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. 2016 Oct 6;16(1):234.
doi: 10.1186/s12866-016-0853-x.

In vivo functional and molecular characterization of the Penicillin-Binding Protein 4 (DacB) of Pseudomonas aeruginosa

Cristian Gustavo Aguilera Rossi  1   2 , Paulino Gómez-Puertas  3 , Juan Alfonso Ayala Serrano  4
Affiliations

In vivo functional and molecular characterization of the Penicillin-Binding Protein 4 (DacB) of Pseudomonas aeruginosa

Cristian Gustavo Aguilera Rossi et al. BMC Microbiol. .

Abstract

Background: Community and nosocomial infections by Pseudomonas aeruginosa still create a major therapeutic challenge. The resistance of this opportunist pathogen to β-lactam antibiotics is determined mainly by production of the inactivating enzyme AmpC, a class C cephalosporinase with a regulation system more complex than those found in members of the Enterobacteriaceae family. This regulatory system also participates directly in peptidoglycan turnover and recycling. One of the regulatory mechanisms for AmpC expression, recently identified in clinical isolates, is the inactivation of LMM-PBP4 (Low-Molecular-Mass Penicillin-Binding Protein 4), a protein whose catalytic activity on natural substrates has remained uncharacterized until now.

Results: We carried out in vivo activity trials for LMM-PBP4 of Pseudomonas aeruginosa on macromolecular peptidoglycan of Escherichia coli and Pseudomonas aeruginosa. The results showed a decrease in the relative quantity of dimeric, trimeric and anhydrous units, and a smaller reduction in monomer disaccharide pentapeptide (M5) levels, validating the occurrence of D,D-carboxypeptidase and D,D-endopeptidase activities. Under conditions of induction for this protein and cefoxitin treatment, the reduction in M5 is not fully efficient, implying that LMM-PBP4 of Pseudomonas aeruginosa presents better behaviour as a D,D-endopeptidase. Kinetic evaluation of the direct D,D-peptidase activity of this protein on natural muropeptides M5 and D45 confirmed this bifunctionality and the greater affinity of LMM-PBP4 for its dimeric substrate. A three-dimensional model for the monomeric unit of LMM-PBP4 provided structural information which supports its catalytic performance.

Conclusions: LMM-PBP4 of Pseudomonas aeruginosa is a bifunctional enzyme presenting both D,D-carboxypeptidase and D,D-endopeptidase activities; the D,D-endopeptidase function is predominant. Our study provides unprecedented functional and structural information which supports the proposal of this protein as a potential hydrolase-autolysin associated with peptidoglycan maturation and recycling. The fact that mutant PBP4 induces AmpC, may indicate that a putative muropeptide-subunit product of the DD-EPase activity of PBP4 could be a negative regulator of the pathway. This data contributes to understanding of the regulatory aspects of resistance to β-lactam antibiotics in this bacterial model.

Keywords: Catalytic function; D,D-peptidase; LMM-PBP4; Macromolecular peptidoglycan; Pseudomonas aeruginosa; Purified muropeptides; Three-dimensional structure.

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Figures

Fig. 1
Fig. 1
a Purification of LMM-PBP4. SDS-PAGE analysis of purified recombinant proteins PBP4HNC (lanes 2, 3, 4, 5), PBP4HN (lanes 6, 7, 8, 9) and PBP4HC (lanes 10, 11, 12, 13). Lane 1, molecular weight marker (51 kDa); lanes 2, 6 and 10, flow-through; lanes 3, 7 and 11, elution with 125 mM imidazole from Ni-NTA; lanes 4, 8 and 12, elution with 250 mM imidazole from Ni-NTA; lanes 5, 9 and 13, elution with 500 mM imidazole from Ni-NTA.. b Identification assays for LMM-PBP4. Pattern of Bocillin™ FL binding to membrane protein extracts prepared from strains E. coli BL21(DE3)/pET-PBP4HNC, E. coli BL21(DE3)/pET-PBP4HN and E. coli BL21(DE3)/pET-PBP4HC. Extracts from non induced cell (NI) and induced by IPTG (IPTG) are shown. Unprocessed and mature forms of the overexpressed proteins are indicated by arrows. PBPs profile model (PBP) for Escherichia coli is shown
Fig. 2
Fig. 2
D,D-peptidase activities on peptidoglycan of Escherichia coli. HPLC chromatograms of peptidoglycan obtained from strains E. coli DV900(DE3)/pET-PBP4HNC, E. coli DV900(DE3)/pET-PBP4HN and E. coli DV900(DE3)/pET-PBP4HC, uninduced (NI) and induced by IPTG (IPTG). Chromatograms for the lysogenized mutant strain E. coli DV900(DE3) and transformed with the expression vector pET-28b(+) are presented as controls. The following muropeptides are identified: M3, monomer disaccharide tripeptide; M4, monomer disaccharide tetrapeptide; M5, monomer disaccharide pentapeptide; M4N, anhydrous monomer disaccharide tetrapeptide; D44, dimer disaccharide tetrapeptide-tetrapeptide; D45, dimer disaccharide tetrapeptide-pentapeptide; D45N, anhydrous dimer disaccharide tetrapeptide-pentapeptide; T445, trimer tetrapeptide-tetrapeptide-pentapeptide; T445N, anhydrous trimer tetrapeptide-tetrapeptide-pentapeptide. A204, absorbance at 204 nm, arbitrary units
Fig. 3
Fig. 3
D,D-peptidase activities on peptidoglycan of Pseudomonas aeruginosa. HPLC chromatograms of peptidoglycan obtained from reference strain UCBPP-PA14 (PA14WT), PA14WT/pHERD-PBP4 induced with L(+)-arabinose 0.2 %, PA14WT treated with cefoxitin (FOX), and PA14WT/pHERD-PBP4 induced with L(+)-arabinose 0.2 % and treated with cefoxitin (FOX). The following muropeptides are identified: M3, monomer disaccharide tripeptide; M4, monomer disaccharide tetrapeptide; M5, monomer disaccharide pentapeptide; M3L, monomer disaccharide tripeptide associated with lipoprotein; D43, dimer disaccharide tetrapeptide-tripeptide; D44, dimer disaccharide tetrapeptide-tetrapeptide; D44N, anhydrous dimer disaccharide tetrapeptide-tetrapeptide; T444, trimer tetrapeptide-tetrapeptide-tetrapeptide; T444N, anhydrous trimer tetrapeptide-tetrapeptide-tetrapeptide. Arab., arabinose; A204, absorbance at 204 nm, arbitrary units
Fig. 4
Fig. 4
D,D-peptidase activity on purified muropeptides. HPLC chromatograms for digestion assays with purified recombinant protein PBP4HC on natural substrate, M5 (monomer disaccharide pentapeptide), D45 (dimer disaccharide tetrapeptide-pentapeptide), M5N (anhydrous monomer disaccharide pentapeptide) and D45N (anhydrous dimer disaccharide tetrapeptide-pentapeptide). The identified products are labeled: M4, monomer disaccharide tetrapeptide; D44, dimer disaccharide tetrapeptide-tetrapeptide; M4N, anhydrous monomer disaccharide tetrapeptide. A204, absorbance at 204 nm, arbitrary units
Fig. 5
Fig. 5
3D model for LMM-PBP4 of PAO1. a Homology modeling of monomer Pseudomonas aeruginosa LMM-PBP4 (surface representation). The different domains are presented in brown (domain I), red (domain II) and green (domain III). b Domain I and conserved motifs in the active site of LMM-PBP4. For the first conserved sequence SxxK (STMK; *S72, catalytic serine at position 72), located at the beginning of α2 helix, residues are represented by an intense, blue, cyan and pale blue color, respectively. The second conserved sequence SxN (SNN), located in a short loop between α3-α4, is represented by a gradient of colors derived from green. The three residues of the third conserved motif KTG, located in a β sheet (β2) are represented on dark red, red and orange, respectively. c Location for synthetic peptide AMV-L-Ala-FGA-L-Lys-D-Ala-D-Ala (linear representation) is indicated within the active site model for LMM-PBP4. Estimated distances for depth, height and width in this cavity are shown. d Putative residues constituents for the specific subsite in the active site of LMM-PBP4. Location of the amino acid lysine (L-Lys) of the synthetic substrate AMV-L-Ala-FGA-L-Lys-D-Ala-D-Ala, the residue equivalent to mesoA2pm in natural muropeptides, is indicated. Each amino acid has been labeled and highlighted by a color (aspartic acid 162, blue; leucine 369, green; threonine 428, yellow; leucine 429, red; asparagine 430, gray). AMV, methyl 2-(acetylamino)-3-O-[(1R)-1-carboxyethyl]-2-deoxy-beta-D-glucopyranoside; FGA, gamma-D-glutamic acid
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