Pseudomonas aeruginosa (a Gram-negative bacterium) is an opportunistic pathogen found in many infected wounds and is known to impair healing. To test the hypothesis that knocking out P. aeruginosa genes that are overexpressed during wound infection can cripple a pathogen's ability to impair healing, we assessed two pathways: the Type III secretion system (T3SS) and alginate biosynthesis. We generated single- and double-mutant strains of ExsA (T3SS activator), AlgD (GDP- mannose 6-dehydrogenase of alginate biosynthesis) and their complemented strains and evaluated their pathogenicity in a rabbit ear full-thickness excision-wound infection model. Wounds were inoculated with different strains (wild type, mutants, and complementary strains) at 106 CFU/wound on post-wounding day 3. After 24 h, 5 days and 9 days post-infection, wounds were harvested for measuring bacterial counts (viable and total) and wound healing (epithelial gap). On day 9 post-infection, the viable counts of the double mutant, (exsA/algD)‾ were 100-fold lower than the counts of the wild type (PAO1), single mutants, or the complement double-mutant, (exsA/algD)‾/+. Also, when compared to wounds infected with wild type or control strains, wounds infected with the double-knockout mutant was less inhibitory to wound healing (p < 0.05). Additionally, the double mutant showed greater susceptibility to macrophage phagocytosis in vitro than all other strains (p < 0.001). In conclusion, compared to single gene knockouts, double knockout of virulence genes in T3SS pathway and alginate biosynthesis pathway is more effective in reducing P. aeruginosa pathogenicity and its ability to impair wound healing. This study highlights the necessity of a dual-targeted anti-virulence strategy to improve healing outcomes of P. aeruginosa-infected wounds.

中文翻译：

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T3SS和铜绿假单胞菌的藻酸盐生物合成削弱了受感染兔子伤口的愈合。

铜绿假单胞菌（革兰氏阴性细菌）是在许多感染伤口中发现的机会病原体，已知会损害愈合。为了检验假说，剔除在伤口感染过程中过表达的铜绿假单胞菌基因会削弱病原体损害愈合的能力，我们评估了两种途径：III型分泌系统（T3SS）和藻酸盐生物合成。我们产生了ExsA（T3SS激活剂），AlgD（藻酸盐生物合成的GDP-甘露糖6-脱氢酶）及其互补菌株的单突变和双突变菌株，并在兔耳全厚度切除伤口感染模型中评估了它们的致病性。在伤口后第3天以106 CFU /伤口接种不同菌株（野生型，突变体和互补菌株）的伤口，在感染后24小时，5天和9天，收获伤口以测量细菌数（存活和总数）和伤口愈合（上皮间隙）。感染后第9天，双突变体（exsA / algD）〜的存活数比野生型（PAO1）单突变体或补体双突变体（exsA / algD）〜/ +。此外，与野生型或对照菌株感染的伤口相比，双敲除突变体感染的伤口对伤口愈合的抑制作用较小（p <0.05）。此外，与所有其他菌株相比，双突变体在体外对巨噬细胞吞噬作用的敏感性更高（p <0.001）。总之，与单基因敲除相比，T3SS途径和藻酸盐生物合成途径中毒力基因的双重敲除在减少铜绿假单胞菌的致病性及其损害伤口愈合的能力方面更为有效。