γ-Glutamyl Spermine Synthetase PauA2 as Potential Target of Antibiotic

22 23 Polyamines are absolute requirements for cell growth. When in excess, P. aeruginosa possesses 24 six γ-glutamylpolyamine synthetases (GPSs) encoded by the pauA1-pauA7 genes to initiate 25 polyamine catabolism. Recently, the pauA2 mutant was reported to lose the capability to grow on 26 spermine (Spm) and spermidine (Spd) as sole carbon and nitrogen sources. Although this mutant 27 grew normally in the defined minimal medium and the LB broth, growth was completely 28 abolished by the addition of Spm or Spd. These two compounds exert a bactericidal effect (Spm 29 > Spd) on the mutants as demonstrated by MIC measurements (over 500-fold reduction) and 30 time-killing curves. Spm toxicity in the pauA2 mutant was attenuated when the major uptake 31 system was further deleted from the strain, suggesting cytoplasmic targets of toxicity. In 32 addition, the synergistic effect of Spm and carbenicillin in the wild type strain PAO1 was 33 diminished in mutants without functional PauA2. Furthermore, Spm MIC was reduced by 8-fold 34 when deleting the Spm uptake system from the wild type strain, suggesting a second target of 35 Spm toxicity in the periplasm. Experiments were also conducted to test the hypothesis that native 36 Spm and Spd in human serum may be sufficient to kill the pauA2 mutant. Growth of the mutant 37 was completely inhibited by 40% (v/v) human serum while the parental strain required 80%. 38 Colony counts indicated that the mutant but not the parent was in fact killed by human plasma. In 39 addition, carbenicillin MIC against the mutant was reduced by 16-fold in the presence of 20% 40 human serum while that of the parental strain remained unchanged. Taking PauA2 as template, 41 sequence comparison indicates that putative PauA2 homologues are wide spread in a variety of 42 Gram-negative bacteria. In summary, this study reveals the importance of GPS in alleviation of 43 on O cber 3, 2017 by gest httpaac.asm .rg/ D ow nladed fom polyamine toxicity when in excess, and provides strong support to the feasibility of GPS as 44 molecular target for new antibiotic development. 45