Open Access

The SAV1322 gene from Staphylococcus aureus: genomic and proteomic approaches to identification and characterization of gene function

  • Jung Wook Kim1,
  • Hyun-Kyung Kim1,
  • Gi Su Kang1,
  • Il-Hwan Kim1,
  • Hwa Su Kim1,
  • Yeong Seon Lee1 and
  • Jae Il Yoo1, 2Email author
BMC MicrobiologyBMC series – open, inclusive and trusted201616:206

https://doi.org/10.1186/s12866-016-0824-2

Received: 12 August 2015

Accepted: 29 August 2016

Published: 6 September 2016

Abstract

Background

Bacterial two-component regulatory systems (TCRS) are associated with the expression of virulence factors and antibiotic susceptibility. In Staphylococcus aureus, 16 TCRS types have been identified. The histidine kinase/response regulator SAV1321/SAV1322 in the S. aureus shares considerable homology with the TCRS DesKR in Bacillus subtilis. However, a function for the SAV1322 locus has not yet been assigned.

Results

Deletion of the SAV1322 locus in S. aureus results in reduced growth when cultured under low (25 °C) and high (46 °C) temperature conditions. The sav1322 deletion mutant is more tolerant to oxidative stress in vitro and is less pathogenic in a murine infection model when compared with wild-type parent strain Mu50. Furthermore, the sav1322 mutant exhibits lower MICs for gentimicin, tetracyclines and glycopeptides, increased autolysis, and a thinner cell wall when compared with the wild-type strain. Microarray and proteomic analyses show that the expression of cell-wall-associated genes glmS and murZ are lower, and the expression of heat shock and stress-related genes (hrcA, ctsR, dnaK, dnaJ, grpE, clpB, and clpC) are higher in the sav1322 mutant when compared with the wild-type strain. In addition, the sav1322 mutant displays altered expression of proteins involved in carbohydrate/energy metabolism, cell wall metabolism, and stress or heat shock response, as well as other metabolic processes including lipid metabolism, amino acid biosynthesis, purine or pyrimidine metabolism, transcription, and protein biosynthesis.

Conclusions

The S. aureus SAV1322 locus plays a pronounced role in temperature adaptation, antibiotic resistance, and virulence by regulating a wide range of genes and proteins involved in metabolism and stress tolerance.

Keywords

Heat shock proteins Microarray analysis Microbial drug resistance Physiological stress Proteomics Staphylococcus aureus

Background

Staphylococcus aureus is a major cause of nosocomial infections, resulting in increased morbidity and mortality worldwide. Commonly reported methicillin-resistant S. aureus (MRSA) exhibits resistance to multiple chemotherapeutic agents, including β-lactams, quinolones, and aminoglycosides, while community-acquired MRSA is associated with serious infectious disease, sepsis, and pneumonia [1, 2]. More recently, vancomycin-intermediate or vancomycin-resistant S. aureus (VISA or VRSA) strains have emerged in several countries. These factors have rendered successful treatment of these infections increasingly problematic.

S. aureus has survived and thrived over the years in part because of its adaptability and stress response capabilities. It is resistant to a variety of environmental factors, including oxidative, pH, osmotic, antibiotic and temperature stressors [3]. Two-component regulatory systems (TCRSs) act in response to a stimulus that allows cells to sense and respond to changes in many different environmental conditions. Most S. aureus strains are endowed with 16 sets of genes that encode TCRSs. An additional TCRS is present in the staphylococcal cassette chromosome mec in MRSA, and this is linked to the induction of methicillin resistance [4]. The well-studied TCRS Agr is a positive regulator of exoproteins, including proteases, hemolysins, and toxins [5, 6]. Other TCRSs, such as SaeSR and ArlSR, influence the expression of some virulence factors [7, 8]. Another system, SrrAB, is homologous to the Bacillus subtilis TCRS ResDE; it is involved the modulation of anaerobic gene expression and sensitivity to oxygen tension [9]. The S. aureus TCRS VraSR is homologous to B. subtilis YvqEC, and it modulates cell wall biosynthesis and increased resistance to vancomycin [10]. Another S. aureus TCRS, NreBC, controls nitrate reductase and nitrite reductase operons [11]. More recently, it was reported that the S. aureus TCRS WalKR is involved in cell wall metabolism, cell survival, and vancomycin resistance [12, 13].

Although most TCRSs identified in S. aureus have been well studied, the functions of a few remain elusive or only partially explained. Of these, the uncharacterized SAV1321/SAV1322 (Mu50) TCRS demonstrates homology with B. subtilis DesKR. DesKR has been described as a regulatory system involved in the maintenance of membrane fluidity in response to temperature downshift. With decreasing temperature, the membrane-bound sensor kinase DesK phosphorylates its corresponding response regulator, DesR, which then binds to a specific recognition sequence in the promoter region of the des gene to activate its transcription [14]. Finally, activity of the membrane-located fatty acid desaturase Des maintains membrane fluidity in the cold [15].

In this study, we generated an S. aureus mutant lacking the SAV1322 locus to examine its role in bacterial physiology and virulence. Comparative microarray and proteomic analyses were performed to determine putative members of the SAV1322 regulon.

Results

Construction and characterization of the S. aureus sav1322 knockout mutant

A sav1322 knockout mutant strain was created from the wild-type (WT) S. aureus Mu50 using homologous recombination. Re-introducing the SAV1322 locus into the sav1322 mutant generated a complementation strain. The WT SAV1322 locus is shown in Fig. 1a. To confirm allelic replacement, chromosomal DNA was isolated from the WT, sav1322 mutant, and complementation strains. PCR analysis, sequencing, and RT-PCR confirmed the mutation (Fig. 1b).
Fig. 1

a Genetic structure of the SAV1322 locus in the wild-type (WT) S. aureus Mu50 strain. b RT-PCR analysis of the sav1322 mutant

To determine whether deletion of the S. aureus SAV1322 locus had any impact on bacterial growth and morphology, growth curves and colony forming units (CFU) were compared at 37 °C. We observed no difference in growth between the WT and the sav1322 mutant strains at this temperature. Similarly, no obvious differences in colony morphology were observed (data not shown).

Temperature stress

To examine the role of the SAV1322 locus in response to heat and cold stress, we compared the growth rates of the WT, sav1322 mutant, and complementation strains at 25 and 46 °C (Fig. 2). The sav1322 mutant grew less at 46 °C when compared with the WT strain. The WT strain exhibited a normal growth curve from inoculation until 3 h, at which point a decline in growth was noted. The complementation strain exhibited a growth curve that was similar to the WT; however, a growth decline was noted after 1 h (Fig. 2a). At 25 °C, growth of the WT and mutant strains started to increase at 3 h, yet the growth rate for the WT strain was faster than that of the sav1322 mutant strain. Growth of the complementation strain increased continuously, beginning immediately after inoculation at 25 °C (Fig. 2b). To determine growth arrest or viability in temperature shifts, we measured the CFU of strains every hour after inoculation (Additional file 1). At 46 °C, mutant strain maintained the CFU from 1 h to 3 h and declined the viability after 4 h. At 25 °C, all strains maintained the CFU during the measurement (7 h). It is suggested that mutant strains exhibited growth arrest in early phase and after declining viability.
Fig. 2

Growth rates of the S. aureus Mu50 (WT, filled triangle), sav1322 mutant (open triangle), and complementation (Com, filled square) strains at (a) 37 °C, (b) 46 °C, and (c) 25 °C. Data are expressed as mean ± SD values for the measurements of absorbance in 600 nm

Oxidative stress

To examine the role of the SAV1322 locus during oxidative stress, a disk diffusion assay with two different concentrations of hydrogen peroxide (H2O2; 15 and 30 %) was performed. When compared with the WT strain, the sav1322 mutant strain displayed greater resistance to both hydrogen peroxide concentrations tested (Fig. 3). Sensitivity of the complementation strain to oxidative stress was similar to that of the WT strain for both hydrogen peroxide concentrations tested (Fig. 3).
Fig. 3

Oxidative stress response of the wild-type (WT) S. aureus Mu50, sav1322 mutant, and complementation strains (Com). Data are expressed as mean ± SD values for the zone of inhibition diameter (mm). Asterisks denote statistical significance (p < 0.001) when compared with the WT strain

Microarray transcriptional analysis

All of the microarray data have been deposited in the NCBI’s Gene Expression Omnibus (GEO) under accession number GSE85824. A comparison of the Mu50 and the sav1322 mutant transcription profiles revealed differential expression of 17 genes (Table 1). Notably, expression of two genes associated with cell wall peptidoglycan synthesis (murZ, encodes UDP-N-acetylglucosamine 1-1carboxylvinyl transferase and glmS, encodes glucosamine-fructose-6-phosphate aminotransferase), was lower in the sav1322 mutant strain. Conversely, transcription of the chaperone genes dnaJ, dnaK, and grpE, as well as the negative heat shock regulator genes, hrcA and ctsR, was higher in the sav1322 mutant strain. Furthermore, transcript levels of two genes encoding Clp ATPases, clpB and clpC, were higher in the sav1322 mutant strain.
Table 1

Representative genes differentially expressed in the WT and sav1322 mutant strains

Gene locus

Gene

Description

Function

Fold change in sav1322 mutant

SAV1147

sdhC

Succinate dehydrogenase cytochrome b-558

 

−1.65

SAV2123

 

Hypothetical protein

 

−1.64

SAV2124

murZ

UDP-N-acetylglucosamine 1-carboxylvinyl transferase

Cell wall biogenesis

−2.02

SAV2125

glmS

Glucosamine-fructose-6-phosphate aminotransferase

Cell wall biogenesis

−1.64

SAV0052

ermA

Adenine N-6-methyltransferase

 

1.55

SAV0522

ctsR

Transcriptional regulator CtsR

Stress response

Transcription regulation

1.76

SAV0523

 

Hypothetical protein

 

2.01

SAV0524

 

ATP: guanido-phosphotransferase

 

1.92

SAV0525

clpC

ATP-dependent Clp protease ATP-binding subunit ClpC

Stress response

1.72

SAV0975

clpB

ClpB chaperone homologue

Stress response

2.13

SAV1350

citB

Aconitate hydratase

Tricarboxylic acid cycle

1.55

SAV1579

dnaJ

Chaperone protein DnaJ

Stress response

1.56

SAV1580

dnaK

Chaperone protein DnaK

Stress response

2.30

SAV1581

grpE

Heat shock protein GrpE

Stress response

3.96

SAV1582

hrcA

Heat-inducible transcriptional repressor

Stress response

3.16

SAV1799

 

Hypothetical protein

 

2.23

SAV2497

 

Putative membrane protein

 

1.82

Two-dimensional gel electrophoresis of the WT and sav1322 mutant strains

Two-dimensional gel electrophoresis was used to examine differences in protein profiles between WT and sav1322 cell lysates (Additional file 2). We observed that proteins associated with carbohydrate metabolism, including those involved in the tricarboxylic acid (TCA) cycle exhibit lower expression in the sav1322 mutant when compared with the WT strain (Table 2). Moreover, several proteins involved in glycolysis/gluconeogenesis were differentially expressed; expression of fructose-biphosphate aldolase (FbaA) and alcohol dehydrogenase (Adh) proteins was greater in the sav1322 mutant, while expression of transketolase, phosphoglyceromutase (GpmA), and pyruvate kinase (PykA) was lower when compared with the WT strain. We also observed a lower expression levels for L-lactate dehydrogenase (LctE) and higher expression of D-lactate dehydrogenase (Ddh) in the mutant; both of these proteins are associated with the interconversion of pyruvate and lactate.
Table 2

Representative proteins differentially expressed in the WT and sav1322 mutant strains

Spot ID

Accessiona

Protein name

Protein Score

Gene

Locusb

Expression change

Carbohydrate metabolism and transport

A023

gi|15924402

Dihydrolipoamide succinyltransferase

714

odhB

SAV1412

Down

A025

gi|15924086

Dihydrolipoamide dehydrogenase

1153

pdhD

SAV1096

Down

A277

gi|15924841

Fumarate hydratase

601

fumC

SAV1851

Down

A043

gi|15924684

Isocitrate dehydrogenase

845

citC

SAV1694

Down

A286

gi|15924412

Glucose-specific enzyme II, PTS system A component

297

crr

SAV1422

Down

A287

gi|15924701

Acetate kinase

750

ackA

SAV1711

Up

Energy metabolism

A029

gi|15925597

Malate:quinone oxidoreductase

1353

mqo2

SAV2607

Down

A055

gi|15923231

L-lactate dehydrogenase

298

lctE

SAV0241

Down

A056

gi|15923246

Xylitol dehydrogenase

78

 

SAV0256

Down

A063

gi|15924332

Transketolase

684

tkt

SAV1342

Down

A071

gi|15925406

Phosphglyceromutase

364

gpmA

SAV2416

Down

A183

gi|15924687

Pyruvate kinase

433

pykA

SAV1697

Down

A008

gi|15923216

Formate acetyltrasferase

1155

pflB

SAV0226

Up

A058

gi|15925514

D-lactate dehydrogenase

972

Ddh

SAV2524

Up

A078

gi|15925115

Fructose-bisphosphate aldolase

62

fbaA

SAV2125

Up

A281

gi|15923595

Alcohol dehydrogenase

1305

Adh

SAV0605

Up

Lipid metabolism

A027

gi|15924291

Glycerol kinase

1037

glpK

SAV1301

Down

A054

gi|15923640

Dihydroxyacetone kinase subunit DhaK

420

 

SAV0650

Down

A067

gi|15924001

Enoyl-(acyl carrier protein) reductase

526

fabI

SAV1011

Down

A072

gi|15923029

Glycerophosphoryl diester phosphodiesterase-like protein

616

 

SAV0039

Down

A089

gi|15925088

(3R)-hydroxymyristoyl-ACP dehydrogenase

125

fabZ

SAV2098

Up

Amino-acid biosynthesis/metabolism

A039

gi|15923948

NAD-specific glutamate dehydrogenase

970

gudB

SAV0958

Down

A046

gi|15923947

Ornithine--oxo-acid transaminase

1027

rocD

SAV0957

Down

A045

gi|15925103

Serine hydroxymethyltransferase

365

glyA

SAV2113

Down

A049

gi|15924192

Carbamoyl phosphate synthase small subunit

923

pyrAA

SAV1202

Up

A051

gi|15924159

Ornithine carbamoyltransferase

556

argF

SAV1169

Up

A060

gi|15924160

Carbamate kinase

990

arcC1

SAV1170

Up

A280

gi|15924727

Bifunctional 3-deoxy-7-phophoheptulonate sunthase/chorismate mutase

434

 

SAV1737

Up

Cell division, cell wall, cell envelope biogenesis

A017

gi|15925144

Glucosamine--fructiose-6-phosphate aminotransferase

167

glmS

SAV2154

Down

A032

gi|15925072

UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D-alanine ligase

792

murF

SAV2082

Down

A033

gi|15925114

UDP-N-acetylglucosamine-1-carboxylvinyl transferase

1062

murZ

SAV2124

Down

A048

gi|15923145

Capsular polysaccharide synthesis enzyme Cap5G

518

capG

SAV0155

Down

A274

gi|15924665

Trigger factor

1278

tig

SAV1675

Down

Purine and pyrimidine metabolism

A079

gi|15924058

Phosphoribosylformylglycinamidine synthase

162

purQ

SAV1068

Down

A035

gi|15924191

Dihydroorotase

866

pyrC

SAV1201

Up

A059

gi|15924190

Aspartate carbamoyltranferase

328

pyrB

SAV1200

Up

A080

gi|15925102

Uracil phosphoribosyltransferase

206

Upp

SAV2112

Up

Transcription and translation

A021

gi|15923537

Elongation factor G

411

fusA

SAV0547

Down

A031

gi|15923518

Glutamyl-tRNA synthetase

264

gltX

SAV0528

Down

A053

gi|15924247

Elongation factor Ts

317

Tsf

SAV1257

Down

A061

gi|15924246

30S ribosomal protein S2

465

rpsB

SAV1256

Down

A065

gi|15923491

50S ribosomal protein L25

424

rplY

SAV0501

Down

A069

gi|15923538

Elongation factor Tu

123

Tuf

SAV0548

Down

A070

gi|15925240

50S ribosomal protein L3

516

rplC

SAV2250

Down

A076

gi|15924709

30S ribosomal protein S4

532

rpsD

SAV1719

Down

A096

gi|15924253

Prolyl-tRNA synthetase

176

proS

SAV1263

Down

A108

gi|15925214

DNA-directed RNA polymerase subunit alpha

905

rpoA

SAV2224

Down

A276

gi|15924256

Transcription elongation factor NusA

931

nusA

SAV1266

Down

A030

gi|15924889

Aspartyl/glutamyl-tRNA amidotrasferase subunit B

336

gatB

SAV1899

Up

A081

gi|15922999

Seryl-tRNA synthetase

379

serS

SAV0009

Up

A087

gi|15923529

50S ribosomal protein L10

292

rplJ

SAV0539

Up

Stress response and heat shock

A066

gi|15923693

Oxidoreductase

242

 

SAV0703

Down

A084

gi|15924414

Methionine sulfoxide reductase A

605

msrA2

SAV1424

Down

A090

gi|15924413

Methionine sulfoxide reductase B

342

msrB

SAV1423

Down

A138

gi|15925129

General stress protein 20U

519

dps

SAV2139

Down

A012

gi|15925538

ATP-dependent Clp proteinase chain

2194

clpL

SAV2548

Up

Others

A042

gi|15924396

Tellurite resistance protein

716

 

SAV1406

Down

A064

gi|15923958

5-oxo-1,2,5-tricarboxilic-3-penten acid decarboxylase

123

 

SAV0968

Down

A088

gi|15924853

Bacterioferritin comigratory protein

959

 

SAV1863

Down

A016

gi|15923990

Thimet oligopeptidase-like protein

733

 

SAV1000

Up

A020

gi|15925280

Urease subunit alpha

862

ureC

SAV2290

Up

A044

gi|15923092

Aminoacylase

628

 

SAV0102

Up

A091

gi|15925281

Urease accessory protein UreE

209

ureE

SAV2291

Up

A114

gi|15925318

Dehydrogenase

389

 

SAV2328

Up

A285

gi|15924697

Metal-dependent hydrolase

609

 

SAV1707

Up

Unknown function

A040

gi|15924926

Hypothetical protein

700

-

SAV1936

Down

A062

gi|15923824

Hypothetical protein SAV0834

537

-

SAV0834

Down

A074

gi|15924667

Hypothetical protein SAV1677

436

-

SAV1677

Down

A075

gi|15925559

Hypothetical protein SAV2569

203

-

SAV2569

Down

A083

gi|15924605

Hypothetical protein SAV1615

91

-

SAV1615

Down

A086

gi|15924342

Hypothetical protein SAV1352

256

-

SAV1352

Down

A085

gi|15925677

Hypothetical protein SA2687

746

-

SAV2687

Up

aAccession numbers are from the NCBI protein database

bDesignate ORF number/gene locus in the S. aureus Mu50 genome

Interestingly, several proteins associated with lipid metabolism and cell wall synthesis also displayed differential expression. In particular, expression of the glycerol kinase, dihydroxyacetone kinase subunit DhaK, enoyl-(acyl carrier protein) reductase and glycerophophoryl diester phophodiesterase-like protein was lower in the sav1322 mutant, whereas expression of (3R)-hydroxymyristoyl-ACP dehydrogenase was higher in the sav1322 mutant when compared with the WT strain. For cell wall synthesis, glucosamine-fructose-6-phophate aminotransferase (GlmS), UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (MurF), and UDP-N-acetylglucosamine-1-carboxylvinyl transferase (MurZ) exhibited lower expression levels in the sav1322 mutant when compared with the WT strain. GlmS is the key enzyme responsible for the synthesis of glucosamine-6-phosphate from fructose-6-phosphate [16], MurF catalyzes the addition of D-Ala-D-Ala to the nucleotide precursor UDP-N-acetylmuramic acid-L-Ala-γ-Glu-meso-diaminopimelate [17], and MurZ catalyzes the condensation of phophoenolpyruvate with UDP-N-acetylglucosamine [18].

Lastly, markedly lower expression levels of proteins involved in the stress response were observed in the sav1322 mutant, including the general stress protein 20U (Dps), methionine sulfoxide reductase A (MsrA2), and methionine sulfoxide reductase B (MsrB). MsrA2 reduces the S isomer of methionine sulfoxide, and MsrB reduces the R form, providing protection against oxidative stress [19]. In contrast, expression of ClpL, a protein involved in thermotolerance, was up-regulated in the sav1322 mutant strain.

In vivo assessment of the sav1322 mutant strain

A mouse model of bacteremia was used to determine how deletion of the SAV1322 locus impacted staphylococcal pathogenicity. Systemic infection following tail vein inoculation of six-week-old female Balb/c mice with the sav1322 mutant produced a 2-log reduction in bacterial burden found in the lung, liver, and kidney when compared with mice inoculated with the WT strain (Fig. 4).
Fig. 4

S. aureus Mu50 (WT) and sav1322 mutant pathogenicity. Mice were infected with the WT or sav1322 mutant strains, and infection progressed for 24 h. CFU (log CFU per mg organ) from infected lungs, livers, and kidneys are shown

Antibiotic susceptibility

Epsilometer (E)-test strips were used to determine if the sav1322 mutant strain exhibited altered susceptibility or resistance to several antibiotics. Minimum inhibitory concentrations (MICs) of vancomycin and teicoplanin which target the bacterial cell wall were lower for the sav1322 mutant when compared to the WT strain (Table 3). We also performed a population analysis to obtain a more detailed evaluation of changes in susceptibility to vancomycin and teicoplanin. Consistent with a lower MIC for both of these antibiotics, we observed a decrease in vancomycin- and teicoplain-resistant subpopulations compared with the WT strain (data not shown). However, those MICs were not recovered in the Com stain. Lower MICs for gentamicin, tigecycline, and tetracycline, which inhibit protein synthesis by binding the 30S subunit of ribosome, were also observed in the sav1322 mutant strain. The MICs of these antibiotics were fully recovered in Com strain. In addition, the MICs for the bacterial cell membrane-targeting lipopeptide antibiotic, daptomycin, were 3 mg/L and 0.19 mg/L for the WT and sav1322 mutant strains, respectively. Electron microscopy was used to determine if cell wall thickness was associated with the observed reduction in glycopeptide resistance in the mutant strain. The sav1322 mutant strain has a thinner cell wall (23.6 ± 3.1 nm) than the WT strain (36.1 ± 5.6 nm) (Fig. 5).
Table 3

Antibiotic susceptibility profiles of the WT, sav1322 mutant, and complementation strains

Antibiotics

MIC (mg/L)

WT

sav1322 mutant

Com

Daptomycin

3

0.19

0.125

Erythromycin

>256

>256

>256

Gentamicin

≥256

1

≥256

Imipenem

>32

1

32

Linezolid

2

2

2

Oxacillin

>256

≥256

≥256

Penicillin

>32

>32

32

Teicoplanin

8

0.5

0.5

Tigecycline

0.5

0.064

0.25

Tetracycline

16

1

8

Vancomycin

8

0.75

0.75

Fig. 5

TEM images of representative S. aureus Mu50 (WT) and sav1322 mutant cells. Magnification: 100,000×. Data are expressed as mean ± SD values for cell wall thickness (nm). Asterisks denote statistical significance (p < 0.001) when compared with the WT strain

Discussion

The SAV1321/SAV1322 TCRS system is a histidine kinase sensor/response regulator in the wild-type S. aureus Mu50. However, the functions of SAV1321/SAV1322 TCRS-associated genes are not yet known. With this study, we elucidate some of these functions by examining phenotypic, transcriptomic, and proteomic changes in a sav1322 mutant strain in response to different environmental stressors.

Heat-shock proteins (Hsps) are essential for stress tolerance and survival under protein-denaturing conditions. Four Hsp classes have been identified in Gram-positive bacteria. Class I Hsps comprise classical Hsps that are encoded by genes within the groESL and dnaK operons, and a cytoplasmic repressor HrcA controls expression of these proteins. Sig B controls Class II Hsps, while class II Hsps are Clp proteases that are typically controlled by the transcriptional repressor CtsR. Class IV Hsps are encoded by genes that are not controlled by HrcA, SigB, or CtsR [3, 20]. Interestingly, our data suggest that the sav1322 mutant strain is more sensitive to heat and cold than the WT strain. Microarray data further demonstrate that the transcription of class I Hsp and III Hsp genes. More specifically, hrcA, ctsR, dnaJ, dnaK, and grpE transcript levels are higher in the sav1322 mutant. However, expression of cold-shock genes (cspA and cspB) is not affected in the mutant. These results suggest that SAV1322 may play a role in temperature adaptation by regulating the expression of chaperone proteins. While CtsR represses the transcription of genes encoding class I and class III Hsps, however, we noted that expression of the class III chaperone genes clpC and clpB is higher in the sav1322 mutant strain.

The clpC operon in S. aureus comprises four genes denoted ctsR, mcsA, mcsB, and clpC. One previous study has reported that a mutation in mcsB results in hypersensitivity to heavy metal, temperature, osmotic pressure, and oxidative stresses [21]. ClpC proteases are implicated in stress resistance, and clpC and clpB transcription increases during acid shock in S. aureus [22]. In this study, the sav1322 mutant exhibited greater resistance to oxidative stress. This may be partially explained by the higher levels of clpC transcription observed in this strain.

Our proteomic data further revealed that the expression of 72 proteins is altered at least two-fold in the sav1322 mutant when compared with the WT strain. Many of these proteins are directly or indirectly involved in important cellular processes, including the TCA cycle. The TCA cycle have been implicated in antibiotic resistance, and, possibly, staphylococcal virulence [23, 24]. Inactivation of the TCA cycle delays the resolution of cutaneous ulcers in soft tissue infections of the mouse [25], and disruption of citZ (citrate synthase), citC (isocitrate dehydrogenase), and citB (aconitate hydratase) genes prevents bacterial capsule formation [26]. Surmann et al. analyzed survival and physiological adaptation of S. aureus after internalization by human lung epitheilial cell lines A549. A number of TCA cycle enzymes including CitZ, CitC, SucC and SdhA increased in level upon internalization by this epithelial cell. Moreover, L-lactate dehydrogenase (LctE) increased in level after internalization by A549 and human embryonic kidney cells HEK 293, indicating the supplementary utilization of fermentative enzymes under microaerobic conditions [27]. Our proteomic results demonstrate that expression of proteins involved in TCA cycle, such as OdhB, CitC, PdhD, and FumC, is lower in the sav1322 mutant. Furthermore, several major fermentative and anaplerotic pathway components were also decreased in the mutant, in particular LctE, malate:quinone oxidoreductase (Mqo2) and pyruvate kinase (PykA). These results suggest that the down-regulation of energy-providing pathways may be energetically less efficient in the mutant compared with the wild type strain, then the recovered CFU were lower in tissue of mice infected with the mutant (Fig. 4).

Furthermore, LctE catalyzes the conversion from pyruvate to lactate. The L-lactate produced by this process allows S. aureus to maintain redox homeostasis during nitrosative stress caused by activated phagocytes and is essential for virulence [28]. In this study, the protein level of LctE was decreased in mutant compared with WT strain. This may resulted in decrease the production of L-lactate in the mutant and cannot survive from the attack of the host, including the radial nitric oxide.

Proteins involved in protein synthesis are also altered upon deletion of SAV1322 including 30S ribosomal protein S2, 30S ribosomal protein S4, and several aminoacyl-tRNA synthetase. In accordance with this proteomic result, the lower MICs of tetracycline, tigecycline, and gentamicin that bind 30S subunit of ribosome were observed in the sav1322 mutant, and fully restored in Com strain. These results suggest that SAV1322 may play a role in antibiotic resistance to tetracycline, tigecycline, and gentamicin, but further studies are needed to elucidate the underlying specific mechanism.

Proteins involved in cell wall biosynthesis are also altered upon deletion of SAV1322. Cell wall synthesis is crucial for bacterial division and growth, and it is an important target for antibiotics, including penicillin, vancomycin, and teicoplanin. As the number of reported MRSA strains increases, vancomycin has become the first-line treatment for staphylococcal infections. However, the use of vancomycin has led to the emergence of VISA strains [1]. Typically, VISA exhibits thick cell walls and reduced autolysis [1, 12]. We speculate that the altered expression of cell wall metabolism-related proteins in the sav1322 mutant results in lower MICs for several antibiotics that inhibit cell wall synthesis. Moreover, the sav1322 mutant had thinner cell walls and is more susceptible to Triton X-100-induced autolysis and lysostaphin-induced cell lysis. Curiously, we have observed that a susceptibility of vancomycin and teicoplanin attenuated by deletion of SAV1322 cannot be readily complemented. Explanations for this phenomenon could be due to: (i) incomplete recovery of the cell wall thickness, (ii) the highly complex glycopeotide resistance regulatory cross-talk in VISA or (iii) the involvement of posttranscriptional mechanisms, as we detected by comparing transciptomic and proteomic dtata, and thus will require further study.

In this study, it was observed that the lack of correlation between transcript level and identified proteins. There is a remarkable series of processes between transcription and translation, including spanning the transcription, processing and degradation of mRNAs to the translation, localization, modification and programmed destruction of proteins. The abundances of protein reflect a dynamic balance among these processes [29]. Other factors include the protein stability. The half-life of different proteins can vary from minutes to days, whereas the degradation rate of mRNA would fall within a much tighter range (several hours). Another factor might be of solubility of proteins in the buffers used in this study. Some highly expressed genes probably encode for highly insoluble proteins that are difficult to analyze even in the presence of detergents.

Conclusions

This study is the first to provide a functional transcriptomic and proteomic analysis of the response regulator SAV1322 in S. aureus. SAV1322 plays a pronounced role in temperature adaptation, resistance to antibiotics, and virulence. In addition, it influences the expression of a large number of genes involved in the heat shock response, cell wall metabolism, energy metabolism, and response to other environmental stressors. Our findings provide valuable insight into antibiotics resistance and staphylococcal pathogenicity. Both may help shape future studies on antibiotic resistance trends and optimal antibacterial treatment strategies.

Methods

Construction of the sav1322 mutant and complementation strains

The S. aureus Mu50 was used to generate a SAV1322 knockout mutant strain using an allelic exchange method described previously [30]. Briefly, sequences flanking SAV1322 were amplified by PCR using primers that contained attB1 (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-) and attB2 (5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-) sites on the respective up- and downstream target sequences. PCR fragments were cloned into the pKOR1 vector, and then introduced into S. aureus Mu50 by electroporation. To select for single-crossover mutants, electroporated clones were cultured overnight at 43 °C. Single-crossover mutants were selected and cultured in antibiotic-free broth medium to facilitate plasmid excision, then subjected to anhydrotetracycline induction to select a non-plasmid-carrying mutant. Successful deletion of SAV1322 was verified by PCR using the specific primers covering target sequences (UP FWD and DOWN REV) and sequence analysis. For the complementation strain, a 1620-bp fragment of the SAV1322 gene was PCR-amplified using Mu50 genomic DNA as a template with the primer sets Com-F and Com-R (Fig. 1a), then cloned into the pYT3 vector and introduced into the sav1322 mutant strain as described previously [31]. To confirm of complementation, reverse transcription-PCR analysis was performed. Total RNA was isolated from the WT, mutant, and complementation strains and reverse-transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase for 60 min at 42 °C. Resulting cDNA fragments were used as templates for PCR amplification of the target gene.

Stress response analyses

Strains were cultured overnight in tryptic soy broth (TSB) at 37 °C and diluted to an OD600 reading of 0.3. Cells were then incubated at 25 and 46 °C with shaking, while OD600 readings were taken at 1 h intervals for 7 h. To investigate the oxidative stress response, WT and sav1322 mutant cultures grown overnight were plated on tryptic soy agar (TSA). A disk containing 10 μl H2O2 (15 % or 30 %) was placed on the prepared agar plates, then incubated at 37 °C for 18 h.

Microarray analysis

RNA extraction, cDNA labeling, hybridization, and microarray data analyses were carried out according to protocols described previously [32]. Cells were grown exponentially to an OD600 of approximately 0.5, then harvested. A customized S. aureus high-density synthetic oligonucleotide array was designed using 982 predicted open reading frames (ORF) with the GenBank accession number NC_002745 (NimbleGen Systems Inc., Madison, WI, US). The ORF listed in Additional file 3. Arrays were scanned with a NimbleGen MS 200 Microarray scanner set at 532 nm with a resolution of 2 μm to produce images (TIFF format) according to the manufacturer’s instructions. Array data export processing and analysis was performed using NimbleScan v2.5 (Gene Expression RMA algorithm). Adjustments for batch effects were made with data-to-filter non-biological experimental variation (http://biosun1.harvard.edu/complab/batch/). A single raw intensity value was determined for each gene in every array by averaging spot replicates of all probes for each of the genes. Gene signal values were log2 transformed. Statistical significance of the expression data was determined using the local-pooled-error test and fold change in which the null hypothesis was that no difference exists between two groups. Adjusting P-values with a Benjamini-Hochberg algorithm controlled the false discovery rate. Hierarchical cluster analysis was performed using complete linkage and Euclidean distance as a measure of similarity.

Two-dimensional gel electrophoresis and protein identification

Cell extracts from overnight WT and sav1322 mutant cultures were separated by two-dimensional gel electrophoresis. Aliquots containing approximately 800-μg protein were diluted in 2D-PAGE rehydration buffer (7 M urea, 2 M thiourea, 4 % CHAPS, 0.4 % DTT) to a final volume of 350 μl and centrifuged at 100,000 g for 30 min to remove insoluble material. For the first dimension, samples were run on pH 4–7 IPG strips (GE Healthcare Life Sciences, UK) on a Multiphor apparatus II instrument (Amersham Biosciences, UK) according to the manufacturer’s instructions. Strips were incubated in equilibration buffer (6 M urea, 20 mM Tris-HCl, pH 8.8, 2 % SDS, 20 % glycerol, 2.5 % acrylamide, and 5 mM tributyl phosphine) for 20 min. The second dimension separation was performed using 8–16 % linear gradient SDS-polyacrylamide gels. Gels were stained with colloidal Coomassie solution (ProteomeTech, Seoul, Korea) according to the manufacturer’s instructions. Differences in the expression profiles of spots were quantified using the ImageMasterTM 2D Platinum software (Amersham Biosciences, UK). In-gel digestion of the protein spots was performed as described previously [33]. Resulting tryptic peptides were analyzed using reversed phase capillary HPLC directly coupled to a Finnigan LCQ ion-trap mass spectrometer (LC-MS/MS) previously described [34]. Individual spectra from MS/MS were processed using TurboSEQUEST software (ThermoQuest, San Jose, CA). The generated peak list files were used to query the MSDB database or NCBI using the MASCOT program (http://www.matrixscience.com).

Antimicrobial susceptibility

MICs for different antimicrobial agents were determined with E-test strips (AB bioMérieux, Marcy I’Etoile, France). A sterile cotton swab was immersed in each bacterial culture grown to a 0.5 McFarland turbidity standard and streaked on Mueller-Hinton agar (Difco, Detroit, MI) plates. Plates were incubated at 37 °C, and MICs were measured after a 24-h incubation period according to the manufacturer’s instructions.

Transmission electron microscopy (TEM)

WT and sav1322 mutant cells were prepared and visualized by TEM as described previously [35]. Morphometric evaluation of cell wall thickness was determined using photographs at 20,000× magnification. At least 30 cells from each strain with almost equatorial cut surfaces were measured and results are expressed as mean ± SD values. The statistical significance of cell wall thickness was evaluated by the Student’s t-test (p < 0.001).

Mouse infection model

All animal experiments were conducted in accordance with guidelines and the approval of the Institutional Animal Care and Use Committee of Korea Centers for Disease Control and prevention (KCDC-030-12-2A). WT and sav1322 mutant strains were grown and harvested at the mid-point of the exponential growth phase, washed in sterile PBS, and resuspended in PBS to a concentration of 1 × 108 cells per 0.1 ml. Female Balb/c mice (aged 5–6 weeks) were inoculated with this suspension or PBS via the tail vein. The experiment was performed with ten mice for each S. aureus strain and five mice for the PBS control. Mice were euthanized 24 h after infection, and lungs, livers, and kidneys were removed and homogenized in sterile PBS. Homogenates were diluted in PBS and plated on TSA, then incubated overnight in 37 °C. The bacterial burden in each organ was assessed by CFU counts. The student’s t-test was used to determine statistical significance between samples.

Abbreviations

CFU: 

Colony-forming unit

MRSA: 

Methicillin-resistant S. aureus

ORF: 

Open reading frame

PBS: 

Phosphate-buffered saline

TCRS: 

Two-component regulatory system

TSA: 

Tryptic soy agar

TSB: 

Tryptic soy broth

VISA: 

Vancomycin-intermediate S. aureus

VRSA: 

Vancomycin-resistant S. aureus

Declarations

Acknowledgements

We thank Keiichi Hiramatsu (Juntendo University, Tokyo, Japan) and Taeok Bae (Indiana University School of Medicine-Northwest, Indiana, USA) for providing the pKOR vector and technical support.

Funding

This study was supported by an intramural research grant awarded to the Korea Centers for Disease Control and Prevention (2010-N44002-00).

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

All of the microarray data have been deposited in the NCBI’s Gene Expression Omnibus (GEO) under accession number GSE85824.

Authors’ contributions

JWK, HKK, and GSK performed laboratory work. JWK and JIY wrote the manuscript. YSL and JIY designed the study. IHK and HSK critically revised the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable

Ethics approval and consent to participate

Not applicable

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Division of Antimicrobial Resistance, Center for Infectious Diseases, National Research Institute of Health, Centers for Disease Control and Prevention
(2)
Korea National Research Institute of Health, Osong Health Technology Administration Complex

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Copyright

© The Author(s). 2016