Evaluating the role of branched chain amino acid transporters in Clostridium perfringens-induced gas gangrene in diabetic and normal mouse models

项目摘要 产气荚膜梭菌A型菌株引起80-90%的气性坏疽（梭菌性肌坏死）， 包括肌肉感染如果不及时治疗，气性坏疽几乎是100%致命的，这通常在- 切断四肢。即使经过治疗，这种感染仍然导致67%的糖尿病患者死亡。然而，在这方面， 只有一小部分伤口感染了C.产气荚膜杆菌进展为气性坏疽，突出了 宿主因素在该病中的重要性。公认的气性坏疽的宿主风险因素是1型或2型糖尿病。 C.产气荚膜杆菌气性坏疽和糖尿病的认识不完全。 然而，我们检测到C.产气荚膜杆菌brnQ 2基因编码一个分支的 当A型菌株与C2 C12分化的肌肉共培养时， 细胞我们假设，这一观察有助于解释为什么糖尿病患者更容易发展气体肝， grene.这一假设得到了其他事实的支持。首先，C.产气荚膜杆菌不能合成自己的支链氨基酸 因此，为了生长，这种细菌必须使用BCAA转运蛋白从宿主获得这些氨基酸。第二、 临床研究发现，糖尿病患者（1型和2型）血液中的BCAA水平升高， 肌肉细胞因此，我们假设，在气性坏疽（特别是糖尿病），C。产气荚膜杆菌生长 在肌肉中使用BCAA转运蛋白从血液和/或毒素损伤的肌肉细胞中摄取BCAA。 为了验证我们的假设，并确定BCAA转运蛋白（S）是重要的C。产气荚膜杆菌生长和 在气性坏疽相关环境中的存活率，目标1将评估C。产气荚膜杆菌在气体中的生长和存活 坏疽相关血液或毒素损伤的分化C2 C12肌细胞的存在。首先，C. 不能产生涉及气性坏疽的毒素的A型产气荚膜杆菌菌株ATCC 3624无效突变体（即， 产气荚膜梭菌溶素O和α毒素）与野生型ATCC 3624的生长/存活进行比较， 和从C2 C12细胞释放BCAA的能力。如果活菌数量和BCAA释放量较少， 毒素突变体，将对其进行补充并重新测定，以排除继发突变效应。目标1将 使用brnQ 2无效突变体和互补菌株来评估BrnQ 2在BCAA摄取中的作用及其重要性 用于使用血液或毒素损伤的C2 C12细胞的ATCC 3624生长/存活。将进行类似的研究 使用不能产生brnQ或brnQ 3的ATCC 3624突变体，其编码以下的另外两种BCAA转运蛋白： C.产气荚膜杆菌目的2将评估BKSdb/db小鼠（具有高血糖的2型糖尿病小鼠模型）是否 血液BCAA水平）比正常小鼠更容易发生气性坏疽。这个目标将使用 目的1 BrnQ家族无效突变体和互补菌株，以鉴定那些BCAA转运蛋白中的哪一种 是糖尿病和/或正常小鼠气性坏疽的重要促成因素。如果我们的假设得到验证， 鉴定了参与气性坏疽毒力的BCAA转运体，BCAA转运体可能是一种 潜在的抑制剂发展的目标，可以改善气性坏疽治疗。

Project Summary Clostridium perfringens type A strains cause 80-90% of all cases of gas gangrene (clostridial myonecrosis), which involves infection of muscle. Gas gangrene is nearly 100% fatal unless promptly treated, which often in- volves amputation of limbs. Even with treatment, this infection still causes 67% mortality in diabetics. However, only a small percentage of wounds infected with C. perfringens progress to gas gangrene, highlighting the im- portance of host factors in this disease. A well-recognized host risk factor for gas gangrene is type 1 or 2 diabetes. The basis for the association between C. perfringens gas gangrene and diabetes is incompletely understood. However, we detected strongly upregulated expression of the C. perfringens brnQ2 gene encoding a branched chain amino acid (BCAA) transporter when a type A strain was co-cultured with C2C12 differentiated muscle cells. We hypothesize that this observation helps to explain why diabetics are more prone to develop gas gan- grene. This hypothesis is supported by additional facts. First, C. perfringens cannot synthesize its own BCAAs so, to grow, this bacterium must obtain these amino acids from the host using a BCAA transporter(s). Second, clinical studies found that diabetics (both type 1 and type 2) have elevated BCAA levels in their blood and, likely, muscle cells. Therefore we postulate that, during gas gangrene (particularly in diabetics), C. perfringens growing in muscle uses a BCAA transporter(s) to take-up BCAAs from blood and/or toxin-damaged muscle cells. To test our hypothesis and identify which BCAA transporter(s) are important for C. perfringens growth and survival in gas gangrene-relevant environments, Aim 1 will evaluate C. perfringens growth and survival in gas gangrene-relevant blood or the presence of toxin-damaged differentiated C2C12 muscle cells. First, C. perfringens type A strain ATCC3624 null mutants unable to produce the toxins involved in gas gangrene (i.e., perfringolysin O and alpha toxin) will be compared against wild-type ATCC3624 for their growth/survival using, and ability to release BCAAs from, C2C12 cells. If viable bacterial numbers and BCAA release are less for the toxin mutants, they will be complemented and re-assayed to rule out secondary mutation effects. Aim 1 will then use the brnQ2 null mutant and a complementing strain to assess BrnQ2’s role in BCAA uptake and its importance for ATCC3624 growth/survival using blood or toxin-damaged C2C12 cells. Similar studies will be performed using ATCC3624 mutants unable to produce brnQ or brnQ3, which encode the other two BCAA transporters of C. perfringens. Aim 2 will evaluate whether BKSdb/db mice (a mouse model for type 2 diabetes that has high blood BCAA levels) are more susceptible than normal mice for developing gas gangrene. This Aim will then use the Aim 1 BrnQ-family null mutants and complementing strains to identify which of those BCAA transporter(s) are important contributors to gas gangrene in diabetic and/or normal mice. If our hypothesis is verified, and a BCAA transporter(s) involved in gas gangrene virulence is identified, that BCAA transporter(s) could be a potential target for inhibitor development that could improve gas gangrene therapy.