巴西铜绿假单胞菌临床分离株蛋白酶的异质性产生

Heterogeneous production of proteases from Brazilian clinical isolates of Pseudomonas aeruginosa.

作者信息

Galdino Anna Clara M, Viganor Lívia, Ziccardi Mariangela, Nunes Ana Paula F, Dos Santos Kátia R N, Branquinha Marta H, Santos André L S

机构信息

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.

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.

出版信息

Enferm Infecc Microbiol Clin. 2017 Dec;35(10):630-637. doi: 10.1016/j.eimc.2016.06.015.

DOI:10.1016/j.eimc.2016.06.015

PMID:27480954

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.

CONCLUSION

The protease production was highly heterogeneous in Brazilian clinical isolates of P. aeruginosa, which corroborates the genomic/metabolic versatility of this pathogen.

摘要

背景

铜绿假单胞菌是一种重要的人类病原体，可在广泛的免疫抑制患者中引起严重感染。在此，我们评估了从不同解剖部位分离出的96株巴西临床铜绿假单胞菌菌株的蛋白水解谱。

方法

通过明胶-SDS-PAGE和可溶性明胶的裂解来证明细胞相关蛋白酶和细胞外蛋白酶。使用肽底物N-琥珀酰-Ala-Ala-Ala-对硝基苯胺测量弹性蛋白酶。通过PCR评估弹性蛋白酶基因（lasA和lasB）的流行情况。

结果

细菌提取物最初应用于明胶-SDS-PAGE，结果显示出四种不同的酶谱：谱I（由145、118和50 kDa的条带组成）、谱II（118和50 kDa）、谱III（145 kDa）和谱IV（118 kDa）。所有蛋白水解酶均被EDTA抑制，确定它们为金属蛋白酶。谱I在细胞提取物（79.2%）和细胞外提取物（84.4%）中检测到的频率最高。总体而言，与细胞提取物相比，在废培养基中测得的明胶酶和弹性蛋白酶活性显著更高（约2倍），且产生水平根据细菌分离部位而有所不同。例如，气管分泌物分离株在细胞和细胞外提取物中测得的明胶酶和弹性蛋白酶量均升高。弹性蛋白酶基因的流行情况显示，100%的分离株lasB呈阳性，85.42%的lasA呈阳性。在明胶酶、弹性蛋白酶产生、分离部位和抗菌药敏性之间显示出一些正/负相关性。