Proteomic analyses of murine macrophages treated with Bacillus anthracis lethal toxin

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MICROBIAL PATHOGENESIS Microbial Pathogenesis 41 (2006) 157–167 www.elsevier.com/locate/micpath

Proteomic analyses of murine macrophages treated with Bacillus anthracis lethal toxin R. Sapraa, S.P. Gauchera, J.S. Lachmanna, G.M. Bufflebena, G.S. Chiricaa, J.E. Comerb, J.W. Petersonc, A.K. Choprac, A.K. Singha, a Sandia National Laboratories, 7011 East Avenue, Livermore, CA 94551, USA Departments of Pathology, Sealy Center for Vaccine Development and Center for Biodefense and Emerging Infectious Diseases, The University of Texas Medical Branch, Galveston, TX 77555-1070, USA c Departments of Microbiology and Immunology, Sealy Center for Vaccine Development and Center for Biodefense and Emerging Infectious Diseases, The University of Texas Medical Branch, Galveston, 301 University Blvd., UTMB, Galveston, TX 77555-1070, USA b

Received 22 March 2006; received in revised form 22 June 2006; accepted 10 July 2006 Available online 1 September 2006

Abstract Bacillus anthracis is the etiological agent of anthrax and the bacterium produces a tripartite anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA represents the binding domain of the toxin and acts in concert with either LF, a metalloprotease, or EF, an adenylate cyclase, to form lethal toxin (LeTx) or edema toxin (EdTx), respectively. We analyzed the proteomics response of two murine macrophage cell lines (J774.1A and RAW264.7) following B. anthracis LeTx treatment to detect unique host proteins involved in anthrax infection using difference in-gel electrophoresis (DIGE) followed by nanoLC-MS for identification of the proteins. The comparative proteomics approach identified a set of proteins in each cell line that was consistently upregulated when the two macrophage cell lines were treated with LeTx. The upregulated proteins include those involved in energy metabolism, cytoskeleton structure and stress response. A subset of five proteins (ATP synthase b subunit, b-actin, Hsp70, vimentin, and Hsp60 homolog) was identified that were commonly upregulated in both cell lines. The proteomic data suggest the involvement of reactive oxygen species (ROS) in cell lysis as seen by the upregulation of proteins that lead to the production of ROS in both the cell lines used in our study. However, proteins that afford protection against ROS may play an important role in the survival of the macrophage to LeTx infection as shown by the differences in proteomic responses of the two cell lines to the action of LeTx. These identified proteins may have the potential to be used as biomarkers for diagnostics and therapeutics. r 2006 Elsevier Ltd. All rights reserved. Keywords: Bacillus anthracis; Lethal toxin; Macrophage cell lines; Difference in-gel electrophoresis (DIGE); Reactive oxygen species (ROS)

1. Introduction Bacillus anthracis, a spore-forming Gram-positive bacterium, is the etiological agent of anthrax, and the toxins produced by this organism are important virulence factors that are responsible for life-threatening sequelae in untreated patients [1]. The bacterium produces a tripartite anthrax toxin, which is composed of the three proteins protective antigen (PA), lethal factor (LF), and edema factor (EF). PA constitutes the binding domain for LF and Corresponding author. Tel.: +925 294 1260; fax: +925 294 3020.

E-mail address: [email protected] (A.K. Singh). 0882-4010/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2006.07.002

EF, and is required for internalization of the holotoxins lethal toxin (LeTx; PA+LF) and edema toxin (EdTx; PA+EF) into the host cell [2]. The PA, which is secreted as an 83 kDa protein by the bacterium, binds to either of two anthrax toxin-receptors, tumor endothelial marker 8 (ATR/TEM8) or capillary morphogenesis protein 2 (CMP2) on the host cell membrane [3,4]. The receptor-bound form of PA is then cleaved by a member of the furin protease family into a 20 kDa fragment and the 63 kDa-active form. Subsequently, the active PA63 moiety undergoes oligomerization forming a heptameric ring on the host cell membrane, which binds three LF and/or EF molecules [3]. LeTx and

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EdTx enter host cells via receptor-mediated endocytosis, with the low endosomal pH changing the conformation of PA, resulting in the formation of a membrane-spanning channel that facilitates the translocation of EF and LF into the cytosol of host cells [5]. EF is a calcium and calmodulin-dependent adenylyl cyclase that converts cytosolic ATP to cAMP [6,7]. It has been suggested that the resulting increase in cAMP might allow bacteria to evade the host’s innate immune response by suppressing polymorphonuclear neutrophil function and impairing host resistance to infection [8]. LF is a zinc-containing endopeptidase that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family of proteins [9]. LeTx has been shown to cleave the N-termini of the MAPKK proteins with the exception of MAPKK5 [10]. While the toxin is active against most cell types, it causes cytolysis only in certain types of murine macrophages [11]. Anthrax infections are categorized into inhalation, gastrointestinal, and subcutaneous forms, with inhalational anthrax being the most severe and is caused by inhalation of B. anthracis spores [12]. For successful infection, spores in the alveoli are phagocytosed by alveolar macrophages and carried to the mediastinal lymph node where they germinate. The bacteria must evade the innate immune cells by altering their functions; human macrophages are not lysed [11,13], but their capacity to kill the bacteria is diminished [14]. Subsequently, the bacteria multiply and spread to the bloodstream. It is imperative from a clinical perspective to detect the infection early and initiate antibiotic treatment; however, the lack of anthrax biomarkers during early infection prevents prompt diagnosis and causes delays in administration of appropriate therapy. Germination of bacterial spores inside macrophages is accompanied by the production of PA, LF, and EF, which reduce the resistance of the host to infection [15]. Since recent genomic studies performed with LeTx-treated murine macrophages [16] indicated global alteration of gene expression, it was logical to identify proteomic changes in the cells in an attempt to discover biomarkers that might aid in early detection of the disease. Therefore, we analyzed the proteomics response of two murine macrophage cell lines to LeTx treatment to detect unique protein biomarkers for anthrax infection. 2. Results 2.1. LeTx treatment of macrophages and host cell lysis We added PA and LF in a molar ratio of 5:1 in murine macrophage cell lysis experiments. This ratio was chosen since it provided an optimal cytotoxic response in murine macrophages [16]. After host cells were treated with the toxin, cell lysis was measured by the release of the cytosolic enzyme lactate dehydrogenase (LDH). When J774A.1 cells were exposed to LeTx, there was no significant or measurable release of LDH during the first 30 min (data

not shown). After the onset of cell lysis at 65 min (20% lysed), macrophages were completely destroyed (
95%) by 140 min (data not shown). However, a slower response was observed when RAW264.7 cells were treated with LeTx. No measurable lysis of RAW264.7 macrophages was noted during the first 90 min of treatment. We noted cell lysis (20–30%) at 120 min, which increased to 80% by 240 min (data not shown). The samples collected for proteomics analysis were at the mid-point of the response to the toxin and corresponded to
50% lysis. To ensure that the cytotoxicity was due to the action of LeTx, we treated control cell cultures with either phosphate-buffered saline (PBS) or PA alone and observed no LDH release. 2.2. Difference in-gel electrophoresis (DIGE) analysis of LeTx-treated macrophages Cells for proteomics analysis were harvested after 85 and 180 min of exposure to LeTx for J774A.1 and RAW264.7 cell lines, respectively. The time points for proteomic analyses were chosen taking two primary factors into account. First, for consistent comparison between the cell lines the samples compared have to be at the same stage of cell lysis to investigate proteins regulated in response to the LeTx effect. Second, since the J774A.1 cell line showed much faster kinetics of lysis as compared to the RAW264.7 cell line, the earliest time point where we could get proteomic data was chosen based on preliminary analyses of a 40 min time point after LeTx exposure which did not show any significant proteomic changes in response to the toxin (data not shown). At short times scales post exposure, the cells are more likely to adapt via posttranslational modification rather than whole proteome changes. The control murine macrophages were exposed to either PA or PBS and harvested at the same time point for analysis. Since it has been shown earlier that host cells do not respond to LF in the absence of PA [9], no experiments were conducted with macrophages exposed to LF alone. When comparisons were made with LeTx-treated host cells, the cell lysates from macrophages treated with PBS or PA alone were always labeled with the Cy3 dye and the cell lysates from LeTx-treated macrophages were always labeled with the Cy5 dye (See Section 5.5). When macrophages (J774.1 or RAW264.7) were exposed to PA alone, no changes were observed in the proteome as compared to PBS-treated macrophages (Fig. 1A). In these experiments, all proteins were within the 1.3–1.5-fold expression range, typical of the inherent noise in the proteomics experiment. However, based on the statistical evaluation of the variation in the baseline, we chose a 2-fold cutoff for data analysis of regulated protein expression. When macrophages were exposed to LeTx, approximately 4.5% (corresponding to 35 protein spots out of an average of 750 detected protein spots on a gel) of the total proteome, as detected by 2-dimensional (2-D) gel electrophoresis, was shown to be regulated (data not shown). Of the 35 regulated proteins, there were 10 spots

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Fig. 1. DIGE images of the proteomic comparison of PBS treated control cells with PA-only treated cells (Fig. 1A). DIGE images of the J774A.1 (1B) and RAW264.7 (1C) murine macrophage cell lines treated with LeTx. The proteins that are common and upregulated in both the cell lines have been circled in the images. (1) Hsp70; (2) Hsp60; (3) ATPase beta subunit; (4) vimentin and (5) actin. Proteins that are present in both treated and control cells appear as yellow spots, proteins that are present only in control samples, labeled with Cy3 fluorochrome dye, appear as green spots and the proteins upregulated when cells are treated with LeTx, labeled with Cy5 fluorochrome dye, appear as red spots. The pH labels on the image are approximate labels based on the pH of the IEF strips used.

that were donwregulated and 25 protein spots that were upregulated more than 2-fold when cells were exposed to LeTx. Only protein spots that were upregulated in response to the LeTx treatment were excised from the gel and digested for mass spectrometry analysis. Since our studies were focused on identifying proteins which could be used for diagnostic purposes, we examined in details only upregulated proteins, which could be more meaningful. The protein samples from macrophages exposed to LeTx were first analyzed using broad pH 3–10 range isoelectricfocusing (IEF) strips, and then we focused on smaller regions where most of the changes were concentrated.

Altogether three different pH gradient gels were performed, pH 3–10, followed by pH 4–7, and pH 3.0–5.6. The same size strips in the acidic range of the proteome resulted in better separation and resolution of proteins (data not shown). Data from both biological and technical repeat experiments were used for statistical analysis using the biological variation module of the DeCyder analysis software (GE Healthcare, Piscataway, NJ). Proteins that were shown to be statistically and consistently upregulated in three independent and different biological experiments were selected for mass spec analysis (see materials and methods for details (Sections 5.7–5.9). A typical 2-D

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electrophoresis pattern with protein spots that were consistently upregulated in J774.1 (Fig. 1B) and RAW264.7 (Fig. 1C) cells are shown. Spots 1–5 represented Hsp70 (#1), Hsp60 (#2), ATPase beta subunit (#3), vimentin (#4), and actin (beta-actin) (#5), as determined by mass spectrometric analysis and are discussed below. 2.3. Protein identification using mass spectrometric analysis Regulated protein spots were selected and digested with trypsin, and the resulting peptides were processed for LCMS analysis. The identified proteins had a score equal to or greater than the 95% confidence limit threshold estimated by Mascot software. The proteins identified from MS analysis were then compared to the DIGE images and their relative positions on the gels, and only the proteins that

were found in analyses from multiple gels were considered significant. We estimated that approximately 75% of the total regulated proteins were identified from our analyses, based on several measured variables such as multiple picks from the same ‘protein gel spot’ and the success rate of protein identification returned from MS analysis. The failure to identify other selected protein spots could be attributed to low concentration of proteins in the gel, low concentration of proteins in the selected protein gel plugs, experimental losses of protein and peptide fragments during processing, mass-spec methods, and low confidence scores of identifications returned from Mascot searches. As shown in Tables 1 and 2, we divided the identified proteins into four major categories—proteins involved in energy production, cytoskeletal protein, stress-response proteins, and miscellaneous other proteins that were either

Table 1 Identity of proteins from J774A.1 cell line that are upregulated in response to LeTx Volume ratioa

MW (Da)b

pIb

NCBI Accession #

Location

Cellular function

2.1 2.0 2.7

56 632 51 171 80 724

5.14 4.92 5.51

gi|23272966 gi|1374715 gi|21704020

Mitochondria Mitochondria Mitochondria

ATP production ATP production Electron transfer; respiration

2.3

53 420

5.75

gi|14548301

Mitochondria

Electron transfer; respiration

Cytoskeleton Actin (Beta-actin)* A–X actin Lamin A

3.0 2.3 2.8

42 053 42 009 74 450

5.29 5.21 6.05

gi|14548301 gi|309090 gi|1346412

Cytoplasm Cytoplasm Nucleus

Cytoskeleton; cell mobility Cytoskeleton Nuclear envelope component

Stress response Hsp70* Vimentin*

2.1 2.4

73 970 53 698

5.70 5.09

gi|2231704 gi|202368

Cytoplasm Cytoplasm

Stress response Stress response cytoskeleton fibers

Miscellaneous Predicted protein

2.6

42 278

5.30

gi|27687455

[Cytoplasm]

[Actin-like protein; RIKEN cDNA 4732495G21] [Chaperonin; Cpn60/ GroEL/Hsp60 family] [Ribosome binding/ chromosome segregation ATPases motif] [RNA-binding/RNA recognition motif] [Stress response/type II keratin motif] [GTP-binding elongation factor]

Protein ID

Energy production ATP5b ATP synthase b subunit* NADH dehydrogenase Fe–S protein Ubiquinol-cytochrome-c reductase complex core protein I

Unnamed protein product* Unnamed protein product

2.2

58 061

5.91

gi|1334284

[Cytoplasm]

2.7

97 441

5.80

gi|12860388

[Membrane]

Unnamed protein product

2.0

30 018

8.31

gi|12851175

[Membrane]

Unnamed protein product

2.2

30 107

5.4

gi|12843914

[Cytoplasm]

Elongation factor 2 (EF-2)

3.4

96 304

6.31

gi|119168

[Cytoplasm]

[ ] indicates the putative function and location of the identified proteins. *Indicates proteins that are found common to the proteomics response of J774A.1 and RAW 264.7 cell lines to LeTx (see results for details). a Average volume ratio of the regulated proteins based on three independent experiments b Predicted MW and pI from sequence analysis.

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Table 2 Identity of proteins from RAW264.7 cell line that are upregulated in response to LeTx Protein ID

Energy production GAPDH ATP synthase b subunit* Dihydrolipoamide dehydrogenase Enolase 1, alpha

Volume ratioa

MW (Da)b

pIb

NCBI Accession #

Location

Cellular function

Glycolysis ATP production Sugar, amino acid metabolism Sugar, amino acid metabolism

2.2 2.7 2.3

36 090 51 171 54 748

8.14 4.92 7.97

gi|37590767 gi|1374715 gi|2078522

Mitochondria Mitochondria Mitochondria

2.2

47 347

6.37

gi|12963491

Mitochondria

Cytoskeleton Actin (Beta-actin)*

3.3

42 053

5.29

gi|1351867

Cytoplasm

Cytoskeleton; cell mobility

Stress response Hsp70* Vimentin*

2.3 2.7

73 970 53 698

5.7 5.09

gi|2231704 gi|202368

Cytoplasm Cytoplasm

GRP 78

4.1

72 526

5.09

gi|1304157

Cytoplasm

Protein disulfide isomerase

2.5

49 058

5.05

gi|58037267

ER lumen

Chaperone/stress response Stress response cytoskeleton fibers [Hsp70-related protein/ stress response] Thioredoxin motif/

Miscellaneous Unnamed protein product* Unnamed protein product

2.3

58 061

5.91

gi|1334284

[Cytoplasm]

2.9

57 507

4.79

gi|54777

[ER lumen]

Unnamed protein product Anx5 [Annexin]

2.2 2.3

11 361 35 773

11.18 4.83

gi|12847763 gi|13277612

[Nucleus] [Cytosol]

Proliferating cell nuclear antigen (PCNA) [40 s Ribosomal protein]

2.1

291 118

4.57

gi|13124447

[Nucleus]

2.3

32 530

4.65

gi|51766344

[Cytosol]

[Chaperonin; Cpn60/ GroEL/Hsp60 family] [Protein disulfide isomerase/ thioredoxin] [Histone 4 protein] Anticoagulant activity/Ca ion binding Regulation of DNA replication/DNA binding [Laminin receptor/protein biosynthesis]

[ ] indicates the putative function or putative location of the identified proteins. *Indicates proteins that are found common to the proteomics response of J774A.1 and RAW 264.7 cell lines to LeTx (see results for details). a Average volume ratio of the regulated proteins based on three independent experiments. b Predicted MW and pI from sequence analysis.

of unknown function or had a broad range of functions. When J774A.1 cells were treated with LeTx, the mitochondrial membrane proteins NADH dehydrogenase Fe–S protein and ubiquinol-cytochrome-c reductase complex core protein-I that are part of the respiratory electron transport chain were shown to be upregulated 2.7- and 2.3-fold, respectively. The other two proteins identified in energy production were ATPase subunits that were also from the mitochondrial membranes. The cytoskeleton proteins b-actin and A–X actin were upregulated 3.0- and 2.3-fold, respectively, and the nuclear membrane component lamin A was shown to be upregulated 2.8-fold when macrophages were exposed to LeTx. Stress response proteins, heat-shock protein Hsp70, and vimentin were expressed more than 2.1-fold under the same conditions after LeTx treatment of macrophages. Although, vimentin is a cytoskeleton protein, we classified it as a stressresponse protein due to its role in the stress response. Interestingly, there were five proteins that were upregulated between 2.0- and 2.7-fold and had no characterized

annotated function. However, based on sequence analysis, these matched proteins were involved in the stress response, cytoskeleton, RNA, and GTP binding (Table 1). When RAW264.7 cells were exposed to LeTx, metabolic proteins like glyceraldehyde-3-phosphate dehydrogenase (GAPDH), dihydrolipoamide dehydrogenase, and enolase 1-alpha were upregulated between 2.2- and 2.3-fold (Table 2). The ATPase b-subunit increased 2.7-fold when compared to macrophages exposed to PBS alone. Only one cytoskeleton protein, b-actin, was upregulated in RAW264.7 cells. There were more stress-response proteins, namely, Hsp70, vimentin, glucose-regulated protein (GRP) 78, and protein disulfide isomerase (PDI) that were identified from RAW264.7 cells compared to J774.1 macrophages. There were six miscellaneous proteins that were upregulated, three were unknown proteins, and one represented annexin 5 (Table 2). Proteins whose levels were increased in both macrophage cell lines include ATP synthase b subunit, b-actin, Hsp70, vimentin, and Hsp60 homolog (Tables 1 and 2).

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3. Discussion 3.1. Proteomic changes in murine macrophages Cytotoxicity in macrophages exposed to LeTx has been correlated with the production of reactive oxygen species (ROS), also commonly known as oxygen radicals [17]. Mutant murine macrophages, deficient in the production of ROS, correspondingly, were found to be insensitive to the effects of LeTx. Our proteomics experiments supported this scenario, where proteins that have been shown to have a role in the production of ROS were upregulated in macrophages exposed to LeTx. Macrophages generate generate ROS [18] for their protection from invading pathogens,however, high levels of ROS pose a risk for the macrophage themselves. Complex-I in the mitochondrial membranes is a major site for the production of ROS and is shown to overproduce oxygen radicals and release cytochrome c into the cytosol [19,20]. LeTx treatment of the J774A.1 cells showed that components of the complexINADH dehydrogenase subunit and ubiquinol-cytochrome-c reductase subunit were differentially overexpressed in LeTx-exposed macrophages (Table 1). Similar observations were reported in recent study by Kuhn et al. [21] who demonstrated that LeTx caused an upregulation of proteins involved in the oxidative stress response in murine macrophages [21]. RAW264.7 macrophages exhibited a slightly different response in the energy pathway proteins that were upregulated when exposed to LeTx (Table 2). The complex-I proteins were not upregulated, as noted with the J774A.1 cells; however, glycolytic pathway proteins, such as GAPDH, dihydrolipoamide dehydrogenase, and enolase 1 were upregulated in response to LeTx exposure. New and additional roles for these proteins have recently been discovered that might explain their roles in the cytolysis of macrophages. For example, GAPDH, in addition to its role as a central glycolytic protein, has recently been implicated in performing other functions such as membrane fusion, RNA binding and nuclear RNA transport [22,23]. However, the most interesting role in relation to LeTx-related toxicity is its suggested role in apoptosis of cultured neurons [23], as GAPDH expression was significantly upregulated in neurons during apoptosis induced by cytotoxic agents. Furthermore, it has been suggested that GAPDH monomer migrates to the nucleus in response to oxidative stress and leads to events that result in cell death by an as yet unknown mechanism [22]. Enolase 1 shares a high degree of homology (495%) with the Myc-binding protein-1 (MBP-1), ectopic expression of which induces cell death and growth suppression [24]. Based on the homology and the interaction of MBP-1 with histone deacetylase, it is possible that Enolase 1 might cause transcriptional suppression in LeTx-exposed macrophages. Dihydrolipoamide dehydrogenase is shared among four mitochondrial complexes, three a-ketoacid-dehydrogeanse complexes and the glycine cleavage system [25].

However, dihydrolipoamide dehydrogeanse, in addition to its role in TCA cycle and other metabolic processes, has also been shown to interact with toxin tetraflourothylcysteine (TEFC), forming an adduct that ultimately results in cell death [26]; a role that could possibly be involved in macrophages treated with LeTx. Cells undergo cytoskeletal rearrangement during cell death due to injury, and actins, which form one of the major cytoskeleton proteins, were upregulated in macrophages treated with LeTx. Actins bind ATP, and the increase in ATPase might also be in response to the increase in actin in the cell [27]. Another class of cytoskeleton proteins is vimentin. Vimentin is a class III intermediate filament, which has been shown to be upregulated when cells are stressed in general [28–30]. Indeed in J774.1 and RAW264.7 cells exposed to LeTx, vimentin levels were increased (Tables 1 and 2). However, other major stress-response proteins, such as Hsp70 and an Hsp60 homolog, were also elevated in J774.1 and RAW264.7 macrophages exposed to LeTx. The latter are molecular chaperones that are coordinately expressed in response to stress. Both Hsp70 and Hsp60 also displayed a requirement for binding of ATP for function. The Hsps are generally considered to be anti-apoptotic due to their role in the inhibition of the activation of caspases [31]. However, the role of Hsp60 remains enigmatic in apotosis with reports of Hsp60 accelerating the activation of caspase 3 during apoptosis, as well as overexpression of Hsp60 preventing apoptosis in cardiac cells [32,33]. The role of Hsp60 may be dependent on the type of cell and the time of expression. Overexpression of Hsp70 proteins, like Hsp72 has been reported to protect cells from apoptosis [34]. The Hsps function as molecular chaperones in aiding protein folding, unfolding and targeting irreversibly denatured proteins for clearance, all of which require ATP [34]. Overall, the upregulation of ATPases in LeTx-treated macrophages (Tables 1 and 2) might be correlated with the need for ATP to produce more stress-response proteins and ATP-binding cytoskeletal proteins that were upregulated at the same time. ATP production might also be associated with its need for cytoskeleton rearrangement during apoptosis as is evidenced by the production of actin, vimentin, and other cytoskeletal proteins, as well as for molecular chaperones PDI, Hsp70 and Hsp60. Furthermore, ATPases are known to be necessary during the action of LeTx, since ATP mediates acidification of vacuoles needed for the translocation of LF in the host cytosol [35]. That ATPases are upregulated is also not surprising given the upregulation of complex-I proteins; complex-I is essential for the formation of membrane potential, which is the driving force for ATP production and is carried out by mitochondrial membrane bound ATPases. 3.2. Differences in response to LeTx between J774A.1 and RAW264.7 macrophages—thioredoxins and ROS The two cell lines J774A.1 and RAW264.7 used in this study are both derived from the BALB/c mice [36,37].

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These cell lines have been shown to display all the normal biochemical and physiological characteristics of primary murine macrophages. The differences between the two lines are that while the J774A.1 cells are derived from female mice and isolated from reticulum cell sarcoma, the RAW264.7 cells are from male mice and derived from abelson murine leukemia virus-induced tumor. As noted, the RAW264.7 cell line was less sensitive to the action of LeTx when compared to the J774.1 cells (Fig. 1). Our proteomics results might provide one explanation. As mentioned earlier, it has been reported that cytolysis in macrophages might result from the generation of ROS. It was seen from our proteomics analyses that in J774A.1 cells, the proteins implicated in overproduction of ROS were upregulated in response to LeTx when compared to RAW264.7 cells, and this might contribute to a ‘stronger burst’ of ROS in J774A.1 cells (Tables 1 and 2). Secondly, in contrast to J774A.1 cells, two proteins that were overproduced in RAW264 cells were thioredoxin and PDI (Table 2). Thioredoxins are a class of proteins that have –CxxC– motif and are implicated in oxidative stress [38–40]. PDI are multifunctional proteins that contain 4 thioredoxin-like folds. One of the recently demonstrated functions of PDI is inactivation of the A1 subunit of cholera toxin [41]. It can also function as an ATPdependent molecular chaperone in apoptotic pathways [22]. Furthermore, PDI can act in concert with thioredoxins in pathways that help cells dispose of toxic free radicals [38,39]. As is seen from our proteomics results, thioredoxin and thioredoxin-like proteins were upregulated in RAW264.7 cells, but not in the J774.1 cells. It is plausible that thioredoxins might provide extra protection to the cells during ROS and reactive nitrogen species (RNS) bursts induced by LeTx in RAW264.7 cells, but not in J774.1 cells. Further, it was also seen from our proteomics analysis that ROS-producing proteins were upregulated in J774A.1 cells and that they might play an additional role in cell toxicity as compared to the RAW264.7 cells. Based on our previous Affymetrix GeneChip analysis, many of the genes that were altered in their expression were also related to energy production and cytoskeletal rearrangement [16]. As noted earlier, the difference between the two cell lines is the original source and method of their generation . Therefore, it is possible that the difference in response of macrophages (RAW264.7 versus J774A.1) to the LeTx treatment may be related to the expression of proteins either due to the sex of the mice or the source from which these cell lines were derived. These differences are currently being studied to elucidate the exact cause of the differential response of the two cell lines to the action of LeTx. Another explanation for the differences in toxin-susceptibility may be a genetic drift between the two mouse colonies from which the cell lines were derived. Murine macrophage susceptibility to LeTx has been mapped to a polymorphic gene, Nalp1b [42]. Therefore, it is possible that polymorphisms in this gene between two distinct mouse colonies may result in the

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differences in LeTx sensitivity of the two representative cell lines. 3.3. Proteomic biomarkers A number of non-immunologic proteins were observed to be consistently and significantly overexpressed in early response (
90 min) to exposure to LeTx. The five proteins that were expressed in both the cell lines in response to LeTx exposure are ATP synthase b-subunit, b-actin, Hsp70, vimentin, and a Hsp60 homolog. Perhaps the most interesting protein is the protein annotated in the genome as ‘unnamed protein product’, which is an Hsp60 homolog (Tables 1 and 2). Its level was upregulated in both the macrophage cell lines. While it is not surprising that an Hsp60 homolog is expressed, since the cells are stressed and undergo apoptosis in response to LeTx, the fact that this specific homolog of Hsp60, annotated in the genome only as an ‘unnamed protein product’, is overexpressed in both cell lines may lend specificity for its use as a biomarker. The stress response proteins are helpful biomarkers since these are broad response biomarkers that could be used for initial screening of samples. Furthermore, these would be expressed in response to even low levels of stressors, i.e., LeTx, and in combination with other biomarkers may provide the specificity for initial screening. The Hsp60 homolog is a highly conserved protein which shows 493% identity to similar sequences from Mus musculus, Rattus norvegicus, Homo sapiens and Bos taurus. However, commercially available antibodies to human Hsp60 were not found to cross react with the Hsp60 homolog and further experiments are underway to generate antibody specific to the protein for future experiments and testing in cell culture and mouse models. The identified proteins, either alone or in combination with the immune markers such as cytokines and chemokines, can be used in the early identification of anthrax infection (further investigations are ongoing for validation of these targets as biomarkers). Murine macrophages (RAW264.7 and J774.1) have been used extensively as a model system for the study of LeTx and a significant amount of information as it pertains to the effects of the toxin on these cells has been published [13,43]. Because of the amount of information on these cells and ease of propagation, these tissue culture models have been used as the first line of investigation. However, they may not be a suitable surrogate for human macrophages, which are inactivated but not lysed by the toxin [13]. Future studies using the proteomics approach detailed here on the effects of LeTx on primary human macrophages will better define the biomarkers of an anthrax infection and form part of our future research. Studies are also currently under way to understand the role of EdTx alone and in combination with LeTx to further elucidate the proteomic response in macrophages to anthrax infection and the validity of the proteins identified in this study as potential biomarkers.

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4. Conclusions Our comparative proteomic approach identified a set of proteins, consistently and significantly upregulated in response to the LeTx treatment, that may suggest a role of ROS in cytolysis of murine macrophages. Furthermore, we identified 5 proteins that were common to both cell lines most intriguing of which was the Hsp60 homolog, which might be valuable as a biomarker. Identification of cellular marker proteins, like the Hsp60 homolog, may also be crucial in the detection of and during treatment of human macrophages with LeTx. The role of this protein will be explored in the future and ongoing work. The proteomic changes observed were for proteins that were produced at high enough levels to be visualized on a 2-D-PAGE and downstream identification by MS analyses. Consequently, we expect that the membrane proteins, which would have an effectively lower concentration in the whole cell lysate, would be under represented in this sample. To get better coverage of the sample, we will incorporate sub-cellular fractionation of the cell sample prior to proteomic analysis in our future studies. The limitations of the technique are that the low abundance proteins, like signal transduction pathway proteins and immune markers like cytokines, are not identified by this approach. While there has been an attempt to identify the cytokine response to LeTx, a proteomic response using the sensitive technique like difference in-gel electrophoresis has been lacking and our study represents a step in that direction. 5. Materials and methods 5.1. Chemicals All chemicals used for cell culture and proteomic analysis were of the highest purity. All reagents used in mass spectrometry analysis were of mass spectrometry grade. 5.2. Growth conditions for macrophage cell lines Murine macrophage cell lines J774A.1 and RAW264.7 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured as monolayers in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% fetal bovine serum, 100 mg/mL penicillin/streptomycin, and 2 mM L-glutamine (Mediatech, Inc., Herndon, VA) at 37 1C in a humidified atmosphere of 5% CO2. The cultured cells were grown to 70–80% confluence in 150 cm2 flasks for preparation of samples that were used in the proteomics analysis. 5.3. LeTx treatment B. anthracis recombinant LF and PA were obtained from either List Biological Laboratories (Campbell, CA) or from

the Biodefense and Emerging Infections Research Resources Repository (BEI Resources, Manassas, VA). The host cells were treated with 12 nM of PA (1.0 mg/mL) and 0.2 mg/mL of LF, which provided optimal lysis of the macrophages [16]. Our controls included macrophages treated with either PBS or PA alone (1.0 mg/mL) with no LF. After the cells were treated with LeTx and appropriate controls, aliquots of the culture medium were removed at regular intervals and assayed for the release of lactate dehydrogenase (LDH) enzyme as an indicator of cell lysis. The LDH released into the supernatant of the macrophage cultures was measured using the CytoTox 96s nonradioactive cytotoxicity assay kit (Promega, Madison, WI) and quantified by measuring wavelength absorbance at 490 nm as recommended by the manufacturer. For the LDH release assay, the host cells were seeded in 96-well tissue culture plates. 5.4. Sample preparation for proteomics analysis The experimental cell cultures were scraped and centrifuged at 2000  g to sediment the cells. The pellet was washed three times with ice cold PBS and lysed in a buffer containing 2% CHAPS in 20 mM Tris–HCl buffer (pH 8.0). The protein lysate was clarified by centrifugation at 8000  g to remove the cell debris, and the supernatants were used for protein measurements and further analysis. The protein content of the supernatant was measured using the BCA assay (Pierce Biochemicals, Rockford, IL). Sample cleanup was performed by precipitating the protein with 4  the volume of acetone containing 50 mM dithiothreitol (DTT). The precipitated protein in each sample was resuspended in a buffer containing 7 M urea, 2 M thiourea and 4% (w/v) CHAPS (Sample Buffer A) and adjusted to a final protein concentration of 2.5 mg/mL. 5.5. Sample labeling and 2-D gel electrophoresis The protein samples in Sample Buffer A were labeled for differential in-gel electrophoresis (DIGE) analysis using the N-hydroxy succinamide (NHS) ester of Cyanine fluorescent dyes (GE Healthcare, Piscataway, NJ) according to the manufacturer’s recommendations. For sample labeling, a total of 400 pmol of the dye was added to a 50 mg aliquot of the protein sample in 20 mM Tris HCl buffer (pH 8.5) according to manufacturer’s recommendation. The samples were vortexed and incubated on ice in the dark for 30 min, and 1 mL of 10 mM lysine was added to quench the reaction. The experimental cell lysates were compared in pairs and for each pair, three sample sets were labeled—the two experimental cell lysates to be compared and a subset of the two samples comprised of equal amounts of the two abovereferred experimental cell lysates, henceforth referred to as the pooled standard. In a pair-wise analysis of the cell lysates, the following samples were compared (the labeleddye used for the cell lysates is shown in parenthesis)—we

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conducted comparison of PBS-treated host cells (Cy3) with PA-treated (Cy5) macrophages, and PA-treated cells (Cy3) with LeTx-treated (Cy5) macrophages. For each pair to be compared, the respective pooled sample was labeled with Cy2 dye. Pooled samples labeled with Cy2 formed the baseline for measurement and served as internal controls. First dimension IEF separation was performed with 18 cm Immobiline pH gradient (IPG) strips (GE Healthcare, Piscataway, NJ). The cell lysates were prepared for IEF by adding 50 mg each of the three labeled samples, 500 mg of the unlabeled protein, 0.5% (v/v) IPG buffer, 1.25% (v/v) of Destreak reagent (GE Healthcare) before adjusting to a final volume of 340 mL in Sample Buffer A. Active strip rehydration at 50 V for 14 h was used for rehydration of the IPG strip with the protein sample. The IEF was performed using IPGphor (GE Healthcare) at 20 1C for a total of 54 000 V h (VHr). Post-IEF, each strip was equilibrated in 10 mL of 6 M urea, 20% glycerol, and 1% (w/v) sodium-dodecyl sulfate buffer (Buffer B) containing 15 mM DTT for 15 min followed by a second equilibration with 55 mM iodoacetamide in Buffer B. The strip was applied to the top of an 18 cm  26 cm 10% polyacrylamide gel (Jule Inc, Milford, CT) and immobilized by overlaying the strip with 0.5% agarose solution. The second dimension was performed at a constant 2 W gel1 for 16 h.

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5.8. Liquid Chromatography—Tandem Mass Spectrometry (LC-MS/MS) A NanoLC-2D system (Eksigent Technologies, Dublin, CA) interfaced with a QTOF Ultima (Waters, Milford, MA) was used for LC-MS/MS. An in-house packed column (Michrom Magic C18 5 mm 200 A˚, 0.1  200 mm) was equilibrated with 5% solvent B (solvent A ¼ 98% H2O, 2% ACN, and 0.2% formic acid and solvent B ¼ 10% H2O, 90% ACN, and 0.2% formic acid). Samples were dissolved in 20 mL solvent A just prior to analysis, and a 10 mL aliquot was loaded directly onto the column at 1 mL/min with 5% solvent B in 11 min. Peptides were eluted with the following gradient: B ¼ 5–60% in 24 min and B ¼ 60–80% in 2 min. Tandem mass spectra (MS/MS) were acquired in datadependent scan mode using the following parameters: 1 s survey scans (m/z 350–1800) were used to select the 3 most abundant species for MS/MS. Only 2+ and 3+ ions with intensity greater than 40 counts per scan were considered. Up to 6, 1.9 s MS/MS scans were acquired for each precursor ion, and the collision energy was determined by charge-state recognition. All MS/MS scans from a given precursor were summed and peaklists generated with MassLynx 4.0. 5.9. Protein identification

5.6. Protein visualization and image analysis The gels were fixed in a solution containing 10% methanol and 5% acetic acid and stained with Sypro Ruby (Invitrogen, Carlsbad, CA) for total protein staining after fluorescence scanning the gels for Cy-dyes. The gels were scanned for Cy2, Cy3, and Cy5 dyes using the Typhoon 9400 gel imager (GE Healthcare) and sypro ruby using preset filters. Gel image analysis was performed using the DeCyder software suite (GE Healthcare) for image overlay and image analysis to identify the regulated protein spots. The identified protein spots were picked and deposited onto 96 well plates for digestion and mass spectrometric analysis.

Peaklists were submitted to a MS/MS ion search against a rodent subset of the NCBInr database using an in-house copy of Mascot v. 1.9 (Matrix Science Ltd., London, UK) [45]. Only tryptic peptides with up to 1 missed cleavage were considered. The mass tolerance for both the precursor and product ions was set at 70.2 Da. Carbamidomethylation of cysteine and optional oxidation of methionine were taken into account. Only protein identifications with a score equal to or greater than the 95% confidence limit threshold estimated by Mascot were accepted. Furthermore, a reverse database search [46] was performed to confirm that the distribution of scores for this reverse search (i.e. false positive scores) did not overlap with the scores of protein identifications included in the results (data not shown).

5.7. Protein digestion

5.10. Statistical analysis

The gel plugs were digested using a modified protocol from Shevchenko et al. [44]. The gel plugs were destained using 50% acetonitrile (ACN) and 100 mM ammonium bicarbonate (ABC) buffer and dehydrated with a final wash of 100% ACN. The resulting gel plugs were vacuumdried and rehydrated with 125 ng of trypsin in 15 mL of 25 mM ABC buffer. The protein plugs were digested overnight at 37 1C, and the peptides were extracted using 50% ACN and 5% formic acid buffer. The extracted peptides were vacuum dried and resuspended in 98% H2O, 2% ACN, and 0.2% formic acid (solvent A).

Wherever applicable, the experiments were performed in triplicate and the data analyzed by either the Student’s t test or multiple group comparison using the Bonferroni ttest. Acknowledgments Financial support for this research was provided by the Sandia National Laboratories Lab Directed Research and Development Program. Sandia is a multi-program laboratory operated by the Sandia Corporation, a Lockheed

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Martin Company, for the United States Department of Energy under Contract DE-AC04-94AL85000. Funding for J. W. Peterson and A. K. Chopra was provided by a contract from NIAID (NO1-AI-30065), a Grant from NIAID (U01AI5385802), and a Grant from the US Army (DAMD170210699). We also acknowledge the support of the Center for Commercialization of Advanced Technology (CCAT) (52109A/7805) and the National Institute of Health, National Institute of Allergy and Infectious Diseases for funding SBIR R43 AI052901-01A1, R43 AI058458-01 and R43 AI058458-02. J.E. Comer, a predoctoral fellow, was supported by National Institutes of Health T32 Predoctoral Training Grants in Emerging and Tropical Infectious Disease (2T32A8007526) and in Biodefense (1T32AI060549).

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