New mechanism of resistance to oxazolidinone antibiotics in Staphylococcus aureus

描述(申请人提供)：利奈唑胺是最新的抗生素之一，用于治疗由革兰氏阳性细菌病原体引起的感染。作为恶唑烷酮类抗生素的第一个临床相关代表，它作用于核糖体并抑制敏感细菌的蛋白质合成。利奈唑胺对包括耐甲氧西林金黄色葡萄球菌(MRSA)在内的许多耐药革兰氏阳性细菌显示出活性，并可能成为对抗MRSA引起的感染的最后一道防线，同时降低了对万古霉素的敏感性。到目前为止，全世界只描述了少数耐利奈唑胺的金黄色葡萄球菌菌株。在几乎所有的病例中，耐药与23S核糖体RNA的V区突变有关。由于金黄色葡萄球菌基因组中存在多个拷贝的rRNA基因，这种类型的耐药性非常罕见，而且发展缓慢。这种耐药性不太可能在革兰氏阳性病原体之间水平传播。本研究旨在探讨金黄色葡萄球菌对利奈唑胺耐药的新机制。我们在分析哥伦比亚一家医院分离的耐利奈唑胺MRSA菌株时发现了这一机制。我们的初步研究表明，MRSA对利奈唑胺的耐药性是由位于核糖体中利奈唑胺结合部位的23S rRNA的一个特殊核苷酸A2503的异常转录后修饰所介导的。CFR甲基转移酶的基因存在于哥伦比亚MRSA分离株的染色体上，对A2503进行修饰，使细胞对利奈唑胺产生抗药性。这是第一例在人类病原体中出现CFR基因的病例。核苷酸序列分析表明，CFR与一种可移动的遗传元件有关。因此，该病例是第一例可能水平传播的恶唑烷酮耐药性。依赖于CFR的rRNA修饰不仅可能对利奈唑胺产生耐药性，而且可能对作用于核糖体肽转移酶中心的其他抗生素产生耐药性。这项建议的主要目的是了解这一新的恶唑烷酮耐药机制的调节、功能和传递的基本原理。新获得的知识将是抗击CFR型耐药性的关键。拟议的研究计划包括检查CFR表达的转录和翻译控制，检查使细胞对利奈唑胺产生抗药性所需的核糖体结构变化，表征CFR酶的结构，研究其功能的分子机制，并分析其在金黄色葡萄球菌染色体中的遗传环境。这项研究的结果将为一种新的、具有医学意义的耐药机制的运作提供关键的见解，并可能为开发更好的恶唑烷酮类抗生素以及避免抗生素耐药性的新方法铺平道路。

DESCRIPTION (provided by applicant): Linezolid is one of the newest antibiotics used for treatment of infections caused by Gram-positive bacterial pathogens. The first clinically-relevant representative of the oxazolidinone class of antibiotics, it acts upon the ribosome and inhibits protein synthesis in sensitive bacteria. Linezolid shows activity against many drug- resistant Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and may serve as the last line of defense against infections caused by MRSA with decreased susceptibility to vancomycin. So far, only a few linezolid-resistant S. aureus strains have been described worldwide. In almost all of the cases, the resistance was associated with mutations in domain V of 23S ribosomal RNA. This type of resistance appears rather rarely and develops slowly because of the presence of multiple copies of rRNA genes in the S. aureus genome. Horizontal transmission of this type of resistance between Gram- positive pathogens is unlikely. This proposal is aimed at investigation of a new mechanism of linezolid resistance in S. aureus. We discovered this mechanism while analyzing a linezolid-resistant MRSA strain isolated in a hospital in Colombia. Our preliminary studies indicate that resistance of the MRSA isolate to linezolid is mediated by an unusual posttranscriptional modification of a specific nucleotide, A2503 of 23S rRNA, which is located in the linezolid binding site in the ribosome. Modification of A2503 by Cfr methyltransferase, whose gene is present on the chromosome of the Colombian MRSA isolate, renders cells resistant to linezolid. This is the first case of appearance of the cfr gene in a human pathogen. The nucleotide sequence analysis suggests association of cfr with a mobile genetic element. Therefore, this case represents the first example of potentially horizontally transmittable oxazolidinone resistance. Cfr-dependent modification of rRNA is likely to confer resistance not only to linezolid but to other antibiotics acting upon the ribosomal peptidyl transferase center. The main goal of this proposal is to understand the fundamental principles of regulation, function and transmission of this new mechanism of oxazolidinone resistance. The newly acquired knowledge will be essential for combating the Cfr-type of drug resistance. The proposed research plan includes examination of transcriptional and translational control of cfr expression, examination of structural changes in the ribosome that are required to render cells resistant to linezolid, characterization of the structure of the Cfr enzyme, study of the molecular mechanisms of its function and analysis of its genetic environment in the S. aureus chromosome. The results of this study should provide critical insights into the operation of a new, medically- significant, mechanism of resistance to one of the newest antibiotics and may pave the way to developing better oxazolidinone antibiotics as well as newer ways for evading antibiotic resistance.