A proteomic approach to cold acclimation of Staphylococcus aureus CECT 976 grown at room and human body temperatures

International Journal of Food Microbiology 144 (2010) 160–168

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

A proteomic approach to cold acclimation of Staphylococcus aureus CECT 976 grown at room and human body temperatures B. Sánchez b, M.L. Cabo a,⁎, A. Margolles b, J.J.R. Herrera a a b

Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (CSIC), Eduardo Cabello, 6. 36208 Vigo, Galicia, Spain Instituto de Productos Lácteos, Consejo Superior de Investigaciones Científicas (CSIC), Crta. de Infiesto s/n. 33300 Villaviciosa, Asturias, Spain

a r t i c l e

i n f o

Article history: Received 6 April 2010 Received in revised form 17 September 2010 Accepted 19 September 2010 Keywords: Staphylococcus aureus Cold acclimation Proteomic

a b s t r a c t Staphylococcus aureus is an important pathogenic microorganism that has been associated with serious infection problems in different fields, from food to clinic. In the present study, we have taken into account that the main reservoirs of this microorganism are the human body and some parts of food processing plants, which have normal temperatures of around 37 and 25 °C, respectively. It can be expected that S. aureus must acclimate its metabolism to colder temperatures before growing in food matrices. Since temperature abuse for foods occurs at approximately 12 °C, it is expected that S. aureus must acclimate its metabolism to colder temperatures before growing in food. For this reason, we have performed a proteomic comparison between exponential- and stationary-phase cultures of S. aureus CECT 976 acclimated to 12 °C after growing at 25 °C or 37 °C. The analysis led to the identification of two different protein patterns associated with cold acclimation, denominated pattern A and pattern B. The first was characteristic of cultures at stationary phase of growth, grown at 25 °C and acclimated to 12 °C. The second appeared in the rest of experimental cases. Pattern A was distinguished by the presence of glycolytic proteins, whereas pattern B was differentiated by the presence of general stress and regulatory proteins. Pattern A was related through physiological experiments with a crossresistance to acid pH, whereas pattern B conferred resistance to nisin. This prompted us to conclude that both molecular strategies could be valid, in vivo, for the process of acclimation of S. aureus to cold temperatures. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Staphylococcus aureus is an opportunistic pathogen causing a wide spectrum of infections, ranging from mild to severe and lifethreatening (Gill et al., 2005; Lowy, 1998; Pamp et al., 2006). In addition, this organism is responsible for toxin-mediated diseases, such as toxic shock syndrome (TSS) and staphylococcal food poisoning (Projan and Novick, 1997; Murray, 2005). For humans, S. aureus is considered as one of the major etiologic agents of food poisoning (EFSA, 2009). The presence of S. aureus in food is generally the result of contamination either from carriers, mostly food handlers (Doyle et al., 2001), or from surfaces in the food environment, where this species has the potential to form biofilms (Herrera et al., 2007). Many foods are stored at low temperatures to preserve/improve quality and to extend shelf life. S. aureus cannot grow at refrigeration temperatures — below 4 °C, since it is a mesophile, but it can grow

⁎ Corresponding author. Eduardo Cabello, 6, Instituto de Investigaciones Marinas (CSIC), Spain. Tel.: + 34 986 231930; fax: +34 986 292762. E-mail address: [email protected] (M.L. Cabo). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.09.015

under temperature abuse conditions of greater than 10 °C by activating molecular mechanisms allowing low temperature acclimation (Holt et al., 1986). Growth of S. aureus in foods requires a drift upwards in refrigeration temperature. As such, the temperature at which S. aureus is initially living defines the gradient of temperature that it is subjected to, and this gradient can have an important effect on low temperature acclimation. It seems clear that physiological studies are needed to design strategies for the control of S. aureus (Kohler et al., 2005). However, studies have focused on pathogenicity (Bronner et al., 2004; Cheung et al., 2004; Novick, 2003), and only a few have addressed differences in protein expression of S. aureus due to biofilm formation (Resch et al., 2006; Bénard et al., 2008), oxidative stress (Wolf et al., 2008) or growth phase (Kohler et al., 2005). Furthermore, the conditions of growth and also the phase of growth can have some influence on the physico-chemical properties of the bacterial cell surface (such as hydrophobicity and surface charge) (Ells and Hansen, 2006). The aim of the present work thus was to analyze the changes in the proteomic signature associated with acclimation low temperature (12 °C) of S. aureus cells previously grown at 37 °C (human body temperature) and 25 °C (typical of some parts of food processing plants), and to correlate the S. aureus physiological state with its proteome fingerprint.

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2. Materials and methods 2.1. Bacteria and culture media Staphylococcus aureus subsp. aureus CECT 976 was obtained from the Spanish Type Culture Collection (Valencia, Spain). The strain was frozen in Triptone Soy Broth (TSB) (Panreac Química S.A., Spain) containing 50% glycerol (v/v) in cryotubes at −80 °C for long term storage. Whenever cells were required, a cryotube was removed and, after the cells were rapidly thawed by warming at 37 °C, they were grown and then subcultured in TSB at 37 °C before use. TSBG (TSB supplemented with 10 g/l of glucose) was used as the medium for the subsequent cultures. 2.2. Experimental design An 8 ml-aliquot of an overnight culture was added to 200 ml of TSBG. The culture was incubated under constant shaking (100 rpm) at 25 °C or 37 °C. When cultures reached exponential or stationary phase of growth, cells were harvested by centrifugation (5000 g, 10 min, 25 °C in a SIGMA 2-16PK centrifuge). In parallel, an 8 ml-aliquot was transferred to 200 ml TSBG and subcultured at 12 °C until the exponential phase of growth. When cultures reached exponential phase at 12 °C, cells were harvested as previously described. All the experiments were carried out in triplicate. Pellets from the cultures grown at 25 °C were divided into two parts. One was used for proteomic studies, whereas the other was used in parallel physiological studies in order to relate some of the proteomic findings at the physiological level. More concisely, we studied if cold acclimation could confer cross-resistance to osmotic stress, acid stress, benzalkonium chloride and nisin.

161

installed locally, and MOWSE score (Pappin, 2003), were used to identify proteins from peptide mass fingerprints, and annotations were made according to the cluster of orthologous genes (COG) functional groups. Enzyme Database BRENDA (http//www.brenda-enzymes. info) was used to identify the metabolic pathway of some of the proteins. A free on-line version of Mascot can be found at http://www. matrixscience.com. 2.4. Physiological assays The sensitivity to NaCl (osmotic stress), pH (acid stress), benzalkonium chloride (BAC) and nisin (chemical and natural biocide, respectively) were determined for exponential- and stationary-phase cultures grown at 25 °C, as well as for their corresponding cultures acclimated to 12 °C. Aliquots of TSB (100 μl) previously fixed at several pHs (2.5–5), or with different concentrations of NaCl (30–250 g/l), BAC (0.5–8 mg/l) or nisin (25–500 IU/ml) were added to 100 μl of a cell suspension in 0.05 M PBS (pH 7.2) adjusted to an absorbance at 700 nm of 0.100. This suspension contained approximately 107 CFU/ml. All assays were carried out in triplicate. Mixtures were incubated in microtiter plates at 25 °C during 24 h and then optical density was measured at 700 nm. Experimental data were adjusted to a modified logistic equation and subsequently lethal dose 50 and lethal dose 90 (LD50 and LD90), corresponding to those values of stress stimuli inhibiting 50 and 90% of the initial population, were determined as previously described in Cabo et al. (1999). Data were subjected to one-way analysis of variance with the SPSS 11.0 software (SPSS Inc., Chicago, IL) using the factor “phase of growth” with four categories: exponential phase (25 °C), stationary phase (25 °C), exponential phase (37 °C), and stationary phase (37 °C). 3. Results and discussion

2.3. Proteomic assays 2.3.1. Extraction of cell-free proteins for 2D electrophoresis Cells harvested at the different phases of growth were washed twice in 0.1 M Tris–HCl buffer, pH 7.5. Pellets were resuspended in 5 ml of 1 M Tris–HCl buffer, pH 7.5, and lysed through a Cell Disruptor (Constant Systems Ltd., Daventry, UK) using a pressure of 2.05 kbar. Unbroken cells and cell debris were removed by centrifugation (4500 g, 4 °C, 15 min) and membrane vesicles were discarded by ultracentrifugation (50,000 g, 4 °C, 20 min). The protein concentration was measured using the BCA protein assay kit (Pierce, Rockford, IL, USA) following the manufacturer's instructions. 2.3.2. Two-dimensional gel electrophoresis statistical analysis and protein identification Two-dimensional (2D) gel electrophoresis was carried out as previously described (Sánchez et al., 2005, 2007). Images from the gels were obtained and compared using an Image Scanner (Amersham Biosciences, Buckinghamshire, United Kingdom). Spots were detected using the Image Master 2D Platinum software (version 5.0; Amersham Biosciences and Geneva Bioinformatics S.A.). Volumes of each were calculated and normalized by referring values to the sum of total spot volumes within each gel. Gels were matched automatically, and groups of spots generated. Student's t-test for paired samples was applied in all cases. In the present work, we considered a protein to be under- or over-produced when the mean of at least four gels from independent cultures was 40% higher or lower than found for a comparison growth condition. It is noteworthy that under certain conditions differential spots appeared, that is, spots that were absent or present in any of the experimental conditions under comparison. Selected spots were excised from the gels and subjected to peptide mass fingerprinting by matrix assisted laser desorption ionizationtime-of-flight mass spectrometry (MS) analysis. Mascot Software

The present study aimed to examine the low temperature acclimation of S. aureus CECT 976 using a proteomic approach (Supplementary Figure 1). These temperatures are characteristic of humans and animals (37 °C) and some parts of the food processing plants (25 °C). In general these temperatures represent two of the most common situations that this bacterium could face in the food industry. Since cold acclimation may depend on the physiological state, this process was examined in both exponential- and stationary-phase cells previously grown at 37 °C and 25 °C. In this work, we have used S. aureus subsp. aureus CECT 976 (also denominated ATCC 13565 or FDA 196E), a variant derived from a strain that caused a food borne outbreak (Casman et al., 1963). Strain CECT 976 may lack for some of the global genetic regulators controlling pathogenesis, although little information is available. This strain, isolated in the laboratory, has been reported to not produce some enterotoxins, such as enterotoxin E, to other many strains from enteritis origin (Borja et al., 1972). 3.1. Cold acclimation of Staphylococcus aureus CECT 976. The importance of the physiological state Acclimation to 12 °C of exponential- and stationary-phase cells of S. aureus previously grown at 37 °C and 25 °C gave rise to two clearly different proteomic patterns (Fig. 1). Pattern A Pattern A resulted from cells acclimated to 12 °C that originally came from stationary-phase cultures at 25 °C (Fig. 1A). This pattern was similar to those of exponentialand stationary-phase cells cultured at 25 °C and 37 °C. Pattern B Pattern B resulted from cells acclimated to 12 °C that originally came from exponential-phase cultures at 25 °C or from cultures at 37 °C either in exponential or stationary phase (Fig. 1B). In these three cases, a different pool of proteins was synthesized for cold acclimation.

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A

Isoelectric Point 4

5

6

7

6

7

kDa 100 SA51

75

SA60

SA76 SA44

SA62

SA72 SA36

50

SA74 SA57 SA42

SA115

SA53 SA41

SA54

SA158

Molecular Mass

SA37

37

SA61 SA62

SA93 SA90 SA77

SA70

25 SA72

B

Isoelectric Point kDa 4 100

5 SA9 SA32

75

SA19 SA14

50

SA7

SA28 SA18

SA30 SA33 SA133

Molecular Mass

37

25 SA165

SA167

SA3

Fig. 1. Protein patterns (A, upper panel and B, lower panel) that were obtained in the different conditions in which S. aureus CECT 976 cultures were analysed. Pattern A corresponded to the following conditions: stationary-phase cultures at 25 °C acclimated to 12 °C, and exponential- and stationary-phase cells cultured at 25 °C and 37 °C. Pattern B was observed in exponential-phase cultures grown at 25 °C or at 37 °C, either in exponential or stationary phase, acclimated to 12 °C. Proteins characteristic from each pattern are labelled.

B. Sánchez et al. / International Journal of Food Microbiology 144 (2010) 160–168

Spots characteristic of each pattern were excised from gels and proteins were identified by peptide mass fingerprinting. As a whole, a total of 24 proteins were identified for pattern A, and 13 for pattern B (Tables 1 and 2, respectively). Half of the proteins of pattern A were related to carbohydrate metabolism (glycolysis, pentose phosphate pathway and tricarboxylic acid cycle). In this way, phosphoglycerate mutase, a protein specific from pattern A, was observed to be induced by cold shock using DNA microarray as described in previous work (Anderson et al., 2006). Ribosomal proteins and elongation factors, which are involved in protein synthesis, were also characteristic of pattern A, as well as some stress proteins. Among them, the chaperone protein HchA (SA37), which is related to the constitutive stressresponse of stationary-phase cells (Mujacic and Baneyx, 2006), a protein similar to the universal stress protein family Mu50 from S. aureus (SA71), and the alkaline shock protein 23 (Asp23, SA48). The latter was up-regulated as a consequence of cold acclimation. Additionally, molecular chaperones, such as CspB, have been already observed to be induced by cold shock in S. aureus (Anderson et al., 2006). Therefore, pattern A corresponded to the main metabolic pathways of S. aureus, and could be representative of the physiological state of the bacteria under optimal growth conditions. In this sense, several transcriptional factors and regulators were also shown to be over-expressed in S. aureus after cold shock, suggesting that this species undergoes certain changes in its genetic regulation as a result of decreases in the temperature (Anderson et al., 2006). In contrast to pattern A, phosphopyruvate hydratase (SA18) was the only glycolytic protein identified among the proteins present in pattern B. This pattern was characterized by the presence of several stress proteins, such as the cold shock like protein CspD (SA9), the 65 kDa heat shock protein Hsp65 (SA28) and the co-chaperonin GroES (SA3), which are involved in the general response of bacteria to environmental stresses (Cailler et al., 2008). In this sense, some proteins involved in the regulation of metabolic pathways, such as the RNA binding S1 domain protein (SA19) or the transcription factor CarD (SA167), were also expressed.

163

A particularly interesting protein in pattern B was 1-L-myo-inositol1-phosphate synthase (SA33). This enzyme catalyzes the conversion of D-glucose 6-phosphate to 1-L-myo-inositol-1-phosphate, which is the first and rate-limiting step in the biosynthesis of inositol-containing compounds, including phospholipids (Majunder et al., 1997). The presence of this enzyme in higher amounts pointed to variations of lipid metabolism as a result of cold acclimation, which can give rise to changes in the composition of membrane phospholipids. It seems that in the three cases associated with pattern B acclimation to 12 °C of S. aureus cells coming from cultures grown at 25 °C (exponential phase) or 37 °C (both exponential and stationary phase of growth) induced a shift in bacterial metabolism. This shift implies a change from a typical glycolytic configuration to a protein arrangement in which proteins involved in stress response, and likely the biosynthesis of new phospholipids, were present. The cold and heat shock responses in S. aureus have been characterized using DNA microarrays. Globally, changes in the optimal growth temperature of the bacterium have been related to deep metabolic reorganizations, including genes involved in carbohydrate, amino acid metabolism and genetic regulators (Anderson et al., 2006; Fleury et al., 2009). 3.2. Cold acclimation of stationary-phase cells of S. aureus grown at 25 °C Acclimation of S. aureus to low temperature proceeded in a particular way in the case of stationary-phase cells grown at 25 °C. As shown in Fig. 2 and Table 3, a total of 19 proteins showed different levels of expression as a result of acclimation at 12 °C in these experimental conditions. Out of these, 5 proteins were down-regulated, whereas 14 proteins were up-regulated. Interestingly, three of the upregulated proteins are involved in the pentose phosphate pathway, namely transketolase (SA50), transaldolase (SA64) and enolase (SA45). Pyruvate dehydrogenase, which converts pyruvate to acetyl CoA, was also up-regulated, as well as superoxide dismutase (SOD) (SA66) and ferritin (SA67). These two latter proteins have roles in

Table 1 Identification of Staphylococcus aureus 976 proteins only expressed in Pattern A. COG funcional groupa

Spot no.

Putative function

MMb

pIc

No. of peptides matchedd

Coveragee (%)

Carbohydrate metabolism

SA38 SA43 SA45 SA50 SA63 SA150 SA56 SA36 SA64 SA47 SA65 SA67 SA48 SA51 SA75 SA49

Fructose-bisphosphate aldolase Phosphoglycerate mutase Enolase Transketolase Triosephosphate isomerase Phosphoglyceromutase Glucose-6-phosphate 1-dehydrogenase GapC Transaldolase Trigger factor Alkyl hydroperoxide reductase subunit C Ferritin Alkaline shock protein 23 ATP-dependent Clp proteinase chain Pyruvate dehydrogenase E1 component alpha subunit Translation elongation factor G:Small GTP-binding protein domain Aspartyl/glutamyl-tRNA amidotransferase subunit B Elongation factor Ts 30 S ribosomal protein S1 Similar to universal stress protein family Cysteine synthase (o-acetylserine sulfhydrylase) homolog Alpha-acetolactate synthase Hypothetical protein Hypothetical protein MW1656

30,931 26,721 47,145 72,206 27,416 56,446 57,043 36,341 25,803 48,579 21,135 19,633 19,180 77,924 41,357 72,977

5.01 5.23 4.55 4.97 4.80 4.74 5.31 4.89 4.72 4.34 4.88 4.67 5.13 4.83 4.9 4.74

9 12 8 18 5 16 7 5 18 14 9 8 8 22 4 10

33 57 25 39 21 31 17 18 75 37 50 46 65 40 10 24

53,680 32,588 43,283 18,521 33,012 67,788 31,734 18,108

5.04 5.05 4.55 5.60 5.39 4.79 5.53 4.56

23 15 12 6 12 16 9 10

60 64 55 50 60 42 34 76

Posttranslational modification, protein turnover, chaperones

Energy production and conversion Translation, ribosomal structure and biogenesis

Signal transduction mechanisms Amino acid transport and metabolism Coenzyme metabolism Unclassified Unclassified a b c d e

SA59 SA40 SA46 SA71 SA39 SA79 SA155 SA161

Cluster of orthologous genes. Theoretical molecular mass. Theoretical isoelectric point. Number of peptides matched that allowed protein identification by peptide fingerprinting. Sequence coverage.

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Table 2 Identification of Staphylococcus aureus 976 proteins only expressed in Pattern B. COG funcional groupa

Spot no.

Putative function

MMb

pIc

No. of peptides matchedd

Coveragee (%)

Carbohydrate metabolism

SA18 SA37 SA33 SA165 SA9 SA167 SA133 SA7 SA14

Fructose-bisphosphate aldolase Chaperone protein HchA Putative 1L-myo-inositol-1-phosphate synthase Single-strand DNA-binding protein Cold shock-like protein CspD Transcription factor CarD K+ channel beta subunit Elongation factor TU Glycyl-tRNA synthetase [marine actinobacterium PHSC20C1] RNA binding S1 domain protein rpsB (ribosomal protein) Polyribonucleotide nucleotidyltransferase Co-chaperonin GroES 65 kDa heat shock protein

30,931 32,270 39,380 – 7594 – – 22,877 52,824

5.01 4.89 4.96 – 8.15 – – 5.03 5.29

9 2 2 – 1 – – 2 5

33 6 4 – 14 – – 15 9

53,717 26,574 79,143 10,338 14,709

4.59 6.88 4.84 4.77 4.45

9 1 7 3 3

17 5 12 45 30

Lipid transport and metabolism DNA replication, recombination and repair Transcription Signal transduction mechanisms Translation, ribosomal structure and biogenesis

Posttranslational modification, protein turnover, chaperones a b c d e

SA19 SA30 SA32 SA3 SA28

Cluster of orthologous genes. Theoretical molecular mass. Theoretical isoelectric point. Number of peptides matched that allowed protein identification by peptide fingerprinting. Sequence coverage.

oxidative stress response, protecting from the deleterious effects of oxygen radicals (Bucker and Martin, 1981; Hebraud and Guzzo, 2000). Interestingly, immunodominant staphylococcal antigen A (IsaA) (SA160), detected as a pre-protein in our experimental conditions, was also up-regulated after cold acclimation. IsaA is a surface protein associated with virulence, which acts as a strong antigen in episodes of human sepsis caused by S. aureus, particularly by methicillinresistant strains (Lorenz et al., 2000). On the contrary, six proteins were down-regulated. Down-regulation of L-lactate dehydrogenase (SA68) and phosphoglycerate mutase (SA43) likely indicated a slowdown of pyruvate metabolism. Alkaline shock protein 23 (Asp23, SA48) was also down-regulated. This result is likely associated with the type of stress response. In this respect, a drastic reduction in the level of this protein in S. aureus subjected to an upshift of pH (up to 10.0) has been shown (Kuroda et al., 1995). In contrast, the content of Asp23 was higher in 48-h biofilms of S. aureus than in planktonic cells (Resch et al., 2006). This protein and some regulators of virulence factors are controlled by the alternative sigma factor σB (Kullik et al., 1998), which regulates the stress response of several Gram-positive bacteria. In short, in addition to modifications in stress-associated proteins, cold acclimation of stationary-phase cells of S. aureus previously grown at 25 °C seems to be enabled by a shift in metabolism from a primarily glycolytic energy metabolism toward the pentose phosphate pathway, together with a modulation of lipid metabolism. This result thus appears to be related to the specific metabolic activity of stationary-phase S. aureus grown at 25 °C. 3.3. Differential protein expression analysis between stationary-phase cells of S. aureus grown at 25 °C and 37 °C Stationary-phase cells of S. aureus grown at 25 °C and 37 °C showed a similar proteomic pattern, which corresponded to pattern A. As discussed, only cultures acclimated to 12 °C coming from stationaryphase cultures grown at 25 °C maintained pattern A. With the aim of identifying proteins responsible for this trait, a comparative analysis of differential protein expression between stationary-phase cells of cultures at 25 °C and 37 °C was carried out. As a result, it was observed that 6-phosphofructokinase was only present in stationary-phase cells cultured at 37 °C. This enzyme catalyzes the interconversion of β-D-fructose 6-phosphate to β-Dfructose 1,6-diphosphate. The absence, or low expression, of 6phosphofructokinase at 25 °C is indicative that, in this situation, the

carbohydrate metabolism shifts toward the pentose phosphate pathway — with respect to growth at 37 °C. Further, cold acclimation of stationary-phase cells grown at 25 °C leads to up-regulation of the pentose phosphate pathway, as discussed previously. It has been shown that fructose diphosphate aldolase and glucose 6-phosphate isomerase, both enzymes of the pentose phosphate pathway, were up-regulated in response to acid stress (Bore et al., 2007). Channelling the metabolism toward the pentose phosphate pathway at 25 °C seems to be physiologically advantageous for cold acclimation of stationary-phase S. aureus. This can be inferred by comparing in Fig. 3 the growth kinetics at 12 °C of each of the four cases under study. It can be seen that the lag phase was really short for cells expressing pattern A, whereas it was extended to 200 h for cells coming from exponential-phase cultures at 25 °C or 37 °C, and to 300 h for cells coming from stationary-phase cultures at 37 °C. It seems that shifting metabolism toward the pentose phosphate pathway at the stationary-phase of growth is energetically favourable for cold acclimation. This shift is probably linked to the fact that the gradient of temperature is lower than for stationary-phase cells cultured at 37 °C. The kinetics of growth of S. aureus CECT 976 at 12 °C have shown the relevance for the food industry of the ecological niche from which the strain comes. However, further studies should elucidate whether other strains of S. aureus also follow different “cold” acclimation metabolic pathways depending on their physiological state. The kinetics of growth also showed that low temperature acclimation of S. aureus CECT 976 was faster when cells were in stationary-phase. Additionally, stationary-phase cells showed a resistance to nisin and acid significantly higher than exponential-phase cells, and this could also enhance the growth of S. aureus in dairy products and other nisincontaining foods, as well as in acid food products. At present, further studies are intended to determine if this metabolic strategy is followed by other strains of S. aureus, and whether it is used by S. aureus to acclimate to other stress conditions. 3.4. Physiological comparisons in cold-acclimated cells Responses of both types of cold-acclimated cells to different stress stimuli were examined to find out possible physiological consequences of the expression of patterns A and B. Acid and osmotic stresses were selected as examples of stimuli provoking some metabolic response, whereas BAC and nisin were taken as representative of stresses that could cause modifications in cell membrane. Cells were collected in both exponential and stationary phase of

B. Sánchez et al. / International Journal of Food Microbiology 144 (2010) 160–168

A

165

Isoelectric Point 4

5

6

7

kDa 100 75

SA50

50 SA45

SA68 SA75 SA152

Molecular Mass

37

SA155

SA52

SA43

SA64

SA160

SA66 SA65

SA48

SA67

25

SA95

SA71 SA163

SA161 SA162

SA92

B

Isoelectric Point 4

5

6

7

kDa 100 75 SA50

50

SA45

SA75

37

Molecular Mass

SA152

SA155 SA43 SA64

SA160

SA65

25

SA66

SA48 SA67 SA161

SA195

SA71 SA163

SA92

SA162

Fig. 2. A, protein profile of stationary-phase S. aureus CECT 976 cells grown at 25 °C. B, protein profiles obtained after cold acclimation to 12 °C S. aureus CECT 976 cells previously grown at 25 °C to stationary-phase.

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Table 3 Identification of proteins from Staphylococcus aureus 976 stationary-phase cells affected by the cold acclimation at 25 °C. COG funcional groupa

Spot no.

Putative function

MMb

pIc

No. of peptides matchedd

Coveragee (%)

25 °C

12 °C

Carbohydrate metabolism

SA50 SA45 SA64 SA152 SA75

72,206 47,145 25,803 36,341 41,357

4.97 4.55 4.72 4.89 4.9

18 8 18 4 4

39 25 75 10 10

a a a a a

aa aa aa aa aa

SA162

Transketolase Enolase Transaldolase GapC Pyruvate dehydrogenase E1 component alpha subunit Elongation factor TU

27,299

4.49

1

6

a

aa

SA66 SA65 SA67 SA163

Superoxide dismutase Alkyl hydroperoxide reductase subunit C Ferritin Signal transduction protein

22,697 21,135 19,633 19,953

5.08 4.88 4.67 6.12

7 9 8 13

56 50 46 58

a a a a

aa aa aa aa

SA155 SA160 SA95 SA161 SA43 SA68 SA48

Hypothetical protein Immunodominant antigen A Hypothetical protein MW1957 Hypothetical protein MW1656 Phosphoglycerate mutase L-lactate dehydrogenase Alkaline shock protein 23

31,734 24,189 23,989

5.53 5.9 5.27

9 3 13

34 19 53

26,721 34,456 19,180

5.23 4.58 5.13

12 13 8

57 47 65

a a a a aa aaaa aa

aa aaaa aaaa aaaa a a a

SA71 SA92

Similar to universal stress protein family Methionine sulfoxide reductase B

18,521 16,367

5.6 4.76

6 6

50 50

aaaa aa

a a

Energy production and conversion Translation, ribosomal structure and biogenesis Inorganic ion transport and metabolism Posttranslational modification, protein turnover, chaperones Signal transduction mechanisms Unclassified

Carbohydrate metabolism Energy production and conversion Posttranslational modification, protein turnover, chaperones Signal transduction mechanisms Unclassified

aa: protein expression between 1.4 and 4 times relative to a. aaaa: protein expression ≥ 4 times relative to a. a Cluster of orthologous genes. b Theoretical molecular mass. c Theoretical isoelectric point. d Number of peptides matched that allowed protein identification by peptide fingerprinting. e Sequence coverage.

3

(A)

37oC

Abs 7OO nm

(A)

0 80 120 160 200 time (hours)

time (hours)

1

time (hours)

500

400

700

(B)

0

600

(B)

0

300

250

200

150

50

100

0

300

250

200

150

100

0

50

1 0,5 0

0

time (hours)

Abs 7OO nm

(A) 1

12oC

1,5

600

0

2 12oC

200

0,5

80

Stationary phase

2 12oC

Abs 7OO nm

(B)

1

20 40 60 time (hours)

Exponential phase

2 12oC

Abs at 7OO nm

Abs at 7OO nm

2

0

300

Stationary phase

0

200

Exponential phase

0

40

100

0

(A)

1

400

1

1,5

(A)

2

2

100

25oC

3

500

4

A) showed a higher resistance to nisin (Fig. 4). As already discussed, pattern A is very similar to the protein patterns of exponential- and stationary-phase cultures, and are supposed to be representative of more active cultures from a metabolic point of view, due mainly to the abundance of glycolytic enzymes. This high metabolic activity may provide cells with the energy equivalents necessary for a more efficient counteracting of the bacteriostatic activity of nisin. Lastly, neither pattern A nor B, conferred cross-resistance to osmotic stress and to

300

Abs 7OO nm

growth from the cultures at 25 °C, and comparisons were established by analyzing the corresponding response of cultures acclimated to 12 °C. A significantly higher resistance (pb 0.05) against acid stress was found in cells expressing pattern B with respect to the other cases (Fig. 4). This could be related to the over-production of molecular chaperones (Cotter and Hill, 2003). On the contrary, cells acclimated to 12 °C originally coming from stationary-phase cultures at 25 °C (pattern

time (hours)

Fig. 3. Growth kinetics of S. aureus CECT 976 under the conditions assayed. Circles represent at which time samples were taken for analysis. The protein profile corresponding to each experimental condition is shown between brackets.

B. Sánchez et al. / International Journal of Food Microbiology 144 (2010) 160–168

100

ID50 pH

ID50 NaCl (g/l)

* 150

6

300

5

5

250

4

*

3 2

50 0

ID50 NISIN (UI/ml)

*

200

6

ID50 BAC (mg/l)

250

167

4 3 2

200 150 100

1

1

50

0

0

0

25oC-EX.

25oC-ST.

12oC-EX.

*

25oC-ST

Fig. 4. Comparison between the resistance (ID50) to different stresses of S. aureus CECT 976 cells expressing Pattern A at 25 °C and 12 °C. 25 °C-Ex: cultures grown at 25 °C at exponential phase of growth; 25 °C-ST: cultures grown at 25 °C at stationary phase of growth; 12 °C-EX: cultures grown at 12 °C at exponential phase from cells grown at 25 °C at exponential phase; 12 °C-ST: cultures grown at 12 °C at exponential phase from cells grown at 25 °C at stationary phase of growth. Vertical lines on the bars represent standard errors. Asterisks indicate significant differences after ANOVA analysis (*, p b 0.05).

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