Abstract
Bacterial persisters are phenotypic variants that tolerate exposure to lethal antibiotics. These dormant cells are responsible for chronic and recurrent infections. Multiple mechanisms have been linked to persister formation. Here, we report that a complex, consisting of an extracellular poly(dC) and its membrane-associated binding protein RmlB, appears to be associated with persistence of the opportunistic pathogen Pseudomonas aeruginosa. Environmental stimuli triggers a switch in the complex physiological state (from poly(dC)/RmlB to P-poly(dC)/RmlB or RmlB). In response to the switch, bacteria decrease proton motive force and intracellular ATP levels, forming dormant cells. This alteration in complex status is linked to a (p)ppGpp-controlled signaling pathway that includes inorganic polyphosphate, Lon protease, exonuclease VII (XseA/XseB), and the type III secretion system. The persistence might be also an adaptive response to the lethal action of the dTDP-l-rhamnose pathway shutdown, which occurs due to switching of poly(dC)/RmlB.
Highlights
We found that a routine procedure for washing cells increased persister levels 102to 103-fold in exponential (i.e., 6 h) and stationary (i.e., 18 h) doses of antibiotics and have been suggested as causative agents of (hereafter called (e) and (s)) wild-type (WT) P. aeruginosa PAO1, chronic and recurrent infections[1]
R tains inorganic polyphosphate (PolyP), Lon protease, exonuclease versely, (e) and (s) supplementation with extracellular single-stranded DNA (ssDNA) extracts recovered the regulatory activities associated with the DNA-free supernatants (Supplementary Fig. 3f). These results suggest a crucial role for (e) and (s) extracellular ssDNA in washing-induced persister formation
Cy5-labeled double-stranded DNA, but did exhibit single strand- (50.5 kDa; locus tag, PA3777), in vitro binds His-tagged XseB to specific 5′ → 3′ cleavage activity, which resulted in the rapid form a complex that can be trapped by Ultracel-30K Centrifugal disappearance of fluorescence in both free and RmlB-bound Filter Units (30-kDa cutoff size) (Supplementary Fig. 9c)
Summary
We subsequently observed that alkaline phosphatase treatment of the resuspended cells neutralized persistence enhancement, suggesting that the elevated persistence levels are associated with the formation of P-poly(dC)/RmlB (Supplementary Fig. 9a). We observed the expected stimulation filtrate, we observed only one potential in-situ switch related of persistence following addition of His-tagged XseB to (s) protein, which has been annotated as a putative exonuclease VII WT cells (Fig. 3b).
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