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Article

Identification of Staphylococcus aureus Cellular Pathways Affected by the Stilbenoid Lead Drug SK-03-92 Using a Microarray

1
Department of Microbiology, University of Wisconsin-La Crosse, La Crosse, WI 54601, USA
2
Emerging Technology Center for Pharmaceutical Development, University of Wisconsin-La Crosse, La Crosse, WI 54601, USA
3
School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA
4
Department of Chemistry and Biochemistry, University of Wisconsin-La Crosse, La Crosse, WI 54601, USA
5
Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA
6
Department of Mathematics and Statistics, University of Wisconsin-La Crosse, La Crosse, WI 54601, USA
*
Author to whom correspondence should be addressed.
Submission received: 28 July 2017 / Revised: 25 August 2017 / Accepted: 7 September 2017 / Published: 11 September 2017

Abstract

:
The mechanism of action for a new lead stilbene compound coded SK-03-92 with bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) is unknown. To gain insight into the killing process, transcriptional profiling was performed on SK-03-92 treated vs. untreated S. aureus. Fourteen genes were upregulated and 38 genes downregulated by SK-03-92 treatment. Genes involved in sortase A production, protein metabolism, and transcriptional regulation were upregulated, whereas genes encoding transporters, purine synthesis proteins, and a putative two-component system (SACOL2360 (MW2284) and SACOL2361 (MW2285)) were downregulated by SK-03-92 treatment. Quantitative real-time polymerase chain reaction analyses validated upregulation of srtA and tdk as well as downregulation of the MW2284/MW2285 and purine biosynthesis genes in the drug-treated population. A quantitative real-time polymerase chain reaction analysis of MW2284 and MW2285 mutants compared to wild-type cells demonstrated that the srtA gene was upregulated by both putative two-component regulatory gene mutants compared to the wild-type strain. Using a transcription profiling technique, we have identified several cellular pathways regulated by SK-03-92 treatment, including a putative two-component system that may regulate srtA and other genes that could be tied to the SK-03-92 mechanism of action, biofilm formation, and drug persisters.

1. Introduction

Staphylococcus aureus is a common inhabitant of the human body that also causes numerous infections, including skin and soft tissue infections as well as more serious infections, such as pneumonia and bacteremia [1]. Presently, around 60% of S. aureus clinical isolates are methicillin-resistant S. aureus (MRSA) [2], and this bacterium is a leading cause of nosocomial infections in the United States [3,4]. In 1997, community-associated methicillin-resistant S. aureus (CA-MRSA) strains emerged in the United States, causing infections in younger people, including necrotizing pneumonia [5,6,7]. Although skin infections caused by CA-MRSA are still prevalent, invasive MRSA infections have decreased [3,8]. In addition to methicillin resistance, CA-MRSA strains are becoming multidrug resistant at an alarming rate [9,10,11]. Heterogeneous vancomycin-intermediate S. aureus and vancomycin-resistant strains of S. aureus have led to vancomycin being less effective against some S. aureus infections [12,13,14,15]. Tolerance to vancomycin now has been reported to be as low as 3% and as high as 47% [16,17]. New drugs are needed to treat MRSA infections; however, most drugs currently in development are derivatives of drugs already being marketed [18,19]. S. aureus is one of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) targeted by the 10 × 20 initiative to develop 10 new, safe and effective antibiotics approved by 2020 [20].
In support of the 10 × 20 initiative, a new antibiotic identified as (E)-3-hydroxy-5-methoxystilbene with promising activity against S. aureus was identified from Comptonia peregrina (L.) Coulter (“sweet fern”) [21]. A structure–activity relationship analysis identified our lead compound, (E)-3-(2-(benzo[b]thiophen-2-yl)vinyl)-5-methoxyphenol; for simplicity, SK-03-92. SK-03-92 was rapidly bactericidal (killing 90% of the population within an hour) against every Gram-positive species that was tested, including MRSA strains [22]. Importantly, a combined safety and pharmacokinetic study demonstrated that the SK-03-92 lead drug was safe in mice [23]. As with all antimicrobials, therapeutic treatment can result in residual bacteria not being killed by that antimicrobial, a phenomenon known as persistence [24,25,26]. Drug persisters are phenotypically different than the parent strain, but are not true drug resistant variants because the MICs of the drug persisters are the same as their parent strains [27,28]. Persisters are thought to be a major component of bacterial biofilms, allowing significant drug tolerance [29,30]. Many drugs used to treat S. aureus infections have drug persister population emerge that are recalcitrant to treatment. To gain insight into the mechanism of action of SK-03-92 and the mechanism of S. aureus persistence to SK-03-92 treatment, the effect of SK-03-92 on S. aureus cells was assessed by transcriptional profiling in the S. aureus strain MW2.

2. Results and Discussion

2.1. General Transcriptome Response of SK-03-92 Treatment

New drugs to treat S. aureus infections are urgently needed, and SK-03-92 holds considerable promise. SK-03-92 has a stilbenoid backbone [22] and is bactericidal within an hour; however, 10% of the population survives as drug persisters that can grow in media containing up to 32 μg/mL of SK-03-92 but with an MIC equivalent to untreated S. aureus cells. The mechanism of action for SK-03-92 is unknown. To ascertain the effects of SK-03-92 treatment on the transcriptome of S. aureus, total RNA was isolated from S. aureus strain MW2 cultures (Table 1) treated for 30 min with 8× the MIC of SK-03-92 and untreated MW2 cultures and an RNA microarray was performed. A total of 52 genes were dysregulated by the SK-03-92 drug treatment (Table 2), representing 2% of the total S. aureus transcriptome. This is remarkable because transcriptional profiling of other bactericidal compounds has shown a larger effect on the S. aureus transcriptome, including ortho-phenylphenol (24%) [31], amicoumacin A (20%) [32] and daptomycin (5% to 32%) [33,34]. Interestingly, the number of downregulated genes (73.1%) greatly surpassed the number of upregulated genes (26.9%).
An examination of genes affected by treatment with other stilbene type compounds demonstrates the disparity in their transcript profile compared to treatment with SK-03-92. Pterostilbene, another stilbenoid compound, in Saccharomyces cerevisiae showed 1189 genes that were dysregulated: 1007 upregulated (85%) and 182 downregulated (15%) [35]. Microarray analysis with resveratrol treated Schizosaccharomyces pombe showed 480 genes dysregulated, 377 genes that were upregulated and 103 that were downregulated [36]. RNA sequence analysis of resveratrol treated S. aureus cells demonstrated 444 dysregulated genes, 201 upregulated and 243 downregulated [37]. The majority of the genes in our study had a two- to four-fold difference in transcript abundance when comparing SK-03-92 treated vs. untreated S. aureus cultures. Very few genes dysregulated by SK-03-92 were previously shown to be dysregulated by resveratrol (e.g., downregulation of the purD, purH, purL, lrgA, and sdhC genes). Only three genes had a 10-fold or higher change in transcript levels, which included two genes annotated to be part of a putative two-component system (TCS) (SACOL2360 (annotated as MW2284 in MW2 strain) = 14.1-fold lower and SACOL2361 (annotated as MW2285 in MW2 strain) = 26.9-fold lower) as well as the glpD gene encoding glycerol-3-phosphate dehydrogenase (10-fold higher).
Dysregulated genes tied to a potential mechanism of action for SK-03-92 included glpD, adhE (SACOL0135), adhP (SACOL0660), and sdhC (SACOL1158). GlpD funnels electrons into the respiratory chain via quinone or menaquinone reduction coupled to the oxidation of glycerol-3-phosphate to glycerone phosphate (dihydroxyacetone phosphate) [40], which can be enzymatically or non-enzymatically transformed into methylglyoxal (MG) [41]. Higher concentrations of MG are thought to halt bacterial growth by damaging proteins by acting as a protein glycating agent that mainly affects arginine residues [42,43]. In Candida albicans, ADH1 catalyzes the NAD+ linked oxidation of MG to pyruvate and disruption of the adh1 gene in C. albicans caused accumulation of MG followed by inhibition of growth [44]. The dysregulation of glpD and adh genes suggests that MG was accumulating and glycation was occurring in SK-03-92-treated S. aureus. MG glycation of proteins, lipids, and DNA generate advanced glycation end products (AGEs) [43]. Importantly, GlpD has been implicated in drug persistence in Escherichia coli [45] and S. aureus [34].
A number of genes involved in metabolism were also dysregulated by SK-03-92 treatment, including the gcvH gene that encodes GcvH, which shuttles the methylamine group of glycine from the P-protein to the T-protein via a lipoyl group [46]. Genes associated with protein degradation and repair had altered transcript abundance in SK-03-92-treated S. aureus. Transcripts encoding a putative repair system for deglycation of Amadori protein adducts derived from ribose-5-P (ptpA) [47] showed altered abundance in SK-03-92-treated S. aureus, as did the transcript encoding the enzyme that produces ribose-5-P (SACOL2605). The formation of Amadori protein adducts occurs spontaneously via a dehydrogenation mechanism when ribose-5-P interacts with an amine, such as the lysine residues of proteins. Amadori glycated proteins undergo further spontaneous reactions to become AGEs. AGEs promote protein aggregation [47,48]. Since ptpA transcript abundance was increased 2.3-fold and the kinase transcript SACOL2605 was decreased 9.6-fold, ribulosamine substrates produced were likely not being deglycated, and protein repair was not occurring. Phase-dark and phase-bright inclusions were observed microscopically in SK-03-92-treated B. subtilis, consistent with perturbation of proteostasis resulting in visible accumulation of protein aggregates [49]. Uncontrolled protein aggregation is toxic to cells [48].
Three genes associated with purine synthesis were downregulated: purD, purH, and purL. Purine metabolism is a necessary part of DNA synthesis and energy production in S. aureus [50]. Genes involved in purine metabolism are often downregulated after treatment with a drug or plant extract [51,52,53]. In addition, one gene associated with pyrimidine synthesis, tdk, was upregulated. Thymidine kinase transfers the terminal phosphate from ATP to thymidine or deoxyuridine [54]. A decrease in the synthesis of purines coupled with an increase in phosphorylation of pyrimidines could result in a dramatic reorganization of the intracellular nucleotide pool. Moreover, less purine metabolism is often tied to drug persister populations [55,56]. Disruption of nucleotide metabolism in a library of S. aureus transposon insertion mutants caused a decrease in persister formation frequency when treated with rifampicin [57].
Consistent with the formation of persister strains, mRNA levels of genes linked to programmed cell death (PCD) were decreased in S. aureus cultures treated with SK-03-92. Specifically, the Cid/Lrg (holin/antiholin) system, which controls autolysis and affects the distribution of extracellular DNA in S. aureus during biofilm development [58,59,60]. This prokaryotic PCD is analogous to the bcl-2 pro-apoptotic effector and anti-apoptotic mediated apoptosis in eukaryotes [61,62].
Twelve putative transport genes were dysregulated encoding for proteins involved in anion transport, a cation efflux family protein, two phosphotransferase system (PTS) transporters, a sodium:alanine symporter, sodium:dicarboxylate symporter family protein, and a copper ion binding protein. The only true virulence factor genes affected by SK-03-92 treatment were the SACOL0151 cap5P, epiE, SACOL2333 gene encoding a YnfA family protein putative transport small multidrug resistance family-3 protein [63], and the srtA gene encoding sortase A that will be described in more detail below [64]. Five genes identified by the microarray were annotated as hypothetical proteins with no known function (three downregulated and two upregulated).

2.2. Genes of a Putative TCS Are Significantly Downregulated by SK-03-92 Treatment

A surprising microarray result that was no known S. aureus global regulatory genes were shown to be affected by the drug treatment. Microarray analysis of daptomycin treated S. aureus demonstrated that the the icaR gene was dysregulated compared to untreated cells [34]. Our microarray showed that a tetR-family transcriptional regulator, SACOL2340, and two genes that comprise a putative TCS in S. aureus annotated as MW2284 (14.1-fold downregulated) and MW2285 in strain MW2 (26.3-fold downregulated) were downregulated. A bioinformatic analysis of the putative MW2284 and MW2285 proteins suggest that they comprise a putative two-component regulatory system where MW2284 (LytTR superfamily regulator protein) is the response regulator protein and MW2285 (membrane protein) is the sensor kinase protein. MW2284 was identified as a 440-bp ORF encoding a putative 14.7-kDa transcriptional regulator protein and MW2285 was identified as a 455-bp ORF encoding a putative 15.1-kDa histidine kinase sensor protein. The MW2285 ORF has a 3-bp overlap with the MW2284 ORF. BLASTP, PSI-BLAST, and BLASTN bioinformatics analyses [65] showed that MW2284 aligned with other two-component regulatory system regulator proteins and MW2285 aligned with other two-component regulatory system sensor proteins. Both proteins have homology with LytTR superfamily proteins involved in the regulation of bacterial genes [66]. LytTR proteins regulate virulence gene expression in a variety of bacterial species including S. aureus. The AgrA transcriptional regulator is one of these LytTR-type proteins [67]. Moreover, the MW2284 and MW2285 ORFs appeared to be conserved across a wide number of Gram-positive species, including all Staphylococcus and Streptococcus species, as well as Bacillus, Clostridium, Lactobacillus, Listeria, and Leuconostoc.
The same LytTR TCS dysregulated in SK-03-92-treated S. aureus was upregulated in purine synthesis deficient mutants in S. aureus [68]. The putative sensor kinase (MW2285) was upregulated in purH mutants and the response regulator (MW2284) was upregulated in purA mutants (adenylosuccinate synthetase involved in purine biosynthesis). The response regulator component transcript was also upregulated during anaerobic growth in another study [69]. A transposon mutant of the sensor kinase component has been previously shown to be viable, capable of producing a more robust biofilm, and had a lower LD50 than the parent strain [70,71]. The mechanistic link between defects in purine synthesis, persister formation, and the LytTR regulatory system remains unclear. Furthermore, RNAseq analysis of resveratrol treated S. aureus cells showed an almost 8-fold downregulation of the MW2284 gene, but no effect on the MW2285 gene [37].

2.3. Validation of Microarray Data by qRT-PCR

The microarray results were confirmed using qRT-PCR analyses on RNAs from 8× the MIC SK-03-92 treated MW2 cells vs. untreated MW2 cells. Transcription of the srtA gene was significantly upregulated almost 6-fold (p < 0.006, Figure 1) and the tdk gene was also upregulated 2.1-fold (p < 0.03) in SK-03-92 treated cells vs. untreated cells. On the other hand, several genes involved in purine biosynthesis (purD, purH, and purL) were shown to be significantly downregulated 2.2- to 2.4-fold (p < 0.01 to 0.04), whereas the MW2284 and MW2285 genes were downregulated 4- (p < 0.01) and 3-fold (p < 0.003), respectively, in the SK-03-92 treated samples. These results confirmed that treatment with the SK-03-92 lead compound caused dysregulation of the srtA, tdk, purD, purH, purL, MW2284, and MW2285 genes.

2.4. SK-03-92 Treatment Causes Alteration of Nucleotide Pool

Because three pur genes involved in purine synthesis and the tdk gene were dysregulated by SK-03-92 treatment, the rapid accumulation of the bacterial alarmone (p)ppGpp and the state of the intracellular nucleotide pool were examined using high-performance liquid chromatography (HPLC, Waters, Milford, MA, USA). Inhibition of isoleucyl tRNA synthetase by mupirocin has been shown to induce production of (p)ppGpp in S. aureus [72,73]. A highly phosphorylated ribonucleotide, (p)ppGpp, can be identified via rapid separation of the S. aureus nucleotide pool using anion-exchange HPLC, where (p)ppGpp elutes as a late peak, which can be detected by absorbance at 254 and 280 nm [74]. In control experiments, this late peak was not detected in untreated cells (Figure 2A), but was detected following treatment with mupirocin (Figure 2B). No (p)ppGpp was detected following treatment with SK-03-92 (Figure 2C). However, the composition and quantity (area under curve) of the nucleotide pool was altered in SK-03-92 treated S. aureus as compared to untreated cells (Figure 2A vs. Figure 2C), suggesting that dysregulation of tdk and the three purine biosynthesis genes by SK-03-92 treatment depleted the nucleotide pool.

2.5. Biofilm Formation Increases as the Concentration of SK-03-92 Increases

With an increase in srtA transcript abundance shown by the qRT-PCR results, an increase in biofilm formation would be expected following SK-03-92 treatment. To further analyze the effects of the increase in srtA transcription, a biofilm assay in microtiter plates was performed after SK-03-92 drug treatment (Figure 3). Wild-type JE2 and MW2 cultures were tested following SK-03-92 drug treatment (range 0.5–0.64 μg/mL). The JE2 culture grown without drug showed an OD570 of 2.41, whereas the MW2 culture had an OD570 of 2.50.
The SK-03-92 drug had a biphasic effect on the wild-type strains. At low concentrations, the drug reduced biofilm formation as exhibited by the 0.5 and 1 μg/mL data points that were significant for both strains that were tested (p < 0.05). As concentrations of SK-03-92 increased, the OD570 readings increased, plateauing at 32 μg/mL for both strains. Strain MW2 showed significant increases in biofilm formation going from 0 μg/mL to 8–64 μg/mL SK-03-92 concentration (p < 0.05). A similar finding was observed when Candida species grown as a biofilm were exposed to varying concentrations of echinocandin [75]. Candida treated with low drug concentrations killed the fungal cells but at concentrations higher than the MIC showed there was an increase in the cell density of the biofilms. Echinocandin acting on the Candida species has the same effect as our SK-03-92 drug, triggering upregulation of a specific gene that increases biofilm formation.
Under normal growth conditions, biofilm formation is not necessary for a cell, but under stressful environmental conditions, such as exposure to the SK-03-92 drug, biofilm formation would greatly benefit the S. aureus population. Formation of a biofilm would benefit cells by allowing for the formation of persister cell populations [76]. When biofilms form, the cells at the base of the biofilm slow or stop most cell metabolism and go into a dormant state, allowing the organisms to survive in the presence of a drug, for example SK-03-92. In addition, cells in a biofilm often undergo quorum sensing, which also can lead to the emergence of persister cells [77].

2.6. A Sortase A Mutant Has a Lower MIC against SK-03-92 Than Wild-Type

Since the putative MW2284/MW2285 TCS appears to repress transcription of the srtA gene, this regulatory effect could be tied to the mechanism of action of the SK-03-92 drug. Sortase A was first described in S. aureus in 1999 [64]. The protein covalently anchors surface proteins (e.g., fibronectin-binding protein, fibrinogen-binding protein, protein A, clumping factors, collagen adhesion protein) to the cell wall of S. aureus and other Gram-positive bacteria [77]. An LPXTG motif [78,79,80] is common among these anchored proteins and many are important for phase I of biofilm formation that allows attachment to biotic or abiotic surfaces [81]. A mutation of the srtA gene caused less expression of several cell wall anchored surface proteins [82,83]. Moreover, srtA mutants are attenuated compared to the wild-type strain in a variety of murine models of infection [82,84,85].
Because srtA and MW2284/MW2285 transcription were affected by SK-03-92 treatment, MICs were performed using the SK-03-92 lead compound on an srtA mutant (NE1787), srtB mutant (control, NE1363), MW2284 mutant (NE671), and MW2285 mutant (NE272) compared to the wild-type strain JE2 [38]. The srtB, MW2284, and MW2285 mutants had MICs that were equal to the wild-type strain (Table 3). However, the srtA mutant had an MIC that was 2-fold lower than the wild-type strain. When a Listeria monocytogenes srtA mutant was tested [39], the MIC for the srtA strain was 8-fold lower than the wild-type strain. A L. monocytogenes srtB mutant had the same MIC as the wild-type bacteria.
Presumably, SK-03-92 treatment causes downregulation of the MW2285 gene with an effect that would be similar to a mutation in the MW2285 gene. The regulatory effect could be derepression of srtA transcription. Either event would create more SrtA protein that in turn would allow greater extracellular presentation of proteins on the surface of S. aureus cells. This result may suggest that something tethered to the cell walls by sortase A that is conserved in both species may be tied to the mechanism of action of the SK-03-92 drug, and we are exploring this possibility.

2.7. Mutations in the MW2284/MW2285 Two-Component Regulatory Genes Cause an Upregulation of the srtA Gene

Since the microarray results showed significant upregulation of the srtA gene and downregulation of the MW2284 and MW2285 genes, we hypothesized that the MW2284 gene product, a putative transcriptional regulator protein, may be repressing the srtA gene. To confirm that the putative two-component regulatory system (MW2284/MW2285) may be involved in repressing the srtA gene, we obtained transposon mutant strains from the Nebraska Transposon Mutant Library [38] with insertion mutations in the MW2284 and MW2285 genes. A qRT-PCR analysis was then undertaken on RNA isolated from the NE272 (MW2285 mutation) and NE671 (MW2284 mutation) strains compared to the wild-type strain JE2, targeting the srtA gene. The results showed that mutations in both the MW2284 and MW2285 genes led to a 9.2-fold (p < 0.005) and 8.1-fold (p < 0.0008) upregulation of srtA transcription, respectively, suggesting that this putative two-component regulatory system may be repressing transcription of the srtA gene (Figure 4).

3. Experimental Section

3.1. SK-03-92 Synthesis

SK-03-92 was synthesized as described previously [22].

3.2. Bacterial Strains and Growth Conditions

The S. aureus MW2 strain [7] used for the initial microarray and confirmatory qRT-PCRs (Table 1) was obtained from Jean Lee (Brigham and Young Hospital, Boston, MA, USA). S. aureus strains JE2 (wild-type), NE671 (MW2284), and NE272 (MW2285) were obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) strain repository (Table 1), representing part of the Nebraska Transposon Mutant Library [38]. Strain JE2 is a plasmid-cured derivative of a USA300 CA-MRSA [86]. Phillip Klebba (Kansas State University, Manhattan, KS, USA) [39] provided the Listeria monocytogenes wild-type strain EGD as well as the isogenic srtA and srtB mutant strains. All strains were grown in brain heart infusion broth (Becton Dickinson, Franklin Lakes, NJ, USA) or trypticase soy broth (Becton Dickinson) shaken 250 rpm at 37 °C. The transposon mutant strains had 5 μg/mL of erythromycin (Sigma-Aldritch, St. Louis, MO, USA) added to the media.

3.3. RNA Extractions

Total RNA was isolated from S. aureus MW2 cells grown to exponential growth phase (OD600 approximately 0.5) either treated with dimethyl sulfoxide (DMSO) or 8× the MIC of SK-03-92 dissolved in DMSO using TRizol extraction (Life Technologies, Carlsbad, CA, USA) according to manufacturer′s instructions with an additional lysostaphin treatment step to help lyse the S. aureus cell walls. The RNA samples were digested with DNase I (New England Biolabs, Ipswich, MA, USA) followed by phenol and chloroform extractions to remove the protein. RNAs were run on 0.8% agarose gels to confirm concentration and integrities of the RNAs. To assess DNA contamination of the samples, PCRs were performed on the RNA samples using SaFtsZ1 and SaFtsZ2 primers (see Table 4). The PCR conditions for amplification with the SaFtsZ1/SaFtsZ2 primers was as follows: 94 °C, 1 min; 55 °C, 1 min; and 72 °C, 1 min for 35 cycles.

3.4. Microarray

Total RNAs from cells treated with DMSO or 8× the MIC of SK-03-92 were converted to cDNAs, biotinylated, and hybridized to S. aureus GeneChips following the manufacturer′s recommendations (Affymetrix, Santa Clara, CA, USA). Agilent GeneSpring G× 7.3 software (Santa Clara, CA, USA) was used to gauge transcript differences and a two-fold or higher difference in the transcript level for one population over the other was considered significant. Nucleic acid sequences with a ≥2-fold change in transcriptional abundance were mapped to the S. aureus COL genome (taxid: 93062) via BLASTN, BLASTX, or PSI-BLAST analysis [65] through the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) website and their putative products were annotated.

3.5. cDNA Synthesis

The cDNAs were synthesized from 5 μg of total RNA from SK-03-92 treated or untreated S. aureus MW2 using a First-Strand Synthesis kit (Life Technologies) according to manufacturer′s instructions.

3.6. Real Time-Quantitative Polymerase Chain Reaction (qRT-PCR)

All of the qRT-PCRs were performed using the LightCycler FastStart DNA MasterPLUS SYBR Green kit according to manufacturer′s instructions (Roche, Indianapolis, IN, USA). Primers used in this study were based off of the MW2 sequenced genome [87] and synthesized by Integrated DNA Technologies (Coralville, IA, USA) that are shown in Table 4. A LightCycler 1.5 machine (Roche) or a CFX96 machine (BioRad, Hercules, CA, USA) were used throughout the study. The guaB and ftsZ housekeeping genes were used as standardization controls. Each RT-qPCR run followed the minimum information for publication of quantitative real-time PCR experiments guidelines [88]. The qRT-PCRs were done at least three times under the following conditions: 94 °C, 20 s; 55 °C, 30 s; and 72 °C, 1 min for 35 cycles. The level of target gene transcripts in MW2 cells was compared to the guaB and ftsZ genes. Crossover points for all genes were standardized to the crossover points for ftsZ and guaB in each sample using the 2−ΔΔCT formula [89].

3.7. HPLC

High-performance liquid chromatography was used to detect the presence of (p)ppGpp in the intracellular nucleotide pools of mid-log phase S. aureus cells following treatment with SK-03-92, mupirocin (positive control), or dimethyl sulfoxide (negative control) [90,91,92]. SK-03-92 was added at 16 µg/mL to 100 mL mid-log (OD600 = 0.4–0.6) culture in cation-adjusted Mueller Hinton broth and incubated for 20 min with shaking at 37 °C. Mupirocin was added at 60 µg/mL. Cells were collected after a 20 min incubation by centrifugation at 10,000× g for 10 min at 4 °C. The supernatant was discarded and the cell pellet suspended in 12 mL of ice cold 0.4 M formic acid (pH 3.5). After 30 min on ice, the cell extract was centrifuged at 10,000× g for 10 min at 4 °C to remove cell debris. The supernatant was evaporated under vacuum and filtered (0.2 µm pore size). Filtered cell extract was stored at −20 °C until use. Fifty microliters of filtered cell extract were loaded on a Hypersil SAX column (Thermo Fisher Scientific, Waltham, MA, USA) (5 µm, 4.6 × 250 mm) at a flow rate of 1.0 mL/min in 0.45 M potassium phosphate 0.05 M magnesium sulfate buffer (pH 3.5) using a Waters 600E pump and 996 photodiode array detector (Waters, Milford, MA, USA). Absorbance of the separated nucleotide pool was monitored at 254 and 280 nm.

3.8. Biofilm Assay

To determine the effect of SK-03-92 treatment on the ability of S. aureus to form a biofilm, a biofilm assay was performed [93]. The S. aureus parent strains MW2 and JE2 were treated with SK-03-92 at concentrations of 0.5–64 µg/mL and those plates were compared to wells with bacteria not treated with the drug. After drying, the remaining dryed crystal violet dye stained biofilm material was extracted with 160 µL 33% glacial acetic acid per well and the OD570 was measured for each well. The total biofilm assay was performed a minimum of 10 times for each strain to achieve statistical significance.

3.9. MICs

In vitro minimum inhibitory concentration (MIC) determinations were performed on the S. aureus strains using SK-03-92 according to the Clinical and Laboratory Standards Institute guidelines [94]. All MICs were done a minimum of three times.

3.10. Statistical Analysis

A two-tailed Student′s t-test was run for the qRT-PCR comparisons and an ANOVA analysis was used for the biofilm assays to assess probabilities. p-values < 0.05 were considered significant.

4. Conclusions

Drug treatment with the stilbenoid compound SK-03-92 caused more genes to be transcriptionally downregulated than upregulated compared to other bactericidal and stilbenoid compounds (e.g., pterostilbene and resveratrol). The methoxy substitution on the main benzene ring at position 5 is likely to be responsible for this effect. A putative TCS, MW2284/MW2285, is clearly downregulated by SK-03-92 treatment. Is the TCS the prime target of the SK-03-92 lead compound and could targeting this TCS be the mechanism of action for SK-03-92 in Gram-positive bacteria? We hypothesize that one of the SK-03-92 targets is this putative TCS. Knockouts of both MW2284 and MW2285 showed substantial upregulation of the srtA gene that encodes sortase A. Sortase A may present something on the exterior of the S. aureus cell that causes rapid cell lysis. Furthermore, the MW2284 and MW2285 ORFs lie just upstream of the MW2286 ORF, which is thought to encode a malate:quinone oxidoreducatase gene important in the electron transport chain. If the MW2284/MW2285 TCS positively regulates this gene, then a mutation in either gene or treatment of S. aureus with a SK-03-92 drug may, in turn, cause downregulation of this gene as well as sdhC and glpD that would disrupt the electron transport chain in S. aureus. Evidence presented in this study also suggests the existence of a conserved bacterial pathway, involving PCD and persister formation, which is triggered by protein glycation and aggregation that may be responsible for the killing mechanism of SK-03-92. Could this putative TCS be tied to these phenomena? Further study may help us determine if the SK-03-92-induced S. aureus cell lysis is caused by a disruption of the electron transport chain, regulation of a conserved prokaryotic PCD pathway, or a combination of both of these events.

Acknowledgments

We thank Phillip Klebba, Jean Lee, and the NARSA for several S. aureus and L. monocytogenes strains used in this study. We also thank Greg Somerville for reviewing the paper. This work was funded by an ARG-WiTAG grant to W.R.S., a WiSys grant to W.R.S. and A.M., a National Institute of Neurological Disorders and Stroke grant NS076517 to J.M.C., a University of Wisconsin-La Crosse (UWL) Undergraduate Research Grant to M.L., a UWL College of Science and Allied Health supply grant to L.L., WisCAMP scholarships to M.L. and S.M.B., and a McNair Scholarship to M.L.

Author Contributions

W.R.S. conceived the experiments, wrote the paper, designed some of the primers, and ran data analysis: P.M.D. ran the microarray analysis and initial microarray annotation; A.M., J.M.C., V.V.N.P.B.T., C.W., and M.T.R. synthesized the SK-03-92 lead compound used in the study; S.M.B., M.L., L.L., and A.B. isolated the RNA samples, designed primers, and ran qRT-PCR; A.W. performed the biofilm assays, D.B. ran the biofilm statistical analysis; and R.P. and M.R. completed the HPLC and some bioinformatic analysis of the microarray results.

Conflicts of Interest

W.R.S., M.R., A.M., and J.M.C. hold a composition of matter and use a patent covering the SK-03-92 lead compound.

References

  1. Suaya, J.A.; Mera, R.M.; Cassidy, A.; O′Hara, P.; Amrine-Madsen, H.; Burstin, S.; Miller, L.G. Incidence and cost of hospitalizations associated with Staphylococcus aureus skin and soft tissue infections in the United States from 2001 to 2009. BMC Infect. Dis. 2014, 14, 296. [Google Scholar] [CrossRef] [PubMed]
  2. Klein, E.Y.; Sun, L.; Smith, D.L.; Laxminarayan, R. The changing epidemiology of methicillin-resistant Staphylococcus aureus in the United States: A national observational study. Am. J. Epidemiol. 2013, 177, 666–674. [Google Scholar] [CrossRef] [PubMed]
  3. Hidron, A.I.; Edwards, J.R.; Patel, J.; Horan, T.C.; Sievert, D.M.; Pollock, D.A.; Fridkin, S.K.; National Healthcare Safety Network Team; Participating National Healthcare Safety Network Facilities. NHSN annual update: Antimicrobial-resistant pathogens associated with healthcare-associated infections: Annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006–2007. Infect. Control Hosp. Epidemiol. 2008, 29, 996–1011. [Google Scholar] [PubMed]
  4. Maree, C.L.; Daum, R.; Boyle-Vavra, S.; Matayoshi, K.; Miller, L. Community associated methicillin-resistant Staphylococcus aureus isolates causing healthcare-associated infections. Emerg. Infect. Dis. 2007, 13, 236–242. [Google Scholar] [CrossRef] [PubMed]
  5. Herold, B.C.; Immergluck, L.C.; Maranan, M.C.; Lauderdale, D.S.; Gaskin, R.E.; Boyle-Vavra, S.; Leitch, C.D.; Daum, R.S. Community-acquired methicillin-resistant Staphylococcus aureus in children with no identified predisposing risk. JAMA 1998, 279, 593–598. [Google Scholar] [CrossRef] [PubMed]
  6. Lina, G.; Piémont, Y.; Godail-Gamot, F.; Bes, M.; Peter, M.O.; Gauduchon, V.; Vandenesch, F.; Etienne, J. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin. Infect. Dis. 1999, 29, 1128–1132. [Google Scholar] [CrossRef] [PubMed]
  7. Center for Disease Control and Prevention. Four pediatric deaths from community-acquired methicillin-resistant Staphylococcus aureus—Minnesota and North Dakota, 1997–1999. Morbid. Mortal. Wkly. Rep. 1999, 52, 88. [Google Scholar]
  8. Dantes, R.; Mu, Y.; Belflower, R.; Aragon, D.; Dumyati, G.; Harrison, L.H.; Lessa, F.C.; Lynfield, R.; Nadle, J.; Petit, S.; et al. National burden of invasive methicillin-resistant Staphylococcus aureus infections, United States, 2011. JAMA Intern. Med. 2013, 173, 1970–1978. [Google Scholar] [PubMed]
  9. Pate, A.J.; Terribilini, R.G.; Ghobadi, F.; Azhir, A.; Barber, A.; Pearson, J.M.; Kalantari, H.; Hassen, G.W. Antibiotics for methicillin-resistant Staphylococcus aureus skin and soft tissue infections: The challenge of outpatient therapy. Am. J. Emerg. Med. 2014, 32, 135–138. [Google Scholar] [CrossRef] [PubMed]
  10. Pendleton, J.N.; Gorman, S.P.; Gilmore, B.F. Clinical relevance of the ESKAPE pathogens. Expert Rev. Anti-Infect. Ther. 2013, 11, 297–308. [Google Scholar] [CrossRef] [PubMed]
  11. Stryjewski, M.E.; Corey, G.R. Methicillin-resistant Staphylococcus aureus: An evolving pathogen. Clin. Infect. Dis. 2014, 58, S10–S19. [Google Scholar] [CrossRef] [PubMed]
  12. Bae, I.G.; Federspiel, J.J.; Miró, J.M.; Woods, C.W.; Park, L.; Rybak, M.J.; Rude, T.H.; Bradley, S.; Bukovski, S.; de la Maria, C.G.; et al. Heterogeneous vancomycin-intermediate susceptibility phenotype in bloodstream methicillin-resistant Staphylococcus aureus isolates from an international cohort of patients with infective endocarditis: prevalence, genotype, and clinical significance. J. Infect. Dis. 2009, 200, 1355–1366. [Google Scholar] [CrossRef] [PubMed]
  13. Gomes, D.M.; Ward, K.E.; LaPlante, K.L. Clinical implications of vancomycin heteroresistant and intermediately susceptible Staphylococcus aureus. Pharmacotherapy 2015, 35, 424–432. [Google Scholar] [CrossRef] [PubMed]
  14. Moise, P.A.; North, D.; Steenbergen, J.N.; Sakoulas, G. Susceptibility relationship between vancomycin and daptomycin in Staphylococcus aureus: Facts and assumptions. Lancet Infect. Dis. 2009, 9, 617–624. [Google Scholar] [CrossRef]
  15. Sader, H.S.; Jones, R.N.; Rossi, K.L.; Rybak, M.J. Occurrence of vancomycin-tolerant and heterogeneous vancomycin-intermediate strains (hVISA) among Staphylococcus aureus causing bloodstream infections in nine USA hospitals. J. Antimicrob. Chemother. 2009, 64, 1024–1028. [Google Scholar] [CrossRef] [PubMed]
  16. Jones, R.N. Microbiological features of vancomycin in the 21st century: Minimum inhibitory concentration creep, bactericidal/static activity, and approved breakpoints to predict clinical outcomes or detect resistant strains. Clin. Infect. Dis. 2006, 42, S13–S24. [Google Scholar] [CrossRef] [PubMed]
  17. Traczewski, M.M.; Katz, B.D.; Steenbergen, J.N.; Brown, S.D. Inhibitory and bactericidal activities of daptomycin, vancomycin, and teicoplanin against methicillin-resistant Staphylococcus aureus isolates collected from 1985–2007. Antimicrob. Agents Chemother. 2009, 53, 1735–1738. [Google Scholar] [CrossRef] [PubMed]
  18. Bassetti, M.; Righi, E. Development of novel antibacterial drugs to combat multiple resistant organisms. Langenbecks Arch. Surg. 2015, 400, 153–165. [Google Scholar] [CrossRef] [PubMed]
  19. Coates, A.R.M.; Halls, G.; Hu, Y. Novel classes of antibiotics or more of the same? Br. J. Pharmacol. 2011, 163, 184–194. [Google Scholar] [CrossRef] [PubMed]
  20. Infectious Diseases Society of America. The 10 × 20 initiative: Pursuing a global commitment to develop 10 new antibacterial drugs by 2020. Clin. Infect. Dis. 2010, 50, 1081–1083. [Google Scholar]
  21. Kabir, M.S.; Engelbrecht, K.; Polanowski, R.; Krueger, S.M.; Ignasiak, R.; Rott, M.; Schwan, W.R.; Stemper, M.E.; Reed, K.D.; Sherman, D.; et al. New classes of Gram-positive selective antibacterials: Inhibitors of MRSA and surrogates of the causative agents of anthrax and tuberculosis. Bioorg. Med. Chem. Lett. 2010, 18, 5745–5749. [Google Scholar] [CrossRef] [PubMed]
  22. Schwan, W.R.; Kabir, M.S.; Kallaus, M.; Krueger, S.; Monte, A.; Cook, J.M. Synthesis and minimum inhibitory concentrations of SK-03-92 against Staphylococcus aureus and other gram-positive bacteria. J. Infect. Chemother. 2012, 18, 124–126. [Google Scholar] [CrossRef] [PubMed]
  23. Schwan, W.R.; Kolesar, J.M.; Kabor, M.S.; Elder, E.J., Jr.; Williams, J.B.; Minerath, R.; Cook, J.M.; Witzigmann, C.M.; Monte, A.; Flaherty, T. Pharmacokinetic/toxicity properties of the new anti-staphylococcal lead compound SK-03-92. Antibiotics 2015, 4, 617–626. [Google Scholar] [CrossRef] [PubMed]
  24. Cohen, N.R.; Lobritz, M.A.; Collins, J.J. Microbial persistence and the road to drug resistance. Cell Host Microbe 2013, 13, 632–642. [Google Scholar] [CrossRef] [PubMed]
  25. Conlon, B.P. Staphylococcus aureus chronic and relapsing infections: Evidence of a role for persister cells: An investigation of persister cells, their formation and their role in S. aureus disease. Bioessays 2014, 36, 991–996. [Google Scholar] [CrossRef] [PubMed]
  26. Lechner, S.; Lewis, K.; Bertram, R. Staphylococcus aureus persisters tolerant to bactericidal antibiotics. J. Mol. Microbiol. Biotechnol. 2012, 22, 235–244. [Google Scholar] [CrossRef] [PubMed]
  27. Lewis, K. Persister cells. Annu. Rev. Microbiol. 2010, 64, 357–372. [Google Scholar] [CrossRef] [PubMed]
  28. Keren, I.; Shah, D.; Spoering, A.; Kaldalu, N.; Lewis, K. Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J. Bacteriol. 2004, 186, 8172–8180. [Google Scholar] [CrossRef] [PubMed]
  29. Spoering, A.L.; Lewis, K. Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J. Bacteriol. 2001, 183, 6746–6751. [Google Scholar] [CrossRef] [PubMed]
  30. Stewart, P.S.; Costerton, J.W. Antibiotic resistance of bacteria in biofilms. Lancet 2001, 358, 135–138. [Google Scholar] [CrossRef]
  31. Jang, H.; Nde, C.; Toghrol, F.; Bentley, W.E. Microarray analysis of toxicogenomic effects of ortho-phenylphenol in Staphylococcus aureus. BMC Genomics 2008, 9, 411. [Google Scholar] [CrossRef] [PubMed]
  32. Lama, A.; Pané-Farré, J.; Chon, T.; Wiersma, A.M.; Sit, C.S.; Vederas, J.C.; Hecker, M.; Nakano, M.M. Response of methicillin-resistant Staphylococcus aureus to amicoumacin A. PLoS ONE 2012, 7, e34037. [Google Scholar] [CrossRef] [PubMed]
  33. Muthaiyan, A.; Silverman, J.A.; Jayaswal, R.K.; Wilinson, B.J. Transcriptional profiling reveals that daptomycin induces the Staphylococcus aureus cell wall stress stimulon and gene responsive to membrane depolarization. Antimicrob. Agents Chemother. 2008, 52, 980–990. [Google Scholar] [CrossRef] [PubMed]
  34. Lechner, S.; Prax, M.; Lange, B.; Huber, C.; Eisenreich, W.; Herbig, A.; Nieselt, K.; Bertram, R. Metabolic and transcriptional activities of Staphylococcus aureus challenged with high-doses of daptomycin. Int. J. Med. Microbiol. 2014, 304, 931–940. [Google Scholar] [CrossRef] [PubMed]
  35. Pan, Z.; Agarwal, A.K.; Xu, T.; Feng, Q.; Baerson, S.R.; Duke, S.O.; Rimando, A.M. Identification of molecular pathways affected by pterostilbene, a natural dimethylether analog of resveratrol. BMC Med. Genomics 2008, 20, 1–7. [Google Scholar] [CrossRef] [PubMed]
  36. Wang, Z.; Gu, Z.; Shen, Y.; Wang, Y.; Li, J.; Lv, H.; Huo, K. The natural product resveratrol inhibits yeast cell separation by extensively modulating the transcriptional landscape and reprogamming the intracellular metabolome. PLoS ONE 2016, 11, e0150156. [Google Scholar] [CrossRef]
  37. Qin, N.; Tan, X.; Jiao, Y.; Liu, L.; Zhao, W.; Yang, S.; Jia, A. RNA-Seq-based transciptome analysis of methicillin-resistant Staphylococcus aureus biofilm inhibition by ursolic acid and resveratrol. Sci. Rep. 2014, 4, 5467. [Google Scholar] [CrossRef] [PubMed]
  38. Fey, P.D.; Endres, J.L.; Yajjala, V.K.; Widhelm, T.J.; Boissy, R.J.; Bose, J.L.; Bayles, K.W. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. mBio 2013, 4, e00537012. [Google Scholar] [CrossRef] [PubMed]
  39. Xiao, Q.; Jiang, X.; Moore, K.J.; Shao, Y.; Pi, H.; Dubail, I.; Charbit, A.; Newton, S.M.; Klebba, P.E. Sortase independent and dependent systems for acquisition of haem and haemoglobin in Listeria monocytogenes. Mol. Microbiol. 2011, 80, 1581–1597. [Google Scholar] [CrossRef] [PubMed]
  40. Yeh, J.I.; Chinte, U.; Du, S. Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism. Proc. Nat. Acad. Sci. USA 2008, 105, 3280–3285. [Google Scholar] [CrossRef] [PubMed]
  41. Ramasamy, R.; Yan, S.F.; Schmidt, A.M. Methylglyoxal comes of AGE. Cell 2006, 124, 258–260. [Google Scholar] [CrossRef] [PubMed]
  42. Ackerman, R.S.; Cozzarelli, N.R.; Epstein, E.W. Accumulation of toxic concentrations of methylglyoxal by wild-type Escherichia coli K-12. J. Bacteriol. 1974, 119, 357–362. [Google Scholar] [PubMed]
  43. Rabbani, N.; Thornalley, P.J. Methylglyoxal, glyoxalase 1 and the dicarbonyl proteome. Amino Acids 2012, 42, 1133–1142. [Google Scholar] [CrossRef] [PubMed]
  44. Kwak, M.; Ku, M.; Kang, S. NAD+-linked alcohol dehydrogenase 1 regulates methylglyoxal concentration in Candida albicans. FEBS Lett. 2014, 588, 1144–1153. [Google Scholar] [CrossRef] [PubMed]
  45. Spoering, A.L.; Vulić, M.; Lewis, K. GlpD and PlsB participate in persister cell formation in Escherichia coli. J. Bacteriol. 2006, 188, 5136–5144. [Google Scholar] [CrossRef] [PubMed]
  46. Stauffer, L.T.; Steiert, P.S.; Steiert, J.G.; Stauffer, G.V. An Escherichia coli protein with homology to the H-protein of the glycine cleavage enzyme complex from pea and chicken liver. DNA Seq. 1991, 2, 13–17. [Google Scholar] [CrossRef] [PubMed]
  47. Gemayel, R.; Fortpied, J.; Rzem, R.; Vertommen, D.; Veiga-da-Cunha, M.; van Schaftingen, E. Many fructosamine 3-kinase homologues in bacteria are ribulosamine/erythrulosamine 3-kinases potentially involved in protein deglycation. FEBS J. 2007, 274, 4360–4374. [Google Scholar] [CrossRef] [PubMed]
  48. Polanowski, R.; Rott, M.; University of Wisconsin-La Crosse, La Crosse, WI, USA. unpublished data. 2016.
  49. Performance Standards for Antimicrobial Susceptibility Testing, 16th Informational Supplement; NCCLS document M100-S16; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2006.
  50. Bednarska, N.G.; Schymkowitz, J.; Rousseau, F.; van Eldere, J. Protein aggregation in bacteria: The thin boundary between functionality and toxicity. Microbiology 2013, 159, 1795–1806. [Google Scholar] [CrossRef] [PubMed]
  51. Wood, R.C.; Steers, E. Study of the purine metabolism of Staphylococcus aureus. J. Bacteriol. 1959, 77, 760–765. [Google Scholar] [PubMed]
  52. Subramanian, D.; Natarajan, J. Network analysis of S. aureus response to ramoplanin reveals modules for virulence factors and resistance mechanisms and characteristic novel genes. Gene 2015, 574, 149–162. [Google Scholar] [CrossRef] [PubMed]
  53. Cuaron, J.A.; Dulal, S.; Song, Y.; Singh, A.K.; Montelongo, C.E.; Yu, W.; Nagarajan, V.; Jayaswal, R.K.; Wilkinson, B.J.; Gustafson, J.E. Tea tree oil-induced transcriptional alterations in Staphylococcus aureus. Phytother. Res. 2013, 27, 390–396. [Google Scholar] [CrossRef] [PubMed]
  54. Shen, F.; Tang, X.; Wang, Y.; Yang, Z.; Shi, X.; Wang, C.; Zhang, Q.; An, Y.; Cheng, W.; Jin, K.; et al. Phenotype and expression prolife analysis of Staphylococcus aureus biofilms and planktonic cells in response to licochalcone A. Appl. Microbiol. Biotechnol. 2015, 99, 359–373. [Google Scholar] [CrossRef] [PubMed]
  55. Blakely, R.L.; Vitols, E. The control of nucleotide biosynthesis. Annu. Rev. Biochem. 1968, 37, 201–224. [Google Scholar] [CrossRef] [PubMed]
  56. Fung, D.K.; Chan, E.W.; Chin, M.L.; Chan, R.C. Delineation of a bacterial starvation stress response network which can mediate antibiotic tolerance development. Antimicrob. Agents Chemother. 2010, 54, 1082–1093. [Google Scholar] [CrossRef] [PubMed]
  57. Maisonneuve, E.; Gerdes, K. Molecular mechanisms underlying bacterial persisters. Cell 2014, 157, 539–548. [Google Scholar] [CrossRef] [PubMed]
  58. Yee, R.; Cui, P.; Shi, W.; Feng, J.; Zhang, Y. Genetic screen reveals the role of purine metabolism in Staphylococcus aureus persistence to rifampicin. Antibiotics 2015, 4, 627–642. [Google Scholar] [CrossRef] [PubMed]
  59. Ranjit, D.K.; Endres, J.L.; Bayles, K.W. Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties. J. Bacteriol. 2011, 193, 2468–2476. [Google Scholar] [CrossRef] [PubMed]
  60. Sadykov, M.R.; Bayles, K. The control of death and lysis in staphylococcal biofilms: A coordination of physiological signals. Curr. Opin. Microbiol. 2012, 15, 211–215. [Google Scholar] [CrossRef] [PubMed]
  61. Yang, S.J.; Rice, K.C.; Brown, R.J.; Patton, T.G.; Liou, L.E.; Park, Y.H.; Bayles, K.W. A LysR-type regulator, CidR, is required for induction of the Staphylococcus aureus cidABC operon. J. Bacteriol. 2005, 187, 5893–5900. [Google Scholar] [CrossRef] [PubMed]
  62. Bayles, K.W. Bacterial programmed cell death: Making sense of a paradox. Nat. Rev. Microbiol. 2014, 12, 63–69. [Google Scholar] [CrossRef] [PubMed]
  63. Tanouchi, Y.; Lee, A.J.; Meredith, H.; You, L. Programmed cell death in bacteria and implications for antibiotic therapy. Trends Microbiol. 2013, 21, 265–270. [Google Scholar] [CrossRef] [PubMed]
  64. Sarkar, S.K.; Bhattacharyya, A.; Mandal, S.S. YnfA, a SMP family efflux pump is abundant in Escherichia coli isolates from urinary infection. Indian J. Med. Microbiol. 2015, 33, 139–142. [Google Scholar] [PubMed]
  65. Mazmanian, S.K.; Liu, G.; Ton-That, H.; Schneewind, O. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 1999, 285, 760–763. [Google Scholar] [CrossRef] [PubMed]
  66. Altschul, S.F.; Madden, T.L.; Schäffer, A.A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D.J. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 1997, 25, 3389–3402. [Google Scholar] [CrossRef] [PubMed]
  67. Nikolskaya, A.N.; Galperin, M.Y. A novel type of conserved DNA-binding domain in the transcriptional regulators of the AlgR/AgrA/LytR family. Nucleic Acids Res. 2002, 30, 2453–2459. [Google Scholar] [CrossRef] [PubMed]
  68. Nicod, S.S.; Weinzierl, R.O.; Burchell, L.; Escalera-Maurer, A.; James, E.H.; Wigneshweraraj, S. Systematic mutational analysis of the LytTR DNA binding domain of Staphylococcus aureus virulence gene transcription factor AgrA. Nucleic Acids Res. 2014, 42, 12523–12536. [Google Scholar] [CrossRef] [PubMed]
  69. Lan, L.; Cheng, A.; Dunman, P.M.; Missiakas, D.; He, C. Golden pigment production and virulence gene expression are affected by metabolisms in Staphylococcus aureus. J. Bacteriol. 2010, 192, 3068–3077. [Google Scholar] [CrossRef] [PubMed]
  70. Fuchs, S.; Pané-Farré, J.; Kohler, C.; Hecker, M.; Engelmann, S. Anaerobic gene expression in Staphylococcus aureus. J. Bacteriol. 2007, 189, 4275–4289. [Google Scholar] [CrossRef] [PubMed]
  71. Kadurugamuwa, J.L.; Sin, L.; Albert, E.; Yu, J.; Francis, K.; DeBoer, M.; Rubin, M.; Bellinger-Kawahara, C.; Parr, T.R., Jr.; Contag, P.R. Direct continuous method for monitoring biofilm infection in a mouse model. Infect. Immun. 2003, 71, 882–890. [Google Scholar] [CrossRef] [PubMed]
  72. Xiong, Y.Q.; Willard, J.; Kadurugamuwa, J.L.; Yu, J.; Francis, K.P.; Bayer, A.S. Real-time in vivo bioluminescent imaging for evaluating the efficacy of antibiotics in a rat Staphylococcus aureus endocarditis model. Antimicrob. Agents Chemother. 2005, 49, 380–387. [Google Scholar] [CrossRef] [PubMed]
  73. Reiß, S.; Pané-Farré, J.; Fuchs, S.; François, P.; Liebeke, M.; Schrenzel, J.; Lidequist, U.; Lalk, M.; Wolz, C.; Hecker, M.; Engelmann, S. Global analysis of the Staphylococcus aureus response to mupirocin. Antimicrob. Agents Chemother. 2012, 56, 787–804. [Google Scholar] [CrossRef] [PubMed]
  74. Anderson, K.L.; Roberts, C.; Disz, T.; Vonstein, V.; Hwang, K.; Overbeck, R.; Olson, P.D.; Projan, S.J.; Dunman, P.M. Characterization of the Staphylococcus aureus heat shock, cold shock, stringent, and SOS responses and their effects on log-phase mRNA turnover. J. Bacteriol. 2006, 188, 6739–6756. [Google Scholar] [CrossRef] [PubMed]
  75. Fischer, M.; Zimmerman, T.P.; Short, S.A. A rapid method for the determination of guanosine 5′-diphosphate-3′-diphosphate and guanosine 5′triphosphate-3′-diphosphate by high performance liquid chromatography. Anal. Biochem. 1982, 121, 135–139. [Google Scholar] [CrossRef]
  76. Melo, A.; Colombo, A.; Arthington-Skaggs, B. Paradoxical growth effect of caspofungin observed on biofilms and planktonic cells of five different Candida species. Antimicrob. Agents Chemother. 2007, 51, 3081–3088. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  77. Wood, T.; Knabels, S.; Kwan, B. Bacterial persister cell formation and dormancy. Appl. Environ. Microbiol. 2013, 79, 7116–7121. [Google Scholar] [CrossRef] [PubMed]
  78. Marraffini, L.A.; DeDent, A.C.; Schneewind, O. Sortases and the art of anchoring proteins to the envelopes of Gram-positive bacteria. Microbiol. Mol. Biol. Rev. 2006, 70, 192–221. [Google Scholar] [CrossRef] [PubMed]
  79. Fischetti, V.A.; Pancholi, V.; Schneewind, O. Conservation of a hexapeptide sequence in the anchor region of surface proteins from gram-positive bacteria. Mol. Microbiol. 1990, 4, 1603–1605. [Google Scholar] [CrossRef] [PubMed]
  80. Boekhorst, J.; de Been, M.W.; Kleerebezem, M.; Siezen, R.J. Genome-wide detection and analysis of cell wall-bound proteins with LPxTG-like sorting motifs. J. Bacteriol. 2005, 187, 4928–4934. [Google Scholar] [CrossRef] [PubMed]
  81. Ton-That, H.; Liu, G.; Mazmanian, S.K.; Faull, K.F.; Schneewind, O. Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc. Natl. Acad. Sci. USA 1999, 96, 12424–12429. [Google Scholar] [CrossRef] [PubMed]
  82. Foster, T.J.; Hook, M. Surface protein adhesins of Staphylococcus aureus. Trends Microbiol. 1998, 6, 484–488. [Google Scholar] [CrossRef]
  83. Mazmanian, S.K.; Liu, G.; Jensen, E.R.; Lenoy, E.; Schneewind, O. Staphylococcus aureus mutants defective in the display of surface proteins and in the pathogenesis of animal infections. Proc. Natl. Acad. Sci. USA 2000, 97, 5510–5515. [Google Scholar] [CrossRef] [PubMed]
  84. Sibbald, M.J.J.; Yang, X.-M.; Tsompanidou, E.; Qu, D.; Hecker, M.; Becher, D.; Buist, G.; Maarten van Dijl, J. Partially overlapping substrate specificities of staphylococcal group—A sortases. Proteomics 2012, 12, 3049–3062. [Google Scholar] [CrossRef] [PubMed]
  85. Jonsson, I.M.; Mazmanian, S.K.; Schneewind, O.; Bremell, T.; Tarkowski, A. The role of Staphylococcus aureus sortase A and sortase B in murine arthritis. Microbes Infect. 2003, 5, 775–780. [Google Scholar] [CrossRef]
  86. Weiss, W.J.; Lenoy, E.; Murphy, T.; Tardio, L.; Burgio, P.; Projan, S.J.; Schneewind, O.; Alksne, L. Effect of srtA and srtB gene expression on the virulence of Staphylococcus aureus in animal infection. J. Antimicrob. Chemother. 2004, 53, 480–486. [Google Scholar] [CrossRef] [PubMed]
  87. Voyich, J.M.; Braughton, K.R.; Sturdevant, D.E.; Whitney, A.R.; Saïd-Salim, B.; Porcella, S.F.; Long, R.D.; Dorward, D.W.; Gardner, D.J.; Kreiswirth, B.N.; et al. Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils. J. Immunol. 2005, 175, 3907–3919. [Google Scholar] [CrossRef] [PubMed]
  88. Baba, T.; Takeuchi, F.; Kuroda, M.; Yuzawa, H.; Aoki, K.; Oguchi, A.; Nagai, Y.; Iwama, N.; Asano, K.; Naimi, T.; et al. Genome and virulence determinants of high virulence community-acquired MRSA. Lancet 2002, 359, 1819–1827. [Google Scholar] [CrossRef]
  89. Bustin, S.S.; Benes, V.; Garson, J.A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller, R.; Nolan, T.; Pfaffl, M.W.; Shipley, G.L.; et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009, 55, 611–622. [Google Scholar] [CrossRef] [PubMed]
  90. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  91. Ochi, K. Occurrence of the stringent response in Streptomyces sp. and its significance for the initiation of morphological and physiological differentiation. J. Gen. Microbiol. 1986, 132, 2621–2631. [Google Scholar] [PubMed]
  92. Ochi, K. Metabolic initiation of differentiation and secondary metabolism by Streptomyces griseus: Significance of the stringent response (ppGpp) and GTP content in relation to A factor. J. Bacteriol. 1987, 169, 3608–3616. [Google Scholar] [CrossRef] [PubMed]
  93. Wilson, J.M.; Oliva, B.; Cassels, R.; O′Hanlon, P.J.; Chopra, I. SB 205952, a novel semisynthetic monic acid analog with at least two modes of action. Antimicrob. Agents Chemother. 1995, 39, 1925–1933. [Google Scholar] [CrossRef] [PubMed]
  94. Stepanovic, S.; Vukovic, D.; Pavlovic, M.; Svabic-Vlahovic, M. Influence of dynamic conditions on biofilm formation by staphylococci. Eur. J. Clin. Microbiol. Infect. Dis. 2001, 20, 502–504. [Google Scholar] [CrossRef] [PubMed]
Figure 1. Quantitative reverse transcribed-polymerase chain reaction results of S. aureus MW2 cells treated with 8× the SK-03-92 MIC vs. untreated cells. The data represents the mean + standard deviation from at least three separate runs.
Figure 1. Quantitative reverse transcribed-polymerase chain reaction results of S. aureus MW2 cells treated with 8× the SK-03-92 MIC vs. untreated cells. The data represents the mean + standard deviation from at least three separate runs.
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Figure 2. Absorbance (254 and 280 nm) of the formic acid extracted nucleotide pool of log-phase S. aureus ATCC 29213 after 20 min with either (A) no treatment, (B) 60 μg/mL mupirocin, or (C) 16 μg/mL SK-03-92. Arrow denotes the (p)ppGpp peak.
Figure 2. Absorbance (254 and 280 nm) of the formic acid extracted nucleotide pool of log-phase S. aureus ATCC 29213 after 20 min with either (A) no treatment, (B) 60 μg/mL mupirocin, or (C) 16 μg/mL SK-03-92. Arrow denotes the (p)ppGpp peak.
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Figure 3. The effects of SK-03-92 drug concentration on 24 h biofilm formation (OD570) for S. aureus strains JE2 (black column) and MW2 (white column). All experiments represent the mean + standard deviation of at least 10 runs done in triplicate.
Figure 3. The effects of SK-03-92 drug concentration on 24 h biofilm formation (OD570) for S. aureus strains JE2 (black column) and MW2 (white column). All experiments represent the mean + standard deviation of at least 10 runs done in triplicate.
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Figure 4. Quantitative reverse transcribed-polymerase chain reaction results of S. aureus srtA transcription in wild-type bacteria compared to MW2284 and MW2285 mutants. The data represents the mean + standard deviation from three separate runs.
Figure 4. Quantitative reverse transcribed-polymerase chain reaction results of S. aureus srtA transcription in wild-type bacteria compared to MW2284 and MW2285 mutants. The data represents the mean + standard deviation from three separate runs.
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Table 1. Bacterial strains used in this study.
Table 1. Bacterial strains used in this study.
Bacterial StrainGenotypeReference
S. aureus
MW2USA400 wild-type[7]
JE2USA300 wild-type[38]
NE272JE2 MW2284 mutant[38]
NE671JE2 MW2285 mutant[38]
NE1363JE2 srtB mutant[38]
NE1787JE2 srtA mutant[38]
L. monocytogenes
EGDWild-type[39]
EGD srtAEGD srtA mutant[39]
EGD srtBEGD srtB mutant[39]
Table 2. Microarray analysis of genes dysregulated in S. aureus MW2 cells treated with 8× the SK-03-92 MIC vs. untreated cells.
Table 2. Microarray analysis of genes dysregulated in S. aureus MW2 cells treated with 8× the SK-03-92 MIC vs. untreated cells.
LocusFold-DifferenceDescription
Stress Response
SACOL1759−2.3universal stress protein family
Transporter
SACOL0086−2.0drug transporter, putative
SACOL0155−5.7cation efflux family protein
SACOL0178−2.9PTS system, IIBC components (scrBC)
SACOL0400−2.6ascorbate-specific PTS system subunit IIC (ulaA)
SACOL0454−2.3sodium:dicarboxylate symporter family protein
SACOL1018−2.3sodium:alanine symporter family protein
SACOL1872−3.0epidermin immunity protein F (epiE)
SACOL2146−2.7PTS system, mannitol-specific IIBC components (mtlA)
SACOL2333−2.8YnfA family protein
SACOL2573−3.2copper ion binding protein (copZ)
SACOL2664−2.3mannose-6-phosphate isomerase (manA)
SACOL2718−4.62-oxoglutarate/malate translocator, sodium sulfate symporter
Signaling/Regulation
SACOL2360−14.1LytTR family regulator protein
SACOL2361−26.9histidine kinase sensor membrane protein
SACOL23402.2transcriptional regulator TetR-family
Cell Wall Associated
SACOL0151−2.7UDP-N-acetylglucosamine 2-epimerase Cap5P (cap5P)
SACOL0247−3.2holin-like protein LrgA (lrgA)
SACOL0612−2.1glycosyl transferase, group 1 family protein
SACOL1071−2.2chitinase-related protein (iraE)
SACOL2554−2.0holin-like protein CidB (cidB)
SACOL25394.2sortase A (srtA)
Anabolism/Nucleic Acids
SACOL013−2.15′ nucleotidase family protein
SACOL1078−3.2phosphoribosylformylglycinamidine synthase II (purL)
SACOL1082−2.5bifunctional purine biosynthesis protein (purH)
SACOL1083−2.6phosphoribosylamine-glycine ligase (purD)
SACOL2329−3.5ribose 5-phosphate isomerase (rpiA)
SACOL21112.2thymidine kinase (tdk)
SACOL23772.3conserved hypothetical protein
Anabolism/Proteostasis
SACOL0085−2.5peptidase, M20.M25/M40 family
SACOL2605−9.6ribulosamine 3-kinase
SACOL04572.6conserved hypothetical protein, heat induced stress
SACOL05902.430S ribosomal protein L7 Ae
SACOL08772.5glycine cleavage system H protein (gcvH)
SACOL19072.4ribosomal large subunit pseudouridine synthase (rluD)
SACOL19392.3phosphotyrosine protein phosphatase (ptpA)
SACOL25962.6metallo-dependent amidohydrolase
Lipid Metabolism
SACOL2091−2.5beta-hydroxyacyl-dehydratase FabZ (fabZ)
SACOL2459−3.8para-nitrobenzyl esterase (pnbA)
SACOL114210.0aerobic glyerol-3-phosphate dehydrogenase (glpD)
Catabolism
SACOL0135−2.4alcohol dehydrogenase, iron-containing (adhE)
SACOL0660−3.4alcohol dehydrogenase, zinc-containing (adhA)
SACOL1158−2.5succinate dehydrogenase, cytochrome b558 subunit (sdhC)
SACOL1604−2.1glucokinase (glk)
SACOL2338−3.5hypothetical protein (putative oxidoreductase)
SACOL17132.3hypothetical protein, putative ammonia monooxygenase
Unknown
SACOL0089−4.4myosin-reactive antigen, 67 kDa
SACOL2315−3.8conserved hypothetical protein
SACOL2338−3.4conserved hypothetical protein
SACOL2491−2.9conserved hypothetical protein
SACOL07423.1conserved hypothetical protein
SACOL17892.4conserved hypothetical protein
Table 3. MIC results for S. aureus and L. monocytogenes mutants and wild-type strains against SK-03-92.
Table 3. MIC results for S. aureus and L. monocytogenes mutants and wild-type strains against SK-03-92.
StrainGenotypeMIC
S. aureus
JE2Wild-type1 a
NE272MW22851
NE671MW22841
NE1363srtB1
NE1787srtA0.5
L. monocytogenes
EGDWild-type1
EGD srtAsrtA0.125
EGD srtBsrtB1
a Mean + standard deviation from three separate runs.
Table 4. Oligonucleotide primers used in this study.
Table 4. Oligonucleotide primers used in this study.
PrimerGeneSequence
SaFtsZ1ftsZ5′-GGTGTAGGTGGTGGCGGTAA-3′
SaFtsZ2 5′-TCATTGGCGTAGATTTGTC-3′
GuaBF1guaB5′-GCTCGTCAAGGTGGTTTAGGTG-3′
GuaBR1 5′-TAAGACATGCACACCTGCTTCG-3′
SrtA1srtA5′-TCGCTGGTGTGGTACTTATC-3′
SrtA2 5′-CAGGTGTTGCTGGTCCTGGA-3′
MW2284AMW22845′-CAATGCAAATGAGACGGAATCT-3′
MW2284B 5′-GAAGAATAGGTGTAGTGTGCAT-3′
MW2285AMW22855′-GTATGTTATTTGCAGACGGCAA-3′
MW2285B 5′-AAAGGCAAGAATCCGACATACG-3′
SA2043Atdk5′-CTTGTTCACTGACAGCCATCA-3′
SA2043B 5′-ACGCACGACTTAACTAATGTTG-3′
SaPurD1purD5′-CAGCCGCTAATTGATGGATTA-3′
SaPurD2 5′-AGCACTTCTGGCTGCTTCAAT-3′
SaPurH1purH5′-CCAGAAATAATGGATGGCCGT-3′
SaPurH2 5′-TGCCGGATGTACAATTGTTGT-3′
SaPurL1purL5′-GTTATGTGGAGTGAACATTGC-3′
SaPurL2 5′-AGCCCCAATAGAGACAATGTC-3′

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MDPI and ACS Style

Schwan, W.R.; Polanowski, R.; Dunman, P.M.; Medina-Bielski, S.; Lane, M.; Rott, M.; Lipker, L.; Wescott, A.; Monte, A.; Cook, J.M.; et al. Identification of Staphylococcus aureus Cellular Pathways Affected by the Stilbenoid Lead Drug SK-03-92 Using a Microarray. Antibiotics 2017, 6, 17. https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics6030017

AMA Style

Schwan WR, Polanowski R, Dunman PM, Medina-Bielski S, Lane M, Rott M, Lipker L, Wescott A, Monte A, Cook JM, et al. Identification of Staphylococcus aureus Cellular Pathways Affected by the Stilbenoid Lead Drug SK-03-92 Using a Microarray. Antibiotics. 2017; 6(3):17. https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics6030017

Chicago/Turabian Style

Schwan, William R., Rebecca Polanowski, Paul M. Dunman, Sara Medina-Bielski, Michelle Lane, Marc Rott, Lauren Lipker, Amy Wescott, Aaron Monte, James M. Cook, and et al. 2017. "Identification of Staphylococcus aureus Cellular Pathways Affected by the Stilbenoid Lead Drug SK-03-92 Using a Microarray" Antibiotics 6, no. 3: 17. https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics6030017

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