Mechanisms of Staphylococcal Co-Resistance to Daptomycin and Host Defense Peptide

描述（由申请人提供）：金黄色葡萄球菌（SA）引起多种临床综合征，并且是全世界血管内感染的主要原因。 SA 特别容易产生多重耐药性，此类菌株的严重感染会导致死亡率增加。自 2003 年 FDA 批准以来，达托霉素 (DAP) 已在许多临床环境中使用，特别是用于顽固性耐甲氧西林 SA (MRSA) 感染。最近有许多关于临床 SA 菌株在 DAP 治疗方案失败的情况下进化出体外 DAP 抗性的报道，特别是在血管内感染中。 DAP-R 菌株的一个一致特征是在相对有限的基因核心中获得一个或多个“功能获得”突变，尤其是 mprF（多肽抗性因子基因）。该基因负责 SA 独特的带正电磷脂 (PL)、赖氨酰磷脂酰甘油 (L-PG) 在其细胞膜 (CM) 内的合成和易位（“翻转”）。因此，mprF 对 SA 的相对正表面电荷有很大贡献。此外，鉴于 DAP 的细菌致死率对钙结合的绝对要求，影响表面电荷的基因（例如 mprF）很可能通过电荷排斥而在 DAP-R 中发挥重要作用。临床和体外衍生的 DAP-R SA 菌株的一个重要特征是 DAP 和阳离子宿主防御肽 (HDP) 之间频繁的交叉耐药性 (13,15,17-19)。因此，我们的中心假设是，MRSA 中 DAP-R 的发展经常与 HDP 耐药性的共同进化相关，并且这一事件影响血管内发病机制和体内治疗结果。我们将解决一些重要问题：i) mprF 功能获得性突变多久伴随 DAP-HDP 交叉耐药表型？ ii) 此类 mprF 突变是否偏向于该基因的合酶或翻转酶结构域，它们是否导致交叉耐药性？； iii) 表现出 DAP-HDP 交叉耐药表型的肽之间是否存在共有的 HDP 特异性结构特征？ iv) 金黄色葡萄球菌菌株对 DAP 和/或 HDP 的时间暴露“时间表”是否会影响交叉耐药性的发展？ vi) mprF 突变和 DAP-HDP 交叉耐药性对先天毒力和 DAP 治疗反应的体内后果是什么？我们预计这些研究将有助于更深入地了解我们的先天宿主防御系统与外源性施用抗菌药物在刺激 SA 适应性生存反应中的相互作用。这应该能够实现未来新型抗 SA 药物的“智能设计”，从而规避这种适应性反应。

DESCRIPTION (provided by applicant): S. aureus (SA) causes a wide spectrum of clinical syndromes, and is the leading cause of endovascular infections world-wide. SA has a particular propensity to develop multi-drug resistance, and serious infections with such strains result in enhanced attributable mortalities. Since FDA approval in 2003, daptomycin (DAP) has been utilized in many clinical settings, especially for recalcitrant methicillin-resistant SA (MRSA) infections. There have been numerous recent reports of clinical SA strains that have evolved in vitro DAP-resistance in the context of failing DAP treatment regimens, especially in endovascular infections. One consistent feature of DAP-R strains is the acquisition of one or more "gain-in-function" mutations in a relatively restricted cadre of genes, especially mprF (multiple peptide resistance factor gene). This gene is responsible for the synthesis and translocation ("flipping") of the SA-unique, positively-charged phospholipid (PL), lysyl-phosphotidylglycerol (L-PG) within its cell membrane (CM). Thus, mprF contributes substantially to the relative positive surface charge of SA. Moreover, in light of the absolute requirement for calcium association for DAP's bacterial lethality, genes such as mprF that impact surface charge are highly likely to be important in DAP-R, potentially via charge repulsion. A seminal feature of both clinical and in vitro-derived DAP-R SA strains is the frequent cross-resistance between DAP and cationic host defense peptides (HDPs) (13,15,17-19). Thus, our central hypothesis is that the development of DAP- R in MRSA is frequently associated with the co-evolution of HDP resistance, and this event impacts endovascular pathogenesis and treatment outcomes in vivo. We will address a number of important questions: i) how often do mprF gain-in-function mutations accompany DAP-HDP cross-resistance phenotypes?; ii) are such mprF mutations biased towards the synthase or flippase domains of this gene, and are they causal in cross-resistance?; iii) are there HDP- specific structural features that are shared amongst those peptides which exhibit the DAP-HDP cross-resistance phenotype?; iv) does the temporal exposure "schedule" of S. aureus strains to DAP and/or HDPs influence the development of cross-resistance?; and vi) what are the in vivo consequences of mprF mutations and DAP-HDP cross resistances upon innate virulence and responses to DAP therapy? We anticipate that these studies will contribute to a deeper understanding of the interactive role of our innate host defense system with exogenously administered antimicrobials in stimulating the adaptive survival response in SA. This should enable 'smart design' of future novel anti-SA agents that circumvent this adaptive response.