Iron-regulated expression of alginate production, mucoid phenotype, and biofilm formation by Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa strains of non-cystic fibrosis (non-CF) origin do not produce significant amounts of extracellular alginate and are nonmucoid. In CF, such isolates can become mucoid through mutation of one of the genes (mucA, mucB, mucC, or mucD) that produce regulatory factors that sequester AlgU, required for increased expression of alginate genes. Mutation of the muc genes in the nonmucoid PAO1, PA14, PAKS-1, and Ps388 strains led to increased levels of extracellular alginate and an obvious mucoid phenotype, but only under iron-limiting growth conditions (≤5 µM), not under iron-replete conditions (≥10 µM). In contrast, >50% of P. aeruginosa isolates from chronic CF pulmonary infections expressed increased levels of alginate and mucoidy both under iron-limiting and iron-replete conditions (i.e., iron-constitutive phenotype). No single iron regulatory factor (e.g., Fur, PvdS) was associated with this loss of iron-regulated alginate expression and mucoidy in these CF isolates. However, the loss of only pyoverdine production, or its uptake, abrogated the ability of P. aeruginosa to produce a robust biofilm that represents the Psl-type of biofilm. In contrast, we show that mutation of the pyoverdine and pyochelin biosynthesis genes and the pyoverdine receptor (FpvA) lead to iron-constitutive expression of the key alginate biosynthesis gene, algD, and an explicitly mucoid phenotype in both iron-limiting and iron-replete conditions. These data indicate that alginate production and mucoidy, in contrast to other types of biofilms produced by P. aeruginosa, are substantially enhanced under iron limitation. These results also have compelling implications in relation to the use of iron chelators in the treatment of P. aeruginosa CF infections.

Importance: Pseudomonas aeruginosa is a leading model for the investigation of biofilms. While data have been generated about the role of iron in alginate-independent (Psl/Pel) biofilm development, there is a paucity of data regarding the role of iron in alginate production and its associated mucoid phenotype. We demonstrate that biologically relevant levels of iron that exist in the airway mucus of cystic fibrosis (CF) patients have a substantial influence on production of alginate and the overt mucoid phenotype, pathognomonic of P. aeruginosa infections in CF. Mucoid mutants of non-CF P. aeruginosa isolates are mucoid only under iron limitation and do not express increased levels of alginate under iron-replete growth conditions. However, a significant number of long-term CF isolates lost their iron-regulated expression of increased alginate production and mucoidy and became iron constitutive for these properties. In contrast to the formation of Psl-type biofilms, increasing iron limitation ultimately leads to an iron-constitutive expression of alginate and mucoidy.

FIG 1

Effect of iron on alginate production by muc mutants of P. aeruginosa PAO1. (A) P. aeruginosa PAO1 ∆mucA mutant was grown on DTSA with or without 100 µM FeCl₃ and imaged after 48 h. (B) The PAO1 strain and ∆mucA, ∆mucB, ∆mucC, and ∆mucD mutants were grown on DTSA containing 5, 10, 25, or 100 µM FeCl₃ for 48 h, and the levels of extracellular alginate were evaluated using a uronic acid assay (see Materials and Methods). Values that are significantly different from the value for PAO1 (wild type [WT]) by Student’s t test are indicated by asterisks as follows: ***, P < 0.0005; **, P < 0.005; *, P < 0.05. (C) Relative expression of algD and algG in wild-type PAO1 and PAO1 ∆mucA mutant was analyzed using qRT-PCR after 24 h of growth on DTSA with and without 100 µM FeCl₃. Values that are significantly different from the value for PAO1 (WT) by Student’s t test are indicated by an asterisk.

FIG 2

Effects of iron levels on alginate production in ∆mucA and ∆mucB mutants of P. aeruginosa PA14, PAKS-1, and Ps388. Alginate production was evaluated by measurement of extracellular uronic acid levels produced by PA14, PAKS-1, and Ps388 with insertion mutations in either mucA or mucB after 48 h of growth on DTSA with and without 100 µM FeCl₃. Values that are significantly different from the value found with 100 µM FeCl₃ by Student’s t test are indicated by asterisks as follows: **, P < 0.005; *, P < 0.05.

FIG 3

Influences of various iron sources on alginate production by P. aeruginosa PAO1 and ∆mucA and ∆mucB mutants. The strains were grown on DTSA supplemented with various sources and concentrations of iron-carrying compounds. Extracellular alginate production was measured by uronic acid assay after 48 h of growth. Values that are significantly different from the value for PAO1 (WT) by Student’s t test are indicated by asterisks as follows: ***, P < 0.0005; **, P < 0.005.

FIG 4

Alginate production and mucoidy of late CF isolates (Late-CFI) in response to variable iron levels. (A) Late-CFI were grown on DSTA with and without 100 µM FeCl₃ and imaged at 48 h. (B) The level of extracellular alginate produced by various Late-CFI grown on DSTA with and without 100 µM FeCl₃ was measured by uronic acid assay at 48 h. Values that are significantly different from the value for late CF isolate 4 (Late-CFI-4) or Late-CFI-7 with 100 µM FeCl₃ by Student’s t test are indicated by asterisks as follows: **, P < 0.005; *, P < 0.05. (C) Relative expression of algD and algG of Late-CFI-1 and Late-CFI-4 was analyzed using qRT-PCR after 24 h of growth on DSTA with and without 100 µM FeCl₃. Values that are significantly different (P < 0.05) from the value for Late-CFI-4 with 100 µM FeCl₃ by Student’s t test are indicated by an asterisk.

FIG 5

Investigation of alginate production in iron acquisition mutants of P. aeruginosa PAO1. (A) P. aeruginosa PAO1 and several isogenic iron acquisition mutants were grown on DTSA with and without 50 µM FeCl₃ and imaged after 48 h. (B) Relative expression of algD in PAO1 (wild type) and several isogenic iron acquisition mutants were analyzed using qRT-PCR after 24 h of growth on DTSA with and without 50 µM FeCl₃. Values that are significantly different from the value for PAO1 (WT) by Student’s t test are indicated by asterisks as follows: ***, P < 0.0005; **, P < 0.005; *, P < 0.05.

FIG 6

Investigation of the role of Fur in alginate production. (A) P. aeruginosa PAO1, ∆mucA mutant, Early-CFI-1, Late-CFI-1, and Late-CFI-4 were grown on DTSA with and without 100 µM FeCl₃. The relative expression of fur was measured by qRT-PCR after 24 h of growth. (B) Strain PAO1, ∆mucA mutant, Early-CFI-1, Late-CFI-1, Late-CFI-2, and Late-CFI-4 were grown on DTSA with and without 100 µM FeCl₃. Fur protein levels (shown as relative intensities within parentheses below the blot) were analyzed by Western blotting and quantified as described in Materials and Methods.