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

评估支链氨基酸转运蛋白在糖尿病和正常小鼠模型中产气荚膜梭菌诱导的气性坏疽中的作用

• 批准号: 10726306
• 负责人: Jihong Li
• 金额: $ 20.53万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-14 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10726306
• 关键词: Amino Acid Transporter; Amino Acids; Anaerobic Bacteria; Bacteria; Biological Assay; Blood; Branched-Chain Amino Acids; Cell Line; Cells; Clinical Research; Clostridium perfringens; Clostridium perfringens theta-toxin; Coculture Techniques; Complement; Development; Diabetes Mellitus; Diabetic mouse; Disease; Enterotoxemia; Environment; Family; Food Poisoning; Gas Gangrene; Gene Expression; Genes; Growth; Human; In Vitro; Individual; Infection; Insulin-Dependent Diabetes Mellitus; Integration Host Factors; Lead; Mediating; Modeling; Mus; Muscle; Muscle Cells; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Nutrient; Pathology; Predisposition; Production; Protein Biosynthesis; Risk Factors; Role; Soft Tissue Infections; Testing; Time; Toxin; Up-Regulation; Virulence; Wound Infection; alpha Toxin; bacteria classification; diabetic; enteritis; fascinate; improved; in vivo; inhibitor; limb amputation; lysin; mortality; mouse model; mutant; novel; pathogen; uptake; wound

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.

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。但是，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。第一，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。第一，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， 鉴定了与气性坏疽毒力有关的BCAA转运蛋白，BCAA转运蛋白可能是一种与气性坏疽毒力有关的BCAA转运蛋白。 潜在的抑制剂发展的目标，可以改善气性坏疽治疗。