Plasmodium falciparum in vitro growth inhibition assays are widely used to evaluate and quantify the functional activity of acquired and vaccine-induced antibodies and the anti-malarial activity of known drugs and novel compounds. However, several constraints have limited the use of these assays in large-scale population studies, vaccine trials and compound screening for drug discovery and development.P. falciparum line was transfected to express green fluorescent protein (GFP). In vitro growth inhibition assays were performed over one or two cycles of P. falciparum asexual replication using inhibitory polyclonal antibodies raised in rabbits, an inhibitory monoclonal antibody, human serum samples, and anti-malarials. Parasitaemia was evaluated by microscopy and flow cytometry.The D10 Transfected parasites expressed GFP throughout all asexual stages and were clearly detectable by flow cytometry and fluorescence microscopy. Measurement of parasite growth inhibition was the same when determined by detection of GFP fluorescence or staining with ethidium bromide. There was no difference in the inhibitory activity of samples when tested against the transfected parasites compared to the parental line. The level of fluorescence of GFP-expressing parasites increased throughout the course of asexual development. Among ring-stages, GFP-fluorescent parasites were readily separated from uninfected erythrocytes by flow cytometry, whereas this was less clear using ethidium bromide staining. Inhibition by serum and antibody samples was consistently higher when tested over two cycles of growth compared to one, and when using a 1 in 10 sample dilution compared to 1 in 20, but there was no difference detected when using a different starting parasitaemia to set-up growth assays. Flow cytometry based measurements of parasitaemia proved more reproducible than microscopy counts.Flow cytometry based assays using GFP-fluorescent parasites proved sensitive and highly reproducible for quantifying the growth-inhibitory activity of antibodies and anti-malarials, with superior reproducibility to light microscopy, and are suitable for high-throughput applications. Plasmodium falciparum, particularly among children [P. falciparum malaria. A number of studies suggest an association between antibody levels to merozoite antigens measured by enzyme linked immunosorbent assays (ELISA) and protection from malaria disease [Malaria is a leading cause of morbidity and mortality globally and the majority of malaria-related mortality is caused by children -3. Antib disease -15. Howe disease -20.in vitro assays [Plasmodium falciparum asexual stage growth inhibition assays have been used extensively in malaria research to measure the levels of growth inhibitory antibodies in clinical and pre-clinical studies [Studies using rabbit polyclonal sera and monoclonal antibodies (MAb) raised by vaccination with recombinant merozoite antigens or affinity purified human antibodies suggest that antibodies against merozoite antigens are capable of interfering with merozoite invasion of the RBC directly in o assays -27. Plas studies ,28-35. GThe most readily available, simplest and most widely used method for measuring parasitaemia in growth inhibition assays is light microscopy ,33,36-40High throughput approaches such as parasite lactate dehydrogenase (pLDH), and amino acid incorporation assays (hypoxanthine and isoleucine uptake assays) allow easier assessment of parasite growth for large sample sets ,39,43,44et al developed transgenic P. falciparum parasites expressing an antigenically distinct 19 kDa fragment of the merozoite surface protein 1 (MSP1-19) orthologue from the rodent malaria Plasmodium chabaudi [P. chabaudi MSP1-19 line versus parasites with the P. falciparum MSP1-19 sequence allowed measurement of P. falciparum MSP1-19-specific inhibitory antibodies. In this study, MSP1-19 specific growth inhibition assays were used as a model as it allowed antigen-specific inhibition to be distinguished from non-specific inhibitory activity. MSP1-19 appears to be a target of protective immunity and is an important vaccine candidate currently under development.In this study, growth inhibition assays were optimized and GFP-labelled fluorescent parasites developed and evaluated in growth inhibition assays using antibodies and anti-malarials. Furthermore, the sensitivity and reproducibility of flow cytometry for measuring parasite growth inhibition by antibodies was examined and compared to light microscopy. Previously, O'Donnell chabaudi ; evaluatPlasmodium falciparum parasites were cultured in Petri dishes using human O+ erythrocytes (RBC) maintained at 2% haematocrit [3, 20 μg/ml gentamicin, 5% (vol/vol) heat inactivated pooled human serum  and 0.25% Albumax II . In some drug inhibition experiments 0.5% Albumax was used with no human serum added. Cultures were maintained in airtight boxes at 37°C in an atmosphere of 1% O2, 4% CO2 and 95% N2. Parasites used in these studies include the D10 clonal line, an isogenic D10-P. chabaudi MSP1-19 parasite line in which the P. chabaudi orthologue replaces the region of P. falciparum msp1 encoding MSP1-19 (known as PcMEGF), and an isogenic D10-P. falciparum MSP1-19 parasite line generated in the same manner as PcMEGF to act as a transfection control (PfM3').matocrit . Parasitet al [The protocol for measurement of antibody inhibition of parasite growth used in this study was modified from Persson et al . PfMSP1-Parasitized RBCs (25 μl to 50 μl) at the appropriate parasitaemia were added to a 96 well U-bottom plate . Inhibitory sample (2.5 μl to 5 μl for a 25 μl or 50 μl assay respectively) was added to the parasitized RBCs giving a 1 in 10 dilution of sample in the assay . All samples were run in duplicate. A panel of non-immune sera from Melbourne donors or PBS were included as non-inhibitory controls. The 96 well plates were placed in a humidified air-tight gassed culture box at 37°C.One cycle ring stage assays for microscopy and flow cytometry were cultured for approximately 24 hours until young rings were visible and no schizont stage parasites remained. Thin smears of cell pellets were methanol fixed and stained with 5% Giemsa . One thousand RBCs were counted for each slide. Only early to late rings (0 to 20 hours old) were counted as parasitized RBCs, with each infected RBC counted as a single invasion event, regardless of how many parasites were in the cell.Growth assays performed over one cycle of erythrocyte invasion were cultured for 48 hours post set up until parasites were between 30-40 hours post invasion, when they were prepared for flow cytometry analysis as appropriate. Growth assays performed over two cycles of erythrocyte invasion were supplemented with 5 μl to 10 μl (25 μl or 50 μl assay respectively) of fresh culture medium warmed to 37°C, 48 hours post set up (parasites 30-40 hours post invasion). Ninety to 96 hours post assay set up the two cycle trophozoite stage assays were prepared for flow cytometry analysis. For non-fluorescent parasite lines, RBCs were resuspended in 100 μl of PBS with 10 μg/ml ethidium bromide , stained for one hour before centrifugation, removal of the supernatant and resuspension in 200 μl of PBS. GFP fluorescent parasite lines were resuspended in PBS to make a final volume of 200 μl. Parasitaemia was measured on a Becton Dickinson FACSCalibur  flow cytometer using a 488 nm laser for excitation of both GFP fluorescent (Fl-1) and EtBr stained (Fl-2) parasites. Typically, 50,000 to 80,000 RBCs were counted in each well. Samples were analysed using FlowJo software  by first gating for intact erythrocytes by side scatter and forward scatter parameters, and subsequently determining the proportion of FL1 or FL2 positive cells.mean) by the mean parasitaemia of two or more wells of non-inhibitory control (Cmean) and multiplying by 100 (G = {Smean/Cmean}*100).In this study, when results for both the parental PcMEGF and PfM3'lines and the GFP fluorescent PcPHG and PfPHG lines (described following) are described they will be referred to as the PcMSP1-19 and PfMSP1-19 lines, respectively. For both the PcMSP1-19 and PfMSP1-19 lines, parasite growth (G) was determined by dividing the mean parasitaemia of duplicate wells for a test sample  were added to the plasmid and then mixed with 200 μl of infected RBC with 5-8% ring stage parasites before being transferred to a 0.2 cm electroporation cuvette. The cytomix and infected RBC mix was electroporated at 950 μF capacitance and 0.31 kV  and the infected red blood cells transferred immediately into 10 ml of culture medium containing 2% haematocrit RBCs, placed into a airtight gassed chamber and incubated at 37°C.Transfection of eviously  using moeviously . BrieflyP. chabaudi MSP1-19 phenotype with up to two months of continuos culture. To explore whether other isolates could be successfully transfected with the same plasmid, isolates CS2 and E8B, derived from the ITG line, were also transfected.Drug cycling commenced two days after transfection using 5 μg/ml of blasticidin-S-HCl to select for fluorescent parasites with 24 ng/ml of pyrimethamine added to maintain integration of the MSP1-19 transfection vectors. After the second cycle of drug selection, fluorescent parasites expressing GFP were cloned by limiting dilution for use in experiments. In order to ensure maintainance of both the MSP1-19 and GFP transfection plasmids for experiments, cloned parasites were cultured with the appropriate drugs . Typically, there was no evidence for the loss of GFP fluorescence or expression of the Successful transfection of parasites with GFP plasmids was confirmed by live parasite fluorescence microscopy using an upright Zeiss Axioskop fluorescence microscope  with parasite nuclei stained with 0.5 μg/ml DAPI . For flow cytometry GFP fluorescent lines were stained with EtBr and the number of fluorescent parasites were quantified using a FACSCalibur and analysed using FlowJo software.P. chabaudi MSP1-19 sequence in place of the endogenous P. falciparum sequence [P. falciparum AMA1 domain 1 common to both the 3D7 and D10 parasite lines showed similar levels of growth inhibition of the PfMSP1-19 and PcMSP1-19 lines at a concentration of 0.5 mg/ml. The reproducibility of PfMSP1-19 specific rabbit antibodies in growth inhibition assays was confirmed in repeat experiments throughout the course of this study in both one cycle and two cycle assays . Two cycle assays set up at 0.05%, 0.1% and 0.3% parasitaemia showed a strong correlation between PfMSP1-19 growth for the 0.05% and 0.1% assays  and the 0.1% and 0.3% assays . These data suggest the start parasitaemia was unlikely to influence the sensitivity of parasite growth inhibition assays over the range of starting parasitaemia tested.MSP1-19 specific rabbit antisera and the AMA1 MAb were compared in one cycle and two cycle growth inhibition assays to determine whether a second cycle of parasite growth would increase the sensitivity of the assay. Growth of the PcMSP1-19 parasite line in the presence of inhibitory antibodies showed that a second cycle increased antibody mediated growth inhibition for both the PcMSP1-19 and AMA1 antibodies but not the PfMSP1-19 antibodies Figure . Similaret al [A number of published studies comparing growth of the PcMSP1-19 line and the PfMSP1-19 line have used a 1 in 20 volume of sample in one cycle microscopy based assays -39. For et al  using a et al [The PfM3' and PcMEGF MSP1-19 lines developed by O'Donnell et al ,51 were The fluorescence profile of both EtBr stained and unstained PfPHG GFP expressing parasites was examined during the course of the asexual parasite lifecycle. GFP fluorescence profiles for early ring stage parasites  separated clearly from the uninfected RBC population Figure . This alThere was a strong correlation between GFP fluorescence and EtBr staining for measurements of PfPHG parasite growth in two cycle assays  in the presence of inhibitory and non-inhibitory samples. Growth of the PfPHG GFP expressing line also correlated strongly with growth of the PfM3' parent line in inhibitory and non-inhibitory samples for two cycle assays .P. chabaudi MSP1-19 specific antiserum, P. falciparum MSP1-19 specific antiserum  and the PfMSP1-19 line  and a flow cytometry based assay. Measurements of parasitaemia between duplicate wells were compared for all samples and control treatments (n = 13) in the experiment for both the PcMEGF and PfM3' parasite lines. The mean percentage difference in parasitaemia counts between duplicates wells for all treatments and both lines in the flow cytometry based assay was 0.2% (range 0% to 0.7%). The mean percentage difference between duplicate wells across all serum treatments for M1 was 0.6% (range 0% to 1.6%). For M2 the mean percentage difference between duplicate wells across all serum treatments was 1.5% (range 0% to 5.5%).Comparison between microscopist M1, microscopist M2 and flow cytometry based counts of parasitaemia (mean of duplicate wells) suggested that the counts for M1  tended to underestimate parasitaemia relative to flow cytometry counts (mean 4.9%), while the counts of M2 (mean 5.7%) tended to overestimate parasitaemia relative to flow cytometry Figure . The comIn this study, flow cytometry-based growth inhibition assays  were furGFP fluorescent PcMSP1-19 and PfMSP1-19 parasites were developed to reduce handling and improve sensitivity in flow cytometry based growth assays; parasites were transfected to express GFP under control of the HSP-86 promoter on both the PcMEGF (PcPHG) and PfM3' (PfPHG) parent line backgrounds and parasites were cloned for subsequent use. Parasites expressing cytoplasmic GFP were easily detectable by flow cytometry and measurements of parasitaemia correlated strongly between GFP fluorescent parasites and parasites detected using the established method of staining with ethidium bromide.in vitro assays. These studies demonstrate that GFP-fluorescent parasites could be used in assays to measure acquired immunity or vaccine-induced responses in the development and testing of blood-stage vaccines, or for screening and evaluation of compounds with anti-malarial activity.There are several advantages of using GFP-fluorescence compared to nucleic acid dyes to detect parasitaemia. The use of the GFP fluorescent parasites removed the need to use a nucleic acid stain for parasite detection by flow cytometry, thereby reducing handling, and gave better resolution between ring stage parasites and uninfected RBCs by flow cytometry compared to EtBr stained rings. Furthermore, fluorescent parasites can be sorted using flow cytometry and used in down-stream applications that require live viable parasites, which can be problematic with nucleic acid dyes ,60. DeteFinally, the measurement of parasitaemia by flow cytometry was found to be more accurate and reproducible than microscopy. Reproducibility of parasitaemia counts between duplicate wells was much higher for flow cytometry than microscopy. Differences in parasitaemia counts between duplicate wells of the same sample were as high as 98% by microscopy, whereas parasitaemia counts by flow cytometry were very similar for duplicate wells of the same sample, suggesting that much of the variation between duplicate wells in microscopy is caused by observer error. There was also substantial variability in results between microscopists.The development and optimisation of flow cytometry based growth inhibition assays using parasite lines transfected to express GFP resulted in a highly reproducible assay that was sensitive and suitable for measuring the inhibitory activity of antibodies and anti-malarial agents. Results were highly reproducible between experiments, between duplicate samples and superior to microscopy.The authors declare that they have no competing interests.DWW - study design, analysis and interpretation of results, performed experiments, wrote the manuscript with JGB; BSC - study design, interpretation of results and contributed to writing the manuscript; JGB - study design, analysis and interpretation of results, wrote the manuscript with DWW. All authors read and approved the final manuscript.


Drosophila morphogenetic protein responsible for patterning the anterior structures in embryos. Recent experimental studies have revealed important insights into the behavior of this morphogen gradient, making it necessary to develop a model that can recapitulate the biological features of the system, including its dynamic and scaling properties.Bicoid (Bcd) is a Drosophila embryos. This model is based on equilibrium binding of Bcd molecules to non-specific, low affinity DNA sites throughout the Drosophila genome. It considers both the diffusion media within which the Bcd gradient is formed and the dynamic and other relevant properties of bcd mRNA from which Bcd protein is produced. Our model recapitulates key features of the Bcd protein gradient observed experimentally, including its scaling properties and the stability of its nuclear concentrations during development. Our simulation model also allows us to evaluate the effects of other biological activities on Bcd gradient formation, including the dynamic redistribution of bcd mRNA in early embryos. Our simulation results suggest that, in our model, Bcd protein diffusion is important for the formation of an exponential gradient in embryos.We present a biologically realistic 2-D model of the dynamics of the Bcd gradient in The 2-D model described in this report is a simple and versatile simulation procedure, providing a quantitative evaluation of the Bcd gradient system. Our results suggest an important role of Bcd binding to non-specific, low-affinity DNA sites in proper formation of the Bcd gradient in our model. They demonstrate that highly complex biological systems can be effectively modeled with relatively few parameters. Drosophila, patterning along the anterior-posterior (A–P) axis exhibits scaling properties such that embryonic structures are patterned in proportion to embryo length Drosophila, is responsible for instructing patterning along the A–P axis Drosophila but they currently remain at a theoretical level Development is a robust process that must achieve a precise and reproducible outcome despite inevitable variations among individual embryos Drosophila embryos. These new findings represent an important foundation for developing and evaluating models of Bcd gradient formation. In particular, a recent live-imaging study demonstrated that Bcd concentrations inside individual nuclei exhibit a striking stability even though the number of nuclei is doubling after each nuclear division in early embryos staufen (stau) females, a loss of Bcd gradient scaling can directly explain the scaling defect of its target expression boundary. These findings suggest that the scaling properties of the Bcd gradient itself are important for scaled patterning along the A–P length in early embryos.Recent experimental studies have significantly advanced our knowledge on Bcd gradient behavior in bcd mRNA from which Bcd is produced. For example, unlike what is assumed in most of the current models, the maternally deposited bcd mRNA is not restricted to a single point at the anterior tip bcd mRNA may affect the shape and formation of the Bcd gradient remains an important question bcd mRNA in early embryos, it was suggested that the shape and formation of the Bcd protein gradient are dictated by bcd mRNA distributions.According to a widely-held view Drosophila embryos. It allows us to evaluate, among other things, the effect of bcd mRNA redistribution on Bcd gradient formation, suggesting that, in our model, Bcd protein diffusion is important for the formation of an exponential protein gradient. We also present simulation results that recapitulate several other important features of the Bcd gradient, including the experimentally observed scaling properties of the Bcd gradient and the stability of nuclear Bcd concentrations between different nuclear cycles.In this report, we develop a biologically realistic 2-D model to simulate the formation of the Bcd gradient in Drosophila embryos, focusing on evaluating the effects of the changing diffusion media within which the Bcd gradient is formed and the molecular properties of bcd mRNA from which Bcd protein is produced. Since stochastic properties of the Bcd gradient system have been extensive investigated recently free, that are in the cytoplasm, and immobile molecules, Bbound, that are bound to low-affinity DNA sites inside the nuclei. The binding/unbinding behavior of Bcd to these DNA sites is characterized by the rate constants 1k and 2k, respectively, and expressed as:AK = 1k/2k:effD can be expressed as:D is the diffusion constant for free-diffusing Bcd molecules inside the cytoplasm.A primary goal of our current study is to establish a biologically realistic model that simulates the dynamics of Bcd protein distribution in early Drosophila embryo, where Bcd molecules are distributed. An ellipse is selected to represent the boundary of the embryo slice, with major axis majora and minor axis minorb. The shape of the ellipse is similar to the mid-coronal section of a typical wt embryo with majora = 550 µm and a minorb/majora ratio of 1/3. A 2-D mesh grid system is used to divide the embryo slice into uniform cubes that are arranged on the coronal 2-D plane, each with a volume of Δx3 µm3. We performed simulations with Δx = 2 and 5 µm, and obtained consistent results based on values for the three established criteria that quantify Bcd gradient properties (see below); Δx3 = 53 µm3 represents a cube volume that approximates the volume of a nucleus in early embryos D to denote the effective diffusion constant in cube  at the cth nuclear cycle. During nuclear cycles 1–9, the entire embryo is treated as a homogeneous medium filled with cytoplasm, in which the nuclei are evenly distributed. The number of nuclei during this period is limited  is selected to be D for all cubes within the embryo boundary during nuclear cycles 1–9. At the onset of nuclear cycle 10, all nuclei become located in the cortex of the embryo in our model, forming the cortical layer  remains to be D for all cubes located within the nucleus-free inner part of the embryo. For cubes within the cortical layer, the doubling of nuclei number after each nuclear division leads to the doubling of the concentration of low-affinity DNA sites for Bcd, with the effective diffusion constant of Bcd molecules expressed as:sites]10 is the concentration of low-affinity DNA sites for Bcd within the cortical layer at nuclear cycle 10.In our 2-D model, we simulate the mid-coronal slice of the bcd mRNA and, thus, produce Bcd protein at a constant rate J (mol/s). In the main model simulations (bcd mRNA sphere is modeled as a disc (orthographic projection) inside the simulated embryo slice, with a uniform density, a radius of 45 µm and a center coordinate of ; the shape, location and amount of bcd mRNA remain constant at all nuclear cycles in these simulations. The J value is identical for all cubes that contain bcd mRNA; thus the effective Bcd production rate for the entire embryo is the aggregate J . At time t, Bcd molecules within each cube are in equilibrium with the low-affinity DNA sites, and we use the Euler forward method  at time t+Δt as a result of protein diffusion to and from its four immediate neighbors and position-independent protein degradation:i,jυ is the volume of cube  and ω is the degradation rate of Bcd molecules. i,jI and i,jk are binary values (0 or 1) depending on the status of the cube:t = 0.2s; we also tested Δt = 0.01, 0.1, 0.5 and 1.0s and obtained consistent results based on the values for the three established criteria to quantify simulated Bcd gradient behavior (see below), indicating that our solutions are numerically stable.In our simulations, we assign a status to each cube in our mesh grid system depending on its center coordinate and developmental time: the cortical layer (I) and the inner part (II); prior to the formation of the cortical layer, all cubes within the embryo boundary are treated as status II as discussed above. We further assign status III to cubes that contain ulations –5, tht = 0 represents the time of egg deposition and our simulations end at 15 minutes into nuclear cycle 14. Nuclear cycles 10–14 start at 81, 95, 110, 125 and 140 minutes after egg deposition, respectively. The Bcd concentration profiles for nuclear cycles 10–14 shown in this report represent simulated data at 94, 109, 124, 139 and 154 minutes after egg deposition unless otherwise stated. The parameter values used in our simulations are either calculated based on estimates  or chosen through a systematical sampling within the biologically reasonable ranges to yield Bcd gradient properties that satisfy three established criteria (see below). In our main model  that we establish to quantify the Bcd gradient behavior. In our simulation results, nuclear Bcd concentrations [Bn] are calculated from the local DNA-bound Bcd concentrations [Bbound]. Since the nuclear volume remains a constant between different nuclear cycles in our main model  values We establish three different criteria to quantify the behavior of the simulated Bcd gradient. First, we measure the stability of nuclear Bcd concentration [Btot]. These simulation results realistically recapitulate the biological system and reveal several important features that are consistent with experimental observations. First, consistent with experimental observations x/L = 0.2 to 0.7 exhibit an excellent exponential fit , the exponential fitting becomes significantly worse when the simulated data points from the most anterior part of the embryo are included . The observed anterior “deviation” of the Bcd gradient profiles in our 2-D model is due to the size and location of bcd mRNA (see bcd mRNA is strictly located at the anterior tip).bound] and nuclear Bcd concentration [Bn], which are shown in n] remain stable  if the amounts of maternally-deposited components, including bcd mRNA, are proportional to the egg volume, i.e., the concentrations of various maternal components remain as constants among individual eggs even though the total maternally-deposited contents  can vary between eggs. In our first analysis, we simulated two individual embryos that have distinct lengths (550 µm and 600 µm). x (in µm) and fractional embryo length x/L. Our results show that, while these two profiles in the anterior and middle portions of the embryo are apart from each other in the x plot, they converge in the x/L plot, most notably in the broad, mid-portion of the embryo. When the same bcd mRNA amount was applied to these two embryos, such a convergence was not observed (data not shown). In a second analysis, we simulated a group of 50 embryos that differ in size (see bound] noise (standard deviation divided by the mean) at nuclear cycle 14 for these embryos as a function of either x or x/L. Our results . In contrast, when there is no correlation between the bcd mRNA amount and embryo size in our simulations, [Bbound] noise actually became higher as a function of x/L than of x . These AK values correspond to a dissociation constant DK range of ∼8×10−5 to ∼3×10−6 M for non-specific DNA binding by Bcd, values that are fully consistent with the biochemical properties of homeodomains sites]10 value of ∼3×10−7 M is most likely an underestimation of the true concentration of the low-affinity sites at nuclear cycle 10 because our calculation assumes that these sites are homogeneously distributed within the entire cortical layer as opposed to the nuclear volume and, furthermore, it is likely that there are other types of low-affinity DNA sites for Bcd in addition to the 5′-TAAT-3′ sites used in our estimates. In our model, both [DNAsites]10 and AK affect the system through the equilibrium ratio of bound to free Bcd molecules within the cortical layer at nuclear cycle 10 (Eqs. 2 and 4). This ratio has an operating range between ∼0.1 and ∼0.36 that is capable of yielding Bcd gradient profiles satisfying all three of the established criteria in our main model. We note that a separate consideration of [DNAsites]10 and AK offers an opportunity to directly evaluate the biological reasonableness of the parameter values used in our simulations. It also allow us to treat embryos from different species based on their distinct physical properties to investigate interspecies scaling of the Bcd gradient profiles (unpublished).Our parameter evaluation analysis also shows that the upper and lower limits of the operating range of ectively . These rprofiles ; it alsofiles  identical to that used in n] remain stable during nuclear cycles 10–14 . These results show that, with modest parameter adjustments, our model can yield, from the redistributed bcd mRNA profiles, nuclear Bcd concentration profiles and dynamics properties that are broadly consistent with experimental observations.A recent study challenges the diffusion model of Bcd gradient formation, raising the question of whether this process is dependent on Bcd protein diffusion es 10–14  and exhiD progressively in our simulations. Our results show that as D is reduced, [Bbound] profiles at nuclear cycle 14 become progressively less exponential . The Adjusted R2 values of the exponential fit (in the region of x/L = 0.2 to 0.7) decreased from 0.9854 at D = 2 µm2s−1, to 0.9708 and 0.8987 at D = 1 and 0.1 µm2s−1, respectively  it enhances the enrichment of Bcd molecules within the cortical layer relative to the inner part of the embryo  terion g . These fn]bound]/nuclear number. Furthermore, our main model assumes that the thickness of the cortical layer remains as a constant between different nuclear cycles. To determine whether changes in nuclear volume and cortical layer may affect the [Bn] stability, we performed simulations where both the cortical layer thickness and nuclear size vary between nuclear cycles. In these simulations, [Bn] calculations were based on estimated nuclear diameter of 10, 10.5, 9.2, 8.2 and 6.5 µm for nuclear cycles 10–14, respectively bound] and [Bn] profiles at different nuclear cycles . These results show that our model, with proper parameter adjustments, can produce stable [Bn] profiles when incorporating changes in both nuclear size and cortical layer thickness between different nuclear cycles  is proportional to the embryo volume. Our simulation results . Second, while the Coppey et al. model assumes a nuclear barrier-mediated equilibrium between Bcd molecules inside and outside the nucleus, our main model assumes a chemical equilibrium between free Bcd molecules and those that are bound to non-specific DNA sites inside the nucleus. Since nuclear localization of a protein is insufficient for proper gradient formation in embryos A recent model n] stability The 2-D model described in this report represents a simple and versatile tool to simulate the Bcd gradient system in a biologically realistic way. In contrast to a recent model that is also sufficient to explain  at nuclear cycle 14 (red and green) and experimentally observed Bcd gradient profile also at early nuclear cycle 14 (blue). The experimental data shown here are from He et al. n] profiles are obtained from simulations identical to the main model simulations expect the center coordinate of bcd mRNA was fine tuned to yield a [Bn] profile that matches the experimentally observed Bcd profile  or the bcd mRNA is restricted to a single cube at the anterior tip of the embryo . The Adjusted R2 values for our experimental data within the fitting ranges of x/L = 0.2 to 0.7 and 0 to 0.7 are 0.9961 and 0.9851, respectively. We note that both simulated Bcd profiles have lower levels in the posterior part of the embryo than the experimentally observed profile, a difference whose biological relevance will require further experimental and modeling investigations. B. Same as in A, except on ln scale. While the simulated red profile matches well with the experimental data and exhibits the experimentally observed anterior “deviation,” the simulated green profile clearly fails to exhibit this property.Comparison between simulated data and experimental data. A. Shown are simulated [B(0.51 MB TIF)Click here for additional data file.Figure S2n] stability as measured by g , gradient shape as measured by length constant λ at nuclear cycle 14 , and cortical enrichment as measured by the ratio of total Bcd molecules in the cortical layer to those in the inner part of the embryo at nuclear cycle 14 . The regions where the Bcd gradient profiles satisfy the established criteria, i.e., |g|<0.1, Ratio>1.5, and 1.5<λ/L<2.0, are shaded. In these analyses, individual parameters are systematically changed in simulations when the other two parameters are at set values at their respective model simulations. Different colors represent different simulation procedures as indicated in the figure.Evaluating the effects of parameter values in different simulations. Parameter values for KA (panels A–C), D (panels D–F) and ω (panels G–I) are systematically altered to evaluate model performance. Three criteria are used in these evaluations: [B(0.76 MB TIF)Click here for additional data file.Figure S3tot] (arbitrary units). In this simulation, the mitotic process is specifically considered at nuclear cycles 10–13, during which all Bcd molecules are allowed to diffuse freely. Other parameter values are identical to those used in the main model. The A–P position is shown as absolute distance x (in µm) from the anterior. At nuclear cycle 14, the ratio of total Bcd molecules in the cortical layer to those in the inner part of the embryo is 1.7975. B. A plot of [Bbound] (arbitrary units) within the cortical layer as a function of x/L, at nuclear cycles 10–14. C. Same as in B, except now showing [Bn] within the cortical layer at nuclear cycles 10–14. Similar to the main model (n] stability (g = 0.0143). The simulated [Bn] profile at nuclear cycle 14 has a length constant λ = 93.3 µm. See 2-D simulation with mitosis. A. A simulated embryo at nuclear cycle 14 showing  is plotted as a function of D. For comparative purposes, results obtained from both the main model simulation (red) and the simulation with bcd mRNA redistribution (blue) are shown. Note the difference in Adjusted R2 sensitivity to D. B. The ratio of total Bcd molecules in the cortical layer to those in the inner part of the embryo plotted as a function of D. Color codes are the same as in A. Regions where Ratio >1.5 are shaded. Note the higher Ratio value obtained in the mRNA redistribution simulation (blue). C. A plot of g as a function of D, with color codes being the same as in A. Regions where |g|<0.1 are shaded. Note the blue curve is within the shaded area under all D values tested, suggesting another potential role of the observed bcd mRNA redistribution.Investigating the effects of bcd mRNA redistribution. A. Adjusted R2 value of the exponential fitting (within the range of x/L = 0.2 to 0.7) of simulated [B(0.54 MB TIF)Click here for additional data file.Figure S5tot] (arbitrary units). In this simulation, non-specific DNA binding site concentrations were calculated based on the following estimates: the thickness of the cortical layer is 15, 20, 20 23, and 25 µm for nuclear cycles 10–14, respectively, corresponding to a cortical layer volume of ∼3.3, 4.2, 4.2 4.7, and 5.0 nl. The concentrations of the non-specific DNA binding sites within the cortical layer are: 5×10−7, 4×10−7, 4×10−7, 3.6×10−7, and 3.4×10−7 M, for nuclear cycles 10–14, respectively. The relative volumes of a single nucleus, which were calculated based on the experimental estimates 2s−1, ω = 0.0004 s−1, KA = 2.4×106 M−1. At nuclear cycle 14, the ratio of total Bcd molecules in the cortical layer to those in the inner part of the embryo is 5.0044. B. A plot of [Bbound] (arbitrary units) within the cortical layer as a function of x/L, at nuclear cycles 10–14. C. Same as in B, except now showing nuclear Bcd concentrations [Bn] at nuclear cycles 10–14. [Bn] is calculated from [Bbound] at each nuclear cycle based on the nuclear number and the volumes of each nucleus and the cortical layer. As seen in the main model (n] profiles exhibit stability between different nuclear cycles (g = −0.050) when changes in nuclear volume and cortical layer between different nuclear cycles are incorporated in our simulation. The [Bn] profile at nuclear cycle 14 has a length constant λ = 105 µm. We note that the results shown in Fig. S5 do not represent an improvement over those obtained in the main model simulations. It is possible that the use of additional parameters may improve the simulation outcome when more biological features are included in our model.Evaluating the effects of nuclear size and cortical layer at different nuclear cycles. A. A simulated embryo at 5 min into nuclear cycle 14 showing  pr(0.75 MB TIF)Click here for additional data file.


Cryphonectria parasitica, a plant pathogen and causative agent of chestnut blight, contains three Gα, one Gβ, one Gγ subunits and phosducin-like protein BDM-1 that have important roles in pigmentation, sporulation and virulence. Deletion of either Gβ subunit or BDM-1 produces identical phenotypes. Additionally, we report that the Gβ subunit is not detectable in absence of BDM-1. Given that the regulatory role of phosducin-like proteins may be influenced by protein kinase 2 (CK2), we confirmed that BDM-1 is a phosphoprotein that can be targeted by CK2 in vitro. Mutagenesis of the five putative CK2 sites revealed that native phosphorylation likely occurs at two locations. Strains bearing a single or double serine to alanine substitutions at those sites were significantly less virulent with only minor phenotypic changes from vegetative colonies. Therefore, CK2 activity appears to mediate key signals that are required for virulence, but not for vegetative growth. Expression of selected CK2 mutants resulted in reduced accumulation of the Gβ subunit, suggesting that phosphorylation of BDM-1 influences Gβ stability.Phosducin-like proteins are conserved regulatory components of G-protein signalling pathways, which mediate many physiological processes. Identified throughout eukaryotic genomes, they are thought to serve as regulators of Gβγ assembly.  Heterotrimeric GTP-binding proteins (G-proteins), consisting of three subunits Gα, Gβ and Gγ, are ubiquitous signalling components that play a well established role in the ability of eukaryotic organisms to react to environmental stimuli  are presSaccharomyces cerevisiae, Dictyostelium discoideum, and various filamentous fungal systems have been shown to utilize the essential features of this system, but with far fewer subunits. With the advent of genome sequence information, it has been possible to determine that Ascomycete fungal systems, for instance, generally contain three Gα subunits, accompanied by a single representative each of Gβ and Gγ. However, even with fewer subunits, the signalling processes are still associated with the control of vital behaviours such as mating . These have been shown to be functionally related to Gβγ complexes in mammalian systems , teleostscoideum . In D. dC. parasitica. We report that BDM-1 is a phosphoprotein that can be a target for CK2 activity and that this phosphorylation is functionally relevant. We have observed that elimination of phosphorylation at these sites caused a significant reduction in virulence and the quantity of both Gβ and BDM-1 proteins, suggesting a role for BDM-1 in protein stability that may be related to its function in mammalian systems as a potential regulator of the CCT complex  spheroplasts, both constructs rescued the distinct phenotype of Δbdm-1 , which revealed putative sites that included five for CK2, two for protein kinase A (PKA), five for protein kinase C (PKC) and one site for N-glycosylation (in vitro (C. parasitica strain EP155 (WT), the migration pattern of BDM-1 was restored to that of BDM-1 prior to CIP treatment  for an unmodified serine, substitution with alanine would cause no change in migration rate but substitution by aspartic acid would retard mobility; (ii) for a modified serine, substitution with alanine would cause an increase in migration rate, whereas an aspartatic acid substitution would not change the mobility. Lastly, treatment with CIP would remove only phosphate moieties and not alter any mobility effects induced by aspartic acid substitution.Since we demonstrated that BDM-1 is post-translationally modified via CK2 activity, we have investigated the nature of this modification in more detail. Previously, QTOF mass spectrometry was used to examine the modifications of PhLP , howeverSingle alanine substitution mutants m2A, m3A, m4A and m5A  we used the completely substituted m12345A and m12345D variants in order to establish any potential relationship between CK2 phosphorylation and Gβ stability. Heterokaryon strains coexpressing myc-Gβ/m12345A and myc-Gβ/m12345D were first verified for presence of both genes by PCR using genomic DNA as template, then observed for distinct phenotypes in comparison to myc-Gβ expressed with non-mutated BDM-1. As with the expression of the BDM-1 mutants in the ckground , the difckground . We noteckground .Western blot analysis of heterokaryon strains revealed differences in the stability of myc-Gβ protein in the presence of BDM-1 phosphorylation mutants. Coexpression of m12345A caused reduced accumulation of myc-Gβ by eightfold compared with m12345D, whereas only a slight decrease of myc-Gβ protein was observed in presence of m12345D, compared with unmodified FLAG-BDM-1 . To ensuC. parasitica have identified components of G-protein signalling, including BDM-1, a PhLP, and CPGB-1, the Gβ-subunit. Elimination of either protein reduces virulence, pigmentation and sporulation in an almost identical manner (C. parasitica with virulence-attenuating dsRNA viruses (hypoviruses) compromises G-protein signalling and causes changes in virulence and phenotype, with aspects that resemble those of the BDM-1 and Gβ deletion strains  or mimic (Ser to Asp) CK2-mediated phosphorylation at single or multiple sites. Western blot analysis of migration patterns of the BDM-1 phosphorylation mutants  allowed Mutants with Ser to Ala substitutions m1A, m34A, m14A, m134A and m12345A showed overall increased mobility in comparison to intact FLAG-BDM-1 , indicatAlthough single mutants m3A and m4A migrated similarly to unmodified FLAG-BDM-1, variant m34A exhibited slightly increased migration. Thus, we suspect that either position m3 or m4 might be targeted by CK2 as well. Bearing in mind that mutants m14A, m134A and m12345A migrated with the same rate, we suggest that CK2 sites m1 and m4 are most likely phosphorylated while sites m2, m3 and m5 are not .It has been reported that phosphorylation within the CK2 sites can be primed in a hierarchical fashion by previously phosphorylated residues . ConsequUnexpectedly, migration of mutant m1234A was equal to that of unmodified FLAG-BDM-1  and coulin vivo since their mobility was retarded . However, the alanine-substituted mutant caused an eightfold reduction of both BDM-1 and CPGB-1 proteins . TherefoThe results presented above provide evidence for the requirement of CK2-mediated phosphorylation of BDM-1, although our current data do not allow us to state with certainty the precise phosphorylation pattern. However, we propose that, in its nascent state, BDM-1 undergoes phosphorylation by CK2, possibly at positions m1 and m4, in a hierarchical manner with m1 being targeted first. Once phosphorylated, we predict that BDM-1 is able to encourage the formation of a functional Gβγ dimer, presumably in the presence of a fungal CCT complex. This would then be followed by association with the Gγ subunit. Substitution of serine residues by alanine at positions m1 and m4 prevents phosporylation at those sites, destabilizes the process such that the signalling pathway becomes impaired to a level that is unable to fully support virulence, but does not greatly impact vegetative growth. In the complete absence of BDM-1, the assembly fails and our data would suggest that this then leads to degradation of the Gβ subunit. This model is consistent with a role for BDM-1 that is analogous to that identified for PhLP-1 in mammalian systems, in which CK2 phosphorylation was required for Gβγ assembly  and accuCryphonectria parasitica EP155 (ATCC 38755), Δcpgb-1 . The fusion protein was recovered from cell lysates using a Ni2+ charged HiTraP column (Amersham Biosciences). The BDM-1 binding portion was recovered from serum by passing over purified BDM-1 attached to a covalently NHS-activated Hi-trap column (Amersham). For all Western blots described below, this purified antiserum was used at 1 : 2500 dilution. Anti-Gβ (Anti-BDM1 antiserum was generated in rabbits by Strategic Biosolutions (now Strategic Diagnostics) using purified BDM-1 isolated from bdm-1 or cpgb-1, respectively, following the start codon while the reverse primers contained either a HindIII or XbaI restriction site. All primer sequences are detailed in bdm-1, which were then subcloned into the same restriction sites of C. parasitica expression vectors pCPX-NBn1 (containing gpd promoter) and pBC6HC1 (containing native bdm-1 promoter) resulting in plasmids pJS-3 and pJS-3X respectively. Excised from plasmid pJS-21 by restriction digest with NotI and HindIII, myc-tagged cpgb-1 was subcloned into the expression vector pCPX-NBn1 resulting in plasmid pJS-26. Both expression vectors pCPX-NBn1 and pBC6HC1 contained a benomyl resistance gene (benr) for selectivity.The coding sequence for the FLAG (DYKDDDDK) or myc (QGKLISQQDL) epitopes was incorporated into 5′ primers for bdm-1 sequences were subcloned into the expression vector as described above and followed by transformation of the Δbdm-1 spheroplasts using the method of Mutagenesis of FLAG-BDM-1 was initially performed using pJS-3 as template according to the Quick Change Site Directed Mutagenesis Kit (Stratagene) and verified by sequencing. Mutated Fusion proteins contained in 35 µg of total protein lystates were bound to 20 µl of ANTI-FLAG M2-agarose beads (Sigma) for 2 h at room temperature, washed and eluted with 2× LDS buffer (Invitrogen) or processed for re-phosphorylation. Samples were run on NuPAGE 10 or 12% Bis-Tris Gels (Invitrogen) in MOPS buffer and probed with anti-BDM-1 serum followed by 1 : 2500 HRP-conjugated anti-rabbit secondary antibody (Bio-Rad).Some 35 µg of clear protein lysates was incubated for 30 min at 37°C with 20 000 units of CIP (Invitrogen) then immunoprecipitated with 20 µl of ANTI-FLAG M2-agarose beads. De-phosphorylated proteins were re-phosphorylated by addition of equal amounts of WT (EP155) protein extracts in presence of 1 mM ATP (Sigma), 100 mM sodium orthovanadate (Sigma) to inhibit residual CIP activity and 20 µM specific Casein Kinase II (CK2) inhibitor 2-dimethylamino-4, 5, 6, 7-tetrabromo-1H-benzimidazole  where indicated. The reaction was performed at room temperature in the dark and protein eluted with 2× LDS buffer followed by separation on NuPAGE 10% Bis-Tris Gels (Invitrogen) in MOPS buffer. Protein detection was carried out as previously described.The virulence assays were performed essentially as described by


Surfactant protein A (SP-A) is a C-type lectin involved in surfactant homeostasis as well as host defense in the lung. We have recently demonstrated that SP-A enhances the killing of bacillus Calmette-Guerin (BCG) by rat macrophages through a nitric oxide-dependent pathway. In the current study we have investigated the role of tyrosine kinases and the downstream mitogen-activated protein kinase (MAPK) family, and the transcription factor NFκB in mediating the enhanced signaling in response to BCG in the presence of SP-A.5 BCG for 30 min at 37°C. Cells were incubated with PBS, SP-A, BCG, or SP-A-BCG complexes for the times indicated. BCG killing was assessed using a 3H-uracil incorporation assay. Phosphorylated protein levels, enzyme assays, and secreted mediator assays were conducted using standard immunoblot and biochemical methods as outlined.Human SP-A was prepared from alveolar proteinosis fluid, and primary macrophages were obtained by maturation of cells from whole rat bone marrow. BCG-SP-A complexes were routinely prepared by incubation of a ratio of 20 μg of SP-A to 5 × 10Involvement of tyrosine kinases was demonstrated by herbimycin A-mediated inhibition of the SP-A-enhanced nitric oxide production and BCG killing. Following infection of macrophages with BCG, the MAPK family members ERK1 and ERK2 were activated as evidence by increased tyrosine phosphorylation and enzymatic activity, and this activation was enhanced when the BCG were opsonized with SP-A. An inhibitor of upstream kinases required for ERK activation inhibited BCG- and SP-A-BCG-enhanced production of nitric oxide by approximately 35%. Macrophages isolated from transgenic mice expressing a NFκB-responsive luciferase gene showed increased luciferase activity following infection with BCG, and this activity was enhanced two-fold in the presence of SP-A. Finally, lactacystin, an inhibitor of IκB degradation, reduced BCG- and SP-A-BCG-induced nitric oxide production by 60% and 80% respectively.These results demonstrate that BCG and SP-A-BCG ingestion by macrophages is accompanied by activation of signaling pathways involving the MAP kinase pathway and NFκB. Mycobacterium tuberculosis, with over three million deaths and eight million new cases per year -Uracil was purchased from NEN . Fetal bovine serum (FBS) for culture of rat bone marrow macrophages (RBMM) was purchased from HyClone Laboratories; all other tissue culture reagents were from GIBCO-BRL . Kinase assay kits, U0126, and antibodies against phosphorylated and non-phosphorylated ERK1 and ERK2 were obtained from Cell Signalling Technologies . All other reagents were purchased from Sigma Chemical . ATP. Labelled oligonucleotide (2 × 105 cmp), single-stranded salmon sperm DNA (200 ng), nuclear extract proteins (10 μg), and binding buffer ) were incubated at room temperature for 20 min. A 10-fold excess of unlabeled oligonucleotide was used in the competition assays. Samples were resolved by electrophoresis on 5% polyacrylamide non-denaturing gels in 0.5× Tris-borate-EDTA (TBE) buffer at 150 volts constant. The gels were dried and bands visualized by autoradiography.Cells were incubated with LPS (100 μg), SP-A, BCG, or SP-A-BCG for 30 min. Nuclear extracts were isolated from cells as follows: cells were suspended in lysis buffer ; 0.5 mM phenylmethylsulfonyl fluoride; and 100 μl protein inhibitor solution (Sigma)), and placed on ice for 10 min. After centrifugation for one minute at 13,000 × g, the nuclei-containing pellet was washed once in lysis buffer, and then suspended in extraction buffer  and vortexed for 15 min at 4°C. Gel shift oligonucleotides containing an NFκB consensus site from the human iNOS promoter (AGTTGAGGGGACTTTCCCAGGC)  were endp value of < 0.05 was considered significant. Data in figures are expressed as mean ± SD.The differences between groups were tested using one-way ANOVA. In all cases, a Activation of intracellular protein tyrosine kinases is a common pathway involved in signalling induced by a variety of pathogens and pathogen-derived products. To determine if BCG-induced production of nitric oxide by rat macrophages in the presence and absence of SP-A involves tyrosine kinase activation, RBMM were incubated with BCG or SP-A-BCG complexes in the presence and absence of 100 nM herbimycin A. As shown in Figure 3H-uracil. After incubation for 5 days, the labelled BCG were collected and the associated radioactivity was quantified. The 3H-uracil assay is useful in this instance since unlike mammalian host cells the parasite (BCG) can utilize the uracil directly for pyrimidine salvage. 3H-Uracil is therefore a valuable counting assay because it allows for pathogen-specific labelling. There should be very little if any labelling of co-purified cellular components. For example, previous studies by Somogyi and Foldes showed that mycobacteria incorporate 80% of 3H-uracil into RNA and 20% into DNA [3H-uracil used in the experiment [We have previously reported that SP-A enhances the killing of BCG by rat macrophages. To determine if intracellular growth of BCG is dependent on protein tyrosine phosphorylation, cells were pre-treated with 100 nM herbimycin A for 30 min, then infected with BCG or SP-A-BCG complexes for 4 hr. The cells were washed, and ingested BCG was metabolically labelled with into DNA . In studperiment .As shown in Figure Several groups have identified MAP kinase family members as key targets of PTKs and participants in signalling cascades leading to the induction of proinflammatory mediators. To determine if two of these family members, ERK-1 and ERK-2, are involved in BCG and SP-A-BCG signalling, immunoblot analysis was used to examine the level of ERK phosphorylation as a measure of ERK activation. Cells were incubated for the indicated times with BCG or SP-A-BCG. At each time point, cells were washed, and then solubilized in immunoprecipitation buffer. Extracts were analyzed by immunoblot analysis, using an antibody specific for the phosphorylated forms of ERK-1 and ERK-2. As shown in Figure To determine if the enhanced phosphorylation of ERK-1 and ERK-2 correlated with increased kinase activity, in vitro kinase assays were performed. Cells were treated with BCG or SPA-BCG for 5 and 15 min. Control cells were incubated for 15 min with SP-A alone. Total cellular protein was extracted, and phosphorylated ERK-1/2 was immunoprecipitated using a polyclonal antibody specific for the phosphorylated forms of both enzymes. The immunoprecipitates were then incubated with kinase buffer and Elk-1-glutathione-S-transferase fusion protein as a substrate in the kinase reaction. ERK activation was then determined by immunoblot analysis of the cell extracts using anti-phospho-Elk-1 antibody. As shown in Figure To determine if ERK activation in response to BCG resulted in production of nitric oxide, cells were pre-treated with U0126, an inhibitor of the upstream kinases MEK-1 and MEK-2 required for ERK activation. U0126 (1 μM) or methanol (vehicle) was added to RBMM 30 min prior to incubation with PBS, SP-A, BCG, or SP-A-BCG. After 24 hr, nitric oxide levels in the media were measured. As shown in Figure Several groups have recently reported activation of NFκB in response to both intact mycobacteria and mycobacterial cell wall components -27. To dMycobacteria are obligate intra-macrophage organisms, and must devise ways to avoid triggering the host response leading to microbe killing. It is therefore likely that interaction of virulent mycobacteria with host macrophages will lead to minimal production of inflammatory mediators and limited activation of anti-microbial processes. In previous studies we have shown that SP-A enhances BCG-induced production of nitric oxide and TNF, resulting in increased BCG killing by the infected macrophages . A commoIn initial experiments we found that a general inhibitor of PTKs (herbimycin A) blocked both the BCG- and SP-A-BCG-induced production of nitric oxide and the killing of internalized BCG, suggesting that one or more cellular kinases was required for signalling. An important downstream target of cellular PTKs is the family of MAP kinases that are activated following phosphorylation. These serine/threonine kinases then phosphorylate and activate downstream targets such as specific transcription factors that lead to modulation of gene expression. In the current study we found that BCG alone activated ERK1/2 with maximal stimulation at 15 min. SP-A enhanced and prolonged this activation with a maximal effect at 5 min. Inhibitors of upstream kinases blocked nitric oxide production in the presence of both BCG and SP-A-BCG, further supporting a role for this pathway during BCG infection. These results suggest that the ability of SP-A to enhance BCG killing as previously described involves activation of the MAP kinases ERK1/2.M. avium-induced TNF production in human monocyte-derived macrophages involved ERK but not p38 [M. avium with mouse bone marrow macrophages resulted in TNF production that was dependent on ERK activation but did not involve stimulation of p38 [M. avium-induced TNF production in mouse bone marrow macrophages [M. avium activated ERK and p38 but not JNK in the same cells [M. tuberculosis activated ERK and JNK but not p38 in RAW cells [These studies are supported by work from other laboratories demonstrating a role of members of the MAP kinase family in mycobacterial signalling, but the specific members of the family that play a role appear to be dependent on the mycobacterial species as well as the source and functional status of the macrophages used for study. For example, Reiling et al. reported that  not p38 . Blumentn of p38 . In controphages , and RoaAW cells . We haveLeishmania have shown that entry of organisms into non-activated macrophages is accompanied by activation of protein tyrosine phosphatases that inactivate MAP kinases through removal of phosphate groups [Leishmania organisms are internalized by stimulated macrophages, MAP kinases are activated with concomitant production of proinflammatory mediators. Ibata-Ombetta reported that Candida was able to prolong survival in macrophages by specific activation of MAP kinase phosphatase (MKP)-1, leading to deactivation of ERK1/2 [There is a growing body of evidence that survival of intra-macrophage pathogens is linked to activation and deactivation of intracellular kinases. Studies with e groups . When Lef ERK1/2 . Henningf ERK1/2 . Thus, aIt has been suggested that the MAP kinase-mediated increase in the production of inflammatory mediators may involve activation of transcription factors such as NFκB, although a direct link leading from MAP kinase activation to NFκB activation has not been established. In the current study we have shown that BCG and SP-A-BCG complexes activate NFκB in addition to members of the MAP kinase family, but we cannot definitely say that NFκB activation is dependent on MAP kinase activity. Manucso et al. reported that the NFκB inhibitor CAPE blocked GBS-stimulated TNF production, however ERK inhibitors did not alter p50/p65 activation, suggesting two independent pathways . Carter M. tuberculosis, M. avium, and BCG – all have high mannose groups exposed on their surfaces, making them good candidates for mannose receptor ligands [M. tuberculosis was internalized by human monocyte-derived macrophages through the mannose receptor in the absence of opsonins. However, there is no report directly linking mycobacterial binding to the mannose receptor to activation of signalling pathways. In fact, Reiling et al. reported that M. avium-induced TNF production by human monocyte-derived macrophages was blocked by anti-CD14 antibodies but not my anti-mannose receptor antibodies [Receptors that might be involved in mediating mycobacterial or SP-A-mycobacterial effects are not yet known. The mycobacteria species that have some clinical relevance – including  ligands . In supptibodies . More retibodies ,27,57,58tibodies ,59. AntiIn the current and previous studies we have found no effect of SP-A alone on RBMM function. Only when attached to a particulate material does SP-A appear to induce signalling in RBMM leading to production of inflammatory mediators. This is somewhat controversial, since other groups have found that SP-A alone has an effect on resident macrophages. For example, early studies from several laboratories reported that SP-A interaction with macrophages and macrophage cell lines resulted in production of reactive oxygen and nitrogen species and inflammatory cytokines, and activated NFκB -64. VazqM. avium. In previous work we have shown that SP-A increases M. avium ingestion by RBMM and enhances production of both TNF and nitric oxide [M. avium. Gomes et al. reported that M. avium growth was enhanced in the presence of nitric oxide [M. avium growth [M. avium, leading to continued and possibly enhanced intracellular growth. The effect of SP-A on pathogen survival may be directly linked to the specific signalling pathways turned on by each pathogen, and SP-A may not be able to overcome alternative cellular pathways activated by certain pathogens.Although we have shown that SP-A enhances killing of BCG by rat macrophages, this does not appear to be the case with ic oxide . Howeveric oxide , and Tsem growth . One migThis is the first report demonstrating that SP-A increases mediator production in response to mycobacteria through activation of MAP kinases and NFκB. Like other intra-macrophage pathogens, mycobacteria have evolved a variety of strategies for evading host defense, including limitation of the ability of the host cell to trigger important signalling pathways. In the lung, during the first insult by mycobacteria, SP-A may play a role in the response of uninfected, non-activated alveolar macrophages by enhancing their capacity to activate signalling pathways, thus turning on necessary defense genes such as iNOS and TNF. The role of SP-A is complex, and may depend directly on the nature of the pathogen and the state of activation of the macrophages. In addition, SP-A may interact differently with mycobacteria released from macrophages as opposed to mycobacteria in the initial onslaught. These questions are currently being addressed in our laboratory.(BCG): bacillus Calmette-Guerin; (PTK): protein tyrosine kinase; (ERK): extracellular signal regulated kinase; (MAP kinase): mitogen-activated protein kinase; (JNK): c-Jun amino terminal kinase; (LPS): lipopolysaccharide; (LAM): lipoarabinomannan; (NFκB): nuclear factor κB; (SP-A): surfactant protein A; (RBMM): rat bone marrow macrophages; (FBS): fetal bovine serum; (TLR): toll-like receptor.The authors declare that they have no competing interests.JL carried out the immunoblot analyses, the inhibitor studies, the NFkB assays, and the enzymatic assays. DV participated in the design and coordination of the study, and helped to draft the manuscript. VS conducted the killing assays, conceived of the study, participated in the design, and supervised the experimental work. All authors read and approved the final manuscript.


Drosophila melanogaster responds to gram-negative bacterial challenges through the IMD pathway, a signal transduction cassette that is driven by the coordinated activities of JNK, NF-κB and caspase modules. While many modifiers of NF-κB activity were identified in cell culture and in vivo assays, the regulatory apparatus that determines JNK inputs into the IMD pathway is relatively unexplored. In this manuscript, we present the first quantitative screen of the entire genome of Drosophila for novel regulators of JNK activity in the IMD pathway. We identified a large number of gene products that negatively or positively impact on JNK activation in the IMD pathway. In particular, we identified the Pvr receptor tyrosine kinase as a potent inhibitor of JNK activation. In a series of in vivo and cell culture assays, we demonstrated that activation of the IMD pathway drives JNK-dependent expression of the Pvr ligands, Pvf2 and Pvf3, which in turn act through the Pvr/ERK MAP kinase pathway to attenuate the JNK and NF-κB arms of the IMD pathway. Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response. Furthermore, given the pleiotropic involvement of JNK in eukaryotic cell biology, we believe that many of the novel regulators identified in this screen are of interest beyond immune signaling. Drosophila melanogaster is an ideal springboard for such studies, as core aspects of innate immune pathways are evolutionarily conserved and novel discoveries in Drosophila often inspire subsequent developments in the characterization of biomedically relevant mammalian pathways. Drosophila responses to certain bacterial invaders proceed through the IMD pathway, which contains partially overlapping signal transduction JNK and NF-κB arms. While substantial efforts have illuminated much of the NF-κB arm, there is a considerable paucity of information on the regulation of the JNK arm. We conducted a survey of the entire Drosophila genome for novel regulators the Imd/dJNK pathway. In this study, we uncovered a novel link between the proliferative Pvr pathway and the IMD pathway.Innate immunity is the sole immune response in the overwhelming majority of multicellular organisms and drives the sophisticated antigen-specific adaptive defenses of vertebrates. Defective regulation of immune signal transduction pathways has disastrous consequences for affected individuals and can result in life-threatening conditions that include cancer, autoimmune and neurological conditions. Thus, there is a major need to identify the regulatory circuits that govern activation of critical innate immune response pathways. The genetically accessible model organism  Drosophila melanogaster is a powerful model for the study of innate immune signaling events owing to the high degree of evolutionary conservation of signal transduction pathways Drosophila led to the characterization of Toll as an essential element of invertebrate immune armories, which prompted the search for and characterization of Toll homologs in humans The adaptive immune response is a recent evolutionary acquisition by vertebrates. In contrast, the innate immune response is highly conserved among metazoans and is the first line of defense against invading pathogens immune deficiency (imd) gene uncovered a distinct immune response to gram-negative bacterial infections in DrosophilaDrosophila immunity to gram-negative bacteria requires an intact IMD signaling pathway, which shares many other similarities with the TNF pathway. Engagement of the IMD pathway requires recognition of diaminopimelic acid-containing peptidoglycan (PGN) by the PGN Receptor Protein (PGRP-LC) Drosophila c-Jun N-terminal Kinase (dJNK) and the NF-κB transcription factor family member Relish (Rel). The Rel arm of the IMD pathway is well characterized thanks to a number of individual studies and complementary genetic and cell culture RNA interference (RNAi) screens. Essentially, Rel activation requires the activities of Imd, the caspase-8 ortholog Dredd, dFADD, dTAB2, dIAP2 and the MAP3 kinase dTAK1 Characterization of a mutation in the attacin (att) and diptericin (dipt).Active dTAK1 drives the subsequent activation of the I-Kappa Kinase (IKK) components Kenny (Key) and Ird5 djnk are lethal due to defective epithelial sheet sealing in the dorsolateral axis of the developing embryo IMD pathway activation of dTAK1 also stimulates a kinase cascade through the MAP2Ks dMAPKK4/7 that leads to dJNK phosphorylation Drosophila tissue culture cells provide an ideal environment to study these events, as PGN-induced activation of the IMD pathway induces a transient dJNK activation that is easily quantified. To understand the regulation of PGN-induced dJNK phosphorylation in the IMD pathway, we performed a high-throughput, quantitative RNAi screen for modulators of dJNK phosphorylation. To this end, we treated the embryonic macrophage-like S2 cell line with 15,683 individual dsRNAs that cover all annotated genes in the Drosophila genome. In contrast to previous RNAi screens of the IMD pathway, our assay did not rely on indirect reporter constructs. Instead, we used phospho-JNK specific monoclonal antibodies in a quantitative plate-based assay to directly quantify the impact of each dsRNA on the extent of PGN-induced dJNK phosphorylation. In this manner, we identified enhancers and suppressors of dJNK activation. As a testament to the accuracy of this screen, we unambiguously identified fifteen established IMD pathway components as modifiers of dJNK activation. In addition, we identified numerous novel regulators of dJNK activation. Given the involvement of dJNK in cellular events as diverse as development, cell migration, immune signaling and cell death, we believe that many of the regulators identified in this screen will be of broad interest to the study of metazoan cell biology.Drosophila enhances the infection-induced expression of att. These data indicate that the Pvr/dERK signal transduction pathway constitutes a novel negative regulator of the Drosophila IMD pathway.We present a comprehensive analysis of a novel regulator of dJNK in IMD pathway signaling – the PDGFR and VEGFR receptor (Pvr) tyrosine kinase. Pvr is primarily known for its role in the guidance of cellular movements Drosophila S2 cells with a library of 15,683 dsRNAs that cover all annotated genes in the Drosophila melanogaster genome and we monitored the subsequent extent of PGN-induced dJNK phosphorylation by In Cell Western (ICW) analysis. We used monoclonal antibodies specific for phosphorylated JNK (P-JNK) and fluorescently labeled secondary antibodies to directly visualize PGN-induced dJNK phosphorylation. We simultaneously monitored filamentous actin (f-actin) levels with fluorescently labeled phalloidin as a control measure of total cell numbers. We then quantified the ratio of P-JNK:f-actin for each well to determine the relative extent of dJNK phosphorylation in each sample. To identify genes that modulate the intensity and duration of dJNK phosphorylation, we screened the entire genome at fifteen and sixty minutes. We reasoned that depletion of gene products that are required for optimal PGN-induced dJNK phosphorylation will decrease dJNK phosphorylation at fifteen minutes and we defined such gene products as enhancers of dJNK phosphorylation. Likewise, we reasoned that depletion of gene products involved in dJNK dephosphorylation will increase the relative intensity and/or duration of dJNK phosphorylation at fifteen and/or sixty minutes and we defined such gene products as suppressors of dJNK phosphorylation.Engagement of the IMD pathway leads to transient dJNK phosphorylation; PGN-induced dJNK phosphorylation peaks at 5min and returns to basal levels by 60min in S2 cells e.g. . We deveA representative 96-well plate from the screen is shown in We then measured the PGN-induced P-JNK:f-actin levels and determined the z-score for all non-lethal dsRNA treatments. We graphed all the z-scores from highest to lowest for both fifteen and sixty minutes PGN-exposures . dsRNA-mDrosophila PDGF/VEGF Receptor (Pvr) homolog appeared as a major node within this branch.To test the validity of the dsRNA screen, we selected a representative cohort of three enhancers and eight suppressors of PGN-induced dJNK phosphorylation for secondary analysis. We monitored the effect of dsRNA treatment for all genes in the cohort on dJNK phosphorylation relative to f-actin at zero, fifteen and sixty minutes PGN-exposure. We compared the eleven putative modifier dsRNAs to two dsRNAs (CG11318 and Toll) that had no effect on dJNK phosphorylation in the primary screen. Secondary dsRNA analysis was consistent with the screen results as nine of the eleven dsRNAs significantly modified dJNK phosphorylation relative to f-actin compared to control dsRNA . Even thTo confirm Pvr as a suppressor of dJNK phosphorylation in the IMD pathway, we depleted S2 cells of Pvr with two independent non-overlapping dsRNAs and monitored relative dJNK phosphorylation upon exposure to PGN at zero, fifteen and sixty minutes. We confirmed that both dsRNAs deplete Pvr by monitoring Pvr protein levels relative to actin in S2 cell lysates using Pvr specific antibodies . TreatmeDrosophila ERK signaling and cell migration. To investigate the involvement of the Pvr pathway in attenuation of dJNK activation, we determined the dJNK:f-actin z-score for each member of the Pvr/dERK axis in the primary screen. As a comparison, we also determined the dJNK:f-actin z-scores for members of the wingless pathway – a signal transduction pathway with no know interaction with the IMD/dJNK module. As expected, our data do not indicate any major interactions between the wingless and IMD/dJNK pathways. In contrast, our data consistently indicate that the Pvr/dERK pathway negatively regulates dJNK activation  from the re to CM . Consistpression . Thus, watt expression. Treatment of S2 cells with the MEK1 inhibitor PD98059 decreased CM-induced dERK phosphorylation 3.2 fold relative to S2 cells treated with CM alone  in adult flies. We then compared the immune response of infected wild type flies to flies that express Pvr-IR. Specifically, we monitored the expression of the Rel-responsive transcript att in uninfected flies (control) and flies that were pricked with a needle coated in E. coli (infection). Strikingly, we noticed that in vivo depletion of Pvr significantly enhanced infection-mediated att expression in three separate experiments in two separate Pvr-IR fly lines , hemipterous  and dMKK7 (MKK7 ortholog) as required for activation of dJNK in the IMD pathway. A recent RNAi-based survey of four hundred eighty two Drosophila genes identified seventy seven core JNK pathway regulators We also consider it likely that we have serendipitously identified general regulators of the JNK pathway with roles that extend beyond immune signaling. For example, we identified core elements of the JNK activation cassette such as We consider the false negative rate for IMD pathway members a more pertinent measure of the success of our screen. In contrast to previous RNAi screens of signal transduction pathways, our assay did not rely on indirect reporter assays. Instead, we measured the contribution of each annotated gene within the fly genome to the IMD-responsive phosphorylation of dJNK. We believe that the direct quantitative nature of our assay combined with the ease of RNAi in S2 cells greatly minimizes the likelihood of false negatives in the primary screen. Indeed, preliminary analysis of our primary screen data identified the bulk of the IMD signal transduction pathway  as essential modifiers of JNK activation in the IMD pathway. In each case, the phenotype was consistent with the established molecular function of the respective IMD pathway element as either negative or positive modifiers of JNK activation. Thus, we are satisfied that false negatives do not obfuscate interpretation of our data in any meaningful manner. Ironically, the only anticipated hit we failed to identify was dJun Drosophila receptor tyrosine kinase Pvr shows considerable similarity to members of the mammalian PDGF and VEGF receptor families and Pvr is considered an evolutionary ancestor of PDGF/VEGF receptors The pvr mutants were originally interpreted to indicate that Pvr detects migratory guidance cues in hemocytes pvr mutants rescues the majority of the migratory phenoptye pvr hemocyte phenotype is the result of cell death and that there are only minor guidance requirements for Pvr in hemocyte migration. Pvr activates the dERK pathway, which induces hemocyte proliferation in vivo and incubation of embryonic mbn-2 hemocytes with Pvr antibodies blocks cellular proliferation in a dose-dependent manner in vivo and a recent study indicated that proliferative signals for hemocytes are preferentially provided by Pvf2 and Pvf3 In addition to requirements for Pvr in cell migration, a parallel body of literature indicates a distinct function for Pvr in the regulation of hemocyte proliferation. The disruptions to embryonic hemocyte migration in Our study reveals a novel role for the Pvr/dERK pathway in the attenuation of the IMD pathway and illuminates our understanding of the network of regulatory checks and balances that fine tune the level of IMD/dJNK activity. Our data are most consistent with a model whereby activation of the IMD pathway results in dJNK-dependent expression of the Pvr ligands Pvf2 and Pvf3. Pvr then signals through dERK to negatively regulate the IMD pathway. On a molecular level, our data show that Pvr signaling dampens the dTAK1-dependent phosphorylation of dJNK and Rel. However, we believe that our data may also uncover an additional physiological role for Pvr. We speculate that the infection-driven production of Pvf2 and Pvf3 engages Pvr receptors on hemocytes and thereby stimulates the Ras/dERK-responsive proliferation of hemocytes. Such an increase in hemocytes numbers would provide a timely measure for the phagocytic elimination of invading extracellular microbes at early stages of infection.Drosophila hemocytes in immunity. Hemocytes are the major phagocytic cell type in Drosophila and are ideally suited for the engulfment of extracellular microbes. We consider it possible that induction of immune responses drives Pvr-mediated proliferation of hemocytes to facilitate rapid neutralization of extracellular microbes through phagocytosis. In this situation, it is advantageous for proliferative signals to suppress JNK activation, as hyper or prolonged activation of JNK in Drosophila often results in cell death. Preliminary data in our lab suggest that links between Pvr and immune signaling may be evolutionarily conserved, as we detected suppression of NF-κB activity through the PDGF receptor superfamily member c-Kit in human cell culture assays .We find it intriguing that proliferative signals inhibit activation of immune pathways. It may be that both processes require major metabolic commitments and that hemocytes preferentially reserve resources for proliferation. An alternative and non-exclusive hypothesis reflects the primary role of Drosophila S2 cells and KC167 cells were cultured at 25°C in HyQ TNM-FH medium (HyClone) supplemented with 10% heat inactivated fetal bovine serum (Invitrogen), 50U/ml of penicillin and 5 µg/ml of streptomycin (GIBCO). Serum-free S2 cells were incubated in SFX-INSECT medium (HyClone) supplemeted with 50U/ml of penicillin and 5 µg/ml of streptomycin (GIBCO). PGN-dependent dJNK activation was inhibited in 106 S2 cells in 1ml of culture media with the addition of 25µM SP600125 for 1h prior to PGN-exposure.5 cells/well in 96 well plate in 20% conditioned media and 80% serum-free culture media with 10µg/ml dsRNAs at 25°C for three days. Cells were exposed to 50µg/ml LPS (Sigma) containing contaminating amounts of PGN for 15 or 60 min. Cell were washed with PBS, fixed in PBS + 3.7% formaldehyde, permeablized in PBS + 0.1% Triton-X 100 and blocked in blocking buffer (LI-COR Biosciences). Cells were probed with mouse anti-active-JNK  and washed with PBS + 0.1% Tween-20. P-JNK staining was detected with fluorescently labeled goat anti-mouse secondary antibodies and f-actin was stained with fluorescently labeled phalloidin (Invitrogen). Cells were washed in PBS + 0.1% Tween and P-JNK and f-actin levels were quantified with an Aerius automated imaging system (LI-COR Biosciences) following the manufacturers recommendations. In secondary ICW analyses P-JNK was monitored relative to JNK by replacing phalloidin staining with rabbit anti-JNK (Santa Cruz Biotechnology) and fluorescently labeled goat anti-rabbit antibodies.The dsRNA library employed in this screen is an extension of a partial-genome library described previously Drosophila interactions database http://www.droidb.org/IMBrowser.jsp). For analysis of att expression in the infection model, the ΔCt values were standardized to an internal control between qRt-PCR runs. The triplicate 0h ΔCt values were averaged and the ΔΔCt values were calculated relative to these values. The fold change was calculated for each sample and the 0h time point was set to one for each fly line. The SEM was calculated for each time point. Statistical significance of experimental values was expressed as p-values of less than .01 (**) or .05 (*), as calculated by a Student's t-test. We performed two-tailed Student's t-tests with two-samples of equal variance to calculate a p-value of experimental values relative to control values.For the RNAi screen, the raw fluorescent trimmed mean level was determined for P-JNK and f-actin channels in each well and the relative P-JNK:f-actin value was calculated. We applied z-score analysis to normalize P-JNK:f-actin values across the entire screen. Z-scores were calculated by subtracting the sample value by the plate median value and dividing by the plate standard deviation. The z-score assumes normal distribution and represents the standard deviation of every P-JNK:f-actin value from the plate median for each dsRNA treatment. Z-scores above 2.58 or below −2.58 represent the 99% confidence interval and z-scores above 1.96 or below −1.96 represent the 95% confidence interval. The f-actin z-scores were also calculated for every well on each plate and dsRNA treatments resulting in f-actin z-scores below −2.58 (99% CI) were excluded from further analysis to eliminate actin modifiers and lethal dsRNAs. We considered dsRNAs that modified P-JNK:actin z-scores outside the 95% confidence interval as hits in the screen. To identify genetic or physical interactions among hits from our screen, all hits were probed in the 6 cells lysed in sample buffer, vortexted and incubated at 95°C for 5min. Proteins were separated by SDS-PAGE electrophoresis and were transfer to nitrocellulose membrane by semidry transfer. Membranes were blocked in blocking buffer (LI-COR Biosciences) and probed with mouse anti-active-JNK (Cell Signailing), rabbit anti-JNK (Santa Cruz Biotechnology), rabbit anti-pan-actin , mouse anti-actin (Sigma), rabbit anti-active MAPK1/2 (Upstate), mouse anti-HA (Sigma) or rat anti-Pvr. Western blot analysis of P-Rel and Rel cleveage was performed as described in Western blot analysis was performed on 106 S2 cells or 10 adult flies using Trizol (Invitrogen) following the manufacturers instructions. cDNA was created from 2µg of RNA using Superscript III (Invitrogen) and oligo-dT primers (Invitrogen), according to the manufacturers instructions. We monitored transcript amplification with a Realplex 2 PCR machine (Eppendorf) using SYBR green as a detection reagent (Invitrogen). We used the following primers to monitor the expression of the corresponding gene products; actin forward 5′-TGCCTCATCGCCGACATAA-3′, actin reverse 5′-CACGTCACCAGGGCGTAAT-3′; att forward 5′-AGTCACAACTGGCGGAC-3′, att reverse 5′-TGTTGAATAAATTGGCATGG-3′; dipt forward 5′-ACCGCAGTACCCACTCAATC-3′, dipt reverse 5′-ACTTTCCAGCTCGGTTCTGA-3′; pvf1 forward 5′-GCGCAGCATCATGAAATCAACCG-3′, pvf1 reverse 5′-TGCACGCGGGCATATAGTAGTAG-3′; pvf2 forward 5′-TCAGCGACGAAACGTGCAAGAG-3′, pvf2 reverse 5′-TTTGAATGCGGCGTCGTTCC-3′; pvf3 forward 5′-AGCCAAATTTGTGCCGCCAAG-3′, pvf3 reverse 5′- CTGCGATGCTTACTGCTCTTCACG-3′. All transcript expression values were normalized to actin and were quantified relative to a control using the ΔΔCt method.Antimicrobial peptide production was monitored in S2 cells and flies by qRT-PCR. Total RNA was extracted from 105′-GGGCGGT-3′ as an anchor sequence; Pvr1 forward 5′-GGGCGGGTGATGACTACATGGAGATGAGCC-3′, Pvr1 reverse 5′-GGGCGGGTATACCTTCGTTGCTCCTTCTCG-3′; Pvr2 forward 5′-GGGCGGGTCTCCTGATTTTGCGGATCTC-3′, reverse 5′-GGGCGGGTGTCTTGGGATCGGTTCTTGA-3′; GFP forward 5′-GGGCGGGTACGTAAACGGCCACAAG-3′, GFP reverse 5′-GGGCGGGTCTCAGGTAGTGGTTGTC-3′. We performed a second PCR amplification of anchor-tagged template DNA with the T7 promoter containing primer 5′-TAATACGACTCACTATAGGGAGACCACGGGCGGGT-3′. dsRNA was amplified from template DNA using T7 RNA polymerase at 25°C for 6h and annealed by cooling from 90°C to 30°C. S2 cells were depleted of Pvr using Pvr1 dsRNA unless stated otherwise. 106 S2 cells were treated with dsRNA for 4 days in 1ml culture media to deplete Pvr. The Pvr pathway was activated in S2 cell using 1∶1 dilution of fresh culture media in conditioned media (CM) collected from 4 day cultures of KC167 cells. Pvr dependent dERK phosphorylation was inhibited in 106 S2 cells in 1ml of culture media with the addition of 50µM PD98059 for 1h prior to CM exposure.We depleted Pvr from S2 cells with two non-overlapping dsRNAs. We designed the following primers to amplify the associated dsRNA template DNA in a two step PCR using Drosophila strains were cultured on standard cornmeal medium (http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/bloomfood.htm) at 25°C. hs-gal4 flies were obtained from Dr. Sarah Hughes and uas-PvrIR flies were obtained from the Vienna Drosophila RNAi Center. For in vivo knock down of Pvr, UAS-PvrIR flies were crossed with hs-gal4 flies or 118w flies. 1 day old flies were heat-pulsed eight times at 37°C for 1h to initiate the expression of the RNAi construct and returned to 25°C for 5h over 48 hours. Infection was monitored in flies that were either uninjured (control), or pricked with a tungsten needle dipped in a pellet of DH5α E. coli bacteria (infection).Figure S1P-JNK screen comparative analysis. Comparative analysis of 15min and 60min PGN-induced P-JNK:f-actin screen results relative to Bakal C. et. al. 2008 (0.50 MB TIF)Click here for additional data file.Table S1z-score analysis of dsRNA effects on 15 and 60 min PGN-induced dJNK phosphorylation. In-cell Western z-scores were calculated from P-JNK:f-actin values from S2 cells incubated with 15,683 dsRNAs and treated with PGN for 15 or 60 min. Z-scores are ordered from highest to lowest 15 min z-score. dsRNAs that modified f-actin levels below the 95% at 15 min PGN treatment were excluded. Each dsRNA is identified by its Celera Genome (CG) number or by its Heidelberg Drosophila Consortium identification number (HCDID) and general function.(0.96 MB PDF)Click here for additional data file.Table S2z-score analysis of dsRNA-mediated depletion of enhancers of PGN-induced dJNK phosphorylation. In-cell Western z-scores were calculated from P-JNK:f-actin values from S2 cells incubated with 15,683 dsRNAs and treated with PGN for 15 min. dsRNAs that modified P-JNK:f-actin z-scores below 1.96 (95% CI) are ordered from smallest to highest z-score. The fold change in dJNK phosphorylation relative to the plate median is shown alongside the z-score values. Each dsRNA is identified by its symbol and Celera Genome (CG) number or by its Heidelberg Drosophila Consortium identification number (HCDID).(0.33 MB DOC)Click here for additional data file.Table S3z-score analysis of dsRNA-mediated depletion of suppressors of 15 min PGN-induced dJNK phosphorylation. In-cell Western z-scores were calculated from P-JNK:f-actin values from S2 cells incubated with 15,683 dsRNAs and treated with PGN for 15 or 60 min. dsRNAs that modified 15 min PGN-induced P-JNK:f-actin z-scores above 1.96 (95% CI) are ordered from highest to lowest z-score. The fold change in dJNK phosphorylation relative to the plate median is shown alongside the z-score values for both 15 and 60 min time points. Each dsRNA is identified by its symbol and Celera Genome (CG) number or by its Heidelberg Drosophila Consortium identification number (HCDID).(1.05 MB DOC)Click here for additional data file.Table S4z-score analysis of dsRNA-mediated depletion of suppressors of 60 min PGN-induced dJNK phosphorylation. In-cell Western z-scores were calculated from P-JNK:f-actin values from S2 cells incubated with 15,683 dsRNAs and treated with PGN for 15 or 60 min. dsRNAs that modified 60 min PGN-induced P-JNK:f-actin z-scores above 1.96 (95% CI) are ordered from highest to lowest z-score. The fold change in dJNK phosphorylation relative to the plate median is shown alongside the z-score values for both 15 and 60 min time points. Each dsRNA is identified by its symbol, Celera Genome (CG) number, Heidelberg Drosophila Consortium identification number (HCDID) and general function.(0.50 MB DOC)Click here for additional data file.