HDAC-driven autophagy repression aids macrophage immune evasion by K. pneumoniae strains displaying classical, MDR, and hypervirulent phenotypes

Summary

The global emergence of multidrug-resistant (MDR) and hypervirulent (hvKp) Klebsiella pneumoniae strains poses a major clinical challenge. Here, we compared representative classical (cKp), MDR-Kp, and hvKp isolates to define host epigenetic responses during infection. At 24 h post-infection (hpi), infection with all three strains induced HDAC2 upregulation and H3K18 deacetylation, with chromatin immunoprecipitation (ChIP) analysis revealing HDAC2 enrichment at the ATG5 promoter, linking epigenetic remodeling to autophagy suppression. Functionally, cKp and MDR-Kp isolates promoted M1-like macrophage polarization, whereas hvKp isolate induced an immunosuppressive M2-like phenotype associated with enhanced intracellular bacterial survival and elevated IL-10 expression. HDAC1/2 gene knockdown reduced intracellular bacterial survival, while pharmacological inhibition using CI994 and SAHA restored autophagy, normalized cytokine responses, enhanced bacterial clearance, and reversed M2-like polarization induced by hvKp-isolate. Collectively, these findings identify HDAC-mediated epigenetic reprogramming as a central mechanism underlying immune evasion during K. pneumoniae infection and support HDAC inhibition as a potential host-directed therapeutic strategy.

Graphical abstract

Highlights

• •Representative cKp, MDR-Kp, and hvKp isolates exhibit distinct host responses
• •HDAC2-mediated autophagy repression and intracellular survival
• •HvKp isolates promote immunosuppressive M2-like macrophage polarization
• •HDAC inhibition restores host defense and enhances intracellular bacterial clearance

Microbiome; Metabolomics; Transcriptomics

Introduction

Klebsiella pneumoniae is a gram-negative opportunistic pathogen that poses a growing threat to global health due to its capacity to cause severe infections, including pneumonia, bacteremia, sepsis, and liver abscesses. Two major variants of K. pneumoniae have heightened concern: multidrug-resistant strains (MDR), which are refractory to most antibiotics, and hypervirulent strains (hvKp), which exhibit enhanced pathogenicity and cause diseases even in immunocompetent individuals.^1,2,3 The recent emergence of convergent MDR-hvKp strains harboring both resistance and virulence determinants represents a critical public health threat. In India, K. pneumoniae remains the second most isolated pathogen from clinical specimens and exhibits alarmingly high levels of carbapenem resistance, with up to 80% of bloodstream isolates resistant to imipenem, underscoring its critical role in healthcare-associated infections and the urgent need for novel therapeutic strategies.⁴

K. pneumoniae pathogenesis is shaped by an array of virulence factors, including capsular polysaccharides, lipopolysaccharides, siderophores, fimbriae, and type VI secretion systems that collectively promote immune evasion, epithelial adherence, and intracellular survival.^5,6 Among these, the ability of K. pneumoniae to persist within macrophages represents a significant advantage in evading early immune clearance. The mechanisms underlying macrophage persistence and immune evasion may differ among classical, hypervirulent and multidrug-resistant K. pneumoniae strains. Comparative analysis of these phenotypes across representative clinical isolates is essential for unraveling strain-specific mechanisms of immune escape.

Macrophages are central players in innate immunity with functional plasticity that enables polarisation into classically activated M1 or alternatively activated M2 phenotypes. K. pneumoniae has been shown to skew macrophage responses away from proinflammatory activation toward regulatory phenotypes that support bacterial survival.⁷ Such alterations in macrophage polarization can influence cytokine responses and compromise host antimicrobial defenses, thereby facilitating bacterial persistence.

Recent evidence points to host epigenetic regulation-particularly histone modification as a key interface manipulated by pathogens to subvert immune responses. Histone deacetylases, HDACs, especially Class I HDACs (HDAC1 and HDAC2) play a central role in silencing the transcription of genes involved in inflammation, autophagy, and pathogen recognition. HDACs act by removing acetyl groups from histone lysine residues, leading to chromatin condensation and repression of gene expression. Their activity is known to suppress proinflammatory signaling pathways, including those downstream of TLRs and type I interferons, and to modulate transcription factors such as NF-κB.^8,9 Intracellular pathogens such as Mycobacterium tuberculosis and Listeria monocytogenes have been shown to exploit HDAC-mediated repression to persist within host cells,^10,11 yet the role of HDACs in K. pneumoniae infection remains underexplored.

In this study, we performed a comparative analysis of representative classical, hypervirulent, and MDR K. pneumoniae strains to identify differential host-pathogen interactions that support intracellular survival. We assessed their ability to resist killing by whole blood and serum, adhere to epithelial cells, and survive within macrophages. We further investigated how these strains differentially modulate host HDAC expression and activity, with a focus on class 1 HDACs. To determine the functional relevance of HDACs in infection, we employed selective HDAC inhibitors (CI994 and SAHA) and evaluated their effects on bacterial clearance, macrophage polarization, cytokine production, and autophagy gene expression. Our findings aim to elucidate the epigenetic mechanisms by which K. pneumoniae modulates host immune responses and to explore HDAC inhibition as a host-directed therapeutic strategy against MDR and hypervirulent infections.

Results

Selection and characterization of representative Klebsiella pneumoniae strains

To enable strain-specific comparative analyses, we first identified clinical K. pneumoniae isolates with hypervirulent and MDR features. Among a panel of clinical strains, U10386 demonstrated pronounced hyper mucoviscosity in the string test (≥5 mm), consistent with a hypervirulent (hvKp) phenotype (Figure S1). PCR amplification and sequencing confirmed the presence of the rmpA2 and iucA genes (Figure S2), both of which are established markers associated with hypervirulent K. pneumoniae (hvKp).^12,13

Whole-genome sequencing (WGS) of U10386 further identified rmpA2 and the aerobactin locus (iucABCD-iutA). Additional loci, including salmecholin (iroE) and the yersiniabactin cluster (ybt genes), were also detected,^14,15 consistent with a hypervirulent genomic profile. Kleborate analysis^16,17,18 classified U10386 as sequence type ST2096 with a K64 capsule locus and O1/O2 serotype, with a virulence score of 4 (Table S1). Meanwhile, strain U10385 was selected as a MDR-Kp isolate via VITEK-based antimicrobial profiling (Table S2). Growth kinetics revealed that hvKp proliferated most rapidly, followed by MDR-Kp, while the classical strain (ATCC 33495, cKp) exhibited the slowest growth (Figure S3), underscoring strain-dependent differences in metabolic activity.

Comparative adherence of various strains to human epithelial cells

To assess early-stage colonization potential, the adherence capabilities of the selected K. pneumoniae strains were evaluated in multiple epithelial cell lines representing distinct tissue origins, including skin (HaCaT), lung (A549), colon (HT29), bladder (T24), and embryonic kidney (HEK293). Notably, both hvKp and MDR-Kp isolates exhibited significantly greater adherence to all cell types compared to cKp (Figures 1A–1E). MDR-Kp isolate showed preferential binding to pulmonary epithelia, whereas the hvKp isolate demonstrated robust adherence across skin, epithelial, bladder, and kidney-derived cells. These findings highlight enhanced epithelial tropism among clinical strains, suggesting their increased ability to establish early colonization compared to the laboratory control.

Figure 1.

Comparative adherence, virulence, and immune resistance of Klebsiella pneumoniae strains

(A–E) Adherence of classical (cKp), hypervirulent (hvKp), and multidrug-resistant (MDR-Kp) strains to HaCaT (skin), A549 (lung), HT29 (colon), T24 (Bladder), and HEK293 (kidney) cells. Both hvKp and MDR-Kp shows significantly higher adherence compared to cKp.

(F) C. elegans survival assay shows rapid killing by hvKp, intermediate virulence of MDR-Kp, and attenuated pathogenicity of cKp comparable to E. coli control.

(G and H) Whole-blood killing assays in active and heat-inactivated blood. HvKp and MDR-Kp exhibited greater survival than cKp in active blood, while all strains proliferated in heat-inactivated blood.

(I and J) Serum killing assays in active and heat-inactivated serum. hvKp showed the highest resistance, followed by MDR-Kp, whereas cKp was most susceptible. Inactivated serum supported growth of all strains.

Data are represented as mean ± SEM from three independent experiments; statistical significance was determined using one-way ANOVA followed by Sidak’s multiple comparison test for (A–E) and log-rank (Mantel-Cox) test for survival analysis in (F) (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant).

Hypervirulent isolate exhibits enhanced in vivo pathogenicity in a C. Elegans infection model

To delineate strain-specific differences in in vivo virulence, a C. elegans survival assay was performed. Infection with the hvKp isolate resulted in rapid and complete host mortality by day 5, underscoring a highly aggressive pathogenic phenotype. In contrast, worms infected with the MDR-Kp isolate exhibited a more gradual decline in survival, reaching complete mortality by day 7. The cKp strain displayed comparatively attenuated virulence, with a slower rate of host killing. Notably, the survival profile of cKp-infected worms closely paralleled that of E. Coli-fed controls (Figure 1F). Taken together, these findings indicate that the hvKp isolate examined in this study exhibits markedly enhanced virulence in vivo, while the cKp strain demonstrates limited pathogenicity comparable to non-pathogenic controls.

Hypervirulent and MDR strains exhibit enhanced resistance to whole blood and serum-mediated killing

To investigate immune evasion strategies, whole blood and serum killing assays were performed to simulate exposure to innate immune defenses. All three K. pneumoniae strains exhibited a time-dependent decline in viability when exposed to fresh human blood, consistent with phagocyte-mediated clearance (Figure 1G). However, both hvKp and MDR-Kp isolates displayed significantly higher survival rates than the classical reference strain, indicating enhanced resistance to cellular immune effectors. In heat-inactivated blood, where immune components are neutralized, all strains proliferated, with hvKp and MDR-Kp isolates showing accelerated growth kinetics (Figure 1H), suggesting greater metabolic adaptability under immune-inert conditions.

Serum killing assays further demonstrated complement-mediated bactericidal activity. While all strains initially expanded, a reduction in CFU was observed after 2 h in active serum, with the hvKp isolate exhibiting the highest resistance, followed by the MDR-Kp isolate. The classical strain showed the greatest susceptibility (Figure 1I). Robust growth in heat-inactivated serum by all strains confirmed that complement components have a significant role in the observed killing (Figure 1J).

K. pneumoniae strains differentially modulate cytokine responses and suppress innate immune signaling

To assess strain-specific effects on inflammatory responses, we quantified cytokines at both protein and transcript levels. At the protein level, cKp infection induced higher IL-10 secretion; hvKp and MDR-Kp isolates showed comparatively reduced IL-10 levels. Notably, the hvKp isolate elevated TNF- α levels, while cKp caused a moderate increase and the MDR-Kp isolate showed reduced TNF-α secretion. Additionally, Il-12p40 was reduced across all strains (Figures 2A–2C). At the transcriptional level, IL-8 was markedly downregulated in infected macrophages, whereas IL-10 was strongly induced, particularly during hvKp and MDR infection. TNF- α expression showed modest upregulation in hvKp and MDR-Kp-infected cells (Figures 2D–2F). Analysis of innate immune sensors revealed consistent downregulation of JAK2, STAT2, TLR2, and TLR4 across strains, with the most pronounced suppression observed in MDR-Kp infection (Figures 2G and 2H). Collectively, these findings indicate that although K. pneumoniae trigger distinct cytokine signatures, they converge on impairing host sensing by repressing TLR-JAK/STAT signaling. Given the central role of these receptors in pathogen sensing and cytokine signaling, their downregulation suggests a coordinated bacterial strategy to impair innate immune activation.

Figure 2.

K. pneumoniae strains differentially modulate cytokine responses and suppress innate immune signaling

(A–C) Cytokine protein levels in macrophage culture supernatants following infection with classical (cKp), hypervirulent (hvKp), or multidrug-resistant (MDR-Kp) strains. cKp increased TNF-α, IL-12p40 and IL-10, hvKp selectively elevated TNF-α, and IL-10, while IL-12p40 was reduced across all strains.

(D–F) Cytokine gene expression by qPCR. IL8 transcripts were significantly downregulated in macrophages upon infection with all three strains, whereas IL10 was strongly induced, particularly during hvKp and MDR-Kp infection. TNF-α expression showed modest upregulation in hvKp and MDR-Kp.

(G–J) Expression of innate immune signaling genes JAK2, STAT2, TLR2, and TLR4. All were suppressed across strains, with the most pronounced reduction observed in MDR-Kp infection. Data are represented as mean ± SEM from three independent experiments; statistical significance was determined by one-way ANOVA followed by the Dunnett test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant).

Upregulation of HDAC1/2 and deacetylation of H3K18 across strains reflect chromatin repression upon Klebsiella infection

We first examined the acetylation status of histone H3 lysine 18 (H3K18ac) by western blot analysis to assess chromatin activity during K. pneumoniae infection. A significant reduction in H3K18 acetylation was observed in macrophages infected with classical, hvKp, and MDR-Kp strains compared to uninfected controls (Figure 3A). This decrease was more pronounced in cells infected with the hvKp and MDR-Kp strains, indicating a strain-dependent repression of transcriptionally active chromatin.

Figure 3.

HDAC upregulation and H3K18 deacetylation in macrophages following Klebsiella pneumoniae infection

(A) Western blot analysis of HDAC2 and H3K18ac levels in uninfected and infected macrophages (cKp, hvKp, MDR-Kp), showing reduced H3K18acetylation and increased HDAC2 expression upon infection.

(B) Western blot analysis of HDAC1 expression with H3 as loading control, showing no significant change across strains.

(C and D) qPCR analysis of HDAC1 (C) and HDAC2 (D) expression levels. HDAC2 showed strain-dependent upregulation, with higher expression in hvKp and MDR-Kp, whereas HDAC1 expression remained largely unchanged. Data are represented as mean ± SEM from three independent experiments; statistical significance was determined using one-way ANOVA followed by Sidak’s multiple comparison test (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant).

To further investigate the potential mechanism underlying this hypoacetylation, we analyzed both protein and transcript levels of HDAC1 and HDAC2. Western blot analysis showed a consistent increase in HDAC2 protein levels across all infected macrophages, regardless of the infecting K. pneumoniae strain (classical, hypervirulent, or MDR) (Figure 3A). In parallel, qPCR analysis revealed strain-dependent differences in HDAC2 transcript levels, with a more pronounced increase observed in hvKp-infected cells, while MDR-Kp showed a similar upward trend and cKp exhibited a slight reduction (Figure 3D). This suggests a broad mechanism of host manipulation through HDAC2.

In contrast, HDAC1 displayed a distinct pattern: Western blotting did not reveal a significant difference in protein levels across strains (Figure 3B). Similarly, qPCR analysis showed only minor variation in HDAC1 transcript levels, with a slight upward trend in hvKp-infected macrophages (Figure 3C).

HDAC inhibition enhances intracellular clearance of K. pneumoniae

To investigate host-directed therapeutic strategies, THP-1 macrophage cells were infected with classical, hypervirulent, and MDR-Kp strains, followed by treatment with the HDAC inhibitors CI994 (15 μM)¹⁰ and SAHA (15 μM)¹⁹ for 24 h. The inhibitor concentrations were based on prior cell-based studies on HDAC inhibition. Both inhibitors significantly reduced intracellular bacterial loads compared to untreated infected controls. Notably, CI994 (15 μM) proved more effective than SAHA (15 μM) in reducing the survival of all three K. pneumoniae strains (Figures 4A and 4B), indicating a more pronounced role of class I HDAC inhibition in restricting intracellular survival.

Figure 4.

HDAC inhibition and knockdown reduce intracellular survival of Klebsiella pneumoniae and restore ROS levels

(A and B) Intracellular survival assays in THP-1 macrophages treated with HDAC inhibitors SAHA (A) and CI994 (B). Both inhibitors significantly reduced bacterial survival across cKp, hvKp, and MDR-Kp, with CI994 showing greater efficacy.

(C and D) PBMC survival assays following treatment with SAHA (C) and CI994 (D), demonstrating consistent reduction in bacterial burden across all strains.

(E and F) siRNA-mediated knockdown of HDAC1 (E) and HDAC2 (F) reduced intracellular survival of cKp, with HDAC2 knockdown showing a strong effect.

(G) Intracellular ROS production, while CI994 treatment partially restored ROS levels. Data are represented as mean ± SEM from three independent experiments; statistical significance was determined using one-way ANOVA followed by Sidak’s multiple comparison test (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant).

This effect was further validated in primary immune cells, where similar reductions in bacterial burden were observed in PBMC-based infection models following treatment with both inhibitors across all strains (Figures 4C and 4D). Collectively, these findings demonstrate that HDAC inhibition consistently reduces intracellular bacterial survival across both macrophage and PBMC models.

siRNA-mediated knockdown of HDAC1 and HDAC2 reduces intracellular survival of K. pneumoniae

To assess the contribution of individual HDACs, siRNA-mediated knockdown of HDAC1 and HDAC2 was carried out in THP-1 macrophages infected with the cKp strain. Reduction of HDAC1 expression resulted in a clear decrease in intracellular bacterial survival (Figure 4E), and a similar reduction was observed following HDAC2 knockdown, with a comparatively greater decrease in bacterial burden (Figure 4F). Together, these findings indicate that the reduction of either HDAC1 or HDAC2 is sufficient to decrease intracellular survival of cKp in THP-1 macrophages.

Intracellular persistence is accompanied by ROS suppression

To explore the underlying immune modulation, intracellular reactive oxygen species (ROS) levels were measured using DCFDA. All strains significantly suppressed ROS production, with MDR-Kp showing the strongest effect. CI994 treatment partially restored ROS levels in all infection conditions (Figure 4G), indicating that HDAC inhibition can reverse infection-associated reductions in intracellular ROS levels.

HDAC inhibition restores cytokine expression at the transcript level during K. pneumoniae infection

To evaluate the role of HDACs in modulating the anti-inflammatory response during K. pneumoniae infection, we quantified IL-10 protein secretion and gene expression in macrophages infected with cKp, hvKp and MDR-Kp isolates in the presence or absence of the HDAC inhibitors CI994 and SAHA.

At the protein level, infection with all three strains increased IL-10 secretion compared with uninfected controls. Interestingly, treatment with SAHA and CI994 further elevated IL-10 protein levels in hvKp and MDR-Kp-infected macrophages, with SAHA showing a comparatively stronger effect. In contrast, both inhibitors reduced IL-10 secretion during cKp infection, with SAHA causing a significant reduction (Figures 5A–5C).

Figure 5.

HDAC inhibition restores cytokine balance during K. pneumoniae infection

(A–C) IL-10 protein levels in macrophages infected with classical (cKp), hypervirulent (hvKp), or multidrug-resistant (MDR-Kp) strains, with or without treatment with CI994 or SAHA (15 μM). cKp infection induced elevated IL-10 secretion, selectively attenuated by SAHA, whereas hvKp and MDR-Kp infections increased IL-10 but remained uncontrolled by HDAC inhibition.

(D–F) IL10 transcript levels measured by qPCR. hvKp and MDR-Kp triggered strong IL10 upregulation (25- to 30-fold), and both inhibitors significantly reduced this induction across strains. Data are represented as mean ± SEM from three independent experiments; statistical significance was determined using one-way ANOVA followed by Sidak’s multiple comparison test (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant).

In contrast to the protein profiles, qPCR analysis revealed marked repression of IL-10 gene expression following HDAC inhibition. While cKp infection maintained near baseline transcript levels, hvKp and MDR-Kp infections induced robust upregulation of IL-10 mRNA approximately 26-fold and 38-fold, respectively. Addition of either CI994 or SAHA markedly reduced this infection-induced transcriptional upregulation, bringing IL-10 transcript levels close to baseline (Figures 5D–5F).

Collectively, these observations raise the possibility that hvKp and MDR-Kp isolates exploit IL-10-associated anti-inflammatory signaling as a potential immune modulatory strategy.

Autophagy is differentially modified by strains and restored by HDAC inhibition

To determine whether K. pneumoniae strains altered host autophagy, we profiled the gene expression of key autophagy-related genes (Beclin, ULK1, and ATG7) in THP-1-derived macrophages. All three genes showed a general reduction following infection with the examined strains, although the decrease in ULK1 expression was less pronounced in cKp-infected macrophages (Figures 6A, 6E, and 6I). Treatment with the HDAC inhibitors CI994 or SAHA partially restored autophagy gene expression in a strain-and inhibitor-dependent manner: CI994 showed greater recovery of Beclin and ULK1 in cKp- and hvKp-infected cells, whereas SAHA significantly enhanced Beclin and ULK1 expression during MDR-Kp infection (Figures 6B–6D and 6F–6H).

Figure 6.

HDAC-dependent regulation of autophagy during Klebsiella pneumoniae infection

(A–D) Beclin1 expression across strains and following HDAC inhibition (CI994, SAHA). Infection reduced Beclin1 levels, which were restored in a strain-and inhibitor-dependent manner.

(E–H) ULK1 expression shows overall downregulation upon infection, with partial recovery upon HDAC inhibition.

(I) ATG7 expression was consistently reduced across all infected conditions.

(J) Western blot analysis of ATG5 and LC3A/B shows differential modulation of autophagy proteins, with reduced levels in hvKp and MDR-Kp infections.

(K) ChIP assay demonstrates the enrichment of HDAC2 at the ATG5 promoter following infection, indicating epigenetic suppression of autophagy. Data are represented as mean ± SEM from three independent experiments; statistical significance was determined using one-way ANOVA followed by Sidak’s multiple comparison test (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant).

Immunoblot analysis of ATG5, a key regulator involved in autophagosome elongation, showed increased protein levels in cKp-infected cells compared with uninfected controls, whereas reduced levels were observed following hvKp and MDR-Kp infection (Figure 6J). Similarly, LC3A/B, a key autophagy-associated protein involved in autophagosomal membrane lipidation and expansion, was elevated in cKp-infected cells, but remained comparable to uninfected controls in hvKp and MDR-Kp infections.

Recruitment of HDAC2 to the promoter of the ATG5 gene in macrophages infected with cKp, hvKp, and MDR-Kp strains

We performed chromatin immunoprecipitation (ChIP) assays to identify HDAC2 target genes associated with transcriptional repression in macrophages during K. pneumoniae infection. To this end, we evaluated HDAC2 recruitment at the promoter regions of key autophagy-related genes as well as selected pro-inflammatory cytokine genes.

Our findings indicate that, at 24 h post-infection (hpi) with cKp, hvKp, and MDR-Kp strains, HDAC2 was significantly enriched at the promoter region of the ATG5 gene, suggesting epigenetic suppression of autophagy under these infection conditions.

Macrophage polarization is differentially modulated by Klebsiella pneumoniae strains

To investigate how different K. pneumoniae strains influence macrophage polarization, we quantified the expression of CD80 (an M1 marker) and CD206 (an M2 marker) in infected macrophages (Figures 7A–7H). MDR-Kp isolate promoted a clear M1-like phenotype, with a significant 2-fold increase in CD80 and only modest changes in CD206. cKp infection also showed a slight increase in CD80 expression, following a trend similar to the MDR-Kp isolate. In contrast, hvKp isolate induced a striking M2-like response, characterized by a 25-fold increase in CD206 and a concomitant reduction in CD80 expression (∼1-fold decrease) compared to controls. Western blot analysis of CD86, an additional M1 marker, revealed reduced expression in hvKp-infected macrophages, with MDR-Kp infection also showing comparatively lower CD86 levels. In contrast, cKp-infected cells exhibited relatively higher CD86 expression (Figure 7I). Importantly, treatment with HDAC inhibitors attenuated this skewing. Both CI994 and SAHA reduced CD206 in hvKP-infected macrophages, thereby counteracting the M2-like phenotype. Furthermore, SAHA exerted a stronger effect than CI994 in restoring CD80 expression, suggesting differential efficacy of HDAC inhibitors in modulating polarization. These findings highlight that the representative K. pneumoniae strains differentially modulate macrophage responses, with cKp and MDR-Kp favoring a pro-inflammatory M1-like program, whereas hvKp promoted a pronounced M2-like phenotype. Epigenetic intervention with HDAC inhibitors partially reverses these effects, underscoring macrophage polarization as an important axis of host-pathogen interaction and a potential therapeutic target.

Figure 7.

Strain-specific modulation of macrophage polarization and its regulation by HDAC inhibition

(A–D) CD80 (M1 marker) expression in macrophages infected with cKp, hvKp, and MDR-Kp ± HDAC inhibitors (CI994, SAHA). MDR-Kp showed a significant increase in CD80, whereas the hvKp isolate showed no significant change or reduction; HDAC inhibition, particularly SAHA, significantly enhanced CD80 expression.

(E–H) CD206 (M2 marker) expression showing a significant increase in hvKp infection, with no significant induction in cKp and MDR-Kp; HDAC inhibition significantly reduced CD206 levels.

(I) Immunoblot analysis of CD86 shows reduced expression in hvKp and MDR-Kp and relatively higher levels in cKp. Data are represented as mean ± SEM from three independent experiments; statistical significance was determined using one-way ANOVA followed by Sidak’s multiple comparison test (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant).

Discussion

The interaction between K. pneumoniae and host immune responses involves multilayered regulation; however, the contribution of epigenetic mechanisms to strain-specific pathogenicity remains incompletely defined. In this study, the representative classical, hypervirulent, and MDR-Kp strains exhibited convergence toward host epigenetic reprogramming, characterized by increased HDAC2 expression and reduced histone acetylation; however, the extent of this modulation varies across strains, resulting in the differential suppression of immune signaling, autophagy, and oxidative responses. Epigenetic control of host defense pathways is increasingly recognized as a central mechanism exploited by intracellular pathogens to promote persistence.^11,20,21

Recent studies have refined the definition of hypervirulent K. pneumoniae, distinguishing it from hyper mucoviscosity and emphasizing its multifactorial genetic basis involving siderophore production, capsule regulation, and immune evasion determinants.^22,23,24,25 Moreover, the emergence of convergent strains combining hypervirulence and antimicrobial resistance highlights the importance of understanding host-pathogen interactions at a mechanistic level.^26,27 In our study, the representative hypervirulent strain exhibited enhanced intracellular persistence and promoted an anti-inflammatory macrophage phenotype, whereas the MDR strain displayed resistance to host-mediated killing despite a pro-inflammatory signature, indicating that distinct virulence programs converge functionally on immune evasion.

A central observation in our study is the preferential involvement of HDAC2 in infection-induced chromatin remodeling. HDACs regulate transcription by removing acetyl groups from histone lysine residues, including H3K18, resulting in chromatin condensation and transcriptional repression.^8,28 We observed consistent elevation of HDAC2 protein levels across the examined infection conditions, accompanied by reduced H3K18 acetylation, indicating active chromatin repression. Moreover, HDAC2 transcriptional upregulation was more pronounced in hypervirulent strain infection, suggesting strain-dependent amplification of this pathway. In contrast, HDAC1 exhibited minimal variation at both transcript and protein levels, supporting the notion that HDAC family members exert non-redundant roles in immune regulation.²⁹

hvKp and MDR-Kp infections induced marked transcriptional upregulation of IL-10, whereas only a non-significant increase was detected at the protein level. In contrast, cKp infection showed significant IL-10 secretion despite minimal transcriptional upregulation. The downstream consequences of this epigenetic modulation were reflected in altered innate immune signaling. Infection led to reduced expression of TLR2/4 and components of the JAK/STAT pathway, which are critical for pathogen recognition and cytokine signaling.^30,31,32 This was accompanied by decreased IL-8 and increased IL-10 production, indicative of an anti-inflammatory environment that may favor bacterial persistence, consistent with previously described immune evasion strategies of K. pneumoniae.^33,34 Notably, we observed a divergence between IL-10 transcript and protein levels following HDAC inhibition. Although hvKp and MDR-Kp isolates induced marked transcriptional upregulation of IL-10, treatment with HDAC inhibitors substantially reduced IL-10 mRNA expression, while extracellular IL-10 protein levels remained elevated or showed only modest changes. In contrast, cKp infection showed significant IL-10 secretion despite minimal transcriptional upregulation, suggesting the involvement of post-transcriptional regulatory mechanisms. Such uncoupling may arise from regulated mRNA turnover mediated by AU-rich element-binding proteins, as well as microRNA-dependent repression that limits ribosome recruitment and translation efficiency.^35,36 In addition, cytokine output is further regulated at the levels of translation and secretion, where infection-induced signaling can influence ribosomal loading or vesicular trafficking, thereby altering protein production independently of mRNA abundance.^37,38 Together, these findings highlight that cytokine expression during infection may be regulated at multiple levels beyond transcription.

Our findings further indicate that epigenetic regulation extends to autophagy, a critical host defense pathway against intracellular pathogens.³⁹ Infection resulted in the reduced expression of Beclin-1, ULK1, and ATG7, consistent with the pathogen-mediated disruption of autophagy initiation.⁴⁰ Importantly, we observed selective recruitment of HDAC2 to the ATG5 promoter, indicating locus-specific chromatin targeting rather than the global repression of autophagy-related genes. ATG5 plays a central role in autophagosome elongation and LC3 lipidation, and its suppression can impair autophagic flux and intracellular pathogen clearance.^41,42

Beyond its role in canonical autophagy, ATG5 has also been implicated in the regulation of innate immune processes, including ROS production and neutrophil extracellular trap (NET) formation.⁴³ In this context, HDAC2-mediated repression of ATG5 may contribute to a coordinated suppression of autophagy-dependent antimicrobial mechanisms. Consistent with this, we observed reduced ROS levels upon infection, which were restored following HDAC inhibition, supporting a functional link between epigenetic regulation, autophagy, and oxidative defense. Together, these findings suggest that the examined K. pneumoniae strains modulate host autophagy through targeted epigenetic mechanisms that may promote intracellular survival while limiting effective antimicrobial responses.

To further evaluate the functional relevance of HDAC-mediated epigenetic regulation during infection, we assessed the impact of pharmacological HDAC inhibition on intracellular bacterial burden, ROS production and autophagy-related gene expression, supporting a role for HDAC activity in regulating host defense pathways. ROS are critical mediators of antimicrobial defense, and their suppression is a well-established mechanism of immune evasion by intracellular pathogens.⁴⁴

Notably, CI994 treatment resulted in a more pronounced reduction in intracellular bacterial burden, supporting a prominent role for class I HDAC-dependent transcriptional regulation in enhancing host-mediated bacterial clearance. The differential effects observed between CI994 and SAHA provide additional mechanistic insight into HDAC-mediated host responses. CI994 is a class I-selective HDAC inhibitor targeting HDAC1/2 and primarily influences transcription through chromatin remodeling complexes such as Sin3A and NuRD,⁴⁵ whereas SAHA (vorinostat), as a pan-HDAC inhibitor, targets both class I and class II HDACs and modulates a broader range of substrates, including non-histone proteins involved in signaling and intracellular trafficking.^46,47,48

In contrast, while both CI994 and SAHA reduced IL-10 transcription, only SAHA attenuated IL-10 protein levels during classical strain infection, indicating an additional layer of regulation beyond transcription. Collectively, these findings suggest that class I HDAC activity primarily governs transcriptional programs associated with bacterial persistence, whereas broader HDAC inhibition may further influence downstream processes such as cytokine translation or secretion, thereby revealing multiple levels of epigenetic control over host immune responses.

These multilayered effects of HDAC inhibition on transcriptional and post-transcriptional regulation prompted us to further examine how such epigenetic modulation influences downstream immune phenotypes, particularly macrophage polarization.

Epigenetic modulation was also closely associated with macrophage polarization.⁴⁹ In our study, the representative hypervirulent strain promoted a pronounced M2-like phenotype characterized by elevated CD206 expression, consistent with an immunosuppressive and tissue-remodeling phenotype. In contrast, the representative MDR strain induced a partial M1-like phenotype with increased CD80 expression but limited bacterial clearance efficiency, suggesting a dysregulated inflammatory response.

Consistent with these observations, additional M1-associated markers further supported this polarization pattern, with hypervirulent strain infection showing reduced expression of pro-inflammatory markers, while classical strain infection maintained relatively higher levels. Importantly, pharmacological inhibition of HDAC activity mitigated this polarization bias, as evidenced by a reduction in M2-associated features and a partial restoration of M1-associated markers. Notably, the broader inhibitory profile of SAHA appeared to exert a stronger effect on restoring M1-associated characteristics, suggesting that the modulation of non-histone targets may contribute to its effects on macrophage functional states.⁵⁰

Taken together, our findings suggest HDAC2-driven epigenetic reprogramming as a central mechanism underlying immune suppression and intracellular persistence during K. pneumoniae infection, with strain-dependent variation in the extent of this regulation. By integrating strain-specific virulence traits with host chromatin dynamics, this study provides a mechanistic framework linking epigenetic regulation to immune evasion and highlights HDACs as promising targets for host-directed therapeutic strategies.

Limitations of the study

This study utilized a single representative classical, hypervirulent, and MDR-Kp strain for comparative mechanistic analyses. Although these strains enabled identification of strain-associated differences in host immune modulation, they do not encompass the full phenotypic and genomic diversity within each K. pneumoniae phenotype. In addition, most experiments were performed using THP-1-derived macrophages, with limited validation in PBMCs and C. elegans infection models, which may not fully reflect mammalian host responses. Although our findings support a role for HDAC-2-associated epigenetic regulation in autophagy and immune modulation, the precise host or bacterial factors involved in HDAC2 recruitment, its interacting chromatin-remodeling partners, and the broader downstream regulatory networks remain to be elucidated. Further studies using larger panels of clinical isolates, primary immune cells, and mammalian infection models will be important to further validate and expand these findings.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Aravind Madhavan (aravindmi@am.amrita.edu).

Materials availability

Material from this study are available through lead contact upon request.

Data and code availability

• •The raw reads generated from the genome sequencing of the bacterial strain U10386 are deposited into the NCBI Sequence Read Archive (SRA) under BioProject: PRJNA1398767.
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact, upon request.

Acknowledgments

A.M. would like to acknowledge the seed grant funding (ASG2022053), Amrita Vishwa Vidyapeetham, and SURE research funding (SUR/2022/003961), Anusandhan National Research Foundation, Government of India, for financial support. A.K. acknowledges the doctoral fellowship from Amrita Vishwa Vidyapeetham. The authors gratefully acknowledge Sri Mata Amritanandamayi Devi (Amma), Chancellor, Amrita Vishwa Vidyapeetham, India, for her inspiration and for providing financial support for the Article Processing Charges (APC) of this publication.

Author contributions

K.A.: conceptualization, data curation, investigation, methodology, resources, software, validation, writing – original draft, and writing – review and editing. S.M.S., A.B.A., S.P., S.S.T., A.M.M., P.M.P.C., and K.R.V.: investigation and methodology. A.P.J.: investigation, methodology, and software. G.B.K., B.G.N., and P.B.: resources, investigation, and writing – review and editing. A.M.: conceptualization, data curation, investigation, methodology, resources, software, and validation.

Declaration of interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the writing process

AI-assisted tools were used only for language editing and improving the clarity of the manuscript. All scientific content, interpretations, and conclusions were developed by the authors and carefully reviewed and verified.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
HDAC1 Rabbit monoclonal	Cell signaling Technology	Cat#34589; RRID: AB_2756821
HDAC2 Rabbit monoclonal	Cell signaling Technology	Cat#57156; RRID: AB_2756828
H3K18 Rabbit polyclonal	Abcam	Cat#ab1191; RRID: AB_298692
H3 Rabbit monoclonal	Cell signaling Technology	Cat#4499; RRID: AB_10544537
ATG5 Rabbit monoclonal	Cell signaling Technology	Cat#12994; RRID: AB_2630393
LC3A/B Rabbit monoclonal	Cell signaling Technology	Cat#12741; RRID: AB_2617131
CD86 Rabbit monoclonal	Cell signaling Technology	Cat#91882; RRID: AB_2797422
BETA ACTIN Mouse monoclonal	Cell signaling Technology	Cat#3700; RRID: AB_2242334
IgG (Rabbit)	Cell signaling Technology	Cat#7074; RRID: AB_2099233
IgG (Mouse)	Cell signaling Technology	Cat#7076; RRID: AB_330924
Bacterialandvirusstrains
K. pneumoniae(ATCC33495)	ATCC	ATCC33495
K. pneumoniae clinical strains	Amrita Institute of Medical Sciences and Research Center (AIMS, Kochi)	N/A
E. coli OP50	Provided by Dr. Sanjay Pal (Amrita Vishwa Vidhyapeetham)	N/A
Chemicals,peptides, andrecombinantproteins
Luria Bertani Broth	Himedia	Cat#M1245
Agar Agar, Type I	Himedia	Cat#GRM666
MacConkey Broth	Himedia	Cat#MH083
RPMI 1640	Gibco	Cat#23400-013
DMEM	Gibco	Cat#12800-058
Penstrep	Invitrogen	Cat#15140122
Amphotericin B	Invitrogen	Cat#15290026
Trypsin- 0.05% EDTA	Invitrogen	Cat#25300062
Gentamicin sulfate	Himedia	Cat#CMS461
Fetal Bovine Serum	Invitrogen	Cat#A5256701
DMSO	Sigma-aldrich	Cat#D2650
BSA	Himedia	Cat#MB083
Triton ×100	Sigma-aldrich	Cat#X100
PVDF membrane	BIO-RAD	Cat#1620177
Nystatin	Himedia	Cat#CMS212
Cholesterol	Himedia	Cat#TC101
Peptone	Himedia	Cat#RM001
PoweUp SYBR Green Master Mix	Thermo Scientific-Applied Biosystems	Cat#A25742
Taq polymerase	Takara	Cat#R001A
Ex Taq	Takara	Cat#RR001A
Histopaque-1077	Sigma-Aldrich	Cat#H8889
PMA	Sigma-Aldrich	Cat#P8139
CI994	Sigma-Aldrich	Cat#C0621
SAHA	Sigma-Aldrich	Cat#SML0061
Criticalcommercialassays
ELISA IL10	Origin	Cat#OPK1142
Primescript 1st strand cDNA synthesis kit	Takara	Cat#6110A
RNA isolation	MN	Cat#740955.50
BCA Protein assay kit	Pierce-Thermo Scientific	Cat#23225
DNeasy ultraclean microbial kit	Qiagen	Cat#12224-50
DNA miniprep Kit	Zymobiomics	Cat#D4300
diagenode ideal chip qpcr	HOLOGIC Diagenode	Cat#C01010180
Depositeddata
Whole genome sequences	NCBI SRA database	BioProject Accession: PRJNA1398767
Experimentalmodels:Cell lines
THP-1	ATCC	TIB-202
A549	NCCS, Pune	N/A
HaCaT	NCCS, Pune	N/A
HT29	NCCS, Pune	N/A
T24	NCCS, Pune	N/A
HEK293	NCCS, Pune	N/A
Experimental models:Organisms/strains
C. elegans (WT Bristol N2 strain)	Provided by Dr. Sanjay Pal (Amrita Vishwa Vidhyapeetham)	N/A
Oligonucleotides
Primers for qPCR (Table S3)	This paper	N/A
Primers for promoters (Table S4)	This paper	N/A
Software andalgorithms
Graphpad prism 10.3.1	Graph pad	10.3.1
ImageLAb	Bio-Rad Laboratories	6.0.1
FastQC	Simon Andrews/Babraham Bioinformatics	v0.12.1
AdapterRemoval	Schubert et al.51	v2.3.4
Unicycler	Wick et al.52	v0.5.1
SPAdes	Prjibelski et al.53	v3.15.5
Kraken2	Wood et al.54	Galaxy Version 2.17.1+galaxy0
KrakenTools	Lu et al.55	Galaxy Version 1.2.1+galaxy0
BUSCO	Manni et al.56; Eddy57; Hyatt et al.58	v6.0.0
QUAST	Mikheenko et al.59	v5.3.0
ABRicate	Seemann14; Chen et al.15	v1.2.0
Kleborate	Lam et al.16; Wyres et al.17; Tsang et al.18	v3.2.4
Other
Quantstudio5 qpcr	Applied biosystems	N/A
Transblot turbo	Bio-Rad	N/A
ChemiDoc XRS+	Bio-RAd	N/A
apotome microscope	Zeiss	N/A

Experimental model and study participant details

Bacterial strains

Clinical isolates of Klebsiella pneumoniae (B330, B12012, B176, B12348, B11542, B8975, B12720, U10385, U10386, and OF10416) were obtained from obtained from Amrita Institute of Medical Sciences and Research Center (AIMS, Kochi). The reference strain K. pneumoniae ATCC33495 was obtained from American Type Culture Collection (ATCC, USA). The bacterial cultures were initiated by inoculating strains into Luria-Bertani (LB) broth (HiMedia, India) and incubated overnight at 37°C with constant agitation at 220 rpm. Subculturing was performed by diluting overnight cultures 1:100 into fresh LB broth and incubating until mid-log phase (OD600 = 0.4), as quantified using a biophotometer (Eppendorf, Germany).

Cell lines

The human monocytic cell line THP-1 (ATCC TIB-202) was purchased from American Type Culture Collection (ATCC), USA and maintained in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, USA) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS; Gibco) and 1% penicillin-streptomycin (Invitrogen) solution. Epithelial cell lines A549 (lung), HaCaT (keratinocyte), T24 (bladder), and HT29 (colon) and HEK293 (human embryonic kidney cell line) were obtained from the National Center for Cell Science (NCCS, Pune, India) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS. Cell lines were authenticated by the respective providers. All Cell lines were tested for mycoplasma contamination and the results were negative. Cells were maintained at 37°C in a humidified incubator with 5% CO₂.

Blood sampling

Peripheral blood was collected from healthy adult donors by venipuncture under aseptic conditions in heparinized vacutainers for whole blood killing assays, and in non-heparinized tubes for serum isolation used in serum killing assays, following written informed consent and institutional ethical approval.

C. elegans, maintenance and synchronization

Caenorhabditis elegans strain N2 was maintained on standard NGM plates seeded with E. coli OP50 at 20°C. Synchronized C. elegans eggs were obtained by hypochlorite treatment (8 min, followed by three washes with M9 buffer and centrifugation at 400 × g for 3 min. Eggs were incubated in M9 buffer at 20°C for 12h to allow hatching to the L1 stage. Larvae were then transferred to NGM plates seeded with E. coli OP50 and cultured to the L4 stage. All analysis were conducted on hermaphrodites.

Ethics considerations

Human peripheral blood samples were obtained from healthy adult donors with written informed consent, under protocols approved by the Institutional Ethics Committee of Amrita School of Medicine (ECASM-AIMS-2023-149), Amrita Vishwa Vidyapeetham (AIMS, Kochi). Experiments involving Caenorhabditis elegans were performed in compliance with institutional biosafety guidelines and are not subject to animal ethics committee approval. All experiments were performed in accordance with relevant guidelines and regulations of Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, India.

Method details

PBMC isolation, processing and survival analysis

Human PBMCs were obtained from peripheral blood collected from healthy volunteers into heparinized tubes. To enrich mononuclear cells, whole blood was diluted 1:1 with RPMI-1640 containing 2% FBS and gently layered over an equal volume of Histopaque-1077 in 50-mL conical tubes. Samples were centrifuged at 600 g for 20 min at room temperature without brake to allow clear separation of cell layers. The PBMC-enriched interface was carefully aspirated and transferred to a fresh tube, followed by three washes in PBS with 2% FBS (400 g, low brake) to remove platelets and residual plasma. The recovered cells were counted, resuspended in RPMI-1640, and seeded for adherence. After a 2-h incubation, non-adherent lymphocytes—including T cells, B cells, and NK cells—were removed, leaving behind an enriched monocyte fraction that was used for all subsequent experiments.

String test for the determination of hypervirulent strains

Clinical isolates of K. pneumoniae were cultured overnight at 37°C on Luria-Bertani (LB) agar, MacConkey agar, and Blood agar plates. The hypermucoid phenotype was evaluated using the string test by gently lifting colonies with a sterile inoculating loop. A string length ≥5 mm was considered positive.

PCR amplification and BLAST analysis of hypervirulent marker genes

Genomic DNA was extracted from the string positive isolates using a commercial DNA extraction kit (Qiagen) according to the manufacturer’s protocol. PCR was performed to detect virulence associated genes (rmpA, rmpA2, iucA, iroB and peg-344) using gene specific primers (IDT technologies) and Taq polymerase enzyme kit (Takara). Thermal cycling was performed following the manufacturer’s protocol. PCR products were analyzed on a 1.5% agarose gel stained with ethidium bromide and visualized under UV illumination. PCR amplicons were purified using a PCR purification kit and sequenced (Sanger sequencing). Resulting sequences were analyzed using NCBI BLAST to confirm gene identity. Isolates with ≥99% identity to known virulence gene sequences were considered positive. Primer sequences used for amplification are listed (Table S3).

Genome extraction and whole genome sequencing

Genomic DNA was extracted using the ZymoBIOMICS DNA Miniprep Kit following the manufacturer’s protocol. Final DNA concentrations were determined via Qubit. Sequencing libraries were prepared using a tagmentation- and PCR-based Illumina DNA Prep workflow with custom IDT 10bp unique dual indices, targeting an insert size of ∼280 bp. Whole-genome sequencing was performed on an Illumina NovaSeq X Plus platform to generate 2 × 151 bp paired-end reads.

Genome assembly and analysis

Raw paired-end sequencing reads were initially subjected to quality assessment using FastQC (v0.12.1) to evaluate per-base sequence quality, GC content distribution, sequence duplication levels, and adapter contamination. Adapter trimming, removal of low-quality bases, and filtering of reads containing ambiguous nucleotides were performed using Adapter Removal (v2.3.4)⁵¹ applying a minimum Phred quality threshold of Q25 to ensure high confidence reads for downstream analysis. The processed high-quality reads were assembled de novo using Unicycler (v0.5.1),⁵² with SPAdes (v3.15.5).⁵³ Taxonomic classification of reads was performed using Kraken2 (Galaxy Version 2.17.1+galaxy0)⁵⁴ against the standard reference database (2021-05-17) to identify potential contamination and confirm species identity, followed by extraction of classified reads of interest (Taxon ID: 570) using KrakenTools (Galaxy Version 1.2.1+galaxy0)⁵⁵ on the Galaxy Platform Galaxy Community (2024). The quality, completeness, and structural integrity of the assembled genome were evaluated using BUSCO (v6.0.0)^56,57,58 with the enterobacterales_odb10lineage dataset and QUAST⁵⁹ (v5.3.0). BUSCO analysis confirmed 96.1% completeness (92.7% single-copy, 3.4% duplicated), with only 0.9% fragmentation. QUASTmetrics demonstrated a high-quality draft assembly comprising 113 contigs with a total genome size of approximately 5.97 Mb, GC content of 56.54%, and an N50 of 206 kb, reflecting good assembly contiguity and coverage. Identification of virulence-associated genes were performed using ABRicate (v1.2.0),^14,15 screening against VFDB to identify virulence-associated genes. Detailed genomic profiling - including MLST, capsule (K) and lipopolysaccharide (O) serotyping, and standardized virulence/resistance scoring was done using Kleborate (v3.2.4).^16,17,18

Minimum inhibitory concentration (MIC) assay

MIC of gentamicin against K. pneumoniae was determined by broth microdilution. Mid-log phase bacteria (∼1 × 10⁵ CFU/mL) were incubated with 2-fold serial dilutions of gentamicin in Luriia-Bertani (LB) broth at 37°C for 16-18h. MIC was defined as the lowest concentration with no visible growth.

Bacterial growth curve assay

To assess growth, K. pneumoniae strains were cultured in LB broth in 96-well plates (200 mL/well). OD600 was recorded at 30 min intervals over a 24h period using a synergy H1 plate reader under continuous shaking at 37°C.

Differentiation of THP-1 monocytes

The human monocytic cell line THP-1 (ATCC-TIB-202) was maintained in RPMI 1640 medium supplemented with 10% FBS at 37ᵒC in a humidified 5% CO₂ incubator. Differentiation into macrophage-like cells was induced by exposing THP-1 monocytes to 20 ng/mL phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich) for 24hrs. Successful differentiation was confirmed by monitoring adherence and morphological changes using phase-contrast microscopy.

Infection of THP-1 derived macrophages and HDAC inhibitor treatment

Differentiated macrophages were infected with Klebsiella pneumoniae strains with a multiplicity of infection (MOI) 1:30 or 1:20 depending on the assay. Following a 30 min incubation, cells were washed thrice with Phosphate-Buffered Saline (PBS) and incubated in medium containing gentamicin (45 μg/mL) for 45 min to eliminate extracellular bacteria. HDAC inhibitors CI994 (15 μM¹⁰; Sigma-Aldrich), or SAHA (15 μM¹⁹; Sigma-Aldrich) were added to appropriate wells, and cells were incubated for 24h. Uninfected macrophages served as baseline controls.

Quantification of intracellular bacteria

After 24h post infection, macrophages were lysed using 0.06% SDS in PBS for 5 min at room temperature. Lysates were serially diluted and plated on LB agar, followed by overnight incubation at 37°C. Colonies were counted manually, and results were expressed as colony forming units (CFU) per mL.

Total protein extraction and quantification

Cells were lysed in RIPA buffer with protease and phosphatase inhibitors (Thermo Fisher Scientific). Total protein was quantified using the Bicinchoninic Acid (BCA) Assay (Pierce BCA Protein Assay Kit) according to the manufacturer’s instructions. Absorbance at 562 nm was measured using a plate reader.

Western blotting

Equal quantities of protein (25 μg) were resolved by SDS-PAGE on 12% polyacrylamide gel and transferred to PVDF membranes. Membranes were blocked in 3% Bovine Serum Albumin (BSA) in TBST for 1h, probed with primary antibodies overnight at 40ᵒC, and then incubated with HRP-conjugated secondary antibodies. Bands were visualized using ECL substrate (Bio-Rad) and imaged on a ChemiDoc XRS+ system.

Cytokine quantification by ELISA

Cell culture supernatants were collected at 24hrs post-treatment. Levels of IL-12p40, TNF-α, and IL-10 were quantified using ELISA kits (Origin) as per the manufacturer’s protocols. Optical density was read at 450 nm using a microplate reader.

RNA extraction and quantitative RT-PCR

Total RNA was isolated from macrophages using the NucleoSpin RNA isolation kit (Macherey-Nagel) and quantified using a Nanodrop spectrophotometer. One microgram of RNA was reverse transcribed into cDNA using the Takara PrimeScript 1^st strand cDNA synthesis kit. Quantitative PCR was performed using TB Green Premix Ex Taq (Takara) on a QuantStudio 3 Real-Time PCR System. Relative expression levels were calculated using the 2ˆ(-ΔΔCt) method, with Beta-actin as the internal control. Primer sequences used for amplification are listed (Table S3).

Measurement of intracellular ROS

Reactive oxygen species (ROS) generation was assessed using the DCFDA dye (Sigma-Aldrich). Infected, treated and control macrophages were incubated with 10micromolar DCFDA for 45 min at 37ᵒC. Fluorescence was measured using a microplate reader at excitation/emission of 485/535 nm.

Whole blood killing assay

Fresh peripheral blood was collected from healthy donors in heparinized vacutainers after written informed consent. The study was reviewed and approved by the Institutional Human Ethics Committee, Amrita Vishwa Vidyapeetham. Blood was aliquoted into two 15 mL falcon tubes; one was incubated at 56ᵒC for 30 min to inactivated, while the other was maintained at 37ᵒC (active blood). K. pneumoniae strains were cultured to OD600 = 0.4, washed twice with PBS, and resuspended in RPMI 1640 medium. Bacterial suspensions were diluted 1:100 in RPMI and mixed with active or inactive blood at a 1:10 ratio. Aliquotes were collected at 0,30,60 and 120 min, serially diluted in PBS, plated on LB agar, and incubated at 37ᵒC overnight. CFU/mL was calculated, and bacterial survival was expressed as log 10(CFU/mL) over time.

Whole serum killing assay

Blood was collected in clot-activator tubes, allowed to clot at room temperature for 30 min, and centrifuged at 1000g for 10 min. The resulting serum was aliquoted into two tubes-one heat inactivated at 56ᵒC for 30 min and other kept active at 37ᵒC. And further assay completed like the blood killing assay.

Adherence assay to host cells

Epithelial cell lines (HaCaT, A549, HT29, T24) were seeded in 24-well plates and infected with K. pneumoniae strains at MOI 1:30. After 20 min, cells were washed thrice with PBS and lysed in 0.1% Triton X-100. Lysates were plated for CFU quantification. Adherence efficiency was expressed as CFU recovered per well.

Chromatin immunoprecipitation and PCR

Chromatin immunoprecipitation (ChIP) was carried out based on the manufacturer’s protocol (iDeal ChIP kit, diagenode). In brief, the uninfected and infected cells were crosslinked with formaldehyde, quenched with glycine, and lysed. Chromatin was sheared by sonication with pulse conditions 30s On and 30s off for 25 cycles to obtain DNA fragments (∼100–600 bp). Sheared chromatin was immunoprecipitated using an anti-HDAC2 antibody with Protein A magnetic beads. After washing, complexes were eluted and reverse crosslinked, and DNA was purified. Enriched DNA was subjected to conventional PCR using primers targeting promoter regions of autophagy-related genes and selected cytokine genes. PCR products were resolved on agarose gels and visualized under UV illumination. Primer sequences used for amplification are listed (Table S4).

HDAC1/2 knockdown and intracellular survival assay

THP-1- derived macrophages (2 × 10⁶ cells) were transfected with siRNAs targeting HDAC1 and HDAC2 (Santa Cruz Biotechnology) using lipofectamine 2000 (Invitrogen) in serum and antibiotic free Opti-MEM medium, according to the manufacturer’s instructions. Briefly, 60 pmol siRNA was used per transfection, and cells were incubated for 36hrs to allow gene silencing. Scrambled siRNA was included as a negative control.

Post-transfection, cells were infected with K. pneumoniae (ATCC 33495) at a multiplicity of infection (MOI) of 1:30. Intracellular bacterial survival was subsequently determined using a gentamicin protection assay followed by CFU enumeration.

C. elegans survival assay

Wild-type Caenorhabditis elegans N2 worms were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20 °C. Synchronized populations were obtained by hypochlorite treatment of gravid adults (8 min), followed by three washes with M9 buffer and centrifugation at 400 × g for 3 min. Eggs were incubated in M9 buffer at 20 °C for 12 h to allow hatching into L1 larvae, which were subsequently transferred to OP50-seeded NGM plates and cultured to the L4 stage. For infection assays, approximately 50 L4-stage hermaphrodites were transferred into 50 μL of M9 buffer and mixed with an equal volume (50 μL) of bacterial suspension prepared from overnight cultures of Klebsiella pneumoniae (ATCC strain, hypervirulent strain, or multidrug-resistant strain) adjusted to OD₆₀₀ = 0.4, pelleted, and resuspended in M9 buffer, resulting in a 1:1 ratio of worms to bacterial inoculum. Control worms were incubated with OP50 under identical conditions. Worms were maintained at 20 °C, and survival was assessed daily by scoring live and dead worms based on responsiveness to gentle mechanical stimulation.

Quantification and statistical analysis

All experiments were performed with a minimum of three independent biological replicates unless otherwise stated. Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism Software (Version 10.3.1).

Comparisons among multiple groups were analyzed using one-way ANOVA followed by Dunnett’s or Sidak’s multiple comparison test, as indicated in the respective figure legends. Kaplan-Meier survival curves for C. elegans assays were analyzed using the log rank (Mantel-Cox) test. Statistical significance was defined as ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001, and ns, not significant.