Functional variation reflects intra-strain diversity of Staphylococcus aureus small colony variants in the host–pathogen interaction

International Journal of Medical Microbiology 303 (2013) 61–69

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Functional variation reflects intra-strain diversity of Staphylococcus aureus small colony variants in the host–pathogen interaction Dina Hilmi a , Marijo Parcina a,b , Konrad Bode a , Jenny Ostrop a , Sabine Schuett a , Klaus Heeg a , Wilma Ziebuhr c , Olaf Sommerburg d , Isabelle Bekeredjian-Ding a,b,∗ a

Department of Infectious Diseases, Medical Microbiology and Hygiene, University Hospital Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany c Institute for Molecular Infection Biology, University of Wuerzburg, Josef-Schneider-Str. 2, D-97080 Wuerzburg, Germany d Department of Pediatrics, University Hospital Heidelberg, Im Neuenheimer Feld 430, D-69120 Heidelberg, Germany b

a r t i c l e

i n f o

Article history: Received 19 August 2012 Received in revised form 26 November 2012 Accepted 2 December 2012 Keywords: Staphylococcus aureus Small colony variants Intrastrain variability Biofilm TLR2 Protein A

a b s t r a c t It is well acknowledged that genetic variation accounts for the intra-species variability in Staphylococcus aureus isolates. Similarly, deficiency in DNA repair and the resulting increase in genomic mutations determine intra-strain variability in S. aureus small colony variants (SCV). The aim of this study was to investigate whether intra-strain diversity would be associated with an alteration of the host–pathogen interaction. To this end, biofilm formation and immune stimulatory capacity were compared in consecutive SCV isolates originating from a single patient. Despite the relatedness of the isolates, the results revealed significant differences in biofilm formation and immune stimulation determined by Toll-like receptor-2 (TLR2) activity. Variation in the extent of biofilm production could be attributed to differences in the expression of protein A (SpA) and agrA. TLR2 activity only partially correlated with these parameters. Although transiently increased functional activity correlated with clinical remission and was abrogated in MRSA superinfection, we can only speculate that changes in the SCV phenotype reflect alterations in the microbial environment and/or treatment. Taken together, our study provides in vivo evidence for the functional consequences of intra-strain variation in S. aureus. © 2013 Elsevier GmbH. All rights reserved.

Introduction Staphylococcus aureus is a versatile microbe that causes severe infections, but can also colonize the respiratory mucosa in the absence of disease manifestation. These contrasting scenarios result from differential regulation of the host–pathogen interaction that is governed by the expression of virulence factors and the quality and intensity of the host immune response elicited. S. aureus is further renown for its superb adaptation to the human host, to certain niches, i.e. the nasal atrium, to the hospital environment, and to antimicrobial therapy. A classic example for this host adaptation is the formation of small colony variants (SCV). These slowly growing and metabolically deficient staphylococcus isolates are commonly cultured from patients with chronic infections such as osteomyelitis, endocarditis, or cystic fibrosis (Kahl et al., 2003; Tuchscherr et al., 2010;

∗ Corresponding author at: Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. Tel.: +49 (0)228 287 15675; fax: +49 (0)228 287 19573. E-mail address: [email protected] (I. Bekeredjian-Ding). 1438-4221/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ijmm.2012.12.008

Proctor et al., 2006). They survive in the intracellular niche and down-regulate key cellular functions such as DNA transcription and peptidoglycan synthesis. SCV formation is further triggered by selective pressure through antibiotics and/or antimicrobial substances derived from other colonizing bacteria such as Pseudomonas aeruginosa (Hoffman et al., 2006; Biswas et al., 2009), thus representing an efficient means to escape their bactericidal effects. The evolvement of the SCV phenotype is frequently accompanied by genetic mutations that stabilize the SCV phenotype and often result from hypermutability due to deficient DNA repair (Besier et al., 2008; Miller, 1996; Schaaff et al., 2003). These mutations confer resistance to antibiotics and changes in the nutritional requirements such as hemin, menadione, and thymidine dependency (Kahl et al., 1998; Gilligan et al., 1987; von Eiff et al., 1997; Proctor et al., 2006). Nevertheless, albeit mutations can accumulate over time, the SCV phenotype is frequently instable and rapidly reverses under nutritious growth conditions. Morphological and biochemical changes may, thus, also occur within the human organism, and may be triggered by acute alterations in the microbial environment, nutritional conditions, and/or the antibiotic regime. The resulting variations in the expression of virulence factors are

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likely to contribute to alternating recurrence and remission phases in chronic infection. To date, it is well acknowledged that genetic variation accounts for intra-species variability in S. aureus isolates and facilitates host adaptation (McCarthy and Lindsay, 2010). In the present study, we sought to investigate the consequences of intra-strain variability for the host–pathogen interaction. To this end, we analyzed the functional properties of a series of S. aureus SCV isolates derived from a single patient, but collected over consecutive time points. The results obtained demonstrate that all isolates originate from the same strain, but differ in biofilm formation and immune stimulation, thus providing in vivo evidence for the functional consequences of intra-strain variation in SCV isolates. Materials and methods Bacteria All clinical isolates were cultured from pharyngeal specimens from a patient with cystic fibrosis (CF). The patient was subject to long-term treatment with meropenem and tobramycin. All SCV and MRSA isolates used in this study were collected over a time period of 2.5 years (Table 1). Respiratory specimens regularly contained ‘tiny’ S. aureus isolates. Susceptibility testing and molecular analysis revealed MLSB phenotype (positive for ermC and msrB) and methicillin sensitivity in SCV (Tables S1 and S2); MRSA isolates additionally harboured ermA and mecA genes. No point mutations conferring macrolide resistance (A2058 or A2059) were detected in the sequenced 23S rDNA PCR products (Besier et al., 2008; Prunier et al., 2002). The accompanying microbial flora contained Aspergillus fumigatus, Candida spp. and ␣-haemolysing streptococci, while Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Haemophilus spp. were only occasionally recovered (Table 1). Notably, the SCV isolates were collected from specimens lacking enterobacteriaceae, i.e. Serratia marcescens, Enterobacter cloacae, Klebsiella oxytoca, that were regularly associated with exacerbation of disease and reduced vital capacity. Bacterial growth conditions and media SA113 (ATCC 35556) and its isogenic mutants, the spa-deficient SA113 spa and the TLR2-inactive SA113 lgt were kindly provided by F. Götz, Tuebingen, Germany, and generated as previously described (Stoll et al., 2005; Herbert et al., 2010). Bacteria were preserved in MAST cryobankTM (Mast Diagnostica, Merseyside, UK). All bacterial colonies that grew on sheep red blood agar upon culture of patient specimens were frozen. For each experiment, bacteria were freshly thawed and grown on sheep red blood agar plates (Columbia agar, BioMerieux). The SCV phenotype was reproducible at all time points. We did not observe revertants within the duration of experiments. As a standardization measure, bacterial cell concentrations were adjusted to a McFarland (McF) of 1 and further diluted as indicated. McF = 1 corresponds to 3 × 108 normal colony-forming units (CFU) per ml (Perry et al., 1999). Thus, bacterial mass was comparable throughout the experiments, but absolute numbers of CFU may have varied depending on the colony or cell size. Genomic PCR and sequencing Genomic PCR was performed using the DreamTaq DNA polymerase (Fermentas, St. Leon-Rot, Germany). All primers and probes are listed in Table 2. Oligonucleotides for PCR amplification of cap5, cap8, mecA, and spa genes were previously described (Goerke et al., 2005; Costa et al., 2005; Klotz et al., 2003; Aires-de-Sousa et al., 2006). spa and 23S PCR products were purified using

QIAquick PCR purification kit (Qiagen, Hilden, Germany). Sequencing was performed at Eurofins/MWG Biotech, Munich, Germany, and sequences were analyzed using the Ridom StaphType software Version 2.0.3 (Oberdorfer et al., 2006; Harmsen et al., 2003; Shopsin et al., 1999) or NCBI blast. RNA isolation and quantitative RT-PCR RNA was extracted with the RNeasy mini kit protocol (Qiagen). Purity was controlled on an Agilent 2100 Bioanalyzer (Waldbronn, Germany); RNA Integrity Numbers were 10. Gene expression levels were quantified by real-time RT-PCR using the Agpath-IDTM one-step kit (Applied Biosystems, Darmstadt, Germany). Primers and probes are listed in Table 2 and in Vaudaux et al. (2002). The hybridization oligonucleotide for spa quantification was specifically designed for spa types t331 and t026. Relative expression (rE) was calculated by normalizing to the 16S cycle threshold values, e.g. rE = 1/(2(cycle threshold of target gene − cycle threshold of 16S) ). Pulsed-field gel electrophoresis Pulsed-field gel electrophoresis (PFGE) of S. aureus strains was performed as described in (Petersdorf et al., 2006; Pfaller et al., 1992). Band patterns were compared using GelCompar software version 4.0 (Applied Maths, Sint-Martems-Latem, Belgium). MALDI-TOF MS spectrum generation and data analysis Sample preparation was performed using a standard ethanol/formic acid protein extraction protocol (Mellmann et al., 2008) and applied with matrix solution (saturated solution of ␣-cyano-4-hydroxy-cinnamic acid in 50% acetonitrile–2.5% trifluoro-acetic acid). MALDI-TOF analysis was performed on a Bruker Microflex I MALDI-TOF mass spectrometer. Seven spectra of each isolate were compared and strain-relatedness calculated using the MALDI Biotyper 2.0 software. Protein lysates and Western blot analysis Bacterial cultures were harvested in the exponential phase of growth (OD600 nm = 1). Cell pellets were resuspended in RIPA lysis buffer containing protease inhibitors and disrupted with glass beads (diameter 0.1 mm, BioSpec Bartlesville, OK, USA). For SDS–PAGE, 10 ␮g of protein were loaded per lane. After Western blotting under semi-dry conditions, membranes were blocked with rabbit serum and incubated with anti-staphylococcal protein A monoclonal antibody (Sigma) or with 1% human serum, followed by HRP-conjugated goat anti-mouse IgG or biotinylated goat anti-human IgG (Jackson ImmunoResearch, Sheffield, UK) and streptavidin–HRP 1:2000 (Millipore). Biofilm assay Biofilm formation was quantified using crystal violet staining as described in Christensen et al. (1985). 0.5 × 106 CFU/ml were plated in a 96-well flat bottom tissue culture plate precoated with 20% human plasma for 24 h. After fixation, staining, and resolubilization in ethanol, the OD570 nm was determined. Based on the assumption that the Lambert–Beer law (i.e. linear correlation of OD to concentration of the absorbing substance) cannot be applied to high OD values, samples yielding an OD ≥ 2 were further diluted. ‘Corrected OD values’ were obtained by multiplying the diluted OD value with the dilution factor. For differentiation between polysaccharide intercellular adhesion (PIA)- or protein-mediated biofilm formation, biofilm dispersal was determined upon treatment of preformed biofilms with proteinase K (Applichem, Darmstadt,

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Table 1 Concomitant microbial flora.

Isolation date (months) Detected microbes MSSA MRSA ˛-Haemolysing streptococci A. fumigatus C. albicans C. glabrata P. aeruginosa S. maltophilia H. influenzae H. parainfluenzae Rothia mucilaginosa

SCV1

SCV2

SCV3

SCV4

SCV5

SCV6

SCV7

SCV8

SCV9

SCV10

MRSA1

MRSA2

MRSA*

0

+2

+6

+11

+13

+22

+22

+24

+29

+30

+29

+30

n.a.

+

+

+

+

+

+

+

+

+ + − − − − − − −

+ − + − − − − − −

+ − − − − − − − −

− + − + − − − − −

− + − − + − − − −

+ + − + − + − − −

+ + − + − + − − −

+ + + + − − − + −

+ + − + + + − + + − +

+ + + + − − − − − − −

+ + − + + + − + + − −

+ + + + − − − − − − −

− + − − − + − + − − −

Germany) or sodium metaperiodate (NaIO4 ) (Merck, Darmstadt, Germany) as described (Hennig et al., 2007). TLR2 assay Human embryonic kidney (HEK) 293 cells were grown in RPMI 1640 (Invitrogen, Karlsruhe, Germany) supplemented with 10% FCS (Invitrogen) and 1% penicillin/streptomycin (Sigma, Munich, Germany). Cells were plated at 5 × 104 cells/well in a 96-well plate.

Twenty-four hours later, the medium was substituted with OptiMEM (Invitrogen) and cells transfected with lipofectamine 2000 (Invitrogen), complexed plasmid encoding TLR2 cDNA (pTLR2; 100 ng/well) (Kirschning et al., 1998), or with lipofectamine alone as in Bekeredjian-Ding et al. (2007). Cells were left unstimulated, stimulated with live bacteria (1 × 106 CFU of normal S. aureus strains or the equivalent bacterial mass for SCV; see explanation above), or stimulated with Pam3 CSK4 (100 ng/ml) (EMC Microcollections, Tuebingen, Germany) as a positive control. IL-8 secretion

Table 2 PCR and sequencing primers. Gene

Primers

Product size (bp)

Ref./GenBank accession number

spa

F (1113) 5 -TAA AGA CGA TCC TTC GGT GAG C-3 R (1514) 5 -CAGCAGTAGTGCCGTTTGCTT-3 Hybridization oligo for t331: 5 -FAM-GAC GGC AAC GGA GTA CAT GTC-3 -BHQ1 F 5 -GAA AGT GAA CGA TTA GTA GAA-3 R 5 -GTA CGA AGC GTT TTG ATA GTT-3 F 5 -GTG GGA TTT TTG TAG CTT TT-3 R 5 -CGC CTC GCT ATA TGA ACT AT-3 F 5 -TCG TTC AAG AAC AAT CAA TAC AGA G-3 R 5 -ATC GTT GAG AAG GGA TTT GC-3 F 5 -GAG TGA AAA GGT ACT CAA CCA AAT AA-3 R 5 -TTG GTG AAT TAA AGT GAC ACG AA-3 F 5 -TGAACATGATAATATCTTTGAAATCG-3 R 5 -CAA TTT TGC GTA TTA TAT CCG TAC TT3 F 5 -TCA TTG GAT GCC TTC ACG TA-3 R 5 -CCAGAATGAAAAAGAAGTTGAGC-3 F 5 -TAT AGCG CTC GTA GGT GCA A-3 R 5 -GTT CTT TCC CCA CCA CTC AA-3 F 5 -TGT GGA TGG CCT AGC TTT TC-3 R 5 -TCG CCA TAA CCC AAT TCT TC-3 PCR F 5 -CGA AAT TCC TTG TCG GGT AA-3 PCR R 5 -GGA ACC ACC GGA TCA CTA AG-3 SEQ F 5 -AGG TAG CGA AAT TCC TTG TC-3 SEQ R 5 -TCA AAG GCT CCT ACC TAT CC-3 F 5 -GAT AAA AAA GAA CCT CTG CT-3 R 5 -ACT GCC TAA TTC GAG TG-3 Hybridization oligo: 5 -FAM-ACA ACT TCA CCA GGT TCA ACT CAA A-BHQ1-3 F 5 -AAT TAA CGA AAT GGG CAG AAA CA R 5 -TGC GCA ACA CCC TGA ACT T Hybridization oligo: 5 -TET-AGA AAT TAA CTG GAT GGT ACG CGC GAA GA-BHQ1-3 F (34) 5 -CAAAGAGAAAACATGGTTACCATTATTAA-3 R (135)5 -CTCAAGCACCTCATAAGGATTATCAG-3 Hybridization oligo: 83T-5 -FAM-AAA AGC CTA TGG AAA TTG CCC TCG CA-3 -BHQ1 F (367) 5 -TTCACTGTGTCGATAATCCA-3 R (436) 5 -TGATTTCAATGGCACAAGAT-3 Hybridization oligo: 388T-5 -FAM-TTT ACT AAG TCA CCG ATT GTT GAA ATG A-3 -BHQ1 F (551) 5 -GGCAAGCGTTATCCGGAATT-3 R (651) 5 -GTTTCCAATGACCCTCCACG-3 Hybridization oligo: 573T-5 -TET-CCTACGCGCGCTTTACGCCCA-3 -BHQ2

200–400

Aires-de-Sousa et al. (2006) J01786 This study

532

Goerke et al. (2005)

437

Goerke et al. (2005)

159

This study X03216 This study U35228 This study M17990 This study M14039 This study X52085 This study NC 009641 This study NC 002951

cap5 cap8 ermA ermB ermC linA/linA’ msrA msrB 23S

mecA

femB

agrA

rnaIII

16S rRNA

208 257 213 270 230 471

273

Costa et al. (2005)

94

Klotz et al. (2003)

102

Vaudaux et al. (2002)

70

Vaudaux et al. (2002)

101

Vaudaux et al. (2002)

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Fig. 1. Molecular typing of SCV. (A, B) Pulsed-field gel electrophoresis (PFGE) of small colony variant isolates compared to S. aureus wild type 113 (SA113) and an unrelated cystic fibrosis (CF) S. aureus isolate (CF-18). (A) Agarose gel. Lanes 1 + 12: ␭DNA marker; lane 2: S. aureus SA113; lane 3: CF-18; lanes 4–11: SCV isolates 1–8. (B) Dendrogram visualizing the estimated genetic relationship calculated by GelComparTM . (C) Amplification products of PCRs for spa (upper panel) and cap8 genes (lower panel) in the SCV isolates. (D) spa type of the SCV isolates. (E, F) MALDI biotyping of SCV isolates compared to the spectra for DSM reference strains and an unrelated CF isolate (CF-03). The graph displays the spectra for every single SCV isolate (E); the dendrogram provides the calculated degree of relationship (F).

was analyzed after 24 h using the BDOpteia ELISA kit (BD Biosciences, Heidelberg, Germany). Statistics Statistical significance was determined using the paired twotailed Student’s t-test (*p ≤ 0.05 and **p ≤ 0.005) in Excel 2003 or the one-way analysis of variance (ANOVA)/Tukey procedure (*p < 0.05) in GraphPad Prism software version 5.0.

Typing of S. aureus SCV isolates To confirm strain relatedness, we performed PFGE, MALDI biotyping and spa typing. The results obtained by PFGE (Fig. 1A and B) and MALDI biotyping (Fig. 1E and 1F) indicated that all SCV isolates are derived from the same ancestral strain. spa typing, however, revealed that SCV3 was typed as t026, a repeat deletion mutant of the ancestral t331 (Fig. 1C and D), a finding well in line with previously described spa deletion mutations in CF patients (Kahl et al., 2005b).

Results Isolation of S. aureus small colony variants

SCV isolates differ in biofilm-forming capacity

Previous reports highlighted the genetic variation that characterizes S. aureus small colony variant isolates (Proctor et al., 2006; Schaaff et al., 2003; Besier et al., 2008). However, little is known about intra-strain variability on a functional level and whether these changes affect the host–pathogen interaction. The scope of the present study was to assess functional intra-strain variation. To this end, we compared consecutive S. aureus SCV isolates collected from one single cystic fibrosis patient over a period of 2.5 years (Table 1) with regard to their functional properties. When compared to a normal S. aureus strain, the SCV isolates displayed the typical morphology, e.g. tiny colonies on solid blood agar, loss of pigmentation, decreased hemolysis, and failure to grow on Mueller–Hinton agar (Fig. S1).

To compare the functional properties of the SCV isolates, we first analyzed their biofilm-forming capacity. Biofilm formation in most of the SCV isolates was low when compared to the reference strain SA113, an excellent biofilm producer (Cramton et al., 1999) (Fig. 2A). However, 2 SCV isolates (SCV3 and SCV4) were superior to the others. Of technical note, biofilm formation was dependent on the presence of human plasma and was lower (SA113) or absent (SCV) when biofilm assays were performed without plasma precoating of the plates (Fig. 2B). Altogether, statistically significant differences in the level of biofilm formation were observed among the SCV isolates. Intra-strain variation may, thus, result in differences in the biofilm-forming capacity.

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Fig. 2. Functional properties of small colony variant isolates. (A, B) Bacteria were plated in plates with and without precoating with human plasma. Biofilm was visualized with crystal violet and quantified by optical density (OD) = 570 nm. NC: negative control (CASO-bouillon). (A) Biofilm formation in SCV isolates. The diagram shows the mean values ± SEM obtained in 9 experiments performed in the presence of plasma. Statistics were calculated using ANOVA/Tukey procedure: *p < 0.05, *p (SCV1:SCV3); *p (SCV2:SCV3); *p (SCV5:SCV3); *p (SCV6:SCV3); *p (SCV7:SCV3); *p (SCV8:SCV3). (B) Biofilm formation in the presence or absence of human plasma. The diagram shows the mean values ± SEM obtained in 5 experiments; *p for SA113 = 0.03; *p for SCV3 = 0.022; *p for SCV4 = 0.015; *p for SCV5 = 0.007. (C, D) TLR2 activity. For assessment of TLR2 activity, HEK293 cells were transiently transfected with or without a plasmid-encoding human TLR2. After a 24-h stimulation, IL-8 concentrations were quantified in the cellular supernatants. (−) refers to unstimulated cells. Both diagrams summarize the results from 3 experiments, given as mean values ± SEM. (C) HEK293 cells were stimulated with 1 × 106 CFU of S. aureus SA113, its isogenic mutant deficient in TLR2-active lipoproteins (SA113 lgt), or with the triacylated lipopeptide Pam3 CSK4 (100 ng/ml) as a positive control. n.d., not detectable. (D) HEK293 cells were stimulated with the SCV isolates. Statistics were calculated using ANOVA/Tukey procedure: *p < 0.05, *p (SCV1:SCV3); *p (SCV2:SCV3); *p (SCV4:SCV3); *p (SCV5:SCV3); *p (SCV6:SCV3); *p (SCV7:SCV3); *p (SCV8:SCV3).

Intra-strain variability results in differences in TLR2 activity We investigated whether the SCV isolates would also differ in their immune stimulatory capacity. Since TLR2-active lipopeptides are typically abundant in the cytoplasmic membrane (Stoll et al., 2005) and TLR2-mediated host defense is essential for the clearance of S. aureus and host survival in infection (Takeuchi et al., 2000), we used TLR2 activity to assess the immune stimulatory potential. To measure TLR2 activity, we used an experimental system based on transient transfection of HEK293 cells with a TLR2-bearing plasmid (Bekeredjian-Ding et al., 2007) where IL-8 secretion is selectively induced upon stimulation with TLR2 ligands such as Pam3 CSK4 or the S. aureus strain SA113 (Fig. 2C), but remains undetectable in the absence of TLR2 or when cells are stimulated with SA113 lgt, a mutant that lacks TLR2-stimulating lipoproteins (Fig. 2C) (Stoll et al., 2005). The comparison of the SCV isolates revealed major differences in their TLR2-stimulatory capacity, one SCV being clearly superior to all other isolates (SCV3) (Fig. 2D). We concluded that intra-strain variation also affects TLR2 recognition.

other isolates (Fig. 3A). This indicated that the genetic variability underlying the spa type results in an alteration of protein conformation or size. What is more, high SpA expression levels in SCV3 and SCV4 seemed to correlate with high biofilm formation (Fig. 2A). Differences in protein A expression levels in SCV correlate with changes in spa, agrA, and rnaIII gene expression levels To investigate whether differences in SpA expression result from differential regulation of gene expression, we analyzed gene expression levels by quantitative real-time RT-PCR. Our findings revealed that differences in spa gene expression correlated with those observed for SpA expression, the relative spa expression in SCV3 and SCV4 being highest (Fig. 3B). Conversely, relative RNA expression levels of the accessory gene regulator (agrA) and its effector RNAIII were down-regulated in SCV3 and SCV4 (Fig. 3C and 3D), while cycle threshold values for 16S rRNA remained constant (Fig. 3E). Differences in agr-dependent regulation of gene expression might, thus, account for the apparent differences in spa expression.

SCV differ in protein A expression levels Protein A contributes to biofilm formation In Western blot analysis, the most eminent finding was the marked differences in protein A expression among the SCV isolates (Fig. 3A). Strikingly, the SpA band in the isolate with the spa repeat deletion mutant (SCV3) was running slightly lower than that in the

This prompted us to ask whether the differences in protein A expression levels could be directly associated with the functional properties of the SCV isolates. To test this hypothesis, we

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Fig. 3. Differences in protein and gene expression levels of spa, agrA, and rnaIII in SCV isolates. (A) Protein lysates were prepared from bacteria harvested in the exponential phase of growth. Protein expression in SA113 is compared to that of SCV. The Western blot shown is representative of 3 experiments. Upper panel: Membranes were incubated with a mouse monoclonal anti-staphylococcal protein A antibody followed by goat anti-mouse IgG. Lower panel: Membranes were re-incubated with 1% human serum and anti-human IgG to control SDS-PAGE loading. (B–E) RNA was extracted from S. aureus SCV in the postexponential phase of growth. Quantitative real-time PCR was performed to determine gene expression levels of spa (B), rnaIII (C), agrA (D), and 16S rRNA (E). The diagrams show the relative expression (rE) of the individual genes normalized to the 16S cycle threshold values obtained in one representative out of 2 experiments performed in triplicates.

performed the functional assays using the S. aureus strain SA113 and its isogenic spa-deficient mutant SA113 spa. The results demonstrated that biofilm formation was significantly reduced in the SpA-deficient strain (Fig. 4A), thus, arguing in favour of a contributory role of SpA in S. aureus-mediated biofilm formation. Further analyses revealed that SCV-induced biofilm is proteindependent and independent of polysaccharides (Fig. 4B): biofilm was abolished by treatment with proteinase K, but was unaffected in the presence of NaIO4 . These results supported our claim that quantitative differences in the expression of host-factor-binding

proteins (such as protein A) may indeed account for the differences observed in the biofilm-forming capacity of the SCV isolates. Correlation of protein A expression with TLR2 activity Similarly, in SCV3, high expression levels of protein A coincided with elevated TLR2 activity (Fig. 2D). However, TLR2-dependent IL-8 induction in SCV4 remained inferior to that induced by SCV3 (Fig. 2D), and SCV2 and SCV7 displayed comparably high TLR2 activity (Fig. 2D) despite low SpA expression (Fig. 3A). Altogether, the

Fig. 4. Contribution of cell wall proteins and SpA to biofilm formation and TLR2 activation. (A, B) Biofilm was quantified by crystal violet staining and measurement optical density (OD) at 570 nm after re-elution in ethanol. NC, negative control (CASO-bouillon). (A) Biofilm formation in the presence and absence of SpA expression. Biofilm formation by SA113 was compared to its isogenic mutant SA113 spa. The diagram shows the mean values ± SEM obtained in 6 experiments; *p (SA113:spa) = 0.022. (B) Qualitative analysis of biofilm in SCV isolates. Biofilm formation was assessed in the absence (−) or presence of proteinase K (PK) or NaIO4 . The diagram shows the mean values ± SEM obtained in 3 experiments performed in triplicates. (C) TLR2 activity. HEK293 cells transfected with or without TLR2 cDNA were stimulated with SA113 or the isogenic spa-deficient mutant SA113 spa; cellular supernatants were harvested after 24 h and IL-8 concentrations determined by ELISA. (−) refers to unstimulated cells. The diagram summarizes the results from 3 experiments, given as mean values ± SEM. *p (SA113: spa) = 0.031.

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Fig. 5. Functional properties and protein expression in co-infecting SCV and MRSA isolates. SCV isolates SCV1, -3, -4, and -5 and new SCV isolates SCV9 and -10 were compared with regard to TLR2 activity (A), biofilm formation (B), and protein expression (C, D). (A) TLR2 assay. HEK293 cells transfected with or without pTLR2 were stimulated with the SCV isolates. IL-8 production was quantified 24 h after stimulation. (−) refers to unstimulated cells. The diagram summarizes the results from 3 experiments, given as mean values ± SEM. Statistics were calculated using ANOVA/Tukey procedure: *p < 0.05, *p (SCV9:SCV3); *p (SCV10:SCV3). (B) Biofilm assay. Biofilm was stained with crystal violet and quantified at an OD of 570 nm. NC, negative control (CASO-bouillon). The diagram summarizes the mean values ± SEM obtained in 7 experiments. Statistics were calculated using ANOVA/Tukey procedure: *p < 0.05, *p (SCV9:SCV3); *p (SCV10:SCV3). (C, D) Western blot analysis. Protein expression was compared in lysates from SA113, SCV, and MRSA isolates. The graphs depict one representative experiment of n = 2. (C) Membranes were incubated with mouse anti-staphylococcal protein A antibody and goat anti-mouse IgG. (D) Subsequently, membranes were incubated with 1% human serum and anti-human IgG to ensure correct loading of the polyacrylamid gel.

association of TLR2 activity with SpA expression levels was less clear, and factors other than SpA obviously control TLR2 activity. Nevertheless, a comparison of SA113 and SA113 spa revealed a decrease in TLR2 activity in the absence of spa expression (Fig. 4C). This implies that SpA serves as a regulatory factor controlling the extent of TLR2 activity.

Superinfection with an unrelated MRSA strain does not affect SCV survival or phenotype The patient history provided us with an interesting coincidence: superinfection with an unrelated MRSA strain. This infection led to rapid clinical aggravation of respiratory symptoms requiring hospitalization and intensified antibiotic therapy. However, it provided us with the unique opportunity to study the development of the methicillin-sensitive SCV strain under the influence of an MRSA. Over a period of 4 months, both strains could be isolated from a pharyngeal specimen; afterwards the SCV could no longer be recovered. Typing of the MRSA isolates by PFGE, MALDI biotyping, spa typing (t014), and cap5+ cap8− demonstrated its unrelatedness to the SCV strain (Fig. S2). Functional analysis of the SCV isolates obtained during MRSA superinfection showed that these isolates displayed low TLR2 activity and low biofilm-forming capacity when compared to SCV3 and SCV4 (Fig. 5A and B). Low SpA expression coincided with low biofilm-producing capacity (Fig. 5C and D) and confirmed the results above. Altogether, co-infection with the unrelated MRSA strain did not affect the persistence of the SCV over 4 months nor did it alter its phenotype and functional properties.

Discussion On a genetic level, both intra-species and intra-strain variability have been described in the S. aureus subspecies (McCarthy and Lindsay, 2010; Monecke et al., 2009). In this study, we provide in vivo evidence for intra-strain variability of S. aureus small colony variants on a functional level (Fig. 2). While the presence of biofilm facilitates colonization and chronic infections, TLR2mediated recognition represents a mainstay of first-line immune defense against S. aureus (Takeuchi et al., 2000; Stoll et al., 2005). We, therefore, reasoned that these 2 parameters could be used to assess global changes in the host–pathogen interaction in vitro. Overall, our findings suggest that differences in functional properties correlate with differential gene expression and might ultimately influence the host–pathogen interaction. Recently, a proteome specifically associated with the SCV phenotype was described (Kriegeskorte et al., 2011). The proteomic SCV footprint mainly consists of proteins involved in the tricarboxylic acid cycle, the folate or purine/pyrimidine pathways. Earlier comparative genetic analyses of SCV isolates derived from the same ancestry neglected differential protein expression, despite its evident consequences for the host–pathogen interaction. Since our findings were obtained in a consecutive series of SCV isolates recovered from the same patient they provide additional insight into the extent of intra-strain variability of protein expression in SCV. What is more, our study links these differences in protein expression levels to functional properties influencing the host–pathogen interaction: increased levels of SpA correlate with increased biofilm formation and at least partially with TLR2 activity, thus, suggesting a positive regulatory role for SpA in these processes.

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Previous studies identified the spa gene as a target gene for mutations in colonizing S. aureus strains (Kahl et al., 2005b). Mutations not only mask the spa type, but actually result in changes in protein size and conformation (Fig. 3A). These changes might be associated with so far unappreciated changes in SpA binding affinities and function. Recent reports describing SpAmediated interaction with surface receptors on host epithelial cells, i.e. TNF and EGF receptors (Gomez et al., 2004, 2007), also provide the notion that increased expression of SpA may represent an advantage in adherence and colonization of epithelial surfaces. To confirm that SpA actually participates in biofilm formation, we had to retrieve a spa-deficient S. aureus mutant SA113 spa. Despite the fact that SA113 does not form SCV, it shares important features with SCV3 and SCV4: low agr expression, high SpA expression, TLR2 activity, and biofilm formation (Cramton et al., 1999; Herbert et al., 2010). Indeed, absence of SpA resulted in a partial loss of the biofilm formation (Fig. 4A). This experiment demonstrated a role for SpA in biofilm formation as previously suggested (Merino et al., 2009). However, SpA is not essential for biofilm formation, and high biofilm producers such as the SA113 and others derived of the CC8 lineage (Croes et al., 2009) make use of several distinct mechanisms to generate biofilm, only one of them being dependent on SpA. Other host-binding factors, extracellular DNA, and polysaccharide intracellular adhesin (PIA) are likely to predominate (Tu Quoc et al., 2007; Götz, 2002; Gross et al., 2001; Jefferson et al., 2003; Cramton et al., 1999), making the biofilm formation attributable to SpA negligible. However, in the SCV and/or in the absence of PIA and other adhesion factors SpA might become relevant for biofilm formation. Moreover, high SpA expression in SCV3 and SCV4 could be attributable to low agr expression in these isolates when compared to the other SCVs, and it has previously been proposed that agr activity counteracts biofilm formation (Herbert et al., 2010; Boles and Horswill, 2008; Vuong et al., 2000). Thus, it is impossible to distinguish whether SpA or loss of agr-mediated inhibitory function or both are responsible for the increase in biofilm formation in SCV3 and SCV4. Although we observed a reduction in TLR2 activity in the absence of SpA when comparing SA113 to the spa-deficient mutant (Fig. 4C), the association of SpA expression levels with TLR2 activity remain less clear. We can only speculate that SpA-mediated host cell adhesion and activation might indirectly costimulate TLR2 responses by increasing TLR2 expression as described in Syed et al. (2007). At present, it is further unknown whether and to what extent agr activity controls expression of core lipoproteins such as SitC or accessory genes encoding putative lipoproteins. Overall, however, our data imply that differences in SpA expression levels may account for functional intra-strain variability in the SCV isolates used in this study. Initial reports on SCV described reduced protein A expression in an hemB-deficient SCV strain (von Eiff et al., 1997). Despite no obvious differences in growth curves and nutritional requirements (data not shown), we can, however, not exclude that mutations affecting the metabolic properties may be responsible for differences in protein A expression. Others reported that downregulation of agr expression in SCV coincides with increased expression of adhesion factors and loss of ␣-hemolysin (Kahl et al., 2005a). Furthermore, quorum sensing via the agr system controls spa gene expression (Cheung et al., 1997; Heinrichs et al., 1996). Comparative gene expression analysis in the SCV isolates revealed that agrA and rnaIII were conversely regulated to spa gene expression (Fig. 3), thus explaining the alterations in spa expression levels. Notably, the functional differences are also explained by differential agr expression, because agr deficiency favours biofilm formation (Boles and Horswill, 2008; Vuong et al., 2000; Beenken et al., 2003).

S. aureus has the unique ability to adapt to the host environment. One prominent example is the formation of SCV. In this study, we describe a series of clinical isolates derived from the same ancestral strain. An interesting question in the context of chronic persistence within the host is whether the bacteria recovered from a specimen represent a single entity. Cultures on solid agar over more than 2 days revealed that only individual colonies revert to normal size (data not shown). Thus, we are rather dealing with a mixture of multiple variants whose functional homogeneity could be preserved by environmental influences. Alternatively, environmental stimuli could select for the variants recovered at single time points. However, small sampling size, cryopreservation, or in vitro culture conditions could also represent an important bias. Unfortunately, the data obtained in this study do not allow a definitive answer. However, next generation sequencing approaches have recently provided new insight into the population dynamics of S. aureus (Harris et al., 2010). Interestingly, a genome-wide analysis of heterogeneity revealed that, indeed, USA300 cultures represent mixtures of multiple genetic variants (Yu, 2010). Taken together, our study on intra-strain variability of S. aureus small colony variants expands previous findings by providing insight into the functional consequences of genetic and proteomic changes in related SCV isolates. Of note, the SCV isolates displaying high spa and low agr expression and functional activity (SCV3 and SCV4) were isolated during a clinical period of convalescence after severe pneumonia, e.g. under stable respiratory function and absence of overt infection. On the contrary, SCV isolates retrieved during clinically manifest MRSA pneumonia (SCV9 and SCV10) were functionally inactive and displayed only low SpA expression levels (Fig. 5). It is well conceivable that changes in the inflammatory response of the host, the microbial environment, and the antibiotic regime not only contribute to the development of SCV, but influence their genetic, proteomic, and functional phenotypes. Nevertheless, we can only speculate that in this patient the absence of infection, the resultant reduction in inflammatory activity, and the concomitant reduction in prescribed antibiotics and other drugs might have favoured a S. aureus phenotype characterized by decreased agr activity and an increase in expression of cell wallassociated proteins such as SpA, thus unleashing SCV immunogenicity, biofilm formation, and possibly extracellular survival. Conflict of interest statement The authors declare no financial conflict of interest. Acknowledgments This study is part of the PhD thesis of D.H. and the Bachelor thesis from J.O. We thank Friedrich Götz, Tuebingen, Germany, for providing the SA113 and SA113 spa and lgt strains, Maria Haensch, Heidelberg, Germany, for help with biofilm assays, and Felix Lasitschka, Heidelberg, Germany, for support with the Agilent bioanalyzer. We would like to acknowledge Angelika Heller and Vanessa Epp for the excellent technical assistance. This study was funded by support of I.B.-D. by the Olympia-Morata grant of the Medical Faculty of the University of Heidelberg, Germany, and the DFG priority programme SPP 1468 IMMUNOBONE grant RI707/8-1. D.H. was supported by a grant from the Pharmaceutical faculty of the University of Damascus, Syria. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.ijmm.2012.12.008.

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