The role of a Brugia malayi p38 MAP kinase ortholog (Bm-MPK1) in parasite anti-oxidative stress responses

Filariasis, caused by thread-like nematode worms, affects millions of individuals throughout the tropics and is a major cause of acute and chronic morbidity. Filarial nematodes effectively evade host immunolog- ical responses and are long lived within their hosts. Recently an emphasis has been placed on enzymatic and non-enzymatic anti-oxidant systems which counteract the generation of reactive oxygen species (ROS) by macrophages and granulocytes, a first line of defense against parasites. We have characterized an anti-oxidant pathway in the filarial parasite Brugia malayi related to the evolutionarily conserved human mitogen-activated p38 protein kinase and the Caenorhabditis elegans PMK-1 protein kinase stress pathways. We have expressed a recombinant p38/PMK-1 ortholog from B. malayi (Bm-MPK1) and have successfully activated the kinase with mammalian upstream kinases. In addition, we have demonstrated inhibition of Bm-MPK1 activity using a panel of known p38 inhibitors. Using the potent and highly selec- tive allosteric p38 inhibitor, BIRB796, we have implicated Bm-MPK1 in a pathway which offers B. malayi protection from the effects of ROS. Our results, for the first time, describe a stress-activated protein kinase pathway within the filarial parasite B. malayi which plays a role in protecting the parasite from ROS. Inhi- bition of this pathway may have therapeutic benefit in treating filariasis by increasing the sensitivity of filarial parasites to ROS and other reactive intermediates.

1. Introduction

Pathogenic filarial nematodes affect the lives of over 120 mil- lion people and place over one billion people at risk of infection [1,2]. Filarial diseases have a significant economic and psychosocial impact in endemic areas, disfiguring and incapacitating millions of individuals. The filarial parasites that pose the most serious pub- lic health threats are Wuchereria bancrofti, Brugia malayi and Brugia timori, the causative agents of lymphatic filariasis (elephantiasis), as well as Onchocerca volvulus and Loa loa, the causative agents of sub- cutaneous filariasis (e.g. river blindness). In spite of some success in treating filiarial diseases with Mectizan® (ivermectin), albenda- zole, and diethylcarbamazine, there is a need for new drugs. Current drugs target the immature stages (microfilariae) but not the long- lived adult worms. There is also significant toxicity associated with using these drugs and drug resistance is emerging [3,4]. Identifica- tion of new therapeutic agents for the treatment of filarial disease requires a better understanding of filarial homeostasis and critical parasite biochemical pathways amiable to drug targeting, an effort greatly enhanced with the sequencing of the B. malayi genome [5]. Our laboratory has been studying protective responses to oxida- tive stress in the filarial parasite B. malayi. Filarial and other parasites exhibit a number of defense mechanisms permitting the parasite to establish infection in the presence of host immune responses, allowing for long term survival within the host [6–9]. One response used by the host to thwart infection is the genera- tion of reactive oxygen species (ROS) by macrophages, neutrophils, eosinophils and basophil granulocytes which can cause severe damage to the parasites through oxidation of proteins, lipids, and nucleic acids. Filarial parasites have elaborated a number of antiox- idant mechanisms and are known to effectively evade oxidative stress mediated by ROS [9,10]. It has been demonstrated that for the distantly related non-parasitic nematode, Caenorhabditis elegans, the critical pathway for dealing with oxidative stress, detoxifica- tion of xenobiotics as well as innate immunity, involves a protein kinase called PMK-1 [12–14]. PMK-1 is a C. elegans ortholog of the human stress-activated protein kinase p38 (p38), and a member of the mitogen-activated protein kinase (MAPK) superfamily. Human p38 is a serine/threonine protein kinase, consisting of four isoforms (p38α, β, γ and 6) that play important roles in cellular responses to external stress signals including osmotic stress, viral infection, ultraviolet light, heat, and inflammatory cytokines [15]. Activation of upstream kinases, such as apoptosis signal-regulating kinase 1 (ASK-1), in response to stress leads to the activation of mitogen- activated protein kinase kinases 3 and 6 (MKK3 and MKK6) which dually phosphorylate the TGY motif within the phosphorylation lip of p38, leading to its activation. Activated p38 subsequently phosphorylates and activates several downstream protein kinases and transcription factors involved in the regulation of genes that mediate a variety of anti-stress responses [15]. Similarly, PMK-1 is activated by dual phosphorylation of a TGY motif by SEK-1, an ortholog of MKK6, in turn leading to the phosphorylation of SKN- 1, a transcription factor involved in mesendodermal specification and oxidative stress responses [12,16]. In this study, we report for the first time the characterization of a closely related p38/PMK- 1 ortholog expressed in the filarial parasite, B. malayi, which we termed Bm-MPK1. Bm-MPK1 orthologs are present in other filarial parasites such as W. bancrofti and L. loa as well as other, non-filarial parasites, such as Echinococcus multilocularis (Fig. 1). Bm-MPK1 and its orthologs exhibit the characteristic 12 domain structure of the mitogen-activated protein kinase family as well as the highly conserved TGY activation motif in domain VIII (Fig. 1). It should be noted that protozoal parasite protein kinases, including stress- activated protein kinases, have been receiving significant attention as potential therapeutic targets [17].

Fig. 1. Clustal W alignment of human and nematode p38 MAPK catalytic domains. Highly conserved segments are highlighted in grey. Roman numerals indicate MAPK subdo- main regions. The TGY dual phosphorylation motif is highlighted in bold font (arrow). The corresponding accession numbers are: Brugia malayi: Bm-MPK1, A8PQS0; Wuchereria bancrofti CMGC/MAPK/p38 protein kinase, WUBG 01668.1/WUBG14523.1(Broad Institute Filarial Database); Loa loa CMGC/MAPK/p38 protein kinase, LOAG 06056.1 (Broad Institute Filarial Database); Caenorhabditis elegans PMK-1, Q17446; Human p38α (MAPK14), Q16539; E. multicularis EmMPK2, B1VK39.

2. Materials and methods

2.1. Generation of vectors expressing Bm-MPK1

The full length Bm-MPK1 sequence was obtained from the UniProt protein database (EMBL-EBI accession no. A8PQS0). The human codon-optimized bm-MPK1 gene was produced syntheti- cally (Blue Heron Biotechnology, Bothell, WA) and cloned into the Gateway® system pDONRTM221 entry vector (Invitrogen, Carls- bad, CA). The bm-MPK1 gene was initially cloned into plasmid pDONRTM221 and then transferred into a pDESTTM27 mam- malian expression vector using in vitro LR recombination reaction as per the manufacturer’s instructions, ultimately producing Bm-MPK1/pDESTTM27. The pDESTTM27 vector is an N-terminal Glutathione S-transferase (GST) fusion vector that generates an N-terminal GST tagged protein. Plasmid DNA concentration was determined using Hoechst 33258 dye assay [18].

2.2. Expression of recombinant Bm-MPK1

FreeStyleTM 293-F cells (HEK 293F, Invitrogen) were maintained in 125 mL polycarbonate Erlenmeyer flasks containing FreestyleTM 293 expression medium (Invitrogen) in a 37 ◦C humidified incu- bator on an orbital shaker platform (135 rpm) with 8% CO2. Cells were subcultured once the cell density reached 2–3 × 106 viable cells/mL ensuring >90% cell viability for all experiments. The cell density and the viability of cells were determined by counting the cells using the trypan blue exclusion method [19]. Cells were trans- fected with the Bm-MPK1/pDESTTM27 expression vector using FreeStyleTM 293fectin Reagent (Invitrogen) or FreeStyleTM MAX Reagent (Invitrogen) according to the manufacturer’s directions. Forty-eight hours later, HEK 293F cells were treated with 400 µM sodium arsenate (Na2HAsO4, MP Biomedicals, Solon, Ohio) for 3 h to activate recombinant Bm-MPK1 [20]. Transfected cells were centrifuged at 100 g for 5 min and washed twice in cold phos- phate buffered saline (PBS) prior to being lysed in 2.0 mL of cold lysis buffer [10 mM HEPES pH 7.4, 50 mM β-glycerolphosphate, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 10 mM NaF and 1:100 HaltTM Protease Inhibitor Cocktail (Thermo-Fisher Scientific, Rockford, IL)] for every 50–100 106 cells. Alternatively, 1.0 mL of commercial M-PER® Mammalian Protein Extraction Reagent (Thermo-Fisher Scientific) per 100 mg ( 100 µL) of cell pellet with 1:100 HaltTM Protease Inhibitor Cock- tail (Thermo-Fisher Scientific) was used. Cell-free lysates were clarified by centrifugation at 14,000 g for 10 min prior to use. Negative control lysates were prepared from cells transfected with pDESTTM27.

2.3. Purification of GST-tagged Bm-MPK1

GST-tagged Bm-MPK1 was purified at 4 ◦C using HighAffinity GST Resin (0.5–1.0 mL resin, GenScript, Piscataway, NJ) in a small column. The column was equilibrated with 10 resin bed volumes of PBS. Cell lysates were added to the GST resin which was re- suspended and incubated for 1 h with mixing on a tube rotator to allow protein binding. The column was washed 4 times with 2 mL of PBS and fractions were monitored by absorbance at 280 nm. The column was washed once with 2 mL of buffer A (25 mM HEPES, 150 mM NaCl, 1 mM DTT, 10% glycerol, 0.1 mM EDTA, 1:100 HaltTM Protease Inhibitor Cocktail). Protein was eluted with 5 resin bed vol- umes of buffer A supplemented with 10 mM reduced L-glutathione (Sigma–Aldrich, St. Louis, MO). Fractions were analyzed for pro- tein using the Bradford assay (Sigma–Aldrich), according to the manufacturer’s instructions, with bovine serum albumin (BSA) as a standard.

2.4. SDS-PAGE and Western blotting of Bm-MPK1 protein

Bm-MPK1 protein samples were analyzed using SDS gel elec- trophoresis (SDS-PAGE). Samples were prepared in NuPAGE® SDS Sample Buffer (Invitrogen) containing NuPAGE® Reducing Agent (Invitrogen) and incubated at 90 ◦C for 5 min prior to loading them on a NuPAGE® 4–12% Bis-Tris gel (Invitrogen). Gels were run at constant voltage (200 V) for 40 min in MOPS-SDS running buffer (Invitrogen) using Novex® Sharp Pre-Stained Protein Standards (Invitrogen) or MagicMarkTM XP Western Standard (Invitrogen) molecular weight markers. Gels were stained for 1 h with stain- ing solution (0.05% Coomassie Blue R-250, 5% acetic acid, 44% methanol) and destained with a 50% methanol–10% acetic acid solution for 30 min followed by a 1 h incubation in 5% acetic acid.

Protein was transferred from SDS-PAGE gels on to polyvinyli- denefluoride (PVDF) membrane using NuPAGE® Transfer Buffer (Invitrogen) containing 10% methanol and 0.25% SDS using a TE77XP Semi-Dry Blotter (Hoefer, Holliston, MA) for 1 h at 54 mA per blot. After transfer, membranes were placed in blocking solu- tion [5% non-fat dry milk in Tris-buffered saline and 0.05% Tween® 20 (TBST)] for 1 h. Blots were either incubated with goat anti- GST primary antibody (1:1000 dilution, GE Healthcare, Piscataway, NJ) or with rabbit Anti-Active® (pTGpY) polyclonal IgG antibody (1:2000, Promega, Madison, WI) in TBST containing 1.0 mg/mL BSA and 0.01% sodium azide for 1 h. Membranes were then washed 3 times for 5 min with TBST and incubated for 1 h with alkaline phos- phatase (AP)-linked secondary antibody. The secondary antibodies used were 1:5000 mouse anti-goat (Santa Cruz Biotechnology, Santa Cruz, CA) or 1:30,000 anti-rabbit (Sigma–Aldrich) when used in conjunction with the anti-GST primary antibody or the Anti- Active® p38 rabbit polyclonal IgG antibody, respectively. Bm-MPK1 was detected using 2.0 mL of Chromogenic Western Blue® Stabi- lized Substrate for AP (Promega).

2.5. p38 inhibitors

Human p38 inhibitors BIRB796 (Axon, Groningen, Netherlands), SB203580 (Selleck, Houston, TX) and RWJ67657 ([21] Dr. Fina Liotta, Montclair State University) were dissolved in 100% DMSO and stored at −20 ◦C prior to use.

2.6. Bm-MPK1 kinase assays

2.6.1. [ -32P] ATP assay

Bm-MPK1 protein kinase activity was assayed by monitoring the incorporation of 32P from [γ-32P] ATP into myelin basic protein (MBP). Alternatively, Bm-MPK1 autophosphorylation was exam- ined in the absence of MBP. Activated Bm-MPK1 (0.48 µM) was incubated with or without 1.0 µg of MBP in 20 µL containing 15 mM HEPES, (pH 7.4), 25 mM β-glycerophosphate, 15 mM NaCl, 10 mM MgCl2, 1 mM EGTA, and 0.02% Tween® 20. Samples were pre- incubated for 10 min at 37 ◦C prior to adding the ATP (50 µM, 0.4 µCi/µL [γ-32P] ATP). Following incubation at 37 ◦C for 20 min, reactions were terminated by adding NuPAGE® SDS sample buffer (Invitrogen) and subjected to SDS-PAGE as described above. Gels were stained, destained and dried onto blotting paper (Sigma) using a vacuum gel dryer prior to performing autoradiography using KodakTM BioMax® Maximum Resolution film. Autoradio- graphs were analyzed using Image-J software [22].

2.6.2. IMAP Bm-MPK1 kinase assay

Bm-MPK1 kinase activity was also assayed using the immo- bilized metal ion affinity-based fluorescence polarization (IMAP, Molecular Devices, Silicon Valley, CA) assay according to the man- ufacturer’s instructions, in the presence of 100 µM ATP, 1.0 mM DTT and where indicated, MKK6 (Sigma–Aldrich) and Bm-MPK1 (un-activated or activated) in reaction buffer – Tween-20 [10 mM Tris–HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3, and 0.01% Tween®20 (RB-T), Molecular Devices]. Reactions were initiated with addi- tion of 100–200 nM fluorescent substrate (FAM-p38tide, Molecular Devices), prepared in RB-T buffer. The fluorescent polarization was read in parallel and perpendicular with an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a Synergy 2 Microplate reader (BioTek, Winooski, VT). Dose–response inhibitor assays were conducted as described above using 160 ng/well of sodium arsenate-activated Bm-MPK1 or 50 ng/well of commer- cial p38α (Millipore, Temecula, CA). The data was analyzed using four parameter logistic curve using Microsoft Excel Solver and dose response curves were generated using Microsoft Excel.

2.7. B. malayi culture, p38 inhibitor treatment and phenotypic analysis

2.7.1. Parasite motility

Female adult B. malayi parasites, harvested from infected jirds, were procured from the NIAID/NIH Filariasis Research Reagent Resource Center (FR3). Adult worms were plated in 24-well plates with 2 mL of Advanced RPMI 1640 medium (Invitrogen) sup- plemented with 25 mM HEPES, 2 mM L-Glutamine (Invitrogen), 100 U/mL Penicillin (Invitrogen), 100 µg/mL Streptomycin (Invitro- gen), 2.5 µg/mL Amphotericin B solution (Invitrogen), and 5% heat
inactivated fetal bovine serum and placed in a 37 ◦C humidified incubator with 5% CO2. After 24 h, adult worms were selected based upon microfilariae release. Microfilariae release was scored as fol- lows: ( ) no microfilariae, ( ) some microfilariae, (+) moderate levels of microfilariae, (++) and (+++) if the parasite secrets large quantities of microfilariae. After the parasites were scored, 6–9 worms were selected for each treatment group based on microfi- lariae release and were transferred to new plates. The microfilariae released in the old plate were pooled together, briefly centrifuged at 5000 g for 5 min, and re-suspended in 2 mL of media. Microfilar- ial density was determined using a hemocytometer and plated in a 96-well plate, 80 microfilariae/well with 200 µL of complete media. Treatment groups received the p38 inhibitor, BIRB796 (1 µM or 10 µM), 5 mM sodium arsenate with 1 µM or 10 µM BIRB796, 0.1% DMSO (control) or 5 mM sodium arsenate vehicle control. Cul- tures were placed in a 37 ◦C humidified incubator with 5% CO2. The worms were transferred into a new plate containing fresh media and drug every 48 h. Parasite and microfilariae motility were given a score from 0 to 4 with 4, rapid movement and largely coiled; 3, moderated movement and uncoiled; 2, slow movement and uncoiled; 1, twitching movement and uncoiled; 0, no motility (dead). The motility of the worms and microfilariae were observed every 24 h and analyzed by a one sided unpaired Student’s t-test using Microsoft Excel. All experiments were performed 2–3 times with similar results.

2.7.2. MTT viability assay

Viability was also assessed colorimetrically using 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The assay was performed as described previously [23] with minor modifications. Briefly, worms were individually placed into a 96- well plate containing 180 µL of PBS, then 20 µL of a MTT solution (5 mg/mL in PBS) was added and the plate incubated at 37 ◦C for 30 min. All the solution was removed from the wells and 200 µL of DMSO was then added to the plate. The plate was incubated at 37 ◦C for 15 min and then placed on a shaker at room temperature for another 15 min. The absorbance of each sample was measured at 550 nm.

3. Results

3.1. Enzymatic activity of recombinant Bm-MPK1 using a fluorescently labeled substrate

Bm-MPK1 was successfully expressed as a GST-tagged protein in human HEK 293F cells. As mentioned previously, p38 MAPKs require activation by dual-phosphorylation of the TGY motif by upstream kinases [15]. All isoforms of human p38 MAP kinase have been shown to be activated by MKK6 [24]. Given the high degree of Bm-MPK1 sequence similarity to other p38 kinases and a conserved TGY motif (Fig. 1), we tested the ability of the upstream kinase acti- vator of human p38, MKK6, to activate Bm-MPK1. Data obtained using the IMAP assay showed that Bm-MPK1 had very low kinase activity unless it was pre-incubated in the presence of active human MKK6 (Fig. 2A). Under the conditions of the assay, a concentration- dependent increase in Bm-MPK1 kinase activity was observed. The addition of the potent p38 kinase inhibitor, RWJ67657 [21] to the assay resulted in virtually complete inhibition of kinase activity (Fig. 2A). We further tested whether Bm-MPK1 could be activated by subjecting HEK 293F cells to sodium arsenate-induced oxida- tive stress, which generates reactive oxygen species. Mammalian proteins comprising the p38 pathway have been shown to be effectively activated using this approach [20]. Sodium arsenate- induced oxidative stress led to a dramatic activation of Bm-MPK1 (Fig. 2B). The addition of recombinant MKK6 to the reaction provided an additional, small, increase in kinase activity indicating that arsenate-induced oxidative stress leads to effective activation of Bm-MPK1. As before, the addition of the human p38 inhibitor, RWJ67657, effectively inhibited kinase activity (Fig. 2(B)). These results illustrate the highly conserved nature of nematode and human p38 kinases both in terms of their mechanism of activation as well as susceptibility to inhibition.

Fig. 2. IMAP Fluorescence Polarization assay of untreated and sodium arsenate- activated Bm-MPK1. The fluorescence polarization response is given as mP and is a measure of the change in substrate polarization due to phosphorylation. (A) Activation of Bm-MPK1 purified from Bm-MPK1/pDESTTM 27-transfected HEK 293-F cells by incubation in the presence (u) or absence (Ç) of MKK6. (B) Bm-MPK1 puri- fied from Bm-MPK1/pDESTTM 27-transfected HEK 293-F cells treated with 400 µM sodium arsenate for 3 h prior to harvesting the cells. Sodium arsenate-activated Bm- MPK1 incubated in the presence (u) or absence (Ç) of MKK6. In both (A) and (B), sodium arsenate-activated Bm-MPK1 treated with 10 µM p38 inhibitor RWJ67657 (●) or MKK6-activated Bm-MPK1 treated with 10 µM p38 inhibitor RWJ67657 (2) is also shown. The data was corrected for the blank (buffer only).

We next examined whether activation of Bm-MPK1 by oxida- tive stress results in dual phosphorylation of the TGY activation loop using a polyclonal Anti-Active® p38 antibody that specifi- cally reacts against pTGpY motif in human p38. Western blots of purified Bm-MPK1 from sodium arsenate stressed HEK 293F cells showed increased phosphorylation compared to unstressed cells (Fig. 3A). An anti-GST antibody showed equivalent amounts of a
67 kDa immunoreactive band in all lanes (Fig. 3B), confirming that oxidative stress enhances phosphorylation. Moreover, these results further attest to the highly conserved nature of the TGY motif between human and B. malayi p38 MAPKs.

3.2. Enzymatic activity of Bm-MPK1 using [ -32P]ATP

The IMAP assay for p38 in the previous experiments utilizes a peptide substrate. We next determined the ability of Bm-MPK1 to phosphorylate a generic MAPK substrate, myelin basic protein (MBP) using [γ-32P]ATP. Bm-MPK1 was capable of efficiently phos- phorylating the 20 kDa MBP protein (Fig. 4, lane 3). Also, Bm-MPK1 was found to be capable of autophosphorylation indicated by the appearance of a radioactive band with a molecular weight of 67 kDa (Fig. 4, lane 2). Auto-phosphorylation is a common property of a number of MAPKs.The effects of two p38 inhibitors exhibiting two distinct inhibitory mechanisms, BIRB796 [25] and RWJ67657 [21], were next examined. BIRB796 is a potent allosteric inhibitor active against all isoforms of human p38 [26]. RWJ67657 is a specific class of human p38 inhibitor that competes with ATP for the occupancy of the nucleotide binding domain of only p38α and β isoforms [21]. Ten micromolar BIRB796 inhibited Bm-MPK1 activity by 94% while 10 µM RWJ67657 only inhibited its activity by 61% (Fig. 5). The one micromolar BIRB796 inhibited Bm-MPK1 activity 73% while 1 µM RWJ67657 inhibited Bm-MPK1 73%. In addition, both inhibitors inhibited Bm-MPK1 autophophorylation (Fig. 5).

Fig. 3. Dual phosphorylation of the TGY motif in Bm-MPK1 by arsenate-induced oxidative stress. (A) Western blot developed with Anti-Active® p38 (pTGpY) rab- bit polyclonal antibody. (B) Western blot developed with anti-GST goat antibody. Bm-MPK1 was expressed in HEK 293-F cells subjected to sodium arsenate-induced stress. Lane 1, Molecular weight markers; Lane 2, purified Bm-MPK1 (100 ng) from control cells; and Lane 3, purified Bm-MPK1 (100 ng) from sodium arsenate (As= Na2 HAsO4 )-stressed cells.

3.3. Comparison of p38 inhibitors dose responses for human p38 and Bm-MPK1

We next determined the relative potency of a small panel of p38 inhibitors against Bm-MPK1 and human p38 using the IMAP assay. To this end, dose response curves were generated for the ATP competitive p38 inhibitors, SB203580 [27] and RWJ67657 and for the allosteric inhibitor BIRB796. The relative potency of these inhibitors was assessed by determining the half maximal inhibitory concen- tration (IC50) for each inhibitor. SB203580, RWJ67657 and BIRB796 exhibited IC50 values of 218, 121 and 141 nM, respectively, against Bm-MPK1. The inhibitors each had much greater potency against human p38, having IC50 values of 33.5, 4.5 and 35.4 nM, respec- tively (Fig. 6 and Table 1), suggesting the existence of differences in the architecture of the inhibitor binding sites between the MAPKs. Nonetheless, these compounds are well within the sub-micromolar range for inhibition making them very attractive precursors for the development of more potent and selective Bm-MPK1 inhibitors.

Fig. 4. Phosphorylation of Myelin Basic Protein (MBP) by Bm-MPK1. MBP was incu- bated with [γ-32 P]ATP in the absence and presence of Bm-MPK1 at 37 ◦C for 20 min. The reaction was terminated by the addition of SDS sample buffer and subjected to SDS-PAGE. Phosphorylation was detected by autoradiography. Lane 1, [γ-32 P]ATP and MBP; Lane 2, [γ-32 P]ATP and Bm-MPK1; and Lane 3, [γ-32 P]ATP, MBP, and Bm-MPK1.

Fig. 5. Inhibition of Bm-MPK1 with p38 inhibitors BIRB796 and RWJ67657. Myelin Basic Protein (MBP) was incubated with [γ-32 P]ATP in the absence and presence of Bm-MPK1 at 37 ◦C for 20 min. The reaction was terminated by the addition of SDS sample buffer and the reaction mixture was subjected to SDS-PAGE. (A) Phospho- rylation was detected by autoradiography. (B) Bands from (A) were scanned using ImageJ program and the area under the curve (AUC) calculated from pixel intensity. Lane 1, control, no inhibitor; Lane 2, 1 µM BIRB796; Lane 3, 10 µM BIRB796; Lane 4, 1 µM RWJ67657; and Lane 5, 10 µM RWJ67657. The percent inhibition was cal- culated based on control and is indicated in parenthesis. The gel was scanned using Image J three times. The bars represent mean ± SD.

3.4. Effects of p38 inhibitors on B. malayi stress responses

It is well documented that filarial parasites are highly resistant to oxidative stress [9,10], therefore we tested whether Bm-MPK1 of its potency and high selectivity against all human p38 isoforms [25,26]. In preliminary experiments we determined that BIRB796 exhibited pronounced effects on parasite motility, an established measure of parasite viability [28], at drug concentrations exceeding 10 µM (data not shown). The relevance of direct effects of BIRB796 on B. malayi motility is unclear. The lack of a defined dose–response may indicate off target effects at high drug concentrations. Drug uptake and distribution in parasitic nematodes is complex and not well defined [29] and the actual concentration of BIRB796 within the parasite is unknown. Upon establishing a concentration of BIRB796 that did not affect motility, we determined whether the combination of 10 µM BIRB796 and 5 mM sodium arsenate affected B. malayi motility. Similar to C. elegans, B. malayi are largely resistant to oxidative stress induced with 5 mM sodium arsenate ([12] and Fig. 7). Interestingly, the combination of 10 µM BIRB796 and 5 mM sodium arsenate resulted in a dramatic decrease in para- site motility by 88% compared to control (Fig. 7). Additionally, B. malayi parasites were also subjected to an MTT assay to determine whether the viability of the parasites on day 6 would correspond to the observed decrease in motility. Addition of 10 µM BIRB796 and 5 mM sodium arsenate reduced the viability of the parasite by >84%, confirming the relationship between parasite motility and viability (Fig. 8). These results support the notion that Bm-MPK1, like the C. elegans ortholog PMK-1, plays a role in protecting B. malayi from toxic oxidative stress.

Fig. 7. Effects of sodium arsenate-induced oxidative stress on adult B. malayi motil- ity in the presence or absence of BIRB796. Female worms were cultured with 0.1% DMSO (control), 1 µM BIRB796, 10 µM BIRB796, 5 mM sodium arsenate alone, 5 mM sodium arsenate with 1 µM BIRB796 and 5 mM sodium arsenate with 10 µM BIRB796 for 6 days. Parasite motility was observed every 24 h and a score 0–4 was given: *p < 0.01, ***p < 0.0001 by a one-tailed unpaired Student’s t-test using sodium arsenate as a vehicle control for sodium arsenate with BIRB796 and 0.1% DMSO as a vehicle control for BIRB796 and sodium arsenate alone. n = 5 or 6 worms. Bars represent mean ± SD.

Fig. 6. Human p38 inhibitor dose–response analysis for p38 and Bm-MPK1. (A) Inhi- bition of human p38 activity by SB203580 (●), RWJ67657 (2), or BIRB796 ( ). (B) Inhibition of Bm-MPK1 activity by SB203580 (●), RWJ67657 (2), or BIRB796 ( ).

We next examined the effects of BIRB796 on the ability of microfilariae to respond to oxidative stress induced by sodium arsenate. Microfilariae are significantly more sensitive to arsenate-induced oxidative stress than adult worms (Fig. 9). This is consistent with observations made by others where microfilariae were shown to be more sensititive to oxidative stress induced with hydrogen per- oxide [11]. Over the course of 6 days, a significant reduction in microfilarial motility was observed in the presence of 5 mM sodium arsenate. Addition of 10 µM BIRB796 resulted in a statistically significant reduction in motility over what was observed with treat- ment with sodium arsenate alone (Fig. 9). These results indicate that, Bm-MPK1 plays a role in microfilarial responses to ROS.

Fig. 8. Effects of BIRB796 on adult B. malayi responses to arsenate-induced oxida- tive stress as assessed by MTT assay on day 6. Female adult worms were cultured with 0.1% DMSO (control), 1 µM and 10 µM BIRB796, 5 mM sodium arsenate and 5 mM sodium arsenate with 1 µM or 10 µM BIRB796 for 6 days: ***p < 0.0001 by a one-tailed unpaired Student’s t-test using sodium arsenate as a vehicle control for sodium arsenate with 1 µM or 10 µM BIRB796 and 0.1% DMSO as a vehicle control for BIRB796 and sodium arsenate alone. n = 2–9 worms. Bars represent mean ± SD.

Fig. 9. Effects of BIRB796 on B. malayi microfilariae motility under sodium arsenate- induced oxidative stress. Microfilariae (80 microfilariae/well) were cultured with 0.1% DMSO (control), 10 µM BIRB796, 5 mM sodium arsenate, and 5 mM sodium arsenate with 10 µM BIRB796 for 6 days in a 96-well plate. The motility of the micro- filariae were observed every 24 h and a score 0–4 was given: *p < 0.01, ***p < 0.0001 by a one sided unpaired Student’s t-test using sodium arsenate as a vehicle control for sodium arsenate with 10 µM BIRB796 and 0.1% DMSO as a vehicle control for BIRB796 and sodium arsenate alone. n = 6 wells. Bars represent mean ± SD.

4. Discussion

This paper describes the biochemical characterization of a B. malayi stress-activated p38/PMK-1 kinase ortholog Bm-MPK1 along with an analysis of the effects of p38 inhibitors on Bm-MPK1 kinase activity and on the ability of B. malayi to respond to ROS. Bm- MPK1 is highly homologous to C. elegans PMK-1 which is known to play a critical role in protection against oxidative stress and innate immunity [12–14]. Genetic deletion of PMK-1, or its upstream acti- vator SEK-1, attenuates C. elegans responses to oxidative stress induced by sodium arsenate [12]. Tissue dwelling filarial parasites such as the lymphatic parasites W. bancrofti and B. malayi and cutaneous O. volvulus strongly modulate host immune responses through several mechanisms [7–9]. These organisms tend to be long lived in their hosts and as such, must deal with host immune responses to avoid elimination while sparing the host from severe, life threatening pathology. An early first line of defense against parasitic infection is the generation of reactive oxygen and nitro- gen species (RNS) by macrophages and granulocytes [30]. ROS represents a class of highly reactive chemical species such as super- oxide anion (O2−) that are formed in these cells through the action of NADPH oxidase [31]. B. malayi, like other parasites, produces several secreted and non-secreted enzymes such as superoxide dis- mutase (SOD), thioredoxin peroxidase and glutathione peroxidase for defense against oxidative damage [30].

The biochemistry of nematode anti-oxidation mechanisms has been extensively studied in the free-living nematode, C. elegans. It has been shown in C. elegans that a mitogen-activated pro- tein kinase pathway (MAPK) is critically important for regulating responses to oxidative stress as well as xenobiotic detoxification and innate immunity [12,13]. In response to oxidative stress, C. ele- gans PMK-1 kinase, an ortholog of human p38 kinase, is activated and directly phosphorylates SKN-1, a transcription factor impor- tant for embryonic mesendodermal development and regulation of oxidative stress responses in the adult (16). Phosphorylation of SKN-1 by PMK-1 results in the translocation of SKN-1 into intesti- nal nuclei where it functions in the regulation of the expression of a variety of antioxidant and phase II detoxification genes such as SOD [16]. Regulation of SKN-1 transcriptional activity through phosphorylation is likely to be more complex since, in addition to PMK-1, it is a substrate for at least four additional protein kinases [32]. Unlike C. elegans, little is known regarding the biochemical and molecular details of the regulation of anti-oxidative responses in parasitic nematodes. In the study presented here, we have char- acterized a PMK-1/p38 ortholog, Bm-MPK1, from the human filarial parasite, B. malayi. Using a chemical biological approach, we have demonstrated inhibition of Bm-MPK1 activity with known ATP competitive and allosteric p38 inhibitors. Furthermore, we have demonstrated that the potent allosteric p38 inhibitor, BIRB796, suppresses B. malayi anti-oxidative responses to ROS induced by sodium arsenate. We have also shown that treatment of adult female worms with BIRB796, under oxidative stress conditions, results in death of the parasites. Additional studies are underway to determine the effects of BIRB796 on stress responses in other devel- opmental stages of B. malayi, especially L3 larvae, the infectious larval form of B. malayi introduced into the host from a mosquito vector.

Fig. 10. Comparison of B. malayi Bm-MPK1 and C. elegans PMK-1 stress-activated signaling pathways.

The B. malayi Bm-MPK1 pathway shares several similarities to the C. elegans PMK-1 pathway (Fig. 10) including the presence of orthologs of upstream activating protein kinases such as SEK-1 [5]. However, there are also differences such as the apparent absence of an SKN-1 ortholog in B. malayi [33]. Additional studies are under- way in our laboratory to further define this pathway. One important aspect of this work is the use of chemical probes to study para- sitic nematode signaling pathways. Unlike C. elegans and protozoal parasites, filarial parasites are frequently resistant to manipulation using standard molecular genetics approaches such as RNAi [34,35]. Synthetic inhibitors, therefore, provide an alternative, chemical biological, approach to study gene function in these organisms. Our results, in addition to defining the biochemical mechanisms involved in parasitic nematode responses to oxidative stress, may offer a potential strategy for treating filarial disease. The design of highly selective and potent Bm-MPK1 inhibitors could potentially be used to interfere with Bm-MPK1 induction of drug detoxification genes such as gamma-glutamylcysteine synthetase (gcs-1) which catalyzes the first, rate-limiting step in the biosynthesis of glu- tathione (GSH). C. elegans (GCS-1), is known to be regulated through the PMK-1/SKN-1 pathway and plays a role in resistance to oxida- tive stress in this organism [12]. Mentioned earlier, GSH, along with GST, glutathione peroxidase (GP) and glutathione reductase (GR), constitute an important antioxidant system in filarial par- asites contributing to their long-term survival in the host [30]. Filarial parasite GST has been targeted for the development of anti-parasitic drugs [36,37]. Disruption of this pathway with Bm- MPK1 inhibitors may compromise the ability of filarial parasites to counteract host oxidative stress responses. In addition, the poten- tial detoxification of known anti-filarial drugs by conjugation with reduced glutathione through the action of GST may be attenuated allowing for an increase in potency and efficacy of known anti- parasitic drugs. In conclusion, we have demonstrated, through a chemical biological approach, that the B. malayi stress-activated MAPK, Bm-MPK1, functions in a signaling pathway important for parasite anti-oxidative responses to ROS. The presence of highly homologous Bm-MPK1 orthologs in other filarial and non-filarial parasites indicates a similar role for these kinases in anti-oxidative and detoxification processes. Further characterization of this path- way will provide additional insights into filarial parasite responses to host-generated ROS and the therapeutic potential BIRB 796 of inhibiting this response.