c-di-AMP Accumulation Impairs Muropeptide Synthesis in Listeria monocytogenes

Abstract

Cyclic di-AMP (c-di-AMP) is an essential and ubiquitous second messenger among bacteria. c-di-AMP regulates many cellular pathways through direct binding to several molecular targets in bacterial cells. c-di-AMP depletion is well known to destabilize the bacterial cell wall, resulting in increased bacteriolysis and enhanced susceptibility to cell wall targeting antibiotics. Using the human pathogen Listeria monocytogenes as a model, we found that c-di-AMP accumulation also impaired cell envelope integrity. An L. monocytogenes mutant deleted for c-di-AMP phosphodiesterases (pdeA pgpH mutant) exhibited a 4-fold increase in c-di-AMP levels and several cell wall defects. For instance, the pdeA pgpH mutant was defective for the synthesis of peptidoglycan muropeptides and was susceptible to cell wall-targeting antimicrobials. Among different muropeptide precursors, we found that the pdeA pgpH strain was particularly impaired in the synthesis of d-Ala-d-Ala, which is required to complete the pentapeptide stem associated with UDP-N-acetylmuramic acid (MurNAc). This was consistent with an increased sensitivity to d-cycloserine, which inhibits the d-alanine branch of peptidoglycan synthesis. Finally, upon examining d-Ala:d-Ala ligase (Ddl), which catalyzes the conversion of d-Ala to d-Ala-d-Ala, we found that its activity was activated by K⁺ Based on previous reports that c-di-AMP inhibits K⁺ uptake, we propose that c-di-AMP accumulation impairs peptidoglycan synthesis, partially through the deprivation of cytoplasmic K⁺ levels, which are required for cell wall-synthetic enzymes.IMPORTANCE The bacterial second messenger c-di-AMP is produced by a large number of bacteria and conditionally essential to many species. Conversely, c-di-AMP accumulation is also toxic to bacterial physiology and pathogenesis, but its mechanisms are largely undefined. We found that in Listeria monocytogenes, elevated c-di-AMP levels diminished muropeptide synthesis and increased susceptibility to cell wall-targeting antimicrobials. Cell wall defects might be an important mechanism for attenuated virulence in bacteria with high c-di-AMP levels.

Keywords: Listeria monocytogenes; c-di-AMP; peptidoglycan.

FIG 1

c-di-AMP accumulation increases susceptibility to cell wall-targeting antimicrobials in L. monocytogenes. (A) PdeA and PgpH are major c-di-AMP phosphodiesterases in L. monocytogenes and B. subtilis. (B) Cefuroxime susceptibilities of L. monocytogenes grown in BHI medium in 96-well plates. Relative growth at varying cefuroxime concentrations was calculated against exponential growth rates in BHI medium only, determined for each strain. (C) Lysozyme susceptibilities of L. monocytogenes. Methods were similar to those used for panel B. The growth medium was noticeably turbid at >1 mg/ml lysozyme, so complete inhibition of the WT could not be assessed. Data are averages from three independent experiments, representative of numerous others. Error bars represent standard deviations.

FIG 2

The L. monocytogenes pdeA pgpH mutant has a reduced peptidoglycan content compared to WT. (A) Cell wall and peptidoglycan quantification. Data are averages from two independent experiments, for a total of four biological replicates. Extracted cell wall was quantified based on total amino acid contents (BCA assay) of the preparations, before or after removal of wall teichoic acid (WTA), and normalized to total protein content of each cell pellet. Normalization to CFU gave the same results. *, P < 0.05; **, P < 0.01 (paired t tests for each biological set). (B) Cell wall thickness, analyzed from transmission electron micrographs of 18 WT cells and 23 pdeA pgpH cells from two independent experiments. Each data point represents the average of 10 random measurements around a bacterial cell. **, P < 0.01 (unpaired t test). (C) Representative TEM images. Arrows delimit a typical cell wall thickness measurement. Error bars represent standard deviations.

FIG 3

The pdeA pgpH mutant harbors reduced intermediates for muropeptide synthesis. (A) Metabolite abundances of the WT and pdeA pgpH strains, determined by untargeted metabolomics. The pink line represents identity between the two strains. The dashed blue lines denote a 2-fold change in abundance. Data represent two independent experiments for four biological replicates. (B) Quantification of UDP-MurNAc and d-Ala–d-Ala, determined from the data in panel A. Relative abundances of Ala are shown as controls. (C) UDP-linked and d-alanine branches of muropeptide synthesis. (D) Abundances of muropeptide precursors in the pdeA pgpH strain relative to the WT, quantified by LC-MS/MS and adjusted to OD₆₀₀ and cell dry weight. Data are averages from four independent experiments, each with three technical replicates. The mean values from each experiment were normalized to corresponding values in the WT. For amino acid quantification, glycine was measured as a control. **, P < 0.05; ****, P < 0.0001 (paired t tests for each biological set).

FIG 4

The pdeA pgpH strain is sensitive to d-cycloserine (DCS), consistent with a weakened cell wall. (A) The d-alanine branch of muropeptide synthesis, with DCS as an inhibitor of Dal and Ddl. (B) DCS susceptibilities determined by the BHI medium microdilution method, performed as for Fig. 1B and C. Data are averages from three independent experiments. (C) A subinhibitory concentration of DCS (3.13 μg/ml), which does not affect growth of the pdeA pgpH mutant, sensitized this strain to cefuroxime. Data are representative of three independent experiments. Bliss interaction scores for DCS and cefuroxime were calculated as [(I_cef + I_DCS) − I_cefI_DCS]/I_cef,DCS, where I_cef is growth inhibition by cefuroxime, I_DCS is growth inhibition by DCS, and I_cef,DCS is observed inhibition by cefuroxime plus DCS. Bliss scores of <1 indicate synergy.

FIG 5

The pdeA pgpH strain has a diminished cytoplasmic K⁺ level. K⁺ was quantified from WT and pdeA pgpH cell pellets by ICP-MS. K⁺ levels were normalized to protein contents, determined by BCA assays on sonicated cell suspensions. Normalization to cell pellet dry weight returned similar results. Data are averages for four biological replicates. *, P < 0.05 (paired t tests for each biological set).

FIG 6

Ddl enzymatic activity is stimulated by K⁺. Continuous enzyme assays were performed using a commercial inorganic phosphate release assay kit. (A) Kinetic assays for ATP, in reaction mixtures containing 10 mM d-Ala. (B) Kinetic assays for d-Ala, in reaction mixtures containing 1 mM ATP. Control assays in which d-Ala, ATP, or Ddl was omitted showed no activity. Reaction rates were calculated as the slopes of the absorbance curves in the first 6 min of reaction.

FIG 7

K⁺ supplementation increases d-cycloserine (DCS) resistance more substantially in the WT than in the pdeA pgpH mutant. Relative growth rates at various DCS or cefuroxime concentrations were calculated as for Fig. 1. BHI medium has a K⁺ content of 10 mM and was supplemented with 100 mM or 200 mM KCl. Data are averages from three experiments.