Investigating the effect of an identified mutation within a critical site of PAS domain of WalK protein in a vancomycin-intermediate resistant Staphylococcus aureus by computational approaches

Background Vancomycin-intermediate resistant Staphylococcus aureus (VISA) is becoming a common cause of nosocomial infections worldwide. VISA isolates are developed by unclear molecular mechanisms via mutations in several genes, including walKR. Although studies have verified some of these mutations, there are a few studies that pay attention to the importance of molecular modelling of mutations. Method For genomic and transcriptomic comparisons in a laboratory-derived VISA strain and its parental strain, Sanger sequencing and reverse transcriptase quantitative PCR (RT-qPCR) methods were used, respectively. After structural protein mapping of the detected mutation, mutation effects were analyzed using molecular computational approaches and crystal structures of related proteins. Results A mutation WalK-H364R was occurred in a functional zinc ion coordinating residue within the PAS domain in the VISA strain. WalK-H364R was predicted to destabilize protein and decrease WalK interactions with proteins and nucleic acids. The RT-qPCR method showed downregulation of walKR, WalKR-regulated autolysins, and agr locus. Conclusion Overall, WalK-H364R mutation within a critical metal-coordinating site was presumably related to the VISA development. We assume that the WalK-H364R mutation resulted in deleterious effects on protein, which was verified by walKR gene expression changes.. Therefore, molecular modelling provides detailed insight into the molecular mechanism of VISA development, in particular, where allelic replacement experiments are not readily available. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-021-02298-9.


Introduction
Vancomycin-intermediate resistant Staphylococcus aureus (VISA), which causes a wide range of life-threatening infections, has been recovered worldwide [1,2]. Nevertheless, the molecular mechanism of VISA development is incompletely understood. The molecular studies on clinical or laboratory-derived VISA isolates have confirmed that VISA phenotype is caused by the diverse nonsynonymous single nucleotide polymorphisms (nsSNPs). However, only a few of these mutations have been verified by allele swapping or complementation experiments. The most relevant nsSNPs reported in VISA isolates are mutations in walKR, graSR, and vraTSR two-component systems (TCSs) [3].
The WalKR system, specific TCS to low G + C Grampositive bacteria, is the only essential TCS in S. aureus viability [4,5]. The WalKR plays a role in cell wall biosynthesis, virulence, and antibiotic resistance due to its extensive interaction network, such as interaction with graSR, vraSR, rpo, agr, and autolysins genes (atlA, sle1, and lytM) [4,6,7]. The walKR mutations may cause destructive regulation of these genes that can arise identical features among VISA isolates, such as reduced autolytic activity and virulence [8].
In S. aureus, the PAS domain (amino acids: from 261 to 375) is conserved among members of Staphylococcus species and contains four critical and highly conserved residues (His 271, Asp 274, His 364, Glu 368) as metalbinding sites, which directly bind to a Zinc ion (Zn 2+ ) [9].
In Bacillus subtilis (B. subtilis), the role of the cytoplasmic PAS (PAS CYTO ) domain in the localization of WalK in the division septum is confirmed [10]. Recently, structural and functional analyses of Zn 2+ -binding residue 271 within the PAS domain of S. aureus WalK showed that the Zn 2+ -binding site regulates S. aureus WalKR [9]. On the other hand, a substitution mutation in H271 was recently reported in a VISA strain [11]. In B. subtilis, residue D274 was confirmed to be related to the regulatory pathways of peptidoglycan synthesis [12].
Missense mutations can disrupt drug-bacterial interaction and protein function via affecting protein stability and interaction with biological molecules such as protein and nucleic acid. The previously deposited crystal structures of the desired bacterial species in the mutation site are utilized to analyze the effect of nsSNP on the protein structure and interactions of protein using molecular computational approaches. These methods for predictions of these impacts usually are used to understand the mechanism of human genetic disease and drug resistance to infections such as Mycobacteria infections [13][14][15][16]. Despite the role of WalKR in VISA development, the molecular modelling of the mutations site and computational approaches in VISA strains requires improved understanding. Therefore, besides the gene expression and complementation experiments to understand the VISA molecular mechanisms, the importance of knowledge about protein structure mapping of mutations and predicting the impact of the mutation on protein function using molecular computational approaches can provide new insight into the mechanisms underlying VISA development and help to design novel therapeutic strategies [17].
In the present study, we detected mutation WalK-H364R, which locates in the Zn 2+ -binding residue of the PAS CYTO domain, in a laboratory-derived VISA strain compared to its wild-type strain. This mutation was also previously reported in a S. aureus strain after nisin selection [18]. In the present study, we attempted to generate a VISA mutant via the selection by vancomycin in a VSSA (vancomycin susceptible S. aureus) strain. The reverse transcriptase quantitative PCR (RT-qPCR) method was used to compare the expression of TCSs (walkR, vraTSR, graSR) genes in the VISA strain compared to the wild-type strain. Using the Sanger sequencing method, we compared the complete sequence of walK and walR genes between VSSA and VISA strains, which showed walKR expression changes. After detection of WalK-H364R mutation within the PAS domain of the VISA strain, this study aimed to predict the possible effects of this mutation on protein function, protein stability, and protein interactions using computational approaches. We supported in silico results by comparing phenotypic features and gene expression of agr RNA III and walK-regulated autolysis genes in the VISA and VSSA strains. Details of the mutation effects can evaluate whether the identified mutations have the potential to change on protein and possible variations of the bacteria characteristics. These findings help to understand the mechanism of VISA development that is an urgent need to identify new drug targets or to design new drugs.

Results
In vitro development, stability, and isogenicity of VAN-I mutant After a serial adaptation process, VAN-I strain (vancomycin MIC = 8 μg/ml) was selected as a VISA strain from a clinical VSSA strain (VAN-S; vancomycin MIC = 1 μg/ml). In this process, the vancomycin MIC of VAN-S was increased to 2, 4, and 8 μg/ml after 6, 10, and 18 days, respectively.
The vancomycin MIC in VAN-I mutant was constant on the vancomycin-free medium over five passages that revealed stability of VISA phenotype at the tested time point.
The same PFGE profile confirmed the isogenicity of VAN-S and VAN-I (Fig. 1).

Transcriptional changes in VAN-I mutant
RT-qPCR analysis to quantify the expression changes of walR, vraR, graR, agr RNAIII, sceD, atlA, sle1, and lytM genes are presented in Fig. 2. The analysis of transcriptional profiles of walKR, vraTSR, and graSR systems indicated that the expression of walKR (0.14-fold; 7.14 times) was significantly (P ≤ 0.05) downregulated in VAN-I mutant. There was no significant statistical difference in the gene expression of vraTSR and graSR systems between VAN-I and VAN-S strains (Fig. 2). Furthermore, the comprising of walKR-dependent peptidoglycan hydrolase genes (lytM, atlA, sle1, and sceD) showed the downregulation (P ≤ 0.05) of lytM (0.14-fold; 7.14 times), atlA (0.04-fold; 25 times), and sle1 (0.45fold; 2.22 times) genes in VAN-I mutant. No significant change was observed in the expression of sceD gene. A notable reduction (P ≤ 0.05) in agr RNAIII expression (0.05-fold; 20 times) of VAN-I strain showed reduced activity of agr locus.
Identification of the nsSNP mutation within Zn 2+ -binding site of Walk in VAN-I mutant After aligning the walK in wail-type (VAN-S) and mutant (VAN-I) strains, one nsSNP was detected in VAN-I mutant (walK nucleotide 1091, base change: A to G). In this mutation, Histidine 364 was replaced by Arginine (WalK-H364R) in WalK protein. Mapping of mutation within WalK protein revealed that WalK-H364R was located in the Zn 2+ -binding site within the PAS CYTO domain [9]. The Zn 2+ -binding sites of WalK protein are displayed in Fig. 3a.

Conservation of Zn 2+ -binding sites in WalK protein
After alignment of WalK sequences (in the selected region) among staphylococci, enterococci, and bacilli genera, the potential conservation of Zn 2+ -binding residues (including H364) among WalK protein of staphylococcus species and enterococci were confirmed (Fig. 3b).

Comparison of protein structural mapping of previously reported nsSNPs in WalK protein
The protein structural mapping of 38 nsSNPs reported in previous studies in WalK of vancomycin nonsusceptible S. aureus strains is shown in Fig. 4. The results displayed that most nsSNPs were located in the PAS CYTO domain (12 nsSNPs) and HAMP domain (9 nsSNPs).
Among the studies reviewed, we observed that one nsSNP (H271D) within the Zn 2+ -binding site (residue 271) of WalK in a VISA strain [11].

Protein stability change upon mutation H364R in WalK
We analyzed the impact of mutation H364R within a zinc 2+ -binding residue of WalK protein using the DUET method. DUET method predicted that the nsSNPs could directly influence protein stability via changes in ΔΔG (kcal/mol) value.
Overall, the presence of mutation H364R was estimated to have a destabilizing effect on WalK protein stability. The results are displayed in Table 1 and include the combined DUET prediction and the mCSM and SDM individually predicted changes.

Protein-protein complex affinity change upon mutation H364R in WalK
The mSCM-PPI2 predicted that the protein-protein affinity of WalK destabilized (Reduced affinity) upon mutation H364R. The ΔΔG affinity against 4mn5 was −0.045 kcal/ mol in chain A and − 0.084 in chain B. The ΔΔG affinity against 4mn6 was −0.069 kcal/mol in chain A and − 0.106 kcal/mol in chain B. The inter-residue non-covalent interaction network of VAN-I mutant and wild-type (PDB ID 4mn6) are displayed in Supplementary Fig. S1.

Protein-nucleic acid binding affinity change upon mutation H364R in WalK
The mCSM-NAv2 displayed that protein-nucleic acid binding affinity was reduced. Therefore, WalK-H364R had a destabilizing effect on protein-nucleic acid  Prediction of the impact of mutation H364R in WalK on protein function SIFT estimated that substitution at position 364 from H to R is predicted to be tolerated with a score of 1.00 and median sequence conservation: 3.02.
Due to the importance of the mutation site, the result of SIFT prediction was surprising.
The SIFT alignment results in FASTA format showed that WalK sequences are homologous to S. aureus SsrB and ResE in B. subtilis in some residues (Fig. 3b). The alignment of WalK showed that an R364 residue (similar to the amino acid substituted in H364R) in protein sensor kinases SrrB in S.aureus and ResE in B. subtilis was matched with residue 364 (Arginine) of VAN-I mutant (Fig. 3b). Therefore, this similarity could lead to predicting a tolerated effect of WalK-H364R by the SIFT method. We searched the binding sites in SsrB protein using PLIP (Protein-Ligand Interaction Profiler) web service (https://projects.biotec.tu-dresden.de/plip-web/plip/ index) (input PDB ID: 6PAJ; data not shown). No Zn 2+binding site was detected in SrrB. Furthermore, residue 348 in SsrB showed no evidence of a ligand-binding site. Therefore, this residue has a different function in SrrB compared to it in WalK. This result showed a low accuracy of the SIFT method in classifying nsSNPs within binding sites of S. aureus WalK. Autolytic activity at the tested time points in VAN-I was significantly decreased (P ≤ 0.05) compared to that in VAN-S (Fig. 5a).
Furthermore, the hemolytic activity of VAN-I was decreased compared to that in VAN-S (23.43% versus 81.03%) (Fig. 5b).

Discussion
Although most reported nsSNPs in VISA strains occur in regulatory TCSs (e.g. walKR), the molecular mechanisms of resistance to vancomycin in VISA remain unclear [3]. In this study, we have reported a VISA strain with a nsSNP, which occurred in a functional residue within the PAS CYTO domain of WalK protein. This residue of WalK protein binds to a specific metal ligand.
The complementation experiments have shown that sometimes only one nsSNP in the walKR system can lead to reduced vancomycin susceptibility. For instance, Walk-G223D or WalK-Q371del could convert VSSA to hetero-resistant VISA (hVISA) [6,19]. WalK-Q371del was located in the PAS CYTO domain.
Recently, the X-ray crystal structure of the PAS CYTO domain of S. aureus WalK has revealed four Zn 2+ -binding sites, including His 271, Asp 274, His 364, Glu 368 within the PAS CYTO domain. Zn 2+ is tetrahedrallycoordinated by four Zn 2+ -binding sites [9]. In the present study, WalK-H364R occurred in residue His 364 in VISA strain VAN-I. This nsSNP was also previously reported to involve nisin resistance in an S. aureus strain after nisin selection [18]. Nisin disrupts the cell membrane as a primary target. Furthermore, it inhibits the peptidoglycan biosynthesis in Gram-positive bacteria through specific lipid II interaction [20]. Lipid II is also a target for vancomycin [21]. Coates-Brown et al. suggested that WalK-H364R could limit pore formation and nisin interaction with lipid II through the increased cell wall thickness, which is a common feature in VISA strain [18]. This hypothesis also provides new insight into the importance of WalK-H364R in developing vancomycin resistance in VISA. High conservation of Zn 2+ -binding sites among staphylococci WalK and the importance of residue H364 in walKR regulation following binding to Zn 2+ ligand can support this hypothesis (Fig. 3b).
In addition to the regulation of cell wall metabolism, the WalKR system regulates pathogenesis and host inflammatory response. Decreased or attenuated virulence in VISA strains is a stealth strategy to evade the host immune and persistent infection [8]. Using the Galleria mellonella model, virulence analysis of the clinical VISA strains and walK mutants (e.g. WalK-G223D) have shown that WalKR mutation can influence pathogenicity and susceptibility to vancomycin in S. aureus [6]. Additionally, Allelic exchange experiments to verify the identified walK mutations in VISA strains using comparative genomics of VSSA/VISA clinical (before and after in vivo vancomycin treatment failure in patients) or laboratory-derived pairs showed attenuated virulence. Those changes include reduced autolytic and hemolytic activities and increased biofilm formation [3,8,22]. Here, walKR and walKR-related autolysins, including sle1, lytM, and atlA, were downregulated in VAN-I compared to those in VAN-S. Moreover, we showed phenotypically reduced autolysis and hemolysis that might be related to dysfunctional walKR. The walK mutations also conferred downregulation of agr locus in VISA strains [6,8]. Agr locus is a global regulatory system in The kinetic data set of autolysis of the S. aureus culture (in 20 ml BHIB) that washed (two times) and adjusted to an OD 600 of 1 (in 10 ml cold distilled water supplemented with 0.1% Triton X-100) was measured at OD 600 in the tested time points. There was a statistically significant difference (P < 0. 01) in the autolysis kinetic activity of VAN-I versus VAN-S (paired-sample Student's t-test) at each tested point. (B) The percent of hemolysis of 300 μl of human red blood cells (RBCs) diluted in 10 ml PBS in exposure to S. aureus culture (adjusted to an OD 600 of 0.3 in BHIB) was measured at OD 543 using the following formula: (A 543 of the sample ˗ A 543 of negative control) / (A 543 of positive control ˗ A 543 of negative control) × 100. Negative control: diluted RBC in PBS without bacteria; Positive control: Triton X-100; the symbol (***) indicates a significant difference (P < 0.001) using the two-tailed Wilcoxon signed-rank test S. aureus that is involved in virulence mechanisms [23]. In the present study, we also observed decreased agr RNAIII expression as an effector molecule of the agr locus. Collectively, these events could be related to H364R, which might negatively regulate the walKR regulon. Subsequently, it led to increased resistance to vancomycin. Thus, the nsSNPs in Zn 2+ -binding sites likely induce different effects on the regulation of walKR regulon. We used computational approaches to support these hypotheses.
Molecular mapping and computational approaches have been used for predicting the effects of nsSNP on the development of drug resistance, including isoniazid and rifampicin resistance in Mycobacterium tuberculosis [14], rifampin resistance in Mycobacterium leprae [15], and carbapenem resistance in Acinetobacter baumannii [16]. However, studies on VISA development are limited to allele swapping and complementation experiments [3]. Although these studies are needed to confirm the effect of nsSNPs in VISA development, the knowledge on molecular modelling of nsSNPs helps to a better insight into the molecular mechanism of VISA development. These approaches are easy and fast, and the only necessity is the published crystal structure of the desired bacterial species in the mutation site.
Here, the molecular computational analyses showed that substitution H364R in the Zn 2+ -binding site of the PAS CYTO domain decreased the stability, flexibility, and interactions (with proteins and nucleic acids) WalK. The destabilizing effect of mutations on protein leads to significant disruption of protein function or regulation [24]. Here, decreasing the affinity of WalK interactions (with proteins and nucleic acids) in VAN-I mutant was confirmed by the downregulation of walKR and walKR-regulated autolysins. Thus, H364 can be a destructing mutation in the WalK and involve in VISA development. However, the allelic replacement experiments and future studies need to complete confirmation. These results showed the importance of nsSNP in the Zn 2+ -binding site of the PAS CYTO domain of WalK.
The molecular models have suggested that the activation of WalK histidine kinase in S.aureus induces through the specific interaction of the PAS EC domain with D-Ala-D-Ala residue, which leads to phosphorylation of the WalR [5]. Recently, Monk et al. have suggested that Zn 2+ -binding sites within the PAS CYTO domain have a regulatory role within S. aureus WalK. They showed that substitution in Zn 2+ -binding residue 271 (WalK-H271Y) led to inhibition of Zn 2+ binding and activation of walKR regulon. H271Y mutant showed the increased hemolysis, atl expression, and vancomycin sensitivity [9]. However, the genome sequencing of a VSSA/VISA pair revealed a mutation in the Zn 2+ -binding site (residue 271: WalK-H271D), which was associated with increased resistance to vancomycin in the VISA strain [11]. Moreover, substitution in Zn2 + −binding (H271Y mutant) displayed reduced size and pigmentation of colonies, reduced growth rate, and increased doubling time similar to VISA strains [9]. In the present study, an overview of the protein structure mapping of several previously reported nsSNPs in VISA strains indicated that WalK nsSNPs frequently occurred in the PAS CYTO domain [6,11,19,[25][26][27]. However, we observed that nsSNP could detect in all WalK protein domains. These results explain the diversity of nsSNPs in VISA strains. This diversity may occur due to the differences in the genetic backgrounds of isolates [28], which needs future analyses.
Finally, the main limitations of the present study need to be mentioned; first, the predicted results of in silico analysis should be verified by strong wet laboratory experimental evidence. Second, whole-genome sequencing and allelic replacement experiments were not performed. Therefore, some phenotypic and transcriptomic changes observed in the VISA strain might not be due to the mutation in the walK gene, but might be due to nsSNPs in other parts of the genome. Third, we have observed that sometimes there may be exceptions to the accuracy of test results. For instance, the low accuracy of the SIFT method in classifying mutation in the metal-binding site of protein was observed.

Conclusion
In the present study, WalK gene sequencing of a VSSA/ VISA pair reveals a mutation H364R in a critical coordinating Zn 2+ site of the PAS CYTO domain. Molecular computational approaches show that this mutation can induce destabilizing effects on WalK protein function in VISA strain through decreasing stability and interactions of protein-protein and protein-nucleic acid. The walKR, agr RNA III, and walKR-related genes that downregulated in VISA strain have verified these results. Mutation H364R might play a role in VISA development due to the importance of its location in protein structure. Collectively, molecular mapping and computational approaches can be beneficial in understanding the mechanism of VISA development and designing novel antimicrobial drugs.

Bacteria parental strain and growth conditions
A clinical MRSA (Methicillin-resistant S. aureus) strain was isolated from a burned patient in Motahhari Hospital, Tehran, Iran, in December 2018. This strain was called VAN-S and served as the parental vancomycin susceptible strain (with a MIC value of vancomycin equal to 1 μg/ml). VAN-S strain harboured type III SCCmec and agr type III.

In vitro selection of VISA strain
One VISA mutant was selected by vancomycin from one S. aureus clinical strain and then compared with its VSSA wild-type to study the mechanism of VISA development [3].
Overnight culture of VAN-S strain was adjusted to 5 × 10 5 CFU/ml concentration into 3 ml of BHIB containing 1/4 × MIC of vancomycin. After 24 h, the culture was serially passaged into BHIB containing 1/2, 1, and 2 × MIC of vancomycin. Then, the process was repeated from 1/4 × MIC of vancomycin. The cultures in each step were speared on BHIA plates to maintain vancomycin tolerance colonies.
This process was repeated for 50 days. The MIC of vancomycin was recorded every 24 h.

Stability of VISA phenotype and isogenicity between parental and mutant strains
After VAN-I mutant selection, it was cultured on vancomycin-free BHIA plates over five passages to evaluate the stability of VAN-I.
To confirm the isogenicity of VAN-S and VAN-I, pulsed-field gel electrophoresis (PFGE) was performed using SmaI endonuclease (Takara, Japan), as previously described [29]. 1% agarose gel containing digested DNA samples were run on the CHEF-Mapper (Bio-Rad Laboratories, CA) for 19 h at 6 V/cm and a field angle of 120°, with switch times of 5 and 40 s at a temperature of 14°C.

Determination of minimum inhibitory concentration of vancomycin
The broth macro dilution method was used to determine the MIC of vancomycin according to the 2018 CLSI guidelines [30]. The cation-adjusted Mueller-Hinton broth medium (Merk, Germany) was used for the MIC assay.

RNA extraction and reverse transcriptase quantitative PCR (RT-qPCR)
The RNA was extracted from VAN-S and VAN-I cells in the mid-logarithmic phase using the GeneAll Hybrid-R™ RNA isolation kit (Geneall Biotechnology, Korea) with DNase I treatment (Thermo Fisher Scientific, USA) of 1 μg of RNA template (final concentration: 0.1 μg/μl).
After cDNA synthesis (Yekta Tajhiz Azma, Iran), RT-qPCR was run on a Rotor-Gene Q (Qiagen, Hilden, Germany) using RealQ Plus 2x Master Mix SYBR Green (Ampliqon, Denmark), and primers (Pishgam Biotech, Iran) (see Supplementary Table S1) to amplify agr RNAIII and TCSs genes, including walR, vraR, graR. If there was a gene expression change in these TCSs (see result section), the RT-qPCR method was also performed to amplify TCSs-regulated genes. Here, the expression change of atlA, lytM, sle1, and sceD genes was examined following changes in walKR expression. The gyrA was used as the internal control. The samples were run at least in triplicate assays.
DNA extraction, gene sequencing, and protein mapping of the mutation Genomic DNA from overnight cultures of VAN-S and VAN-I was extracted using the Gene Transfer kit (Pioneers, Iran).
PCR was performed to amplify TCS genes that showed gene expression changes by the RT-qPCR method (see result section). Therefore, walK and walR genes were amplified using the primers (see Supplemental Material Table S1 online) within a T100TM Thermal cycler (Bio-Rad, USA).
The PCR products were confirmed by electrophoresis on a 1% agarose gel, purified, and then sequenced (by Microsynth AG Co, Switzerland) using the Sanger sequencing method (forward and reverse reads).

Protein stability analysis
The sequence alignment of WalK protein VAN-S was performed to obtain the crystal structure of S. aureus WalK protein using the Protein Blast tool (https://blast. ncbi.nlm.nih.gov/Blast.cgi) among protein data bank (PDB) database (wwpdb.org). Finally, 4mn5 and 4mn6 PDB IDs were selected based on the coverage of the mutation site in VAN-S (residue 364) with 96.03 and 100% identifies, respectively, as wild-type templates for future protein analyses. 4mn5 and 4mn6 display the crystal structure of the PAS CYTO domain from S. aureus WalK based on the X-ray diffraction method at 2 Å and 2.1 Å resolutions, respectively.
The DUET web server (http://biosig.unimelb.edu.au/ duet/) was applied to predicting the effects of mutation H364R on the WalK protein stability.
DUET predicts the changes in protein stability upon the introduction of nsSNP using consolidating mCSM (mutation Cutoff Scanning Matrix) and SDM (Site Directed Mutator) computational approaches [37].

Protein-Protein interactions analysis
Predicting the impact of mutation H364R on the protein-protein affinity of WalK was carried out by the mSCM-PPI2 web server (http://biosig.unimelb.edu.au/ mcsm_ppi2/). The mSCM-PPI2 applied an optimized graph-based signature approach to better evaluate the molecular mechanism of the nsSNP using modelling the effects of variations in the inter-residue non-covalent interaction network [38].
Prediction of the impact of nsSNP mutation on protein function SIFT (sorts intolerant from tolerant) webserver (http:// sift-dna.org) was used to predict changes in the protein function. SIFT performs PSI-BLAST search and predicts changes based on sequence homology and the physical properties of amino acid residues.

Doubling time calculation
The doubling time was calculated using an obtained graph of the growth kinetic curve, as previously described with modifications [40]. Briefly, the S. aureus culture was adjusted to 0.01 at an optical density at 660 nm (OD 660 ) in 20 ml BHIB and incubated for 15 h. The OD 660 of these cultures was recorded every 1 h using a spectrophotometer (Biochrom WPA Biowave II, UK). The formula for calculating the doubling time (DT) was [(t2 -t1) × log 2] / [log OD 660 at t2 -log OD 660 at t1]. The abbreviations t1 and t2 are the start andend times of the exponential phase, respectively [40]. The experiment was performed in triplicates.
Triton X-100 induced autolysis kinetic assay 20 ml of the S. aureus culture was collected by centrifugation (6800 × g for 10 min), washed twice with 20 ml of cold distilled water, and adjusted to an OD 600 of 1 in 10 ml cold distilled water supplemented with 0.1% Triton X-100. The absorbance decline at 600 nm within 0, 1, 2, 3, 4, and 16 h of incubation time was measured (using a spectrophotometer) as autolysin activity [41]. The experiments were done on three independent occasions.

Hemolysis assay
The α-hemolysin activity was examined as previously described [42], with minor modifications. The human RBCs were separated from plasma using centrifugation (900 × g for 2 min), washed three times with 1X PBS solution, and diluted (300 μl of RBCs in 10 ml PBS). 200 μl of S.aureus cells (OD 600 = 0.3) added into diluted RBCs, incubated for 1 h (with shaking at 250 rpm), and centrifuged at 6000 × g for 10 min. In three independent experiments, the absorbance of the supernatant was measured (using the spectrophotometer) at OD 543 nm and analyzed as hemolytic activity.
Triton X-100 and diluted RBCs in PBS without bacteria were used as positive and negative controls, respectively. The percent of hemolysis activity was calculated from the average of three experiments by the following formula: (A 543 of the sample -A 543 of negative control) / (A 543 of positive control -A 543 of negative control) × 100.

Statistical analysis
SPSS24 statistical software (SPSS inc., Chicago, IL) was used for statistical analyses of data. A P value ≤0.05 was considered as a significant difference between the isogenic pair of VAN-S/VAN-I. The results were reported as mean ± standard deviation (SD).
The autolysis activity of the isogenic pair of VAN-S/ VAN-I was compared using parametric paired-sample Student's t-test. Data of hemolysis assay and doubling time were analyzed using the nonparametric two-tailed Wilcoxon signed-rank test.