Membrane alterations and daptomycin tolerance

 描述(申请人提供)：在过去的二十年里，医院一直在与一个重大危机作斗争：抗药性细菌的增加。除了这场危机，还有 在开发治疗这些感染的新疗法方面收效甚微。然而，药物达托霉素最近被批准用于治疗耐药的革兰氏阳性细菌。虽然只在医院环境中使用，但在达托霉素获准使用后两年内就发现了耐药细菌。通常情况下，细菌通过耐药盒的基因交换而产生抗药性；然而，这些对达托霉素耐药的细菌并没有获得新的基因，而是在涉及细胞膜形成或修饰的基因上发生了突变。因此，膜的改变导致了抗性的产生。细菌病原体粪肠球菌是医院获得性感染的主要原因，它可以结合宿主体液中的脂肪酸，进而导致对达托霉素的耐药性增加。这不是由于基因突变，而是由于脂肪酸来源生长时发生的生理变化。此外，达托霉素耐药性的增加并不是由于先前描述的通路激活所致。这项建议旨在解决改变膜成分如何导致粪肠球菌对达托霉素敏感性降低的问题。具体地说，它将研究不同的脂肪酸如何影响达托霉素的耐药性，以及膜成分改变如何影响与抗生素的相互作用(目标1，3)。这项提案还将评估新发现的脂肪酸结合所需蛋白质的作用，以及这些蛋白质如何 有助于改变达托霉素的敏感性。随着脂肪酸来源的增加导致磷脂心磷脂水平的增加，将进行实验，以确定哪种因素、脂肪酸组成或磷脂含量对达托霉素敏感性降低的影响最大(AIMS 2，3)。这项分析将与对野生型和特定基因突变菌株的整体转录和代谢组谱分析相结合，这些菌株在有或没有脂肪酸的情况下培养，并在接触达托霉素时(目标3)。总之，这些研究将阐明脂肪酸和磷脂的变化如何影响对达托霉素的敏感性，以及脂肪酸转运抑制剂用于抗生素开发的潜在用途。

 DESCRIPTION (provided by applicant): Within the past two decades, hospitals have been combating a major crisis: a rise in antibiotic resistant bacteria. To add to this crisis, there has been little success in the development of new therapeutics to treat these infections. The drug daptomycin, however, was recently approved to treat antibiotic-resistant Gram-positive bacteria. Although only used in hospital settings, resistant bacteria were discovered within two years after daptomycin was approved for use. Oftentimes, bacteria become resistant to antibiotics by genetic exchange of drug resistance cassettes; however, these daptomycin resistant bacteria did not acquire new genes but instead had mutations in genes involved in cell membrane formation or modification. Thus, membrane alterations contribute to resistance. The bacterial pathogen Enterococcus faecalis, a major cause of hospital-acquired infections, can incorporate fatty acids from host fluids which in turn leads to increased daptomycin resistance. This is not due to genetic mutation but rather physiological changes that occur upon growth in fatty acid sources. Furthermore, this increased daptomycin resistance is not due to activation of previously described pathways. This proposal is designed to address how altered membrane compositions leads to decreased sensitivity of E. faecalis to daptomycin. Specifically, it will examine how different fatty acids impact daptomycin resistance and how altered membrane composition influences interactions with the antibiotic (Aim 1, 3). This proposal will also evaluat the roles of newly identified proteins required for fatty acid incorporation and how these proteins contribute to alterations in daptomycin sensitivity. As growth in fatty acid sources leads to increased levels of the phospholipid cardiolipin, experiments will be performed discern which factor, fatty acid composition, or phospholipid content, contributes the most to decreased sensitivity to daptomycin (Aims 2, 3). This analysis will be combined with whole transcriptomic and metabolomic profiling on wild type and specific genetic mutant strains cultured in the presence and absence of fatty acids and upon exposure to daptomycin (Aim 3). Together, these studies will elucidate how fatty acid and phospholipid alterations can impact sensitivity to daptomycin and the potential use of fatty acid transport inhibitors for antibiotic development.