Neuraminidase A controls pneumococcal recognition and fate: deficiency enhances immune sensing and intracellular survival, while treatment promotes phagocytic clearance
Kristine Farmen, Georgia Yfanti, Fabienne Geers, Johanna Hollenbeck, Miguel Tofiño Vian, Thomas P Kohler, Sven Hammerschmidt, Federico Iovino
TL;DR
This study shows that a bacterial enzyme called Neuraminidase A (NanA) helps pneumococci evade immune detection, and blocking it could improve treatment for meningitis.
Contribution
The study reveals that NanA modulates phagocytic responses in microglia and macrophages, with secreted NanA uniquely enhancing immune sensing.
Findings
NanA deficiency increases bacterial adhesion but paradoxically promotes intracellular survival.
Recombinant NanA treatment restores phagocytic activation and bacterial clearance.
The effect of NanA is dependent on its sialidase activity and is specific to the secreted form.
Abstract
Streptococcus pneumoniae (pneumococcus) is a leading cause of bacterial meningitis worldwide and is associated with cerebrovascular complications and long-term neurological sequelae. These outcomes are largely driven by an excessive yet ineffective neuroinflammatory response. Although pneumococci express multiple virulence factors that enable immune evasion, how these factors modulate phagocytic responses in the central nervous system remains unclear. Here, we identify neuraminidase A (NanA) as a critical regulator of phagocytic activation in microglia and macrophages. Using murine and human microglia and RAW 264.7 macrophages, we show that NanA deficiency increases bacterial adhesion, indicating enhanced immune recognition, but paradoxically promotes intracellular bacterial survival. Despite elevated expression of the phagocytic marker CD68, microglia infected with NanA-deficient…
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Figure 5- —Vetenskapsrådet10.13039/501100004359
- —The Swedish Research Council
- —The Strategic Research Area Neuroscience StratNeuro
- —Crown Princess Lovisa's Association for Children's Healthcare10.13039/501100009757
- —Axel Tielman Memorial Fund
- —INCATE
- —Clas Groschinskys Memorial Fund
- —Magnus Bergvall Foundation10.13039/501100006285
- —Åhlén Foundation10.13039/501100005701
- —Tore Nilson Foundation
- —Tysta Skolan Foundation10.13039/100010799
- —Wera Ekström Foundation
- —Gun & Bertil Stohne Foundation10.13039/100009673
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TopicsPneumonia and Respiratory Infections · Bacterial Infections and Vaccines · Carbohydrate Chemistry and Synthesis
Introduction
Streptococcus pneumoniae (the pneumococcus) is the predominant causative agent of bacterial meningitis, a life-threatening condition associated with high mortality and frequent long-term neurological sequelae in up to 50% survivors [1–4]. In the central nervous system (CNS), resident microglia are among the first responders to the invading pathogens and are responsible for the elimination and the generation of the immune response towards these [5]. The recognition of the pneumococcus causes the release of inflammatory mediators, including cytokines, chemokines, and matrix metalloproteinases, which causes the influx of peripheral monocytic-derived macrophages and neutrophils to aid the elimination of the pathogen. Invading pneumococci is recognized by phagocytic immune cells [5, 6]. Importantly, microglia are professional phagocytes and adopt an amoebic shape associated with clearance of the pneumococcus [5, 6].
Microglial phagocytosis is fundamental for the control and resolution of CNS infections [7]. It not only facilitates the direct clearance of pathogens but also limits excessive inflammation and neuronal damage by preventing the accumulation of bacterial debris and toxins [8, 9]. However, S. pneumoniae has evolved multiple strategies to evade this immune response, including its polysaccharide capsule, which impairs microglial recognition and uptake, and pneumolysin (Ply), a pore-forming toxin, which disrupts microglial cytoskeletal dynamics and suppresses phagocytic efficiency [9–13]. Despite this, microglia retain the potential to mount effective antibacterial responses when appropriately activated.
It is therefore fundamental to understand how the pneumococcus modulates the microglial phagocytic machinery to promote the development of novel immunotherapeutic strategies. By identifying molecular cues that boost microglial antimicrobial activity, without exacerbating neuroinflammation, it may be possible to enhance bacterial clearance in the CNS and improve outcomes for patients with pneumococcal meningitis. Unlike traditional antibiotics, which do not directly modulate host immune functions, such approaches could complement existing therapies and help overcome the limitations posed by antibiotic resistance and treatment delays [5, 14, 15].
Our study reveals that the pneumococcal secreted form of neuraminidase A (NanA) activates the recognition of the pathogen by microglia, which are prone to take up pneumococci lacking NanA in higher amounts compared to NanA-expressing S. pneumoniae. On the other hand, this high number of bacteria interacting with microglia in the first place does not lead to an increased bacterial elimination but, instead, to the survival of NanA-lacking pneumococci inside phagocytic immune cells, as this was observed in both microglia and macrophages. This finding not only uncovers a key immune strategy for protection of the host but also points to NanA as a promising target for future immunomodulatory therapies against pneumococcal meningitis.
Methods & materials
Streptococcus pneumoniae deletion mutants and bacterial cultivation
For the cultivation of S. pneumoniae T4 (T4), its isogenic mutants, and D39, bacteria were grown at 37°C with 5% CO_2_ to an OD_620nm_ of 0.2–0.3 (exponential phase) in Todd-Hewitt broth (THY) and stored in a solution of THY supplemented with 20% glycerol at −80°C. For growth curve experiments, cultures were initiated by diluting thawed pre-grown aliquots 1:40 in THY, incubated at 37°C with 5% CO_2,_ and growth was measured by OD measurement at 620 nm with Novaspec III + spectrophotometer every 30 min for 24 h.
Streptococcus pneumoniae single-gene deletion mutants were generated in the T4 genetic background [16] and bacteria were therefore transformed with plasmid pQSV22Spec [17] for deletion of pspC, or with plasmid pMiniTΔnanA::erm_T4 [18] for deletion of nanA, as previously described.
Cell lines
The murine microglial cell line BV-2 (Cytion) was maintained in DMEM supplemented with 5% fetal bovine serum (FBS, Merck) and 1× penicillin-streptomycin. BV-2 cells were seeded for experimental assays at a density of 2 × 10^5^ to 5 × 10^5^ cells per well in 12- or 6-well plates and used within 3 days. The murine macrophage cell line RAW 264.7 (ATCC TIB-71) was maintained in DMEM supplemented with GlutaMAX™, D-glucose (4.5 g/L), and pyruvate with 10% FBS, 1× penicillin-streptomycin, and seeded at 1 × 10^5^ cells per well in 12-well plates for experimental assays. The human microglial cell line HMC3 (ATCC CRL-3304) was maintained in EMEM containing 10% FBS and 1× penicillin–streptomycin and seeded at 1 × 10^5^ cells per well in 12-well plates, used within 2 days in EMEM supplemented with 5% FBS. The cells were incubated at 37°C with 5% CO_2_, with media changed every 2nd–3rd day. The cells were incubated at 37°C with 5% CO_2_, with media changed every 2nd–3rd day. The day prior to infection, the cell media was replaced with DMEM containing 2.5% FBS without antibiotics.
Production of recombinant NanA and RrgA
Coding fragments of soluble NanA (Q54-N800) and soluble RrgA (E39-G868) were PCR-amplified from S. pneumoniae T4 genomic DNA and subcloned into the pNIC28-Bsa4 plasmid (Addgene #26103). Expression plasmids were transformed into Escherichia coli BL21(DE3) Rosetta. Expression cultures were grown in Terrific Broth (TB) supplemented with appropriate antibiotics and 0.1% v/v antifoam 204 (Sigma-Aldrich). Briefly, 750 ml TB was inoculated with 20 ml overnight starter culture and incubated at 37°C under agitation by the LEX minibioreactor system (Harbinger Biotech). The temperature was lowered to 18°C when OD_620nm_ reached 2.0, and 0.5 mM IPTG was added after 1 h, and target protein expression continued overnight. Bacteria were harvested 20 h post-induction by centrifugation at 4000 g and resuspended in at least 1.5 times cell pellet volume of lysis buffer (100 mM HEPES, 500 mM NaCl, 10 mM Imidazole, 10% glycerol, 0.5 mM TCEP, pH 8.0, supplemented with benzonase and non-EDTA protease inhibitor cocktail). Bacterial suspensions were sonicated on ice and clarified by centrifugation at 47 000 g. Lysate supernatants were filtered and loaded onto ÄKTAxpress systems (Cytiva) for Ni-NTA purification followed by size exclusion chromatography. The latter was performed on HiLoad 16/600 Superdex 200 prep grade columns (Cytiva) equilibrated in gel filtration buffer (20 mM HEPES, 300 mM NaCl, 10% (v/v) glycerol, 0.5 mM TCEP, pH 7.5). Eluted fractions were analysed on SDS-PAGE. Pure proteins were pooled, added with more TCEP to 2.0 mM, and concentrated to 5–20 mg/ml prior to freezing and storage. For heat inactivation, purified NanA was incubated at 60 °C for 15 min to abolish sialidase activity.
Invasion and intracellular survival assays
On the day of infection, the cells were washed with PBS and incubated for 1 h in DMEM containing 2.5% FBS for the murine cell lines (BV-2 and RAW 264.7) and in EMEM containing 5% FBS for HMC3 cells. Bacteria were added at a multiplicity of infection (MOI) of 50 to 100 for BV-2 and HMC3 cells, and at an MOI of 200 for RAW 264.7 macrophages. When indicated, treatments were included at this step, including recombinant NanA or RrgA protein at 500 or 2000 ng/ml or 10 µM cytochalasin. Plates were centrifuged at 50 × g for 5 min and incubated at 37 °C for 2 h for BV-2 cells or 1.5 h for RAW 264.7 cells. After infection, supernatants containing non-adherent bacteria were collected for enumeration, and the cells were washed twice with PBS and treated with 200 µg/ml gentamicin and 10 µg/ml penicillin-G for 1 h for BV2-cells, and 300 µg/ml gentamicin and 0.12 mg/ml penicillin G for 15 min for RAW 264.7 cells to kill extracellular bacteria. The cells were gently washed in pre-warmed PBS to remove unbound bacteria, and either lysed immediately with a 1:1 mixture of 0.1% saponin and 1× trypsin for 15 min at 37 °C or incubated with fresh medium for investigation into bacterial intracellular survival before lysis. Lysates were collected and plated for enumeration of the intracellular bacteria. For investigation of adhesion, the antibiotic treatment step was omitted, and the cells were lysed directly as described above for quantification of the adherent bacteria.
Lactate Dehydrogenase (LDH) cytotoxicity assays
LDH release was quantified using a colorimetric assay to assess cytotoxicity (Invitrogen). Upon plasma membrane damage, cytosolic LDH is released into the culture medium and detected spectrophotometrically at 450 nm following addition of the kit’s coupled enzymatic reaction mixture. The assay was used to evaluate the cytotoxicity of S. pneumoniae infection toward microglial cells. Reagents were prepared according to the manufacturer’s instructions. Supernatants and the positive control were centrifuged (10 000 × g, 3 min), and 50 µl of clarified supernatants were transferred to a 96-well plate. An equal volume of reaction mixture was added, mixed, and incubated for 30 min at room temperature in the dark. The reaction was stopped by adding 50 µl of stop solution, and bubbles were removed prior to measurement. Absorbance at 450 nm was recorded, and cytotoxicity was calculated as:
Immunofluorescence staining
The BV-2 cells cultured on coverslips in 12-well plates were infected and treated with antibiotics as described above. One hour after antibiotic treatment, cells were fixed in 4% paraformaldehyde (HistoLab) for 10 min at room temperature (RT), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min and blocked in 5% bovine serum albumin (BSA; Sigma-Aldrich) and 100 mM glycine in PBS for 30 min at RT, before incubated with unconjugated primary antibody type 4 rabbit polysaccharide capsule antiserum (SSI Diagnostica; 1:200) overnight at 4 °C followed by incubation with antibodies Alexa Fluor 594 anti-rabbit IgG (Invitrogen; 1:500) at RT for 1 h. For staining with the conjugated antibodies, anti-mouse CD45 rabbit polyclonal antibody labeled with Zenon Alexa 488 (Invitrogen; 1:250), APC conjugated anti-mouse CD68 (BioLegend; 1:100), FITC conjugated anti-mouse CD45 (BioLegend; 1:200), and GFP-lectin (Vector Laboratories; 1:100), caspase-3 (R&D Systems; 1:200), cathepsin B (Cell Signaling Technology; 1:1000), Iba1 (Abcam; 1:1000), Lectin (Vector Laboratories; 1:250), cells were incubated in the staining solution immediately after blocking for 2 h at RT. For nuclear staining, cells were incubated with DAPI (Abcam) for 10 min at the same conditions. Coverslips were mounted with Dako mounting medium (Agilent Technologies) and stored at 4 °C until imaging.
Confocal microscopy analysis and image processing
To minimize bias, all microscopy and image analyses were performed under blinded conditions, and the acquisition settings were kept constant within each experiment. For serotype 4 rabbit polysaccharide capsule and CD45 staining, images were acquired at 63× magnification on a Zeiss LSM 880-Airy confocal microscope using 5 × 5 tile scans, with four technical replicates per biological replicate analysed. For CD68, CD45, Iba1, caspase-3, lectin and cathepsin B analyses, images were acquired on a Zeiss LSM 900-Airy confocal microscope at 40× magnification, and only the bacteria inside microglia were detected; all the images represent z-stacks (spanning 10 μm) using z-stacks spanning 10 μm; with two technical replicates per biological replicate analysed.
Image analysis was performed in ImageJ (Fiji, NIH). For bacterial quantification, microglial cells and intracellular bacterial signal was counted manually by using the Point Tool; invaded bacteria per cell were calculated as bacterial signal divided by microglial cell number. For CD68, caspase-3, cathepsin B and lectin analysis, z-stacks were merged, background subtracted, and the mean fluorescence intensity per microglial cell was calculated by dividing the total signal by the number of microglial nuclei, quantified from DAPI images converted to grayscale, thresholded, and analysed using the ‘Analyze Particles’ function.
Statistical analysis
GraphPad Prism-9 was used for statistical analyses (α5%). Outliers were identified by Grubb's test. Normality was tested with D'Agostino–Pearson test. Ordinary one-way ANOVA or Kruskal–Wallis test was performed with Tukey or Dunn's multiple comparisons, as indicated in the figure legends. Unpaired two-tailed t-tests were used for pairwise comparisons and for experiments involving two independent variables, two-way ANOVA followed by Bonferroni correction for multiple comparisons was performed.
Results
NanA deficiency enhances microglial recognition but promotes pneumococcal intracellular survival
To identify which virulence factors of S. pneumoniae contribute to evasion of microglial phagocytosis, we examined a panel of isogenic mutant strains generated in the laboratory strain TIGR4 (T4), specifically targeting the genes encoding Ply, pilus-1, pneumococcal surface protein C (PspC = CbpA), NanA, all virulence factors known for pneumococcal pathogenicity during meningitis and invasive pneumococcal disease [19–23]. Notably, deletion of these virulence factors did not affect bacterial growth (Supplementary Figure S1). BV-2 microglial cells were infected with these pneumococcal strains, and the first bacterial adhesion was quantified as an indication of the capacity of microglia to recognize the pathogen treat. Among the tested mutants, the ΔnanA displayed significantly enhanced adhesion to microglia compared to wild-type T4 and other NanA-expressing mutant strains (Fig. 1a). NanA can exist in either a membrane-bound or released form, dependent on the pneumococcal strain. In T4, NanA is secreted, a form more commonly associated with invasive clinical isolates [18, 24, 25]; furthermore, recently published data reported that the absence of the sortase A motif in TIGR4 promotes the secretion of NanA rather than the expression of a surface-anchored form, present in the D39 pneumococcal strain [18]. To investigate whether membrane-anchored NanA also influences microglial interaction, we used the serotype 2 strain D39 wild type and its nanA-mutants: ΔnanA 721 (Janus insertion), ΔnanA 736 (sialidase domain deletion), and ΔnanA 737 (lectin domain deletion) [26]. In contrast to T4, no significant differences in adhesion were observed among the D39-derived strains (Fig. 1b), suggesting that only the released form of NanA facilitates microglial recognition of the pneumococcal strain.
Adhesion and intracellular survival capabilities of S. pneumoniae with and without NanA in microglia. Lack of released NanA caused increased adhesion of the T4 strain to BV-2 cells (a), while the membrane-bound form of NanA had no effect on adhesion rates for the pneumococcus in the D39 strains (b). (c) Intracellular survival of wild-type T4 and its isogenic mutant strains 5 h post-infection (2 h post-antibiotic treatment). Values of biological replicates represent the percentage of strain-specific adherent (a, b) or intracellular (c) CFUs relative to total recovered CFUs, normalized to wild-type T4 (a, c) or D39 (b). (a-c) Statistical analysis was performed using a one-way ANOVA, followed by Dunn’s multiple comparisons test, comparing each mutant strain to T4. Columns represent mean values; error bars indicate standard deviations (SD), n = 3–4. (d) Uptake of S. pneumoniae T4 and its isogenic ΔnanA mutant in HMC3 human microglia; values of biological replicates represent the percentage of strain-specific intracellular CFUs relative to total recovered CFUs, normalized to wild-type T4. Statistical analysis was performed using an unpaired two-tailed t-test to compare wild-type T4 and the ΔnanA mutant; columns represent mean values; error bars indicate standard deviations (SD), n = 3.
We next evaluated the impact of virulence factor expression on intracellular bacterial survival in microglial cells. At 5 h post-infection, corresponding to 2 h of bacterial intracellular survival after antibiotic treatment, the ΔnanA mutant showed significantly increased intracellular bacterial loads compared to T4 wild-type, while intracellular levels of all mutant strains declined over time (Fig. 1c); furthermore, pneumococci lacking NanA had higher survival also in HMC3 human microglia (Fig. 1d). Immunofluorescence confocal microscopy with tile function further confirmed a significant increase in bacterial presence inside microglia for the ΔnanA mutant compared to wild-type T4 (Fig 2a and b). After a 2 h recovery period post-antibiotic treatment, the elevated bacterial signal persisted only for ΔnanA (Fig 2c and d). Importantly, the viability levels of intracellular bacteria were not dependent on the viability of infected microglia; in fact, the infected microglia showed the same levels of both cytotoxicity, assessed by LDH measurement (Supplementary Figure S2), and apoptosis, assessed by caspase 3 staining (Supplementary Figure S3). Together, these findings highlight NanA’s key role in triggering microglial degradation of the intracellular bacteria.
Microglia infected with S. pneumoniae depicted an increased bacterial signal when infected with pneumococci lacking NanA. Confocal microscopy analysis (63 × magnification) was performed to detect BV2 microglia (CD45, magenta) with intracellular pneumococci (T4 capsule, cyan). (a) Invasion of S. pneumoniae wild-type T4 and deletion mutants after antibiotic treatment (assessment of bacterial invasion), and respective semi-quantification analysis of bacterial signal (b). (c) Intracellular survival of S. pneumoniae mutants at 1 h timepoint after antibiotic treatment (assessment of bacterial intracellular survival), semi-quantification analysis of bacterial signal (d). In a and c, scale bar 50 µm, images shown are representative of three independent experiments. In b and d, values of biological replicates represent the percentage of strain-specific intracellular CFUs relative to total recovered CFUs, normalized to wild-type T4. Statistical analysis was performed using one-way ANOVA, followed by Dunn’s multiple comparisons test, comparing each experimental group to T4. Columns represent mean values; error bars indicate SD, n = 3.
Pneumococcal intracellular viability is due to uptake by microglia rather than bacterial invasion of host cells.
To determine whether microglial phagocytosis was responsible for the uptake of S. pneumoniae T4 and its isogenic deletion mutants by microglia, cytochalasin D was used to inhibit actin polymerization and thereby block phagocytic activity. Microglial cells treated with cytochalasin D displayed significantly reduced numbers of intracellular bacteria, also for the T4ΔnanA strain (Fig. 3a). This clearly demonstrates that bacterial presence inside microglia is driven by actin-mediated uptake of bacteria by microglia rather than the bacteria actively invading immune cells.
Intracellular presence of bacteria is due to the immune cell phagocytosis process, and the viability of pneumococci lacking NanA inside microglia is associated with increased phagocytic activity. (a) Invasion assay of BV-2 infected cells with S. pneumoniae T4 and its isogenic mutant strains after treatment with cytochalasin D shows decreased invasion of the T4 strains, indicating that bacterial presence inside microglia is the result of an active phagocytic process of microglia towards bacteria. Values of biological replicates represent the percentage of strain-specific intracellular CFUs relative to total recovered CFUs. (b) Confocal microscopy analysis of BV-2 microglial cells infected with S. pneumoniae T4 or ΔnanA mutant strain showed an increase of phagocytic activity after infection with ΔnanA; 40 × magnification objective was used to detect phagocytic activity of BV2 microglia (CD68, red), microglial cells (CD45, green) and the nuclei (DAPI, blue), scale bar 25 µm; each image is representative of three images (fields of view) taken per each fluorescence channel per each experimental group. (c) Quantification of the mean intensity of CD68 normalized to T4 after infection with wild-type T4 and ΔnanA or uninfected. Statistical analysis was performed using two-way ANOVA with Bonferroni correction for multiple comparisons, n = 3 (a), and one-way ANOVA followed by Dunn’s multiple comparisons test, comparing each experimental group to T4, n = 4 (c). Columns represent mean values; error bars indicate SD.
To further investigate the link between bacterial uptake and phagocytic activity in microglia, we performed confocal microscopy analysis with tile function of BV-2 microglial cells after infection with S. pneumoniae T4 wild-type or its isogenic ΔnanA mutant to assess microglial phagocytic activity in the presence and in the absence of NanA. Microglia infected with the ΔnanA mutant surprisingly exhibited elevated CD68 expression, indicating increased phagocytic activity in response to the mutant strain (Fig. 3b and c). However, confocal microscopy analysis of cathepsin B, a lysosomal protease involved in cellular degradation, showed no detectable differences in cathepsin B expression within microglia infected with the various TIGR4 strains (Fig. 4a and b). We therefore observed an apparent paradox: microglia containing more surviving bacteria over a longer time-course displayed a strongly increased phagocytic activity [27]. This scenario indicates that, in the absence of NanA, microglia may increase phagocytic uptake or lysosomal mass but fail to complete downstream digestion, resulting in elevated bacterial uptake with inefficient bactericidal processing.
Increased intracellular presence of pneumococci lacking NanA is not associated with enhanced degrading capability of microglia. (a) Confocal microscopy analysis of BV-2 microglial cells infected with S. pneumoniae T4 or mutant strains after antibiotic treatment; 40 × magnification objective was used to detect lysosomal proteolytic activity of BV-2 cells (Cathepsin B, green), microglial cells (Iba1, red) and the nuclei (DAPI, blue), scale bar 20 µm; each image is representative of three images (fields of view) taken per each fluorescence channel per each experimental group. (b) Quantification of the mean intensity of Cathepsin B normalized to T4 after infection with wild-type T4 and ΔnanA or non-infected. Statistical analysis was performed using one-way ANOVA followed by Dunn’s multiple comparisons test, comparing each experimental group to T4-infected cells, n = 3. Columns represent mean values; error bars indicate SD.
Treatment with NanA triggers microglial elimination of pneumococci lacking NanA.
To determine whether treatment with recombinant NanA could restore microglial uptake against pneumococci lacking endogenous NanA, BV-2 microglia were infected with either wild-type S. pneumoniae T4 wild-type or a ΔnanA mutant, with or without treatment with purified recombinant NanA protein. Because the absence of pilus-1 (rrgA-srtD pilus-1 genetic islet) was not affecting microglial phagocytic elimination of pneumococci, as shown in Fig. 1a, treatment with recombinant RrgA, the tip protein of the pilus-1 [20–22], was used as a control. Importantly, treatment with recombinant NanA prior to infection with ΔnanA pneumococci significantly enhanced bacterial elimination, restoring uptake activity to levels comparable to those observed with T4 wild-type (Fig. 5a). Moreover, treatment with recombinant RrgA did not restore the phagocytic elimination of ΔnanA to T4 wild-type levels, as expected (Fig. 5a). Additionally, we abolished sialidase activity by heat-inactivating NanA; consistent with this, treatment with heat-inactivated NanA failed to enhance intracellular bacterial survival (Fig. 5a). These findings demonstrate that treatment with NanA, with retained sialidase activity, is sufficient to reactivate the microglial phagocytic machinery and promote the elimination of NanA-deficient pneumococci.
Recombinant and endogenous NanA enhance microglial and macrophage clearance of S. pneumoniae. (a) Uptake of S. pneumoniae T4, its isogenic ΔnanA mutant, and ΔnanA infections in the presence of recombinant NanA, active or heat-inactivated (HI), or RrgA. Values of biological replicates represent the percentage of experimental group intracellular CFUs relative to the total recovered CFUs, normalized to wild-type T4. Statistical analysis was performed using one-way ANOVA followed by Dunn’s multiple comparisons test. Columns represent mean values; error bars indicate standard deviations (SD), n = 3–6. (b) Uptake of S. pneumoniae T4 and its isogenic mutant strains in RAW 264.7 macrophages. Values of biological replicates represent the percentage of experimental group intracellular CFUs relative to the total recovered CFUs, normalized to wild-type T4. Statistical analysis was performed using one-way ANOVA, with a mixed-effect model for (a), followed by Dunn’s multiple comparisons test. Columns represent mean values; error bars indicate SD, n = 3–6.
NanA maintains its key role to trigger immune cell phagocytosis also in macrophages.
Next, we assessed whether the immunomodulatory role of NanA was retained upon infection of peripheral macrophages. In RAW 264.7 macrophages infected with S. pneumoniae wild-type T4 and isogenic mutant strains revealed that the ΔnanA mutant exhibited a significant increase in uptake by microglial cells, compared to the wild-type T4 strain, confirming an enhanced bacterial survival also within macrophages in the absence of NanA (Fig. 5b). Interestingly, the ΔnanA mutant also presented significantly higher intracellular levels compared to the wild-type T4 strain (Fig. 5b). These findings demonstrate that NanA, not only in microglia but also in macrophages, plays a pivotal role in modulating immune cell phagocytosis of intracellular S. pneumoniae. Additionally, the Ply-deficient pneumococci also possessed increased intracellular levels, confirming the reduced macrophage LC3-associated phagocytic activity when S. pneumoniae lacks pneumolysin [28]. To further assess whether NanA-mediated activity generates cryptic glycoconjugates on the immune cell surface, as previously described [29], we performed lectin staining on microglia infected with: (i) D39, (ii) TIGR4, (iii) TIGR4ΔnanA, (iv) TIGR4ΔnanA supplemented with NanA, and (v) TIGR4ΔnanA supplemented with RrgA, as a control. Under our experimental conditions, we detected no measurable differences in lectin staining across these groups (Supplementary Figure S4). These findings suggest that, in microglia and within the context of our infection model, NanA activity does not induce a strong or persistent alteration in the lectin-detectable cell-surface glycan landscape.
Conclusion
This study identifies the secreted form of pneumococcal NanA as a critical immunomodulatory factor aiding bacterial degradation through the phagocytic machinery of both microglia and macrophages. While NanA has long been recognized as a virulence determinant contributing to colonization and tissue invasion during pneumococcal infections [23–25, 30], our findings uncover a new role of NanA in the immunomodulation of the phagocytic response of innate immune cells. Intracellular survival of S. pneumoniae has been linked to NanA, which suppresses Ply activity inside host cells and thereby limits bactericidal autophagy [31]. Strikingly, our findings reveal that immune sensing remains unchanged regardless of Ply’s presence, whereas the presence or absence of NanA markedly reshapes microglial uptake, phagocytosis, and the bacterium’s ability to persist inside cells.
Using both microglial (BV-2 and HMC3) and macrophage (RAW264.7) models, we demonstrated that genetic ablation of NanA results in increased bacterial adhesion, indicating the immune cell recognition of the pathogen treat. In particular, the absence of NanA was associated with higher CD68 expression in microglia and an increased intracellular bacterial load, indicating elevated phagocytic activity without an efficient bacterial elimination, as demonstrated by the levels of cathepsin degrading enzyme, which were not increased when microglia were infected with pneumococci lacking NanA. CD68 is a lysosome/endosome-associated glycoprotein whose expression and glycosylation increase with phagocytic activation and lysosomal expansion; thus, high CD68 often reports enlarged or more abundant phagolysosomal compartments rather than assured microbicidal competence [27]. Second, several models of dysfunctional microglia/macrophages show the same signature, abundant CD68-positive cells coexisting with reduced phagocytic efficiency or impaired clearance. For example, primary microglia from α-synuclein knockout mice exhibit increased CD68 and a strongly impaired ability to clear bacterial bioparticles, indicating that CD68 upregulation can coincide with defective killing [32]. Likewise, macrophage-rich lesions such as atherosclerotic plaques contain many CD68 + macrophages but show defective efferocytosis/phagocytosis in situ (large numbers of ‘free’ apoptotic bodies), consistent with a state of increased uptake/lysosomal burden but reduced degradative efficiency [33]. Our results therefore provide novel evidence that microglia can display markedly elevated CD68 expression without a corresponding increase in lysosomal cathepsin activity, which may compromise their ability to effectively eliminate intracellular bacteria; these new findings provide further support reinforcing prior observations of inefficient phagocytosis in macrophages and extending them to the microglial context, thereby urging caution in interpreting CD68 as a straightforward indicator of effective microbial clearance.
Pneumococcal NanA remodels host cell-surface glycans, exposing N-acetylglucosamine and D-galactose while reducing sialic acids in specific host compartments, such as the chinchilla eustachian tube, promoting colonization and invasion [29]. In contrast, NanA-deficient pneumococci induced no such changes. In microglia, using our cellular models, lectin staining of cells infected with D39, TIGR4, or ΔnanA, with or without NanA supplementation, revealed no detectable alterations in surface glycans. These results suggest that NanA-driven glycan remodeling is tissue-specific, significantly affecting epithelial surfaces but not microglia.
Moreover, our findings are in line with previous reports suggesting that phagocytosis by microglia and macrophages is not a passive event but tightly regulated by pathogen-associated molecular patterns and host-pathogen interaction cues [5, 6, 34].
Importantly, treatment with purified recombinant NanA protein, with active sialidase activity, was sufficient to restore bacterial degradation in microglia infected with ΔnanA pneumococci, whereas recombinant RrgA, the tip protein of the pilus-1, had no effect. Additionally, this effect extended beyond microglia to peripheral macrophages, indicating that NanA-induced phagocytic priming is a conserved and systemic immune activation mechanism. In both cell types, the absence of NanA conferred a significant advantage to the bacteria.
Previous studies have reported that the secreted form of NanA is more frequently associated with invasive pneumococcal strains [23–25, 30]. Interestingly, our results demonstrate that this same secreted form of NanA, as found in strain T4, promotes phagocytic elimination, whereas the membrane-anchored version present in strain D39 does not elicit this effect.
In conclusion, we reveal a novel function of pneumococcal NanA as a critical virulence factor in favor of host immunity by stimulating immune cell recognition and elimination in both CNS and peripheral immune compartments. Our findings shed light on the possibility that modulating NanA activity can directly enhance immune clearance mechanisms. These insights open avenues for the development of novel therapeutics aimed at exploiting NanA to prevent bacterial evasion or leveraging NanA-mediated pathways to boost immune cell phagocytic function. Ultimately, NanA represents a promising target to improve host defense and reduce disease burden associated with pneumococcal pathogenesis.
Supplementary Material
ltag002_Supplementary_Data
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