The Role of PaFicT in Pseudomonas aeruginosa Persister Cell Formation.

Abstract

The opportunistic pathogen Pseudomonas aeruginosa (Pa) is a major concern for immunocompromised and cystic fibrosis patients. Chronic lung infections caused by Pa are generally considered incurable, in part, due to the bacteria's ability to form persister cells. These variants are categorized as being phenotypically dormant and highly tolerant to antibiotic treatment. Currently, the mechanisms involved in Pa persister cell formation is poorly understood. One promising candidate is the Pa filamentation induced by cAMP (FIC) domain containing toxin (PaFicT), which like other FIC toxins transiently inhibits cell growth. Genetic knockout and complementation by single copy chromosomal insertion was used to characterize paficT involvement in Pa persister cell formation. Toxicity and the PaFicT active site were examined by overexpression of wild-type and mutant protein variants. Antibiotic tolerance of PaFicT-induced Pa persister cells, was measured by minimum inhibitory concentration (MIC) analysis and compared to parental mostly non-persister populations. Deletion of paficT resulted in a 7.2-fold reduction in persister cell formation, which was fully complemented by re-insertion of the gene. Expression of PaFicT significantly increased persister cell formation by 5.9-fold, and this phenotype required a functional FIC active site motif. Unlike growing cell populations, PaFicT-induced persister cells were unaffected by 4 h treatment with 10 × MIC meropenem and showed an increased survival of 6.2 × 10⁵-fold to tobramycin under the same conditions. Alternatively, survival of both persisters and parental, mostly non-persister, populations were below detectable levels following amikacin treatment. Results indicate a potential major involvement of PaFicT in Pa persister cell formation and multidrug tolerance.