Targeting Type 3 Secretion System And Alginate Biosynthesis May Improve Wound Healing In Pseudomonas Aeruginosa Infected Wounds
SAI LAKSHMI RAJASEKH KARNA1, J.Q Nguyen1, S.J Evani1, Q Li-Wu1, P Chen1, A.B Fourcaudot1, J.J Abercrombie1, E.A Sebastian1, P D’Arpa2, K.P Leung1.
1US Army Institute of Surgical Research, JBSA FORT SAM HOUSTON, TX, USA, 2The Geneva Foundation, Tacoma, WA, USA.
Pseudomonas aeruginosa (gram-negative bacterium) is an opportunistic pathogen found in many infected-wounds, impairing their healing. We tested hypothesis that knocking out Pseudomonas genes that are overexpressed during wound infection can cripple pathogen’s ability to cause inflammation and impair healing. We assessed two pathways, Type 3 secretion system (T3SS) and alginate-biosynthesis. We generated single- and double-mutant strains of ExsA (T3SS activator), AlgD (GDP-mannose 6-dehydorogenase of alginate-biosynthesis) and their complementary strains (control strains), evaluated their pathogenicity in rabbit ear full-thickness excision-wound infection model. Strains were inoculated (106CFU) into wounds on post-wounding day-3. After 24hrs, at 5 and 9 days, wounds were harvested to measure viable, total bacterial counts, inflammation (cytokines), and wound healing (epithelial-gap). On day-9 post-infection (mature-biofilm in wild-type (PAO1)), viable counts of exsA‾/algD‾ double-mutant were 100-fold less than counts of single-mutants, wild-type, or double-mutant complement. Compared to wound beds infected with wild-type and control-strains, wound beds infected with mutant, exsA‾, algD‾, or exsA‾/algD‾, produced higher levels of TNF-α, IL1-β, and IL-6 at 24hrs and day-5. However, at day-9, wound beds infected with wild-type, mutant and control strains produced similarly low levels of TNF-α and IL1-β, while IL-6 was elevated in algD‾-infected wound beds. Only strains with both mutations knocked out were more susceptible to macrophage phagocytosis in vitro, were less viable in wound bed, and had less effect on impairment of wound healing. Thus, simultaneous targeting of a gene in T3SS and a gene in alginate-biosynthesis attenuated P. aeruginosa pathogenicity, supporting the possibility of dual-targeted anti-virulence strategy.
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