Dynamic Protein Phosphorylation in Streptococcus pyogenes during Growth, Stationary Phase, and Starvation.

Phosphorylation of proteins at serine, threonine, and tyrosine residues plays an important role in physiological processes of bacteria, such as cell cycle, metabolism, virulence, dormancy, and stationary phase functions. Little is known about the targets and dynamics of protein phosphorylation in Streptococcus pyogenes, which possesses a single known transmembrane serine/threonine kinase belonging to the class of PASTA kinases. A proteomics and phosphoproteomics workflow was performed with S. pyogenes serotype M49 under different growth conditions, stationary phase, and starvation. The quantitative analysis of dynamic phosphorylation, which included a subset of 463 out of 815 identified phosphorylation sites, revealed two main types of phosphorylation events. A small group of phosphorylation events occurred almost exclusively at threonine residues of proteins related to the cell cycle and was enhanced in growing cells. The majority of phosphorylation events occurred during stationary phase or starvation, preferentially at serine residues. PASTA kinase-dependent cell cycle regulation processes found in related bacteria are conserved in S. pyogenes. Increased protein phosphorylation during the stationary phase has also been described for some other bacteria, and could therefore be a general feature in the physiology of bacteria, whose functions and the kinases involved need to be elucidated in further analyses.

Synthetic mRNA delivered to human cells leads to expression of Cpl-1 bacteriophage-endolysin with activity against Streptococcus pneumoniae.

Endolysins are bacteriophage-encoded hydrolases that show high antibacterial activity and a narrow substrate spectrum. We hypothesize that an mRNA-based approach to endolysin therapy can overcome some challenges of conventional endolysin therapy, namely organ targeting and bioavailability. We show that synthetic mRNA applied to three human cell lines (HEK293T, A549, HepG2 cells) leads to expression and cytosolic accumulation of the Cpl-1 endolysin with activity against Streptococcus pneumoniae. Addition of a human lysozyme signal peptide sequence translocates the Cpl-1 to the endoplasmic reticulum leading to secretion (hlySP-sCpl-1). The pneumococcal killing effect of hlySP-sCpl-1 was enhanced by introduction of a point mutation to avoid N-linked-glycosylation. hlySP-sCpl-1N215D, collected from the culture supernatant of A549 cells 6 h post-transfection showed a significant killing effect and was active against nine pneumococcal strains. mRNA-based cytosolic Cpl-1 and secretory hlySP-sCpl-1N215D show potential for innovative treatment strategies against pneumococcal disease and, to our best knowledge, represent the first approach to mRNA-based endolysin therapy. We assume that many other bacterial pathogens could be targeted with this novel approach.

Knockdown of coagulation factor VII has potential anticoagulant and anti-inflammatory effects in a mouse model of group A streptococcal sepsis.

Infections originating from subcutaneous tissues are among the most common invasive infections caused by group A streptococcus (GAS) and associated with systemic coagulation activation. The role of intrinsic coagulation factors on GAS virulence has recently been determined, but the role of the extrinsic coagulation factor VII is unknown. Using a mouse model, in which GAS-sepsis emerges from a subcutaneous infection, we show that FVII is a negative acute phase protein. F7 knockdown using antisense oligonucleotides resulted in an attenuated systemic coagulation activation and inflammatory response in septic animals. The findings indicate FVII's ability to modify the host response.

Immunogenicity and contraceptive efficacy of plant-produced putative mouse-specific contraceptive peptides.

Rodent population control through contraception requires species-specific oral contraceptive vaccines. Therefore, in this study, we produced putative mouse-specific contraceptive peptides, mZP2 (from oocyte) and mIzumo1 (from sperm), in plants using Agrobacterium-mediated transient expression. Peptides were produced separately in Nicotiana benthamiana using constructs encoding antigens containing three copies of each peptide. We also determined the immunogenicity and contraceptive effects of the plant-produced antigens in female BALB/c mice. Mice immunized subcutaneously with a relatively low amount of antigen (5 µg/dose of each peptide in a mixture) showed systemic immune responses against mZP2-3 and mIzumo1-3 antigens. Moreover, the mean litter size of mice treated with the plant-produced antigens was reduced by 39% compared to that of the control mice. Notably, there was a significant negative correlation between the number of pups born and individual antibody levels against both antigens. Immunofluorescence assays demonstrated the binding of induced antibodies to the oocytes of BALB/c and wild-type mice in vivo and in vitro, respectively. Our study demonstrate the feasibility of producing small contraceptive peptides in plants that can be further used to develop oral contraceptive vaccines against mouse populations.

Integrative analysis of the salt stress response in cyanobacteria.

Microorganisms evolved specific acclimation strategies to thrive in environments of high or fluctuating salinities. Here, salt acclimation in the model cyanobacterium Synechocystis sp. PCC 6803 was analyzed by integrating transcriptomic, proteomic and metabolomic data. A dynamic reorganization of the transcriptome occurred during the first hours after salt shock, e.g. involving the upregulation of genes to activate compatible solute biochemistry balancing osmotic pressure. The massive accumulation of glucosylglycerol then had a measurable impact on the overall carbon and nitrogen metabolism. In addition, we observed the coordinated induction of putative regulatory RNAs and of several proteins known for their involvement in other stress responses. Overall, salt-induced changes in the proteome and transcriptome showed good correlations, especially among the stably up-regulated proteins and their transcripts. We define an extended salt stimulon comprising proteins directly or indirectly related to compatible solute metabolism, ion and water movements, and a distinct set of regulatory RNAs involved in post-transcriptional regulation. Our comprehensive data set provides the basis for engineering cyanobacterial salt tolerance and to further understand its regulation.

Periodontal pathogens alter the synovial proteome. Periodontal pathogens do not exacerbate macroscopic arthritis but alter the synovial proteome in mice.

Rheumatoid arthritis (RA) and periodontitis (PD) are chronic inflammatory diseases that appear to occur in tandem. However, the mutual impact PD exerts on RA and vice versa has not yet been defined. To address this issue, we set up an animal model and analyzed how two prime inducers of periodontitis-Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa)-differ in their pathogenic potential. Our experimental setup included collagen induced arthritis (CIA) in the mouse, oral inoculation with Pg or Aa to induce alveolar bone loss and the combination of both diseases in inverted orders of events. Neither pathobiont impacted on macroscopic arthritis and arthritis did not exacerbate alveolar bone loss. However, there were subtle differences between Pg and Aa with the former inducing more alveolar bone loss if PD was induced before CIA. On a molecular level, Pg and Aa led to differential expression patterns in the synovial membranes that were reminiscent of cellular and humoral immune responses, respectively. The Pg and Aa specific signatures in the synovial proteomes suggest a role for oral pathogens in shaping disease subtypes and setting the stage for subsequent therapy response.

Memory-Enhancing Effects of Origanum majorana Essential Oil in an Alzheimer's Amyloid beta1-42 Rat Model: A Molecular and Behavioral Study.

Origanum L. (Lamiaceae) is an important genus of medicinal and aromatic plants used in traditional medicine since ancient times as culinary herbs and remedies. The aim of the present study was to evaluate the chemical composition, as well as the biochemical and cellular activities of freshly prepared Origanum majorana L. essential oil (OmEO) in an Alzheimer's disease (AD) amyloid beta1-42 (Aß1-42) rat model. OmEO (1% and 3%) was inhaled for 21 consecutive days, while Aß1-42 was administered intracerebroventricularly to induce AD-like symptoms. Our data demonstrate that OmEO increased antioxidant activity and enhanced brain-derived neurotrophic factor (BDNF) expression, which in concert contributed to the improvement of cognitive function of animals. Moreover, OmEO presented beneficial effects on memory performance in Y-maze and radial arm-maze tests in the Aß1-42 rat AD model.

Kininogen supports inflammation and bacterial spreading during Streptococccus Pyogenes Sepsis.

BACKGROUND: High-molecular-weight kininogen is a cofactor of the human contact system, an inflammatory response mechanism that is activated during sepsis. It has been shown that high-molecular-weight kininogen contributes to endotoxemia, but is not critical for local host defense during pneumonia by Gram-negative bacteria. However, some important pathogens, such as Streptococcus pyogenes, can cleave kininogen by contact system activation. Whether kininogen causally affects antibacterial host defense in S. pyogenes infection, remains unknown. METHODS: Kininogen concentration was determined in course plasma samples from septic patients. mRNA expression and degradation of kininogen was determined in liver or plasma of septic mice. Kininogen was depleted in mice by treatment with selective kininogen directed antisense oligonucleotides (ASOs) or a scrambled control ASO for 3 weeks prior to infection. 24 h after infection, infection parameters were determined. FINDINGS: Data from human and mice samples indicate that kininogen is a positive acute phase protein. Lower kininogen concentration in plasma correlate with a higher APACHE II score in septic patients. We show that ASO-mediated depletion of kininogen in mice indeed restrains streptococcal spreading, reduces levels of proinflammatory cytokines such as IL-1ß and IFNγ, but increased intravascular tissue factor and fibrin deposition in kidneys of septic animals. INTERPRETATION: Mechanistically, kininogen depletion results in reduced plasma kallikrein levels and, during sepsis, in increased intravascular tissue factor that may reinforce immunothrombosis, and thus reduce streptococcal spreading. These novel findings point to an anticoagulant and profibrinolytic role of kininogens during streptococcal sepsis. FUNDING: Full details are provided in the Acknowledgements section.

Stoichiometry of two plant glycine decarboxylase complexes and comparison with a cyanobacterial glycine cleavage system.

The multienzyme glycine cleavage system (GCS) converts glycine and tetrahydrofolate to the one-carbon compound 5,10-methylenetetrahydrofolate, which is of vital importance for most if not all organisms. Photorespiring plant mitochondria contain very high levels of GCS proteins organised as a fragile glycine decarboxylase complex (GDC). The aim of this study is to provide mass spectrometry-based stoichiometric data for the plant leaf GDC and examine whether complex formation could be a general property of the GCS in photosynthesizing organisms. The molar ratios of the leaf GDC component proteins are 1L2 -4P2 -8T-26H and 1L2 -4P2 -8T-20H for pea and Arabidopsis, respectively, as determined by mass spectrometry. The minimum mass of the plant leaf GDC ranges from 1550 to 1650 kDa, which is larger than previously assumed. The Arabidopsis GDC contains four times more of the isoforms GCS-P1 and GCS-L1 in comparison with GCS-P2 and GCS-L2, respectively, whereas the H-isoproteins GCS-H1 and GCS-H3 are fully redundant as indicated by their about equal amounts. Isoform GCS-H2 is not present in leaf mitochondria. In the cyanobacterium Synechocystis sp. PCC 6803, GCS proteins concentrations are low but above the complex formation threshold reported for pea leaf GDC. Indeed, formation of a cyanobacterial GDC from the individual recombinant GCS proteins in vitro could be demonstrated. Presence and metabolic significance of a Synechocystis GDC in vivo remain to be examined but could involve multimers of the GCS H-protein that dynamically crosslink the three GCS enzyme proteins, facilitating glycine metabolism by the formation of multienzyme metabolic complexes. Data are available via ProteomeXchange with identifier PXD018211.

Comparison of accompanying proteins in different therapeutic human serum albumin preparations.

Pharmaceutical human serum albumin products are manufactured from donated human plasma and may contain up to 5% accompanying non-albumin proteins. It has been reported that albumin preparations manufactured by different pharmaceutical companies differed in the degree of posttranslational modifications, the redox state as well as antioxidant properties of albumin, whereas the composition of the accompanying proteins has never been comparatively analyzed. In this study, a non-targeted mass spectrometric approach was used for label-free quantification and comparison of different pharmaceutical albumin preparations. Haptoglobin and a few other proteins accounted for approximately 80% of the accompanying proteins in all products tested. Low abundance proteins were enriched by means of a combinatorial peptide ligand library (ProteoMiner, Bio-Rad). Significant differences between the amounts of several mainly low abundance proteins, such as complement factors, were observed indicating differences in the manufacturing processes of the pharmaceutical companies. The removal of the stabilizers octanoate and N-acetyltryptophan from albumin solutions using the charcoal-based Hepalbin adsorbent simultaneously reduced the accompanying proteins. For therapy evaluation of albumin preparations, the variable composition of the accompanying proteins in different albumin products should be taken into account in addition to the known heterogeneity of the albumin protein itself.

The contact system proteases play disparate roles in streptococcal sepsis.

Sepsis causes an activation of the human contact system, an inflammatory response mechanism against foreign surfaces, proteins and pathogens. The serine proteases of the contact system, factor XII and plasma kallikrein, are decreased in plasma of septic patients, which was previously associated with an unfavorable outcome. However, the precise mechanisms and roles of contact system factors in bacterial sepsis are poorly understood. We, therefore, studied the physiological relevance of factor XII and plasma kallikrein in a mouse model of experimental sepsis. We show that decreased plasma kallikrein concentration in septic mice is a result of reduced mRNA expression plasma prekallikrein gene, indicating that plasma kallikrein belong to negative acute phase proteins. Investigations regarding the pathophysiological function of contact system proteases during sepsis revealed different roles for factor XII and plasma kallikrein. In vitro, factor XII decelerated bacteria induced fibrinolysis, whereas plasma kallikrein supported it. Remarkably, depletion of plasma kallikrein (but not factor XII) by treatment with antisense-oligonucleotides, dampens bacterial dissemination and growth in multiple organs in the mouse sepsis model. These findings identify plasma kallikrein as a novel host pathogenicity factor in Streptococcus pyogenes sepsis.

Cytosine N4-Methylation via M.Ssp6803II Is Involved in the Regulation of Transcription, Fine- Tuning of DNA Replication and DNA Repair in the Cyanobacterium Synechocystis sp. PCC 6803.

DNA methylation plays a crucial role for gene regulation among eukaryotes, but its regulatory function is less documented in bacteria. In the cyanobacterium Synechocystis sp. PCC 6803 five DNA methyltransferases have been identified. Among them, M.Ssp6803II is responsible for the specific methylation of the first cytosine in the frequently occurring motif GGCC, leading to N4-methylcytosine (GGm4CC). The mutation of the corresponding gene sll0729 led to lowered chlorophyll/phycocyanin ratio and slower growth. Transcriptomics only showed altered expression of sll0470 and sll1526, two genes encoding hypothetical proteins. Moreover, prolonged cultivation revealed instability of the initially obtained phenotype. Colonies with normal pigmentation and wild-type-like growth regularly appeared on agar plates. These colonies represent suppressor mutants, because the sll0729 gene was still completely inactivated and the GGCC sites remained unmethylated. The suppressor strains showed smaller cell size, lowered DNA content per cell, and decreased tolerance against UV compared to wild type. Promoter assays revealed that the transcription of the sll0470 gene was still stimulated in the suppressor clones. Proteomics identified decreased levels of DNA topoisomerase 4 subunit A in suppressor cells. Collectively, these results indicate that GGm4CC methylation is involved in the regulation of gene expression, in the fine-tuning of DNA replication, and DNA repair mechanisms.

Membrane Protein Identification in Rodent Brain Tissue Samples and Acute Brain Slices.

Acute brain slices are a sample format for electrophysiology, disease modeling, and organotypic cultures. Proteome analyses based on mass spectrometric measurements are seldom used on acute slices, although they offer high-content protein analyses and explorative approaches. In neuroscience, membrane proteins are of special interest for proteome-based analysis as they are necessary for metabolic, electrical, and signaling functions, including myelin maintenance and regeneration. A previously published protocol for the enrichment of plasma membrane proteins based on aqueous two-phase polymer systems followed by mass spectrometric protein identification was adjusted to the small sample size of single acute murine slices from newborn animals and the reproducibility of the results was analyzed. For this, plasma membrane proteins of 12 acute slice samples from six animals were enriched and analyzed by liquid chromatography-mass spectrometry. A total of 1161 proteins were identified, of which 369 were assigned to membranes. Protein abundances showed high reproducibility between samples. The plasma membrane protein separation protocol can be applied to single acute slices despite the low sample size and offers a high yield of identifiable proteins. This is not only the prerequisite for proteome analysis of organotypic slice cultures but also allows for the analysis of small-sized isolated brain regions at the proteome level.

Drug Repositioning Inferred from E2F1-Coregulator Interactions Studies for the Prevention and Treatment of Metastatic Cancers.

Metastasis management remains a long-standing challenge. High abundance of E2F1 triggers tumor progression by developing protein-protein interactions (PPI) with coregulators that enhance its potential to activate a network of prometastatic transcriptional targets. Methods: To identify E2F1-coregulators, we integrated high-throughput Co-immunoprecipitation (IP)/mass spectometry, GST-pull-down assays, and structure modeling. Potential inhibitors of PPI discovered were found by bioinformatics-based pharmacophore modeling, and transcriptome profiling was conducted to screen for coregulated downstream targets. Expression and target gene regulation was validated using qRT-PCR, immunoblotting, chromatin IP, and luciferase assays. Finally, the impact of the E2F1-coregulator complex and its inhibiting drug on metastasis was investigated in vitro in different cancer entities and two mouse metastasis models. Results: We unveiled that E2F1 forms coactivator complexes with metastasis-associated protein 1 (MTA1) which, in turn, is directly upregulated by E2F1. The E2F1:MTA1 complex potentiates hyaluronan synthase 2 (HAS2) expression, increases hyaluronan production and promotes cell motility. Disruption of this prometastatic E2F1:MTA1 interaction reduces hyaluronan synthesis and infiltration of tumor-associated macrophages in the tumor microenvironment, thereby suppressing metastasis. We further demonstrate that E2F1:MTA1 assembly is abrogated by small-molecule, FDA-approved drugs. Treatment of E2F1/MTA1-positive, highly aggressive, circulating melanoma cells and orthotopic pancreatic tumors with argatroban prevents metastasis and cancer relapses in vivo through perturbation of the E2F1:MTA1/HAS2 axis. Conclusion: Our results propose argatroban as an innovative, E2F-coregulator-based, antimetastatic drug. Cancer patients with the infaust E2F1/MTA1/HAS2 signature will likely benefit from drug repositioning.

Proteome analysis reveals that de novo regenerated mucosa over fibula flap-reconstructed mandibles resembles mature keratinized oral mucosa.

AIM: The aim of this study was to determine whether intra-oral de novo regenerated mucosa (D) that grew over free fibula flap reconstructed-mandibles resembled the donor tissue i.e. external skin (S) of the lateral leg, or the recipient site tissue, i.e. keratinized oral mucosa (K). MATERIALS AND METHODS: Differential proteome analysis was performed with ten tissue samples from each of the three groups: de novo regenerated mucosa (D), external skin (S), and keratinized oral mucosa (K). Expression differences of cornulin and involucrin were validated by Western blot analysis and their spatial distributions in the respective tissues were ascertained by immunohistochemistry. RESULTS: From all three investigated tissue types a total of 1188 proteins were identified, 930 of which were reproducibly and robustly quantified by proteome analysis. The best differentiating proteins were assembled in an oral mucosa proteome signature that encompasses 56 differentially expressed proteins. Principal component analysis of both, the 930 quantifiable proteins and the 56 oral mucosa signature proteins revealed that the de novo regenerated mucosa resembles keratinized oral mucosa much closer than extra-oral skin. Differentially expressed cornification-related proteins comprise proteins from all subclasses of the cornified cell envelope. Prominently expressed in intra-oral mucosa tissues were (i) cornifin-A, cornifin-B, SPRR3, and involucrin from the cornified-cell-envelope precursor group, (ii) S100A9, S100A8 and S100A2 from the S100 group, and (iii) cornulin which belongs to the fused-gene-protein group. CONCLUSION: According to its proteome signature de novo regenerated mucosa over the free fibula flap not only presents a passive structural surface layer but has adopted active tissue function.

Deletion of the L-Lactate Dehydrogenase Gene ldh in Streptococcus pyogenes Leads to a Loss of SpeB Activity and a Hypovirulent Phenotype.

Streptococcus pyogenes uses lactic acid fermentation for the generation of ATP. Here, we analyzed the impact of a deletion of the L-lactate dehydrogenase gene ldh on the virulence of S. pyogenes M49. While the ldh deletion does not cause a general growth deficiency in laboratory media, the growth in human blood and plasma is significantly hampered. The ldh deletion strain is furthermore less virulent in a Galleria mellonella infection model. We show that the ldh deletion leads to a decrease in the activity of the cysteine protease SpeB, an important secreted virulence factor of S. pyogenes. The reduced SpeB activity is caused by a hampered autocatalytic activation of the SpeB zymogen into the mature SpeB. The missing SpeB activity furthermore leads to increased plasmin activation and a reduced activation of the contact system on the surface of S. pyogenes. All these effects can be reversed when ldh is reintroduced into the mutant via a plasmid. The results demonstrate a previously unappreciated role for LDH in modulation of SpeB maturation.

The Regulatory Small RNA MarS Supports Virulence of Streptococcus pyogenes.

Small regulatory RNAs (sRNAs) play a role in the control of bacterial virulence gene expression. In this study, we investigated an sRNA that was identified in Streptococcus pyogenes (group A Streptococcus, GAS) but is conserved throughout various streptococci. In a deletion strain, expression of mga, the gene encoding the multiple virulence gene regulator, was reduced. Accordingly, transcript and proteome analyses revealed decreased expression of several Mga-activated genes. Therefore, and because the sRNA was shown to interact with the 5' UTR of the mga transcript in a gel-shift assay, we designated it MarS for m ga-activating regulatory sRNA. Down-regulation of important virulence factors, including the antiphagocytic M-protein, led to increased susceptibility of the deletion strain to phagocytosis and reduced adherence to human keratinocytes. In a mouse infection model, the marS deletion mutant showed reduced dissemination to the liver, kidney, and spleen. Additionally, deletion of marS led to increased tolerance towards oxidative stress. Our in vitro and in vivo results indicate a modulating effect of MarS on virulence gene expression and on the pathogenic potential of GAS.

Ethanol, glycogen and glucosylglycerol represent competing carbon pools in ethanol-producing cells of Synechocystis sp. PCC 6803 under high-salt conditions.

Cyanobacteria are photoautotrophic micro-organisms, which are increasingly being used as microbial cell factories to produce, for example, ethanol directly from solar energy and CO2. Here, we analysed the effects of different salt concentrations on an ethanol-producing strain of Synechocystis sp. PCC 6803 that overexpresses the pyruvate decarboxylase (pdc) from Zymomonas mobilis and the native alcohol dehydrogenase (adhA). Moderate salinities of 2 % NaCl had no negative impact on ethanol production, whereas the addition of 4 % NaCl resulted in significantly decreased ethanol yields compared to low-salt conditions. Proteomic analysis identified a defined set of proteins with increased abundances in ethanol-producing cells. Among them, we found strong up-regulation of α-1,4 glucan phosphorylase (GlgP, Slr1367) in the producer strain, which consistently resulted in a massive depletion of glycogen pools in these cells regardless of the salinity. The salt-induced accumulation of the compatible solute glucosylglycerol was not affected by the ethanol production. Glycogen and probably compatible solutes could present competing pools with respect to organic carbon, explaining the decreased ethanol production at the highest salinity.

A 2D gel electrophoresis-based snapshot of the phosphoproteome in the cyanobacterium Synechocystis sp. strain PCC 6803.

Cyanobacteria are photoautotrophic prokaryotes that occur in highly variable environments. Protein phosphorylation is one of the most widespread means to adjust cell metabolism and gene expression to the demands of changing growth conditions. Using a 2D gel electrophoresis-based approach and a phosphoprotein-specific dye, we investigated the protein phosphorylation pattern in cells of the model cyanobacterium Synechocystis sp. strain PCC 6803. The comparison of gels stained for total and phosphorylated proteins revealed that approximately 5 % of the protein spots seemed to be phosphoproteins, from which 32 were identified using MALDI-TOF MS. For eight of them the phosphorylated amino acid residues were mapped by subsequent mass spectrometric investigations of isolated phosphopeptides. Among the phosphoproteins, we found regulatory proteins, mostly putative anti-sigma factor antagonists, and proteins involved in translation. Moreover, a number of enzymes catalysing steps in glycolysis or the Calvin-Benson cycle were found to be phosphorylated, implying that protein phosphorylation might represent an important mechanism for the regulation of the primary carbon metabolism in cyanobacterial cells.

Molecular adaptations of Helicoverpa armigera midgut tissue under pyrethroid insecticide stress characterized by differential proteome analysis and enzyme activity assays.

Helicoverpa armigera is an insect that causes important economic losses in crops. To reduce this loss, pyrethroids have been commonly used against H. armigera in farming areas. However, excess and continuous usage of pyrethroids cause resistance in H. armigera. Therefore, expressions of midgut proteins of two H. armigera field populations were compared to those of a susceptible strain by 2-D PAGE and MALDI-ToF-MS. Our results indicate that H. armigera reacts to pyrethroid-induced stress mainly by increasing the expression of energy metabolism-related proteins, such as ATP synthase and arginine kinase. NADPH cytochrome P450 reductase, also up-regulated, could play a role in detoxification of toxic pyrethroid metabolites, such as 3-phenoxybenzaldehyde. Interestingly, while GSTs were not found up-regulated in the comparative proteome analysis, biochemical assays showed significant increases of enzyme activities in both field populations as compared to the susceptible strain. Similarly, although esterases were not found differentially expressed, biochemical assays showed significant increases of esterase activities in both field populations. Thus, esterases are also proposed to be involved in metabolic responses towards pyrethroid insecticide-induced stress. In conclusion, we suggest increased energy metabolism in the midgut tissue of H. armigera as a general prerequisite for compensating the costs of energy-consuming detoxification processes.