Department of Microbiology, Military Institute of Hygiene and Epidemiology, Warsaw, Poland.
Curr Microbiol. 2010 May;60(5):360-4. doi: 10.1007/s00284-009-9550-4. Epub 2009 Dec 3.
Over 100 of Pseudomonas aeruginosa isolates representing the two TTSS genotypes (exoU (-)/exoS (+) or exoU (+)/exoS (-)) were cultured in different media in order to evaluate their proteolytic activities and find a relationship between proteolytic activity and the cytotoxic and/or invasive phenotypes displayed by the strains upon infection of RAW 264.7 murine macrophage-like cells and pulmonary microvascular endothelial cells (PME). The elastolytic activity, protein concentration, and total proteolytic activity (TPA) were measured in culture supernatants. No significant differences were observed in the median elastolytic activities among cytotoxic/noninvasive, noncytotoxic/invasive, and cytotoxic/invasive phenotypes displayed by P. aeruginosa strains. The only significant difference was noted when isolates of the two different TTSS genotypes were grown in a calcium-depleted minimal medium for induction of TTSS (MI). The exoU (-)/exoS (+) isolates showed significant higher levels of the median elastolytic activity when compared to the exoU (+)/exoS (-) isolates. These two groups of isolates secreted the elastase B (LasB) with distinct molecular masses 158 or 116 kD, respectively. The strains of the two TTSS genotypes secreted similar amount of total proteins; however, the higher values of TPA were observed for the isolates of the exoU (+) /exoS (-) genotype when grown in MI medium. We concluded that there is no direct relationship between secretion of proteases with elastolytic activity and the cytotoxic and/or invasive phenotypes of the isolates observed upon infection of both RAW 264.7 and PME monolayers. Further studies are needed to find out whether others factors beside proteases could influence the mechanism of host cells intoxication mediated by the P. aeruginosa TTSS-delivered toxins.
超过 100 株铜绿假单胞菌分离株代表两种 TTSS 基因型（exoU(-)/exoS(+)或 exoU(+)/exoS(-)），分别在不同的培养基中培养，以评估它们的蛋白水解活性，并寻找蛋白水解活性与菌株感染 RAW 264.7 鼠巨噬细胞样细胞和肺微血管内皮细胞（PME）时显示的细胞毒性和/或侵袭表型之间的关系。在培养上清液中测量弹性酶活性、蛋白浓度和总蛋白水解活性（TPA）。在细胞毒性/非侵袭性、非细胞毒性/侵袭性和细胞毒性/侵袭性表型的铜绿假单胞菌菌株中，弹性酶活性的中位数没有显著差异。只有当两种不同 TTSS 基因型的分离株在钙耗尽的最小培养基中生长以诱导 TTSS（MI）时，才观察到唯一的显著差异。exoU(-)/exoS(+)分离株的中位弹性酶活性明显高于 exoU(+)/exoS(-)分离株。这两组分离株分别分泌分子质量为 158 或 116 kD 的弹性蛋白酶 B（LasB）。两种 TTSS 基因型的菌株分泌相似量的总蛋白；然而，当在 MI 培养基中生长时，exoU(+)/exoS(-)基因型的分离株观察到更高的 TPA 值。我们得出的结论是，在感染 RAW 264.7 和 PME 单层时观察到的分离株的细胞毒性和/或侵袭表型与具有弹性酶活性的蛋白酶的分泌之间没有直接关系。需要进一步的研究来找出除蛋白酶以外的其他因素是否会影响铜绿假单胞菌 TTSS 输送的毒素介导的宿主细胞中毒机制。
