Article Figures & Data
Figures
- FIG 1
Effect of phoPQ, pmrAB, or phoPQ and pmrAB deletion on Pm resistance in a PAK ΔcprRS cprRS21 background. For this and subsequent figures, Pm resistance experiments were performed twice; if discrepancies were seen, the experiment was performed a third time. Each panel shows a representative experiment, with the results expressed as means from three technical replicates. The error bars represent the standard deviations (SD). The nonitalicized allele name indicates the presence of an episomal version, i.e., an inducible expression strain (ctrl, empty vector control). Results from a PMB plate assay of strains induced with 0.1% l-Ara for 24 h are shown for strain 4537 (ΔphoPQ ΔcprRS cprRS21) with strain 4240 (ΔcprRS cprRS21) as a positive control and strain 4541 (ΔphoPQ ΔcprRS ctrl) as a negative control (A), strain 4472 (ΔpmrAB ΔcprRS cprRS21) with strain 4240 (ΔcprRS cprRS21) as a positive control and strain 4470 (ΔpmrAB ΔcprRS ctrl) as a negative control (B), and strain 4543 (ΔphoPQ ΔpmrAB ΔcprRS cprRS21) with strain 4240 (ΔcprRS cprRS21) as a positive control and strain 4547 (ΔphoPQ ΔpmrAB ΔcprRS ctrl) as a negative control (C).
- FIG 2
Effect of cprA2 deletion on Pm resistance in various PAK strain backgrounds. Shown are the results of a PMB plate assay of strain 4374 (ΔcprA2 ΔphoQ) and strain 4614 (ΔcprA2 ΔphoQ cprA2) induced with 0.1% l-Ara for 24 h, with strain 2326 (ΔphoQ) as a positive control (A), strain 4519 (ΔcprA2 ΔpmrAB pmrAB12) induced with 0.1% l-Ara for 24 h, with strain 2735 (ΔpmrAB pmrAB12) as a positive control (B), and strain 4425 (ΔcprA2 ΔcprRS cprRS21) induced with 0.1% l-Ara for 24 h, with strain 4240 (ΔcprRS cprRS21) as a positive control (C). Empty vector control strains 4497 (ΔcprA2 ΔphoQ pJN105), 4521 (ΔcprA2 ΔpmrAB pJN105), and 4447 (ΔcprA2 ΔcprRS pJN105) had Pm susceptibility values similar to those of the corresponding parental strains (not shown).
- FIG 3
Repair of the cprA1 mutant allele promotes high-level Pm resistance in the PAO1 ΔpmrAB strain expressing pmrAB12 in trans. Shown are the results of a PMB plate assay for strain 4763 (ΔcprA1::cprA+ ΔpmrAB pmrAB12) induced with 0.1% l-Ara for 24 h, with strains 4734 (ΔpmrAB pmrAB12) and 4765 (ΔcprA1::cprA+ ΔpmrAB ctrl) as negative controls.
- FIG 4
Deletion of cprA2 does not affect l-Ara4N modification of lipid A from the PAK strain. (A) Structure of lipid A with baseline hexa-acylation (m/z, 1,617), single l-Ara4N addition (m/z, 1,748), and double l-Ara4N addition (m/z, 1,879). Also shown are the MALDI-TOF spectra of lipid A isolated from strain 1026 (WT) (B), strain 2326 (ΔphoQ) (C), and strain 4374 (ΔcprA2 ΔphoQ) (D).
- FIG 5
Effect of arnC deletion in the PAK ΔcprRS cprRS21 strain. Shown are the results of a PMB plate assay of strain 4581 (ΔarnC ΔcprRS cprRS21) induced with 0.1% l-Ara for 24 h, with strain 4240 (ΔcprRS cprRS21) as a positive control and strain 4586 (ΔarnC ΔcprRS ctrl) as a negative control.
- FIG 6
Two-component regulatory systems mediating resistance to Pm and other CAPs modulate expression of arn and eptA, loci that enable specific lipid A modifications, and cprA, a locus potentially implicated in other LPS modification(s) and vesiculation from the outer membrane.
Tables
- TABLE 1
Strains of P. aeruginosa used in this work
Strain Strain description (source/resistance profile) Reference 1026 PAK laboratory-adapted WT (S. Lory) 65 1027 PAO1 laboratory-adapted WT (S. Lory) 46 1555 PAO1 laboratory-adapted WT (M. Franklin) 66 2114 PAO1 laboratory-adapted WT (C. Manoil) 67 2178 PAO1 laboratory-adapted WT (M. Vasil) 68 1016 Clinical isolate (Aarhus CF patient 2, 1996) 12 1020 Clinical isolate (Copenhagen CF patient 6, 1998) 12 1603 Clinical isolate (Copenhagen CF patient 14, 2003) 12 1611 Clinical isolate (Copenhagen CF patient 15, 2001) 12 4240 PAK ΔcprRS pJN105D::cprR+S21 (GENr) 29 4537 PAK ΔphoPQ ΔcprRS pJN105D::cprR+S21(GENr) This study 4541 PAK ΔphoPQ ΔcprRS pJN105 (GENr) This study 4472 PAK ΔpmrAB ΔcprRS pJN105D::cprR+S21 (GENr) This study 4470 PAK ΔpmrAB ΔcprRS pJN105 (GENr) This study 4543 PAK ΔphoPQ ΔpmrAB ΔcprRS pJN105D::cprR+S21 (GENr) This study 4547 PAK ΔphoPQ ΔpmrAB ΔcprRS pJN105 (GENr) This study 4232 PAK ΔpmrAB ΔcprRS pJN105D::pmrAB12 (GENr) This study 4234 PAK ΔpmrAB ΔcprRS pJN105 (GENr) This study 4343 PAK ΔcprA2 This study 4458 PAK ΔcprA2 pJN105 (GENr) This study 4460 PAK ΔcprA2 pJN105D::cprA2 (GENr) This study 2326 PAK ΔphoQ 24 4374 PAK ΔcprA2 ΔphoQ This study 4614 PAK ΔcprA2 ΔphoQ pJN105D::cprA2 (GENr) This study 4497 PAK ΔcprA2 ΔphoQ pJN105 (GENr) This study 3420 PAK ΔpmrAB pJN105 (GENr) This study 2735 PAK ΔpmrAB pJN105D::pmrAB12 (GENr) 12 4519 PAK ΔcprA2 ΔpmrAB pJN105D::pmrAB12 (GENr) This study 4521 PAK ΔcprA2 ΔpmrAB pJN105 (GENr) This study 4425 PAK ΔcprA2 ΔcprRS pJN105D::cprR+S21 (GENr) This study 4447 PAK ΔcprA2 ΔcprRS pJN105 (GENr) This study 4200 PAK ΔcprRS ΔphoQ pJN105D::cprR+S21 (GENr) 12 4608 PAK ΔcprA2 ΔcprRS ΔphoQ pJN105D::cprR+S21 (GENr) This study 4612 PAK ΔcprA2 ΔcprRS ΔphoQ pJN105 (GENr) This study 4581 PAK ΔarnC ΔcprRS pJN105D::cprR+S21(GENr) This study 4586 PAK ΔarnC ΔcprRS pJN105 (GENr) This study 4734 PAO1 ΔpmrAB pJN105D::pmrAB12 (GENr) This study 4763 PAO1 ΔpmrAB ΔcprA1::cprA+ pJN105D::pmrAB12 (GENr) This study 4765 PAO1 ΔpmrAB ΔcprA1::cprA+ pJN105 (GENr) This study - TABLE 2
Effect of cprA2 deletion in the PAK strain on pmrAB12-induced cationic antimicrobial peptide resistance
Strain/genotype PMBa Protegrin-1 C18G EC50 (mg/liter) FIR EC50 (mg/liter) FIR EC50 (mg/liter) FIR PAK WT <0.125 1 0.125 1 2 1 PAK ΔpmrAB pJN105 <0.125 1 0.125 1 2 1 PAK ΔpmrAB pJN105D::pmrAB12 32 >256 12 96 256 128 PAK ΔcprA2 ΔpmrAB pJN105D::pmrAB12 2 >16 2 16 64 32 ↵a EC50, effective concentration that kills 50% of CFU; FIR, fold increase in resistance to the indicated antimicrobial peptide of each strain with respect to PAK WT (ratio of EC50s).
- TABLE 3
Relative abundance of cprA and arnC mRNA transcripts in phoQ deletion and pmrAB12 or cprRS21 expression backgrounds, determined by RT-qPCR analysis (n = 3 biological replicates)
Genotype Relative mRNA abundance ± SDa cprA arnC PAK ΔphoQ 1 ± 0.4 34 ± 17 PAK ΔpmrAB pJN105D::pmrAB12 94 ± 26 21 ± 7 PAK ΔcprRS pJN105D::cpRS21 165 ± 37 34 ± 18 PAK ΔcprA2 ΔcprRS pJN105D::cpRS21 NA 30 ± 9 PAK ΔarnC ΔcprRS pJN105D::cpRS21 172 ± 70 NA PAK ΔphoQ ΔcprRS pJN105D::cprRS21 236 ± 162 51 ± 40 ↵a Normalized to mRNA abundance in the PAK WT strain. NA, not applicable.
Supplemental material
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Supplemental file 1 -
Supplemental Tables S1 and S2 and Figures S1 to S4.
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Supplemental file 1 -
