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Serum Proteomic Analysis from Bacteremic and Leucopenic Rabbits
Journal of Surgical Research 171, 749–754 (2011) doi:10.1016/j.jss.2010.04.056
Serum Proteomic Analysis from Bacteremic and Leucopenic Rabbits Zheng Zhou, M.D.,* Jianan Ren, M.D.,*,1 Haiyan Liu, M.D.,† Guosheng Gu, M.D.,* and Jieshou Li, M.D.* *Research Institute of General Surgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, P.R. China; and †Intensive Care Unit, First Affiliated Hospital of Anhui Medical University, Anhui, Hefei, P.R. China Submitted for publication January 25, 2010
Background. Bacteremia causes mild to lifethreatening illnesses as a consequence of inflammatory pathway activation, and leucopenia can increase mortality rates and treatment costs. The identification of novel biomarkers for leucopenia in the context of bacteremia would facilitate early detection and provide insights into the identification of novel therapeutic targets. Methods. Rabbits were infected with Klebsiella pneumoniae to induce bacteremia with leucopenia, and sera from infected and noninfected rabbits was subjected to proteomic analysis using twodimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to compare serum protein profiles. Results. Five differentially-expressed protein spots were identified in bacteremic rabbits with leucopenia compared to protein expression profiles prior to infection. The proteins were identified as desmin, cystic fibrosis transmembrane conductance regulator (CFTR), interleukin (IL) 4, and the interleukin 6 precursor. The increased levels of desmin and CFTR in serum were further confirmed by Western blot analysis. Conclusions. These results indicated that desmin, CFTR, IL-4, and the IL-6 precursor may be potential molecular biomarkers for leucopenia occurring with bacteremia. Ó 2011 Elsevier Inc. All rights reserved. Key Words: bacteremia; leucopenia; serum proteomics; two-dimensional gel electrophoresis.
Bacteremia is typically associated with prolonged hospital stay and increased cost, particularly if accompanied with leucopenia, since patients with leucopenia have a worse prognosis than infected but non-leucopenic patients [1–4]. For example, patients hospitalized as a consequence of community-acquired, health careassociated, or hospital-acquired pneumonia have increased mortality rates if they were also leucopenic [5]. Although studies have indentified C-reactive protein (CRP) and procalcitonin (PCT) as markers of bacteremia, little work examining markers in the context of leucopenia have been carried out. Therefore, discovery of previously unidentified host factors associated with the pathophysiology of leucopenia in the context of bacteremia can be used as diagnostic biomarkers. Since proteomic analyses were introduced by Wasinger et al. in 1995 [6], they have been widely utilized in biomarker identification. In this study, proteomic analyses (two-dimensional gel electrophoresis [2-DE], image analysis, and mass spectrometry [MS]) were utilized to identify proteins or peptides present at low concentrations in rabbits with bacteremia/leucopenia to identify novel biomarkers. MATERIALS AND METHODS Animals
INTRODUCTION
Bacteremia causes mild to life-threatening illnesses through the activation of a series of pro-inflammatory, anti-inflammatory, and apoptotic pathways that ultimately results in a disruption of physiologic homeostasis. 1 To whom correspondence and reprint requests should be addressed at Research Institute of General Surgery, Jinling Hospital, Medical School of Nanjing University, Zhongshan Road #305, Nanjing, P.R. China. E-mail: [email protected].
Ten New Zealand rabbits with a mean weight of 3.44 6 0.26 kg were used for the infection studies. Animals were housed in a specific pathogen-free animal care facility with ad libitum access to tap water and standard balanced rabbit chow and 12 h light/dark cycles. This study was approved by the Institutional Animal Care and Use Committee of Nanjing University and the Principles of Laboratory Animal Care were followed.
Bacterial Isolates Klebsiella pneumoniae obtained from a human nosocomial infection was used for the rabbit infections. The minimal inhibitory
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concentrations (MICs) for this isolate (according to the Clinical and Laboratory Standards Institute [CLSI]) are amoxicillin/clavulanate (4 mg/L), piperacillin (8 mg/L), ceftazidime (4 ml/L), ciprofloxacin 1 mg/L), amikacin (2 mg/mL), and imipenem (2 mg/mL). PCR (polymerase chain reaction) was used to define the K. pneumoniae strain and confirmed by amplification of the SHV (Super Hybrid Vehicle) gene. The K. pneumoniae concentration was determined by measuring the optical density (OD) 600 and comparing values to a predetermined standard curve. Bacteria were then diluted to the desired concentration prior to inoculation.
Bacteremia Model Rabbits were sedated by an intramuscular injection of 20 mg/kg of ketamine. Anesthesia was maintained by the intravenous administration of 20 mg/kg of lidocaine at 20 min intervals. A 2-cm-long midline incision was made on the neck to expose the right inner jugular vein and an 18 G catheter was inserted. 1 3 108 cfu/kg of K. pneumoniae was injected in a volume of 2.0 mL. Blood was taken from ear veins for proteomic analysis twice, once prior to infection and the second 3 h post-infection when leucocyte achieved nadir. Blood was immediately centrifuged at 2000 rpm for 10 min, the sera collected, and then frozen at –70 C until used. The catheter was removed and the incision was sutured with 3-0 stitches. WBC values between 4000 and 11,000 mL were considered normal, and leucopenia was defined by values <4000 mL.
Removal of High Abundance Proteins from Serum Samples Fifty mL of each of the two groups of pooled rabbit serum samples were processed with the ProteoExtract Albumin/IgG Removal Kit (Calbiochem, San Diego, CA) that selectively removed albumin and IgG from the serum samples. After albumin and IgG depletion, excess salts were eliminated using the ProteoExtract Protein Precipitation Kit (Calbiochem). Samples were processed according to the manufacturer’s instructions. The final serum sample protein concentrations were determined by the Bradford method (Bio-Rad Protein Assay; Bio-Rad, Carlsbad, CA).
Two-Dimensional Gel Electrophoresis Serum proteins (60 mg) were diluted with rehydration solution (8 M urea, 2% CHAPS, 65 mM DTT, 0.5% vol/vol [pH 4-7] IEF [isoelectric focusing] buffer, trace bromophenol blue) in 250 mL. IEF was performed using a Protean IEF system (Amersham Bioscience, Uppsala, Sweden). The gels were rehydrated at 30 V for 6 h and at 60 V for 6 h. Proteins were focused subsequently for 1 h at 500 V and 1 h at 1000 V; then a gradient was applied from 1000 to 8000 V for 1 h and finally at 8000 V to give a total of 30,000 V h. All IEFs were carried out at 20 C. After the first-dimension IEF, IPG strips were placed in an equilibration solution (6 M urea, 2% SDS, 30% glycerol, 1.5 M Tris-HCl, pH 8.8) containing 2% DTT and were shaken for 15 min at 50 rpm. The strips were then transferred to equilibration solution containing 2.5% iodoacetamide and shaken for another 15 min before being subjected to 12.5% homogeneous polyacrylamide gel electrophoresis. Separation in the second dimension was carried out using an Ettan-Dalt II system (Amersham Biosciences, San Francisco, CA), the gels fixed and then silver stained.
Image Acquisition and Analysis Stained gels were scanned and images were analyzed using the ImageMaster 2-D Platinum Software (Amersham Bioscience, Geneva, Switzerland) for spot identification. Match sets containing two groups (bacteremia with leucopenia or controls) were created for total gel images and differentially expressed protein spots were evaluated. To minimize variation in sample loading and staining,
2-DE analysis was repeated three times. Consistently differentially expressed spots (5-fold increase) were identified by MALDI-TOF-MS.
In-Gel Tryptic Digestion and MALDI-TOF MS Each differentially expressed spot obtained from respective gels was dehydrated with 50 ml ACN for 5 min, incubated in 50 mL 10 mM DTT at 56 C for 1 h and then incubated in 50 mL of 55 mM iodoacetamide (alkylating solution) for 45 min. Subsequently, respective spots were dehydrated with 50 mL of ACN, rehydrated in 5 mL porcine trypsin followed by the addition of 10 mL 25 mM ammonium bicarbonate. Proteolysis was carried out overnight at 37 C and stopped by adding 10 mL 2% formic acid. The resulting peptides were concentrated and mixed with a-cyano-4-hydroxycinammic acid (Sigma, St. Louis, MO), deposited on a 384-well MALDI target and air-dried. Analyses were performed using a Biflex IV (Bruker Daltonics, Bremen, Germany). The MS data were compared against tryptic peptide sequences from the SWISS-PROT database using Mascot (Matrix Sciences, London, UK) search algorithms.
Western Blot Analysis Each serum sample was loaded at the same concentration. Serum proteins were subjected to 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (Millipore, Billerica, MA) membrane. The membranes were blocked for 30 min with 5% nonfat milk in Tris-buffered saline with 0.05% Tween-20 (TBST). The samples were incubated overnight with goat anti-rabbit-desmin (1:400) or goat anti-rabbit-CFTR (1:400) antibodies at 4 C. The membranes were washed three times in TBST for 5 min each and then blotted for 2 h with horseradish peroxidase-conjugated secondary rabbit anti-goat IgG (1:500) at room temperature. The membranes were then washed twice with TBST followed by another wash in TBS. Immunoreactive bands were visualized using the Western Blotting Detection System (Pierce, Rockford, IL). Protein mass was compared after quantifying the intensity of protein bands by Quantity One software (Bio-Rad).
RESULTS Quantitative Comparison and 2-DE Proteomic Analysis
Serum from 10 rabbits with a similar weight distribution was pooled for 2-DE analysis. The average neutrophil counts were 2290 mL and the lymphocyte counts were 2380 mL in the rabbits prior to infection. In contrast, the average neutrophil counts were 540 mL and the lymphocyte counts were 960 mL 3 h post infection. Animals were monitored for 14 d during which time seven infected rabbits died. Gel images of serum harvested from rabbits either post-infection or after the onset of bacteremia/leucopenia were compared (Fig. 1). Approximately 200 protein spots localized in the 4–7 pI range and between 10 and 150 kDa were detected on the 2-DE gels. Five differentially expressed protein spots (defined by more than a 5-fold change in spot density) were identified. Protein Identification
Ten proteins in the bacteremic/leucopenic group with a differential expression of greater than 5-fold were
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differentially-expressed protein spots is shown in Fig. 2. Western Blot Analysis Validation of Up-Regulated Proteins
To confirm the 2-DE data, animals were infected as above and sera analyzed by Western blot. Six bacteremic/leucopenic rabbits had high levels of desmin expression (Fig. 3A) and CFTR expression was detected in 100% (6/6) of the serum samples examined (Fig. 3B). Multiple CFTR bands were observed in bacteremic/leucopenic samples at 3 h, indicating protein modification or degradation. Comparison of serum from the two treatment groups demonstrated that both of these proteins were up-regulated in sera from bacteremic/leucopenic animals. DISCUSSION
FIG. 1. 2-DE analysis of serum proteins from rabbits with bacteremia and leucopenia. Ten different-expressed protein spots are labeled with arrows. (Color version of figure is available online.)
subjected to in-gel digestion for MALDI-TOF MS analysis. Three over-expressed spots were identified that corresponded to desmin (spot 118, 165) and one spot corresponded to CFTR (cystic fibrosis transmembrane conductance regulator) (spot 304) (Table 1). Protein spots that could not be identified were excluded. Two proteins in the bacteremic/leucopenic group were down-regulated and corresponded to IL-4 (spot 22) and the IL-6 precursor (spot 35). Spots 92 and 155 could not be identified. Quantitative comparison of the five
Analysis of serum proteins represents an ideal means of establishing the diagnosis of different infections and pathologies due to the ease by which serum can be obtained [7]. Furthermore, new technologies have been developed for the identification of novel serumassociated biomarkers. For example, ovarian cancer can be diagnosed by carrying out TOF spectra of serum using a low-resolution mass spectrometer [8], and an extensive reference serum proteome database was established from trauma patients using a combination of major protein depletion, target protein enrichment, and multidimensional LC and MS [9]. In this study, we used comparative proteomic approaches (2-DE and MS) that identified 10 proteins whose expression profile was altered by more than 5-fold in rabbits with bacteremia and leucopenia, and five of these targets were identified. Intrinsic differences between rabbits with respect to differences in protein expression profiles was negated by pooling the serum from rabbits before and after infection, further strengthening the association of these five candidate biomarkers with bacteremia/leucopenia. Furthermore, the presence in serum of the two significantly up-regulated biomarkers (desmin and CFTR) was confirmed by Western blot analysis.
TABLE 1 Differentially Expressed Proteins Identified by MALDI-TOF-MS Spot
Accession number
Score
Sequence coverage
pI/Mr
Protein name
22 35 118 165 304
EDL33576 NP-112445 P02541 P02541 ABI93671
133 269 218 176 84
52% 55% 45% 44% 13%
9.21/10610 6.96/24597 5.21/53463 5.21/53463 8.98/168924
IL-4 IL-6 precursor Desmin Desmin CFTR*
*
CFTR ¼ cystic fibrosis transmembrane conductance regulator.
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FIG. 2. Quantitative protein analysis. Protein expression levels between the five differentially expressed protein spots identified in bacteremic/leucopenic rabbits (compared with healthy rabbits) were characterized. The volume of spots was normalized and quantified as relative intensity using ImageMaster 2D Software. (Color version of figure is available online.)
Desmin is the main intermediate filament (IF) protein expressed in cardiac, skeletal, and smooth muscles and is a primary cytoskeletal component that interacts with other proteins to maintain cellular integrity. Desmin is much more abundant in cardiac muscle (2% of total protein) than in skeletal muscle (0.35%) and is a major component of Purkinje fibers that facilitate heart function and coordination [10]. Previous reports suggested that desminopathy is associated with childhood-onset cardioskeletal myopathy, pure skeletal myopathy, adult-onset skeletal myopathy with cardiac involvement, cardiomyopathy with distal weakness, pure dilated cardiomyopathy, and distal myopathy with cardiac, respiratory, and smooth muscle involvement [11–17]. However, to the best of our knowledge,
FIG. 3. Western blot analysis. Pooled rabbit serum prior to and after the onset of bacteremia/leucopenia was examined by Western blot analysis and probed for either (A) desmin or (B) CFTR. Note the higher expression levels of desmin in bacteremia and leucopenia sera than that in pre-infection sera. The molecular weight of CFTR is about 168 kDa. Multiple bands corresponding to CFTR were observed in rabbits with bacteremia and leucopenia suggesting protein degradation or modification. (Color version of figure is available online.)
there are few reports linking desmin to bacteremia and leucopenia. Our Western blot analysis demonstrated that serum from bacteremic/leucopenic rabbits had higher desmin levels than control animals confirming the 2-DE gel experiment data. This result indicated that the myocardium was likely severely damaged in animals with bacteremia and leucopenia. CFTR is an cellular ion chloride channel that also regulates bicarbonate and sodium transporter channels [18], which has been know to be secreted in airway passages during Pseudomonas aeruginosa infections. In addition, it was demonstrated that CFTR dysfunction might lead to inefficient antimicrobial peptide function at the apical airway membrane surface epithelia [19, 20]. Pier et al. proposed that CFTR was an epithelial cell receptor for P. aeruginosa, with a CFTR peptide binding to the conserved outer-core oligosaccharide of the bacterium [21, 22]. Recently, this hypothesis was supported by Schroeder et al., who found that transgenic cystic fibrosis mice had a reduced capacity to clear an inoculum of P. aeruginosa applied to the nares. This reduced clearance rate was associated with decreased cellular internalization of P. aeruginosa [23]. The up-regulation of CFTR in this study supports work by Painter et al. who examined how extracellular chloride concentrations affected P. aeruginosa killing by normal neutrophils and found that polymorphonuclear neutrophil (PMN)-mediated bacterial killing was strongly dependent on extracellular chloride concentration i.e., neutrophils in a chloride-deficient medium inefficiently killed P. aeruginosa. This phenomenon can be explained by the fact that the chloride anion is essential for converting myeloperoxidase (MPO) into hypochlorous acid (HOCl) in PMNs [24]. Like other infectious agents, K. pneumoniae can induce the activation of immune responses and provoke the activation of a series of inflammatory processes associated with cytokine production. It is therefore imperative that a balance between immunity, pathogen clearance and maintenance of tissue integrity be maintained [25]. IL-4 (down-regulated in bacteremic/leucopenic rabbits) is acute-phase protein [26, 27] and has long been known as a cytokine with the capacity to rescue B cells from apoptosis and enhance their survival [26]. For example, transgenic C3H mice overexpressing IL-4 developed an autoimmune-type disorder resembling lupus [28]. Another important biologic function of IL-4 is T cell down-regulation [27]. In a previous study, Singh showed that the concomitant administration of a recombinant adenovirus vector encoding murine IL-4 and a T-cell activating peptide reduced IgG anti-dsDNA antibody production in a model of lupus [29]. Thus, neutrophil respiratory burst activity, like the occurrence of bacteremia with leucopenia, can
ZHOU ET AL.: SERUM PROTEOMIC ANALYSIS
be inhibited by IL-4, suggesting protective roles for IL-4 during inflammatory responses. IL-6, also down-regulated in bacteremic/leucopenic animals, is a cytokine with various biological activities, which includes the induction of inflammatory responses, immune regulation, and hematopoiesis [30]. Overproduction of IL-6 is associated with inflammatory diseases pathology, e.g., sepsis [31–33]. For example, it was demonstrated that IL-6 levels correlated with APACHE II scores, i.e., mortality rates increased significantly in patients who presented with IL-6 serum levels above 1000 pg/mL. In a murine colitis model, mice that developed Th1 cell-mediated colitis presented with wasting. Rat anti-mouse IL-6R antibody treatment suppressed colitis symptoms and wasting by inducing apoptosis of lamina propria T cells [34]. However, in the present study, both IL-4 and IL-6 were decreased, suggesting that the bacteremic/leucopenic animals were immunosuppressed. In conclusion, proteomic analysis identified serum components associated with the pathophysiology of leukopenia in the context of bacteremia. Although our results did not uncover all proteins likely associated with this disease manifestation, we identified two proteins that were increased in our infection group. To our knowledge, this is the first report linking desmin and CFTR together to infectious disease, suggesting that these proteins can be used as markers to predict clinical outcomes particularly during cases of bacteremia with leucopenia. ACKNOWLEDGMENTS The authors acknowledge support for this work by grants from the National Natural Science Foundation of China (30872456).
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9. Liu T, Qian WJ, Mottaz HM, et al. Evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry. Mol Cell Proteomics 2006;5:2167. 10. Price MG. Molecular analysis of intermediate filament cytoskeleton — A putative load-bearing structure. Am J Physiol 1984; 246:H566. 11. Goldfarb LG, Park KY, Cervena´kova´ L, et al. Missense mutations in desmin associated with familial cardiac and skeletal myopathy. Nat Genet 1998;19:402. 12. Park KY, Dalakas MC, Goebel HH, et al. Desmin splice variants causing cardiac and skeletal myopathy. J Med Genet 2000; 37:851. 13. Dalakas MC, Dagvadorj A, Goudeau B, et al. Progressive skeletal myopathy, a phenotypic variant of desmin myopathy associated with desmin mutations. Neuromuscul. Disord 2003; 13:252. 14. Schro¨der R, Goudeau B, Simon MC, et al. On noxious desmin: Functional effects of a novel heterozygous desmin insertion mutation on the extrasarcomeric desmin cytoskeleton and mitochondria. Hum Mol Genet 2003;12:657. 15. Li D, Tapscoft T, Gonzalez O, et al. Desmin mutation responsible for idiopathic dilated cardiomyopathy. Circulation 1999; 100:461. 16. Ariza A, Navaspalacios JJ. Desmin myopathy: A multisystem disorder involving skeletal, cardiac, and smooth muscle. Hum Pathol 1995;26:1032. 17. Mun˜oz-Ma´rmol AM, Strasser G, Isamat M, et al. A dysfunctional desmin mutation in a patient with severe generalized myopathy. Proc Natl Acad Sci USA 1998;95:11312. 18. Sheppard DN, Welsh MJ. Structure and function of the CFTR chloride channel. Physiol Rev 1999;79:S23. 19. Travis SM, Conway BA, Zabner J, et al. Activity of abundant antimicrobials of the human airway. Am J Respir Cell Mol Biol 1999;20:872. 20. Bals R, Weiner DJ, Moscioni AD, et al. Augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide. Infect Immun 1999;67:6084. 21. Goldberg JB, Pier GB. The role of the CFTR in susceptibility to Pseudomonas aeruginosa infections in cystic fibrosis. Trends Microbiol 2000;8:514. 22. Pier GB. Role of the cystic fibrosis transmembrane conductance regulator in innate immunity to Pseudomonas aeruginosa infections. Proc Natl Acad Sci USA 2000;97:8822. 23. Schroeder TH, Reiniger N, Meluleni G, et al. Transgenic cystic fibrosis mice exhibit reduced early clearance of Pseudomonas aeruginosa from the respiratory tract. J Immunol 2001; 166:7410. 24. Painter RG, Bonvillain RW, Valentine VG, et al. The role of chloride anion and CFTR in killing of Pseudomonas aeruginosa by normal and CF neutrophils. J Leukocyte Biol 2008; 83:1345. 25. Wan J, Sun W, Li X, et al. Inflammation inhibitors were remarkably up-regulated in plasma of severe acute respiratory syndrome patients at progressive phase. Proteomics 2006; 6:2886. 26. Mori M, Morris SC, Orekhova T, et al. IL-4 promotes the migration of circulating B cells to the spleen and increases splenic B cell survival. J Immunol 2000;164:5704. 27. Morris SC, Gause WC, Finkelman FD. IL-4 suppression of in vivo T cell activation and antibody production. J Immunol 2000;164:1734. 28. Erb KJ, Ru¨ger B, von Brevern M, et al. Constitutive expression of interleukin-4 (IL-4) in vivo causes autoimmune-type disorders in mice. J Exp Med 1997;185:329. 29. Singh RR. IL-4 and many roads to lupuslike autoimmunity. Clin Immunol 2003;108:73. 30. Nishimoto N, Kishimoto T. Interleukin-6: From bench to bedside. Nat Clin Pract Rheumatol 2006;2:619.
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31. Remick DG, Bolgos GR, Siddiqui J, et al. Six at six: Interleukin-6 measured 6 h after the initiation of sepsis predicts mortality over 3 days. Shock 2002;17:463. 32. Oda S, Hirasawa H, Shiga H, et al. Sequential measurement of IL-6 blood levels in patients with systemic inflammatory response syndrome (SIRS)/sepsis. Cytokine 2005;21:169.
33. Damas P, Ledoux D, Nys M, et al. Cytokine serum level during severe sepsis in human IL-6 as a marker of severity. Ann Surg 1992;215:356. 34. Ito H, Takazoe M, Fukuda Y, et al. A pilot randomized trial of human anti-interleukin-6 receptor monoclonal antibody in active Crohn’s disease. Gastroenterology 2004;126:989.
Serum Proteomic Analysis from Bacteremic and Leucopenic Rabbits Zheng Zhou, M.D.,* Jianan Ren, M.D.,*,1 Haiyan Liu, M.D.,† Guosheng Gu, M.D.,* and Jieshou Li, M.D.* *Research Institute of General Surgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, P.R. China; and †Intensive Care Unit, First Affiliated Hospital of Anhui Medical University, Anhui, Hefei, P.R. China Submitted for publication January 25, 2010
Background. Bacteremia causes mild to lifethreatening illnesses as a consequence of inflammatory pathway activation, and leucopenia can increase mortality rates and treatment costs. The identification of novel biomarkers for leucopenia in the context of bacteremia would facilitate early detection and provide insights into the identification of novel therapeutic targets. Methods. Rabbits were infected with Klebsiella pneumoniae to induce bacteremia with leucopenia, and sera from infected and noninfected rabbits was subjected to proteomic analysis using twodimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to compare serum protein profiles. Results. Five differentially-expressed protein spots were identified in bacteremic rabbits with leucopenia compared to protein expression profiles prior to infection. The proteins were identified as desmin, cystic fibrosis transmembrane conductance regulator (CFTR), interleukin (IL) 4, and the interleukin 6 precursor. The increased levels of desmin and CFTR in serum were further confirmed by Western blot analysis. Conclusions. These results indicated that desmin, CFTR, IL-4, and the IL-6 precursor may be potential molecular biomarkers for leucopenia occurring with bacteremia. Ó 2011 Elsevier Inc. All rights reserved. Key Words: bacteremia; leucopenia; serum proteomics; two-dimensional gel electrophoresis.
Bacteremia is typically associated with prolonged hospital stay and increased cost, particularly if accompanied with leucopenia, since patients with leucopenia have a worse prognosis than infected but non-leucopenic patients [1–4]. For example, patients hospitalized as a consequence of community-acquired, health careassociated, or hospital-acquired pneumonia have increased mortality rates if they were also leucopenic [5]. Although studies have indentified C-reactive protein (CRP) and procalcitonin (PCT) as markers of bacteremia, little work examining markers in the context of leucopenia have been carried out. Therefore, discovery of previously unidentified host factors associated with the pathophysiology of leucopenia in the context of bacteremia can be used as diagnostic biomarkers. Since proteomic analyses were introduced by Wasinger et al. in 1995 [6], they have been widely utilized in biomarker identification. In this study, proteomic analyses (two-dimensional gel electrophoresis [2-DE], image analysis, and mass spectrometry [MS]) were utilized to identify proteins or peptides present at low concentrations in rabbits with bacteremia/leucopenia to identify novel biomarkers. MATERIALS AND METHODS Animals
INTRODUCTION
Bacteremia causes mild to life-threatening illnesses through the activation of a series of pro-inflammatory, anti-inflammatory, and apoptotic pathways that ultimately results in a disruption of physiologic homeostasis. 1 To whom correspondence and reprint requests should be addressed at Research Institute of General Surgery, Jinling Hospital, Medical School of Nanjing University, Zhongshan Road #305, Nanjing, P.R. China. E-mail: [email protected].
Ten New Zealand rabbits with a mean weight of 3.44 6 0.26 kg were used for the infection studies. Animals were housed in a specific pathogen-free animal care facility with ad libitum access to tap water and standard balanced rabbit chow and 12 h light/dark cycles. This study was approved by the Institutional Animal Care and Use Committee of Nanjing University and the Principles of Laboratory Animal Care were followed.
Bacterial Isolates Klebsiella pneumoniae obtained from a human nosocomial infection was used for the rabbit infections. The minimal inhibitory
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JOURNAL OF SURGICAL RESEARCH: VOL. 171, NO. 2, DECEMBER 2011
concentrations (MICs) for this isolate (according to the Clinical and Laboratory Standards Institute [CLSI]) are amoxicillin/clavulanate (4 mg/L), piperacillin (8 mg/L), ceftazidime (4 ml/L), ciprofloxacin 1 mg/L), amikacin (2 mg/mL), and imipenem (2 mg/mL). PCR (polymerase chain reaction) was used to define the K. pneumoniae strain and confirmed by amplification of the SHV (Super Hybrid Vehicle) gene. The K. pneumoniae concentration was determined by measuring the optical density (OD) 600 and comparing values to a predetermined standard curve. Bacteria were then diluted to the desired concentration prior to inoculation.
Bacteremia Model Rabbits were sedated by an intramuscular injection of 20 mg/kg of ketamine. Anesthesia was maintained by the intravenous administration of 20 mg/kg of lidocaine at 20 min intervals. A 2-cm-long midline incision was made on the neck to expose the right inner jugular vein and an 18 G catheter was inserted. 1 3 108 cfu/kg of K. pneumoniae was injected in a volume of 2.0 mL. Blood was taken from ear veins for proteomic analysis twice, once prior to infection and the second 3 h post-infection when leucocyte achieved nadir. Blood was immediately centrifuged at 2000 rpm for 10 min, the sera collected, and then frozen at –70 C until used. The catheter was removed and the incision was sutured with 3-0 stitches. WBC values between 4000 and 11,000 mL were considered normal, and leucopenia was defined by values <4000 mL.
Removal of High Abundance Proteins from Serum Samples Fifty mL of each of the two groups of pooled rabbit serum samples were processed with the ProteoExtract Albumin/IgG Removal Kit (Calbiochem, San Diego, CA) that selectively removed albumin and IgG from the serum samples. After albumin and IgG depletion, excess salts were eliminated using the ProteoExtract Protein Precipitation Kit (Calbiochem). Samples were processed according to the manufacturer’s instructions. The final serum sample protein concentrations were determined by the Bradford method (Bio-Rad Protein Assay; Bio-Rad, Carlsbad, CA).
Two-Dimensional Gel Electrophoresis Serum proteins (60 mg) were diluted with rehydration solution (8 M urea, 2% CHAPS, 65 mM DTT, 0.5% vol/vol [pH 4-7] IEF [isoelectric focusing] buffer, trace bromophenol blue) in 250 mL. IEF was performed using a Protean IEF system (Amersham Bioscience, Uppsala, Sweden). The gels were rehydrated at 30 V for 6 h and at 60 V for 6 h. Proteins were focused subsequently for 1 h at 500 V and 1 h at 1000 V; then a gradient was applied from 1000 to 8000 V for 1 h and finally at 8000 V to give a total of 30,000 V h. All IEFs were carried out at 20 C. After the first-dimension IEF, IPG strips were placed in an equilibration solution (6 M urea, 2% SDS, 30% glycerol, 1.5 M Tris-HCl, pH 8.8) containing 2% DTT and were shaken for 15 min at 50 rpm. The strips were then transferred to equilibration solution containing 2.5% iodoacetamide and shaken for another 15 min before being subjected to 12.5% homogeneous polyacrylamide gel electrophoresis. Separation in the second dimension was carried out using an Ettan-Dalt II system (Amersham Biosciences, San Francisco, CA), the gels fixed and then silver stained.
Image Acquisition and Analysis Stained gels were scanned and images were analyzed using the ImageMaster 2-D Platinum Software (Amersham Bioscience, Geneva, Switzerland) for spot identification. Match sets containing two groups (bacteremia with leucopenia or controls) were created for total gel images and differentially expressed protein spots were evaluated. To minimize variation in sample loading and staining,
2-DE analysis was repeated three times. Consistently differentially expressed spots (5-fold increase) were identified by MALDI-TOF-MS.
In-Gel Tryptic Digestion and MALDI-TOF MS Each differentially expressed spot obtained from respective gels was dehydrated with 50 ml ACN for 5 min, incubated in 50 mL 10 mM DTT at 56 C for 1 h and then incubated in 50 mL of 55 mM iodoacetamide (alkylating solution) for 45 min. Subsequently, respective spots were dehydrated with 50 mL of ACN, rehydrated in 5 mL porcine trypsin followed by the addition of 10 mL 25 mM ammonium bicarbonate. Proteolysis was carried out overnight at 37 C and stopped by adding 10 mL 2% formic acid. The resulting peptides were concentrated and mixed with a-cyano-4-hydroxycinammic acid (Sigma, St. Louis, MO), deposited on a 384-well MALDI target and air-dried. Analyses were performed using a Biflex IV (Bruker Daltonics, Bremen, Germany). The MS data were compared against tryptic peptide sequences from the SWISS-PROT database using Mascot (Matrix Sciences, London, UK) search algorithms.
Western Blot Analysis Each serum sample was loaded at the same concentration. Serum proteins were subjected to 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (Millipore, Billerica, MA) membrane. The membranes were blocked for 30 min with 5% nonfat milk in Tris-buffered saline with 0.05% Tween-20 (TBST). The samples were incubated overnight with goat anti-rabbit-desmin (1:400) or goat anti-rabbit-CFTR (1:400) antibodies at 4 C. The membranes were washed three times in TBST for 5 min each and then blotted for 2 h with horseradish peroxidase-conjugated secondary rabbit anti-goat IgG (1:500) at room temperature. The membranes were then washed twice with TBST followed by another wash in TBS. Immunoreactive bands were visualized using the Western Blotting Detection System (Pierce, Rockford, IL). Protein mass was compared after quantifying the intensity of protein bands by Quantity One software (Bio-Rad).
RESULTS Quantitative Comparison and 2-DE Proteomic Analysis
Serum from 10 rabbits with a similar weight distribution was pooled for 2-DE analysis. The average neutrophil counts were 2290 mL and the lymphocyte counts were 2380 mL in the rabbits prior to infection. In contrast, the average neutrophil counts were 540 mL and the lymphocyte counts were 960 mL 3 h post infection. Animals were monitored for 14 d during which time seven infected rabbits died. Gel images of serum harvested from rabbits either post-infection or after the onset of bacteremia/leucopenia were compared (Fig. 1). Approximately 200 protein spots localized in the 4–7 pI range and between 10 and 150 kDa were detected on the 2-DE gels. Five differentially expressed protein spots (defined by more than a 5-fold change in spot density) were identified. Protein Identification
Ten proteins in the bacteremic/leucopenic group with a differential expression of greater than 5-fold were
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differentially-expressed protein spots is shown in Fig. 2. Western Blot Analysis Validation of Up-Regulated Proteins
To confirm the 2-DE data, animals were infected as above and sera analyzed by Western blot. Six bacteremic/leucopenic rabbits had high levels of desmin expression (Fig. 3A) and CFTR expression was detected in 100% (6/6) of the serum samples examined (Fig. 3B). Multiple CFTR bands were observed in bacteremic/leucopenic samples at 3 h, indicating protein modification or degradation. Comparison of serum from the two treatment groups demonstrated that both of these proteins were up-regulated in sera from bacteremic/leucopenic animals. DISCUSSION
FIG. 1. 2-DE analysis of serum proteins from rabbits with bacteremia and leucopenia. Ten different-expressed protein spots are labeled with arrows. (Color version of figure is available online.)
subjected to in-gel digestion for MALDI-TOF MS analysis. Three over-expressed spots were identified that corresponded to desmin (spot 118, 165) and one spot corresponded to CFTR (cystic fibrosis transmembrane conductance regulator) (spot 304) (Table 1). Protein spots that could not be identified were excluded. Two proteins in the bacteremic/leucopenic group were down-regulated and corresponded to IL-4 (spot 22) and the IL-6 precursor (spot 35). Spots 92 and 155 could not be identified. Quantitative comparison of the five
Analysis of serum proteins represents an ideal means of establishing the diagnosis of different infections and pathologies due to the ease by which serum can be obtained [7]. Furthermore, new technologies have been developed for the identification of novel serumassociated biomarkers. For example, ovarian cancer can be diagnosed by carrying out TOF spectra of serum using a low-resolution mass spectrometer [8], and an extensive reference serum proteome database was established from trauma patients using a combination of major protein depletion, target protein enrichment, and multidimensional LC and MS [9]. In this study, we used comparative proteomic approaches (2-DE and MS) that identified 10 proteins whose expression profile was altered by more than 5-fold in rabbits with bacteremia and leucopenia, and five of these targets were identified. Intrinsic differences between rabbits with respect to differences in protein expression profiles was negated by pooling the serum from rabbits before and after infection, further strengthening the association of these five candidate biomarkers with bacteremia/leucopenia. Furthermore, the presence in serum of the two significantly up-regulated biomarkers (desmin and CFTR) was confirmed by Western blot analysis.
TABLE 1 Differentially Expressed Proteins Identified by MALDI-TOF-MS Spot
Accession number
Score
Sequence coverage
pI/Mr
Protein name
22 35 118 165 304
EDL33576 NP-112445 P02541 P02541 ABI93671
133 269 218 176 84
52% 55% 45% 44% 13%
9.21/10610 6.96/24597 5.21/53463 5.21/53463 8.98/168924
IL-4 IL-6 precursor Desmin Desmin CFTR*
*
CFTR ¼ cystic fibrosis transmembrane conductance regulator.
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FIG. 2. Quantitative protein analysis. Protein expression levels between the five differentially expressed protein spots identified in bacteremic/leucopenic rabbits (compared with healthy rabbits) were characterized. The volume of spots was normalized and quantified as relative intensity using ImageMaster 2D Software. (Color version of figure is available online.)
Desmin is the main intermediate filament (IF) protein expressed in cardiac, skeletal, and smooth muscles and is a primary cytoskeletal component that interacts with other proteins to maintain cellular integrity. Desmin is much more abundant in cardiac muscle (2% of total protein) than in skeletal muscle (0.35%) and is a major component of Purkinje fibers that facilitate heart function and coordination [10]. Previous reports suggested that desminopathy is associated with childhood-onset cardioskeletal myopathy, pure skeletal myopathy, adult-onset skeletal myopathy with cardiac involvement, cardiomyopathy with distal weakness, pure dilated cardiomyopathy, and distal myopathy with cardiac, respiratory, and smooth muscle involvement [11–17]. However, to the best of our knowledge,
FIG. 3. Western blot analysis. Pooled rabbit serum prior to and after the onset of bacteremia/leucopenia was examined by Western blot analysis and probed for either (A) desmin or (B) CFTR. Note the higher expression levels of desmin in bacteremia and leucopenia sera than that in pre-infection sera. The molecular weight of CFTR is about 168 kDa. Multiple bands corresponding to CFTR were observed in rabbits with bacteremia and leucopenia suggesting protein degradation or modification. (Color version of figure is available online.)
there are few reports linking desmin to bacteremia and leucopenia. Our Western blot analysis demonstrated that serum from bacteremic/leucopenic rabbits had higher desmin levels than control animals confirming the 2-DE gel experiment data. This result indicated that the myocardium was likely severely damaged in animals with bacteremia and leucopenia. CFTR is an cellular ion chloride channel that also regulates bicarbonate and sodium transporter channels [18], which has been know to be secreted in airway passages during Pseudomonas aeruginosa infections. In addition, it was demonstrated that CFTR dysfunction might lead to inefficient antimicrobial peptide function at the apical airway membrane surface epithelia [19, 20]. Pier et al. proposed that CFTR was an epithelial cell receptor for P. aeruginosa, with a CFTR peptide binding to the conserved outer-core oligosaccharide of the bacterium [21, 22]. Recently, this hypothesis was supported by Schroeder et al., who found that transgenic cystic fibrosis mice had a reduced capacity to clear an inoculum of P. aeruginosa applied to the nares. This reduced clearance rate was associated with decreased cellular internalization of P. aeruginosa [23]. The up-regulation of CFTR in this study supports work by Painter et al. who examined how extracellular chloride concentrations affected P. aeruginosa killing by normal neutrophils and found that polymorphonuclear neutrophil (PMN)-mediated bacterial killing was strongly dependent on extracellular chloride concentration i.e., neutrophils in a chloride-deficient medium inefficiently killed P. aeruginosa. This phenomenon can be explained by the fact that the chloride anion is essential for converting myeloperoxidase (MPO) into hypochlorous acid (HOCl) in PMNs [24]. Like other infectious agents, K. pneumoniae can induce the activation of immune responses and provoke the activation of a series of inflammatory processes associated with cytokine production. It is therefore imperative that a balance between immunity, pathogen clearance and maintenance of tissue integrity be maintained [25]. IL-4 (down-regulated in bacteremic/leucopenic rabbits) is acute-phase protein [26, 27] and has long been known as a cytokine with the capacity to rescue B cells from apoptosis and enhance their survival [26]. For example, transgenic C3H mice overexpressing IL-4 developed an autoimmune-type disorder resembling lupus [28]. Another important biologic function of IL-4 is T cell down-regulation [27]. In a previous study, Singh showed that the concomitant administration of a recombinant adenovirus vector encoding murine IL-4 and a T-cell activating peptide reduced IgG anti-dsDNA antibody production in a model of lupus [29]. Thus, neutrophil respiratory burst activity, like the occurrence of bacteremia with leucopenia, can
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be inhibited by IL-4, suggesting protective roles for IL-4 during inflammatory responses. IL-6, also down-regulated in bacteremic/leucopenic animals, is a cytokine with various biological activities, which includes the induction of inflammatory responses, immune regulation, and hematopoiesis [30]. Overproduction of IL-6 is associated with inflammatory diseases pathology, e.g., sepsis [31–33]. For example, it was demonstrated that IL-6 levels correlated with APACHE II scores, i.e., mortality rates increased significantly in patients who presented with IL-6 serum levels above 1000 pg/mL. In a murine colitis model, mice that developed Th1 cell-mediated colitis presented with wasting. Rat anti-mouse IL-6R antibody treatment suppressed colitis symptoms and wasting by inducing apoptosis of lamina propria T cells [34]. However, in the present study, both IL-4 and IL-6 were decreased, suggesting that the bacteremic/leucopenic animals were immunosuppressed. In conclusion, proteomic analysis identified serum components associated with the pathophysiology of leukopenia in the context of bacteremia. Although our results did not uncover all proteins likely associated with this disease manifestation, we identified two proteins that were increased in our infection group. To our knowledge, this is the first report linking desmin and CFTR together to infectious disease, suggesting that these proteins can be used as markers to predict clinical outcomes particularly during cases of bacteremia with leucopenia. ACKNOWLEDGMENTS The authors acknowledge support for this work by grants from the National Natural Science Foundation of China (30872456).
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