Molecular Biology and Virulence of CTX Phage

描述(申请人提供)：可移动的遗传元素已经深刻地影响了细菌病原体的进化。毒力和抗生素耐药性的许多决定因素是由能够水平传播的可移动元件携带的，如质粒和噬菌体。在这个赠款周期中，我们研究了CTXF生命周期的几个方面，CTXF是一种编码霍乱毒素的丝状噬菌体，霍乱毒素是霍乱病原体霍乱弧菌的主要毒力因子。我们描述了介导CTXF整合到霍乱弧菌染色体并控制CTXF基因表达的新机制。我们还扩大了工作范围，探索了SXT分子生物学的几个方面，SXT是霍乱弧菌衍生的整合接合元件(ICE)，编码多个抗生素耐药基因。近年来，SXT及其密切相关成分在霍乱弧菌临床分离株中高度流行。我们的长期目标是破译CTXF和SXT传播的分子机制。我们建议探索CTXF和SXT控制其传播和进化的5个方面的分子生物学。实验一旨在阐明控制CTXF抑制因子RstR表达的机制，将揭示两个宿主因子lexA和rpos如何参与控制CTXF产生的新的遗传电路。在AIMS II-V中，我们将探索SXT生命周期某些方面的分子基础，这些方面将这种可移动元件与噬菌体和质粒区分开来。研究SXT的这些新特征将增强我们对ICES的理解，ICES是一组广泛的、医学上重要的可移动元件，尚未进行深入研究，并将有助于揭示与SXT相关的ICE在伽马蛋白细菌中的流行和多样性的过程。特别是，我们将探索II)限制其转移到新宿主的SXT接合步骤，III)在供体细胞中维持SXT所需的基因，IV)防止SXT向已存在的宿主的冗余传播的机制，以及V)产生新的/杂交SXT相关ICE的过程。这些研究的完成将加强我们对水平基因转移控制机制的理解，并产生原核生物细胞生物学和病原体进化的基本方面的知识。此外，这些研究可能为创造新的抗菌剂和疫苗提供有价值的信息。

DESCRIPTION (provided by applicant): Mobile genetic elements have profoundly influenced the evolution of bacterial pathogens. Many determinants of virulence and antibiotic resistance are borne by mobile elements, such as plasmids and bacteriophages, which are capable of horizontal transmission. During this grant cycle, we studied several aspects of the life cycle of CTXF, a filamentous phage that encodes cholera toxin, the principal virulence factor of the cholera pathogen Vibrio cholerae. We described novel mechanisms that mediate the integration of CTXF into the V. cholerae chromosome and that govern CTXF gene expression. We also expanded the scope of our work to explore several aspects of the molecular biology of SXT, a V. cholerae-derived integrating conjugative element (ICE) that encodes multiple antibiotic resistance genes. In recent years, SXT and closely related elements have become highly prevalent in V. cholerae clinical isolates. Our long-term goal is to decipher the molecular mechanisms that enable the dissemination of CTXF and SXT. We propose to explore 5 aspects of the molecular biology of CTXF and SXT that govern their transmission and evolution. Experiments in Aim I to elucidate mechanisms that control expression of RstR, the CTXF repressor, will reveal how two host factors, LexA and RpoS, contribute to the novel genetic circuitry governing CTXF production. In Aims II-V, we will explore the molecular bases for aspects of the SXT life-cycle that distinguish this mobile element from phages and plasmids. Studying these novel features of SXT will enhance our understanding of ICEs, an extensive and medically important set of mobile elements that have not been subject to intensive study, and will shed light on processes that have contributed to the prevalence and diversity of SXT-related ICEs in the gamma proteobacteria. In particular, we will explore II) steps in SXT conjugation that limit its transfer to new hosts, III) genes required for SXT maintenance in donor cells, IV) mechanisms for preventing redundant transmission of SXT to hosts in which it is already present and V) processes by which new/hybrid SXT-related ICEs are generated. Completion of these studies will enhance our understanding of the mechanisms that control of horizontal gene transfer and yield knowledge of fundamental aspects of prokaryote cell biology and pathogen evolution. Furthermore, these studies may provide valuable information for the creation of new antimicrobial agents and vaccines.