Cyclic-di-GMP regulates lipopolysaccharide modification and contributes to Pseudomonas aeruginosa immune evasion

Abstract

Pseudomonas aeruginosa is a Gram-negative bacterial pathogen associated with acute and chronic infections. The universal cyclic-di-GMP second messenger is instrumental in the switch from a motile lifestyle to resilient biofilm as in the cystic fibrosis lung. The SadC diguanylate cyclase is associated with this patho-adaptive transition. Here, we identify an unrecognized SadC partner, WarA, which we show is a methyltransferase in complex with a putative kinase, WarB. We established that WarA binds to cyclic-di-GMP, which potentiates its methyltransferase activity. Together, WarA and WarB have structural similarities with the bifunctional Escherichia coli lipopolysaccharide (LPS) O antigen regulator WbdD. Strikingly, WarA influences P. aeruginosa O antigen modal distribution and interacts with the LPS biogenesis machinery. LPS is known to modulate the immune response in the host, and by using a zebrafish infection model, we implicate WarA in the ability of P. aeruginosa to evade detection by the host.

Figure 1. WarA interacts with SadC.

a, Bacterial two hybrid assay to validate interaction between WarA and the cytoplasmic domain (CD) of SadC. A graphical representation of β-galactosidase activity from E. coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or T18 subunits. Corresponding spots on LB X-Gal plates are shown. Activity was measured in Miller units. b, Purified SadC (CD)_His (16 μg) and WspR_His (16 μg) spotted on a nitrocellulose membrane probed with purified WarA_HisGST overnight. A GST antibody was used to detect binding. c, Purified WarA_His (10 μg) and WspR_His (10 μg) spotted on a nitrocellulose membrane probed with purified WarB_HisGST overnight. Standard deviation error bars shown and representative images/data of at least 3 replicates shown. Significance testing performed by Student’s t test. *, P<0.05; **, P<0.01.

Figure 2. Structural homology of WbdD and WarAB.

a, Superposition of WbdD (Magenta) secondary structure (PDB:4AZW) with that of the Phyre 2.0 predicted secondary structure of WarAB (Cyan). S-Adenosyl methionine (SAM) in blue (WarA) and ATP in yellow (WarB). Selected residues highlighted. b, Model of LPS O antigen biosynthesis in E. coli O9a. The glycosyltransferases WbdA, WbdB and WbdC are responsible for the synthesis of the O antigen. WbdD adds a phosphoryl and a methyl group to the terminal-reducing end of the O antigen, this terminates synthesis and primes the chain for export through the inner membrane via the ABC transporter Wzm/Wzt. Components of model individually labelled with corresponding names.

Figure 3. WarA impact LPS modal distribution.

a, Model of the heteropolymeric OSA biosynthesis via the Wzy/Wzx-dependent pathway. Individual repeating units are assembled in the cytoplasm and transported via a flippase across the inner membrane to the periplasmic space. Here they are assembled into the O antigen polymer by the Wzy polymerase. Wzz₁ and Wzz₂ regulate chain length. b, Model of CPA biosynthesis via the ABC transporter pathway. Like LPS biosynthesis in E.coli O9a, synthesis and assembly occur in the cytoplasm via the glycosyltransferases WbpX, WbpY and WbpZ. The assembled O antigen component is then transported via the Wzm/Wzt ABC transporter across the inner membrane. c, Western blot analysis using OSA specific antibody MF83-1 on PAK wild type with an empty vector (+ Ev), PAK wild type overexpressing warA (+ warAOe), a sadC and a warA mutant. d, Western blot analysis using CPA specific antibody (N1F10) on PAK wild type with an empty vector (+ Ev), PAK wild type overexpressing warA (+ warAOe), a sadC and a warA mutant. Representative images of at least two biological replicates shown.

Figure 4. WarA is a functional methyltransferase and binds c-di-GMP.

a, Bacterial two hybrid screen of the interaction between WarA and the glycosyltransferases of the primary CPA biosynthesis cluster WbpX, WbpY and WbpZ. A graphical representation of β-galactosidase activity from E. coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or T18 subunits. Corresponding spots on LB X-Gal plates are shown. Activity was measured in Miller units. b, DRaCALA analysis of WarA using increasing concentrations of WarA. Competition experiment (Middle Panel) using unlabelled c-di-GMP (100 μM), various other nucleotides (100μM) were used to test specificity of WarA (50 μM) binding (bottom panel). c, A fluorescent-based methyltransferase activity assay was used to determine WarA activity in increasing concentrations of LPS. d, Addition of c-di-GMP (15 μM) to the reaction mix at a range of different WarA concentrations. Representative images/data of at least 3 replicates shown. Standard deviation error bars shown. Significance testing performed by Student’s t test. *, P<0.05; **, P<0.01; ***; P<0.001.

Figure 5. Role of SadC and WarA in infection and immune evasion.

a, Survival analysis of zebrafish 3 days post fertilization (dpf) infected in the hindbrain ventricle with P. aeruginosa at a dose of 5 x 10⁴ CFU. Zebrafish challenged with either mutant displayed a reduced mortality at 12 hpi (12 hpi, ANOVA, *, P<0.05). Data is representative of at least 3 independent biological replicates with a minimum of at least 24 fish per replicate. b, Representative images of lyz:dsRed larvae infected in the hindbrain with PAK::gfp, PAKΔsadC::gfp and PAKΔwarA::gfp from panel C. For each treatment, the same larva was imaged at 0, 6 and 12 hpi using a fluorescent stereomicroscope, where neutrophils were quantified at the site of infection (as defined by the white box, enlarged as inset image) (See also Video S1, S2 and S3).c, Neutrophils were counted around the site of infection at 0 and 6 hours hpi for zebrafish larvae infected in the hindbrain ventricle with 5 x 10⁴ CFU of PAK::gfp, PAKΔsadC::gfp and PAKΔsadC::gfp +psadC. Data is from 4 independent biological replicates with 3-6 larvae per strain per replicate. Standard deviation error bars are shown. Significance testing performed by Student’s t test: *, P<0.05; **, P<0.01. d, TNF-α mRNA levels in larvae infected with 5 x 10⁴ CFU for 6h. TNF-α mRNA are relative to the house keeping gene ef1α. Mean ± SEM (horizontal bars) from 3 independent experiments with 5 pooled larvae per strain per experiment. Significance testing performed by Student’s t test: *, P<0.05; **, P<0.01.

Figure 6. Role of WarA in CPA biosynthesis.

SadC recruits WarA to the inner membrane where it interacts with the CPA O antigen machinery by recruiting WbpX to the assembly interface and also by potentiating the methyltransferase activity of WarA. This fine tuning regulation plays a key role in allowing the cell to escape detection by the host innate immune system.