A proteome reference map and proteomic analysis of Bifidobacterium longum NCC2705

A comprehensive proteomic study was carried out to identify and characterize proteins expressed by Bifidobacterium longum NCC2705. A total of 708 spots representing 369 protein entries were identified by MALDI-TOF-MS and/or ESI-MS/MS. Isoelectric point values estimated by gel electrophoresis matched closely with their predicted ones, although some discrepancies exist suggesting that post-translational protein modifications might be common in B. longum. The identified proteins represent 21.4% of the predicted 1727 ORFs in the genome and correspond to 30% of the predicted proteome. Moreover 95 hypothetical proteins were experimentally identified. This is the first compilation of a proteomic reference map for the important probiotic organism B. longum NCC2705. The study aimed to define a number of cellular pathways related to important physiological processes at the proteomic level. Proteomic comparison of glucose- and fructose-grown cells revealed that fructose and glucose are catabolized via the same degradation pathway. Interestingly the sugar-binding protein specific to fructose (BL0033) and Frk showed higher levels of expression in cells grown on fructose than on glucose as determined by semiquantitative RT-PCR. BL0033 time course and concentration experiments showed that the induction time and fructose concentration correlates to increased expression of BL0033. At the same time, an ABC (ATP-binding cassette) transporter ATP-binding protein (BL0034) was slightly up-regulated in cells grown on fructose compared with glucose. All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific transport system in which BL0033 might play an important role.     

Sugar transport systems of Bifidobacterium longum NCC2705

Here we present the complement of the carbohydrate uptake systems of the strictly anaerobic probiotic Bifidobacterium longum NCC2705. The genome analysis of this bacterium predicts that it has 19 permeases for the uptake of diverse carbohydrates. The majority belongs to the ATP-binding cassette transporter family with 13 systems identified. Among them are permeases for lactose, maltose, raffinose, and fructooligosaccharides, a commonly used prebiotic additive. We found genes that encode a complete phosphotransferase system (PTS) and genes for three permeases of the major facilitator superfamily. These systems could serve for the import of glucose, galactose, lactose, and sucrose. Growth analysis of NCC2705 cells combined with biochemical characterization and microarray data showed that the predicted substrates are consumed and that the corresponding transport and catabolic genes are expressed. Biochemical analysis of the PTS, in which proteins are central in regulation of carbon metabolism in many bacteria, revealed that B. longum has a glucose-specific PTS, while two other species (Bifidobacterium lactis and Bifidobacterium bifidum) have a fructose-6-phosphate-forming fructose-PTS instead. It became obvious that most carbohydrate systems are closely related to those from other actinomycetes, with a few exceptions. We hope that this report on B. longum carbohydrate transporter systems will serve as a guide for further in-depth analyses on the nutritional lifestyle of this beneficial bacterium.     

Bile-Inducible Efflux Transporter from Bifidobacterium longum NCC2705, Conferring Bile Resistance

Bifidobacteria are normal inhabitants of the human gut. Some strains of this genus are considered health promoting or probiotic, being included in numerous food products. In order to exert their health benefits, these bacteria must overcome biological barriers, including bile salts, to colonize and survive in specific parts of the intestinal tract. The role of multidrug resistance (MDR) transporters in bile resistance of probiotic bacteria and the effect of bile on probiotic gene expression are not fully understood. In the present study, the effect of subinhibitory concentrations of bile on the expression levels of predicted MDR genes from three different bifidobacterial strains, belonging to Bifidobacterium longum subsp. longum, Bifidobacterium breve, and Bifidobacterium animalis subsp. lactis, was tested. In this way, two putative MDR genes whose expression was induced by bile, BL0920 from B. longum and its homolog, Bbr0838, from B. breve, were identified. The expression of the BL0920 gene in Escherichia coli was shown to confer resistance to bile, likely to be mediated by active efflux from the cells. To the best of our knowledge, this represents the first identified bifidobacterial bile efflux pump whose expression is induced by bile.In the gut, commensal bacteria, including those that have health-promoting or probiotic activity, are challenged by the presence of several toxic compounds of intestinal origin, such as bile salts. Bile is secreted into the duodenum to an estimated concentration of 0.2 to 2% for a given bile salt (14). Bile salts are detergent-like compounds with strong antimicrobial activity (2). Therefore, intestinal microorganisms have developed strategies to tolerate physiological concentrations of bile salts during passage through, or colonization of, the gut.Bifidobacteria are common inhabitants of the human gastrointestinal tract (GIT) (19, 39). Some strains of this genus are known to have probiotic activity and are used as functional ingredients in food products around the world (40). The health-promoting effects attributed to these microorganisms are numerous (20, 42). To exert beneficial actions, these bacteria must overcome biological barriers including acid in the stomach and bile in the intestine in order to, at least temporarily, colonize specific parts of the GIT. Thus, understanding the mechanisms of resistance of a given commensal or probiotic microorganism to toxic substances, such as bile salts, is important in the context of its physiology in the GIT. Although bifidobacterial bile tolerance mechanisms are currently poorly understood, bifidobacteria with increased resistance to bile salts have been obtained by progressive adaptation to gradually increasing concentrations of these compounds (27, 29). Through the analysis of such bile-tolerant derivatives, it was shown that the acquisition of bile resistance coincides with changes in membrane protein profiles (27) and carbohydrate metabolism (35, 38), while also conferring cross-resistance to other environmental stresses (29, 36). This indicates that the bifidobacterial response to bile entails a complex cellular activation/repression process, which impacts on general metabolic pathways (37).Specific bile resistance mechanisms have been described in intestinal bacteria, with bile efflux and bile salt hydrolysis being the most prevalent (31). In this respect, multidrug resistance (MDR) transporters seem to play a crucial role in conferring a bile resistance phenotype. MDR proteins are present in all organisms and frequently confer resistance against several structurally unrelated toxic compounds (31). Research on these transporters has mainly been focused on their role in antibiotic resistance. However, given their ubiquitous presence this does not seem to be their primary function. In fact, recent studies support a role for various MDR transporters in allowing microorganisms to survive, establish, and persist in their (human) host (31).It has been shown elsewhere that MDR proteins confer resistance to bile in different enteric bacteria (32). Bile was found to upregulate the expression of the multidrug efflux system cmeABC in Campylobacter jejuni (22), and inhibition of this pump was found to reduce the colonization ability of the microorganism by reducing bile resistance (21). In vitro and in vivo induction of acrAB expression by bile was observed in Vibrio cholerae (6), and also other MDR proteins appear to be induced in this microorganism (5). In the intestinal bacterium Bacteroides fragilis, the expression of different MDR pumps was also found to be upregulated by bile (34).Several MDR systems have been identified in gram-positive bacteria (including probiotic bacteria) (8, 28), but their role in conferring resistance to intestinal toxic compounds, such as bile salts, and the effect of bile on gene expression have not received much scientific attention. Transporters able to extrude bile salts have been found in gram-positive bacteria such as Lactococcus lactis (44, 45) and Lactobacillus johnsonii (7). In Lactobacillus plantarum a membrane protein whose expression is induced by bile, both in vitro and in vivo, was previously identified (3). Proteins conferring resistance to bile, and whose expression is induced by it, have also been reported in other lactobacilli (30, 43). Just a couple of studies have been published on MDR transporters in bifidobacteria. Margolles and coworkers identified and characterized two MDR transporters from Bifidobacterium breve, BbmAB and BbmR, conferring resistance to antimicrobials (25, 26). In Bifidobacterium longum an MDR transporter, Ctr, was found to export cholate from the cell, conferring resistance to this compound when cloned in a heterologous bacterial host (33). However, there is still a knowledge gap with regard to possible effects of bile on MDR gene expression and the potential role of MDR transporters in bile resistance in bifidobacteria.In the present study, the effect of subinhibitory concentrations of bile on the expression levels of genes encoding bifidobacterial MDR protein homologs was tested. For this purpose, known or putative MDR genes were selected from the genomes of different Bifidobacterium strains belonging to the species B. longum, B. breve, and Bifidobacterium animalis. A putative MDR-encoding gene, present as a homolog in both B. breve and B. longum and whose expression was strongly induced by bile, was identified, and the B. longum gene was then characterized.     

Fructose uptake in Bifidobacterium longum NCC2705 is mediated by an ATP-binding cassette transporter

Recently, a putative ATP-binding cassette (ABC) transport system was identified in Bifidobacterium longum NCC2705 that is highly up-regulated during growth on fructose as the sole carbon source. Cloning and expression of the corresponding ORFs (bl0033-0036) result in efficient fructose uptake by bacteria. Sequence analysis reveals high similarity to typical ABC transport systems and suggests that these genes are organized as an operon. Expression of FruE is induced by fructose, ribose, or xylose and is able to bind these sugars with fructose as the preferred substrate. Our data suggest that BL0033-0036 constitute a high affinity fructose-specific ABC transporter of B. longum NCC2705. We thus suggest to rename the coding genes to fruEKFG and the corresponding proteins to FruE (sugar-binding protein), FruK (ATPase subunit), FruF, and FruG (membrane permeases). Furthermore, protein-protein interactions between the components of the transporter complex were determined by GST pulldown and Western blot analysis. This revealed interactions between the membrane subunits FruF and FruG with FruE, which in vivo is located on the external side of the membrane, and with the cytoplasmatic ATPase FruK. This is in line with the proposed model for bacterial ABC sugar transporters.     

Genome comparison of Bifidobacterium longum strains NCC2705 and CRC-002 using suppression subtractive hybridization

Because probiotic effects are strain dependent, genomic explanations of these differences will contribute to understanding their mechanisms of action. The genomic sequence of the Bifidobacterium longum probiotic strain NCC2705 was determined, but little is known about the genetic diversity between strains of this species. Suppression subtractive hybridization (SSH) is a powerful method for generating a set of DNA fragments differing between two closely related bacterial strains. The purpose of this study was to identify genetic differences between genomes of B. longum strains NCC2705 and CRC-002 using PCR-based SSH. Strain CRC-002 produces exopolysaccharides whereas NCC2705 is not known for reliable exopolysaccharide production. Thirty-five and 30 different sequences were obtained from the SSH libraries of strains CRC-002 and NCC2705, respectively. Specific CRC-002 genes found were predicted to be involved in the biosynthesis of exopolysaccharides and metabolism of other carbohydrates, and these genes were not present in the genome of strain NCC2705. The identification of an endo-1,4-beta-xylanase gene in the CRC-002 SSH library is an important difference because xylanase genes have previously been proposed as a defining characteristic of the NCC2705 strain. The results demonstrate that the SSH technique was useful to highlight potential genes involved in complex sugar metabolism that differ between the two probiotic strains.     

Combination of heterogeneous catalase and superoxide dismutase protects Bifidobacterium longum strain NCC2705 from oxidative stress

Bifidobacteria are generally sensitive to oxidative stress caused by reactive oxygen species (ROS). To improve oxidative-stress tolerance, the superoxide dismutase (SOD) gene from Streptococcus thermophilus (StSodA) and the heme-dependent catalase (KAT) gene from Lactobacillus plantarum (LpKatL) were heterologously expressed in Bifidobacterium longum strain NCC2705. Three types of strain NCC2705 transformants were obtained: with transgenic SOD expression, with transgenic KAT expression, and with coexpression of the two genes. Intracellular expression of the genes and their functional role in oxidative-stress resistance were evaluated. In response to oxidative stress, B. longum NCC2705/pDP401-LpKatL (expressing LpKatL) and NCC2705/pDP-Kat-Sod (coexpressing LpKatL and StSodA) rapidly degraded exogenous H₂O₂ and the peroxides generated as a byproduct of aerobic cultivation, preventing oxidative damage to DNA and RNA. Individual expression of StSodA or LpKatL both improved B. longum NCC2705 cell viability. Survival rate of strain NCC2705 was further improved by combining SOD and KAT expression. The two enzymes played complementary roles in ROS-scavenging pathways, and coexpression led to a synergistic beneficial effect under conditions of intensified oxidative stress. Our results illustrate that heterogeneous expression of heme-dependent KAT and Mn²⁺-dependent SOD is functional in the B. longum oxidative-stress response, and synergistic protection is achieved when their expressions are combined.     

长双歧杆菌NCC2705株果糖结合蛋白BL0033基因的克隆及其在大肠杆菌中的表达

目的：克隆长双歧杆菌NCC2705株果糖结合蛋白BL0033的基因,利用大肠杆菌表达GST-BL0033融合蛋白并纯化。方法：以长双歧杆菌NCC2705株基因组为模板,PCR扩增BL0033基因,并将其插入pGEX-4T-1表达载体,转化至大肠杆菌DH5α;提取质粒,经PCR、质粒双酶切及测序鉴定后,转入大肠杆菌BL21,并对表达条件进行摸索;用谷胱甘肽-Sepharose4B树脂对可溶性GST-BL0033融合蛋白进行纯化。结果：PCR扩增的BL0033基因长度接近1000bp,与预期值一致;重组菌在IPTG浓度为0.05mmoL/L的条件下,于16℃诱导过夜后,SDS-PAGE分析可见可溶性表达条带,相对分子质量约60×103,与预期值一致;亲和纯化后,SDS-PAGE结果显示单一的表达条带。结论：克隆了BL0033蛋白的基因,并表达纯化了融合蛋白GST-BL0033,为进一步研究长双歧杆菌NCC2705株BL0033蛋白功能奠定了基础。     

长双歧杆菌NCC2705群体感应系统信号分子AI-2的检测

目的:用生物学方法检测长双歧杆菌NCC2705是否产生群体感应系统信号分子AI-2。方法:将长双歧杆菌NCC2705不同时间点的培养上清分别加至AI-2特异报告系统哈氏弧菌BB170中,以空白培养基上清为对照,用荧光光度计对哈氏弧菌发光强度进行计量,推测出长双歧杆菌NCC2705上清中是否含有分泌的AI-2,并由此推断AI-2的活性。结果:通过微孔板检测系统对加入长双歧杆菌NCC2705培养上清的哈氏弧菌BB170进行检测,发现双歧杆菌上清的加入增强了哈氏弧菌BB170发出的荧光强度。结论:长双歧杆菌NCC2705中存在依赖于luxS/AI-2的群体感应系统,并能够分泌有活性的AI-2,为进一步研究长双歧杆菌NCC2705AI-2及luxS基因的功能打下基础。     

长双歧杆菌NCC2705高效转化系统的建立及GFP表达

目的:建立长双歧杆菌NCC2705高效稳定的电转化系统,并以其为宿主表达绿色荧光蛋白(GFP),构建一个稳定的带报告基因的表达载体。方法:从双歧杆菌克隆载体pDG7中切取其在双歧杆菌中复制所必需的pMB1复制子插入质粒pUC19的多克隆位点区,并将gfp基因在双歧杆菌中进行表达;设计双歧杆菌电击转化体系,研究在不同电击电压条件下的电转效率。结果:首先构建了大肠杆菌-长双歧杆菌穿梭质粒pUB1和带有gfp基因的表达质粒pUB2,用电击法将之转化至双歧杆菌,当细菌生长到D600nm约为0.5时制备感受态细胞,在电容25μF、电阻200Ω、电压12.5 kV/cm的电击条件下进行完整质粒转化,可得到较高的转化率。结论:构建了以长双歧杆菌NCC2705为宿主的高效稳定的表达系统,为进一步研究益生菌的分子生物学和功能特性提供了基础。     

Analysis of proteome changes in doxorubicin-treated adult rat cardiomyocyte

Doxorubicin-induced cardiomyopathy in cancer patients is well established. The proposed mechanism of cardiac damage includes generation of reactive oxygen species, mitochondrial dysfunction and cardiomyocyte apoptosis. Exposure of adult rat cardiomyocytes to low levels of DOX for 48h induced apoptosis. Analysis of protein expression showed a differential regulation of several key proteins including the voltage dependent anion selective channel protein 2 and methylmalonate semialdehyde dehydrogenase. In comparison, proteomic evaluation of DOX-treated rat heart showed a slightly different set of protein changes that suggests nuclear accumulation of DOX. Using a new solubilization technique, changes in low abundant protein profiles were monitored. Altered protein expression, modification and function related to oxidative stress response may play an important role in DOX cardiotoxicity.     

Aluminum induced proteome changes in tomato cotyledons

Cotyledons of tomato seedlings that germinated in a 20 µM AlK(SO₄)₂ solution remained chlorotic while those germinated in an aluminum free medium were normal (green) in color. Previously, we have reported the effect of aluminum toxicity on root proteome in tomato seedlings (Zhou et al.¹). Two dimensional DIGE protein analysis demonstrated that Al stress affected three major processes in the chlorotic cotyledons: antioxidant and detoxification metabolism (induced), glyoxylate and glycolytic processes (enhanced), and the photosynthetic and carbon fixation machinery (suppressed).Key words: aluminum, cotyledons, proteome, tomatoDifferent biochemical processes occur depending on the developmental stages of cotyledons. During early seed germination, before the greening of the cotyledons, glyoxysomes enzymes are very active. Fatty acids are converted to glucose via the gluconeogenesis pathway.^2,3 In greening cotyledons, chloroplast proteins for photosynthesis and leaf peroxisomal enzymes in the glycolate pathway for photorespiration are metabolized.^2–4 Enzymes involved in regulatory mechanisms such as protein kinases, protein phosphatases, and mitochondrial enzymes are highly expressed.^3,5,6The chlorotic cotyledons are similar to other chlorotic counterparts in that both contains lower levels of chlorophyll, thus the photosynthetic activities are not as active. In order to understand the impact of Al on tomato cotyledon development, a comparative proteome analysis was performed using 2D-DIGE following the as previously described procedure.¹ Some proteins accumulated differentially in Al-treated (chlorotic) and untreated cotyledons (Fig. 1). Mass spectrometry of tryptic digestion fragments of the proteins followed by database search has identified some of the differentially expressed proteins (Open in a separate windowFigure 1Image of protein spots generated by Samspot analysis of Al treated and untreated tomato cotyledons proteomes separated on 2D-DIGE.

Table 1

Proteins identified from tomato cotyledons of seeds germinating in Al-solution

Spaceflight induced changes in the human proteome

Introduction: Spaceflight is one of the most extreme conditions encountered by humans: Individuals are exposed to radiation, microgravity, hypodynamia, and will experience isolation. A better understanding of the molecular processes induced by these factors may allow us to develop personalized countermeasures to minimize risks to astronauts.

Areas covered: This review is a summary of literature searches from PubMed, NASA, Roskosmos and the authors’ research experiences and opinions. The review covers the available proteomic data on the effects of spaceflight factors on the human body, including both real space missions and ground-based model experiments.

Expert commentary: Overall, the authors believe that the present background, methodology and equipment improvements will enhance spaceflight safety and support accumulation of new knowledge on how organisms adapt to extreme conditions.     

Cellular and extracellular proteome analysis of Streptococcus mutans grown in a chemostat

The oral pathogen, Streptococcus mutans, was grown under glucose limitation in a chemostat at pH 7.0 and a dilution rate of 0.1 h(-1) to mimic the conditions prevailing in a healthy human oral cavity in between meal times. Solubilized cellular and extracellular proteins were separated by two-dimensional gel electrophoresis (2-DE) and, following tryptic digestion, 421 protein spots analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry or electrospray ionization-tandem mass spectrometry. Analyses of the mass spectral data showed that the proteins matched the translation products of 200 different open reading frames (ORFs) deduced from contigs of the S. mutans UA159 genome and thus represented proteins derived from approximately 11% of the total ORFs of the bacterium. Of the identified proteins, 172 (including one surface protein) were characterized in the cellular fraction, and the remaining 28 (including two surface proteins) were uniquely identified from the culture fluid. The expression and therefore the existence of 30 proteins previously designated as 'hypothetical' or with no known function was confirmed. 2-DE of whole cell lysates revealed only a single intrinsic membrane protein. This is consistent with proteomic analyses of other Gram-positive bacteria where hydrophilic proteins represent the vast majority of those characterized.     

Partial hepatectomy induced liver proteome changes in mice

Acceleration of liver regeneration could be of great clinical benefit in various liver-associated diseases. However, at present little is known about therapeutic interventions to enhance this regenerative process. Our limited understanding and the complexity of the mechanisms involved have prevented the identification of new targets for treatment. Here we propose a broad-range proteomic approach to this problem that makes possible the simultaneous study of different signaling and metabolic pathways on the liver proteome. Changes in protein expression in mouse livers (n = 5 per group) at 6 h and 12 h after partial hepatectomy and sham operation, as compared to untreated controls, were analyzed using two-dimensional gel electrophoresis, mass spectrometry (MS), and mass fingerprinting. Twelve proteins, identified by MS, were up-regulated by at least 2-fold after partial hepatectomy. These included adipose differentiation-related protein, gamma-actin, enoyl coenzyme A hydratase 1, serum amyloid A and eukaryotic translation initiation factor 3. These results indicate that liver regeneration following partial hepatectomy affects various signaling and metabolic pathways.     

Analysis of the proteome of Pseudomonas putida KT2440 grown on different sources of carbon and energy

Using 2D electrophoresis the protein expression pattern during growth on carbon sources with different impact on carbon catabolite repression of phenol degradation was analysed in a derivative of Pseudomonas putida KT2440. The cytosolic protein pattern of cells growing on phenol or the non-repressive substrate pyruvate was almost identical, but showed significant differences to that of cells growing with the repressive substrates succinate or glucose. Proteins, which were mainly expressed in the presence of phenol or pyruvate, could be assigned to the functional groups of transport, detoxification, stress response, amino acid, energy, carbohydrate and nucleotide metabolism. The addition of succinate to cells growing with phenol ('shift-up') resulted in the inhibition of the synthesis of these proteins. Proteins with enhanced expression at growth with succinate or glucose were proteins for de novo synthesis of nucleotides, amino acids and enzymes of the TCA cycle. The synthesis of proteins, necessary for phenol catabolism was regulated in different manners following the addition of succinate. Whereas the synthesis of Phl-proteins (subunits of the phenolhydroxylase) only decreased slowly, was the translation of the Cat-proteins (catechol 1,2-dioxygenase, cis,cis-muconate cycloisomerase and muconolactone isomerase) repressed immediately and the synthesis of the Pca-proteins (beta-ketoadipate enolactone hydrolase, beta-ketoadipate succinyl-CoA transferase and beta-ketoadipyl CoA thiolase) remained unaffected.     

Glial proteome changes in response to moderate hypothermia

Reactive glia plays a central role in neuroinflammation associated with secondary damage after brain injury. In order to understand the global effects of therapeutic hypothermia on glial activation and neuroinflammation, we performed proteomic profiling of glial cultures following inflammatory stimulation and hypothermic exposure. Primary mixed glial cultures prepared from mouse brains were stimulated with lipopolysaccharide and interferon-γ under normothermic (37°C) or moderate hypothermic (29°C) conditions, and their proteome profiles were compared by LC-ESI-MS/MS. Differentially expressed proteins were determined by high-throughput label-free quantification. Under hypothermic conditions, 64 and 16 proteins were upregulated (≥1.5-fold) and downregulated (≤ 0.7-fold), respectively, compared to normothermic conditions. More importantly, hypothermia altered the abundance of 143 proteins that were either increased or decreased by inflammatory stimulation. The results were validated for several proteins (ICAM-1, STAT-1, YWHAB, and IFIT-3) by Western blot analysis. Pathway and network analysis indicate that hypothermia influences various biological functions of glia such as molecular transport, cell movement, immune response, cell death, and stress response. In conclusion, moderate hypothermia seems to have a significant effect on the protein expression profiles of brain glia and possibly ensuing neuroinflammation. These proteins may be involved in the protective mechanism of hypothermia against brain injuries.     

Analysis of signal-dependent changes in the proteome of Drosophila blood cells during an immune response

Innate immunity is based on the recognition of cell-surface molecules of infecting agents. Microbial substances, such as peptidoglycan, lipopolysaccharide, and beta-1,3-glucans, produce functional responses in Drosophila hemocytes that contribute to innate immunity. We have used two-dimensional gel electrophoresis and MS to resolve lipopolysaccharide-induced changes in the protein profile of a Drosophila hemocytic cell line. We identified 24 intracellular proteins that were up- or down-regulated, or modified, in response to immune challenge. Several proteins with predicted immune functions, including lysosomal proteases, actin-binding/remodeling proteins, as well as proteins involved in cellular responses to oxidative stress, were affected by the immune assault. Intriguingly, a number of the proteins identified in this study have recently been implicated in phagocytosis in higher vertebrates. We suggest that phagocytosis is activated in Drosophila hemocytes by the presence of microbial substances, and that this activation constitutes an evolutionarily conserved arm of innate immunity. In addition, a number of proteins involved in calcium-regulated signaling, mRNA processing, and nuclear transport were affected, consistent with a possible role in reprogramming of gene expression. In conclusion, the present proteome analysis identified many proteins previously not linked to innate immunity, demonstrating that differential protein profiling of Drosophila hemocytes is a valuable tool for identification of new players in immune-related cellular processes.