Expression of Pseudomonas aeruginosa CupD fimbrial genes is antagonistically controlled by RcsB and the EAL-containing PvrR response regulators

Abstract

Pseudomonas aeruginosa is a gram-negative pathogenic bacterium with a high adaptive potential that allows proliferation in a broad range of hosts or niches. It is also the causative agent of both acute and chronic biofilm-related infections in humans. Three cup gene clusters (cupA-C), involved in the assembly of cell surface fimbriae, have been shown to be involved in biofilm formation by the P. aeruginosa strains PAO1 or PAK. In PA14 isolates, a fourth cluster, named cupD, was identified within a pathogenicity island, PAPI-I, and may contribute to the higher virulence of this strain. Expression of the cupA genes is controlled by the HNS-like protein MvaT, whereas the cupB and cupC genes are under the control of the RocS1A1R two-component system. In this study, we show that cupD gene expression is positively controlled by the response regulator RcsB. As a consequence, CupD fimbriae are assembled on the cell surface, which results in a number of phenotypes such as a small colony morphotype, increased biofilm formation and decreased motility. These behaviors are compatible with the sessile bacterial lifestyle. The balance between planktonic and sessile lifestyles is known to be linked to the intracellular levels of c-di-GMP with high levels favoring biofilm formation. We showed that the EAL domain-containing PvrR response regulator counteracts the activity of RcsB on cupD gene expression. The action of PvrR is likely to involve c-di-GMP degradation through phosphodiesterase activity, confirming the key role of this second messenger in the balance between bacterial lifestyles. The regulatory network between RcsB and PvrR remains to be elucidated, but it stands as a potential model system to study how the equilibrium between the two lifestyles could be influenced by therapeutic agents that favor the planktonic lifestyle. This would render the pathogen accessible for the immune system or conventional antibiotic treatment.

Figure 1. Genetic organization and proposed signaling mechanism.

A) Genetic organization of the cupD/rcs/pvr locus. cupD1, cupD2 and cupD3 encode the major fimbrial subunit, a chaperone and an usher respectively, whereas cupD4 and cupD5 encode a putative adhesin and a second chaperone. rcsC and pvrS encode putative two-component sensors, and rcsB and pvrR are predicted to encode their cognate response regulators. B) Left: Domain organization and signaling mechanism of the RocS1/A1/R two-component system that controls expression of the cupB and cupC gene clusters. Right: Domain organization and proposed signaling mechanism of the Rcs and Pvr two component systems that control expression of the cupD gene cluster. RocS1 and RcsC are unorthodox sensors with a transmitter domain, a receiver domain and an Hpt phosphorelay. PvrS is a hybrid sensor that requires an external Hpt module to relay the signal to the response regulator. Like RocA1, RcsB is likely to activate transcription directly via its helix-turn-helix motif that allows DNA binding. Conversely, RocR and PvrR are likely to repress transcription indirectly via the EAL domain, which degrades the second messenger cyclic-di-GMP.

Figure 2. Overexpression of rcsB induces cupD gene expression.

Growth and β-galactosidase activity of PA14::cupD1-lacZ/pBBR1MCS-5 (black diamonds and bars, respectively) and PA14::cupD1-lacZ/pBBR1MCS-5-rcsB (white diamonds and bars, respectively).

Figure 3. Overexpression of rcsB leads to the production of CupD1 and assembly of CupD fimbriae.

Western blot of fimbrial protein using a CupD1 antibody. 1) PA14/pBBR1MCS-5-rcsB, 2) PA14/pBBR1MCS-5 and 3) PA14::rcsC. Upper panel: Whole cell extract. Lower panel: Sheared extracellular fimbriae as described in Materials and Methods.

Figure 4. Biofilm formation phenotypes in PA14 and derivative strains.

Biofilms were grown in 24 well plates and stained with crystal violet (CV). A) CV stained biofilms after 6 h incubation. PA14 or PA14ΔcupD carrying pBBR1MCS-5 (vector), pBBR1MCS-5-rcsB (rcsB) or pBBR1MCS-4-pvrR (pvrR) (only for PA14) are shown. B) and C) Quantification of biofilm formation at different times (2 to 24 hours) using crystal violet staining. PA14 wild type (B) or PA14ΔcupD (C) carrying pBBR1MCS-5 (white bars), pBBR1MCS-5-rcsB (light grey bars) and pBBR1MCS-5-pvrR (dark grey bars, only for PA14). M63 is a negative control containing cell free M63 medium (black bars).

Figure 5. rcsB overexpression leads to a small colony morphotype in PA14.

PA14 (A and B), PA14ΔcupD (C and D) or PAO1 (E and F) strains freshly conjugated with pBBR1MCS-5 (A, C and E) or pBBR1MCS-5-rcsB (B, D and F) and plated on Pseudomonas isolation agar supplemented with appropriate antibiotics. Images are presented at the same scale. Insets: Enlarged image of a typical colony from each strain.

Figure 6. rcsB overexpression reduces motility in PA14.

Swimming (A, C and E) and twitching (B, D and F) motility of P. aeruginosa PA14 (A and B), PA14ΔcupD (C and D) or PAO1 (E and F) strains carrying either pBBR1MCS-5 (vector) or pBBR1MCS-5-rcsB (rcsB). Twitching zones have been visualized using crystal violet staining.

Figure 7. Sedimentation of bacterial cells.

P. aeruginosa cultures, normalized for OD_{600 nm}, were incubated at room temperature without shaking for 24 hours. PA14 (A), PA14ΔcupD (B) and PAO1 (C) carry pBBR1MCS-5 (vector) or pBBR1MCS-5-rcsB (rcsB), while PA14::rcsC (D) carries pBBR1MCS-4 (vector) or pBBR1MCS-4-pvrR (pvrR).

Figure 8. Overexpression of pvrR reduces cupD gene expression.

Growth and β-galactosidase activity of PA14::rcsC::cupD1-lacZ/pBBR1MCS-4 (black diamonds and bars, respectively) and PA14::rcsC::cupD1-lacZ/pBBR1MCS-4-pvrR (white diamonds and bars, respectively).

Figure 9. pvrR overexpression reduces biofilm formation.

Biofilms of PA14::rcsC carrying either the empty vector pBBR1MCS-4 (vector) or the pvrR-overexpressing plasmid pBBR1MCS-4-pvrR (pvrR) were analyzed. A) Image of crystal violet stained biofilms after 4 hours. B) Quantification of crystal violet stained biofilms at different times of growth (2–6 hours). PA14::rcsC carrying pBBR1MCS-4 (white bars) and pBBR1MCS-4-pvrR (black bars) are shown. M63 is a negative control containing cell free M63 medium (dark grey bars).