Purification, conformational analysis and cytotoxic activities of host-defense peptides from the Tungara frog Engystomops pustulosus (Leptodactylidae; Leiuperinae).

The amphibian family Leptodactylidae is divided into three sub-families: Leiuperinae, Leptodactylinae, and Paratelmatobiinae. Host-defense peptides (HDPs) present in the skins of frogs belonging to the Leptodactylinae have been studied extensively, but information is limited  regarding peptides from Leiuperinae species. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Tungara frog Engystomops pustulosus (Leiuperinae) collected in Trinidad led to the isolation and structural characterization of previously undescribed pustulosin-1 (FWKADVKEIG KKLAAKLAEELAKKLGEQ), [Q28E] pustulosin-1 (pustulosin-2), and pustulosin-3 (DWKETAKELLKKIGAKVAQVISDKLNPAPQ). The primary structures of these peptides do not resemble those of previously described frog skin HDPs. In addition, the secretions contained tigerinin-1EP (GCKTYLIEPPVCT) with structural similarity to the tigerinins previously identified in skin secretions from frogs from the family Dicroglossidae. Pustulosin-1 and -3 adopted extended α-helical conformations in 25% trifluoroethanol-water and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). Pustulosin-1 and -3 displayed cytotoxic activity against a range of human tumor-derived cell lines (A549, MDA-MB-231, and HT29), but their therapeutic potential for development into anti-cancer agents is limited by their comparable cytotoxic activity against non-neoplastic human umbilical vein endothelial cells. The peptides also displayed weak antimicrobial activity against Escherichia coli (MIC = 125 µM) but were inactive against Staphylococcus aureus. Tigerinin-1EP was inactive against both the tumor-derived cells and bacteria.

Impact of Carbon Source Supplementations on Pseudomonas aeruginosa Physiology.

Pseudomonas aeruginosa is an opportunistic pathogen highly resistant to a wide range of antimicrobial agents, making its infections very difficult to treat. Since microorganisms need to perpetually adapt to their surrounding environment, understanding the effect of carbon sources on P. aeruginosa physiology is therefore essential to avoid increasing drug-resistance and better fight this pathogen. By a global proteomic approach and phenotypic assays, we investigated the impact of various carbon source supplementations (glucose, glutamate, succinate, and citrate) on the physiology of the P. aeruginosa PA14 strain. A total of 581 proteins were identified as differentially expressed in the 4 conditions. Most of them were more abundant in citrate supplementation and were involved in virulence, motility, biofilm development, and antibiotic resistance. Phenotypic assays were performed to check these hypotheses. By coupling all this data, we highlight the importance of the environment in which the bacterium evolves on its metabolism, and thus the necessity to better understand the metabolic pathways implied in its adaptative response according to the nutrient availability.

Venom Peptide Repertoire of the European Myrmicine Ant Manica rubida: Identification of Insecticidal Toxins.

Using an integrated transcriptomic and proteomic approach, we characterized the venom peptidome of the European red ant, Manica rubida. We identified 13 "myrmicitoxins" that share sequence similarities with previously identified ant venom peptides, one of them being identified as an EGF-like toxin likely resulting from a threonine residue modified by O-fucosylation. Furthermore, we conducted insecticidal assays of reversed-phase HPLC venom fractions on the blowfly Lucilia caesar, permitting us to identify six myrmicitoxins (i.e., U3-, U10-, U13-, U20-MYRTX-Mri1a, U10-MYRTX-Mri1b, and U10-MYRTX-Mri1c) with an insecticidal activity. Chemically synthesized U10-MYRTX-Mri1a, -Mri1b, -Mri1c, and U20-MYRTX-Mri1a irreversibly paralyzed blowflies at the highest doses tested (30-125 nmol·g-1). U13-MYRTX-Mri1a, the most potent neurotoxic peptide at 1 h, had reversible effects after 24 h (150 nmol·g-1). Finally, U3-MYRTX-Mri1a has no insecticidal activity, even at up to 55 nmol·g-1. Thus, M. rubida employs a paralytic venom rich in linear insecticidal peptides, which likely act by disrupting cell membranes.

LasB and CbpD Virulence Factors of Pseudomonas aeruginosa Carry Multiple Post-Translational Modifications on Their Lysine Residues.

Pseudomonas aeruginosa is a multi-drug resistant human pathogen largely involved in nosocomial infections. Today, effective antibacterial agents are lacking. Exploring the bacterial physiology at the post-translational modifications (PTM) level may contribute to the renewal of fighting strategies. Indeed, some correlations between PTMs and the bacterial virulence, adaptation, and resistance have been shown. In a previous study performed in P. aeruginosa, we reported that many virulence factors like chitin-binding protein CbpD and elastase LasB were multiphosphorylated. Besides phosphorylation, other PTMs, like those occurring on lysine, seem to play key roles in bacteria. In the present study, we investigated for the first time the lysine succinylome and acetylome of the extracellular compartment of P. aeruginosa by using a two-dimensional immunoaffinity approach. Some virulence factors were identified as multimodified on lysine residues, among them, LasB and CbpD. Lysine can be modified by a wide range of chemical groups. In order to check the presence of other chemical groups on modified lysines identified on LasB and CbpD, we used 1- and 2- dimensional gel electrophoresis approaches to target lysine modified by 7 other modifications: butyrylation, crotonylation, dimethylation, malonylation, methylation, propionylation, and trimethylation. We showed that some lysines of these two virulence factors were modified by these 9 different PTMs. Interestingly, we found that the PTMs recovered on these two virulence factors were different than those previously reported in the intracellular compartment.

Global Dynamic Proteome Study of a Pellicle-forming Acinetobacter baumannii Strain.

For several decades, many bacteria, among which A. baumannii, have shown their ability to colonize the upper surface of static liquids, forming a biofilm at the air-liquid interface named pellicle. Despite the ubiquity of these pellicles in both natural and artificial environments, few studies have investigated this biofilm type. The present data set provides the first description of the whole proteome of A. baumannii cells grown as pellicle, using a label-free mass spectrometry approach. Results are in accord with the general findings reporting that sessile bacteria are far more resistant to detrimental conditions than their planktonic counterparts, by the accumulation of stress proteins. The present investigation also confirmed previous studies suggesting a correlation between the pellicle forming ability and the bacterial virulence. Indeed, we showed the up-regulation of numerous virulence factors during the pellicle growth, e.g. phospholipases, adhesion factors, as well as those of the GacAS Two-Component System (TCS) and Type 6 Secretion System (T6SS). We also highlighted that Bam and Tam systems, both related to the OM insertion machinery, play a critical role during pellicle biogenesis. Moreover, sessile bacteria activate several pathways, e.g. iron, magnesium, phosphate pathways, which allows for increasing the panel of nutrient sources.

Neuroprotective effect of grape seed extract on brain ischemia: a proteomic approach.

Stroke is one of the leading causes of long-lasting disability in human and oxidative stress an important underlying cause. Molecular insights into pathophysiology of ischemic stroke are still obscure, and the present study investigated the protective effect of high dosage Grape Seed Extract (GSE 2.5 g/kg) on brain ischemia-reperfusion (I/R) injury using a proteomic approach. Ischemia was realized by occlusion of the common carotid arteries for 30 min followed by 1 h reperfusion on control or GSE pre-treated rats, and a label-free quantification followed by mass spectrometry analysis used to evaluate I/R induced alterations in protein abundance and metabolic pathways as well as the protection afforded by GSE. I/R-induced whole brain ionogram dyshomeostasis, ultrastructural alterations, as well as inflammation into hippocampal dentate gyrus area, which were evaluated using ICP-OES, transmission electron microscopy and immuno-histochemistry respectively. I/R altered the whole brain proteome abundance among which 108 proteins were significantly modified (35 up and 73 down-regulated proteins). Eighty-four proteins were protected upon GSE treatment among which 27 were up and 57 down-regulated proteins, suggesting a potent protective effect of GSE close to 78%of the disturbed proteome. Furthermore, GSE efficiently prevented the brain from I/R-induced ion dyshomeostasis, ultrastructural alterations, inflammatory biomarkers as CD56 or CD68 and calcium burst within the hippocampus. To conclude, a potent protective effect of GSE on brain ischemia is evidenced and clinical trials using high dosage GSE should be envisaged on people at high risk for stroke.

Lysine Succinylation and Acetylation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a multi-drug-resistant human opportunistic pathogen largely involved in nosocomial infections. Unfortunately, effective antibacterial agents are lacking. Exploring its physiology at the post-translational modifications (PTMs) level may contribute to the renewal of combat tactics. Recently, lysine succinylation was discovered in bacteria and seems to be an interesting PTM. We present the first succinylome and acetylome of P. aeruginosa PA14 cultured in the presence of four different carbon sources using a 2D immunoaffinity approach coupled to nanoliquid chromatography tandem mass spectrometry. A total of 1520 succinylated (612 proteins) and 1102 acetylated (522 proteins) lysine residues were characterized. Citrate was the carbon source in which we identified the higher number of modified proteins. Interestingly, 622 lysine residues (312 proteins) were observed either acetylated or succinylated. Some of these proteins, were involved in virulence, adaptation, resistance, and so on. A label-free quantification points out the existence of different protein forms for a same protein (unmodified, succinylated or acetylated) and suggests different abundance as a function of the carbon sources. This work is a promising starting point for further investigations on the biological role of lysine succinylation in P. aeruginosa.

SAG12, a Major Cysteine Protease Involved in Nitrogen Allocation during Senescence for Seed Production in Arabidopsis thaliana.

SAG12 is the most widely used senescence-associated reference gene for characterizing leaf senescence, and the increase in SAG12 protein during leaf senescence is remarkable. However, the role of this cysteine protease in N remobilization and the leaf senescence process remains unclear. The role of SAG12 has been poorly investigated and the few reports dealing with this are somewhat controversial. Indeed, sag12 Arabidopsis mutants have not shown any phenotype, while OsSAG12-1 and OsSAG12-2 overexpression in rice moderates senescence progression. Therefore, this study aims at clarifying the role of the SAG12 cysteine protease during the entire plant life span and during leaf senescence. Arabidopsis thaliana plants knocked-out for the SAG12 gene (sag12) did not exhibit any special phenotypic traits when grown under optimal nitrogen supply (HN), suggesting that other cysteine proteases could provide compensatory effects. Moreover, for the first time, this study shows that aspartate protease activity is significantly increased in sag12. Among the putative aspartate proteases involved, a CND41-like aspartate protease has been identified. Under low nitrogen (LN) availability, when inducible proteolytic systems are not sufficient to cope with SAG12 depletion, a decrease in yield is observed. Altogether, these results show that SAG12 (and perhaps also aspartate proteases) could be involved in RuBisCO degradation during the leaf senescence associated with seed filling.

Proteomics of Pseudomonas aeruginosa: the increasing role of post-translational modifications.

INTRODUCTION: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen widely involved in human infections. The high occurrence of this bacterial species in the clinical field is due to its high ability to adapt to detrimental environments, in particular its strong inherent antibiotic resistance, its ability to form biofilms and to produce virulence factors. The application of proteomics to clinical microbiology is probably one of the most innovative strategies of the last decades to understand complex microbial systems, by providing individual proteome charts of pathogens. Areas covered: In the last decade, proteomic advances have allowed in high-throughput the screening of proteins modified by diverse co- and post-translational modifications in P. aeruginosa. This review will present the current state of the art for the characterization of PTMs in P. aeruginosa by proteomics approaches. We will then discuss on the involvement of PTMs in P. aeruginosa physiology. Expert commentary: Modified proteins and enzymes involved in the addition/removal of modifications will surely constitute targets of interest to develop new therapeutic drugs to fight against P. aeruginosa.

Identification by mass spectrometry of glucosaminylphosphatidylglycerol, a phosphatidylglycerol derivative, produced by Pseudomonas aeruginosa.

RATIONALE: Pseudomonas aeruginosa is an opportunistic pathogen bacterium widely considered to be an excellent research model in several areas of molecular studies, namely genomics and proteomics. However, its lipid metabolism is still not totally decrypted. While it is known that this bacterium has the particularity to produce phosphatidylcholine, a lipid mainly found in eukaryotes, other singularities are still to be discovered. METHODS: P. aeruginosa was grown as planktonic cultures to the stationary state. Membrane pellets were collected and lipids were extracted using the Bligh and Dyer protocol. Lipid extracts were analyzed by Electrospray Ionization Mass Spectrometry (ESI-MS) using high-resolution mass spectrometer (LTQ Orbitrap Elite, Thermo Scientific) in the negative mode. MSn spectra were recorded both in the Orbitrap and in the ion trap analyzer (collision-induced dissociation (CID) or higher energy collision-induced dissociation (HCD) mode). RESULTS: We observed by mass spectrometry and thin layer chromatography that P. aeruginosa produced an unreferenced lipid in classical growth conditions. MS2 analysis of the unknown ion indicates that it is a phosphatidylglycerol derivative. The exact mass shift corresponds to glucosamine which is largely found in the metabolism of this bacterium. MS3 analysis of secondary ions allowed us to conclude that this lipid is a glucosaminylphosphatidylglycerol, a phosphatidylglycerol derivative containing a glucosamine substituted at C4. CONCLUSIONS: We show here that P. aeruginosa is able to produce glucosaminylphosphatidylglycerols via a probable esterification of phosphatidylglycerols by glucosamine.

Unsaturated Fatty Acids Affect Quorum Sensing Communication System and Inhibit Motility and Biofilm Formation of Acinetobacter baumannii.

The increasing threat of Acinetobacter baumannii as a nosocomial pathogen is mainly due to the occurrence of multidrug-resistant strains that are associated with the real problem of its eradication from hospital wards. The particular ability of this pathogen to form biofilms contributes to its persistence, increases antibiotic resistance, and promotes persistent/device-related infections. We previously demonstrated that virstatin, which is a small organic compound known to decrease virulence of Vibrio cholera via an inhibition of T4-pili expression, displayed very promising activity to prevent A. baumannii biofilm development. Here, we examined the antibiofilm activity of mono-unsaturated chain fatty acids, palmitoleic (PoA), and myristoleic (MoA) acids, presenting similar action on V. cholerae virulence. We demonstrated that PoA and MoA (at 0.02 mg/mL) were able to decrease A. baumannii ATCC 17978 biofilm formation up to 38% and 24%, respectively, presented a biofilm dispersing effect and drastically reduced motility. We highlighted that these fatty acids decreased the expression of the regulator abaR from the LuxIR-type quorum sensing (QS) communication system AbaIR and consequently reduced the N-acyl-homoserine lactone production (AHL). This effect can be countered by addition of exogenous AHLs. Besides, fatty acids may have additional non-targeted effects, independent from QS. Atomic force microscopy experiments probed indeed that PoA and MoA could also act on the initial adhesion process in modifying the material interface properties. Evaluation of fatty acids effect on 22 clinical isolates showed a strain-dependent antibiofilm activity, which was not correlated to hydrophobicity or pellicle formation ability of the tested strains, and suggested a real diversity in cell-to-cell communication systems involved in A. baumannii biofilm formation.

Pseudomonas aeruginosa produces phosphatidyltris(hydroxymethyl)aminomethane and derivatives when grown in Tris-buffered medium.

For optimal growth of a microorganism, the pH of the culture medium should be set at an optimum value. For that reason, growth media require buffering agents. We show in this study that, when grown in a medium supplemented with tris(hydroxymethyl)aminomethane (Tris), Pseudomonas aeruginosa is able to use this organic compound to produce new phospholipids. We thus pointed out that phosphatidyltris(hydroxymethyl)aminomethane as well as diphosphatidyltris(hydroxymethyl)aminomethane was detected in membrane lipid extracts of bacteria grown in Tris-buffered medium. Moreover, the amounts of lysoglycerophospholipids in the lipidome of P. aeruginosa grown in Tris-buffered medium increased leading to the presence of lysophosphatidylglycerol and lysophosphatidyltris(hydroxymethyl)aminomethane as well as other lysophospholipid derivatives. Finally, we investigated the effect of the presence of these exogenous phospholipids on the susceptibility of P. aeruginosa to some antibiotics. We observed a decrease of the minimal inhibitory concentrations of different antibiotic families, i.e., fluoroquinolones, aminoglycosides, ß-lactams and polymyxins, proving the importance of the buffer choice for growth medium and its impact on the lipidome.

Brain proteomic modifications associated to protective effect of grape extract in a murine model of obesity.

In recent years, the obesity epidemic has developed into a major health crisis worldwide. With current treatments limited to expensive, high-risk surgery and minimally efficacious pharmacotherapy, new therapeutic options are urgently needed to fight against this alarming trend. Though brain dysfunction has been studied linked to high fat diet (HFD) and grape seed and skin extract (GSSE) correction, the proteomic modifications linking the two effects on brain lipotoxicity are not well understood. To this end rats were exposed for 8 weeks to HFD treatment, to GSSE (500mg/kg BW) and to binary mixture of HFD and GSSE to gain insight into the potential pathways altered with metabolic disease and the protection afforded by GSSE. Significant modifications of brain proteins were detected using mass spectrometry-based differential proteomics. These proteins were mainly related to oxidative stress, glycolysis and calcium signaling. Additionally, proteins involved in the cytoskeleton were also affected by HFD treatment. Interestingly, whether up- or down regulated protein abundances, GSSE corrected most of the disturbances of HFD treatment. These findings provide impetus for future therapeutic investigation on GSSE against other metabolic disorders.

Cytotoxic peptides with insulin-releasing activities from skin secretions of the Italian stream frog Rana italica (Ranidae).

Peptidomic analysis of norepinephrine-stimulated skin secretions from Italian stream frog Rana italica led to the purification and characterization of two host-defense peptides differing by a single amino acid residue belonging to the brevinin-1 family (brevinin-1ITa and -1ITb), a peptide belonging to the temporin family (temporin-ITa) and a component identified as prokineticin Bv8. The secretions contained relatively high concentrations of the methionine-sulphoxide forms of brevinin-1ITa and -1ITb suggesting that these peptides may have a role as antioxidants in the skin of this montane frog. Brevinin-1ITa (IVPFLLGMVPKLVCLITKKC) displayed potent cytotoxicity against non-small cell lung adenocarcinoma A549 cells (LC50  = 18 µM), breast adenocarcinoma MDA-MB-231 cells (LC50  = 8 µM) and colorectal adenocarcinoma HT-29 cells (LC50  = 18 µM), but the peptide was also strongly hemolytic against mouse erythrocytes (LC50  = 7 µM). Temporin-ITa (VFLGAIAQALTSLLGKL.NH2 ) was between three and fivefold less potent against these cells. Brevinin-1ITa inhibited growth of both Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli as well as a strain of the opportunist yeast pathogen Candida parapsilosis, whereas temporin-ITa was active only against S. epidermidis and C. parapsilosis. Both peptides stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, but brevinin-1ITa was cytotoxic to the cells at concentrations ≥3 µM. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Two novel peptides with angiotensin I converting enzyme inhibitory and antioxidative activities from Scorpaena notata muscle protein hydrolysate.

Fish protein hydrolysate was prepared from muscle of small red scorpionfish (Scorpaena notata) by treatment with a protease from the fungus Penicillium digitatum. Protein hydrolysate was found to strongly inhibit the angiotensin I converting enzyme and exhibited high antioxidative activity through 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assay. After ultrafiltration, peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high-performance liquid chromatography with a high purification yield of 2.5 mg of peptide per gram of initial protein. Two major peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS), corresponding to the following sequences: Leu-Val-Thr-Gly-Asp-Asp-Lys-Thr-Asn-Leu-Lys (1,204.665 Da) and Asp-Thr-Gly-Ser-Asp-Lys-Lys-Gln-Leu (992.511 Da). These peptides, mainly composed of hydrophilic amino acids, showed high antioxidative and angiotensin I converting enzyme inhibitory activities. These data suggest that the two novel peptides isolated from the muscle hydrolysate of small red scorpionfish can be a beneficial ingredient for functional foods or pharmaceuticals against hypertension and oxidative stress.

Proteomics dedicated to biofilmology: What have we learned from a decade of research?

Advances in proteomics techniques over the past decade, closely integrated with genomic and physicochemical approach, have played a great role in developing knowledge of the biofilm lifestyle of bacteria. Despite bacterial proteome versatility, many studies have demonstrated the ability of proteomics approaches to elucidating the biofilm phenotype. Though these investigations have been largely used for biofilm studies in the last decades, they represent, however, a very low percentage of proteomics works performed up to now. Such approaches have offered new targets for combating microbial biofilms by providing a comprehensive quantitative and qualitative overview of their protein cell content. Herein, we summarized the state of the art in knowledge about biofilm physiology after one decade of proteomic analysis. In a second part, we highlighted missing research tracks for the next decade, emphasizing the emergence of posttranslational modifications in proteomic studies stemming from recent advances in mass spectrometry-based proteomics.

Purification, Conformational Analysis, and Properties of a Family of Tigerinin Peptides from Skin Secretions of the Crowned Bullfrog Hoplobatrachus occipitalis.

Four host-defense peptides belonging to the tigerinin family (tigerinin-1O: RICTPIPFPMCY; tigerinin-2O: RTCIPIPLVMC; tigerinin-3O: RICTAIPLPMCL; and tigerinin-4O: RTCIPIPPVCF) were isolated from skin secretions of the African crowned bullfrog Hoplobatrachus occipitalis. In aqueous solution at pH 4.8, the cyclic domain of tigerinin-2O adopts a rigid amphipathic conformation that incorporates a flexible N-terminal tail. The tigerinins lacked antimicrobial (MIC > 100 µM) and hemolytic (LC50 > 500 µM) activities but, at a concentration of 20 µg/mL, significantly (P < 0.05) inhibited production of interferon-γ (IFN-γ) by peritoneal cells from C57BL/6 mice without affecting production of IL-10 and IL-17. Tigerinin-2O and -4O inhibited IFN-γ production at concentrations as low as 1 µg/mL. The tigerinins significantly (P ≤ 0.05) stimulated the rate of insulin release from BRIN-BD11 clonal ß-cells without compromising the integrity of the plasma membrane. Tigerinin-1O was the most potent (threshold concentration 1 nM) and the most effective (395% increase over basal rate at a concentration of 1 µM). Tigerinin-4O was the most potent and effective peptide in stimulating the rate of glucagon-like peptide-1 release from GLUTag enteroendocrine cells (threshold concentration 10 nM; 289% increase over basal rate at 1 µM). Tigerinin peptides have potential for development into agents for the treatment of patients with type 2 diabetes.

Unraveling the effects of static magnetic field stress on cytosolic proteins of Salmonella by using a proteomic approach.

The present study investigated the adaptation of Salmonella enterica subsp. enterica serovar Hadar to static magnetic field (SMF) exposure (200 mT, 9 h). The proteomic analysis provides an overview of potentially important cytosolic proteins that Salmonella needs to regulate to survive and adapt to magnetic stress. Via 2-dimensional electrophoresis and liquid chromatography tandem mass spectrometry, we compared cytosolic proteomes before and after exposure to magnetic field. A total of 35 proteins displaying more than a 2-fold change were differentially expressed in exposed cells, among which 25 were upregulated and 10 were downregulated. These proteins can be classified mainly into 6 categories: (i) proteins involved in metabolic pathways of carbohydrates, (ii) chaperones and proteins produced in response to oxidative stress, (iii) proteins involved in energy homeostasis, (iv) elongation factors (EF-Tu and EF-Ts), (v) proteins involved in motility, and (vi) proteins involved in molecules transport. Many of the presented observations could be explained, while some represent still-unknown mechanisms. In addition, this study reveals 5 hypothetical proteins. It seems that the stress response to SMF (200 mT) is essentially set up to avoid oxidative damages, with the overexpression of proteins directly involved in oxidative stress response and metabolic switches to counteract oxidative stress. Interestingly, several proteins induced under SMF exposure are found to overlap with those induced by other stresses, such as heat shock and starvation.

Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins.

Protein lysine acetylation is a reversible and highly regulated post-translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology.

Antimicrobial Peptide LL-37 Is Both a Substrate of Cathepsins S and K and a Selective Inhibitor of Cathepsin L.

Lung cysteine cathepsins B, K, L, and S contribute to physiological and pathological processes including degradation of antimicrobial peptides/proteins (AMPs) such as surfactant protein SP-A, lactoferrin, secretory leukocyte peptidase inhibitor, and beta-defensins-2 and -3. Substantial amounts of uncleaved LL-37, a 37-mer cationic AMP, were observed in the sputum of patients with cystic fibrosis (CF). Nevertheless LL-37 was degraded after prolonged incubation in CF sputum, and the hydrolysis was blocked by E-64, a selective inhibitor of cysteine proteases. Cathepsins K and S, expressed in human alveolar macrophages, thoroughly hydrolyzed LL-37 in vitro, whereas it competitively inhibited cathepsin L (Ki = 150 nM). Cleavage of LL-37 by cathepsins S and K impaired its antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus, in a time- and concentration-dependent manner. The exchange of residues 67 and 205 in the S2 pockets of cathepsins L (Leu67Tyr/Ala205Leu) and K (Tyr67Leu/Leu205Ala) switched the specificity of these mutants toward LL-37. Molecular modeling suggested that LL-37 interacted with the active site of cathepsin L in both forward (i.e., substrate-like) and reverse orientations with similar binding energies. Our data support the hypothesis that cysteine cathepsins modulate the innate immunity response by degrading distinct and representative members of the AMP family.