Three pathways feed the folate-dependent one carbon pool for growth and virulence of Listeria monocytogenes

Reviewer #1 (Public review):

Summary:

This study identifies three redundant pathways-glycine cleavage system (GCS), serine hydroxymethyltransferase (GlyA), and formate-tetrahydrofolate ligase/FolD-that feed the one-carbon tetrahydrofolate (1C-THF) pool essential for Listeria monocytogenes growth and virulence. Reactivation of the normally inactive fhs gene rescues 1C-THF deficiency, revealing metabolic plasticity and vulnerability for potential antimicrobial targeting

Strengths:

(1) Novel evolutionary insight - reversible reactivation of a pseudogene (fhs) shows adaptive metabolic plasticity, relevant for pathogen evolution.

(2) They systematically combine targeted gene deletions with suppressor screening to dissect the folate/one-carbon network (GCS, GlyA, Fhs/FolD).

Weaknesses:

(1) The study infers 1C-THF depletion mostly genetically and indirectly (growth rescue with adenine) without direct quantification of folate intermediates or fluxes. Biochemical confirmation, LC-MS-based metabolomics of folates/1C donors, or isotopic tracing would strengthen mechanistic claims.

(2) In multiple result sections, the authors report data from technical triplicates but do not mention independent biological replicates (e.g., Figure 2C, Figure 4A-B, Figure 6D). In addition, some results mention statistical significance but without a detailed description of the specific statistical tests used or replicates, such as Figure 2A-C, Figure 2E, and Figure 2G-I.

Reviewer #2 (Public review):

Summary:

The manuscript by Freier et al examines the impact of deletion of the glycine cleavage system (GCS) GcvPAB enzyme complex in the facultative intracellular bacterial pathogen Listeria monocytogenes. GcvPAB mediates the oxidative decarboxylation of glycine as a first step in a pathway that leads to the generation of N5, N10-methylene-Tetrahydrofolate (THF) to replenish the 1-carbon THF (1C-THF) pool. 1C-THF species are important for the biosynthesis of purines and pyrimidines as well as for the formation of serine, methionine, and N-formylmethionine, and the authors have previously demonstrated that gcvPAB is important for bacterial replication within macrophages. A significant defect for growth is observed for the gcvPAB deletion mutant in defined media, and this growth defect appears to stem from the sensitivity of the mutant strain to excess glycine, which is hypothesized to further deplete the 1C-THF pool. Selection of suppressor mutations that restored growth of gcvPAB deletion mutants in synthetic media with high glycine yielded mutants that reversed stop codon inactivation of the formate-tetrahydrofolate ligase (fhs) gene, supporting the premise that generation of N10-formyl-THF can restore growth. Mutations within the folk, codY, and glyA genes, encoding serine hydroxymethyltransferase, were also identified, although the functional impact of these mutations is somewhat less clear. Overall, the authors report that their work identifies three pathways that feed the 1C-THF pool to support the growth and virulence of L. monocytogenes and that this work represents the first example of the spontaneous reactivation of a L. monocytogenes gene that is inactivated by a premature stop codon.

Strengths:

This is an interesting study that takes advantage of a naturally existing fhs mutant Listeria strain to reveal the contributions of different pathways leading to 1C-THF synthesis. The defects observed for the gcvPAB mutant in terms of intracellular growth and virulence are somewhat subtle, indicating that bacteria must be able to access host sources (such as adenine?) to compensate for the loss of purine and fMet synthesis. Overall, the authors do a nice job of assessing the importance of the pathways identified for 1C-THF synthesis.

Weaknesses:

(1) Line 114 and Figure 1: The authors indicate that the gcvPAB deletion forms significantly fewer plaques in addition to forming smaller plaques (although this is a bit hard to see in the plaque images). A reduction in the overall number of plaques sounds like a bacterial invasion defect - has this been carefully assessed? The smaller plaque size makes sense with reduced bacterial replication, but I'm not sure I understand the reduction in plaque number.

(2) Do other Listeria strains contain the stop codon in fhs? How common is this mutation? That would be interesting to know.

(3) Based on the observation that fhs+ ΔgcvPAB ΔglyA mutant is only possible to isolate in complex media, and fhs is responsible for converting formate to 1C-THF with the addition of FolD, have the authors thought of supplementing synthetic media with formate and assessing mutant growth?

Reviewer #3 (Public review):

Summary:

In this study, Freier et al. demonstrate that 3 distinct metabolic pathways are critical for the synthesis of 1C-THF, a metabolite that is crucial for the growth and virulence of Listeria monocytogenes. Using an elegant suppressor screen, they also demonstrate the hierarchical importance of these metabolic pathways with respect to the biosynthesis of 1C-THF.

Strengths:

This study uses elegant bacterial genetics to confirm that 3 distinct metabolic pathways are critical for 1C-THF synthesis in L. monocytogenes, and the lack of either one of these pathways compromises bacterial growth and virulence. The study uses a combination of in vitro growth assays, macrophage-CFU assays, and murine infection models to demonstrate this.

Weaknesses:

(1) The primary finding of the study is that the perturbation of any of the 3 metabolic pathways important for the synthesis of 1C-THF results in reduced growth and virulence of L. monocytogenes. However, there is no evidence demonstrating the levels of 1C-THF in the various knockouts and suppressor mutants used in this study. It is important to measure the levels of this metabolite (ideally using mass spectrometry) in the various knockouts and suppressor mutants, to provide strong causality.

(2) The story becomes a little hard to follow since macrophage-CFU assays and murine infection model data precede the in vitro growth assays. The manuscript would benefit from a reorganization of Figures 2,3, and 4 for better readability and flow of data.