Graphical abstract

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Highlights

     * •
       MAC4 shows potent antibacterial activity against multiple bacterial
       strains
     * •
       MAC4 prevents biofilm formation and disrupts bacterial adhesion
     * •
       MAC4 targets bacterial membranes, metabolism, and DNA
     * •
       MAC4 reduces lung inflammation and bacterial load in in vivo
       studies
     __________________________________________________________________

   Pharmacology; Medical Microbiology; Immunology

Introduction

   Bacterial infections remain a critical global health challenge, with
   Escherichia coli being a prominent example of a pathogen causing a wide
   spectrum of diseases, from mild gastroenteritis to life-threatening
   conditions such as septicemia and hemolytic uremic
   syndrome.[40]^1^,[41]^2^,[42]^3^,[43]^4^,[44]^5 The burden of E. coli
   infections on healthcare systems worldwide is substantial, leading to
   increased morbidity and mortality.[45]^6^,[46]^7 Current treatment
   strategies, heavily dependent on antibiotics, face mounting challenges
   due to antimicrobial resistance (AMR), with some bacterial strains
   exhibiting resistance to multiple drug classes.[47]^8^,[48]^9^,[49]^10
   This crisis necessitates the urgent development of novel therapeutic
   approaches that can effectively combat bacterial infections while
   minimizing resistance development.[50]^11

   One particularly challenging aspect of bacterial pathogenesis is
   biofilm formation - structured communities of microorganisms encased in
   a self-produced polymer matrix.[51]^12^,[52]^13 Within biofilms,
   bacteria exhibit altered phenotypes with enhanced resistance to
   antimicrobial agents and host immune defenses.[53]^14 For species such
   as E. coli, biofilm formation significantly increases virulence and
   complicates treatment outcomes.[54]^15^,[55]^16 The ability of biofilms
   to resist conventional antibiotics highlights the need for novel
   therapeutic strategies that can both prevent biofilm formation and
   eliminate established biofilms.

   Traditional Chinese Medicine (TCM) offers a promising avenue for
   discovering novel antimicrobial compounds. Recent studies have
   demonstrated that natural compounds derived from traditional medicines
   possess significant antimicrobial and anti-inflammatory
   properties.[56]^17^,[57]^18^,[58]^19 For instance, Rashed and Butnariu
   (2014) identified potent antimicrobial compounds in Bauhinia
   racemosa,[59]^11 while similar studies on Eriobotrya japonica revealed
   bioactive compounds with both antimicrobial and antioxidant
   properties.[60]^20 These findings suggest that traditional medicine
   could be a valuable source of novel therapeutic agents against
   bacterial infections.

   Our metabolomic analysis of Squama manitis, a TCM staple traditionally
   used for infectious diseases, revealed 78 distinct metabolites.[61]^21
   Through systematic screening and analysis, we identified a novel Malic
   Acid Combination (MAC4), comprising malic acid, fumaric acid, glycine,
   and hippuric acid, that showed promising antibacterial
   efficacy.[62]^22^,[63]^23 MAC4 represents a convergence of traditional
   medicine and modern scientific approaches, distinguishing itself from
   conventional synthetic antimicrobials through its natural origin and
   complex composition. The present study aims to evaluate MAC4’s dual
   antimicrobial and immunomodulatory properties through comprehensive
   in vitro and in vivo studies. Using a multi-faceted approach combining
   Minimum Inhibitory Concentration (MIC) assays, biofilm formation
   analyses, whole-transcriptome sequencing, and a mouse model of
   E. coli-induced lung infection, we investigated both MAC4’s direct
   antibacterial effects and its ability to modulate host immune
   responses. This comprehensive evaluation addresses critical gaps in
   current antimicrobial research by exploring the compound’s capacity to
   combat bacterial infections while potentially minimizing inflammatory
   damage. Our findings could provide valuable insights into developing
   novel therapeutic strategies against bacterial infections, particularly
   those complicated by biofilm formation and inflammatory responses,
   contributing to the global effort to combat antimicrobial resistance
   through natural product discovery.

Results

Broad-spectrum antibacterial action of malic acid combination against key
pathogens

   To evaluate the antibacterial potential of MAC4, we assessed
   its efficacy against four critical pathogenic bacteria: E. coli,
   S. aureus, P. aeruginosa, and S. marcescens. Comprehensive analyses,
   including minimum inhibitory concentration (MIC), growth curve
   assessment, and minimum bactericidal concentration (MBC), were
   conducted with varying concentrations of MAC4 ([64]Figures 1A–1D). At
   optimally determined concentrations (0.1C for E. coli and S. aureus;
   0.2C for P. aeruginosa and S. marcescens), MAC4 exhibited significant
   inhibitory effects. The MICs were established at these concentrations,
   with growth inhibition rates at the MIC showing notable efficacy: 80%
   for E. coli, 67% for S. aureus, 71% for P. aeruginosa, and 73% for
   S. marcescens ([65]Figures 1A–1D). In 24-h antimicrobial assays,
   ciprofloxacin (CIP) demonstrated excellent antibacterial activity with
   inhibition rates exceeding 97% against all four tested strains
   (E. coli, S. aureus, P. aeruginosa, and S. marcescens), compared to
   MAC4’s inhibition rates of 67–80%. Longitudinal monitoring over 24 h at
   the MIC revealed that MAC4 consistently suppressed bacterial
   proliferation across all tested species, affirming its potent
   inhibitory capability ([66]Figures 1E–1H).

Figure 1.

   [67]Figure 1
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   MAC4 exhibits broad-spectrum antibacterial activity

   (A–D) Minimum inhibitory concentration (MIC) of MAC4 and CIP against
   E. coli, S. aureus, P. aeruginosa, and S. marcescens determined by
   broth microdilution assay after 24-h exposure. Data points represent
   mean ± SEM. (n = 3 independent experiments).

   (E–H) Growth curves of E. coli, S. aureus, P. aeruginosa, and
   S. marcescens treated with various concentrations of MAC4 and CIP over
   time. Data points represent mean ± SEM. (n = 3 independent
   experiments).

   (I) Antibacterial activity of MAC4 against a panel of Gram-positive and
   Gram-negative bacteria.

   To further elucidate the bactericidal properties of MAC4, MBC assays
   were conducted at multiples of the MIC (1MIC, 2MIC, and 4MIC). The
   bactericidal thresholds for E. coli and S. aureus were identified at
   4MIC (0.4C), highlighting a robust bactericidal effect. Conversely, for
   P. aeruginosa and S. marcescens, the effective MBC was at 1MIC (0.2C),
   underscoring the compound’s broad-spectrum bactericidal properties
   against diverse bacterial strains ([69]Figure 1I).

   The findings from this study underscore the remarkable antibacterial
   activity of MAC4 against a spectrum of pathogenic bacteria, presenting
   a notable inhibitory and bactericidal capacity. The ability of MAC4 to
   effectively inhibit and eradicate bacterial cells at specific
   concentrations suggests its utility as a potent broad-spectrum
   antibacterial agent. These results not only affirm MAC4’s therapeutic
   potential but also contribute to the global effort in combating
   bacterial infections, offering a promising avenue for the development
   of antimicrobial strategies.

Disrupting the shield: malic acid combination’s multidimensional assault on
E. coli and S. aureus biofilms, morphology, and genetic integrity

   To evaluate the potential of MAC4 in inhibiting biofilm formation by
   both E. coli and S. aureus, we conducted biofilm formation assays,
   metabolic activity of biofilms assays, and scanning electron microscopy
   (SEM) analysis. Biofilm formation was evaluated using a crystal violet
   staining method, which quantitatively measures the total biomass of the
   biofilm. The absorbance at 595 nm was used to assess biofilm mass after
   treatment with MAC4, and the inhibition was calculated as a percentage
   of the untreated control. Our results revealed that the biofilm
   formation of both E. coli and S. aureus was inhibited across all tested
   concentrations of MIC when compared to untreated controls
   ([70]Figures 2A and 2B). Notably, treatment groups with concentrations
   at 2-fold and 4-fold the MIC of MAC4 resulted in a marked inhibition of
   biofilm formation. We observed a positive correlation between higher
   concentrations of MAC4 and greater inhibition of biofilm formation in
   both bacterial species. This outcome aligns with the results of
   metabolic activity within the biofilms as determined by the MTT assay,
   which evaluates the metabolic activity of live cells within the
   biofilm. After treatment with MAC4 at 2MIC and 4MIC concentrations, the
   metabolic activity of the biofilms decreased by over 94.97%, suggesting
   a reduction in vitality or biofilm density ([71]Figure 2C).
   Interestingly, MAC4 showed comparable efficacy to ciprofloxacin at 2MIC
   concentration, particularly in biofilm formation inhibition and
   metabolic activity reduction assays ([72]Figures 2A–2C). Both compounds
   achieved >90% reduction in biofilm metabolic activity, suggesting
   MAC4’s particular effectiveness against bacterial biofilms.

Figure 2.

   [73]Figure 2
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   MAC4 inhibits biofilm formation in E. coli and S. aureus

   (A, B) Inhibition of E. coli and S. aureus biofilm formation by
   different concentrations of MAC4 and CIP, assessed by crystal violet
   (CV) staining. Data represent mean ± SEM (n = 3 independent
   experiments; ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, t-test).

   (C) Metabolic activity of E. coli and S. aureus biofilms treated with
   MAC4 and CIP, determined by MTT assay. Data represent mean ± SEM. (n =
   3 independent experiments; ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001, t-test).

   (D) Scanning electron microscopy (SEM) images of E. coli and S. aureus
   biofilms after 24 h treatment with 0.1× MIC MAC4 compared to untreated
   controls. Representative scale bars: 50 μm (left column), 5 μm (middle
   column), and 1 μm (right column).

   (E) TEM images of E. coli and S. aureus treated with 0.1× MIC MAC4
   compared to untreated controls. MAC4 treatment resulted in 1. uneven
   cytoplasm distribution, 2. blurred cell edges, 3. content overflow, 4.
   increased cell edge transparency, 5. plasmolysis, and 6. DNA
   condensation (red arrows). Representative scale bars: 1 μm (left
   column), 500 nm (middle column), and 200 nm (right column).

   Further SEM analysis showed a reduction in the biofilm density and
   disruption of the cell structure of both E. coli and S. aureus after
   MAC4 treatment when compared to control groups. ([75]Figure 2D).
   Notably, in the E. coli control group, bacterial cells were connected
   by filamentous structures, likely composed of extracellular DNA (eDNA)
   or matrix fibers,[76]^24 components of the biofilm matrix that provide
   a framework for bacterial survival and motility.[77]^25^,[78]^26
   However, we observed a significant reduction in the connectivity of
   these structures in E. coli and S. aureus after MAC4 treatment
   ([79]Figure 2D). This suggests a possible direct action of MAC4 on the
   eDNA, leading to its denaturation and inactivation, as well as
   affecting the synthesis of matrix fibers, thereby diminishing their
   connectivity. These findings indicate that MAC4 exhibits a capacity to
   effectively mitigate biofilm formation and may disrupt interbacterial
   interactions within biofilms.

Morphological alterations in E. coli and S. aureus cells

   Transmission electron microscopy (TEM) was employed to discern the
   ultrastructural impacts of MAC4 on E. coli and S. aureus. Comparative
   analysis between untreated controls and MAC4-treated bacteria unveiled
   stark morphological contrasts. In controls, E. coli and S. aureus
   preserved their characteristic rod and cocci shapes, respectively, with
   intact cell walls, clearly defined membranes, and homogeneously dense
   cytoplasm ([80]Figure 2E). Conversely, MAC4 treatment instigated
   significant morphological disruptions, including uneven cytoplasm
   distribution, edge blurring, content overflow, increased cell edge
   transparency, plasmolysis, and DNA condensation. These structural
   alterations suggest compromised cellular integrity, potentially
   hindering critical bacterial functions, diminishing viability, and
   leading to cell destruction.

Reduced bacterial adhesion post-malic acid combination treatment

   Further investigations into MAC4’s effects on bacterial adhesion
   employed FITC-labeled E. coli to quantify adhesion efficiency to human
   HaCaT cells before and after MAC4 exposure. Our results demonstrated a
   dramatic decrease in E. coli adhesion post-treatment ([81]Figure 3A).
   Initially, untreated E. coli showed a 6.85% adhesion rate to HaCaT
   cells, which significantly dropped to 0.81% following MAC4 treatment,
   marking an 8.5-fold reduction in adhesion capability. This pronounced
   decline in adhesion efficiency after 24 h of MAC4 exposure underscores
   a substantial impairment in the bacterial adhesion process.

Figure 3.

   [82]Figure 3
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   MAC4 induces reduces bacterial adhesion and alters gene expression in
   E. coli

   (A) Adhesion rate of E. coli to HaCaT cells following MAC4 treatment
   (mean ± SEM, n = 3 independent experiments; ^∗∗∗∗p < 0.0001, t-test).

   (B) Volcano plot of DEGs in MAC4-treated E. coli. Gray dots:
   non-significant genes; red dots: upregulated DEGs; blue dots:
   downregulated DEGs. Horizontal axis: log2(fold change); vertical axis:
   -log10(q-value).

   (C) GO term enrichment and (D) KEGG pathway analysis of downregulated
   DEGs in MAC4-treated E. coli.

   (E) Correlation between RNA-seq and RT-PCR data for selected DEGs. R^2:
   coefficient of determination for the linear regression.

Transcriptomic insights unveil malic acid combinations antimicrobial
mechanism against E. coli

   To study how bacteria respond to MAC4 at the transcriptomic level, we
   sequenced and compared the gene expression profiles of MAC4-treated
   E. coli cells to untreated controls, using four biological replicates
   for each. We identified 1,150 differentially expressed genes (DEGs),
   with 490 being upregulated and 660 downregulated ([84]Figure 3B and
   [85]Table S1). Gene Ontology (GO) enrichment for downregulated genes
   revealed a focus on membrane-related components, specifically integral
   components of the membrane (GO:0005886), plasma membrane (GO:0005886),
   and outer membrane (GO:0019867) ([86]Figure 3C). This suggests the
   MAC4’s targeted disruption of membrane integrity and function.
   Moreover, downregulated DEGs showed enrichment in DNA binding and
   recombination activities, indicating an impairment in genetic processes
   critical for bacterial survival and adaptation.

   KEGG pathway analysis via Kyoto Encyclopedia of Genes and Genomes
   (KEGG) revealed significant enrichment in pathways such as
   Glycerophospholipid metabolism (ko00564), ABC transporters (ko02010),
   DNA replication (ko03030), Lipopolysaccharide biosynthesis (ko00540),
   and Bacterial invasion of epithelial cells (ko05100) ([87]Figure 3D).
   Notably, the ABC transporter pathway, crucial for nutrient import and
   toxin export,[88]^27 emerged as a major target of downregulation,
   alongside pathways involved in energy metabolism and biofilm formation.
   These insights suggest that MAC4’s bactericidal mechanism encompasses
   broad-spectrum interference with essential bacterial functions, from
   membrane integrity to energy metabolism and genetic processes.

   To ascertain the accuracy of our DEG findings, we conducted RT-qPCR
   validation on a random subset of 10 genes, employing the rrsG (16S rRNA
   gene) as a normalization control. The high correlation coefficient
   (R^2 = 0.77) between RT-qPCR and RNA-Seq data validates the reliability
   of our identified DEGs ([89]Figure 3E and [90]Table S2). Our study
   elucidates the complex antimicrobial mechanism of MAC4, demonstrating
   its dual action in not only preventing biofilm formation and bacterial
   adhesion but also disrupting bacterial morphology and cellular
   processes. The observed transcriptomic alterations further reveal
   MAC4’s capacity to target critical pathways essential for bacterial
   viability and communication.

Malic acid combination modulates immune responses and preserves lung
integrity against E. coli infection

   To assess MAC4’s antibacterial effectiveness in vivo, we utilized an
   ELI model in mice. Intratracheal inoculation with E. coli at a
   concentration of 1 × 10^9 CFU/mL was followed by the administration of
   MAC4 at dosages ranging from 0.5 to 1.5 g/kg/d. Lung bacterial counts,
   conducted three days post-infection, revealed a significant decrease in
   bacterial load in mice treated with MAC4 compared to controls
   ([91]Figure 4A). This demonstrates MAC4’s potent antibacterial activity
   in a live animal model, providing a promising outlook for its
   therapeutic application.

Figure 4.

   [92]Figure 4
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   MAC4 treatment reduces bacterial load and modulates inflammatory
   cytokine expression in an E. coli-induced pneumonia (ELI) mouse model

   (A) Bacterial load in lung tissues of ELI mice treated with MAC4 or
   CIP. Data represent mean ± SEM (n = 6 mice per group; compared with
   control group, ^###p < 0.001, t-test; compared with model group,
   ^∗∗∗p < 0.001, t-test).

   (B) Representative hematoxylin and eosin (HE) stained lung sections
   displaying histopathological changes. Scale bars: 100 μm for the upper
   row of images and 200 μm for the lower row of magnified images.

   (C) mRNA levels of inflammatory cytokines IL-10, IL-6, IL-1β, and TNF-α
   in mouse lungs analyzed by RT-qPCR, normalized to GAPDH. Data represent
   mean ± SEM (n = 4 mice per group; ^∗∗p < 0.01; t-test).

   (D) Protein expression levels of IL-10, IL-6, IL-1β, and TNF-α detected
   by western blotting, with β-Actin as a loading control. Quantification
   of normalized protein expression levels is shown on the right. Data
   represent mean ± SD (n = 4 mice per group; ^∗p < 0.05, ^∗∗p < 0.01,
   ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; t-test).

   Further insights were gained through the histopathological examination
   of lung tissues to evaluate structural changes and inflammation levels.
   Normal lung architecture characterized the control group, with intact
   alveolar spaces and minimal inflammatory cell infiltration
   ([94]Figure 4B). In contrast, the model group displayed signs of
   infection-induced alterations, such as increased inflammatory cell
   infiltration (evident as dense accumulations of dark purple cells,
   indicated by blue arrows), obscured alveolar spaces indicative of edema
   or exudate, and thickened alveolar walls reflecting inflammation.
   Treatment with MAC4, similar to ciprofloxacin (a known drug to treat
   pneumonia), showed a notable improvement in lung tissue condition. All
   treated groups exhibited reduced cellularity, with a clear
   dose-dependent effect observed in the MAC4-treated groups. The extent
   of inflammatory cell infiltration was determined by the presence and
   density of dark purple cell clusters, which were substantially reduced
   in the treatment groups, particularly at medium and high doses of MAC4.
   All treated groups displayed clearer alveolar structures, and
   diminished vascular and bronchiole wall thickening, with MAC4 treatment
   at medium and high doses markedly alleviating inflammation (as
   evidenced by the near-absence of dark cell clusters) and preserving
   lung integrity in the E. coli-induced pneumonia mouse model.

   Analysis of endogenous lung RNA levels of inflammatory markers in an
   ELI model showed a significant decrease in pro-inflammatory cytokines
   (IL-6, IL-1β, TNF-α) following MAC4 treatment ([95]Figures 4C and 4D
   and [96]Data S1). However, the increase in the anti-inflammatory
   cytokine IL-10 was not statistically significant (p > 0.05). These
   results, supported by both RT-PCR and western blot analyses, suggest
   that MAC4 not only acts as an antibacterial agent but also modulates
   the host immune response, reducing inflammation. Altogether, our data
   showed that MAC4 demonstrates potential as an effective antibacterial
   and immunomodulatory agent for bacterial pneumonia, with implications
   for managing various bacterial lung infections and associated
   inflammation.

Malic acid combination-induced immunomodulation enhances host defense against
lung infection: transcriptomic evidence

   To investigate the host response to MAC4 treatment in an
   E. coli-induced pneumonia (ELI) mouse model, we performed
   transcriptomic analysis of lung tissues before and after MAC4
   administration. RNA-Seq analysis identified 535 differentially
   expressed genes (DEGs), with 310 upregulated and 225 downregulated
   genes post-MAC4 treatment ([97]Figure 5A and [98]Table S3). The
   validation of selected DEGs via RT-qPCR demonstrated a strong positive
   correlation (R^2 = 0.85) with RNA-Seq data, confirming the reliability
   of the transcriptomic findings ([99]Table S4 and [100]Figure 5F).
   Notably, MAC4 treatment upregulated genes involved in immune response
   and neutrophil production, such as Csf3 and Alox15, suggesting an
   enhancement of host defense mechanisms[101]^28 Concurrently, the
   downregulation of genes such as Ccl7 and Adtrp indicated a strategic
   modulation of immune responses, potentially reducing excessive
   inflammation and tissue damage.

Figure 5.

   [102]Figure 5
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   MAC4 treatment alters gene expression in the ELI mouse model

   (A) Volcano plot of DEGs in lung tissues of MAC4-treated ELI mice
   compared to controls. Red dots: upregulated genes; blue dots:
   downregulated genes. The top 10 up-and-down-regulated genes by fold
   change are labeled. GO enrichment analysis of (B) upregulated and
   (C) downregulated DEGs. KEGG pathway enrichment analysis of
   (D) upregulated and (E) downregulated DEGs.

   (F) Correlation between RNA-Seq and RT-qPCR data for selected DEGs. R2:
   coefficient of determination for the linear regression. Data points
   represent mean ± SEM. (n = 4 independent experiments).

   Gene enrichment analyses revealed that upregulated genes were
   predominantly involved in immune response processes (GO:0002376 and
   GO:0045087), transcriptional regulation (GO:0006357 and GO:0000122),
   and metabolic adjustments (GO:0006629) essential for infection control
   ([104]Figure 5B). Conversely, downregulated genes were associated with
   cell adhesion (GO:0007155) and apoptotic pathways (GO:0006915),
   suggesting mechanisms to restrict pathogen dissemination and cell death
   ([105]Figure 5C). The decreased expression in inflammatory response
   (GO:0006954) could potentially reduce post-infection inflammation.
   Moreover, alterations in the lipid metabolic process (GO:0006629) may
   affect the integrity and functionality of cell membranes, crucial for
   defending against pathogen invasion ([106]Figure 5C). KEGG pathway
   analysis identified upregulation in immune-related pathways, including
   cytokine-cytokine receptor interaction (mmu04060) and Toll-like
   receptor signaling (mmu04620), indicating enhanced immune response
   ([107]Figure 5D). Downregulation of pathways such as the PI3K-Akt
   signaling pathway (mmu04151) suggested reduced inflammatory cell
   activation and survival, contributing to decreased inflammation
   ([108]Figure 5E). Altogether, our data demonstrate that MAC4 inhibits
   pathogen proliferation and modulates the host’s immune response during
   lung infection, highlighting its therapeutic potential against
   bacterial pathogens.

Discussion

   Bacterial infections pose a significant global health challenge,
   particularly due to increasing antibiotic resistance and the
   limitations of current therapeutic approaches. Our study introduces
   MAC4, a novel therapeutic compound derived from traditional Chinese
   medicine, which demonstrates both potent antibacterial activity and
   immunomodulatory effects. This dual-action mechanism distinguishes MAC4
   from conventional antibiotics, which typically focus solely on
   bacterial elimination.

   MAC4’s broad-spectrum activity is particularly noteworthy when compared
   to conventional antibiotics, which often target specific bacterial
   groups. The concentration-dependent bactericidal effect of MAC4
   suggests a mechanism of action that could potentially overcome
   antibiotic resistance, a growing concern in current antimicrobial
   therapies.[109]^29 Moreover, MAC4’s ability to disrupt biofilm
   formation addresses a critical challenge in infection treatment, as
   biofilms are major contributors to antibiotic resistance and treatment
   failure.[110]^30^,[111]^31^,[112]^32

   Mechanistically, our transcriptomic analysis revealed that MAC4 targets
   multiple cellular processes simultaneously. The observed downregulation
   of genes (e.g., evgA, yihL, fimB, fimE) involved in membrane
   components, DNA binding, and recombination activities indicates that
   MAC4 may target these crucial processes, leading to the disruption of
   bacterial cell integrity, impaired DNA replication and repair, and
   ultimately, cell death.[113]^33^,[114]^34 Furthermore, the
   downregulation of metabolic pathways, such as glycerophospholipid
   metabolism and ABC transporters, suggests that MAC4 may interfere with
   energy production and nutrient acquisition, hindering bacterial growth
   and survival.[115]^35^,[116]^36 These findings suggest MAC4 acts
   through multiple complementary mechanisms affecting bacterial cellular
   processes.

   Our structural investigations have provided additional mechanistic
   insights. TEM analysis demonstrated clear morphological alterations,
   including disrupted cell wall integrity, altered cytoplasmic
   distribution, and DNA condensation. Similarly, SEM analysis showed
   significant disruption of biofilm architecture and bacterial
   connectivity. These structural changes are further supported by
   functional evidence, including an 8.5-fold decrease in bacterial
   adhesion capacity, significant inhibition of biofilm formation, and
   reduced metabolic activity in biofilms by more than 94.97%.

   The immunomodulatory properties of MAC4 represent a particularly
   promising aspect of its therapeutic potential. Our findings demonstrate
   that MAC4 can effectively balance the host’s pro- and anti-inflammatory
   responses, as evidenced by the modulation of cytokines and immune cell
   receptors. The upregulation of genes associated with cytokine signaling
   (e.g., Csf3, Il4i1, Ccl4, Tnf)[117]^37^,[118]^38^,[119]^39 and immune
   cell receptor function (e.g., Cd180, Fcrl1, Fcer1g)[120]^40^,[121]^41
   suggests that MAC4 promotes host immune responses. Additionally, MAC4
   downregulated genes associated with cell adhesion molecules (e.g.,
   Itga3, Itga8),[122]^42 extracellular matrix (e.g., Col12a1,
   Col6a1),[123]^43 and cell cycle/apoptosis (e.g., Bcl2l1),[124]^44
   suggesting the potential suppression of excessive inflammation and
   tissue damage. These findings suggest that MAC4’s therapeutic effect
   extends beyond direct bacterial killing, encompassing a strategic
   modulation of host immune responses to favor recovery and minimize
   collateral tissue damage.

   The implications of our findings extend beyond acute bacterial
   infections. MAC4’s antibacterial, antibiofilm, and anti-inflammatory
   properties make it particularly promising for treating conditions where
   infection and inflammation are interlinked, such as chronic infections
   or inflammatory diseases.[125]^45^,[126]^46^,[127]^47 By reducing
   bacterial load, preventing infection-induced inflammatory responses,
   and inhibiting biofilm formation, MAC4 may help mitigate disease
   progression. However, given the complex nature of many such conditions,
   which often involve multiple factors,[128]^48^,[129]^49^,[130]^50
   additional therapeutic approaches targeting these contributing factors
   may be necessary to complement MAC4’s effects.

   In conclusion, MAC4 represents a promising therapeutic agent that
   addresses both the direct challenges of bacterial infection and the
   associated inflammatory responses. Its mechanism of action, combined
   with its origin in traditional medicine, exemplifies the potential of
   integrating ancient medical knowledge with modern scientific
   approaches. Further development of MAC4 could lead to more effective
   treatments for bacterial infections and inflammatory conditions,
   particularly in cases where conventional antibiotics alone may be
   insufficient.

Limitations of the study

   Several limitations of our study warrant consideration. First, while we
   tested MAC4 against key pathogens, future research should evaluate its
   efficacy against a broader spectrum of bacteria, particularly
   drug-resistant strains. Second, although we demonstrated MAC4’s
   immunomodulatory effects, the complete mechanisms underlying its
   interaction with host immune pathways require further investigation.
   Third, all experiments were conducted using six-week-old female ICR
   mice. The exclusive use of female mice may limit the generalizability
   of our findings.

   Long-term studies in animal models of chronic conditions would be
   valuable in assessing MAC4’s safety and efficacy for extended use.
   Future research should focus on First structure-activity relationship
   studies to optimize MAC4’s composition. Second, the exploration of
   synergies with conventional antibiotics for combination therapies.
   Third, deeper mechanistic studies using knockout mutants, the
   investigation of the potential synergistic effects between MAC4
   components, metabolomic profiling (to track cellular metabolic
   changes), and advanced imaging techniques for studying membrane
   integrity. Fourth, the inclusion of both male and female animal models
   to evaluate the influence of sex on MAC4’s efficacy and safety. These
   studies would facilitate MAC4’s development toward clinical
   applications.

Resource availability

Lead contact

   Requests for further information and resources should be directed to
   and will be fulfilled by the lead contact, Siew Woh Choo
   (cwoh@wku.edu.cn).

Materials availability

   This study did not generate new unique reagents.

Data and code availability

     * •
       Data: Raw sequence reads data have been deposited at NCBI Sequence
       Read Archive (SRA) and are publicly available as of the date of
       publication. Accession numbers are listed in the [131]key resources
       table.
     * •
       Code: This article does not report the original code.
     * •
       All other requests: Any additional information required to
       reanalyze the data reported will be shared by the [132]lead contact
       upon request.

Acknowledgments