Andrejko Mariola, Zdybicka-Barabas Agnieszka, Wawrzoszek Maria, Cytryńska Małgorzata
Department of Immunobiology, Institute of Biology and Biochemistry, Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, 19 Akademicka St., Lublin 20-033, Poland.
Zoolog Sci. 2013 May;30(5):345-51. doi: 10.2108/zsj.30.345.
We investigated the effects of extracellular proteinases of two Pseudomonas aeruginosa clinical isolates on the essential humoral immune response parameters in hemolymph of the insect model organism Galleria mellonella in vitro. Two culture media, rich LB and minimal M9, known to induce synthesis of different sets of proteinases secreted by P. aeruginosa were used. Changes in lysozyme, antibacterial and antifungal activities, as well as protein and peptide patterns in hemolymph treated with proteolytic fractions were evaluated. The effect of the proteolytic fractions on the apoLp-III level in hemolymph was determined by immunoblotting with antibodies against G. mellonella apolipophorin III (apoLp-III). We found that apoLp-III is hardly degraded by the proteinases of the proteolytic fractions of both clinical P. aeruginosa strains, in contrast to the high susceptibility of the protein to the proteinases of the entomopathogenic strain. The detected differences, together with the changes in the hemolymph protein and peptide patterns caused by the studied fractions, reflected the distinct composition of secreted proteinases of the entomopathogenic P. aeruginosa strain and the clinical strains tested. Our results also suggest the involvement of alkaline protease, the main proteinase of proteolytic fractions of P. aeruginosa grown in minimal medium, in the degradation of G. mellonella antimicrobial factors, such as lysozyme, antibacterial polypeptides, and proteins with antifungal activity. The diverse effects of the P. aeruginosa proteolytic fractions studied on the parameters of G. mellonella immune response indicate that this model insect may be useful in the analysis of the virulence factors of different P. aeruginosa strains.
我们在体外研究了两种铜绿假单胞菌临床分离株的胞外蛋白酶对昆虫模式生物大蜡螟血淋巴中重要体液免疫反应参数的影响。使用了两种已知可诱导铜绿假单胞菌分泌不同蛋白酶组的培养基，即丰富的LB培养基和基本的M9培养基。评估了经蛋白水解组分处理的血淋巴中溶菌酶、抗菌和抗真菌活性以及蛋白质和肽谱的变化。通过用抗大蜡螟载脂蛋白III（apoLp-III）的抗体进行免疫印迹，测定了蛋白水解组分对血淋巴中apoLp-III水平的影响。我们发现，与该蛋白对昆虫病原性菌株蛋白酶的高敏感性相反，两种临床铜绿假单胞菌菌株的蛋白水解组分中的蛋白酶几乎不会降解apoLp-III。检测到的差异，以及所研究的组分引起的血淋巴蛋白质和肽谱的变化，反映了昆虫病原性铜绿假单胞菌菌株和所测试临床菌株分泌的蛋白酶的不同组成。我们的结果还表明，在基本培养基中生长的铜绿假单胞菌蛋白水解组分的主要蛋白酶碱性蛋白酶参与了大蜡螟抗菌因子的降解，如溶菌酶、抗菌多肽和具有抗真菌活性的蛋白质。所研究的铜绿假单胞菌蛋白水解组分对大蜡螟免疫反应参数的不同影响表明，这种模式昆虫可能有助于分析不同铜绿假单胞菌菌株的毒力因子。
