Mechanisms of propagation of antibiotic resistance-conferring mobile genetic elements in Enterobacteriaceae and Vibrionaceae

了解驱动抗生素耐药基因流动性的遗传决定因素和分子机制对于对抗多重耐药细菌的出现和传播至关重要。SXT/R391家族的整合元件和接合元件（SRI）以及A/C不相容组的接合质粒（ACP）是致病性肠杆菌科和弧菌科多重耐药基因的主要载体。SRI是霍乱弧菌第七大流行谱系的抗生素耐药性的原因，霍乱弧菌是霍乱的病原体，它仍然是全球范围内死亡和发病的主要原因。同时，在全球范围内，从环境、食用动物、食品和人类中回收的肠道病原体中，ACP与超广谱β-内酰胺酶和碳青霉烯酶相关的报告令人不安地增加。ACP还促进多药耐药沙门氏菌基因组岛1（SGI 1）的繁殖。由于ACP和SRI来自共同的祖先，它们编码高度相似的接合转移机制，介导遗传物质从供体细胞到受体细胞的易位。目前，介导这些转移的分子机制还不清楚。ACP和SRI DNA的易位由ACP和SRI编码的松弛酶TraI和辅助蛋白MobI在转移起点（oriT）启动。TraI属于松弛酶的非典型家族，其被预测为HD磷酸二酯酶，并且似乎需要MobI样辅助蛋白来发挥功能，这表明接合转移起始的完全独特和新颖的机制。我们预计TraI和MobI是参与ACP介导的基因组岛（如SGI 1）转移的关键因素。该提案的主要目标是表征ACP和SRI的接合转移启动机制，以及ACP的接合机制与它们动员的基因组岛之间的分子相互作用。更具体地说，我们建议确定所有的伙伴蛋白参与DNA加工在oriT。此外，我们的目标是发现蛋白质易位信号，允许通过由ACP和SRI编码的IV型分泌系统识别和分泌蛋白质。我们还将功能地图与单核苷酸的准确性oriT，以确定哪些特定的序列特征促进组装的高阶复合物在这个位点。最后，我们将研究ACP编码的松弛体和ACP动员的基因组岛之间的相互作用。我们的研究结果将产生关于基础生物学ACP和SRI的基本见解，从而为科学界提供强大的基础和新的潜在分子靶点，可用于开发旨在解决多药耐药进展的工具。

Knowledge of the genetic determinants and molecular mechanisms that drive the mobility of antibiotic resistance genes is essential to fight the emergence and spread of multidrug resistant bacteria. The integrative and conjugative elements of the SXT/R391 family (SRIs) and the conjugative plasmids of the A/C incompatibility group (ACPs), are major vectors of multidrug resistance genes in pathogenic Enterobacteriaceae and Vibrionaceae. SRIs is responsible for antibiotic resistance of the seventh pandemic lineage of Vibrio cholerae, the etiologic agent of cholera, which remains a major cause of mortality and morbidity on a global scale. Meanwhile, reports associating ACPs with extended-spectrum β-lactamases and carbapenemases are unsettlingly rising worldwide in enteric pathogens recovered from the environment, food-producing animals, food products and humans. ACPs also facilitate the propagation of the multidrug resistance-conferring Salmonella Genomic Island 1 (SGI1). Because ACPs and SRIs derive from a common ancestor, they code for highly similar conjugative transfer machineries, which mediate the translocation of genetic material from donor to recipient cells. Currently, the molecular mechanisms mediating these transfers are not well understood. Translocation of ACP and SRI DNA is initiated at the origin of transfer (oriT) by the relaxase TraI and the auxiliary protein MobI, both encoded by ACPs and SRIs. TraI belongs to an atypical family of relaxases, which are predicted HD phosphodiesterases and seems to require a MobI-like auxiliary protein to function, suggesting a completely unique and novel mechanism of conjugative transfer initiation. We anticipate that TraI and MobI are key factors involved in the ACP-mediated transfer of genomic islands such as SGI1.***The main goal of this proposal is to characterize the mechanism of conjugative transfer initiation of ACPs and SRIs, and the molecular interactions between the conjugative machinery of ACPs and genomic islands that they mobilize. More specifically, we propose to identify all the partner proteins that are involved in DNA processing at oriT. Moreover, we aim at discovering the protein translocation signals that allow recognition and secretion of proteins through the type IV secretion system encoded by ACPs and SRIs. We will also functionally map with single-nucleotide accuracy the oriT to determine what specific sequence features promote assembly of a high-order complex at this locus. Finally, we will investigate the interactions between the ACP-encoded relaxosome and genomic islands that are mobilized by ACPs.***Our results will yield fundamental insights about the basic biology ACPs and SRIs, thereby providing the scientific community with strong foundations and new potential molecular targets that could be used for the development of tools aimed at tackling the progression of multidrug resistance.