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

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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.