title: Heterogeneous production of proteases from Brazilian clinical isolates of Pseudomonas aeruginosa.
abstract: BACKGROUND
  Pseudomonas aeruginosa is an important human pathogen that causes severe infections in a wide range of immunosuppressed patients. Herein, we evaluated the proteolytic profiles of 96 Brazilian clinical isolates of P. aeruginosa recovered from diverse anatomical sites.
  
  
  METHODS
  Cell-associated and extracellular proteases were evidenced by gelatin-SDS-PAGE and by the cleavage of soluble gelatin. Elastase was measured by using the peptide substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide. The prevalence of elastase genes (lasA and lasB) was evaluated by PCR.
  
  
  RESULTS
  Bacterial extracts were initially applied on gelatin-SDS-PAGE and the results revealed four distinct zymographic profiles as follows: profile I (composed by bands of 145, 118 and 50kDa), profile II (118 and 50kDa), profile III (145kDa) and profile IV (118kDa). All the proteolytic enzymes were inhibited by EDTA, identifying them as metalloproteases. The profile I was the most detected in both cellular (79.2%) and extracellular (84.4%) extracts. Overall, gelatinase and elastase activities measured in the spent culture media were significantly higher (around 2-fold) compared to the cellular extracts and the production level varied according to the site of bacterial isolation. For instance, tracheal secretion isolates produced elevated amount of gelatinase and elastase measured in both cellular and extracellular extracts. The prevalence of elastase genes revealed that 100% isolates were lasB-positive and 85.42% lasA-positive. Some positive/negative correlations were showed concerning the production of gelatinase, elastase, isolation site and antimicrobial susceptibility.
  
  
  CONCLUSIONS
  The protease production was highly heterogeneous in Brazilian clinical isolates of P. aeruginosa, which corroborates the genomic/metabolic versatility of this pathogen.
authors.affiliation: Programa de Pós-Graduação em Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Anna Clara M
authors.initials: ACM
authors.lastname: Galdino
authors.affiliation: Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; The Inorganic Pharmaceutical and Biomimetic Research Centre, Focas Research Institute, Dublin Institute of Technology, Dublin, Ireland.
authors.forename: Lívia
authors.initials: L
authors.lastname: Viganor
authors.affiliation: Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
authors.forename: Mariangela
authors.initials: M
authors.lastname: Ziccardi
authors.affiliation: Departamento de Patologia e Programa de Pós-Graduação em Doenças Infecciosas, Universidade Federal do Espírito Santo, Espírito Santo, Brazil.
authors.forename: Ana Paula F
authors.initials: APF
authors.lastname: Nunes
authors.affiliation: Departamento de Microbiologia Médica, Instituto de Microbiologia Paulo de Góes, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Kátia R N
authors.initials: KRN
authors.lastname: Dos Santos
authors.affiliation: Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Marta H
authors.initials: MH
authors.lastname: Branquinha
authors.affiliation: Programa de Pós-Graduação em Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Electronic address: andre@micro.ufrj.br.
authors.forename: André L S
authors.initials: ALS
authors.lastname: Santos
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D001826:Body Fluids / Q000382:microbiology; D001938:Brazil; D003550:Cystic Fibrosis / Q000150:complications; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D018093:Gelatinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D006801:Humans; D045726:Metalloproteases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D009928:Organ Specificity; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification; D018410:Pneumonia, Bacterial / Q000382:microbiology; D011480:Protease Inhibitors / Q000494:pharmacology; D011552:Pseudomonas Infections / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D012007:Rectum / Q000382:microbiology; D012137:Respiratory System / Q000382:microbiology; D014774:Virulence; D014946:Wound Infection / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D011480:Protease Inhibitors; D004492:Edetic Acid; D045726:Metalloproteases; D010196:Pancreatic Elastase; D018093:Gelatinases
keywords: Aislamientos clínicos brasileños; Brazilian clinical isolates; Elastasa; Elastase; Factores virulentos; Gelatinasas; Gelatinases; Proteasas; Proteases; Pseudomonas aeruginosa; Virulence factors