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The exoproteomes of clonally related Staphylococcus aureus strains are diverse

The exoproteomes of clonally related Staphylococcus aureus strains are diverse Ann Microbiol (2015) 65:1809–1813 DOI 10.1007/s13213-015-1064-7 SHORT COMMUNICATION The exoproteomes of clonally related Staphylococcus aureus strains are diverse Yun Khoon Liew & Rukman Awang Hamat & Syafinaz Amin Nordin & Pei Pei Chong & Vasanthakumari Neela Received: 15 May 2014 /Accepted: 18 February 2015 /Published online: 7 March 2015 Springer-Verlag Berlin Heidelberg and the University of Milan 2015 Abstract Several studies have shown that protein expression erogeneity in the exoproteome of closely related S. aureus patterns vary in unrelated bacterial strains due to genomic strains observed in the current study postulates that pre- plasticity and gene regulation, resulting in enhanced heteroge- existing antibodies are not very protective during recurrent neity in the infection potential. However, exoprotein expres- infection with the same strain. Therefore, our findings under- sion patterns of closely related clonal strains have not been score the importance of taking all clonally related strains into well characterized. Here, we used medium-range (pH 4–7) account during proteome analyses. immobilized pH gradient–two-dimensional gel electrophore- sis to investigate the exoproteome from closely related . . . Keywords Staphylococcus aureus Exoprotein 2-DGE Staphylococcus aureus clonal isolates. Interestingly, we found Closely related clonal that, under identical in vitro experimental conditions, a num- ber of protein spots were uniquely present in samples from each clonal isolate irregardless of the similarity of the geno- type and the same virulence gene profile. Only a few abundant Despite the large numbers of studies which have been carried invariant proteins were found among identical genotypic iso- out over the past 130 years on Staphylococcus aureus, the lates. Our results clearly shown that heterogeneity in the emergence of multidrug-resistant strains with a heterogeneous exoproteome was present even among clonally related strains. virulence potential is still on the rise (Sakoulas and Moellering We suggest that this heterogeneity may contribute to the de- 2008; Hongo et al. 2009; Ventura et al. 2010;Muthukrishnan gree of virulence even within one clonal genotype. The het- et al. 2011; Alonzo III et al. 2012). Heterogeneity in exoprotein expression patterns has been observed for distinct genotypes of S. aureus (Bernardo et al. 2002;Ziebandtetal. 2010). This variation in exoproteins increases S. aureus fitness Y. K. Liew Department of Life Sciences, School of Pharmacy, International by enhancing its pathogenicity potential, including attachment Medical University, 57000 Kuala Lumpur, Malaysia and invasion/evasion of host defenses, as well as by promot- ing growth in different niches in the environments of the hu- : : : Y. K. Liew R. A. Hamat S. A. Nordin V. Neela man host. However, the question of whether clonally related Department of Medical Microbiology and Parasitology, Universiti Putra Malaysia, 43400 Serdang, Malaysia S. aureus strains [as defined by a pulsed-field gel electropho- resis (PFGE) profile with>80 % similarity and sharing of the P. P. Chong same sequence type) display a homogeneous pattern at the Department of Biomedical Sciences, Universiti Putra Malaysia, protein level once subjected to medium-range immobilized 43400 Serdang, Malaysia pH gradient (IPG)–2-DGE remains unanswered. To address V. Neela (*) this question, we randomly selected six clinical isolates of Department of Medical Microbiology and Parasitology, Faculty of three S. aureus clones, namely, ST1, ST30 and ST8 (2 isolates Medicine and Health Sciences, Universiti Putra Malaysia, per clonal type), for 2-DGE comparison. PFGE patterns and 43400 Serdang, Malaysia e-mail: vasantha@upm.edu.my virulence gene profiles for each isolate were also determined 1810 Ann Microbiol (2015) 65:1809–1813 (Fig. 1). This pilot study was not designed to identify the peptides were then extracted according to standard techniques. various spots on the gel due to the small number of clones Electrospray ionization mass spectrometry analysis of tested, but rather to characterize the exoprotein expression peptides was carried out using the Ultimate 3000 nano patterns from closely related S. aureusstrains through HPLC system (Dionex Corp., Sunnyvale, CA) coupled medium-range (pH 4–7) IPG–2-DGE and a modified silver to a 4000 Q TRAP mass spectrometer (Applied staining technique for the clear visualization of protein Biosystems, Foster City, CA). Briefly, tryptic peptides variants. were loaded onto a C18 PepMap100, 3 μmcolumn Briefly, S. aureus culture (0.03–0.04 OD )was (LC Packings–Dionex Corp.) and separated with a linear grown in Tryptic Soy Broth supplemented with gradient of water/acetonitrile/0.1 % formic acid (v/v). 0.001 M 2,2′-dipyridyl (Sigma-Aldrich, St. Louis, MO) Spectra were analyzed to identify proteins of interest at 37 °C under constant agitation (150 rpm). The culture using Mascot sequence matching software (Matrix supernatant was precipitated using the ice-cold ethanol– Science, London, UK) with the Ludwig NR database. TCA method when the growth reached the Ludwig NR is a comprehensive, audited database which postexponential phase. Exoproteins isolated from a cul- contains non-identical protein sequence information ture supernatant often comprise a wide variety of viru- based on all major publicly available datasets. The fol- lence factors. In our study, the exoproteins were subject- lowing search parameters were applied for liquid chro- ed to 2-DGE with a pH 4–7 IPG strip (Bio-Rad, matography–tandem mass (MS/MS) analysis on the Hercules, CA) as described previously (Liew et al. 4000 QTRAP mass spectrometer: (1) oxidation (M) 2013). The stained gels were scanned with a calibrated was set with variable modifications; a peptide mass tol- densitometer (model GS-800; Bio-Rad) and analyzed erance of±1.2 Da and MS/MS tolerance of±06 Da were using PDQuest Advanced 8.0.1 2D Gel Analysis soft- used; (2) a peptide charge state of +1+2 or +3 was ware (Bio-Rad), which determined the densities (volume accepted; (3) the maximum number of missed cleavages by Gaussian integration) of each of the protein spots on was set to 1. An individual ion score of >60 was con- the gels. Technical triplicates were performed for each sidered to indicate significant identification of a protein sample. The correlation coefficients between technical spot, which corresponds to a P≤0.05. replicates of gels ranged from 0.79 to 0.95, which is Interestingly, there was much more variation in exoprotein normal for 2-DGE analyses (Vydra et al. 2008). Mean patterns than expected—even among the isolates that shared values for spot intensity were subjected to analysis the same clonal lineage (Fig. 2). Isolate pairs showing a high using the Student t test to determine whether there were similarity in the PFGE analysis did not necessarily show a any significant individual differences in exoprotein ex- homogeneous exoproteome pattern. Moreover, 40–50 % of protein spots within the similar exoprotein pattern showed pression between paired isolates. A P value of≤0.05 was considered to indicate statistical significance. more than a twofold variation in spot intensity. Only a few The abundant invariant protein spots were manually ex- invariant proteins were abundantly expressed by same clonal cised from the 2-DGE gels and digested with trypsin; the lineage isolates, such as staphylococcal secretory antigen Fig. 1 Pulsed-field gel electrophoresis (PFGE) and virulence factor Goering 2010; Alibayov et al. 2014). It should be noted that the PFGE patterns. Most of the closely related isolates showed similar PFGE pattern of ST1 isolates showed high similarity to an identical virulence patterns after restriction with SmaI (Sigma-Aldrich, St. Louis, MO; profile Ann Microbiol (2015) 65:1809–1813 1811 Fig. 2 Exoproteome of closely related isolates. Pairs A/B, C/D and E/F, level of heterogeneity in exoprotein expression can be seen in isolates of respectively, were compared. Comparative pseudo-colored images of clone ST1 harboring the same virulence gene combination (c, d). The exoprotein expression between the closely related isolates of each pair similarity between spot patterns was only as high as (approximately)50 % is illustrated at the right. Both images were superimposed by PDQuest even when both isolates had the same clonal lineage. Arrows indicate Advanced 8.0.1 2D Gel Analysis software (Bio-Rad, Hercules, CA). abundant spots corresponding to the absence of any significantly Protein spots whose intensity did not vary between closely related differentially expressed proteins. a, b Exoprotein expression by clones strains are indicated in yellow; green or red spots represent the of genotype ST30, c, d exoprotein expression by clones of genotype ST1, exoproteome of closely related strains that were very heterogeneous. e, f, exoprotein expression by clones of genotype ST8. Only one of the Exoprotein spot patterns varied in each comparison. In particular, a high three technical replicates is shown (SsaA), immunodominant antigen A (IsaA) and alkyl relatively small number of strains, heterogeneity in the hydroperoxidereductase subunit C (AhpC). Isolates with the exoproteome was clearly present, even among clonally related same sequence type and same combination of virulence genes strains. (for example, ST1 isolates with an identical virulence factor Our results differ from those of earlier studies which report- profile of sea, seh, cna, fnbA, icaA, icaD, pvl) also exhibited ed that closely related strains exhibit highly similar variation in their exoprotein expression pattern (Figs. 1, 2). exoproteome patterns (Nakano et al. 2002; Ziebandt et al. While we do emphasize that our results are based on a 2010). Ziebandt et al. (2010) demonstrated that S. aureus 1812 Ann Microbiol (2015) 65:1809–1813 Table 1 Identification of invariant and abundant proteins between closely related Staphylococcus aureus strains Open reading frame name Protein name pI/kDa Score Sequence coverage (%) Average protein intensity ratio within strain ST30 ST1 ST8 SAOUHSC_00365 Alkyl hydroperoxide reductase subunit C 4.88/20.963 403 70 2.5 1.4 0.9 SAO46_0518 Immunodominant antigen A 5.91/24.219 179 20 1.3 1.6 1.1 ST398NM01_2354 Staphylococcal secretory antigen 6.01/17.415 91 10 1.2 1.5 1.1 The mean intensity change in the protein of a spot between two isolates per clonal type in terms of relative intensity to one another. The majority of these abundant protein spots showed non-significant differences of less than a twofold and more than a 0.5-fold ratio isolates with the same sequence type (ST80) collected from not be excluded during any proteomic study as the results different patients displayed an identical exoprotein pattern. from such studies may contribute to our knowledge of the Similarly, Nakano et al. (2002) showed that protein expression degree of virulence even within a clonal genotype. Along with profiles were similar among strains of same coagulase type. genotype analysis (multilocus sequence typing and PFGE), However, most of these earlier studies used an IPG strip with a the pattern of exoprotein spots provides a more discriminatory wide pH range (pH 3–10) instead of the relatively narrower picture and facilitates further subdivisions of S. aureus strains. pH range used in our study (pH 4–7); it must be noted that in However, the 2-DGE approach, when used as a discriminatory tool, still needs to be refined for practical applications. Further these earlier studies most of the spots at pH 4–6 were poorly resolved and low-abundance proteins were not analyzed fur- investigations should address our lack of knowledge on pro- ther. In contrast, in our study, the careful detection of low- tein expression patterns of S. aureus during the in vivo abundance protein spots after the enrichment of spots and condition. the enhancement of staining sensitivity by medium-range Acknowledgments This study was supported by Research University Grant Scheme (04-01-09-0795RU) from Universiti Putra Malaysia. IPG, as well as the application of the modified silver staining method of Liew et al. (2013), clearly revealed variation in the Conflict of interest None. exoproteome patterns. Similarly, our findings also differ from the results of more recent DNA microarray or PCR studies that revealed very similar virulence gene expression profiles in isolates with the same sequence type (Jamrozy et al. 2012; References Shambatetal. 2012). Our results do agree with those of Munsky et al. (2012) and Le Maréchal et al. (2011) who found Alibayov B, Zdeňková K, Purkrtová S, Demnerová K, Karpíšková R variable gene expression in identical environments even when (2014) Detection of some phenotypic and genotypic characteristics of Staphylococcus aureus isolated from food items in the the cells were genetically identical. One explanation of this Czech Republic. Ann Microbiol 64:1587–1596 variability is the possible accumulation of truncations and oth- Alonzo F III, Benson MA, Chen J, Novick RP, Shopsin B, Torres VJ er nucleotide differences in certain genes. In our study, only (2012) Staphylococcus aureus leucocidin ED contributes to system- three of the abundant conserved proteins showed no distinct ic infection by targeting neutrophils and promoting bacterial growth difference in spot intensity between clonally related isolates in vivo. Mol Microbiol 83:423–435 Bernardo K, Fleer S, Pakulat N, Krut O, Hünger F, Krönke M (2002) such as SsaA and IsaA, which are responsible for S. aureus Identification of Staphylococcus aureus exotoxins by combined so- cell-wall metabolism, and AhpC, a stress response protein that dium dodecyl sulfate gel electrophoresis and matrix-assisted laser efficiently provides resistance to hydrogen peroxide, cumene desorption/ionization-time of flight mass spectrometry. Proteomics hydroperoxide, tert-butyl hydroperoxide, paraquat and 2:740–746 peroxynitrite (Fig. 2; Table 1). Based on these results, we Goering RV (2010) Pulsed field gel electrophoresis: a review of applica- tion and interpretation in the molecular epidemiology of infectious suggest that clonally related S. aureus strains are able to pro- disease. Infect Genet Evol 10:866–875 duce a variable proteome, which in turn causes developments Hongo I, Baba T, Oishi K, Morimoto Y, Ito T, Hiramatsu K (2009) in antibiotic therapy and immunotherapy to be more challeng- Phenol-soluble modulin α3 enhances the human neutrophil lysis ing and complicated, especially in the context of recurrent mediated by panton-valentine leukocidin. J Infect Dis 200:715–723 infection with the same strain. Jamrozy DM, Fielder MD, Butaye P, Coldham NG (2012) Comparative genotypic and phenotypic characterisation of methicillin-resistant In conclusion, we observed high heterogeneity in Staphylococcus aureus ST398 isolated from animals and humans. exoprotein expression that was irrespective of genotypic relat- PLoS One 7:e40458 edness. These findings suggest that the comparison of Le Maréchal C, Seyffert N, Jardin J, Hernandez D, Jan G, Rault L, exoproteomes from closely related S. aureus strains should Azevedo V, François P, Schrenzel J, van de Guchte M, Even S, Ann Microbiol (2015) 65:1809–1813 1813 Berkova N, Thiéry R, Fitzgerald JR, Vautor E, Le Loir Y (2011) Shambat S, Nadig S, Prabhakara S, Bes M, Etienne J, Arakere G (2012) Clonal complexes and virulence factors of Staphylococcus aureus Molecular basis of virulence in Staphylococcus aureus mastitis. PLoS One 6:e27354 from several cities in India. BMC Microbiol 12:64 Liew YK, Neela V, Hamat RA, Nordin SA, Chong PP (2013) Modified Ventura CL, Malachowa N, Hammer CH, Nardone GA, Robinson MA, silver staining in 2DE improves protein detection even at extremely Kobayashi SD, DeLeo FR (2010) Identification of a novel low sample concentration. Electrophoresis 34:397–400 Staphylococcus aureus two-component leukotoxin using cell sur- Munsky B, Neuert G, van Oudenaarden A (2012) Using gene expression face proteomics. PLoS One 5:e11634 noise to understand gene regulation. Science 336:183–187 Vydra J, Selicharová I, Smutná K, Sanda M, Matousková E, Muthukrishnan G, Quinn GA, Lamers RP, Diaz C, Cole AL, Chen S, Bursíková E, Prchalová M, Velenská Z, Coufal D, Jirácek J Cole AM (2011) Exoproteome of Staphylococcus aureus reveals (2008) Two-dimensional electrophoretic comparison of meta- putative determinants of nasal carriage. J Proteome Res 10:2064– static and non-metastatic human breast tumors using in vitro 2078 cultured epithelial cells derived from the cancer tissues. BMC Nakano M, Kawano Y, Kawagishi M, Hasegawa T, Iinuma Y, Ohta M Cancer 8:107 (2002) Two-dimensional analysis of exoproteins of methicillin- Ziebandt A, Kusch H, Degner M, Jaglitz S, Sibbald MJJB, Arends JP, resistant Staphylococcus aureus (MRSA) for possible epidemiolog- Chlebowicz MA, Albrecht D, Pantucek R, Doskar J, Ziebuhr W, ical applications. Microbiol Immunol 46:11–22 Bröker BM, Hecker M, van Dijl JM, Engelmann S (2010) Sakoulas G, Moellering RC (2008) Increasing antibiotic resistance Proteomics uncovers extreme heterogeneity in the Staphylococcus among methicillin-resistant Staphylococcus aureus strains. Clin aureus exoproteome due to genomic plasticity and variant gene Infect Dis 46:S360–S367 regulation. Proteomics 10:1634–1644 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

The exoproteomes of clonally related Staphylococcus aureus strains are diverse

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Springer Journals
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Copyright © 2015 by Springer-Verlag Berlin Heidelberg and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1869-2044
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Abstract

Ann Microbiol (2015) 65:1809–1813 DOI 10.1007/s13213-015-1064-7 SHORT COMMUNICATION The exoproteomes of clonally related Staphylococcus aureus strains are diverse Yun Khoon Liew & Rukman Awang Hamat & Syafinaz Amin Nordin & Pei Pei Chong & Vasanthakumari Neela Received: 15 May 2014 /Accepted: 18 February 2015 /Published online: 7 March 2015 Springer-Verlag Berlin Heidelberg and the University of Milan 2015 Abstract Several studies have shown that protein expression erogeneity in the exoproteome of closely related S. aureus patterns vary in unrelated bacterial strains due to genomic strains observed in the current study postulates that pre- plasticity and gene regulation, resulting in enhanced heteroge- existing antibodies are not very protective during recurrent neity in the infection potential. However, exoprotein expres- infection with the same strain. Therefore, our findings under- sion patterns of closely related clonal strains have not been score the importance of taking all clonally related strains into well characterized. Here, we used medium-range (pH 4–7) account during proteome analyses. immobilized pH gradient–two-dimensional gel electrophore- sis to investigate the exoproteome from closely related . . . Keywords Staphylococcus aureus Exoprotein 2-DGE Staphylococcus aureus clonal isolates. Interestingly, we found Closely related clonal that, under identical in vitro experimental conditions, a num- ber of protein spots were uniquely present in samples from each clonal isolate irregardless of the similarity of the geno- type and the same virulence gene profile. Only a few abundant Despite the large numbers of studies which have been carried invariant proteins were found among identical genotypic iso- out over the past 130 years on Staphylococcus aureus, the lates. Our results clearly shown that heterogeneity in the emergence of multidrug-resistant strains with a heterogeneous exoproteome was present even among clonally related strains. virulence potential is still on the rise (Sakoulas and Moellering We suggest that this heterogeneity may contribute to the de- 2008; Hongo et al. 2009; Ventura et al. 2010;Muthukrishnan gree of virulence even within one clonal genotype. The het- et al. 2011; Alonzo III et al. 2012). Heterogeneity in exoprotein expression patterns has been observed for distinct genotypes of S. aureus (Bernardo et al. 2002;Ziebandtetal. 2010). This variation in exoproteins increases S. aureus fitness Y. K. Liew Department of Life Sciences, School of Pharmacy, International by enhancing its pathogenicity potential, including attachment Medical University, 57000 Kuala Lumpur, Malaysia and invasion/evasion of host defenses, as well as by promot- ing growth in different niches in the environments of the hu- : : : Y. K. Liew R. A. Hamat S. A. Nordin V. Neela man host. However, the question of whether clonally related Department of Medical Microbiology and Parasitology, Universiti Putra Malaysia, 43400 Serdang, Malaysia S. aureus strains [as defined by a pulsed-field gel electropho- resis (PFGE) profile with>80 % similarity and sharing of the P. P. Chong same sequence type) display a homogeneous pattern at the Department of Biomedical Sciences, Universiti Putra Malaysia, protein level once subjected to medium-range immobilized 43400 Serdang, Malaysia pH gradient (IPG)–2-DGE remains unanswered. To address V. Neela (*) this question, we randomly selected six clinical isolates of Department of Medical Microbiology and Parasitology, Faculty of three S. aureus clones, namely, ST1, ST30 and ST8 (2 isolates Medicine and Health Sciences, Universiti Putra Malaysia, per clonal type), for 2-DGE comparison. PFGE patterns and 43400 Serdang, Malaysia e-mail: vasantha@upm.edu.my virulence gene profiles for each isolate were also determined 1810 Ann Microbiol (2015) 65:1809–1813 (Fig. 1). This pilot study was not designed to identify the peptides were then extracted according to standard techniques. various spots on the gel due to the small number of clones Electrospray ionization mass spectrometry analysis of tested, but rather to characterize the exoprotein expression peptides was carried out using the Ultimate 3000 nano patterns from closely related S. aureusstrains through HPLC system (Dionex Corp., Sunnyvale, CA) coupled medium-range (pH 4–7) IPG–2-DGE and a modified silver to a 4000 Q TRAP mass spectrometer (Applied staining technique for the clear visualization of protein Biosystems, Foster City, CA). Briefly, tryptic peptides variants. were loaded onto a C18 PepMap100, 3 μmcolumn Briefly, S. aureus culture (0.03–0.04 OD )was (LC Packings–Dionex Corp.) and separated with a linear grown in Tryptic Soy Broth supplemented with gradient of water/acetonitrile/0.1 % formic acid (v/v). 0.001 M 2,2′-dipyridyl (Sigma-Aldrich, St. Louis, MO) Spectra were analyzed to identify proteins of interest at 37 °C under constant agitation (150 rpm). The culture using Mascot sequence matching software (Matrix supernatant was precipitated using the ice-cold ethanol– Science, London, UK) with the Ludwig NR database. TCA method when the growth reached the Ludwig NR is a comprehensive, audited database which postexponential phase. Exoproteins isolated from a cul- contains non-identical protein sequence information ture supernatant often comprise a wide variety of viru- based on all major publicly available datasets. The fol- lence factors. In our study, the exoproteins were subject- lowing search parameters were applied for liquid chro- ed to 2-DGE with a pH 4–7 IPG strip (Bio-Rad, matography–tandem mass (MS/MS) analysis on the Hercules, CA) as described previously (Liew et al. 4000 QTRAP mass spectrometer: (1) oxidation (M) 2013). The stained gels were scanned with a calibrated was set with variable modifications; a peptide mass tol- densitometer (model GS-800; Bio-Rad) and analyzed erance of±1.2 Da and MS/MS tolerance of±06 Da were using PDQuest Advanced 8.0.1 2D Gel Analysis soft- used; (2) a peptide charge state of +1+2 or +3 was ware (Bio-Rad), which determined the densities (volume accepted; (3) the maximum number of missed cleavages by Gaussian integration) of each of the protein spots on was set to 1. An individual ion score of >60 was con- the gels. Technical triplicates were performed for each sidered to indicate significant identification of a protein sample. The correlation coefficients between technical spot, which corresponds to a P≤0.05. replicates of gels ranged from 0.79 to 0.95, which is Interestingly, there was much more variation in exoprotein normal for 2-DGE analyses (Vydra et al. 2008). Mean patterns than expected—even among the isolates that shared values for spot intensity were subjected to analysis the same clonal lineage (Fig. 2). Isolate pairs showing a high using the Student t test to determine whether there were similarity in the PFGE analysis did not necessarily show a any significant individual differences in exoprotein ex- homogeneous exoproteome pattern. Moreover, 40–50 % of protein spots within the similar exoprotein pattern showed pression between paired isolates. A P value of≤0.05 was considered to indicate statistical significance. more than a twofold variation in spot intensity. Only a few The abundant invariant protein spots were manually ex- invariant proteins were abundantly expressed by same clonal cised from the 2-DGE gels and digested with trypsin; the lineage isolates, such as staphylococcal secretory antigen Fig. 1 Pulsed-field gel electrophoresis (PFGE) and virulence factor Goering 2010; Alibayov et al. 2014). It should be noted that the PFGE patterns. Most of the closely related isolates showed similar PFGE pattern of ST1 isolates showed high similarity to an identical virulence patterns after restriction with SmaI (Sigma-Aldrich, St. Louis, MO; profile Ann Microbiol (2015) 65:1809–1813 1811 Fig. 2 Exoproteome of closely related isolates. Pairs A/B, C/D and E/F, level of heterogeneity in exoprotein expression can be seen in isolates of respectively, were compared. Comparative pseudo-colored images of clone ST1 harboring the same virulence gene combination (c, d). The exoprotein expression between the closely related isolates of each pair similarity between spot patterns was only as high as (approximately)50 % is illustrated at the right. Both images were superimposed by PDQuest even when both isolates had the same clonal lineage. Arrows indicate Advanced 8.0.1 2D Gel Analysis software (Bio-Rad, Hercules, CA). abundant spots corresponding to the absence of any significantly Protein spots whose intensity did not vary between closely related differentially expressed proteins. a, b Exoprotein expression by clones strains are indicated in yellow; green or red spots represent the of genotype ST30, c, d exoprotein expression by clones of genotype ST1, exoproteome of closely related strains that were very heterogeneous. e, f, exoprotein expression by clones of genotype ST8. Only one of the Exoprotein spot patterns varied in each comparison. In particular, a high three technical replicates is shown (SsaA), immunodominant antigen A (IsaA) and alkyl relatively small number of strains, heterogeneity in the hydroperoxidereductase subunit C (AhpC). Isolates with the exoproteome was clearly present, even among clonally related same sequence type and same combination of virulence genes strains. (for example, ST1 isolates with an identical virulence factor Our results differ from those of earlier studies which report- profile of sea, seh, cna, fnbA, icaA, icaD, pvl) also exhibited ed that closely related strains exhibit highly similar variation in their exoprotein expression pattern (Figs. 1, 2). exoproteome patterns (Nakano et al. 2002; Ziebandt et al. While we do emphasize that our results are based on a 2010). Ziebandt et al. (2010) demonstrated that S. aureus 1812 Ann Microbiol (2015) 65:1809–1813 Table 1 Identification of invariant and abundant proteins between closely related Staphylococcus aureus strains Open reading frame name Protein name pI/kDa Score Sequence coverage (%) Average protein intensity ratio within strain ST30 ST1 ST8 SAOUHSC_00365 Alkyl hydroperoxide reductase subunit C 4.88/20.963 403 70 2.5 1.4 0.9 SAO46_0518 Immunodominant antigen A 5.91/24.219 179 20 1.3 1.6 1.1 ST398NM01_2354 Staphylococcal secretory antigen 6.01/17.415 91 10 1.2 1.5 1.1 The mean intensity change in the protein of a spot between two isolates per clonal type in terms of relative intensity to one another. The majority of these abundant protein spots showed non-significant differences of less than a twofold and more than a 0.5-fold ratio isolates with the same sequence type (ST80) collected from not be excluded during any proteomic study as the results different patients displayed an identical exoprotein pattern. from such studies may contribute to our knowledge of the Similarly, Nakano et al. (2002) showed that protein expression degree of virulence even within a clonal genotype. Along with profiles were similar among strains of same coagulase type. genotype analysis (multilocus sequence typing and PFGE), However, most of these earlier studies used an IPG strip with a the pattern of exoprotein spots provides a more discriminatory wide pH range (pH 3–10) instead of the relatively narrower picture and facilitates further subdivisions of S. aureus strains. pH range used in our study (pH 4–7); it must be noted that in However, the 2-DGE approach, when used as a discriminatory tool, still needs to be refined for practical applications. Further these earlier studies most of the spots at pH 4–6 were poorly resolved and low-abundance proteins were not analyzed fur- investigations should address our lack of knowledge on pro- ther. In contrast, in our study, the careful detection of low- tein expression patterns of S. aureus during the in vivo abundance protein spots after the enrichment of spots and condition. the enhancement of staining sensitivity by medium-range Acknowledgments This study was supported by Research University Grant Scheme (04-01-09-0795RU) from Universiti Putra Malaysia. IPG, as well as the application of the modified silver staining method of Liew et al. (2013), clearly revealed variation in the Conflict of interest None. exoproteome patterns. Similarly, our findings also differ from the results of more recent DNA microarray or PCR studies that revealed very similar virulence gene expression profiles in isolates with the same sequence type (Jamrozy et al. 2012; References Shambatetal. 2012). Our results do agree with those of Munsky et al. (2012) and Le Maréchal et al. (2011) who found Alibayov B, Zdeňková K, Purkrtová S, Demnerová K, Karpíšková R variable gene expression in identical environments even when (2014) Detection of some phenotypic and genotypic characteristics of Staphylococcus aureus isolated from food items in the the cells were genetically identical. One explanation of this Czech Republic. Ann Microbiol 64:1587–1596 variability is the possible accumulation of truncations and oth- Alonzo F III, Benson MA, Chen J, Novick RP, Shopsin B, Torres VJ er nucleotide differences in certain genes. In our study, only (2012) Staphylococcus aureus leucocidin ED contributes to system- three of the abundant conserved proteins showed no distinct ic infection by targeting neutrophils and promoting bacterial growth difference in spot intensity between clonally related isolates in vivo. Mol Microbiol 83:423–435 Bernardo K, Fleer S, Pakulat N, Krut O, Hünger F, Krönke M (2002) such as SsaA and IsaA, which are responsible for S. aureus Identification of Staphylococcus aureus exotoxins by combined so- cell-wall metabolism, and AhpC, a stress response protein that dium dodecyl sulfate gel electrophoresis and matrix-assisted laser efficiently provides resistance to hydrogen peroxide, cumene desorption/ionization-time of flight mass spectrometry. Proteomics hydroperoxide, tert-butyl hydroperoxide, paraquat and 2:740–746 peroxynitrite (Fig. 2; Table 1). Based on these results, we Goering RV (2010) Pulsed field gel electrophoresis: a review of applica- tion and interpretation in the molecular epidemiology of infectious suggest that clonally related S. aureus strains are able to pro- disease. Infect Genet Evol 10:866–875 duce a variable proteome, which in turn causes developments Hongo I, Baba T, Oishi K, Morimoto Y, Ito T, Hiramatsu K (2009) in antibiotic therapy and immunotherapy to be more challeng- Phenol-soluble modulin α3 enhances the human neutrophil lysis ing and complicated, especially in the context of recurrent mediated by panton-valentine leukocidin. J Infect Dis 200:715–723 infection with the same strain. Jamrozy DM, Fielder MD, Butaye P, Coldham NG (2012) Comparative genotypic and phenotypic characterisation of methicillin-resistant In conclusion, we observed high heterogeneity in Staphylococcus aureus ST398 isolated from animals and humans. exoprotein expression that was irrespective of genotypic relat- PLoS One 7:e40458 edness. These findings suggest that the comparison of Le Maréchal C, Seyffert N, Jardin J, Hernandez D, Jan G, Rault L, exoproteomes from closely related S. aureus strains should Azevedo V, François P, Schrenzel J, van de Guchte M, Even S, Ann Microbiol (2015) 65:1809–1813 1813 Berkova N, Thiéry R, Fitzgerald JR, Vautor E, Le Loir Y (2011) Shambat S, Nadig S, Prabhakara S, Bes M, Etienne J, Arakere G (2012) Clonal complexes and virulence factors of Staphylococcus aureus Molecular basis of virulence in Staphylococcus aureus mastitis. PLoS One 6:e27354 from several cities in India. BMC Microbiol 12:64 Liew YK, Neela V, Hamat RA, Nordin SA, Chong PP (2013) Modified Ventura CL, Malachowa N, Hammer CH, Nardone GA, Robinson MA, silver staining in 2DE improves protein detection even at extremely Kobayashi SD, DeLeo FR (2010) Identification of a novel low sample concentration. 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Journal

Annals of Microbiology – Springer Journals

Published: Mar 7, 2015

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