Staphylococcus aureus counters organic acid anion-mediated inhibition of peptidoglycan cross-linking through robust alanine racemase activity

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    In this valuable study, the authors use Staphylococcus aureus to understand how organic acids inhibit bacterial growth. They provide convincing evidence that acetic acid specifically inhibits the activity of the Ddl enzyme and that S. aureus maintains a high intracellular D-ala concentration to circumvent acetate-mediated growth inhibition. This work will be of interest to researchers studying bacteria and antimicrobials.

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Abstract

Weak organic acids are commonly found in host niches colonized by bacteria, and they can inhibit bacterial growth as the environment becomes acidic. This inhibition is often attributed to the toxicity resulting from the accumulation of high concentrations of organic anions in the cytosol, which disrupts cellular homeostasis. However, the precise cellular targets that organic anions poison and the mechanisms used to counter organic anion intoxication in bacteria have not been elucidated. Here, we utilize acetic acid, a weak organic acid abundantly found in the gut to investigate its impact on the growth of Staphylococcus aureus . We demonstrate that acetate anions bind to and inhibit D-alanyl-D-alanine ligase (Ddl) activity in S. aureus . Ddl inhibition reduces intracellular D-alanyl-D-alanine (D-Ala-D-Ala) levels, compromising staphylococcal peptidoglycan cross-linking and cell wall integrity. To overcome the effects of acetate-mediated Ddl inhibition, S. aureus maintains a high intracellular D-Ala pool through alanine racemase (Alr1) activity and additionally limits the flux of D-Ala to D-glutamate by controlling D-alanine aminotransferase (Dat) activity. Surprisingly, the modus operandi of acetate intoxication in S. aureus is common to multiple biologically relevant weak organic acids indicating that Ddl is a conserved target of small organic anions. These findings suggest that S. aureus may have evolved to maintain high intracellular D-Ala concentrations, partly to counter organic anion intoxication. Under mildly acidic conditions, weak organic acids like acetic acid accumulate to high concentrations within the cytosol as organic anions. However, the physiological consequence of organic anion accumulation is poorly defined. Here we investigate how the acetate anion impacts S. aureus . We show that acetate anions directly bind Ddl and inhibit its activity. The resulting decrease in intracellular D-Ala-D-Ala pools impacts peptidoglycan integrity. Since acetate is a weak inhibitor of Ddl, mechanisms that maintain a high intracellular D-Ala pools are sufficient to counter the effect of acetate-mediated Ddl inhibition in S. aureus .

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  1. eLife assessment

    In this valuable study, the authors use Staphylococcus aureus to understand how organic acids inhibit bacterial growth. They provide convincing evidence that acetic acid specifically inhibits the activity of the Ddl enzyme and that S. aureus maintains a high intracellular D-ala concentration to circumvent acetate-mediated growth inhibition. This work will be of interest to researchers studying bacteria and antimicrobials.

  2. Reviewer #1 (Public Review):

    Summary:

    The manuscript entitled "Staphylococcus aureus counters organic acid anion-mediated inhibition of peptidoglycan cross-linking through robust alanine racemase activity" by Panda, S et al. reports an extensive biochemical analysis of the result from a Tn screen that identified alr1 as being required for acetic acid tolerance. In the end, they demonstrate that reduced D-Ala pools in the ∆alr1 mutant lead to a drastic reduction in D-Ala-D-Ala dipeptide. They show that this is due to the ability of organic acid anions to limit the D-Ala-D-Ala ligase enzyme Ddl. They demonstrate that:

    (1) Acetate exposure in the ∆alr1 results in reduced D-Ala-D-Ala dipeptide, but not the monomers.

    (2) Acetate can bind to purified Ddl in vitro.

    (3) This binding results in reduced enzyme activity.

    (4) Other organic acid anions such as lactate, proprionate, and itaconitate can also inhibit Ddl.

    The experiments are clearly described and logically laid out. I have only a few minor comments to add.

    Strengths:

    The most significant strength is the exceptional experimental data that supports the authors' hypotheses.

    Weaknesses:

    Only minor weaknesses were identified by this reviewer.

    (1) Which allele is alr1, the one upstream of MazEF or the one in the Lysine biosynthetic operon?

    (2) Figure 3B. Where does the C3N2 species come from in the WT and why is it absent in the mutants? It is about 25% of the total dipeptide pool.

    (3) Figure 3D could perhaps be omitted. I understand that the authors attained statistical significance in the fitness defect, but biologically this difference is very minor. One would have to look at the isotopomer distribution in the Dat overexpressing strain to make sure that increased flux actually occurred since there are other means of affecting activity (e.g. allosteric modulators).

    (4) In Figure 4A, why is the complete subunit UDP-NAM-AEKAA increasing in each strain upon acetate challenge if there was such a stark reduction in D-Ala-D-Ala, particularly in the ∆alr1 mutant? For that matter, why are the levels of UDP-NAM-AEKAA in the ∆alr1 mutant identical to that of WT with/out acetate?

    (5) Figure 4B. Is there no significant difference between ddl and murF transcripts between WT and ∆alr1 under acetate stress? This comparison was not labeled if the tests were done.

    (6) Although tricky, it is possible to measure intracellular acetate. It might be of interest to know where in the Ddl inhibition curve the cells actually are.

  3. Reviewer #2 (Public Review):

    Summary:

    In this manuscript, using Staphylococcus aureus as a model organism, Panda et al. aim to understand how organic acids inhibit bacterial growth. Through careful characterization and interdisciplinary collaboration, the authors present valuable evidence that acetic acid specifically inhibits the activity of Ddl enzyme that converts 2 D-alanine amino acids into D-ala-D-ala dipeptide, which is then used to generate the stem pentapeptide of peptidoglycan (PG) precursors in the cytoplasm. Thus, a high concentration of acetic acid weakens the cell wall by limiting PG-crosslinking (which requires a D-ala portion). However, S. aureus maintains a high intracellular D-ala concentration to circumvent acetate-mediated growth inhibition.

    Strengths:

    The authors utilized a well-established transposon mutant library to screen for mutants that struggle to grow in the presence of acetic acid. This screen allowed authors to identify that a strain lacking intact alr1, which encodes for alanine racemase (converts L-ala to D-ala), is unable to grow well in the presence of acetic acid. This phenotype is rescued by the addition of external D-ala. Next, the authors rule out the contribution of other pathways that could lead to the production of D-ala in the cell. Finally, by analyzing D-ala and D-ala-D-ala concentrations, as well as muropeptide intermediates accumulation in different mutants, the authors pinpoint Ddl as the specific target of acetic acid. In fact, the synthetic overexpression of ddl alone overcomes the toxic effects of acetic acid. Using genetics, biochemistry, and structural biology, the authors show that Ddl activity is specifically inhibited by acetic acid and likely by other biologically relevant organic acids. Interestingly, this mechanism is different from what has been reported for other organisms such as Escherichia coli (where methionine synthesis is affected). It remains to be seen if this mechanism is conserved in other organisms that are more closely related to S. aureus, such as Clostridioides difficile and Enterococcus faecalis.

    Weaknesses:

    Although the authors have conclusively shown that Ddl is the target of acetic acid, it appears that the acetic acid concentration used in the experiments may not truly reflect the concentration range S. aureus would experience in its environment. Moreover, Ddl is only significantly inhibited at a very high acetate concentration (>400 mM). Thus, additional experiments showing growth phenotypes at lower organic acid concentrations may be beneficial. Another aspect not adequately discussed is the presence of D-ala in the gut environment, which may be protective against acetate toxicity based on the model provided.

  4. Thank you so much for your study on the important bacterial pathogen Staphylococcus aureus. I get very excited about D-amino acids so thank you also for contributing to the field of chiral chemistry and to our understanding of the enzymes that mediate these types of reactions. I was wondering if I could ask you a few questions about the findings in your paper. First, I was wondering if you have more information on the function of the Alr2 enzyme? Is there any information about its expression levels? Or information about its activity towards different amino acids? I wonder if this enzyme may be a broad racemase with activity towards amino acids such as D-Arg and D-Lys? In my PhD studies, I characterized just such a broad racemase enzyme found in Pseudomonas putida, and that enzyme was also annotated as an Alanine racemase! Second, do you know if the cell wall of the wild-type S. aureus becomes weaker upon acetate intoxication? I see that you describe there is less cross-linking but I’m wondering if you have considered challenging the wild-type strain with an osmotic shock, after exposing to acetate? Third, do you think an acetate treatment could be combined with an antibiotic to act as an adjuvant? I was wondering if a treatment with acetate may allow us to use some antibiotics that S. aureus is typically resistant against. Thank you once more for your hard work and your excellent paper!