Molecular mechanisms of spermine on its synergistic effect with beta-lactams against Staphylococcus aureus

Spermine (Spm), a potent bactericidal polyamine, exerts a strong synergistic effect with βlactams against methicillin-resistant Staphylococcus aureus (MRSA) in a pH-dependent manner. At high pH (>8) Spm is a potent nucleophile, and able to form Spm-β-lactam adduct. At physiological pH (or lower), Spm carries positive charges, and can bind to DNA through charge interactions. The potential of Spm interfering with cell wall was first investigated. A spontaneous mutant of MRSA Mu50 selected for Spm resistance conferred resistance to Spm/β-lactam synergy. This phenotype was due to the presence of a 7-bp deletion in pbpB as identified by genome resequencing and confirmed by complementation. Analysis of cell wall composition by HPLC revealed the combination of Spm and β-lactam can reduce the cross-linkage of peptidoglycan. These two lines of evidence suggest Spm may perturb cell wall integrity in favor of β-lactam efficacy with PBPs as a promising target. However, from the results of microarray analysis and fluorescent Bocillin labeling, Spm did not appear to suppress the PBPs expression or alter their interactions with β-lactams. Next, transcriptome analyses reveal the genes responsive to the synergy effect overlap extensively with those to high Spm challenge, implying the enhanced detrimental effect of Spm facilitated by β-lactams in inhibition on cell growth. In particular the induction of iron transport and reduction of energy production under synergy were depicted in this study, and high dose Spm was found to turn off the SigB regulon. Of interest, the tetM gene encoding a ribosomal protection protein for tetracycline (Tc) resistance exhibited the most significant fold change and high signals by both high and low dose Spm. Further analysis by qRT-PCR demonstrated the tetM expression was specifically induced by Tc and Spm to a comparable level but not by other polyamines, suggesting a similar mode of action by Spm and Tc in interactions with the ribosome to initiate tetM induction. Collectively, these data indicated the role of Spm could be multifarious with more than one target, and a combination of Spm and β-lactams may inhibit growth of MRSA in a more complicated manner than just potentiating β-lactam inhibition on PBPs. INDEX WORDS: Spermine, MRSA, Synergy, β-lactams MOLECULAR MECHANISMS OF SPERMINE ON ITS SYNERGISTIC EFFECT WITH ΒETA-LACTAMS AGAINST STAPHYLOCOCCUS AUREUS