Activation of Neutrophils by the Two-Component Leukotoxin LukE/D from <i>Staphylococcus aureus</i>: Proteomic Analysis of the Secretions

<i>Staphylococcus aureus</i> is responsible for severe bacterial infections in hospitals and healthcare facilities. It produces single and bicomponent toxins (leukotoxins and hemolysins) that hinder innate immune function. Leukotoxin subunits bind to leukocyte cell membrane thus inducing transmembrane pores and subsequently, cell lysis. Leukotoxin LukE/D is a member of the bicomponent toxin family, but to date, no study concerning its involvement in host-pathogen interactions has been reported. In the present study, we performed the proteomic analysis of the secretions recovered after activation of human neutrophils by leukotoxin LukE/D. The neutrophil secretions were purified by RP-HPLC and different fractions were analyzed by Edman sequencing, LC-MS/MS, immunoblotted for chromogranin-derived peptides and further analyzed for antimicrobial properties. Proteomic analysis revealed that neutrophil secretions constitute a large number of proteins related with immune boosting mechanisms, proteolytic degradation, inflammatory process and antioxidant reactions

Activation of Neutrophils by the Two-Component Leukotoxin LukE/D from <i>Staphylococcus aureus</i>: Proteomic Analysis of the Secretions

<i>Staphylococcus aureus</i> is responsible for severe bacterial infections in hospitals and healthcare facilities. It produces single and bicomponent toxins (leukotoxins and hemolysins) that hinder innate immune function. Leukotoxin subunits bind to leukocyte cell membrane thus inducing transmembrane pores and subsequently, cell lysis. Leukotoxin LukE/D is a member of the bicomponent toxin family, but to date, no study concerning its involvement in host-pathogen interactions has been reported. In the present study, we performed the proteomic analysis of the secretions recovered after activation of human neutrophils by leukotoxin LukE/D. The neutrophil secretions were purified by RP-HPLC and different fractions were analyzed by Edman sequencing, LC-MS/MS, immunoblotted for chromogranin-derived peptides and further analyzed for antimicrobial properties. Proteomic analysis revealed that neutrophil secretions constitute a large number of proteins related with immune boosting mechanisms, proteolytic degradation, inflammatory process and antioxidant reactions

Activation of Neutrophils by the Two-Component Leukotoxin LukE/D from <i>Staphylococcus aureus</i>: Proteomic Analysis of the Secretions

<i>Staphylococcus aureus</i> is responsible for severe bacterial infections in hospitals and healthcare facilities. It produces single and bicomponent toxins (leukotoxins and hemolysins) that hinder innate immune function. Leukotoxin subunits bind to leukocyte cell membrane thus inducing transmembrane pores and subsequently, cell lysis. Leukotoxin LukE/D is a member of the bicomponent toxin family, but to date, no study concerning its involvement in host-pathogen interactions has been reported. In the present study, we performed the proteomic analysis of the secretions recovered after activation of human neutrophils by leukotoxin LukE/D. The neutrophil secretions were purified by RP-HPLC and different fractions were analyzed by Edman sequencing, LC-MS/MS, immunoblotted for chromogranin-derived peptides and further analyzed for antimicrobial properties. Proteomic analysis revealed that neutrophil secretions constitute a large number of proteins related with immune boosting mechanisms, proteolytic degradation, inflammatory process and antioxidant reactions

Activation of Neutrophils by the Two-Component Leukotoxin LukE/D from <i>Staphylococcus aureus</i>: Proteomic Analysis of the Secretions

<i>Staphylococcus aureus</i> is responsible for severe bacterial infections in hospitals and healthcare facilities. It produces single and bicomponent toxins (leukotoxins and hemolysins) that hinder innate immune function. Leukotoxin subunits bind to leukocyte cell membrane thus inducing transmembrane pores and subsequently, cell lysis. Leukotoxin LukE/D is a member of the bicomponent toxin family, but to date, no study concerning its involvement in host-pathogen interactions has been reported. In the present study, we performed the proteomic analysis of the secretions recovered after activation of human neutrophils by leukotoxin LukE/D. The neutrophil secretions were purified by RP-HPLC and different fractions were analyzed by Edman sequencing, LC-MS/MS, immunoblotted for chromogranin-derived peptides and further analyzed for antimicrobial properties. Proteomic analysis revealed that neutrophil secretions constitute a large number of proteins related with immune boosting mechanisms, proteolytic degradation, inflammatory process and antioxidant reactions

The bacterial killing kinetics of the bCTL against the <i>S. aureus</i> ATCC 25923.

<p>Different concentrations (MIC 40 µg/mL and 2× MIC 80 µg/mL) of bCTL were used. C+, represents antibiotic control (Cefotaxime 0.1 µg/mL + Tetracycline 10 µg/mL) and C-, represents phosphate buffer saline control. (A): <i>S. aureus</i> killing kinetics at time zero to 60 min. (B): <i>S. aureus</i> killing kinetics over 24 h time period.</p

The proteolytic cleavage sites induced after treatment of bCAT, hCAT and CTL with different <i>S. aureus</i> strains (ATCC25923, ATCC49775, S1 and S2).

<p>The experimental molecular masses obtained after MALDI-TOF analysis, are indicated.</p

Antibacterial assays of catestatin (bovine, bCAT and human, hCAT) and cateslytin (bovine, bCTL) against different <i>S. aureus</i> strains, compared to the cathelicidin antimicrobial peptide-18 (LL-37).

<p>Results are presented as MIC (µg/mL) of each peptide against four <i>S. aureus</i> strains. Values represent the means of the triplicate (n = 3) wells. Means with same letters are not significantly different (<i>p</i><0.05). Small letters (a, b, c) represents significance between different <i>S. aureus</i> strains, while capital letters (A, B, C) represent significantly difference between peptides.</p

Sequence alignment of bovine catestatin (CgA_344–364) with corresponding fragments from several species.

<p>For each position predominant identical residues are indicated in bold letters. Homology sequence is indicated (%).The data base used is UniProtKB. (+, basic residue; a, for A/G/T/P).</p

HPLC chromatograms of bCAT, hCAT and CTL alone or with different bacterial strain supernatants, with or without protease inhibitors.

<p>(A): Alignment of the HPLC chromatograms corresponding to: (1) bCAT, (2) bCAT+MHB, (3) bCAT+S49775, (4) bCAT+S25923 (5) bCAT+S1 (6) bCAT+S2. (B): Alignment of the HPLC chromatograms corresponding to: (1) bCAT, (2) bCAT+Pi+S49775, (3) bCAT+Pi+S25923 (4) bCAT+Pi+S1 (5) bCAT+Pi+S2. (C): Alignment of the HPLC chromatograms corresponding to: (1) hCAT, (2) hCAT+MHB, (3) hCAT+S49775, (4) hCAT+S25923 (5) hCAT+S1 (6) hCAT+S2. (D): Alignment of the HPLC chromatograms corresponding to: (1) hCAT, (2) hCAT+Pi+S49775, (3) hCAT+Pi+S25923 (4) hCAT+Pi+S1 (5) hCAT+Pi+S2. (E): Alignment of the HPLC chromatograms corresponding to: (1) bCTL, (2) bCTL+MHB, (3) bCTL+S49775, (4) bCTL+S25923 (5) bCTL+S1 (6) bCTL+S2. (F): Alignment of the HPLC chromatograms corresponding to: (1) bCTL, (2) bCTL+Pi+S49775, (3) bCTL+Pi+S25923 (4) bCTL+Pi+S1 (5) bCTL+Pi+S2.</p