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 A型产气荚膜梭菌导致80%-90%的气性坏疽(梭状肌坏死)， 这涉及到肌肉感染。如果不及时治疗，气体坏疽几乎是100%致命的，通常发生在- 田鼠截肢。即使接受治疗，这种感染仍会导致67%的糖尿病患者死亡。然而， 感染产气荚膜梭菌的伤口中只有一小部分进展为气性坏疽，这突显了这种感染的严重性。 寄主因素在该病中的重要性。一种公认的气体坏疽的宿主危险因素是1型或2型糖尿病。 产气荚膜梭菌气体坏疽和糖尿病之间的联系的基础还不完全清楚。 然而，我们检测到编码分支的产气荚膜梭菌brnQ2基因的表达强烈上调。 A型菌株与C2C12分化肌肉共培养时的BCAA转运蛋白 细胞。我们推测，这一观察结果有助于解释为什么糖尿病患者更容易患上气体甘油。 格伦。这一假设得到了更多事实的支持。首先，产气荚膜梭菌不能合成自己的支链氨基酸 因此，为了生长，这种细菌必须使用支链氨基酸转运体(S)从宿主那里获得这些氨基酸。第二, 临床研究发现，糖尿病患者(1型和2型)血液中支链氨基酸水平升高，很可能， 肌肉细胞。因此，我们推测，在气性坏疽(尤其是糖尿病患者)期间，产气假单胞菌不断生长。 在肌肉中使用支链氨基酸转运体(S)从血液和/或受毒素破坏的肌肉细胞中提取支链氨基酸。 为了验证我们的假设并确定哪些支链氨基酸转运体(S)对产气荚膜梭菌的生长和 在气体坏疽相关环境中的存活，Aim 1将评估产气假单胞菌在气体中的生长和存活 坏疽相关血液或存在毒素损伤的分化的C2C12肌肉细胞。首先，C. 产气荚膜杆菌A型菌株ATCC3624缺失突变株不能产生与气性坏疽有关的毒素(即， 产气荚膜溶血素O和α毒素)将与野生型ATCC3624进行生长/存活比较， 以及从C2C12细胞释放支链氨基酸的能力。如果活细菌数量和支链氨基酸释放较少 毒素突变后，将对它们进行补充和重新检测，以排除二次突变的影响。到那时，目标1将 利用BrnQ2缺失突变体和互补菌株评估BrnQ2‘S在支链氨基酸吸收中的作用及其重要性 使用血液或毒素损伤的C2C12细胞进行ATCC3624的生长/存活。将进行类似的研究 使用不能产生brnQ或brnQ3的ATCC3624突变体，它们编码另外两个BCAA转运蛋白 产气荚膜假单胞菌。Aim 2将评估BKSdb/db小鼠(一种高血糖的2型糖尿病小鼠模型 (血液中支链氨基酸水平)比正常小鼠更容易发生气性坏疽。然后，这个目标将使用 目的1BrnQ家族零突变体及其互补株鉴定支链氨基酸转运蛋白(S) 是糖尿病小鼠和/或正常小鼠发生气性坏疽的重要因素。如果我们的假设得到验证，并且 参与气性坏疽毒力的支链氨基酸转运体(S)被鉴定，支链氨基酸转运体(S)可能是一种 潜在的靶点是开发可以改善气性坏疽治疗的抑制剂。

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.