Journal of Microbiological Methods 107 (2014) 74–79

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Application of zwitterionic detergent to the solubilization of Klebsiella pneumoniae outer membrane proteins for two-dimensional gel electrophoresis I. Bednarz-Misa a,⁎, P. Serek a, B. Dudek b, A. Pawlak b, G. Bugla-Płoskońska b, A. Gamian a,c a b c

Department of Medical Biochemistry, Wroclaw Medical University, Chałubińskiego 10, 50-368 Wrocalaw, Poland Department of Microbiology, Institute of Genetics and Microbiology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wroclaw, Poland

a r t i c l e

i n f o

Article history: Received 14 July 2014 Received in revised form 17 September 2014 Accepted 17 September 2014 Available online 28 September 2014 Keywords: Klebsiella pneumoniae Outer membrane proteins Two-dimensional electrophoresis Zwitterionic detergent

a b s t r a c t Klebsiella pneumoniae is a frequent cause of nosocomial respiratory, urinary and gastrointestinal tract infections and septicemia with the multidrug-resistant K. pneumoniae being a major public health concern. Outer membrane proteins (OMPs) are important virulence factors responsible for the appropriate adaptation to the host environment. They constitute of the antigens being the first in contact with infected organism. However, K. pneumoniae strains are heavily capsulated and it is important to establish the OMPs isolation procedure prior to proteomics extensive studies. In this study we used Zwittergent Z 3-14® as a detergent to isolate the OMPs from K. pneumoniae cells and resolve them using two-dimensional electrophoresis (2-DE). As a result we identified 134 protein spots. The OMPs identified in this study are possible candidates for the development of a protein-based vaccine against K. pneumoniae infections. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Klebsiella pneumoniae is an opportunistic pathogen which causes many nosocomial infections including the most common urinary tract infections, pneumonia and septicemia especially in immunocompromised patients. Recent investigations connect K. pneumoniae infections with inflammatory bowel diseases or liver abscesses. Also, they rank them second to Escherichia coli cause of the bacteremia due to biliary infection (Ebringer et al., 2007; ECDC Surveillance report, 2007; Yang et al., 2009; Lee et al., 2011; Ortega et al., 2012). K. pneumoniae is (among ten) the most frequently detected pathogen in infections related to hospitalization (Hidron et al., 2008). Despite the use of appropriate antibiotic therapy, the morbidity and mortality due to K. pneumoniae remain extremely high, with mortality rates up to 60% (Coovadia et al., 1992; Sahly and Podschun, 1997; Yadav et al., 2005). Moreover, multiresistant strains are frequently isolated, emphasizing the need to find new ways to prevent and treat K. pneumoniae infections (Lytsy et al., 2008; Chagas et al., 2011). Unfortunately, no vaccine capable of preventing infection by K. pneumoniae has been developed yet. Among the different cell structures, surface components are being discussed as candidates for vaccine: siderophores, exotoxins, lipopolysaccharides (LPS) and capsular polysaccharides (CPS) (Podschun and Ullmann 1998; Yadav et al. 2005). However, the LPS released from the ⁎ Corresponding author. E-mail address: [email protected] (I. Bednarz-Misa).

http://dx.doi.org/10.1016/j.mimet.2014.09.004 0167-7012/© 2014 Elsevier B.V. All rights reserved.

bacterial cells may cause renal scaring, even after one episode of bacterial infection (Cryz et al., 1984). The best characterized virulence factor of K. pneumoniae is CPS, which protects bacteria from complement attack and antimicrobial peptide-mediated killing (Campos et al., 2004; Moranta et al., 2010). Also, bacterial CPS prevents phagocytosis by host cells (Athamna et al., 1991). Most of the studies have been focused on the role of LPS and CPS in pathogenicity of K. pneumoniae. LPS has nine different O-type antigens, but its possible application for vaccination is hampered by high toxicity during immunization. CPS, in turn, has proved to be highly immunogenic and non-toxic. However, it has a large variety of K-type antigens (77 different) and hence requiring the application of multivalent vaccines. A vaccine with 24 different K-types has been reported, but there is no information of any further development (Cryz et al., 1986; Yadav et al. 2005). Due to the disadvantages of these antigens as vaccine components, research has been directed toward more conservative proteins, such as extracellular toxins, outer membrane proteins (OMPs) or fimbriae proteins (Klipstein et al., 1983; Goetsch et al., 2001; Lavender et al., 2005; Struve et al., 2009). Of these, FepA, OmpA OmpK17, OmpK36 and colicin have been selected as potential vaccine candidates (Lai et al., 2001; Struve et al., 2003; Kurupati et al., 2006). Positive results have been reported for a DNA vaccine based on outer membrane proteins in a study conducted on a mouse model (Kurupati et al., 2011). Data supporting the hypothesis on surface-related proteins being involved in bacterial pathogenesis, for example in adhesion, invasion and protection against host immune response, has been recently

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accumulated. Proteins attached to the outer membrane are important for membrane integrity and provide transport of molecules from the environment (Lin et al., 2002). Also OMPs are receptors for bacteriophages and play a key role in signal transduction, intracellular transport, and energy transformation processes ensuring proper bacterial cell functioning (Navarre and Schneewind, 1999; Cabanes et al., 2002; Fluegge et al., 2004; Maione et al., 2005). Therefore, identifying novel OMPs may aid research on new strategies of preventing colonization and infection with K. pneumoniae. OMPs are hard to purify and solubilize for general analysis due to their hydrophobic nature. They are often associated with other substances such as membrane lipids. In this investigation we used Zwittergent Z 3-14® as an effective detergent to isolate the OMPs of K. pneumoniae and resolve them using two-dimensional electrophoresis (2-DE). The Zwittergent 3-14® has already been used for isolation of OMPs from a number of bacterial species (Kokeguchi et al., 1991; Tagawa et al., 1993; Kokeguchi et al., 1994; Pal et al., 1997; Halling and Koster, 2001; Gatto et al., 2002; Siritapetawee et al., 2004; Augustyniak et al., 2010; Bugla-Płoskońska et al., 2009; Bugla-Płoskońska et al., 2011; Futoma-Kołoch et al., 2009; Zhang et al., 2011), but its application for OMPs of K. pneumoniae has not been tried yet. Rationale of these studies on Klebsiella was thick capsule produced by these strains and the need to establish the 2-DE conditions prior to proteomics extensive studies. 2. Material and methods 2.1. Bacterial strains and growth conditions K. pneumoniae strain PCM 2713 from the Polish Collection of Microorganisms (PCM) at Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Science, Wroclaw, Poland was selected for this study. Culture was grown for 18 h at 37 °C, under shaking (200 rpm) in 50 ml of Brain Heart Infusion Broth (Difco). 2.2. Isolation of outer membrane proteins Outer membrane proteins were isolated using Murphy and Bartos' method (1989) with minor modifications (Bugla-Płoskońska et al., 2009; Futoma-Kołoch et al., 2009; Bugla-Płoskońska et al., 2011). Overnight cultures were centrifuged (4000 rpm, 4 °C, 20 min) and bacteria were resuspended in 1.25 ml of 1 M sodium acetate (POCH) with 1 mM β-mercaptoethanol (Merck). Subsequently, 11.25 ml of 5% (w/v) Zwittergent Z 3-14® (Calbiochem) and 0.5 M CaCl2 (POCh) in water were added and stirred for 1 h at room temperature (RT). To precipitate nucleic acids, 3.13 ml of 96% (v/v) cold ethanol (POCH) was added very slowly and centrifuged (12,300 rpm, 4 °C, 10 min). Proteins were precipitated from supernatant after centrifugation (12,300 rpm, 4 °C, 10 min) by addition of 46.75 ml of 96% (v/v) cold ethanol. Pellet was dried at RT, then suspended in 2.5 ml of 50 mM Trizma-Base (SigmaAldrich) buffer, pH 8.0 with 0.05% (w/v) Zwittergent Z 3-14® and 10 mM EDTA (Sigma-Aldrich), stirred for 1 h at RT and subsequently kept at 4 °C overnight. Insoluble material was removed from OMPs solution by centrifugation (8700 rpm, 4 °C, 10 min). Protein content was quantified by BCA Protein Assay according to Smith et al. (1985) with bovine serum albumin (BSA) (Thermo Scientific) as the standard. Prior to 2-DE, OMPs preparations were pretreated with ReadyPrep™ 2-D Cleanup Kit (Bio-Rad) according to the manufacturer's instructions to reduce interference with substances contaminating samples for isoelectric focusing (IEF) and 2-DE. To assure that soluble fractions of buffer Z are not contaminated with cytosolic proteins, the enzymatic activity of succinic dehydrogenase (marker of cytoplasmic proteins) was checked in previous preparations of OMPs from E. coli and Salmonella spp. (Bugla-Płoskońska et al., 2009;

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Futoma-Kołoch et al., 2009; Bugla-Płoskońska et al., 2011) using Rockwood et al.'s method (1987) and the result was negative. 2.3. Two-dimensional electrophoresis The OMPs from K. pneumoniae were separated with 3–10 immobilized gradient IPG strips and 4–7 pH immobilized gradient IPG strips (7 cm) (Bio-Rad). 2-DE was carried out with the MiniPROTEAN® Tetra Cell System (Bio-Rad). The main reagents for 2-DE were purchased from Bio-Rad and used according to the manufacturer's instructions (Bio-Rad). Prior to the first dimension, precast IPG strips were rehydrated with 120 μl rehydration buffer (Bio-Rad), 15 μg of proteins and 3 μl of proteome marker for 2-DE (Serva) (16 h, RT). The rehydrated strips were positioned in the focusing tray and covered with 2 ml of mineral oil (Bio-Rad) to prevent evaporation. Isoelectric focusing (IFE) was conducted by stepwise increase of the voltage as follows: 250 V, 20 min, 4000 V, 120 min (linear) and 4000 V (rapid) until the total volt-hours reached 14 kVh. After IEF separation, the strips were equilibrated in 6 M urea, 0.375 M Tris, 2% SDS, and pH 8.8, reduced with 2% (w/v) DTT and alkylated with 0.135 M iodoacetamide. IPG strips were then loaded onto the top of a 9–12.5% gradient polyacrylamide gel (10 × 8 cm, 1.0 mm thick) using 0.5% agarose (Bio-Rad) in the running buffer. Molecular mass standards — Precision Plus Protein™ Standards (Bio-Rad) were applied at the basic end of the IPG strips. Modified Laemmli (1970) system with tricine (Calbiochem) instead of glycine in the electrophoresis buffer (50 mM tricine, 0.1% (w/v) sodium dodecyl sulfate, pH 8.2) was used as previously described (Bugla-Płoskońska et al., 2009). Electrophoresis was performed at 4 °C with constant power (1 W) until the dye front reached the bottom. Following separation in the second dimension, the gels were stained using Gromova and Celis' method (2006). Spot patterns were visualized under white light and photographed using a GelDoc XR camera system (Bio-Rad). Spots of proteins were analyzed by PDQuest software (Bio-Rad). To create master gel three repetitions of 2-DE procedures were performed. Master or reference gel is a virtual representative profile for match-set among the sample gels. The master gel summarizes all identified spots and serves as the basic image. Molecular masses of analyzed proteins were calculated by comparing with that of molecular mass markers for 2-DE and the pI values were calculated according to the linearity of IPG strips using the software. 3. Results and discussion In this investigation we used Zwittergent Z 3-14® as an effective detergent to isolate the OMPs of K. pneumoniae and resolve them using two-dimensional electrophoresis. OMPs from K. pneumoniae were separated by 2-DE in two pH ranges: 3–10 and 4–7 (Figs. 1A and 2A). More accurate separation was achieved when pH 4–7 IPG strips were used. Three replicates of 2-DE gels were stained for this strain and showed similar results. After 2-DE, silver stained gels revealed about 85 spots within the 3–10 range and 134 spots within the 4–7 pH range (Figs. 1B and 2B). Most of the proteins had molecular mass between 25 and 50 kDa. The pI and mass of spots are shown in Table 1. In order to obtain the highest concentration and the best quality of proteins that will promote the likelihood of identifying as many proteins in 2-DE as possible, another method was employed, including isolations using Triton X-100. However, this method yielded poor quality of separation (Figs. 3A, B and 4A, B). Isolation by Zwittergent Z 3-14® produced substantially higher concentration and better quality of protein spots and was therefore selected for use in this study to obtain the soluble and insoluble bacterial outer membrane proteins for analysis. Zwittergent Z 3-14® was successfully applied for highly efficient isolation of OMPs from Neisseria gonorrhoeae (Blake and Gotschlich, 1984),

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Fig. 1. A. Two-dimensional electrophoresis (2-DE) gel profiles of outer membrane proteins of Klebsiella pneumoniae. Separation of total proteins (15 μg) was extracted from bacteria with Zwittergent Z 3-14® on 2-DE over the pH range of 3–10 (silver stained). Resolution of isoelectric focusing within the specified pH range was insufficient. B. Master gel profiles of outer membrane proteins of Klebsiella pneumoniae were isolated with the use of Zwittergent Z 3-14® after two-dimensional electrophoresis (2-DE) over the pH range of 3–10. 85 protein spots were identified.

Legionella pneumophila (Gabay et al., 1985), Haemophilus influenzae (Kyd et al., 1994), Pseudomonas aeruginosa (Carnoy et al., 1994), E. coli (Bugla-Płoskońska et al., 2009), and Salmonella (Futoma-Kołoch et al., 2009). However, there are no studies describing the use of zwitterionic detergents in OMPs isolation from K. pneumoniae, for which sodium lauroyl sarcosinate had previously been used (Albertí et al., 1995; Hernández-Allésa et al., 2000). It is well known that Klebsiella strains are heavily capsulated, thus it was important to perform a separate study on usefulness of that method for strain of this genus. In contrast to other amphoteric surfactants, synthetic zwitterionic detergent maintains its unique active amphoteric character at a wide pH range due to the presence of a strongly basic ammonium ion and equally strong sulfate ion. This balance probably prevents irreversible binding of detergent to other anionic or cationic compounds. Micelles formed by detergent are analogous to the bilayer of biological membranes, through hydrophobic interactions. This detergent lacks conductivity and electrophoretic mobility and it is not bound to the ion exchange resin that is an advantageous feature during protein purification process (Bhairi and Mohan, 2001; McLachlan et al., 2010). 2-DE methodology has a potential as a new way to develop the research of specific proteins for vaccines against infections caused by K. pneumoniae in humans. 2-DE methods have been already used for separation of outer membrane proteins in Enterobacteriaceae: E. coli (Molloy et al., 2000), Salmonella typhimurium (Hamid and Jain, 2008), Edwardsiella tarda (Kawai et al., 2004), and Shigella flexneri (Peng

Fig. 2. A. Two-dimensional electrophoresis (2-DE) gel profiles of Klebsiella pneumoniae outer membrane proteins. Separation of total proteins (15 μg) was extracted from bacteria with Zwittergent Z 3-14® on 2-DE over the pH range of 4–7 (silver stained). B. Master gel profiles of outer membrane proteins of Klebsiella pneumoniae after two-dimensional electrophoresis (2-DE) were extracted from bacteria with Zwittergent Z 3-14®. 134 protein spots were identified. C. Master gel profiles of outer membrane proteins of Klebsiella pneumoniae were extracted with Zwittergent Z 3-14® after two-dimensional electrophoresis (2-DE) over the pH range of 4–7. Numbers of spots with Mr and pI are given in Table 1.

et al., 2004) and bacteria from families other than Enterobacteriaceae: Leptospira interrogans (Cullen et al., 2002), Riemerella anatipestifer (Hu et al., 2012), and Campylobacter jejuni (Zhang et al., 2013). However, sodium carbonate, urea-CHAPS, sodium lauroyl sarcosinate, Triton X-100, and Triton X-114 have been applied for OMPs isolation. These detergents can be used to OMPs isolation but they differ in their

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Table 1 Mr and pI values for all identified spots on master gel profiles (Fig. 2C) of outer membrane proteins of Klebsiella pneumoniae after two-dimensional electrophoresis (2-DE). Spot number

Mr

pI

Spot number

Mr

pI

Spot number

Mr

pI

1 101 103 105 106 108 203 204 402 601 702 703 1003 1102 1104 1401 1502 1603 1703 1705 1706 1707 1806 2002 2003 2101 2102 2201 2301 2402 2403 2404 2502 2503 2603 2606 2607 2608 2609 2610 2701 3002 3003

10.97 19.72 20.88 19.59 21.01 18.82 23.68 23.87 32.00 44.74 55.89 56.16 12.57 20.85 18.40 32.28 39.97 44.84 50.53 61.62 61.68 61.67 77.00 12.90 13.14 20.62 20.93 22.21 25.97 32.55 32.46 38.07 39.97 39.80 44.78 46.56 44.87 42.19 42.41 44.54 50.23 15.46 13.36

5.20 5.34 5.38 5.40 5.43 5.45 5.38 5.34 5.40 5.45 5.40 5.36 5.52 5.48 5.49 5.53 5.51 5.48 5.51 5.50 5.47 5.46 5.50 5.70 5.61 5.57 5.70 5.72 5.62 5.71 5.62 5.72 5.68 5.59 5.61 5.69 5.53 5.56 5.65 5.69 5.57 5.87 5.86

3202 3402 3403 3404 3405 3501 3502 3601 3603 3701 3702 3801 3803 3901 3903 4001 4002 4103 4104 4201 4203 4401 4402 4501 4502 4601 4602 4603 4701 4702 4703 4801 4802 4803 4804 4805 4902 4903 5001 5105 5201 5202 5203

21.42 37.98 32.44 36.85 34.33 40.07 40.15 42.24 46.68 65.72 66.01 81.27 81.16 89.99 89.78 9.80 13.11 20.64 18.69 22.17 20.93 34.39 33.94 40.61 40.82 46.26 44.41 44.77 58.19 57.90 57.98 82.47 82.19 82.12 74.15 74.11 89.68 89.59 13.79 18.56 22.51 21.06 24.40

5.84 5.81 5.81 5.90 5.80 5.77 5.86 5.74 5.85 5.79 5.74 5.87 5.92 5.86 5.90 6.11 6.10 6.11 6.14 5.97 6.03 6.00 6.18 5.96 6.07 5.95 6.08 6.16 6.18 6.09 6.00 6.04 6.08 6.13 6.08 6.15 5.99 5.95 6.21 6.36 6.28 6.35 6.32

5301 5401 5402 5403 5404 5405 5501 5601 5602 5702 5802 6001 6002 6202 6203 6301 6302 6303 6401 6402 6403 6404 6601 6701 6702 6805 7101 7201 7205 7301 7302 7305 7306 7307 7403 7404 7405 7601 7602 7603 7604 7605 7701

28.87 34.85 31.72 37.37 37.53 37.54 41.27 44.93 41.66 53.70 74.58 9.89 15.89 24.57 23.66 25.40 31.41 29.43 34.16 37.58 34.41 31.88 42.07 54.06 54.16 67.00 19.09 23.26 21.57 26.14 25.59 30.92 30.81 31.25 36.70 35.34 33.95 41.36 41.85 41.68 44.12 45.37 54.96

6.25 6.25 6.29 6.29 6.36 6.20 6.19 6.24 6.35 6.33 6.23 6.53 6.43 6.46 6.42 6.39 6.41 6.52 6.43 6.44 6.48 6.47 6.46 6.42 6.52 6.40 6.55 6.53 6.58 6.54 6.60 6.81 6.73 6.64 6.79 6.68 6.55 6.55 6.64 6.75 6.62 6.61 6.62

properties. For example Triton X-100 and Triton X-114 are non-ionic surfactants, however their usefulness in membrane protein extraction is limited, as these detergents have a strong absorbance interfering protein concentration determinations (Arachea et al., 2012). Triton X-100 and Triton X-114 are non-ionic detergents with a short chain (C7– C10), which often leads to deactivation of the protein (Seddon et al., 2004). Triton X-114 is also reported not to be efficient in OMPs extraction, as this detergent interacts uncharacteristically with OMP porins. This detergent also does not solubilize OMPs completely (Pinne and Haake, 2009). Samples of OMPs extracted with Triton X-114 are often contaminated with other types of proteins, e.g. cytosolic proteins. Also urea-CHAPS has its limitations in OMPs isolation as it was described as a compatible detergent with the isoelectric focusing, however it does not solubilize all proteins in a sample (Fountoulakis and Takács, 2001). Sodium carbonate and sodium lauroyl sarcosinate are not compatible with 2-DE technique, as ionic detergents should be avoided, when separating proteins by isoelectric focusing (Bhairi and Mohan, 2001). Zwitterionic detergents are unique as they possess properties of ionic and non-ionic detergents. Zwittergent Z 3-14® improves protein solubilization, is suited for breaking protein–protein interactions and lacks conductivity and electrophoretic mobility (Bhairi and Mohan, 2001). Zwitterionic detergents are compatible with 2-DE technique. Those detergents improve the quality of 2-DE gels because of more spots detection (Chevallet et al., 1998).

Fig. 3. A. Two-dimensional electrophoresis (2-DE) gel profiles of outer membrane proteins of K. pneumoniae. Separation of total proteins (15 μg) was extracted from bacteria with Triton X-100 on 2-DE over the pH range of 3–10 (silver stained). Resolution of isoelectric focusing within the specified pH range was insufficient. B. Master gel profiles of outer membrane proteins of K. pneumoniae were isolated with the use of Triton X-100 after two-dimensional electrophoresis (2-DE) over the pH range of 3–10. 53 protein spots were identified.

Hydrophobic proteins such as membrane integral proteins are generally hard to solubilize under the conditions of isoelectric focusing step, that is, low ionic strength without ionic detergents. Proteins that are partially or completely masked by high abundance spots with similar mass and pI values might be difficult to visualize. However, low abundance proteins may be reliably visualized if fewer amounts are used. Proteins with relative concentration differing by several orders of magnitude pose yet another problem. In such situations, strategies including the removal of high abundance proteins or the use of narrow-range IPG strips could be employed to enable the detection of low abundance proteins (Greenough et al., 2004; Chevalier, 2010; Rabilloud, 2009). Therefore, in our research strips with pH 4–7 range were used. Strips with a range of 3–10 pH may be used only to evaluate the effectiveness of isolation procedure and to establish optional electrophoresis settings. The resolution of isoelectric focusing within the pH 3–10 IPG strips was, however, insufficient. 2-DE has commonly been used to study bacterial proteins because it allows a large scale qualitative and quantitative comparison and generates a reproducible pattern of separated proteins with high resolution. It is a powerful tool for the delineation of bacterial proteins expressed in different conditions and isolated from patients with various diseases. This allows one to compare and correlate the biological pathways of infection (Henzel et al., 2003; Völker and Hecker, 2005). In combination with mass spectrometry (MS) it is often possible to identify almost every single protein spot on the 2-DE gel. Bacterial physiology has been viewed as a very important discipline in microbiology

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In conclusion, we optimized the method of isolation of OMPs from K. pneumoniae with Zwittergent Z 3-14® and obtained clearly visualized profiles in 2-DE with 134 identified proteins. It gives new approach for the control of K. pneumoniae infection because many antimicrobial active factors against of Gram-negative bacteria are directed to cellenvelope targets. The proteins identified in this study could be candidates for serological diagnosis markers and the development of novel, protein-based vaccine against K. pneumoniae.

Acknowledgments The research was supported by the Wroclaw Research Center EIT+ under the project “Biotechnologies and advanced medical technologies— BioMed” (POIG 01.01.02-02-003/08-00) financed from the European Regional Development Fund (Operational Programme Innovative Economy, 1.1.2).

References

Fig. 4. A. Two-dimensional electrophoresis (2-DE) gel profiles of outer membrane proteins of K. pneumoniae. Separation of total proteins (15 μg) was extracted from bacteria with Triton X-100 on 2-DE over the pH range of 4–7 (silver stained). B. Master gel profiles of outer membrane proteins of K. pneumoniae were isolated with the use of Triton X-100 after two-dimensional electrophoresis (2-DE) over the pH range of 4–7. 108 protein spots were identified.

(El-Sharoud and Rowbury, 2006). It is associated with the study of subcellular structure, metabolic activities, growth and survival of microorganisms, which is largely connected with structure and function of outer membrane proteins. The use of 2-DE techniques in bacterial proteomics can be used to shed some light on entire bacterial cell. 2-DE has notable limitations for the analysis and resolution of OMPs resulting from isolation methods. Detergents for the isolation and reconstitution of OMPs need to be optimized prior to two-dimensional electrophoresis. 2-DE has contributed to the general understanding of bacterial proteomics. Despite its intrinsic limitations, it remains a practical and straightforward method facilitating understanding of a role played by the outer membrane proteins in bacteria (Curreem et al, 2012). Klebsiella strains are nowadays very often the etiological factors of intestinal infections. They are heavily capsulated what renders the establishing the 2DE conditions prior to proteomics extensive studies difficult. In this study, the profiles of OMPs expressed by K. pneumoniae in vitro were characterized. Results combined with identification techniques such as mass spectrometry (e.g. liquid chromatography LC-MS) could be useful for determining the protein function (Brotz-Oesterhelt et al, 2005; Bandow, 2005). Future studies will deal with the roles of isolated OMPs in K. pneumoniae pathogenicity and modulation of host immune response during infection.

Albertí, S., Rodríquez-Quiñones, F., Schirmer, T., Rummel, G., Tomás, J.M., Rosenbusch, J.P., Benedí, V.J., 1995. A porin from Klebsiella pneumoniae: sequence homology, threedimensional model, and complement binding. Infect. Immun. 63 (3), 903–910. Arachea, B.T., Sun, Z., Potente, N., Malik, R., Isailovic, D., Viola, R.E., 2012. Detergent selection for enhanced extraction of membrane proteins. Protein Expr. Purif. 86, 12–20. Athamna, A., Ofek, I., Keisari, Y., Markowitz, S., Dutton, G.G., Sharon, N., 1991. Lectinophagocytosis of encapsulated Klebsiella pneumoniae mediated by surface lectins of guinea pig alveolar macrophages and human monocyte-derived macrophages. Infect. Immun. 5, 1673–1682. Augustyniak, D., Mleczko, J., Gutowicz, J., 2010. The immunogenicity of the liposomeassociated outer membrane proteins (OMPs) of Moraxella catarrhalis. Cell. Mol. Biol. Lett. 15 (1), 70–89. http://dx.doi.org/10.2478/s11658-009-0035-z. Bandow, J.E., 2005. Proteomic approaches to antibiotic drug discovery. Curr. Protoc. Microbiol. http://dx.doi.org/10.1002/9780471729259.mc01f02s00 (Chapter 1:Unit 1 F.2.). Bhairi, S.M., Mohan, C., 2001. Detergents — a guide to the properties and uses of detergents in biological system. Calbiochem. http://wolfson.huji.ac.il/purification/PDF/ detergents/CALBIOCHEM-DetergentsIV.pdf. Accessed 20 June 2014. Blake, M.S., Gotschlich, E.C., 1984. Purification and partial characterization of the opacityassociated proteins of Neisseria gonorrhoeae. J. Exp. Med. 159 (2), 452–462 (1). Brotz-Oesterhelt, H., Bandow, J.E., Labischinski, H., 2005. Bacterial proteomics and its role in antibacterial drug discovery. Mass Spectrom. (24, 549‐65-549-65). Bugla-Płoskońska, G., Futoma-Kołoch, B., Skwara, A., Doroszkiewicz, W., 2009. Use of zwitterionic type of detergent in isolation of Escherichia coli O56 outer membrane proteins improves their two-dimensional electrophoresis (2-DE). Pol. J. Microbiol. 58 (3), 205–209. Bugla-Płoskońska, G., Korzeniowska-Kowal, A., Guz-Regner, K., 2011. Reptiles as a source of Salmonella O48 — clinically import bacteria for children: the relationship between resistance to normal cord serum and outer membrane protein patterns. Microb. Ecol. 61, 41–51. Cabanes, D., Dehoux, P., Dussurget, O., Frangeul, L., Cossart, P., 2002. Surface proteins and the pathogenic potential of Listeria monocytogenes. Trends Microbiol. 10, 238–245. Campos, M.A., Vargas, M.A., Regueiro, V., Llompart, C.M., Albertí, S., Bengoechea, J.A., 2004. Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides. Infect. Immun. 72, 7107–7114. http://dx.doi.org/10.1128/IAI.72.12.7107-7114.2004. Carnoy, C., Scharfman, A., Van Brussel, E., Lamblin, G., Ramphal, R., Roussel, P., 1994. Pseudomonas aeruginosa outer membrane adhesins for human respiratory mucus glycoproteins. Infect. Immun. 62 (5), 1896–1900. Chagas, T.P., Seki, L.M., Cury, J.C., Oliveira, J.A., Dávila, A.M., Silva, D.M., Asensi, M.D., 2011. Multiresistance, beta-lactamase-encoding genes and bacterial diversity in hospital wastewater in Rio de Janeiro, Brazil. J. Appl. Microbiol. 111 (3), 572–581. http:// dx.doi.org/10.1111/j.1365-2672.2011.05072.x. Chevalier, F., 2010. Highlights on the capacities of “gel-based” proteomics. Proteome Sci. 8, 23. http://dx.doi.org/10.1186/1477-5956-8-23. Chevallet, M., Santoni, V., Poinas, A., Rouquib, D., Fuchs, A., Kieffer, S., Rossigno, M., Lunardi, J., Garin, J., Rabilloud, T., 1998. New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. Electrophoresis 19, 1901–1909. Coovadia, Y.M., Johnson, A.P., Bhana, R.H., Hutchinson, G.R., George, R.C., Hafferjee, I.E., 1992. Multiresistant Klebsiella pneumoniae in a neonatal nursery: the importance of maintenance of infection control policies and procedures in the prevention of outbreaks. J. Hosp. Infect. 22 (3), 197–205. Cryz Jr., S.J., Fürer, F., Germanier, R., 1984. Experimental Klebsiella pneumoniae burn wound sepsis: role of capsular polysaccharide. Infect. Immun. 43 (1), 440–441. Cryz Jr., S.J., Mortimer, P., Cross, A.S., Fürer, E., Germanier, R., 1986. Safety and immunogenicity of a polyvalent Klebsiella capsular polysaccharide vaccine in humans. Vaccine 4, 15–20. Cullen, P.A., Cordwell, S.J., Bulach, D.M., Haake, D.A., Adler, B., 2002. Global analysis of outer membrane proteins from Leptospira interrogans serovar Lai. Infect Immun. 70 (5), 2311–2318.

I. Bednarz-Misa et al. / Journal of Microbiological Methods 107 (2014) 74–79 Curreem, S.O., Watt, R.M., Lau, S.K., Woo, P.C., 2012. Two-dimensional gel electrophoresis in bacterial proteomics. Protein Cell 3 (5), 346–363. http://dx.doi.org/10.1007/ s13238-012-2034-5. Ebringer, A., Rashid, T., Tiwana, H., Wilson, C., 2007. A possible link between Crohn's disease and ankylosing spondylitis via Klebsiella infections. Clin. Rheumatol. 26 (3), 289–297. ECDC Surveillance report, 2007. Surveillance of healthcare-associated infections in Europe 2007. http://www.ecdc.europa.eu/en/publications/Publications/120215_SUR_HAI_ 2007.pdf (Accessed 20 June 2014). El-Sharoud, W.M., Rowbury, R.J., 2006. Recent insights into microbial physiology. Sci. Prog. 89, 141–149. Fluegge, K., Schweier, O., Schiltz, E., Batsford, S., Berner, R., 2004. Identification and immunoreactivity of proteins released from Streptococcus agalactiae. Eur. J. Clin. Microbiol. Infect. Dis. 23, 818–824. Fountoulakis, M., Takács, B., 2001. Effect of strong detergents and chaotropes on the detection of proteins in two-dimensional gels. Electrophoresis 22 (9), 1593–1602. Futoma-Kołoch, B., Bugla-Płoskońska, G., Doroszkiewicz, W., 2009. Isolation of outer membrane proteins (OMP) from Salmonella cells using zwitterionic detergent and their separation by two-dimensional electrophoresis (2-DE). Pol. J. Microbiol. 4, 363–366. Gabay, J.E., Blake, M., Niles, W.D., Horwitz, M.A., 1985. Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin. J. Bacteriol. 162 (1), 85–91. Gatto, N.T., Dabo, S.M., Hancock, R.E., Confer, A.W., 2002. Characterization of, and immune responses of mice to, the purified OmpA-equivalent outer membrane protein of Pasteurella multocida serotype A:3 (Omp28). Vet. Microbiol. 87 (3), 221–235. Goetsch, L., Gonzalez, A., Plotnicky-Gilquin, H., Haeuw, J.F., Aubry, J.P., Beck, A., Bonnefoy, J.Y., Corvaïa, N., 2001. Targeting of nasal mucosa-associated antigen-presenting cells in vivo with an outer membrane protein A derived from Klebsiella pneumoniae. Infect. Immun. 69, 6434–6444. Greenough, C., Jenkins, R.E., Kitteringham, N.R., Pirmohamed, M., Park, B.K., Pennington, S.R., 2004. A method for the rapid depletion of albumin and immunoglobulin from human plasma. Proteomics 4 (10), 3107–3111. Gromova, I., Celis, J.E., 2006. Protein detection in gels by silver staining: a procedure compatible with mass spectrometry, In: Celis, J.E., Carter, N., Hunter, T., Simons, K., Small, J.V., Shotton, D. (Eds.), 3rd ed. Cell Biology: A Laboratory Handbook vol. 4. Elsevier Academic Press, pp. 219–223 (Ch27). Halling, S.M., Koster, N.A., 2001. Use of detergent extracts of Brucella abortus RB51 to detect serologic responses in RB51-vaccinated cattle. J. Vet. Diagn. Invest. 13 (5), 408–412. Hamid, N., Jain, S.K., 2008. Characterization of an outer membrane protein of Salmonella enterica serovar typhimurium that confers protection against typhoid. Clin. Vaccine Immunol. 15 (9), 1461–1471. Henzel, W.J., Watanabe, C., Stults, J.T., 2003. Protein identification: the origins of peptide mass fingerprinting. J. Am. Soc. Mass Spectrom. 14, 931–942. Hernández-Allés, S., Md, Conejo, Pascual, A., Tomás, J.M., Benedí, V.J., Martínez-Martínez, L., 2000. Relationship between outer membrane alterations and susceptibility to antimicrobial agents in isogenic strains of Klebsiella pneumoniae. J. Antimicrob. Chemother. 46 (2), 273–277. Hidron, A.I., Edwards, J.R., Patel, J., Horan, T.C., Sievert, D.M., Pollock, D.A., Fridkin, S.K., National Healthcare Safety Network Team, Participating National Healthcare Safety Network Facilities, 2008. NHSN annual update: antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006–2007. Infect. Control Hosp. Epidemiol. 11, 996–1011. http://dx. doi.org/10.1086/591861. Hu, Q., Ding, C., Tu, J., Wang, X., Han, X., Duan, Y., Yu, S., 2012. Immunoproteomics analysis of whole cell bacterial proteins of Riemerella anatipestifer. Vet. Microbiol. 157 (3-4), 428–438. http://dx.doi.org/10.1016/j.vetmic.2012.01.009 (15). Kawai, K., Liu, Y., Ohnishi, K., Oshima, S., 2004. A conserved 37 kDa outer membrane protein of Edwardsiella tarda is an effective vaccine candidate. Vaccine 22 (25-26), 3411–3418 (3). Klipstein, F.A., Engert, R.F., Houghten, R.A., 1983. Immunological properties of purified Klebsiella pneumoniae heat-stable enterotoxin. Infect. Immun. 42, 838–841. Kokeguchi, S., Kato, K., Nishimura, F., Kurihara, H., Murayama, Y., 1991. Isolation and partial characterization of a 39 kDa major outer membrane protein of Actinobacillus actinomycetemcomitans Y4. FEMS Microbiol. Lett. 61 (1), 85–89. Kokeguchi, S., Miyamoto, M., Ohyama, H., Hongyo, H., Takigawa, M., Kurihara, H., Murayama, Y., Kato, K., 1994. Biochemical properties of the major outer membrane proteins of Porphyromonas gingivalis. Microbios 77 (313), 247–252. Kurupati, P., Teh, B.K., Kumarasinghe, G., Poh, C.L., 2006. Identification of vaccine candidate antigens of an ESBL producing Klebsiella pneumoniae clinical strain by immunoproteome analysis. Proteomics 6, 836–844. Kurupati, P., Ramachandran, N.P., Poh, C.L., 2011. Protective efficacy of DNA vaccines encoding outer membrane protein A and OMPK36 of Klebsiella pneumoniae in mice. Clin. Vaccine Immunol. 18, 82–88. Kyd, J.M., Taylor, D., Cripps, A.W., 1994. Conservation of immune responses to proteins isolated by preparative polyacrylamide gel electrophoresis from the outer membrane of nontypeable Haemophilus influenza. Infect. Immun. 62 (12), 5652–5658. Laemmli, K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lai, Y.C., Peng, H.L., Chang, H.Y., 2001. Identification of genes induced in vivo during Klebsiella pneumoniae CG43 infection. Infect. Immun. 69, 7140–7145. Lavender, H., Jagnow, J.J., Clegg, S., 2005. Klebsiella pneumoniae type 3 fimbria-mediated immunity to infection in the murine model of respiratory disease. Int .J. Med. Microbiol. 295, 153–159.

79

Lee, N.K., Kim, S., Lee, J.W., Jeong, Y.J., Lee, S.H., Heo, J., Kang, D.H., 2011. CT differentiation of pyogenic liver abscesses caused by Klebsiella pneumoniae vs non-Klebsiella pneumoniae. Br. J. Radiol. 1002, 518–525. http://dx.doi.org/10.1259/bjr/23004588. Lin, J., Huang, S., Zhang, Q., 2002. Outer membrane proteins: key players for bacterial adaptation in host niches. Microbes Infect. 4, 325–331. Lytsy, B., Sandegren, L., Tano, E., Torell, E., Andersson, D.I., Melhus, A., 2008. The first major extended-spectrum beta-lactamase outbreak in Scandinavia was caused by clonal spread of a multiresistant Klebsiella pneumonia producing CTX-M-15. APMIS 116 (4), 302–308. http://dx.doi.org/10.1111/j.1600-0463.2008.00922.x. Maione, D., Margarit, I., Rinaudo, C.D., Masignani, V., Mora, M., Scarselli, M., Tettelin, H., Brettoni, C., Iacobini, E.T., Rosini, R., D'Agostino, N., Miorin, L., Buccato, S., Mariani, M., Galli, G., Nogarotto, R., Nardi-Dei, V., Vegni, F., Fraser, C., Mancuso, G., Teti, G., Madoff, L.C., Paoletti, L.C., Rappuoli, R., Kasper, D.L., Telford, J.L., Grandi, G., 2005. Identification of a universal group B Streptococcus vaccine by multiple genome screen. Science 309, 148–150. McLachlan, A.A., Singh, K., Marangoni, D.G., 2010. A conformational investigation of zwitterionic surfactants in the micelle via 13C chemical shift measurements and 2D NOESY spectroscopy. Colloid Polym. Sci. 288, 653–666. Molloy, M.P., Herbert, B.R., Slade, M.B., Rabilloud, T., Nouwens, A.S., Williams, K.L., Gooley, A.A., 2000. Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem. 267 (10), 2871–2881. Moranta, D., Regueiro, V., March, C., Llobet, E., Margareto, J., Larrate, E., Garmendia, J., Bengoechea, J., 2010. Klebsiella pneumoniae capsule polysaccharide impedes the expression of β-defensins by airway epithelial cells. Infect. Immun. 78, 1135–1146. http://dx.doi.org/10.1128/IAI.00940-09. Murphy, T.F., Bartos, L.C., 1989. Surface-exposed and antigenically conserved determinants of outer membrane proteins of Branhamella catarrhalis. Infect Immun. 57 (10), 2938–2941. Navarre, W.W., Schneewind, O., 1999. Surface proteins of gram‐positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol. Mol. Biol. Rev. 63, 174–229. Ortega, M., Marco, F., Soriano, A., Almela, M., Martínez, J.A., López, J., Pitart, C., Mensa, J., 2012. Epidemiology and prognostic determinants of bacteraemic biliary tract infection. J. Antimicrob. Chemother. 67, 1508–1513. http://dx.doi.org/10.1093/jac/dks062. Pal, S., Theodor, I., Peterson, E.M., de la Maza, L.M., 1997. Immunization with an acellular vaccine consisting of the outer membrane complex of Chlamydia trachomatis induces protection against a genital challenge. Infect. Immun. 65 (8), 3361–3369. Peng, X., Ye, X., Wang, S., 2004. Identification of novel immunogenic proteins of Shigella flexneri 2a by proteomic methodologies. Vaccine 22 (21-22), 2750–2756 (29). Pinne, M., Haake, D.A., 2009. A comprehensive approach to identification of surfaceexposed, outer membrane-spanning proteins of Leptospira interrogans. PLoS One 4 (6), e6071. http://dx.doi.org/10.1371/journal.pone.0006071. Podschun, R., Ullmann, U., 1998. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin. Microbiol. Rev. 11, 589–603. Rabilloud, T., 2009. Membrane proteins and proteomics: love is possible, but so difficult. Electrophoresis 30, 174–180. http://dx.doi.org/10.1002/elps.200900050. Rockwood, D., Wilson, M.T., Darley-Usmar, V.M., 1987. Isolation and characteristic of intact mitochondria. In: Darley-Usmar, V.M., Rickwood, D., Wilson, M.T. (Eds.), Mitochondria: A Practical Approach. IRL Press, Oxford, pp. 1–16. Sahly, H., Podschun, R., 1997. Clinical, bacteriological, and serological aspects of Klebsiella infections and their spondylarthropathic sequelae. Clin. Diagn. Lab. Immunol. 4 (4), 393–399. Seddon, A.M., Curnow, P., Booth, P.J., 2004. Membrane proteins, lipids and detergents: not just a soap opera. Biochim. Biophys. Acta 1666, 105–117. Siritapetawee, J., Prinz, H., Krittanai, C., Suginta, W., 2004. Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion. Biochem. J. 384 (Pt 3), 609–617. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85. Struve, C., Forestier, C., Krogfelt, K.A., 2003. Application of a novel multi-screening signature-tagged mutagenesis assay for identification of Klebsiella pneumoniae genes essential in colonization and infection. Microbiology 149, 167–176. Struve, C., Bojer, M., Krogfelt, K.A., 2009. Identification of a conserved chromosomal region encoding Klebsiella pneumoniae type 1 and type 3 fimbriae and assessment of the role of fimbriae in pathogenicity. Infect. Immun. 77, 5016–5024. http://dx.doi.org/10. 1128/IAI.00585-09. Tagawa, Y., Ishikawa, H., Yuasa, N., 1993. Purification and partial characterization of the major outer membrane protein of Haemophilus somnus. Infect. Immun. 61 (1), 91–96. Völker, U., Hecker, M., 2005. From genomics via proteomics to cellular physiology of the Gram-positive model organism Bacillus subtilis. Cell. Microbiol. 7, 1077–1085. Yadav, V., Sharma, S., Harjai, K., Mohan, H., Chhibber, S., 2005. Lipopolysaccharidemediated protection against Klebsiella pneumoniae-induced lobar pneumonia: intranasal vs. intramuscular route of immunization. Folia Microbiol. (Praha) 50, 83–86. Yang, Y.S., Siu, L.K., Yeh, K.M., Fung, C.P., Huang, S.J., Hung, H.C., Lin, J.C., Chang, F.Y., 2009. Recurrent Klebsiella pneumoniae liver abscess: clinical and microbiological characteristics. J. Clin. Microbiol. 47 (10), 3336–3339. http://dx.doi.org/10.1128/JCM.00918-09. Zhang, W., Shao, J., Liu, G., Tang, F., Lu, Y., Zhai, Z., Wang, Y., Wu, Z., Yao, H., Lu, C., 2011. Immunoproteomic analysis of bacterial proteins of Actinobacillus pleuropneumoniae serotype 1. Proteome Sci. 9 (1), 32. http://dx.doi.org/10.1186/1477-5956-9-32. Zhang, M.J., Gu, Y.X., Di, X., Zhao, F., You, Y.H., Meng, F.L., Zhang, J.Z., 2013. In vitro protein expression profile of Campylobacter jejuni strain NCTC11168 by two-dimensional gel electrophoresis and mass spectrometry. Biomed. Environ. Sci. 26 (1), 48–53. http:// dx.doi.org/10.3967/0895-3988.2013.01.006.

Application of zwitterionic detergent to the solubilization of Klebsiella pneumoniae outer membrane proteins for two-dimensional gel electrophoresis.

Klebsiella pneumoniae is a frequent cause of nosocomial respiratory, urinary and gastrointestinal tract infections and septicemia with the multidrug-r...
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