The mechanism of the beta-lactam resistance in Staphylococcus aureus

项目摘要 细胞壁靶向β-内酰胺抗生素是最大的一类抗菌剂， 是治疗细菌感染的主要药物。然而，大多数β-内酰胺抗生素不能用于 治疗耐甲氧西林金黄色葡萄球菌（MRSA）感染，因为MRSA产生一种细胞- 壁合成酶PBP 2A，大多数β-内酰胺不能降解。虽然PBP 2A是主要的 作为MRSA β-内酰胺耐药性的决定因素，MRSA的总体β-内酰胺耐药性受以下因素的影响： 另外的细菌因子如FtsH，细胞膜中的蛋白酶。FtsH的过表达使得 对β-内酰胺类药物敏感的MRSA。FtsH的β-内酰胺致敏作用是由于其 YpfP的降解，YpfP是合成脂磷壁酸（LTA）的锚分子的酶。YpfP- FtsH的降解导致产生异常大的LTA。另一方面，产量增加 通过缺失ftsH基因使正常的LTA减少，使MRSA对β-内酰胺类抗生素更具耐药性。当生产 通过抑制LTA合成酶LtaS的产生来抑制正常LTA的合成，MRSA成为 对β-内酰胺过敏基于这些结果，本研究假设，大的LTA降低了 通过抑制细胞壁合成酶的酶活性来产生MRSA β-内酰胺耐药性 而正常的LTA是他们所需要的。即使在β-内酰胺致敏条件下，MRSA也可以 恢复对β-内酰胺类药物的耐药性。对26个突变体的基因组测序表明，16个基因是关键的 耐甲氧西林金黄色葡萄球菌β-内酰胺耐药性本项目的长期目标是开发新型β-内酰胺增效剂 抗MRSA本研究的目的是了解FtsH和干扰在LTA合成中的作用 使MRSA对β-内酰胺类药物敏感，以及MRSA如何恢复β-内酰胺类药物耐药性。在目标1中，使用MRSA菌株 产生大的LTA或减少量的正常LTA，细胞壁合成步骤受 将确定LTA分子。LTA对细胞壁合成的影响将通过一个直接测试。 肽聚糖合成测定。此外，细菌双杂交分析将确定降解是否 YpfP的表达受FtsH结合蛋白的调节。在目标2中，通过在细胞中产生每种抗性突变， 在MRSA的染色体上，将确定对MRSA β-内酰胺耐药至关重要的突变。还有， 转座子介导的突变将鉴定MRSA恢复β-内酰胺抗性所必需的基因。 最后，为了验证这16个基因的功能是否在S.金黄色葡萄球菌菌株，耐药 将从另外两种MRSA菌株中鉴定出突变株，并对它们的基因组进行测序。完成 这项研究将揭示MRSA中β-内酰胺耐药调节因子的复杂网络， 本发明涉及开发新的β-内酰胺增效剂， MRSA感染。这些药物有望减少抗生素耐药性的负担。

Project Summary Cell-wall targeting beta-lactam antibiotics are the largest group of antibacterial agents and have been the mainstay for treatment of bacterial infections. However, most beta-lactam antibiotics cannot be used for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections because MRSA produces a cell- wall synthesis enzyme PBP2A, which most beta-lactams cannot inactivate. Although PBP2A is the primary determinant of MRSA beta-lactam resistance, the overall beta-lactam resistance of MRSA is affected by additional bacterial factors such as FtsH, a protease in the cell membrane. Overexpression of FtsH makes MRSA sensitive specifically to beta-lactams. The beta-lactam-sensitizing effect of FtsH is due to its degradation of YpfP, an enzyme synthesizing the anchor molecule for lipoteichoic acid (LTA). The YpfP- degradation by FtsH causes the production of abnormally large LTA. On the other hand, increased production of normal LTA by deletion of the ftsH gene makes MRSA more resistant to beta-lactams. When the production of normal LTA is suppressed by inhibiting the production of the LTA synthesis enzyme LtaS, MRSA becomes hypersensitive to beta-lactams. Based on these results, this study hypothesizes that the large LTA lowers MRSA beta-lactam resistance by suppressing the enzyme activities of the cell-wall synthesis enzymes whereas the normal LTA is required for them. Even under the beta-lactam sensitizing conditions, MRSA can regain resistance to beta-lactams. Genome-sequencing of 26 such mutants showed that 16 genes are critical for MRSA beta-lactam resistance. The long-term goal of this project is to develop novel β-lactam potentiators against MRSA. The objective of this study is to understand how FtsH and the disturbance in LTA synthesis sensitize MRSA to β-lactams, and how MRSA regains β-lactam resistance. In aim 1, with the MRSA strains producing either the large LTA or reduced amount of the normal LTA, the cell wall synthesis steps affected by the LTA molecules will be determined. The effect of LTA on cell wall synthesis will be directly tested by a peptidoglycan-synthesis assay. Also, the bacterial two-hybrid analysis will determine whether the degradation of YpfP is modulated by FtsH-binding proteins. In aim 2, by generating each of the resistance mutations in the chromosome of MRSA, the mutations critical for MRSA β-lactam resistance will be determined. Also, transposon-mediated mutagenesis will identify the genes essential for MRSA to regain the β-lactam resistance. Finally, to test whether the roles of the 16 genes are universally conserved among S. aureus strains, resistant mutants will be identified from two more MRSA strains, and their genomes will be sequenced. Completion of this study will reveal the intricate network of the beta-lactam resistance regulators in MRSA and open the door to developing novel beta-lactam potentiators with minimal risk of resistance development in the treatment of MRSA infections. Such drugs are expected to reduce the burdens of antibiotic resistance.