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. 2009 Apr;72(2):368-79.
doi: 10.1111/j.1365-2958.2009.06654.x. Epub 2009 Mar 4.

Control of Pseudomonas aeruginosa AlgW protease cleavage of MucA by peptide signals and MucB

Brent O Cezairliyan  1 , Robert T Sauer
Affiliations

Control of Pseudomonas aeruginosa AlgW protease cleavage of MucA by peptide signals and MucB

Brent O Cezairliyan et al. Mol Microbiol. 2009 Apr.

Abstract

The ability of a pathogen to survive the defensive attacks of its host requires the detection of and response to perturbations in its own physiology. Activation of the extracytoplasmic stress response in the pathogen Pseudomonas aeruginosa results in higher tolerance against immune defences as well as in the production of alginate, a surface polysaccharide that also confers resistance to many host defences and antibiotic treatments. The alginate response is regulated by proteolytic cleavage of MucA, a transmembrane protein that inhibits the transcription factor AlgU, and by the periplasmic protein MucB. Here we show that specific peptides bind to the periplasmic AlgW protease and activate its cleavage of MucA. We demonstrate that tight binding of MucB to MucA strongly inhibits this cleavage. We also probe the roles of structural features of AlgW, including a key regulatory loop and its PDZ domain, in regulating substrate binding and cleavage.

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Figures

Figure 1
Figure 1
Cleavage of MucA by AlgW. (A) Sequence alignment of the periplasmic domains of P. aeruginosa MucA and E. coli RseA. Conserved residues are highlighted. (B) Circular dichroism spectra of MucAperi (3 μM) at different temperatures. (C) Cleavage of MucAperi by AlgW assayed by SDS-PAGE. MucAperi (20 μM) was incubated with AlgW (0.5 μM trimer) in the absence or presence of MucE peptide (35 μM) for the times indicated. (D) Rates of cleavage of 35S-labeled MucAperi by AlgW (0.5 μM trimer) in the presence of MucE peptide (35 μM). The curve is fit to the Hill form of the Michaelis-Menten equation with Km = 159 μM, Vmax = 1.2 s−1, and Hill constant = 1.3.
Figure 2
Figure 2
Cleavage sites and specificities. (A) Reverse-phase HPLC demonstrated that AlgW initially cleaves MucAperi to produce two discrete fragments. Mass spectrometry and peptide sequencing revealed that these fragments corresponded to MucAperi residues 53–110 (first peak) and 2–52 (second peak). (B) Sequence alignment of periplasmic domains of MucA from P. aeruginosa and closely related species. The large arrow indicates the initial AlgW cleavage site. Small arrows indicate subsequent cleavages by AlgW. (C) DegS (11 μM trimer) cleavage of RseAperi (20 μM) and MucAperi (20 μM). Reactions were performed in the absence or presence of YYF peptide (60 μM) at room temperature for 16 hours. (D) AlgW (0.5 μM trimer) cleavage of RseAperi (20 μM) in the absence or presence of MucE peptide (35 μM). Cleavage was less efficient than observed for MucAperi (see Fig. 1C).
Figure 3
Figure 3
Peptide binding and activation of AlgW. (A) AlgW binding to fluorescently labeled peptides (50 nM) with different C termini was assayed by changes in fluorescence anisotropy. (B) Peptide activation of cleavage of 35S-labeled MucAperi (74 μM) by AlgW (0.5 μM trimer). Cleavage rates were determined by change in TCA-soluble radioactive counts over time. (C) Rates of cleavage of MucAperi (592 μM) by AlgW, AlgWΔPDZ, and AlgW R279A (0.5 μM trimer). When present, the MucE peptide concentration was 35 μM. Numbers above the bars are substrate molecules cleaved per second per enzyme trimer.
Figure 4
Figure 4
Sequence alignment of AlgW and homologous proteases. The LA loop, L2 loop, and PDZ domains are labeled.
Figure 5
Figure 5
Role of the LA loop in proteolysis. (A) Cleavage of MucAperi (20 μM) by AlgWΔLA (0.5 μM trimer) in the absence or presence of MucE peptide (35 μM) assayed by SDS-PAGE. Disappearance of the upper band over the time course is due to cleavage at secondary sites. (B) Rates of cleavage of different concentrations of 35S-labeled MucAperi by AlgWΔLA (0.25 μM trimer) in the presence of MucE peptide (35 μM). The curve is fit to the Hill form of the Michaelis-Menten equation with Km = 6.3 μM, Vmax = 1.2 s−1, and Hill constant = 1.2. (C) Time course of cleavage of 35S-labeled MucAperi (592 μM) by AlgW or AlgWΔLA (0.5 μM trimer) in the absence of MucE peptide. The linear fits correspond to 0.0013 and 0.16 molecules of MucAperi cleaved per protease trimer per second for AlgW and AlgWΔLA respectively.
Figure 6
Figure 6
Interactions between MucA and MucB. (A) MucB (80 μM) inhibited cleavage of MucAperi (20 μM) by AlgW (0.5 μM trimer) in the presence of MucE peptide (35 μM). (B) Binding of MucB to fl-MucAperi (50 nM). The curve is a fit to the quadratic form of a hyperbolic binding equation (KD = 120 nM). (C) Dissociation of fl-MucAperi (50 nM) from MucB (3.5 μM) upon addition of unlabeled MucAperi (38 μM). The curve is an exponential fit with a rate constant of 0.0014 s−1. (D) Gel filtration of MucB orthologs (4.3 nmol monomer). MucB = P. aeruginosa MucB, RseB = E. coli RseB, HiB = H. influenzae RseB. (E) Gel filtration of fl-MucAperi (0.15 nmol) or the fl-MucAperi/MucB complex (0.15 nmol fl-MucAperi mixed with 1.35 nmol MucB) was monitored by fluorescein absorbance.
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