Interaction and modulation of two antagonistic cell wall enzymes of mycobacteria

Abstract

Bacterial cell growth and division require coordinated cell wall hydrolysis and synthesis, allowing for the removal and expansion of cell wall material. Without proper coordination, unchecked hydrolysis can result in cell lysis. How these opposing activities are simultaneously regulated is poorly understood. In Mycobacterium tuberculosis, the resuscitation-promoting factor B (RpfB), a lytic transglycosylase, interacts and synergizes with Rpf-interacting protein A (RipA), an endopeptidase, to hydrolyze peptidoglycan. However, it remains unclear what governs this synergy and how it is coordinated with cell wall synthesis. Here we identify the bifunctional peptidoglycan-synthesizing enzyme, penicillin binding protein 1 (PBP1), as a RipA-interacting protein. PBP1, like RipA, localizes both at the poles and septa of dividing cells. Depletion of the ponA1 gene, encoding PBP1 in M. smegmatis, results in a severe growth defect and abnormally shaped cells, indicating that PBP1 is necessary for viability and cell wall stability. Finally, PBP1 inhibits the synergistic hydrolysis of peptidoglycan by the RipA-RpfB complex in vitro. These data reveal a post-translational mechanism for regulating cell wall hydrolysis and synthesis through protein-protein interactions between enzymes with antagonistic functions.

Figure 1. M. tuberculosis RipA identifies bifunctional synthase PBP1.

(A) RipA, a 472 residue protein, contains a domain of unknown function (COG3883) as well as a predicted endopeptidase domain. The region used to screen for interacting proteins in a yeast two-hybrid screen is shown, consisting of amino acids 350–472. (B) PBP1, a 678 residue protein encoded by ponA1, is a bifunctional peptidoglycan synthase. PBP1 contains a transglycosylase domain at the N-terminus and a penicillin-sensitive transpeptidase domain at the C-terminus.

Figure 2. C-terminal regions are required for interaction.

(A) Data from quantitative LacZ assays of four overlapping regions of 200 amino acids each of PBP1 tested for interaction with the C-terminal 123 amino acids of RipA. The C-terminal 259 amino acids of PBP1 were found to be sufficient for interaction with RipA, while regions lacking the C-terminal 150 amino acids failed to interact. Data shown are from a representative experiment done in triplicate. Data are represented as mean +/− SEM. (B) Data from quantitative LacZ assays of several different deletion constructs for interaction with the C-terminal 259 amino acids of PBP1 in the yeast two-hybrid assay. Deletion of the C-terminal of RipA decreased the intensity of interaction and the C-terminal 25 amino acids of RipA were sufficient for interaction with PBP1. Data shown are from a representative experiment done in triplicate. Data are represented as mean +/− SEM.

Figure 3. Recombinant PBP1 coprecipitates with RipA in vitro.

(A) Fusion proteins were co-expressed in E. coli and GST fusion proteins were purified directly from the lysate. Co-purifying MBP fusion proteins were detected by Western blotting using anti-MBP antibody (top panel). Unfused GST was used to test the specificity of the interaction. A Coomassie-stained PAGE gel containing lysates obtained prior to GST purification (bottom panel) and after GST purification (middle panel) is shown to demonstrate that similar amounts of proteins were available for pulldown. (B) Proteins were separately purified from E. coli, combined as indicated in equimolar amounts, incubated, then purified on amylose resin. Samples were taken before (middle and bottom panels) and after (top panel) MBP purification. A Coomassie-stained PAGE gel containing protein mixtures prior to MBP purification is shown to demonstrate that similar amounts of proteins were available for pulldown. Co-purifying GST fusion proteins were detected by Western blotting using anti-GST antibody. Unfused GST was used to test the specificity of the interaction.

Figure 4. PBP1 localizes to the poles and septa of M. smegmatis.

Fluorescence microscopy of M. smegmatis expressing M. tuberculosis PBP1 fused to monomeric red fluorescent protein (RFP). PBP1-RFP fusion protein is under the control of a tetracycline-inducible promoter. PBP1 localized to the poles and also to septa. Arrows indicate narrow septa where PBP1 does not localize and arrowheads indicate larger septa where PBP1 localizes.

Figure 5. Depletion of PBP1 blocks cell division.

(A) Diagram showing the strategy used to replace the native promoter of the ponA1 operon (PponA1) in M. smegmatis with a tetracycline-inducible promoter (Ptet) through homologous recombination (strategy and diagram adapted from [12]). OriE: E. coli origin of replication. (B) PBP1 (ponA1) depletion strain of M. smegmatis was grown with inducer (ATC = anhydrotetracycline), then inoculated into media with decreasing amounts of inducer and followed by OD₆₀₀ over time. Data are represented as mean +/− standard deviation. (C) Micrographs of M. smegmatis PBP1 (ponA1) depletion strain with membranes imaged by staining with TMA-DPH. Bacteria were grown with no anhydrotetracycline inducer (No ATC), 10, or 100 ng/ml ATC. Bacteria grown with 100 ng/ml ATC grew as wildtype, while No ATC and 10 ng/ml ATC grew slowly, with bulbous poles and round-shaped regions (indicated by white arrows). Bacteria were visualized with a 100× objective.

Figure 6. M. smegmatis ponA1 complements ponA1 operon depletion.

(A) The M. smegmatis ponA1 depletion strain was complemented with either a vector expressing an operon of M. smegmatis ponA1 and red fluorescent protein (RFP) or RFP alone. Complemented strains were grown in the presence (50 ng/ml ATC) or absence of inducer and growth was determined by OD₆₀₀. While the RFP complemented strain only grew in the presence of inducer, the PBP1 complemented strain grew normally in both the absence and presence of inducer. (B) At 36 hours post-induction, cultures from RFP and ponA1 complemented strains were photographed. Data is representative of several biological replicates for both strains. (C) Microscopic analysis of complemented strains—either the RFP complemented strain grown with inducer or the ponA1 complemented strain without inducer—showed that both strains expressed RFP from the complementation vector and appear morphologically normal.

Figure 7. PBP1 inhibits the synergistic hydrolysis of cell wall by RipA-RpfB complex.

N-terminal GST fusion proteins were expressed and purified from E. coli. Equal amounts of individual or combinations of proteins were incubated with insoluble FITC-labeled substrate: M. luteus cell wall material (A) or Streptomyces peptidoglycan (B). The extent of hydrolysis was determined by measuring the amount of soluble FITC remaining after centrifugation, and thus released during hydrolysis of the insoluble substrate. GST and buffer alone were used to determine background release of FITC and were subtracted from final values. Data are from representative experiments, each done in triplicate. Data are represented as mean +/− SEM. p-values for one-tailed, unpaired t-tests: (A) 1 vs. 2: 0.027 significant, 2 vs. 4: 0.016 significant, 1 vs. 3: 0.074 not significant, 1 vs. 4: 0.188 not significant, (B): 1 vs. 2: 0.009 significant, 2 vs. 4: 0.018 significant, 1 vs. 3: 0.279 not significant, 1 vs. 4: 0.240 not significant (significant to p<0.05). (C) Schematic diagram of the basic structure of mycobacterial peptidoglycan, indicating where RpfB and RipA are predicted to have hydrolytic activity and where PBP1 is predicted to have synthetic activity. Lines connecting NAG to NGM represent β-1,4-glycosidic bonds, while lines connecting NGM to NGM represent peptide cross-linkages. NAG: N-acetyl glucosamine, NGM: N-glycolyl muramic acid, NAM: N-acetyl muramic acid (note that mycobacteria have a mixture of NGM and NAM, with the NGM structure shown here).

Figure 8. Model of RipA regulation by PBP1 during septation.

A RipA-PBP1 complex that exists at the initiation of septation may synthesize a thick layer of peptidoglycan (PG) between daughter cells during the process of cytokinesis. After PG synthesis and fission are finished, RipA may exchange PBP1 for autolysin binding partners like RpfB. These new complexes are highly efficient at PG hydrolysis and will separate the cell walls of the two mature daughter cells.