OMP proteomic analysis of benzalkonium chloride and ciprofloxacin adapted biofilm cells

Adaptive resistance to antimicrobials has been widely reported in planktonic studied trough phenotypic characterization and proteomic analysis. Concerning biofilm adaptation, the response of biofilm-entrapped cells to chemical stress conditions is not yet well studied. There is evidence that proteins involved in oxidative stress response, cell envelope synthesis, as well as in synthesis of EPS become up- or down-regulated in biofilms, indicating that these altered phenotypes might contribute to antimicrobial tolerance. This work aimed to examine whether exposure of Pseudomonas aeruginosa biofilms to benzalkonium chloride (BC) and ciprofloxacin (CIP) could induce an adaptive response in bacteria. This was attained by inspection of proteome alterations of the outer membrane (OMP) in biofilm cells. Biofilms were formed in 6-well plates for 24h being after submitted to the presence of 0.9 mM BC and 6.0 ug/ml CIP, during 13 days. The obtained biofilm-cells were separated and the OMP extracted. Protein patterns were analysed by 2-DE and gels by the SameSpot software. Biofilm-proteome showed that P. aeruginosa adaptation to BC promoted the down-regulation of 36 OMP and the up-regulation of only one. OMP 2DE of P. aeruginosa adapted to CIP revealed the down-regulation of 29 OMP. Six OMPs were changed in common by both antimicrobials, revealing a possible similar stress response. Proteins identification is in progress. This study highlighted that there might be an OMP regulation when bacteria within biofilms are submitted to chemical adaptation. This particular response to the environment can be one of the causes of the well-known biofilm resistance phenotype

Membranome analysis of benzalkonium chloride adapted planktonic and biofilm cells

Este resumo faz parte de: Book of abstracts of the Meeting of the Institute for Biotechnology and Bioengineering, 2, Braga, Portugal, 2010. A versão completa do livro de atas está disponível em: http://hdl.handle.net/1822/1096

Proteomic characterization of Benzalkonium Chloride- and Ciprofloxacin-adapted Pseudomonas aeruginosa biofilms

Bacteria are able to adapt to several environmental stresses such as the presence of antimicrobial molecules and, as consequence, bacterial resistance may increase with increasing exposure to antimicrobials. The most impressive mechanism of the bacterial mode of life is their grow as part of a sessile community referred to as biofilm [1]. Biofilm formation is an important aspect of many bacterial diseases, especially those related with medical devices [2]. When biofilms are identified as the cause of infection, treatment becomes very difficult since bacteria within biofilms demonstrate peculiar features, that confer them increased resistance to biocides. The adaptive response to antimicrobial stresses of sessile bacteria is more effective than the corresponding planktonic populations. Adaptive resistance to antimicrobials has been widely reported in planktonic state and characterized in terms of phenotypic traits and proteomic analysis [3,4]. Concerning biofilm adaptation, the response of the biofilm-entrapped cells to chemical stress conditions is not yet well studied. This work aimed to examine whether exposure of Pseudomonas aeruginosa biofilms to benzalkonium chloride (BC) and ciprofloxacin (CIP) during a laboratory adaptation process could induce any proteomic alterations in the outer membrane (OM) of the biofilm cells. Biofilms were formed in 6-well plates for 24 h being after submitted to the presence of 324 mg/L of BC and 6.0 mg/L of CIP, during 13 days. The obtained biofilm-cells were separated from the biofilm matrix and the OM proteins extracted. Protein patterns were analyzed by 2-DE and gels by Progenesis SameSpot software. Protein spots from the bacterial populations were considered to display significant quantitative differences if they fulfilled the following criteria: p values ≤ 0.05 (t-test); detection threshold, average volume ≥ 20 (n = 3); differential tolerance, fold change ≥ 2. Excised spots from three different gels of each adapted bacteria were identified by LC-MS/MS. Biofilm proteome analysis showed that P. aeruginosa adaptation to BC and CIP changed the expression of six proteins. The biofilm exposure to both antimicrobials generated common down-regulation of three proteins: GroEL, major capsid protein and putative tail sheath protein, revealing a possible similar stress response. The type 4 fimbrial biogenesis outer membrane protein PilQ precursor was over-expressed only in biofilms submitted to BC, while the probable bacteriophage protein and the hypothetical protein PA0537 were overexpressed in CIP exposed biofilms. When bacteria are within biofilms and exposed to chemical stress, the regulation of OM proteins expression can contribute to increase the biofilm resistance. The proteins involved in adhesion, oxidative stress response, as well as in synthesis of lipopolysaccharide, were upor down-regulated in adapted P. aruginosa biofilms. These acquired proteomic profiles may be associated with antimicrobial tolerance

Proteomic approach to Pseudomonas aeruginosa adaptive resistance to benzalkonium chloride

This study aimed to assess the membrane modifications in Pseudomonas aeruginosa after continuous exposure to increasing doses of benzalkonium chloride (BC). Two different concentrations were used, 0.9 and 12.0 mM. Proteomic investigations revealed that the range of the outermembrane proteome alterations following continuous exposure is very low, i.e. about 10% and BC concentration dependent. Adapted cells revealed different expressions of key proteins frequently reported as involved in acquired resistance mechanisms. Porins (OprF and OprG) and lipoproteins (OprL and OprI) were underexpressed when the higher adaptation concentration (12 mM) was used. Some of thesemembrane alterations have been described as involved in the acquired resistance to antibiotics, suggesting possible commonmechanisms between these two types of resistance.The authors would like to acknowledge the financial support from IBB-CEB and Fundacao para a Ciencia e Tecnologia (FCT) and European Community fund FEDER, through Program COMPETE, in the ambit of the Project PTDC/SAUESA/64609/2006/FCOMP-01-0124-FEDER-00702 and Idalina Machado PhD Grant (SFRH/BD/31065/2006)

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.Peer reviewe

Purification, Conformational Analysis and Cytotoxic Activities of Host-Defense Peptides from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae)

International audienceFrogs from the extensive amphibian family Hylidae are a rich source of peptides with therapeutic potential. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae) collected in Trinidad led to the isolation and structural characterization of five host-defense peptides with limited structural similarity to figainin 2 and picturin peptides from other frog species belonging to the genus Boana. In addition, the skin secretions contained high concentrations of tryptophyllin-BN (WRPFPFL) in both C-terminally α-amidated and non-amidated forms. Figainin 2BN (FLGVALKLGKVLG KALLPLASSLLHSQ) and picturin 1BN (GIFKDTLKKVVAAVLTTVADNIHPK) adopt α-helical conformations in trifluroethanol–water mixtures and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). The CD data also indicate contributions from turn structures. Both peptides and picturin 2BN (GLMDMLKKVGKVALT VAKSALLP) inhibited the growth of clinically relevant Gram-negative and Gram-positive bacteria with MIC values in the range 7.8–62.5 µM. Figainin 2BN was potently cytotoxic to A549, MDA-MB-231 and HT-29 human tumor-derived cells (LC50 = 7–14 µM) but displayed comparable potency against non-neoplastic HUVEC cells (LC50 = 15 µM) indicative of lack of selectivity for cancer cells

Peptidomic Analysis of Skin Secretions of the Caribbean Frogs Leptodactylus insularum and Leptodactylus nesiotus (Leptodactylidae) Identifies an Ocellatin with Broad Spectrum Antimicrobial Activity

International audienceOcellatins are peptides produced in the skins of frogs belonging to the genus Leptodactylus that generally display weak antimicrobial activity against Gram-negative bacteria only. Peptidomic analysis of norepinephrine-stimulated skin secretions from Leptodactylus insularum Barbour 1906 and Leptodactylus nesiotus Heyer 1994, collected in the Icacos Peninsula, Trinidad, led to the purification and structural characterization of five ocellatin-related peptides from L. insularum (ocellatin-1I together with its (1-16) fragment, ocellatin-2I and its (1-16) fragment, and ocellatin-3I) and four ocellatins from L. nesiotus (ocellatin-1N,-2N,-3N, and-4N). While ocellatins-1I,-2I, and-1N showed a typically low antimicrobial potency against Gram-negative bacteria, ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH 2) was active against an antibiotic-resistant strain of Klebsiella pneumoniae and reference strains of Escherichia coli, K. pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium (minimum inhibitory concentrations (MICs) in the range 31.25-62.5 µM), and was the only peptide active against Gram-positive Staphylococcus aureus (MIC = 31.25 µM) and Enterococcus faecium (MIC = 62.5 µM). The therapeutic potential of ocellatin-3N is limited by its moderate hemolytic activity (LC 50 = 98 µM) against mouse erythrocytes. The peptide represents a template for the design of long-acting, non-toxic, and broad-spectrum antimicrobial agents for targeting multidrug-resistant pathogens

Growth of Acinetobacter baumannii in Pellicle Enhanced the Expression of Potential Virulence Factors

BACKGROUND: Interestingly, Acinetobacter baumannii presents an enhanced capacity to form biofilms (also named pellicles) at the air-liquid interface as compared to the other Acinetobacter species. This characteristic questions the contribution of this phenotype to an increased risk of clinical infections by this pathogen. METHODOLOGY/PRINCIPAL FINDINGS: By a proteomic approach using 2-D gel electrophoresis-LC-MS/MS mass spectrometry, we compared the membrane protein patterns of A. baumannii 77, a pellicle-forming clinical isolate, grown in planktonic and in sessile modes. We identified 52 proteins with a differential expression, including 32 up-regulated and 20 down-regulated in the pellicle state. Several proteins, differentially expressed during pellicle development, were of particular interest. We determined the over-expression of four siderophore iron uptake systems including the acinetobactin and enterobactin receptors and confirmed that the development of this type of biofilm is promoted by ferric ions. Two over-expressed proteins, CarO and an OprD-homologue, putative carbapenem-resistance associated porins, would be involved in the transport of specific compounds, like ornithine, a biosynthesis precursor of a siderophore from the hydroxamate family. We evidenced the overexpression of a lipase and a transporter of LCFA that may be involved in the recycling of lipids inside the pellicle matrix. Finally, we demonstrated both by proteomic and by AFM studies that this particular type of biofilm required multiple pili systems to maintain this cohesive structure at the air-liquid interface; two of these systems have never been described in A. baumannii. CONCLUSIONS/SIGNIFICANCE: Our study demonstrated that several proteins, overexpressed at a late state of pellicle development, could be potentially involved in virulence processes. Therefore, regarding the number of potential virulence factors that are over-expressed in this growth mode, the pellicle-forming clinical isolates should be kept under survey

Host-defense peptides isolated from the skin secretions of the Northern red-legged frog Rana aurora aurora

Abstract Antimicrobial peptides in the skin secretions of anurans constitute a component of the innate immunity that protects the organism against invading pathogens. Four peptides with antimicrobial activity were isolated in high yield from norepinephrine-stimulated skin secretions of the Northern red-legged frog Rana aurora aurora and their primary structures determined. Ranatuerin-2AUa (GILSSFKGVAKGVAKNLAGKLLDELKCKITGC) showed potent growth-inhibitory activity against a range of Gram-positive and Gram-negative bacteria (minimum inhibitory concentrations , 20 mM) but low hemolytic activity against human erythrocytes (50% hemolysis at 290 mM). Brevinin-1AUa (FLPILAGLAAKLVPKVFCSITKKC) and brevinin-1AUb (FLPILAGLAANILPKVFCSITKKC) also showed potent antimicrobial activity but were strongly hemolytic (HC 50 , 10 mM). Temporin-1AUa (FLPIIGQLLSGLL.NH 2 ) atypically lacked a basic amino acid residue and showed very weak antimicrobial and hemolytic activity. Its biological function remains to be established. The primary structures of the antimicrobial peptides are consistent with a close phylogenetic relationship between R. aurora, Rana boylii and Rana luteiventris.