Characterization of serine hydroxymethyltransferase GlyA as a potential source of D-alanine in Chlamydia pneumoniae

Abstract

For intracellular Chlamydiaceae, there is no need to withstand osmotic challenges, and a functional cell wall has not been detected in these pathogens so far. Nevertheless, penicillin inhibits cell division in Chlamydiaceae resulting in enlarged aberrant bodies, a phenomenon known as chlamydial anomaly. D-alanine is a unique and essential component in the biosynthesis of bacterial cell walls. In free-living bacteria like Escherichia coli, penicillin-binding proteins such as monofunctional transpeptidases PBP2 and PBP3, the putative targets of penicillin in Chlamydiaceae, cross-link adjacent peptidoglycan strands via meso-diaminopimelic acid and D-Ala-D-Ala moieties of pentapeptide side chains. In the absence of genes coding for alanine racemase Alr and DadX homologs, the source of D-Ala and thus the presence of substrates for PBP2 and PBP3 activity in Chlamydiaceae has puzzled researchers for years. Interestingly, Chlamydiaceae genomes encode GlyA, a serine hydroxymethyltransferase that has been shown to exhibit slow racemization of D- and L-alanine as a side reaction in E. coli. We show that GlyA from Chlamydia pneumoniae can serve as a source of D-Ala. GlyA partially reversed the D-Ala auxotrophic phenotype of an E. coli racemase double mutant. Moreover, purified chlamydial GlyA had racemase activity on L-Ala in vitro and was inhibited by D-cycloserine, identifying GlyA, besides D-Ala ligase MurC/Ddl, as an additional target of this competitive inhibitor in Chlamydiaceae. Proof of D-Ala biosynthesis in Chlamydiaceae helps to clarify the structure of cell wall precursor lipid II and the role of chlamydial penicillin-binding proteins in the development of non-dividing aberrant chlamydial bodies and persistence in the presence of penicillin.

Keywords: D-alanine; D-cycloserine; GlyA; aberrant bodies; alanine racemase; chlamydial anomaly; penicillin; persistence.

Figure 1

Proposed lipid II pathway in Chlamydiaceae. A complete cycle of lipid II biosynthesis, including translocation to the periplasm, processing and bactoprenol carrier recycling is required for coordinated function of the divisome machinery and modulation of Nod1 and Nod2 mediated host immune response to chlamydial muropeptides. Biosynthesis of lipid II takes place in the cytoplasm and at the inner leaflet of the cytoplasmic membrane. In two consecutive biosynthesis steps of L-Ala racemization and D-Ala ligation, catalyzed by GlyA and the MurC/Ddl fusion protein, the D-Ala-D-Ala dipeptide is produced. MurF adds the dipeptide to the nascent peptide chain to complete synthesis of the soluble precursor UDP-MurNAc-pentapeptide and to provide D-Ala-D-Ala moieties in the cell wall precursors for transpeptidation activity of the penicillin-binding proteins PBP2 and PBP3. Actin-ortholog MreB functionally organizes MurF, MraY, and MurG (Gaballah et al., 2011), the last three enzymes in lipid II biosynthesis, at the septum. The synthesized precursor is translocated to the periplasm and processed by the concerted activity of the PBP enzymes and amidase AmiA to allow for bactoprenol-P recycling. In the process, the rudimentary by-product found by Liechti et al. (2013), in which the peptide side chains are cross-linked by peptide bonds, might result (Ghuysen and Goffin, 1999) and released muropeptides might contribute to modulation of the host immune response (McCoy and Maurelli, 2006). Chlamydiaceae lack transglycosylases as well as endopeptidases and pyrophosphorylases described so far to link lipid II sugar units to form glycan chains, to cleave peptide bridges between cross-linked glycan chains and to dephosphorylate bactoprenol-PP, respectively. Moreover, the L-Glu racemase MurI is absent. Question marks and dashed arrows highlight steps of the pathway that remain to be clarified. (GlcNAc: N-acetylglucosamine; MurNAc: N-acetylmuramic acid).

Figure 2

GlyA_Cp exhibits in vivo activity in an E. coli racemase double mutant. A temperature sensitive Δ alrΔ dadX E. coli double mutant was transformed with pET21b-glyA_Cp to allow for the expression of GlyA_Cp in the cytoplasm. Independently generated transformants (1–3 containing pET21b-glyA_Cp and 4–6 containing the empty vector) were grown on solid (A) or in liquid (B) LB medium under limited D-Ala growth conditions at 42°C. LB medium was supplemented with 5 mg/L D-Ala, 50 μM of cofactor PLP, 25 μg/ml thymine and 50 μg/ml ampicillin. Expression of GlyA_Cp was induced by the addition of 0.1 mM IPTG.

Figure 3

The purified serine hydroxymethyltransferase GlyA_Cp has alanine racemase activity. In vitro activity of recombinant GlyA_Cp was tested in a coupled enzymatic assay containing L-Ala and cofactor PLP. D-Ala that was produced by 1 μg GlyA or Bst racemase was converted to pyruvate by the activity of DAAO and colorimetrically quantified. (DCS: D-cycloserine; Bst racemase: alanine racemase from B. stearothermophilus).