therapeutics for the treatment of S. aureus infections.

Measurement of bacterial growth
S. aureus strains were inoculated in TSB and incubated at 37°C overnight on a shaker incubator adjusted at a speed of 150 rpm.Thereafter, an aliquot from each previously grown cultures was subcultured into fresh TSB and subsequently incubated with shaking at 37°C.The bacterial cell density and growth rate were monitored by determining the optical density of the cultures at 600 nm (OD600) every 60 minutes to establish the growth curve and to determine the bacterial growth phases.

Real-time PCR (rt-PCR)
S. aureus strains were inoculated in TSB and incubated at 37°C with shaking, and RNA isolation was established during the log phase of bacterial growth.Cell pellets were harvested by centrifugation at 10000 rpm for 5 minutes at 4°C.Then, the resultant cell pellets were mixed with 300 µL of triazole reagent (TRI reagent ® , Sigma-Aldrich, USA) and 150 mg glass beads (212-300 μm, 50-70 U.S. sieve, unwashed, Sigma-Aldrich, USA).The reaction mixtures were heated in a heating block adjusted at 60°C for 20 minutes with vortexing every 5 minutes, and then were vortexed for 30 minutes.Afterwards, the cells were incubated in a -80°C freezer for 3 minutes, then incubated at 37°C for another 3 minutes.This cycle of freezing and thawing was repeated 3 times to disrupt the cell integrity and was followed by centrifugation at 6000 rpm for 5 minutes at 4°C.Following the TRI reagent ® manufacturer's protocol, RNA was precipitated, air dried for 10 minutes and resuspended in 20 µL of RNase free water.RNA samples were treated with DNase set (Qiagen, Hilden, Germany), according to the manufacturer's protocol, and then were kept at -80°C.The quality of isolated RNA was checked and its concentration was measured using NanoDrop (Thermo Fisher Scientific inc., Massachusetts, USA).Using SensiFAST™ cDNA synthesis kit (Bioline, Bloomberg, USA), synthesis of cDNA was performed according to the manufacturer's guidelines from the purified RNA.Real-time PCR (rt-PCR) was conducted using 5x HOT FIREPol ® EvaGreen HRM Mix (Solis BioDyne, Tartu, Estonia) according to the manufacturer's guidelines and the Rotor-Gene Q (Qiagen, Hilden, Germany).Each reaction mixture consisted of 4 μL of 5x HOT FIREPol ® EvaGreen HRM Mix, 2 μL of template cDNA, 12 μL of sterile water (PCR grade), 1 μL of forward primer (250 nM) and 1 μL of reverse primer (250 nM) (Table 2).In every PCR run, negative control reaction mixtures were included and were prepared by excluding the reverse transcriptase enzyme or cDNA template from the reaction mixture.In each run, a pool (8 µL) was created from the cDNA of tested strains, and 4 dilution points from this pool were included in each run to establish a standard curve.PCR was carried out according to the conditions; primary activation for 15 minutes at 95°C.Then, 45 cycles of denaturation for 12 seconds at 95°C, annealing for 20 seconds at the appropriate temperature and extension for 20 seconds at 72°C.Amplification specificity was determined from the amplicon melting curves.16S rRNA was used as a house-keeping gene to normalize the expression of tested genes.The RNA concentration of each sample was calculated through comparing Ct of each gene sample to the established standard curve.In these experiments, the housekeeping gene was used as an endogenous reference gene, where the level of expression of each gene in different samples was calculated compared to the house-keeping gene by the comparative method (Ct) using UAMS-1 as a calibrator sample (Schmittgen & Livak, 2008).0.4 mL of the supernatants.The reaction mixtures were incubated for an appropriate period of time at 30°C.Thereafter, 1.4 mL of 5% trichloroacetic acid (TCA) was added to cease the reactions.The reaction mixtures were centrifuged at 5000 rpm for 5 minutes at 4°C and the obtained supernatants were added to equal volumes of 0.5 M NaOH.Finally, the absorbance was determined at 440 nm.In these experiments, TSB was used as a negative control.

Assessment of the lipase activity
Reaction mixtures were prepared by mixing 20 µL of the prepared bacterial supernatants (as described above in protease assay), 75 µL of 20 mM p-nitro phenyl palmitate (P-NPP) and 0.1 M tris HCl buffer (pH 8.5) to a final volume of 3 mL.The reactions were incubated for 10 minutes at 37°C, and then were frozen at -20°C for another 10 minutes to cease the lipolytic action.The yellow color of the reaction product was spectrophotometrically measured at 410 nm (Sarkar et al., 2012; El-baz et al., 2017).In these experiments, TSB was used as a negative control.

Assessment of the staphylokinase activity
Staphylokinase extraction was carried out according to (Shagufta Naseer et al., 2014).In a 10 mL of TSB, colonies of S. aureus stains were inoculated and incubated on a shaker incubator at 150 rpm overnight at 37°C.Then, OD600 of the cultures was adjusted to 0.2-0.275.In a 10 mL of TSB, 200 μL of the adjusted bacterial suspensions were subcultured and incubated for 24 hours at 37°C on a shaker incubator at 200 rpm.Then, the cultures were centrifuged for 15 minutes at a speed of 3000 rpm at 4°C.To the separated supernatants, 40% (v/v) cold absolute alcohol was added and they were frozen at -20°C for two days to completely precipitate the staphylokinase enzyme.Staphylokinase enzyme was collected as pellets after centrifugation of the enzyme precipitate at 10000 rpm for 20 minutes at 4°C.The pellets were subsequently dissolved in 0.1 M phosphate buffer solution (pH 7.2) (one-tenth of the original supernatant volume) and stockpiled at -20°C for later use.Thereafter, staphylokinase enzyme quantitative assay was established.Initial weights of Eppendorf tubes were measured (X1) and 500 μL of freshly collected human blood was transferred to the Eppendorf tubes, and incubated for 60 minutes at 37°C to enable blood clotting.After complete clotting of blood samples, the produced serum was aspirated and the Eppendorf tubes were reweighed (X2).The clot weight was determined by calculating the difference between the second and the first weights (X2 -X1).To each Eppendorf tube, 250 μL of the previously prepared enzyme extract was added, and then the tubes were incubated at 37°C for 2 hours to evaluate clot lysis.After lysis, every Eppendorf tube was weighed subsequent to fluid removal.For each sample, the difference in clot weights (before and after lysis) was calculated.Afterwards the clot lysis percentage, which represents the staphylokinase plasmolytic action was calculated.In these experiments, TSB was used as a negative control.

Assessment of the hyaluronidase activity
Hyaluronidase quantitative assay was executed on previously separated bacterial supernatants (as described above in protease assay) using 1% agarose plates containing 0.4 mg/mL of hyaluronic acid (Sigma, H-1504) in 0.3 M sodium phosphate buffer (pH 5.3) and 1% bovine serum albumin (BSA).Under aseptic conditions, wells were cut in agarose to embody 100 µL of the supernatant of tested strains.After 24 hours of incubation at 37°C, 2 M acetic acid was added to flood the plates.The undigested hyaluronic acid was detected as opaque precipitation in conjugation with BSA behind the clear zones of the hydrolyzed hyaluronic acid.The clear zone diameters were measured in millimeters (mm) (Kumar et al., 2012;Abdelkader et al., 2018).In these experiments, TSB was used as a negative control.

Statistical analysis
Significance of difference was assessed in the results by repeating the experiments three independent times and comparing the data using student's ttest.P values lower than 0.05 were regarded significantly different.

Effect of sarA disruption on protease activity
The UAMS-1 wild type strain and its sarA mutant strain as well as the revertant strain were compared for protease toxins gene expression and toxin production.Expression of sspA, aur, scpA and sspB was significantly increased in the sarA mutant strain as compared to the wild type and revertant strains (Figure 1A).The relative gene expression of the mutant strain versus the wild type strain was 32.10, 81.30, 5.85 and 9.81 for sspA, aur, scpA and sspB, respectively.Moreover, protease production was significantly higher in the sarA mutant strain than the wild type and revertant strains (Figure 1B).The protease activity of the mutant strain was 2.87 of that of the wild type strain.
Figure 1 Effect of sarA disruption on sspA, aur, scpA and sspB gene expression levels (A) and on protease activity (B).Data was represented as mean ± S.D. The asterisk (*) indicates significant difference (P < 0.05) between sarA mutant strain (black bar) and both UAMS-1 wild type strain (white bar) and revertant strain (grey bar).

Effect of sarA disruption on lipase activity
Mutation of sarA was noticed to downregulate lip1 and lip2 expression (Figure 2A).The relative gene expression of the mutant strain versus the wild type strain was 0.11 and 0.12 for lip1 and lip2, respectively.This result was confirmed by the quantitative assay of lipase, where, lipase production was significantly altered in the sarA mutant strain compared to both the wild type and revertant strains (Figure 2B).The lipase activity of the mutant strain was 0.11 of that of the wild type strain.
Figure 2 Effect of sarA disruption on lip1 and lip2 gene expression levels (A) and on lipase activity (B).Data was represented as mean ± S.D. The asterisk (*) indicates significant difference (P < 0.05) between sarA mutant strain (black bar) and both UAMS-1 wild type strain (white bar) and revertant strain (grey bar).

Effect of sarA disruption on staphylokinase activity
Expression of sak was significantly upregulated in the sarA mutant strain in contrast to the wild type and revertant strains (Figure 3A).The relative gene expression of the mutant strain versus the wild type strain was 3.35 for sak.In addition, staphylokinase production level increased noticeably in response to the sarA mutation compared to the wild type and revertant strains (Figure 3B).The staphylokinase activity of the mutant strain was 1.55 of that of the wild type strain.
Figure 3 Effect of sarA disruption on sak gene expression level (A) and on staphylokinase activity (B).Data was represented as mean ± S.D. The asterisk (*) indicates significant difference (P < 0.05) between sarA mutant strain (black bar) and both UAMS-1 wild type strain (white bar) and revertant strain (grey bar).

Effect of sarA disruption on hyaluronidase activity
Expression of hysA1 and hysA2 was upregulated due to the sarA mutation in comparison to the wild type and revertant strains (Figure 4A).The relative gene expression of the mutant strain versus the wild type strain was 9.89 and 230.89 for hysA1 and hysA2, respectively.Furthermore, quantitative assay of hyaluronidase activity revealed lower production levels in the wild type and revertant strains, and a higher level in the sarA mutant strain (Figure 4B).The hyaluronidase activity of the mutant strain was 2.06 of that of the wild type strain.

CONCLUSION
To sum up, SarA increased the lipase production.On the other hand, staphylococcal serine protease, metalloprotease, staphopain protease, cysteine protease, staphylokinase and hyaluronate lyases were found to be downregulated by SarA.Therefore, it can be concluded that SarA is an important global regulator that controls the expression and production of many toxins in S. aureus.Our data support previous literature that SarA may act either as a positive regulator (may be through increasing the stability of mRNA of the genes) or a negative regulator (may be through decreasing the transcription of target genes) or may act through other regulatory mechanisms in S. aureus (Figure 5).This can open the door to set novel therapeutic approaches, targeting SarA, for the treatment of certain bacterial infections and help in tackling the overwhelming problem of antibiotic resistance.This can be achieved through using either new key inhibitors or modulators of SarA to control the production of virulence factors that are responsible for the clinical characteristics of each infection.
Figure 5 Proposed action of SarA as a global regulator in S. aureus.SarA may act either as a positive regulator (may be through increasing the stability of mRNA of the genes) or a negative regulator (may be through decreasing the transcription of target genes) or may act through other regulatory mechanisms.

(
Lacey et al., 2016).The regulation of virulence determinant production in S. aureus involves several global regulatory loci; of these, SarA, a regulatory nucleic acid binding protein, is particularly important (Arvidson & Tegmark, 2001; Jones et al., 2008; Mauro et al., 2016; Jenul & Horswill, 2019).S. aureus secretes many types of proteases that play a crucial role in the staphylococcal proteolytic effects, breaking down host peptide bonds.Protease production is mediated by different operons encoding seven serine proteases (sspA) and serine protease-like proteins (spls), two cysteine proteases (staphopain A, scpA and staphopain B, sspB) and a metalloprotease (aureolysin, aur) (Singh & Phukan, 2019).These proteases are able to lyse many host proteins such as fibrinogen and fibronectin (Pietrocola et al., 2017).Besides protease production, S. aureus is known as a potential lipase producer.Lipase production by this pathogen enables immune evasion by interfering with the phagocytosis by human granulocytes (Chen & Alonzo, 2019).Lipases can inactivate antimicrobial lipids, and also facilitate host cell lysis by helping degradation of cell membrane lipids.Lipase effect on host lipids results in release of free fatty acids from lipid stores (Cadieux et al., 2014; Chen & Alonzo, 2019).There are two types of lipases; Sal1 and glycerol ester hydrolase (Geh = Sal2) encoded by gehA and gehB, respectively (Nguyen et al., 2018; Chen & Alonzo, 2019

Figure 4
Figure 4 Effect of sarA disruption on hysA1 and hysA2 gene expression levels (A) and on hyaluronidase activity (B).Data was represented as mean ± S.D. The asterisk (*) indicates significant difference (P < 0.05) between sarA mutant strain (black bar) and both UAMS-1 wild type strain (white bar) and revertant strain (grey bar).DISCUSSION S. aureus is an important opportunistic Gram-positive pathogen that is responsible for a number of clinical infections (Chuang-Smith & Schlievert, 2021).Previous studies showed that S. aureus is implicated in many clinical cohorts (Mehraj et al., 2016).It is a major cause of skin and soft tissue infections worldwide as it is the causative agent of more than 80% of the skin and soft tissue infections.It is the most commonly isolated pathogen from infections in surgical sites, purulent cellulitis, wounds and chronic ulcers (Mehraj et al., 2016).It can systemically infect most tissues in the host such as eyes, brain, bones, joints, kidneys, lungs, hearts and muscles (Kuehl et al., 2020).S. aureus is armed with various types of virulence factors that support its pathogenicity and allow its evasion from immune system (Haddad et al., 2018).Expression of virulence factors genes in S. aureus is controlled by global regulators such as Agr, SarA, Rot and MgrA (Jenul & Horswill, 2019).SarA is a 124 amino acid long winged helix protein encoded by sarA locus, which is composed of three overlapped transcripts (sarA P1, sarA P3 and sarA P2).It is encoded by a 372 bp sarA open reading frame (ORF) that forms the final SarA protein form (Liu et al., 2006; Cheung et al., 2008).SarA is known to regulate the expression of about 120 genes, of which 76 genes are upregulated, while 44 genes are downregulated either by direct DNA binding or by indirect pathways (Liu et al., 2006; Jones et al., 2008).Murine models infected with sarA mutant S. aureus strain showed a suppressed virulence pattern, and reduced susceptibility to septic arthritis and osteomyelitis.Moreover, these models revealed that sarA mutant strain was less likely to produce biofilm, and therefore was more susceptible to antibiotics (Gordon et al., 2013).Our findings in this study demonstrated that proteases levels [staphylococcal serine protease (SspA), metalloprotease (aureolysin; Aur), staphopain (ScpA) and cysteine protease (SspB)] were increased in the sarA mutant strain when compared to the wild type and revertant strains.This was found to be accordant

Table 1
Bacterial strains used in this study

Table 2
Oligonucleotide primers used in this study (Elgaml et al., 2013;Elgaml et al., 2014;Abdel-Sattar et al., 2016) shaking at 150 rpm for an appropriate time.Thereafter, cultures were centrifuged at 10000 rpm for 10 minutes at 4°C.The obtained supernatants were filtered through 0.2 µM Millipore filter and subsequently used in the assay of protease activity.Protease quantitative assay was carried out on bacterial supernatants according to(Elgaml et al., 2013;Elgaml et al., 2014;Abdel-Sattar et al., 2016).Azocasein (1.0 mg) (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 50 mM Tris HCl (pH 8.0) in volume of 0.6 mL.From this solution, 0.2 mL was mixed with