Proteomics analysis of liver samples from puffer fish Takifugu rubripes exposed to excessive fluoride: An insight into molecular response to fluorosis

Comparative proteomics was performed to identify proteins in the liver of Takifugu rubripes in response to excessive fluoride exposure. Sixteen fish were randomly divided into a control group and an experimental group. The control group was raised in soft water alone (F^? = 0.4 mg/L), and the experimental group was raised in the same water with sodium fluoride at a high concentration of 35 mg/L. After 3 days, proteins were extracted from the fish livers and then subjected to two‐dimensional polyacrylamide gel electrophoresis analysis. The matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) was applied to identify the proteins that were differentially expressed from the two groups of fish. Among an average of 816 and 918 proteins detected in the control and treated groups, respectively, 16 proteins were upregulated and 35 were downregulated (P < 0.01) in the fluoride‐treated group as compared with those in the control group. Twenty‐four highly differentially expressed proteins were further analyzed by MALDI‐TOF/TOF‐MS, and eight were identified by Mascot. These eight proteins include disulfide isomerase ER‐60, 4SNc‐Tudor domain protein, SMC3 protein, Cyclin D1, and mitogen‐activated protein kinase 10, as well as three unknown proteins. Consistent with their previously known functions, these identified proteins seem to be involved in apoptosis and other functions associated with fluorosis. These results will greatly contribute to our understanding of the effects of fluoride exposure on the physiological and biochemical functions of Takifugu and the toxicological mechanism of fluoride causing fluorosis in both fish and human. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:21–28, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20308     

Discovery and identification of serum potential biomarkers for pulmonary tuberculosis using iTRAQ‐coupled two‐dimensional LC‐MS/MS

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis is a chronic disease. Currently, there are no sufficiently validated biomarkers for early diagnosis of TB infection. In this study, a panel of potential serum biomarkers was identified between patients with pulmonary TB and healthy controls by using iTRAQ‐coupled 2D LC‐MS/MS technique. Among 100 differentially expressed proteins screened, 45 proteins were upregulated (>1.25‐fold at p < 0.05) and 55 proteins were downregulated (<0.8‐fold at p < 0.05) in the TB serum. Bioinformatics analysis revealed that the differentially expressed proteins were related to the response to stimulus, the metabolic and immune system processes. The significantly differential expression of apolipoprotein CII (APOCII), CD5 antigen‐like (CD5L), hyaluronan‐binding protein 2 (HABP2), and retinol‐binding protein 4 (RBP4) was further confirmed using immunoblotting and ELISA analysis. By forward stepwise multivariate regression analysis, a panel of serum biomarkers including APOCII, CD5L, and RBP4 was obtained to form the disease diagnostic model. The receiver operation characteristic curve of the diagnostic model was 0.98 (sensitivity = 93.42%, specificity = 92.86%). In conclusion, APOCII, CD5L, HABP2, and RBP4 may be potential protein biomarkers of pulmonary TB. Our research provides useful data for early diagnosis of TB.     

Disulfide–bridging PEGylation during refolding for the more efficient production of modified proteins

Proteins that are modified by chemical conjugation require at least two separate purification processes. First the bulk protein is purified, and then after chemical conjugation, a second purification process is required to obtain the modified protein. In an effort to develop new enabling technologies to integrate bioprocessing and protein modification, we describe the use of disulfide‐bridging conjugation to conduct PEGylation during protein refolding. Preliminary experiments using a PEG‐mono‐sulfone reagent with partially unfolded leptin and unfolded RNAse T1 indicated that the cysteine thiols underwent disulfide‐bridging conjugation to give the PEGylated proteins. Interferon‐β1b (IFN‐β1b) was then expressed in E.coli as inclusion bodies and found to undergo disulfide bridging‐conjugation during refolding. The PEG‐IFN‐β1b was isolated by ion‐exchange chromatography and displayed in vitro biological activity. In the absence of the PEGylation reagent, IFN‐β1b refolding was less efficient and yielded protein aggregates. No PEGylation was observed if the cysteines on IFN‐β1b were first modified with iodoacetamide prior to refolding. Our results demonstrate that the simultaneous refolding and disulfide bridging PEGylation of proteins could be a useful strategy in the development of affordable modified protein therapeutics.     

Proteomic analysis of doxorubicin‐induced changes in the proteome of HepG2cells combining 2‐D DIGE and LC‐MS/MS approaches

HepG‐2 cells are widely used as a cell model to investigate hepatocellular carcinomas and the effect of anticancer drugs such as doxorubicin, an effective antineoplastic agent, which has broad antitumoral activity against many solid and hematological malignancies. To investigate the effect of doxorubicin on the protein pattern, we used complementary proteomic workflows including 2‐D gel‐based and gel‐free methods. The analysis of crude HepG2 cell extracts by 2‐D DIGE provided data on 1835 protein spots which was then complemented by MS‐centered analysis of stable isotope labeling by amino acids in cell culture‐labeled cells. The monitoring of more than 1300 distinct proteins, including proteins of the membrane fraction provides the most comprehensive overview on the proteome of the widely used model cell line HepG2. Of the proteins monitored in total, 155 displayed doxorubicin‐induced changes in abundance. Functional analysis revealed major influences of doxorubicin on proteins involved in protein synthesis, DNA damage control, electron transport/mitochondrial function, and tumor growth. The strongest decrease in level was found for proteins involved in DNA replication and protein synthesis, whereas proteins with a function in DNA damage control and oxidative stress management displayed increased levels following treatment with doxorubicin compared with control cells. Furthermore, the doxorubicin‐associated increase in levels of multiple forms of keratins 8, 18, and 19 and other structural proteins revealed an influence on the cytoskeleton network.     

PUGNAc induces protein ubiquitination in C2C12 myotube cells

O‐linked β‐N‐acetylglucosaminylation (O‐GlcNAcylation) regulates many cellular processes including the cell cycle, cell signaling, and protein trafficking. Dysregulation of O‐GlcNAcylation may be involved in the development of insulin resistance and type 2 diabetes. Therefore, it is necessary to identify cellular proteins that are induced by elevated O‐GlcNAcylation. Here, using adenosine 5′‐triphosphate affinity chromatography, we employed a proteomic approach in order to identify differentially expressed proteins in response to treatment with the O‐GlcNAcase inhibitor, O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), in mouse C2C12 myotube cells. Among 205 selected genes, we identified 68 nucleotide‐binding proteins, 14 proteins that have adenosinetriphosphatase activity, and 10 proteins with ligase activity. Upregulation of proteins, including ubiquitin‐activating enzyme E1, proteasome subunit 20S, cullin‐associated NEDD8‐dissociated protein 1, ezrin, and downregulation of the protein nucleoside diphosphate kinase B, were confirmed by western blot analysis. In particular, we found that the protein ubiquitination level in C2C12 cells was increased by PUGNAc treatment. This is the first report of quantitative proteomic profiles of myotube cells after treatment with PUGNAc, and our results demonstrate the potential to enhance understanding of the relationship between insulin resistance, O‐GlcNAc, and PUGNAc in the future. Copyright © 2015 John Wiley & Sons, Ltd.     

The Expression of Cyclin D1 during Adipogenesis in Pig Primary Stromal–Vascular Cultures

Objective: Our objective in this study was to measure the expression of cyclin D1 in pig primary stromal‐vascular (S‐V) cells as they differentiate into adipose cells and to identify which factors may alter cyclin D1 expression. Research Methods and Procedures: Western blot analysis was performed on cultured S‐V cells using 8% sodium dodecyl sulfate‐polyacrylamide gels, mouse monoclonal cyclin D1 antibody, and anti‐mouse IgG secondary labeled with horseradish peroxidase. For immunocytochemistry, cultures were fixed with 4% paraformaldehyde and incubated with anti‐CCAAT/enhancer binding protein (C/EBPα) and anti‐cyclin D1 together. Cyclin D1 expression was evaluated in 105‐day fetal dorsal subcutaneous tissues using paraffin sections. Results: Our results with Western blot analysis showed that cyclin D1 was found in freshly isolated S‐V cells and continued to be expressed during the first 3 days of adipose cell development with a significant increase in late development at day 9. Elevated cyclin D1 levels were colocalized with C/EBPα beginning at day 3 and remained colocalized with C/EBPα through day 9. Removing insulin from cultures resulted in a reduction in differentially elevated levels of cyclin D1. Discussion: The elevated level of cyclin D1 expression colocalized with C/EBPα expression is unexpected because differentiated adipocytes would be expected to have reduced proliferative potential. The elevated levels of cyclin D1 expression we observed in mature adipocytes depend on insulin. In addition, cyclin D1 is absent from lipid‐filled fetal adipose cells in vivo, where insulin levels are very low. The activity of cyclin D1 in differentiated adipocytes may be directed toward proteins outside of the cell cycle.     

Expression of proteins containing disulfide bonds in an insect cell-free system and confirmation of their arrangements by MALDI-TOF MS

Escherichia coli alkaline phosphatase (AP) and human lysozyme (h-LYZ), which contain two and four disulfide bonds, respectively, were expressed in a cell-free protein synthesis system constructed from Spodoptera frugiperda 21 (Sf21) cells. AP was expressed in a soluble and active form using the insect cell-free system under non-reducing conditions, and h-LYZ was expressed in a soluble and active form under non-reducing conditions after addition of reduced glutathione (GSH), oxidized glutathione (GSSG), and protein disulfide isomerase (PDI). The in vitro synthesized proteins were purified by means of a Strep-tag attached to their C termini. Approximately 41 microg AP and 30 microg h-LYZ were obtained from 1 mL each of the reaction mixture. The efficiency of protein synthesis approached that measured under reducing conditions. Analysis of the disulfide bond arrangements by MALDI-TOF MS showed that disulfide linkages identical to those observed in the wild-type proteins were formed.     

Proteomics of mouse liver microsomes: Performance of different protein separation workflows for LC‐MS/MS

The mouse liver microsome proteome was investigated using ion trap MS combined with three separation workflows including SDS‐PAGE followed by reverse‐phase LC of in‐gel protein digestions (519 proteins identified); 2‐D LC of protein digestion (1410 proteins); whole protein separation on mRP heat‐stable column followed by 2‐D LC of protein digestions from each fraction (3‐D LC; 3703 proteins). The higher number of proteins identified in the workflow corresponded to the lesser percentage of run‐to‐run reproducibility. Gel‐based method yielded a number of predicted membrane proteins similar to LC‐based workflows.     

Proteomic evaluation of wound‐healing processes in potato (Solanum tuberosum L.) tuber tissue

Proteins from potato (Solanum tuberosum L.) tuber slices, related to the wound‐healing process, were separated by 2‐DE and identified by an MS analysis in MS and MS/MS mode. Slicing triggered differentiation processes that lead to changes in metabolism, activation of defence and cell‐wall reinforcement. Proteins related to storage, cell growth and division, cell structure, signal transduction, energy production, disease/defence mechanisms and secondary metabolism were detected. Image analysis of the 2‐DE gels revealed a time‐dependent change in the complexity of the polypeptide patterns. By microscopic observation the polyalyphatic domain of suberin was clearly visible by D4, indicating that a closing layer (primary suberisation) was formed by then. A PCA of the six sampling dates revealed two time phases, D0–D2 and D4–D8, with a border position between D2 and D4. Moreover, a PCA of differentially expressed proteins indicated the existence of a succession of proteomic events leading to wound‐periderm reconstruction. Some late‐expressed proteins (D6–D8), including a suberisation‐associated anionic peroxidase, have also been identified in the native periderm. Despite this, protein patterns of D8 slices and native periderm were still different, suggesting that the processes of wound‐periderm formation are extended in time and not fully equivalent. The information presented in this study gives clues for further work on wound healing‐periderm formation processes.     

A comparative proteomic analysis of HepG2 cells incubated by S(−) and R(+) enantiomers of anti‐coagulating drug warfarin

Warfarin is a commonly prescribed oral anti‐coagulant with narrow therapeutic index. It interferes with vitamin K cycle to achieve anti‐coagulating effects. Warfarin has two enantiomers, S(?) and R(+) and undergoes stereoselective metabolism, with the S(?) enantiomer being more effective. We reported that the intracellular protein profile in HepG2 cells incubated with S(?) and R(+) warfarin, using iTRAQ‐coupled 2‐D LC‐MS/MS. In samples incubated with S(?) and R(+) warfarin alone, the multi‐task protein Protein SET showed significant elevation in cells incubated with S(?) warfarin but not in those incubated with R(+) warfarin. In cells incubated with individual enantiomers of warfarin in the presence of vitamin K, protein disulfide isomerase A3 which is known as a glucose‐regulated protein, in cells incubated with S(?) warfarin was found to be down‐regulated compared to those incubated with R(+) warfarin. In addition, Protein DJ‐1 and 14‐3‐3 Proteinσ were down‐regulated in cells incubated with either S(?) or R(+) warfarin regardless of the presence of vitamin K. Our results indicated that Protein DJ‐1 may act as an enzyme for expression of essential enzymes in vitamin K cycle. Taken together, our findings provided molecular evidence on a comprehensive protein profile on warfarin–cell interaction, which may shed new lights on future improvement of warfarin therapy.     

Verification of protein disulfide bond arrangement by in‐gel tryptic digestion under entirely neutral pH conditions

To develop a concise proteomic procedure to verify the protein disulfide bond arrangement, non‐reductive trypsin digestion of neuregulin 1‐β1 (176–246), a model disulfide‐containing protein, was assessed by a proteolytic ¹⁸O‐labeling analysis. As a result, the commonly used in‐gel tryptic digestion method has been improved for use entirely under neutral pH conditions. With this procedure, the disulfide arrangement of proteins could represent a clinical index candidate in pathological proteomic studies.     

Altered protein expression pattern in colon tissue of mice upon supplementation with distinct selenium compounds

The essential trace element selenium (Se) is controversially discussed concerning its role in health and disease. Its various physiological functions are largely mediated by Se incorporation in the catalytic center of selenoproteins. In order to gain insights into the impact of Se deficiency and of supplementation with different Se compounds (selenite, selenate, selenomethionine) at defined concentrations (recommended, 150 μg/kg diet; excessive, 750 μg/kg diet) in murine colon tissues, a 20‐week feeding experiment was performed followed by analysis of the protein expression pattern of colon tissue specimens by 2D‐DIGE and MALDI‐TOF MS. Using this approach, 24 protein spots were identified to be significantly regulated by the different Se compounds. These included the antioxidant enzyme peroxiredoxin‐5 (PRDX5), proteins with binding capabilities, such as cofilin‐1 (COF1), calmodulin, and annexin A2 (ANXA2), and proteins involved in catalytic processes, such as 6‐phosphogluconate dehydrogenase (6PGD). Furthermore, the Se compounds demonstrated a differential impact on the expression of the identified proteins. Selected target structures were validated by qPCR and Western blot which mainly confirmed the proteomic profiling data. Thus, novel Se‐regulated proteins in colon tissues have been identified, which expand our understanding of the physiologic role of Se in colon tissue.     

High throughput screening of disulfide‐containing proteins in a complex mixture

The formation of disulfide bonds between cysteine residues is crucial for the stabilization of native protein structures and, thus, determination of disulfide linkages is an important facet of protein structural characterization. Nonetheless, the identification of disulfide bond linkages remains a significant analytical challenge, particularly in large proteins with complex disulfide patterns. Herein, we have developed a new LC/MS strategy for rapid screening of disulfides in an intact protein mixture after a straightforward reduction step with tris(2‐carboxyethyl)phosphine. LC/MS analysis of reduced and nonreduced protein mixtures quickly revealed disulfide‐containing proteins owing to a 2 Da mass increase per disulfide reduction and, subsequently, the total number of disulfide bonds in the intact proteins could be determined. We have demonstrated the effectiveness of this method in a protein mixture composed of both disulfide‐containing and disulfide‐free proteins. Our method is simple (no need for proteolytic digestion, alkylation, or the removal of reducing agents prior to MS analysis), high throughput (fast on‐line LC/MS analysis), and reliable (no S–S scrambling), underscoring its potential as a rapid disulfide screening method for proteomics applications.     

Mapping of proteins in human saliva using two-dimensional gel electrophoresis and peptide mass fingerprinting

Human saliva contains a large number of proteins that can be used for diagnosis and are of great potential in clinical and epidemiological research. The aim of this work was to map the proteins in saliva by two-dimensional gel electrophoresis (2-DE), and to identify abundant proteins by peptide mass fingerprinting using trypsin cleavage and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry analysis. One hundred proteins were identified representing 20 different identities according to accession numbers. Abundant proteins expressed in different forms were: alpha-amylase, immunoglobulin A, prolactin-inducible protein, zinc-alpha(2)-glycoprotein and cystatins (S, SA, D and SN). Other proteins found were interleukin-1 receptor antagonist, von Ebner's gland protein (lipocalin-1) and calgranulin A and B (S100A8 and A9). Furthermore, apolipoprotein A-I, beta(2)-microglobulin, glutathione S-transferase P and fatty acid-binding protein were also identified. Our results show that human saliva contains a large number of proteins that are involved in inflammatory and immune responses. The 2-DE protein map constructed opens the possibility to investigate protein changes associated with disease processes.     

Plasma profiles in active systemic juvenile idiopathic arthritis: Biomarkers and biological implications

Systemic juvenile idiopathic arthritis (SJIA) is a chronic arthritis of children characterized by a combination of arthritis and systemic inflammation. There is usually non‐specific laboratory evidence of inflammation at diagnosis but no diagnostic test. Normalized volumes from 89/889 2‐D protein spots representing 26 proteins revealed a plasma pattern that distinguishes SJIA flare from quiescence. Highly discriminating spots derived from 15 proteins constitute a robust SJIA flare signature and show specificity for SJIA flare in comparison to active polyarticular juvenile idiopathic arthritis or acute febrile illness. We used 7 available ELISA assays, including one to the complex of S100A8/S100A9, to measure levels of 8 of the15 proteins. Validating our DIGE results, this ELISA panel correctly classified independent SJIA flare samples, and distinguished them from acute febrile illness. Notably, data using the panel suggest its ability to improve on erythrocyte sedimentation rate or C‐reactive protein or S100A8/S100A9, either alone or in combination in SJIA F/Q discriminations. Our results also support the panel's potential clinical utility as a predictor of incipient flare (within 9 wk) in SJIA subjects with clinically inactive disease. Pathway analyses of the 15 proteins in the SJIA flare versus quiescence signature corroborate growing evidence for a key role for IL‐1 at disease flare.     

Comparative proteomic analysis between fifth‐instar nymphs and adults of Asian citrus psyllid Diaphorina citri

Asian citrus psyllid Diaphorina citri Kuwayama is extremely problematic worldwide, particularly where Huanglongbing (HLB) disease, the most serious and devastating of citrus diseases, is found. The threat is a result of its ability to transmit the causal agent of HLB, Candidatus Liberibacter asiaticus (CLas) bacterium. Improvements in proteomics, mass spectrometry, bioinformatics tools and gene ontology annotation facilitate the mapping and large‐scale identification and quantification of proteins. To date, only a few comparative proteomic studies report the developmental proteomic changes of hemimetabolous and plant–disease vector insects. Two‐dimensional gel electrophoresis analysis of D. citri total protein is able to detect qualitative and quantitative developmental differences. Liquid chromatography‐tandem mass spectrometry identifies 89 protein spots. Most proteins are metabolism and bioenergetics‐related. Nineteen protein spots are found to be implicated in stress/defence/immunity; 7 in development regulation; 9 in nervous system functions; 4 in the reproductive system; 23 in cytoskeleton and muscle organization; and 4 in movement, flight and other processes. Significant increases in the level of proteins related to structural constitution of the skeleton, stress/defence/immunity, reproduction system, muscles, locomotion and flight are found in adults, consistent with the fact that D. citri is a hemimetabolous insect, whereas proteins involved in developmental regulation are higher in the nymphal stage. The identification of these variably expressed proteins between the nymph and adult stages, linked with the basis of their physiological roles, will lead to a better understanding of the factors influencing development in D. citri and the regulation of some crucial metabolic pathways. It may also help to identify targets for genetic manipulation using RNA interference or other techniques to disrupt Asian citrus psyllid development, lifespan or its ability to transmit CLas.     

iTRAQ‐coupled 2‐D LC‐MS/MS analysis of protein profile associated with HBV‐modulated DNA methylation

The development of hepatocellular carcinoma (HCC) is believed to be associated with multiple risk factors, including the infection of hepatitis B virus (HBV). Based on the analysis of individual genes, evidence has indicated the association between HCC and HBV and has also been expanded to epigenetic regulation, with an involvement of HBV in the DNA methylation of the promoter of cellular target genes leading to changes in their expression. Proteomic study has been widely used to map a comprehensive protein profile, which in turn could provide a better understanding of underlying mechanisms of disease onset. In the present study, we performed a proteomic profiling by using iTRAQ‐coupled 2‐D LC/MS‐MS analysis to identify cellular genes down‐regulated in HBV‐producing HepG2.2.15 cells compared with HepG2 cells. A total of 15 proteins including S100A6 and Annexin A2 were identified by our approach. The significance of these cellular proteins as target of HBV‐mediated epigenetic regulation was supported by our validation assays, including their reactivation in cells treated with 5‐aza‐2′‐deoxycytidine (a DNA methyltransferase inhibitor) by real‐time RT‐PCR and Western blot analysis, as well as the DNA methylation status analysis by bisulfite genome sequencing. Our approach provides a comprehensive analysis of cellular target proteins to HBV‐mediated epigenetic regulation and further analysis should facilitate a better understanding of its involvement in HCC development.     

Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis

Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one-dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP-dissociation inhibitor 1, tropomyosin, fibroblast type, IL-18 and annexin I were significantly up-regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down-regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense/IL-18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, while IL-18 antisense remarkably decreased cell invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play a role in metastasis by inhibiting E-cadherin expression.     

Proteomic profiling of Cronobacter turicensis 3032, a food‐borne opportunistic pathogen

Members of the genus Cronobacter are opportunistic pathogens for neonates and are often associated with contaminated milk powder formulas. At present little is known about the virulence mechanisms or the natural reservoir of these organisms. The proteome of Cronobacter turicensis 3032, which has recently caused two deaths, was mapped aiming at a better understanding of physiology and putative pathogenic traits of this clinical isolate. Our analyses of extracellular, surface‐associated and whole‐cell proteins by two complementary proteomics approaches, 1D‐SDS‐PAGE combined with LC‐ESI‐MS/MS and 2D‐LC‐MALDI‐TOF/TOF MS, lead to the identification of 832 proteins corresponding to a remarkable 19% of the theoretically expressed protein complement of C. turicensis. The majority of the identified proteins are involved in central metabolic pathways, translation, protein folding and stability. Several putative virulence factors, whose expressions were confirmed by phenotypic assays, could be identified: a macrophage infectivity potentiator involved in C. turicensis persistence in host cells, a superoxide dismutase protecting the pathogen against reactive oxygen species and an enterobactin‐receptor protein for the uptake of siderophore‐bound iron. Most interestingly, a chitinase and a metalloprotease that might act against insects and fungi but no casein hydrolysing enzymes were found, suggesting that there is an environmental natural habitat of C. turicensis 3032.