3.4 DISCUSSION
3.4.5 The working model
Shown in Fig. 3.4.1 is a possible working model that attempts to explain how PhoU mutation can lead to polyP overaccumulation and counteracts spermine toxicity in P. aeruginosa. During growth in the presence of low phosphate levels, PhoR can phosphorylate PhoB, and the phosphorylated PhoB in turns activates the Pho regulon, including the phosphate transporter, pstSCAB. When phosphate is in excess, PhoU may prevent the phosphorylation of PhoB by PhoR, and thereby the induction of the Pho regulon. The PhoU599 mutant might have lost its regulatory ability due to the C599T substitution, therefore leading to constitutive expression of Pho regulon. Under the spermine stress conditions tested in this study, the phoU gene serves as a hotspot for mutations that cause constitutive induction of the Pho regulon. This observation suggests that PhoU plays a negative regulatory role in control of the Pho regulon. An elevated level of the PstSCAB transporter may increase the uptake of phosphate ions from the medium, possibly leading to increased ATP synthesis. However, the presence of polyphosphate granules in the phoU mutants suggests a significant flow of ATP into polyphosphate synthesis, which could consequently result in ATP deficiency. The hemostasis of polyphosphate is controlled by two enzymes, PPK for synthesis and PPX for degradation. We demonstrated that increasing PPX expression reduces the formation of polyphosphate granules and reverses the phenotypeof the phoU mutants from the spermine-resistant to spermine-sensitive. While more work is needed to elucidate the molecular mechanism of enhanced polyphosphate granule formation, findings of this study support the hypothesis that spermine toxicity can be attenuated by polyphosphate granules through charge neutralization.
In summary, without the main detoxification enzyme PauA2, P. aeruginosa seems to pay a high metabolic price for the development of suppressors against spermine stress. Therefore, the
polyamine pathway might still be a good target for drug discovery to enhance efficacy of β- lactam antibiotics. Many reports indicate the involvement of phoU and polyphosphate on various aspects in bacterial physiology, including persister formation, virulence, and tolerance to
multiple antibiotics and stresses (Amado & Kuzminov, 2009; Y. Li & Zhang, 2007; Proctor et al., 2014). It has been reported that endogenous polyamines can affect polyphosphate
accumulation in E. coli (Kei Motomura, 2006). The results of our study further support the physiological significance of interactions between polyamine and polyphosphate.
Figure 3.4.1 Schematic representations of a working model for polyphosphate accumulation against spermine toxicity.
PstS, phosphate binding protein; PstABC: the phosphate ABC transporter; PhoBR, the phosphate two-component regulator and sensor proteins; SpuE, spermine/spermidine binding protein; SpuFGH, the spermine/spermidine ABC transporter; PPK, polyphosphate kinase; PPX, exopolyphosphate phosphatase. PolyP, polyphosphate
SpuE SpuF SpuF SpuG Spu H spm H+ ATP Synthetase
Periplasm
Cytoplasm
Pi v P st A P st C PstS PstB PstB PhoU P h o R P h o R PhoB PhoB PiPho Regulon
pstSCAB-phoU phoBR phnCDEFGHIJKLMNOP vreAIR ATP ADP (PolyP)n (PolyP)n+1PPK
PPX
Pi Pi Pi Pi P Pi spm Spermine spm PolyP spm spm spm spm spm spmREFERENCES
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4 APPEDEX