Fig 1.
Deletion of ECA synthesis or enterobactin transport pathway genes lower ciclopirox MICs.
(a) Ciclopirox and 1,10-phenanthroline MICs of the E. coli Keio parent strain BW25113 and the isogenic deletion mutant strains. Student’s t-test was used to assess significance of each mutant relative to BW25113 (** p < 0.005). (b) Schematic of the Enterobacterial Common Antigen synthesis pathway (adapted from a combination of [54–56]). The Wec and Rff family proteins assemble the ECA trisaccharide unit, which consists of repeating N-acetyl-D-glucosamine, N-acetyl-D-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-D-galactose on the C55 isoprenoid lipid carrier undecaprenol diphosphate (C55-PP). ECA polymers are assembled using a designated flippase (WzxE) and polymerase (WzyE), the length of which is controlled by a chain-length regulator protein (WzzE). ECA is covalently linked to the phosphoglyceride (ECAPG), in the periplasm in a cyclic form (ECACyc) or is attached to the core LPS by the RfaL protein (ECALPS). (c) Schematic of the Enterobactin Transport Pathway (adapted from a combination of [57–60]). Enterobactin, synthesized by the Ent family proteins, is transported extracellularly by the transport proteins EntS and TolC. The channel protein TolC is complexed with RND transporters AcrAB, AcrAD or MdtABC (not shown). Ironbound enterobactin is then transported back into the bacterial cell through an outer membrane gated channel protein, FepA, a periplasmic protein, FepB, and an inner membrane ABC transporter, FepC-FepD-FepG. FepA is powered by an inner membrane protein complex, TonB-ExbB-ExbD. Once in the cytoplasm, iron is freed from the siderophore by the enterochelin esterase Fes.
Fig 2.
Ciclopirox stimulates enterobactin production.
Catechol siderophore production by the E. coli Keio parent strain BW25113 as measured using a modified Arnow chemical assay. A student’s t-test was used to assess significance of the mutants relative to the isogenic parent strain (**, p <0.005).
Fig 3.
Ciclopirox-mediated enterobactin production is altered by deletion of galE or galT.
(a) Schematic of galactose salvage pathway (adapted from a combination of [27,49,50]). (b) Catechol siderophore production measured using the Arnow chemical assay for the Keio parent BW25113 and select Keio mutant strains with deletions in genes encoding proteins involved in the galactose salvage pathway grown with sub-inhibitory concentrations of ciclopirox in MH medium (c) A repeat of the catechol siderophore production measurement described in (b) for a subset of strains except in the presence of sub-inhibitory concentrations of 1,10-phenanthroline. (d) A repeat of the catechol siderophore production measurement described in (c) except the bacteria were grown in low iron M9 minimal medium with no drug. A student’s t-test was used to assess significance of the mutants relative to the isogenic parent strain (**, p <0.005). The Δfur strain and the ΔentC strain served as positive and negative controls, respectively.
Fig 4.
Ciclopirox-mediated enterobactin production is unaffected by disruption of the ECA synthesis pathway.
Catechol siderophore production was measured as above. A Student’s t-test was used to assess significance of the mutants relative to the isogenic parent strain (**p <0.005). The Δfur strain and the ΔentC strain served as positive and negative controls, respectively.
Fig 5.
Sub-inhibitory concentrations of ciclopirox increase swarming of E. coli strain BW25113.
(a) Representative data. Bacterial cultures were dotted on the surface of LB Eiken agar containing various concentrations of ciclopirox. (b) Swarming on agar containing 2.5 or 5 μg/mL ciclopirox was normalized relative to swarming on agar without ciclopirox. (c) Area of swarming motility for E. coli grown without or with 5 μg/mL ciclopirox or 5 μg/mL 1,10-phenanthroline, as well as without or with 1,000 μM FeCl3, to examine combinatory effects. Student’s t-tests were used to assess statistical significance (*p < 0.05; **p < 0.005).
Fig 6.
Sub-inhibitory concentrations of ciclopirox increase P. aeruginosa swarming.
(a) Representative data of P. aeruginosa strain ATTC 27853 swarming after ~18 or ~42 hours. Yellow indicates pyoverdine production; bluish-green indicates pyocyanin production. (b) Quantification of swarming motility in cm2. A Student’s t-test was used to assess significance of swarming with ciclopirox compared to the absence of ciclopirox (* p < 0.05, **p < 0.005).
Fig 7.
Ciclopirox does not increase swarming or biofilm production in Rcs effector deletion strains.
(a) Bacteria swarming in 0, 2.5, or 5 μg/mL ciclopirox for the ΔrcsA strain (b) Bacteria swarming in 0, 2.5, or 5 μg/mL ciclopirox for the ΔrcsB strain (c) An MTT assay was used to assess absorbance at 560 nm, indicative of biofilm production, for the parent E. coli strain BW25113 and the ΔrcsA or ΔrcsB deletion mutant strains. Student’s t-tests were used to assess significance (* p < 0.05, ** p < 0.005).