Pp6-Pfkfb1 axis modulates intracellular bacterial proliferation by orchestrating host-pathogen metabolic crosstalk

TL;DR

The study reveals how intracellular bacteria manipulate host metabolism through the Pp6-Pfkfb1 pathway to enhance their survival.

Contribution

The discovery of the Pp6-Pfkfb1 axis as a key regulator of host-pathogen metabolic interactions during intracellular bacterial infection.

Findings

Pp6 deficiency increases bacterial growth in macrophages by altering metabolic pathways.

Pfkfb1 promotes bacterial proliferation by modulating nitric oxide and arginine metabolism.

miR-31 deletion enhances bacterial clearance by upregulating Pp6.

Abstract

Intracellular bacterial pathogens exhibit heterogeneous replication rates within host macrophages, but the mechanisms by which they manipulate host factors for survival remain incompletely understood. Using a fluorescence-dilution reporter system in Salmonella Typhimurium (Salmonella)-infected macrophages, we found that Protein Phosphatase 6 (Pp6) was downregulated in macrophages harboring growing bacteria. Conditional knockout of Pp6 elevated host susceptibility to Salmonella-mediated lethality due to compromised antimicrobial defenses. MicroRNA-31 (miR-31) was identified as a negative regulator of Pp6, and its conditional ablation enhanced bacterial clearance. Yeast two-hybrid screening identified 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 (Pfkfb1), a metabolic regulator, as a substrate of Pp6. Pp6 deficiency resulted in significantly elevated expression of Pfkfb1, which was highly expressed in macrophages containing replicating Salmonella. Pfkfb1 deletion restricted bacterial proliferation by promoting nitric oxide (NO) production while concurrently suppressing arginase-1 (Arg-1) expression and impairing arginine metabolism in macrophages. Collectively, these results establish the Pp6-Pfkfb1 axis as a key regulator of host metabolic adaptation and intracellular bacterial survival, offering potential therapeutic targets against multidrug-resistant pathogens.

Intracellular pathogens secrete effector proteins that intercept and modify host cells to usurp host defenses and establish habitable intracellular niches, yet the host factors that are critical for intracellular bacterial survival are not fully understood. Using a fluorescence-dilution reporter system in Salmonella Typhimurium (Salmonella)-infected macrophages, we found Protein Phosphatase 6 (Pp6) was decreased in macrophages harboring replicating Salmonella and Pp6 deficiency increased bacterial growth in macrophages. On the other hand, the deletion of microRNA-31 (miR-31), a negative regulator of Pp6, enhanced Salmonella clearance. Furthermore, through yeast two-hybrid screening, we identified 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 (Pfkfb1) is a substrate of Pp6. And we demonstrated that Pfkfb1, abundantly expressed in macrophages containing growing bacteria, is crucial for Salmonella proliferation by regulating nitric oxide (NO) levels, arginase-1 (Arg-1) expression and arginine metabolism. Overall, the molecular changes in Pp6-Pfkfb1 axis drive host metabolic adaptations that enable intracellular bacterial survival.

Figures

Pp6 expression is downregulated in macrophages harboring replicating Salmonella.(A) Experimental workflow of BMDMs infected with Salmonella carrying the dual-reporter plasmid (pFCcGi). Three distinct macrophage populations were identified: (i) uninfected bystanders, (ii) BMDMs with non-growing bacteria, and (iii) BMDMs with growing bacteria. (B) The populations were validated using FACS and further characterized through immunocytochemistry imaging. Scale bar, 3 μm. (C-F) Different cell subpopulations were isolated at 18 h post infection (hpi) and the expression of Pp6 was respectively detected in non-infected, bystander, non-growing and growing group by immunocytochemistry (C) and western blot (E); corresponding statistical data were shown in D and F (n = 4). Scale bar, 25 μm. P-values were determined by two-tailed unpaired t-test (mean ± SEM). P < 0.01.

Pp6 deficiency promotes intracellular hyperproliferation of Salmonella in macrophages.(A-B) Flow cytometry analysis of Salmonella proliferation in BMDMs from LysMCrePp6fl/fl mice and Pp6fl/fl mice (n = 4) (A); statistical data were shown in (B). P-values were determined by two-tailed unpaired t-test (mean ± SEM). ns, not significant, *P < 0.05. (C) Kaplan-Meier survival curves of LysMCrePp6fl/fl mice and Pp6fl/fl mice (n = 10) infected intraperitoneally with 1 × 103 colony-forming units (CFU) WT Salmonella. P-value was determined by Log-rank (Mantel-Cox) test. *P < 0.001. (D) Schematic illustration of whole-mount staining of the mouse liver following in vivo infection. This figure was created in BioRender. Wang, L. (2026) https://BioRender.com/xrp6bgl. (E-H) Representative fluorescence labeling of GFP-labeled Salmonella and F4/80 in liver tissues from LysMCrePp6fl/fl mice and Pp6fl/fl mice following 3-day infection (E). The colocalization fluorescence intensity on the yellow dashed line of zoom in images (F, G) and the GFP signal intensity (H) were quantified using ImageJ software. Scale bar, 250 μm or 50 μm. P-value was determined by two-tailed unpaired t-test (mean ± SEM). P < 0.01.

miR-31 deficiency impairs Salmonella proliferation in macrophages.(A) Schematics of the WT and point-mutated 3’ UTR reporter constructs. TS, target site. (B) Luciferase activity assessment in RAW264.7 cells transfected with miR-31 mimics and either the WT or point-mutated 3′ UTR reporter construct (designated as WT UTR or mutant UTR) (n = 4). P-values were determined by two-tailed unpaired t-test (mean ± SEM). ns, not significant, P < 0.0001. (C) Western blot analysis of Pp6 protein levels in BMDMs from WT and miR-31-/- mice. (D-E) Flow cytometry analysis of infected BMDMs from LysMCremiR-31fl/fl and miR-31fl/fl mice (n = 4) (D). And statistics of differential cell groups with non-growing or growing bacteria (E). P-values were determined by two-tailed unpaired t-test (mean ± SEM). ns, not significant, *P < 0.05. (F) Kaplan-Meier survival curves of LysMCremiR-31fl/fl and miR-31fl/fl mice (n = 14) infected intraperitoneally with 1 × 103 CFU WT Salmonella. P-value was determined by Log-rank (Mantel-Cox) test. *P < 0.05. (G-J) Representative whole-mount fluorescence images of GFP-labeled Salmonella and F4/80 in liver tissues from LysMCremiR-31fl/fl mice and miR-31fl/fl mice 3 days post-infection (G). The colocalization fluorescence intensity on the yellow dashed line of zoom in images (H, I) and the GFP signal intensity (J) were quantified using ImageJ software. Scale bar, 100 μm or 50 μm. P-values were determined by two-tailed unpaired t-test (mean ± SEM). *P < 0.05.

PFKFB1 is a substrate of PP6.(A) Identification of Pp6-interacting protein using the yeast two-hybrid system. (B) Cell lysates from 293FT cells expressing myc-tagged PP6 were immunoprecipitated with antibodies against AURORA A, PFKFB1, PFKFB3, IκB-ε, or rabbit IgG control, and then incubated with anti-Myc. AURORA A [36] and IκB-ε [15] have been reported to interact with PP6, serving as positive controls in this study. (C) Lysates were immunoprecipitated with IgG or anti-Myc, and blotted with anti-PFKFB1 and anti-PP6. (D) Endogenous PP6 removal in 293FT cells using the CRISPR/Cas9 system and target protein expression analysis by western blot. (E) The phosphorylation sites of Ser-2, Ser-33 and Ser-178 were mutated with Ala substitution. Target protein levels were measured in WT and PP6-deficient 293FT cells transfected with these mutant plasmids.

Pfkfb1 promotes bacteria growth and regulates macrophage polarization.(A) Expression of Pfkfb1 and Pp6 in non-infected BMDMs, bystander BMDMs and BMDMs containing growing and non-growing bacteria was detected by western blot. (B-C) Flow cytometry analysis of Salmonella replication in BMDMs from WT and Pfkfb1-/- mice (n = 4-5) (B). And statistics of differential cell groups with non-growing or growing bacteria (C). P-values were determined by two-tailed unpaired t-test (mean ± SEM). ns: not significant, *P < 0.05. (D) Kaplan-Meier survival curves of WT and Pfkfb1-/- mice (n = 16-18) infected intraperitoneally with 1 × 103 CFU WT Salmonella. P-value was determined by Log-rank (Mantel-Cox) test. *P < 0.05. (E) Heatmap of differentially expressed genes in arginine biosynthesis and metabolism (n = 3). (F) GSEA of differentially expressed genes in mouse BMDMs with arginine biosynthesis-associated genes enriched (n = 3). (G-H) Flow cytometry analysis (G) and quantification (H) of Arg-1 in WT and Pfkfb1-/- BMDMs (n = 6). P-value was determined by two-tailed unpaired t-test (mean ± SEM). *P < 0.001. (I) The NO levels in WT and Pfkfb1-/- BMDMs were measured using Griess regents (n = 6). P-value was determined by two-tailed unpaired t-test (mean ± SEM). P < 0.01.

Graphical summary.The Pp6-Pfkfb1 axis, modulated by miR-31, orchestrates macrophage metabolic adaptation by targeting Pfkfb1 to regulate NO and arginine metabolism, thereby affecting Salmonella intracellular survival. This figure was created in BioRender. Wang, L. (2026) https://BioRender.com/cm1ly6b.

Full text

Introduction

Macrophages serve essential functions in preserving tissue homeostasis and providing host defense against pathogens [1]. Upon exposure to microbial signals, damaged tissue components, or activated lymphocyte-derived stimuli, macrophages undergo metabolic and functional reprogramming, resulting in distinct phenotypic states that dictate their capacity to regulate immune response [2]. When activated by lipopolysaccharide (LPS) or interferon (IFN)-γ, macrophages are polarized to a pro-inflammatory M1 phenotype, which harbor elevated intracellular nitric oxide (NO) levels and strong bactericidal activity. Under alternative conditions, interleukin-4 (IL-4) and interleukin-13 (IL-13) stimulation induces macrophage polarization toward a M2 phenotype, which exhibits increased arginase-1 (Arg-1) expression accompanied by metabolic reprogramming of arginine metabolism toward ornithine and polyamine biosynthesis. M2-polarized macrophages primarily mediate tissue remodeling and anti-inflammatory response [3–5].

Salmonella Typhimurium (Salmonella) is an invasive, facultative enteric pathogen responsible for gastroenteritis and life-threatening systemic diseases in humans and animals [6]. Macrophages engulf intracellular bacterial pathogens, such as Salmonella, which subsequently replicates within host cells and causes difficult-to-eradicate infections. Once internalized, Salmonella triggers multiple host cell responses, including specific metabolic alterations mediated by certain virulence factors to provide energy sources and suitable environments for bacterial growth [7,8]. Multiple bacteria-centered mechanisms required for intracellular survival have been unveiled. For instance, the ZnuABC transporter and its associated glycolytic activity in Salmonella contribute to antinitrosative defenses against NO [9]; reactive persulfides from Salmonella inhibit autophagy-mediated bacterial clearance in macrophages [10]; Salmonella Pathogenicity Island 2-encoded type III secretion system confers resistance to reactive oxygen species [6]. Nevertheless, host cellular components manipulated by Salmonella to indulge bacterial replication remain poorly defined. A recent single-cell transcriptomic analysis revealed that macrophages harboring non-growing Salmonella display hallmarks of pro-inflammatory M1 polarization, whereas those containing growing bacteria undergo metabolic reprogramming toward anti-inflammatory M2-like state [11,12]. Therefore, deciphering the mechanistic basis underlying macrophage state transitions between non-growing and growing bacteria will uncover potential therapeutic targets and advance the treatment strategies for intracellular bacterial infections.

Protein phosphatase 6 (Pp6), a member of the type 2A Ser/Thr protein phosphatase family, is a master regulator of cellular defense, serving as a critical brake on NF-κB-driven inflammation [13–15] and a key facilitator of DNA damage repair [16–18]. The relevance of this phosphatase family in antibacterial defense is underscored by the finding that PP2A regulates Rho GTPase [19,20] and that its regulatory subunit PPP2R3C modulates the replication of Salmonella within macrophages [21]. Despite these compelling connections, the specific role of Pp6 in macrophage-mediated antimicrobial immunity remains a critical knowledge gap. This study therefore aims to define the function and elucidate the molecular mechanisms by which Pp6 shapes the host antibacterial response to Salmonella.

Here, our study demonstrates that during Salmonella infection, Pp6 exhibits marked reduction within macrophages containing replicating bacteria and its absence facilitates Salmonella proliferation and shortens the survival of infected mice. In contrast, knockout of microRNA-31 (miR-31), a negative regulator of Pp6, suppresses Salmonella replication. Critically, phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 (Pfkfb1), an enzyme crucial for metabolism-oriented macrophage polarization [22], is identified as a substrate of Pp6 and distinctly expressed in macrophages containing growing pathogens. Pp6 deficiency impairs Pfkfb1 dephosphorylation and increases Pfkfb1 expression levels. Genetic ablation of Pfkfb1 increases NO production and downregulates Arg-1 expression as well as arginine biosynthesis and metabolism, thereby restricting Salmonella replication in infected cells. Collectively, these findings highlight the Pp6-Pfkfb1 axis as a pivotal regulator of host-pathogen interactions, indicating that therapeutic targeting of this pathway may potentiate host antibacterial defenses.

Results

Pp6 deficiency enhances bacterial replication in macrophages

Given the significant heterogeneity in growth rates exhibited by intracellular bacterial pathogens within host macrophages [11], a Salmonella strain harboring a dual-reporter plasmid (pFCcGi) was developed to monitor bacterial replication following internalization [23]. This system utilizes constitutively expressed mCherry and conditionally expressed GFP, permitting differentiation of three distinct states: (i) actively dividing bacteria (increasing mCherry/stable GFP), (ii) non-replicating bacteria (stable dual fluorescence), and (iii) bystander macrophages (double-negative) [11,12] (Fig 1A). Fluorescence-activated cell sorting (FACS) successfully resolved the three macrophage populations: bystander, and those containing growing/non-growing bacteria (Fig 1B). We further validated that the gating strategy effectively isolated macrophages with distinct intracellular bacterial loads by reanalyzing the sorted cells using fluorescence microscopy (Fig 1B). Immunocytochemistry analysis revealed a diminished level of Pp6 in macrophages harboring growing Salmonella, relative to non-infected controls, bystander macrophages and macrophages containing non-replicating bacteria (Figs 1C, 1D and S1). This observation received further validation via western blot analysis (Fig 1E and 1F).

*Pp6 expression is downregulated in macrophages harboring replicating Salmonella.(A) Experimental workflow of BMDMs infected with Salmonella carrying the dual-reporter plasmid (pFCcGi). Three distinct macrophage populations were identified: (i) uninfected bystanders, (ii) BMDMs with non-growing bacteria, and (iii) BMDMs with growing bacteria. (B) The populations were validated using FACS and further characterized through immunocytochemistry imaging. Scale bar, 3 μm. (C-F) Different cell subpopulations were isolated at 18 h post infection (hpi) and the expression of Pp6 was respectively detected in non-infected, bystander, non-growing and growing group by immunocytochemistry (C) and western blot (E); corresponding statistical data were shown in D and F (n = 4). Scale bar, 25 μm. P-values were determined by two-tailed unpaired t-test (mean ± SEM). *P < 0.01.

To investigate the role of Pp6 in bacterial replication, LysM^Cre^Pp6^fl/fl^ mice were generated (S2A Fig), with macrophage-specific Pp6 depletion confirmed by western blot (S2B Fig). In vitro infection assays using bone marrow-derived macrophages (BMDMs) demonstrated significantly elevated bacterial loads in LysM^Cre^Pp6^fl/fl^ macrophages compared to Pp6^fl/fl^ controls during the replicative phase of pFCcGi-expressing Salmonella, as quantified by flow cytometry analysis (Fig 2A and 2B). For in vivo assessment, intraperitoneal challenge with a lethal dose of wild-type (WT) Salmonella resulted in markedly decreased survival in LysM^Cre^Pp6^fl/fl^ mice compared with Pp6^fl/fl^ mice (Fig 2C). Whole-mount imaging of liver tissues at 3 days post-infection enabled spatial mapping of bacterial dissemination (Fig 2D), revealing substantially enhanced bacterial colonization within the livers of LysM^Cre^Pp6^fl/fl^ mice versus littermate controls (Fig 2E-2H). These results collectively establish Pp6 as a critical host regulatory factor limiting intracellular bacterial proliferation.

*Pp6 deficiency promotes intracellular hyperproliferation of Salmonella in macrophages.(A-B) Flow cytometry analysis of Salmonella proliferation in BMDMs from LysMCrePp6fl/fl mice and Pp6fl/fl mice (n = 4) (A); statistical data were shown in (B). P-values were determined by two-tailed unpaired t-test (mean ± SEM). ns, not significant, *P < 0.05. (C) Kaplan-Meier survival curves of LysMCrePp6fl/fl mice and Pp6fl/fl mice (n = 10) infected intraperitoneally with 1 × 103 colony-forming units (CFU) WT Salmonella. P-value was determined by Log-rank (Mantel-Cox) test. ***P < 0.001. (D) Schematic illustration of whole-mount staining of the mouse liver following in vivo infection. This figure was created in BioRender. Wang, L. (2026) https://BioRender.com/xrp6bgl. (E-H) Representative fluorescence labeling of GFP-labeled Salmonella and F4/80 in liver tissues from LysMCrePp6fl/fl mice and Pp6fl/fl mice following 3-day infection (E). The colocalization fluorescence intensity on the yellow dashed line of zoom in images (F, G) and the GFP signal intensity (H) were quantified using ImageJ software. Scale bar, 250 μm or 50 μm. P-value was determined by two-tailed unpaired t-test (mean ± SEM). *P < 0.01.

miR-31 deficiency rescues the expression of Pp6 and facilitates Salmonella clearance

Our previous work identified that Pp6 as a key target of miR-31 in keratinocytes, with miR-31 deletion restoring the expression of Pp6 [24]. Therefore, we constructed a luciferase-expressing plasmid to investigate the post-transcriptional regulation mediated by miR-31 binding to the 3’ untranslated region (UTR) of Pp6 mRNA in macrophages (Fig 3A). As a result, forced miR-31 expression in RAW264.7 cells and BMDMs significantly reduced luciferase activity driven by the WT 3’ UTR plasmid, whereas no reduction occurred with the mutated plasmid lacking the target sequence (Figs 3B and S3A). This confirmed direct targeting of Pp6 by miR-31 in macrophages. Measurement of Pp6 protein levels in BMDMs from miR-31^-/-^ mice and littermate controls at different time points after infection revealed progressive Pp6 reduction from 2 to 12 hours in WT controls. Conversely, miR-31^-/-^ macrophages exhibited partial Pp6 restoration during this period (Figs 3C and S3B). We next generated LysM^Cre^miR-31^fl/fl^ mice by crossing miR-31 floxed mice with LysM Cre transgenic mice [24,25] (S3C and S3D Fig). Infection of BMDMs from LysM^Cre^miR-31^fl/fl^ and miR-31^fl/fl^ mice with Salmonella carrying pFCcGi revealed a decreased proportion of LysM^Cre^miR-31^fl/fl^ macrophages containing replicating bacteria compared to miR-31^fl/fl^ controls (Fig 3D and 3E). Moreover, following intraperitoneal challenge with a lethal dose of WT Salmonella, LysM^Cre^miR-31^fl/fl^ mice exhibited a significantly enhanced survival rate than controls (Fig 3F). To further assess bacterial proliferation in macrophages, we infected mice with GFP-labeled Salmonella and observed a significantly reduced colonization in LysM^Cre^miR-31^fl/fl^ mice at day 3 after Salmonella infection (Fig 3G-3J). Taken together, our findings demonstrate that miR-31 deletion rescues Pp6 expression, reduces the proportion of macrophages harboring growing Salmonella, and enhances host resistance to infection.

*miR-31 deficiency impairs Salmonella proliferation in macrophages.(A) Schematics of the WT and point-mutated 3’ UTR reporter constructs. TS, target site. (B) Luciferase activity assessment in RAW264.7 cells transfected with miR-31 mimics and either the WT or point-mutated 3′ UTR reporter construct (designated as WT UTR or mutant UTR) (n = 4). P-values were determined by two-tailed unpaired t-test (mean ± SEM). ns, not significant, ****P < 0.0001. (C) Western blot analysis of Pp6 protein levels in BMDMs from WT and miR-31-/- mice. (D-E) Flow cytometry analysis of infected BMDMs from LysMCremiR-31fl/fl and miR-31fl/fl mice (n = 4) (D). And statistics of differential cell groups with non-growing or growing bacteria (E). P-values were determined by two-tailed unpaired t-test (mean ± SEM). ns, not significant, *P < 0.05. (F) Kaplan-Meier survival curves of LysMCremiR-31fl/fl and miR-31fl/fl mice (n = 14) infected intraperitoneally with 1 × 103 CFU WT Salmonella. P-value was determined by Log-rank (Mantel-Cox) test. *P < 0.05. (G-J) Representative whole-mount fluorescence images of GFP-labeled Salmonella and F4/80 in liver tissues from LysMCremiR-31fl/fl mice and miR-31fl/fl mice 3 days post-infection (G). The colocalization fluorescence intensity on the yellow dashed line of zoom in images (H, I) and the GFP signal intensity (J) were quantified using ImageJ software. Scale bar, 100 μm or 50 μm. P-values were determined by two-tailed unpaired t-test (mean ± SEM). P < 0.05.

Pfkfb1 is a substrate of Pp6

To identify Pp6 substrates critical for macrophage functional regulation, a mouse universal cDNA library was employed in a yeast two-hybrid screening using Pp6 as bait. This approach identified 95 unique candidate substrates, with Pfkfb1 exhibiting robust binding affinity for Pp6 (S1 Table and Fig 4A). Co-immunoprecipitation assays performed in 293FT cells expressing PFKFB1 and myc-tagged PP6 further validated the PP6-PFKFB1 interaction (Fig 4B and 4C). Moreover, this interaction was also verified in BMDMs (S4A Fig). CRISPR/Cas9-mediated PP6 ablation in 293FT cells significantly increased PFKFB1 protein expression (Fig 4D). To investigate the PP6-regulated phosphorylation sites on PFKFB1, LC-MS/MS analysis of PFKFB1 immunoprecipitates from WT and PP6-deficient 293FT cells was performed. The results detected significantly enhanced phosphorylation at Ser-2, Ser-33, and Ser-178 residues in PP6-depleted cells (S4B-S4D Fig). Subsequent evaluation of PFKFB1 protein levels following alanine (Ala) substitution mutations at these phosphorylation sites revealed that PFKFB1^S33-mut^ and PFKFB1^S178-mut^ maintained expression patterns consistent with the control group (Fig 4E). In contrast, the PFKFB1^S2-mut^ mutation abolished PP6-mediated regulation of PFKFB1 protein expression (Fig 4E). These findings identify PFKFB1 as a primary substrate of PP6 and establish that PP6 governs PFKFB1 protein stability through site-specific dephosphorylation at Ser-2.

PFKFB1 is a substrate of PP6.(A) Identification of Pp6-interacting protein using the yeast two-hybrid system. (B) Cell lysates from 293FT cells expressing myc-tagged PP6 were immunoprecipitated with antibodies against AURORA A, PFKFB1, PFKFB3, IκB-ε, or rabbit IgG control, and then incubated with anti-Myc. AURORA A [36] and IκB-ε [15] have been reported to interact with PP6, serving as positive controls in this study. (C) Lysates were immunoprecipitated with IgG or anti-Myc, and blotted with anti-PFKFB1 and anti-PP6. (D) Endogenous PP6 removal in 293FT cells using the CRISPR/Cas9 system and target protein expression analysis by western blot. (E) The phosphorylation sites of Ser-2, Ser-33 and Ser-178 were mutated with Ala substitution. Target protein levels were measured in WT and PP6-deficient 293FT cells transfected with these mutant plasmids.

Pfkfb1 is essential for Salmonella growth and influences macrophage polarization

To determine whether Pfkfb1 functionally regulates intracellular bacterial replication within macrophages, Pfkfb1 expression was first assessed across FACS-sorted macrophage subsets: non-infected cells, infected cells harboring growing or non-growing bacteria, and bystander cells. Notably, Pfkfb1 expression was exclusively detected in macrophages containing replicating bacteria, concomitant with reduced Pp6 levels (Figs 5A and S5A). Subsequently, we generated a Pfkfb1 knockout (Pfkfb1^-/-^) mouse model to further explore its role (S5B and S5C Fig). Infection of WT and Pfkfb1^-/-^ macrophages with pFCcGi-expressing Salmonella mediated a pronounced reduction in bacterial load within Pfkfb1^-/-^ macrophages relative to WT controls (Fig 5B and 5C). Furthermore, intraperitoneal infection with a lethal dose of Salmonella resulted in significantly increased survival of Pfkfb1^-/-^ mice compared to WT littermate controls (Fig 5D****).

*Pfkfb1 promotes bacteria growth and regulates macrophage polarization.(A) Expression of Pfkfb1 and Pp6 in non-infected BMDMs, bystander BMDMs and BMDMs containing growing and non-growing bacteria was detected by western blot. (B-C) Flow cytometry analysis of Salmonella replication in BMDMs from WT and Pfkfb1-/- mice (n = 4-5) (B). And statistics of differential cell groups with non-growing or growing bacteria (C). P-values were determined by two-tailed unpaired t-test (mean ± SEM). ns: not significant, *P < 0.05. (D) Kaplan-Meier survival curves of WT and Pfkfb1-/- mice (n = 16-18) infected intraperitoneally with 1 × 103 CFU WT Salmonella. P-value was determined by Log-rank (Mantel-Cox) test. *P < 0.05. (E) Heatmap of differentially expressed genes in arginine biosynthesis and metabolism (n = 3). (F) GSEA of differentially expressed genes in mouse BMDMs with arginine biosynthesis-associated genes enriched (n = 3). (G-H) Flow cytometry analysis (G) and quantification (H) of Arg-1 in WT and Pfkfb1-/- BMDMs (n = 6). P-value was determined by two-tailed unpaired t-test (mean ± SEM). ***P < 0.001. (I) The NO levels in WT and Pfkfb1-/- BMDMs were measured using Griess regents (n = 6). P-value was determined by two-tailed unpaired t-test (mean ± SEM). *P < 0.01.

To explore the mechanistic basis of these findings, RNA-seq analysis was performed on BMDMs derived from WT and Pfkfb1^-/-^ mice. Interestingly, we found that most genes implicated in arginine biosynthesis and metabolism exhibited reduced expression in BMDMs from Pfkfb1^-/-^ mice compared with WT controls (Fig 5E). Gene set enrichment analysis (GSEA) further revealed the diminished activity in arginine biosynthesis pathway within Pfkfb1^-/-^ macrophages (Fig 5F). Arginine serves as a common substrate for both arginase and nitric oxide synthase (NOS). Through flow cytometry analysis, we observed the expression of arginase-1 (Arg1), a characteristic marker of M2 macrophages, was markedly reduced in Pfkfb1^-/-^ macrophages (Fig 5G and 5H). Concurrent assessment of NO expression levels demonstrated a marked increase in NO production in Pfkfb1^-/-^ cells relative to WT counterparts (Fig 5I). Collectively, these results highlight Pfkfb1 as a critical metabolic regulator that modulates macrophage functionally and orchestrates host antimicrobial defense against bacterial infections.

Discussion

Macrophage polarization is a dynamic process driven by extensive metabolic reprogramming, with the balance between pro-inflammatory M1 and anti-inflammatory M2 phenotypes playing a critical role in immune response [2,4]. Although heterogeneity in macrophage polarization and Salmonella growth rate has been extensively investigated [11,12,26], the underlying mechanisms remain incompletely characterized. This study demonstrated that the host Pp6/Pfkfb1 axis drives macrophage polarization toward to M2 phenotype, creating a permissive niche for Salmonella survival and replication within macrophages. These findings suggest a potential therapeutic target for infectious diseases mediated by intracellular multidrug-resistant bacteria.

Pp6 exhibits high conservation across all eukaryotic species from yeast to humans, highlighting its fundamental biological significance. Mutations in Pp6 exist in 9-12.4% of melanomas and potentially drive melanoma development [27,28]. Sit4/Ppe1, a Pp6 yeast homologue, is required for G1/S progression [29]. Human Pp6 plays critical roles in DNA damage responses via modulating DNA-dependent protein kinase (DNA-PK) signaling and homologous recombination-mediated repair of DNA double-strand breaks (DSBs) [16,30], and interacts with Aurora A kinase [31]. Furthermore, recent studies indicate a broader role of Pp6 in pre-mRNA splicing [32], apoptosis regulation in immune cells [33], and Hippo pathway signaling modulation [34]. Collectively, these studies reveal that Pp6 integrates multiple signaling pathways. However, the intrinsic function of Pp6 in bacterial replication within infected macrophages has not been explored. Here, our research provides the first mechanistic evidence for Pp6-mediated regulation of intracellular bacterial growth in macrophages. We demonstrate that Pp6 expression is downregulated in macrophages during Salmonella infection, and that conditional deletion of Pp6 enhances intracellular bacterial growth and significantly reduces host survival. These results establish Pp6 as a crucial regulator of immune responses against intracellular bacterial infections.

A previous study reported that miR-31 directly targets Pp6, with miR-31 knockout rescuing the expression of Pp6 [24]. This regulatory relationship was confirmed within macrophages. Using genetic knockout mouse models, we further show that conditional ablation of miR-31 attenuates intracellular bacterial proliferation in both in vitro and in vivo settings, thereby validating the functional role of Pp6 in macrophages during Salmonella infection.

Pfkfb1, expressed in M2 macrophages [35], remains poorly understood in the context of intracellular bacterial infections. Here, for the first time we identify Pfkfb1 is a direct substrate of Pp6, and Pp6 deficiency increases Pfkfb1 expression and phosphorylation levels. Notably, Pfkfb1 expression is specifically detected in macrophages containing replicating bacteria. Experimental evidence confirms significant reduction in Salmonella growth and dissemination in Pfkfb1-deficient macrophages. Furthermore, Pfkfb1 is shown to regulate macrophage polarization through modulating NO and Arg-1 expression, thereby controlling bacterial replication. While these findings establish involvement of Pfkfb1 in bacterial pathogenesis, the underlying mechanisms governing Pfkfb1-mediated regulation of intracellular bacterial growth require further studies.

In summary (Fig 6), this study reveals that Pp6 scavenges Salmonella from infected macrophages by negatively regulating Pfkfb1. During infection, Pfkfb1 regulates macrophages metabolic reprogramming to support bacteria proliferation within host cells. This regulatory mechanism represents a potential therapeutic target for interventions against infectious diseases caused by intracellular bacterial pathogens.

Graphical summary.The Pp6-Pfkfb1 axis, modulated by miR-31, orchestrates macrophage metabolic adaptation by targeting Pfkfb1 to regulate NO and arginine metabolism, thereby affecting Salmonella intracellular survival. This figure was created in BioRender. Wang, L. (2026) https://BioRender.com/cm1ly6b.

Materials and methods

Ethics statement

All experimental mice were maintained under specific pathogen-free (SPF) conditions in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals with the approval (SYXK-2003–0026) by the Scientific Investigation Board of Shanghai Jiao Tong University School of Medicine (Shanghai, China).

Bacterial strains and bacteria culture

The Salmonella strains used in this study were wild-type (WT) Salmonella Typhimurium S14028 (ATCC) strains along with two genetically modified derivatives: Salmonella carrying pFCcGi [11,12,23] and GFP-labeled Salmonella [21]. WT strains were cultured for 16 h at 37°C in LB medium. The pFCcGi-carrying strains were cultured for 16 h at 37°C in a MgMES pH 5.0 minimum medium containing 5 mM KCl, 7.5 mM (NH4)2_SO_4, 1 mM KH_2_PO_4_, 8 mM MgCl_2_, 38 mM glycerol, 0.1% (w/v) casamino acids, 0.5 mM K_2_SO_4_, 170 mM MES pH 5.0 in distilled water (dH_2_O), supplemented with 0.2% L-arabinose and 100 μg/mL ampicillin. GFP-labeled Salmonella was cultured for 16 h at 37°C in LB medium, supplemented with 15 μg/mL Gentamycin. Before infecting cells, bacteria were opsonized with 10% normal mouse serum (YEASEN) for 20 min at room temperature to obtain a phagocytosis rate of one bacterium per macrophage.

Cell culture

Primary murine bone marrow-derived macrophages (BMDMs) were obtained from femoral and tibial bones of C57BL/6 WT mice or transgenic mice aged 8–10 weeks-old. The cells were cultured for 7 days in DMEM/high glucose medium (HyClone) containing 10% fetal bovine serum (FBS) (Gibco), 55 μM 2-Me (Gibco), 1% Penicillin/streptomycin/Amphotericin B (Solarbio), 100 μM Non-Essential amino acids (Solarbio), 10 mM HEPES (Solarbio), 1 mM sodium pyruvate (Solarbio) and 20 ng/mL macrophage colony-stimulating factor (M-CSF) (PeproTech), under standard culture conditions (37°C, 5% CO_2_). The culture medium was partially refreshed every 2 days. RAW264.7 cells were cultured in RPMI-1640 medium (Gibco) containing 5% FBS. HEK293FT cells were cultivated in DMEM/high glucose medium containing 10% FBS.

Mice

C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China). LysM cre mice (stock number: 004781) were obtained from The Jackson Laboratory. The miR-31^fl/fl^ mice were generated as previously reported [24] and subsequently crossed with LysM cre transgenic mice. Pp6^fl/fl^ transgenic mice, provided by Dr. Wufan Tao, were similarly bred with LysM cre mice. The Pfkfb1^+/-^ mice were procured from Cyagen Biosciences.

In vitro infection

To identify replicating and non-replicating intracellular Salmonella, BMDMs were plated at a density of 1.5x10^6^ cells per well in 12-well plate. Following overnight culture, the cells were infected with Salmonella-pFCcGi at a multiplicity of infection (MOI) of approximately 5 after washed with PBS. Centrifuge plate at 1600rpm for 5 min, followed by 30 min incubation at 37°C with 5% CO_2_. After incubation, the infected cells were washed three times with PBS and supplemented with fresh medium containing 100 μg/mL gentamicin for 30 min to eliminate extracellular bacteria. BMDMs were washed three times and incubated in medium with 5 μg/mL gentamicin for the remainder of the infection. After 18 h (Pp6 mice groups) or 22 h (other mice groups) of infection, cells were detached with 0.5% trypsin-EDTA (Gibco), stained with DAPI (BD Biosciences, 1:1000) for 10 min, and analyzed by flow cytometry (BD Fortessa) or sorted (BD FACS Aria III). FlowJo software was employed for data analysis.

FACS sorting

To discriminate different subsets of BMDMs with bacteria carrying PFCcGi plasmid, cells were sorted post-infection. All experimental analyses excluded apoptotic macrophages and cellular doublets through appropriate gating strategies. Cell populations were isolated using a BD FACS Aria III sorter. Subsequently, sorted samples were processed for either western blot or immunofluorescence assay.

Immunofluorescence

Infected-BMDMs were sorted into three groups according to intracellular bacterial fluorescence intensity. Each subpopulation was plated separately into 10 mm culture dishes. Bacterial contents were confirmed using fluorescence microscopy. Cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature (RT). Subsequent processing included two washes with 0.1% PBST, followed by incubation with primary anti-PPP6C antibody (Abcam, 1:100) for 2 h at RT. After two additional 0.1% PBST washes and one PBS wash, samples were incubated with fluorochrome-conjugated secondary antibodies (Life Technologies, 1:500) for 1 h at RT in dark. Then incubated with DAPI (BD Biosciences, 1:1000) for 5 min at RT in dark. Following two final 0.1% PBST washes and one PBS wash, samples were mounted using fluorescent mounting medium (Dako cat. S3023) and visualized with a Zeiss Leica TCS SP8 confocal microscope.

In vivo infection

For survival rate assessment, WT Salmonella infection was established by infecting 8–9 weeks-old mice with 1 × 10^3^ CFU bacteria administered intraperitoneally. Mortality was recorded daily. To monitor in vivo bacterial proliferation, GFP-labeled Salmonella infection was established by infecting 8–9 weeks-old mice with 1 × 10^3^ CFU bacteria administered intraperitoneally. At 3 days post-infection, animals were euthanized and liver tissues were collected for whole-mount immunofluorescence. For these experiments, mice were maintained in the P2 laboratory of Prof. X. Jia Group, Clinical Medicine Scientific and Technical Innovation Park, Shanghai Tenth People’s Hospital.

Whole-mount immunofluorescence

Whole-mount samples were prepared following the Rapiclear 1.52 Solution protocol. Briefly, C57BL/6 mice were euthanized and subjected to cardiac perfusion with PBS. Liver samples were subsequently isolated and fixed with a 4% paraformaldehyde solution. Following three PBS washes, permeabilization was conducted using 2% PBST (2% Triton X-100, 0.05% sodium azide in PBS) for 3 days at RT. Samples were then rinsed with PBS and incubated in blocking buffer on an orbital shaker for 2 days at 4°C. Immunostaining proceeded with F4/80 (Santa Cruz Biotechnology, clone A3-1, 1:200) for 4 days at 4°C. After incubation, samples were washed with washing buffer 3 times in RT and then overnight at 4°C. Afterward, samples were then incubated with a secondary antibody (Life Technologies, 1:500) for 2 days at 4°C and washed with washing buffer 3 times in RT then overnight at 4°C. Following three PBS washes, nuclear staining was performed using DAPI for 1.5 h at RT. Subsequent PBS washing preceded tissue clearing with Rapiclear reagent (SunJin Lab, cat: RC152001). Cleared specimens were mounted in Glass Bottom Cell Culture Dish (NEST) for imaging. Images were acquired using a confocal microscopy system(Leica, TSC SP8) with HC PL APO CS2 10 × /0.40 DRY objective, with a scanning speed of 200 Hz and a step size of 2.0 μm. The X/Y acquiring format was set as 1024 × 1024, and Z-stack size was determined by specimen thickness respectively.

Luciferase reporter assay

The Pp6 3’ UTR was cloned using primers: Pp6 Forward, 5’-CCGCTCGAGCTC AAATGCTGCCTCTTGCCTTTTTTTTTAAT-3’, Reverse, 5’-ATAAGAATGCGG CCGCGAGGTTTACAGCCGGGTTGA-3’. The mutagenic primers used for Pp6 were Mutant Forward, 5’-CCGCTCGAGCTCAAATGCTGCCGCGTACATTTTTT TTTAAT-3’. A genomic fragment corresponding to the Pp6 3’ UTR was cloned into the multiple cloning site of the psiCheck-2 synthetic firefly luciferase reporter plasmid (Promega, #C8021). Plasmid integrity was verified through DNA sequencing.

RAW264.7 cells were seeded in 48-well plates and transfected with a complex containing 100 ng 3’ UTR luciferase reporter vector and 50 pmol miR-31 mimics or miRNA NC. Following 24-hour incubation post-transfection, cells lysis was performed.

BMDMs were seeded in 24-well plates and infected with Ppp6c 3’ UTR (WT or Mut) lentivirus (Shanghai Quan Yang Biological Technology), following the manufacturer’s protocol. At 72 hours post-infection, the cells were transfected with miR-31 mimics or miRNA NC. Cell lysis was carried out after a further 24 hours of incubation.

Luciferase activity quantification was conducted using the Dual-Luciferase Reporter Assay System (Promega) with measurements taken on a Lumat^3^ LB 9508 Single Tube Luminometer instrument (Berthold Technologies). Each experiment was repeated four times. The ratio of Renilla-to-Firefly luciferase was calculated for each well.

Yeast-two hybrid screening

Sequence encoding mouse Pp6 was inserted into the pGBKT7 vector (BD-Pp6) and transformed into Y2H Gold yeast strain using Yeastmaker Yeast Transformation System 2 (Clontech, Cat. 630440). A normalized universal mouse cDNA library transformed into Y187 yeast strain was purchased from Clontech (Cat. 630482). Library screening was performed using Matchmaker Gold Yeast Two-Hybrid System (Clontech, Cat. 630489) according to the manufacturer’ s instructions. Selected clones were isolated and sequenced. The prey plasmids were rescued and the positive interactions were confirmed using minimal media quadruple dropout plus X-α-Gal and Aureobasidin A (QDO/X/A).

Co-Immunoprecipitation

293FT cells were transfected with PFKFB1 and myc-tagged pp6 plasmids. The cells were harvested and lysed on ice for 20 minutes in ProteinExt Mammalian Total Protein Extraction Kit (TransGen Biotech) supplemented with protease inhibitor (Thermo Scientific) and phosphatase inhibitor A&B (Thermo Scientific). The lysates were incubated with anti-Myc (proteintech, 5 μg) and rabbit IgG (Invitrogen, 5 μg) or anti-PP6C (Merck, 5 μg) and rabbit IgG (Invitrogen, 5 μg) overnight at 4°C, followed by binding of protein A/G magnetic beads (selleck) for 4h at 4°C. The beads were rinsed five times with ice-cold lysis buffer and then eluted with SDS loading buffer. The immunoprecipitates were then subjected to immunoblotting.

Western blotting

Cells lysis was performed using a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 250 mM mannitol, 1% (v/v) Triton X-100, supplemented with protease inhibitor (Thermo Scientific) and phosphatase inhibitor A&B (Thermo Scientific). The proteins (10–30 μg) were loaded onto SDS-PAGE. Immunoblotting was performed with the following antibodies: Rabbit anti-PP6C (Merck, 1:1000; Proteintech, 1:1000), Rabbit anti-PFKFB1 (abcam, 1:500; Invitrogen, 1:500), Mouse anti-β-actin (Proteintech, 1:10000), HRP-labeled goat anti-mouse IgG (H + L) (Beyotime, 1:2000), HRP-labeled goat anti-rabbit IgG (H + L) (Beyotime, 1:2000), and anti-rabbit IgG for IP, AlpSdAbs VHH(HRP) (1:10000). Protein signals were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore Sigma) and captured with GE Amersham Imager 600 (GE Healthcare).

Untargeted metabolomics analysis

WT and PP6-deficient 293FT cells were cultivated. The samples were snap frozen and metabolites were extracted with 80% ice-cold methanol. Liquid chromatography-mass spectrometry (LC-MS) was performed on an Ultimate 3000-Velos Pro system equipped with a binary solvent delivery manager and a sample manager, coupled with a LTQ Orbitrap Mass Spectrometer equipped with an electrospray interface (ThermoFisher Scientific). The raw data were processed with progenesis QI (Walters Corporation) and Principal component analysis (PCA) and hierarchical clustering of metabolites were analyzed.

Detection of NO production

Cells were lysed with cell lysis buffer for Western and IP (P1003, Beyotime) and the concentration of NO was determined using an NO assay kit (S0021, Beyotime) based on the Griess reaction. All samples were prepared according to the manufacturer’s protocol.

Flow cytometry

BMDMs were initially digested using 0.5% trypsin-EDTA solution (Gibco) and were stained with Fc blocker and Live/dead Fixable Blue Dead Cell Stain Kit (Thermo Scientific cat. L34962) for 20 min at 4°C. Cells were then washed with PBS containing 2% FBS. Cell fixation and permeabilization were performed using the Cytosis/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences cat. 554714). Cells stained with antibody AF-700 anti-hu/mo Arginase 1(Invitrogen, 1:200) for 30 min at 4°C. Samples were subsequent run on a BD Fortessa instrument and the data were analyzed using FlowJo software.

RNA-seq and transcriptome analysis

BMDMs isolated from WT and Pfkfb1^-/-^ mice underwent two PBS washes prior to RNA extraction. Total RNA isolation was performed using RNAiso Plus (Takara Bio), followed by purification with magnetic oligo (dT) beads after denaturation. Purified mRNA samples were reverse-transcribed into fragmented DNA samples and adenylated at the 3′ ends. Library preparation involved adapter ligation, followed by DNA quantification using Qubit (Invitrogen). After cBot cluster generation, DNA samples were sequenced by an Illumina NovaSeq 6000 instrument from Shanghai Xu ran Biological Technology Co., LTD. Raw sequencing data were converted into FASTQ format, and transcript per million fragments mapped (fragments per kilobase) was calculated and log2-transformed using Cuffnorm. Differential gene transcripts were analyzed with DESeq, followed by gene set enrichment analysis (GSEA).

Statistical analysis

All data were analyzed using GraphPad Prism version 8.0, and are presented as the mean ± SEM. Statistical comparisons employed Student’s t-test for parametric data and Log-rank test for survival analysis. The probability values of P < 0.05 were considered statistically significant, with the following notation scheme applied: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Non-significant results were designated as “ns”. The error bars depict the SEM.

Supporting information

S1 FigThe expression of Pp6 in BMDMs after infection (unsorted).(A-B) The expression of Pp6 in BMDMs after infection (unsorted) was detected by immunocytochemistry (A) and statistical data were shown in (B). Scale bar, 25 μm. P-values were determined by two-tailed unpaired t-test (mean ± SEM). **P < 0.01.(DOCX)

S2 FigStrategies for Pp6 conditional knockout mouse construction.(A-B) Schematic of Pp6 deletion in LysM^Cre^Pp6^fl/fl^ mice (A) and western blot analysis of Pp6 expression in BMDMs from LysM^Cre^Pp6^fl/fl^ and Pp6^fl/fl^ mice (B).(DOCX)

S3 FigmiR-31 directly targets Pp6 in BMDMs and strategies for miR-31 conditional knockout mouse construction.(A) Luciferase activity assessment in BMDMs transfected with miR-31 mimics and either the WT or point-mutated 3’ UTR reporter construct (n = 4). P-values were determined by two-tailed unpaired t-test (mean ± SEM). ns, not significant, ***P < 0.001. (B) Quantification of Pp6 relative expression in BMDMs from WT and miR-31^-/-^ mice (n = 4). P-value was determined by two-tailed unpaired t-test (mean ± SEM). *P < 0.05, **P < 0.01. (C-D) Schematic of miR-31 deletion in LysM^Cre^miR-31^fl/fl^ mice (C) and western blot analysis of Pp6 expression in BMDMs from LysM^Cre^miR-31^fl/fl^ and miR-31^fl/fl^ mice (D).(DOCX)

S4 FigSer-2, Ser-33 and Ser-178 sites were hyper-phosphorylated in PP6-deficient 293FT cells.(A) Lysates from Salmonella-infected BMDMs were immunoprecipitated with IgG or anti-PP6C, and blotted with anti-PFKFB1 and anti-PP6C. (B-D) LC-MS/MS-based detection of phosphorylation sites on PFKFB1 immunoprecipitates from WT and PP6-deficient 293FT cells. Intensity of Ser-2 (B), Ser-33 (C), Ser-178 (D) in WT and PP6-deficient 293FT cells. P-values were determined by two-tailed unpaired t-test (mean ± SEM). ns: not significant, *P < 0.05.(DOCX)

S5 FigStrategies for Pfkfb1 knockout mouse construction.(A) Quantification of Pfkfb1 in non-infected BMDMs, bystander BMDMs and BMDMs containing growing and non-growing bacteria (n = 3). P-value was determined by two-tailed unpaired t-test (mean ± SEM). **P < 0.01. (B-C) Schematic of Pfkfb1 deletion in Pfkfb1^-/-^ mice (B) and Pfkfb1 genotyping of the toes derived from WT and Pfkfb1^-/-^ mice (C).(DOCX)

S1 TableGenes in yeast two-hybrid screening.(XLSX)