Machin Alexandre, Telleria Jenny, Brizard Jean-Paul, Demettre Edith, Séveno Martial, Ayala Francisco José, Tibayrenc Michel
Unité Mixte de Recherche, Institut de Recherche pour le Développement/Centre National de la Recherche Scientifique/Universités Montpellier 1 and 2, Génétique et Evolution des Maladies Infectieuses, n° 5290, Maladies Infectieuses et Vecteurs Ecologie, Génétique, Evolution et Contrôle (MIVEGEC) Institut de Recherche pour le Développement, Montpellier, France.
Unité Mixte de Recherche, Institut de Recherche pour le Développement/Centre National de la Recherche Scientifique, n° 5096, Centre Institut de Recherche pour le Développement, Montpellier, France.
PLoS One. 2014 Apr 18;9(4):e95442. doi: 10.1371/journal.pone.0095442. eCollection 2014.
We have analyzed the comportment in in vitro culture of 2 different genotypes of Trypanosoma cruzi, the agent of Chagas disease, pertaining to 2 major genetic subdivisions (near-clades) of this parasite. One of the stocks was a fast-growing one, highly virulent in mice, while the other one was slow-growing, mildly virulent in mice. The working hypothesis was that mixtures of genotypes interact, a pattern that has been observed by us in empirical experimental studies. Genotype mixtures were followed every 7 days and characterized by the DIGE technology of proteomic analysis. Proteic spots of interest were characterized by the SAMESPOT software. Patterns were compared to those of pure genotypes that were also evaluated every 7 days. One hundred and three spots exhibited changes in time by comparison with T = 0. The major part of these spots (58%) exhibited an under-expression pattern by comparison with the pure genotypes. 32% of the spots were over-expressed; 10% of spots were not different from those of pure genotypes. Interestingly, interaction started a few minutes after the mixtures were performed. We have retained 43 different proteins that clearly exhibited either under- or over-expression. Proteins showing interaction were characterized by mass spectrometry (MALDI-TOF). Close to 50% of them were either tubulins or heat shock proteins. This study confirms that mixed genotypes of T. cruzi interact at the molecular level. This is of great interest because mixtures of genotypes are very frequent in Chagas natural cycles, both in insect vectors and in mammalian hosts, and may play an important role in the transmission and severity of Chagas disease. The methodology proposed here is potentially applicable to any micropathogen, including fungi, bacteria and viruses. It should be of great interest in the case of bacteria, for which the epidemiological and clinical consequences of mixed infections could be underestimated.
我们分析了恰加斯病病原体克氏锥虫2种不同基因型在体外培养时的行为，这2种基因型属于该寄生虫的2个主要遗传亚群（近分支）。其中一个菌株生长迅速，对小鼠具有高致病性，而另一个菌株生长缓慢，对小鼠致病性较弱。我们的工作假设是基因型混合物会相互作用，这是我们在实证实验研究中观察到的一种模式。每隔7天对基因型混合物进行监测，并通过蛋白质组分析的差异凝胶电泳（DIGE）技术进行表征。感兴趣的蛋白质斑点通过SAMESPOT软件进行表征。将这些模式与同样每隔7天评估一次的纯基因型模式进行比较。与T=0时相比，有103个斑点随时间发生了变化。与纯基因型相比，这些斑点中的大部分（58%）呈现表达下调模式。32%的斑点表达上调；10%的斑点与纯基因型的斑点没有差异。有趣的是，混合物形成后几分钟就开始了相互作用。我们鉴定出43种明显表现出表达下调或上调的不同蛋白质。对表现出相互作用的蛋白质进行了基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）表征。其中近50%是微管蛋白或热休克蛋白。这项研究证实克氏锥虫的混合基因型在分子水平上会相互作用。这一点非常重要，因为在恰加斯病的自然循环中，无论是在昆虫媒介还是在哺乳动物宿主中，基因型混合物都很常见，并且可能在恰加斯病的传播和严重程度方面发挥重要作用。这里提出的方法可能适用于任何微生物病原体，包括真菌、细菌和病毒。对于细菌来说，这种方法应该会非常有用，因为混合感染的流行病学和临床后果可能被低估了。
