Targeting phenol-soluble modulin α3-driven M1 macrophage polarization and necroptosis mitigates MRSA infection in mice

Domain: biology
Published: 2026-03-27
Editorial outcome: minor_revision

Abstract
The growing antibiotic resistance and high mortality rates associated with methicillin-resistant
Staphylococcus aureus (MRSA) pose a global health threat, highlighting the urgent need for novel
therapeutic strategies. Phenol-soluble modulin α3 (PSMα3) is a critical virulence factor in MRSA
pathogenesis and immune evasion. However, its underlying mechanisms remain unclear. Here, we
demonstrate that PSMα3 promotes both M1 macrophage polarization and necroptosis. These processes
are mechanistically linked through an interaction between the interferon-stimulated gene factor 3 (ISGF3)
and necrosome complexes, with formyl peptide receptor 2 (FPR2) serving as the key receptor. Based on
this mechanism, we show that targeting signal transducer and activator of transcription 1 (STAT1), a key
component of the ISGF3 complex, with the clinically approved drug fludarabine effectively mitigates
MRSA infection in murine sepsis and pneumonia models. These findings reveal the mechanisms of
MRSA pathogenesis and highlight the potential of anti-virulence strategies as innovative therapeutic
approaches against MRSA infections.

Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a significant global health threat,
characterized by its high mortality rates and widespread antibiotic resistance. As reported by The Lancet
(2024), deaths caused by MRSA increased by 127% between 1990 and 2021, rising from 57,200 to
130,000. This makes MRSA the antimicrobial-resistant bacterium with the highest increase in mortality
1. In response to this growing threat, the World Health Organization (WHO) has classified MRSA as a
“Priority 1: Critical” pathogen in its ESKAPE list, highlighting the urgency of addressing this issue 2.
However, the treatment of MRSA infections is considerably complicated by bacterial rapidly evolving
antibiotic resistance. Traditional antibiotics like vancomycin, which is often reserved as a last-line defense,
have shown treatment failure rates exceeding 50% in MRSA infections 3. Even newer agents, such as
ceftaroline and tedizolid, are facing emerging resistance 4,5. This serious challenge is partly attributable
to the excessive use of traditional antibiotics, which exerts selective pressure on bacteria, leading to the
evolution of hypervirulent and multidrug-resistant variants 6. Given the current challenges and limitations
of traditional antibiotics, there is an urgent need to explore innovative and alternative treatment strategies.

Virulence factors are key determinants of bacterial pathogenicity, enabling pathogens to evade host
defenses and establish infections 7. Targeting virulence factors and their associated pathways offers a
promising alternative to traditional antibiotics, as it reduces bacterial virulence and immune evasion
without exerting selective pressure on bacteria, thereby minimizing the risk of resistance development 8.
Importantly, this strategy relies on facilitating clearance by the host immune defense, making it a more
sustainable approach to combat infections 9. Given these advantages, it is essential to identify the critical
virulence factors of pathogenic bacteria and elucidate their mechanisms of action in order to discover new
therapeutics. Among the various MRSA virulence factors, phenol-soluble modulins (PSMs) have
attracted much attention due to their critical role in MRSA pathogenicity 10. Unlike other virulence factors
that are often encoded by mobile genetic elements (MGEs) and restricted to specific Staphylococcus
aureus isolates, PSMs are encoded within the core genome and produced by virtually all strains 10.
Research has elucidated several mechanisms of action of PSMs, including membrane disruption leading
to cell lysis and tissue damage, biofilm development, as well as modulation of the host immune response
by interfering with neutrophil function and promoting pro-inflammatory cytokine release 11-15. MRSA
produces PSMs, including PSMα1–α4, PSMβ1–β2, and δ-toxin. Among these, PSMα3 stands out for its
potent cytotoxic and lytic activity, which is attributed to its α-helical structure 11. Interestingly, PSMα3
also exhibits a unique ability to catalyze the degradation of β-lactam antibiotics 16. As a critical virulence
factor with dual functionality in promoting both bacterial virulence and antibiotic resistance, PSMα3
emerges as a highly promising target for therapeutic intervention. Therefore, further research into the
mechanisms of PSMα3-mediated damage and immune modulation is crucial for developing new strategies
to combat MRSA infections.

Macrophages play a central role in host defense against bacterial infections. They are capable of
phagocytosing and killing pathogens, as well as modulating the immune response 17. In infected tissues,
macrophages initially polarize into a pro-inflammatory M1 phenotype, releasing mediators that trigger
inflammation and recruit other inflammatory cells, such as neutrophils, to fight pathogens 18. M1
macrophages are induced by pro-inflammatory cytokines such as interferon-γ (IFN-γ) and tumor necrosis
factor-α (TNF-α), as well as bacterial components like lipopolysaccharide (LPS). The M1 phenotype can
produce high levels of pro-inflammatory cytokines (e.g., interleukin-1β (IL-1β), IL-6, TNF-α) and
reactive oxygen species (ROS), which are crucial for pathogen killing 19. Conversely, the M2 phenotype
is more involved in tissue repair and immune regulation. M2 macrophages are induced by cytokines such
as IL-4 and IL-13, and they produce high levels of anti-inflammatory cytokines (e.g., IL-10, TGF-β),
which promote tissue repair and suppress the immune response 18,19. Nevertheless, in the ongoing battle
between bacteria and the host immune system, bacteria have evolved various mechanisms to evade the
host immune response by modulating macrophage polarization and inducing cell death 19,20. For instance,
Escherichia coli can induce excessive M1 macrophage polarization and pyroptosis via LPS, leading to
excessive and prolonged inflammatory responses, tissue damage, and even sepsis 21,22. In contrast,
Mycobacterium tuberculosis secretes the virulence factor heparin-binding hemagglutinin (HBHA), which
suppresses the antimicrobial immune response by promoting M2 polarization and inhibiting autophagy,
thereby facilitating immune evasion 23. However, the specific effects of PSMα3 on macrophage
polarization and cell death during MRSA infection remain poorly understood.

In the present study, we investigated the impact of PSMα3 on macrophage polarization and cell death,
aiming to uncover mechanisms that could inform the development of innovative antimicrobial strategies.
Our results demonstrated that PSMα3 played a pivotal role in driving M1 macrophage polarization and
necroptosis by facilitating the crosstalk between the ISGF3 complex and necrosome complex, with these
processes modulated by the activation of the FPR2 receptor. The ISGF3 complex, composed of IRF9,
STAT1, and STAT2, is a critical transcription factor complex involved in the regulation of macrophage
polarization and plays a key role in the immune response. The necrosome complex, consisting of RIP1,
RIP3, and MLKL, is a signaling complex that drives necroptosis. Based on these findings, we showed
that targeting STAT1, a crucial component of the ISGF3 complex, with the clinically approved drug
fludarabine could effectively mitigate MRSA infection. It also reduced the cytokine storm and enhanced
host immune defense in vivo. These results reveal the mechanisms underlying PSMα3-mediated MRSA
pathogenesis and highlight the potential of fludarabine as a candidate compound against MRSA infections.

Results
PSMα3 triggers M1 macrophage polarization
To investigate the effect of PSMα3 on macrophage polarization, we analyzed the expression levels of
M1/M2 markers in both human THP-1-derived macrophages and mouse bone marrow-derived
macrophages (BMDMs) following treatment with PSMα3. Remarkably, qRT-PCR analysis revealed that
PSMα3 treatment induced a concentration-dependent upregulation of M1 marker genes (TNF-α, IL-1β,
IL-6, IL-12, and CXCL-10), while downregulating M2 marker genes (IL-1RA, FN-1, and CD206) in
THP-1-derived macrophages (Figure 1a–h). These changes were consistent with the effects observed with
LPS + IFN-γ treatment, which served as a positive control for M1 macrophage polarization, whereas IL-
4 treatment, used as a positive control for M2 macrophage polarization, had the opposite effect. Similar
results were also observed in mouse BMDMs, where 3 μM PSMα3 significantly increased M1-associated
genes (iNOS, TNF-α, IL-12, IL-6, IL-1β) and suppressed M2 markers (CD206, Arg-1, FN-1)
(Supplementary Figure 1a–h). Further validation was provided by Luminex multiplex cytokine analysis.
The results indicated that PSMα3 treatment induced a concentration-dependent increase in the secretion
of M1 macrophage polarization-related pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8, IL-12p70,
IFN-α, IFN-β, IFN-γ) and chemokines (CCL-2, CCL-3, CCL-5, CXCL-10) in the supernatants of THP-
1-derived macrophages (Figure 1i). Similar results were also obtained in mouse BMDMs (Supplementary
Figure 1i–k). In addition, fluorescence analysis revealed a significant increase in the surface marker CD86
of THP-1-derived macrophages following PSMα3 exposure (Figure 1j). These findings indicate that
PSMα3 can upregulate M1 macrophage polarization markers at both the mRNA and protein levels.
Meanwhile, we compared the effects of PSMα3 with other PSMs on M1 macrophage polarization. THP-
1-derived macrophages were treated with 3 μM of PSMα1, PSMα2, PSMα3, PSMα4, PSMβ1, PSMβ2, or
δ-toxin, and the secretion of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) was analyzed. The results
demonstrated that PSMα3 induced the highest levels of cytokine release among all tested PSMs
(Supplementary Figure 2a–c). Furthermore, we compared cytokine production in THP-1-derived
macrophages infected with the wild-type strain MRSA USA300 strain (LAC), an isogenic PSMα3
knockout strain (△psmα3), and the complemented mutant strain △psmα3 (pEX-psmα3) at a multiplicity
of infection (MOI) of 10:1. Notably, infection with the △psmα3 strain resulted in significantly reduced
secretion of TNF-α, IL-1β, and IL-6 compared to the wild-type MRSA (Figure 1k–m). The complemented
mutant strain restored the secretion of these cytokines to levels comparable to those induced by the wild-
type strain (Figure 1k–m). These results collectively demonstrate that PSMα3 plays a crucial role in
driving M1 macrophage polarization.

PSMα3 induces macrophage necroptosis
Emerging evidence strongly suggests that M1-polarized macrophages are closely associated with various
modes of cell death 24-26. To investigate whether PSMα3 induces cell death in macrophages and to identify
the specific mode of cell death involved, THP-1-derived macrophages were treated with PSMα3. The
results showed that PSMα3 caused a concentration-dependent decline in cell viability, with an IC50 value
of 2.496 ± 0.0513 μM (Figure 2a). These results indicate that PSMα3 has significant cytotoxic effects on
macrophages. Next, the plasma membrane and nuclei of THP-1-derived macrophages were stained with
CellMask Green, and Hoechst 33342, as well as the membrane-impermeant dye PI. In specific modes of
cell death, such as necroptosis and pyroptosis, pores open in the cell plasma membrane, allowing PI to
enter and stain the nuclei. Fluorescence analysis revealed a significant increase in PI-positive nuclei
following PSMα3 exposure, indicating damage to the cell plasma membrane (Figure 2b). Thus, these
findings suggest that PSMα3-induced cell death is associated with membrane disruption. To further
determine the specific type of cell death involved, chemical inhibitors targeting different cell death
pathways were used. Notably, necroptosis inhibitors necrostatin-1 (Nec-1, RIP1 inhibitor),
necrosulfonamide (NSA, MLKL inhibitor), and GSK’872 (RIP3 inhibitor) conferred significant
protection against PSMα3-induced cytotoxicity (Figure 2c). In contrast, apoptosis inhibitors (Z-VAD-
FMK, Z-IETD-FMK, and Ac-DEVD-CHO), pyroptosis inhibitor belnacasan (VX-765), autophagy
modulators 3-Methyladenine (3-MA), wortmannin, chloroquine (CQ), and ferroptosis inhibitor
ferrostatin-1 (Fer-1) showed minimal protective effect (Figure 2c). Consistent results were also observed
in BMDMs treated with PSMα3 in the presence of Nec-1 and NSA (Supplementary Figure 3). To further
confirm that PSMα3 drives necroptosis, MLKL-knockout (MLKL-/-) THP-1-derived macrophages were
treated with 3 µM PSMα3 for 24 h. The result showed that PSMα3-induced cytotoxicity was significantly
attenuated in MLKL-/- macrophages compared to wild-type cells (Figure 2d). These results indicate that
necroptosis, rather than apoptosis, pyroptosis, autophagy or ferroptosis, plays a crucial role in PSMα3-
induced cell death in macrophages.
Meanwhile, we compared the effects of PSMα3 with those of other PSMs on cytotoxicity and
necroptosis. The results showed that PSMα3 exhibited the highest cytotoxicity, followed by PSMα2,
PSMα1, and δ-toxin, whereas PSMα4, PSMβ1, and PSMβ2 showed minimal cytotoxicity (Figure 2e).
Fluorescence analysis indicated that PSMα3 induced the highest expression of the necroptosis marker p-
MLKL (Figure 2f, Supplementary Figure 2d). Furthermore, to verify the contribution of PSMα3 to the
cytotoxicity of MRSA, THP-1-derived macrophages were infected with the wild-type LAC strain, the
Δpsmα3 mutant, and the complemented mutant strain △psmα3 (pEX-psmα3). Infection with the △psmα3
strain caused a marked reduction in cytotoxicity relative to LAC, whereas cytotoxicity was restored in the
complemented mutant (Figure 2g). Consistent results were also observed in THP-1-derived macrophages
treated with the supernatants from these strains (Figure 2h). Additionally, both Nec-1 and NSA protected
THP-1-derived macrophages from LAC-induced cytotoxicity (Figure 2i, j). Collectively, these results
indicate that PSMα3 is a key virulence factor responsible for MRSA-induced macrophage necroptosis.

Proteomic analysis reveals crosstalk between M1 macrophage polarization and necroptosis
Although macrophage polarization and necroptosis have been extensively studied individually, the
interplay between macrophage polarization and necroptosis, as well as the underlying regulatory
mechanisms, remains poorly understood. To investigate the molecular mechanisms by which PSMα3
promotes M1 macrophage polarization and necroptosis in THP-1-derived macrophages, we conducted a
proteomic analysis. A total of 5208 proteins were identified (Supplementary Data 1). The heat map
revealed a clear separation and distinct protein signatures among the control group and the groups treated
with 1 μM and 3 μM PSMα3 (Figure 3a). The differentially expressed proteins (DEPs) with a 1.3-fold
change cutoff were analyzed in three comparisons: 1 μM PSMα3 vs. control (22 proteins up-regulated,
20 down-regulated); 3 μM PSMα3 vs. control (63 proteins up-regulated, 56 down-regulated); and 3 μM
PSMα3 vs. 1 μM PSMα3 (45 proteins up-regulated, 29 down-regulated) (Supplementary Data 2). We then
focused on these DEPs for functional analysis. Biological process analysis revealed that an obvious
enrichment of DEPs was linked to the JAK-STAT pathway and inflammation response, including
“positive regulation of receptor signaling pathway via JAK-STAT”, “positive regulation of receptor
signaling pathway via STAT”, “positive regulation of acute inflammatory response”, “the cellular
response to type I interferon”, and “the positive regulation of tumor necrosis factor production”, etc.
(Figure 3b). Notably, the JAK-STAT pathway, type I interferon, and TNF-α were all highly relevant to
M1 macrophage polarization 27. These findings suggest that PSMα3-induced M1 macrophage polarization
may be closely associated with these pathways and factors. Moreover, KEGG enrichment analysis of
DEPs between the 3 μM PSMα3-treated and control groups identified “Necroptosis” as a significant
functional pathway (Figure 3c). Key proteins enriched in necroptosis included SQSTM1/p62, IRF9,
CHMP6, PYCARD, and CYBB (Figure 3d, Supplementary Figure 4). Among these proteins, protein-
protein interaction (PPI) analysis revealed a potential relationship between SQSTM1/p62 and IRF9
(Figure 3e). IRF9 is a key protein in the formation of ISGF3 complex within the JAK-STAT1 pathway,
facilitating the recruitment and activation of downstream signaling molecules 28. Additionally,
SQSTM1/p62 has been reported to be involved in the necroptosis pathway, where it regulates the
formation of the necrosome complex 29. The relationship between SQSTM1/p62 and IRF9 suggested a
potential crosstalk between the JAK-STAT1 pathway and necroptosis signaling, implying their
coordinated role in modulating PSMα3-induced M1 polarization and necroptosis in macrophages.

PSMα3 activates ISGF3 and necrosome complexes in THP-1-derived macrophages
Based on the above analysis, we further investigated whether PSMα3 activates the IRF9-related ISGF3
complex and modulates necroptosis through SQSTM1/p62 in THP-1-derived macrophages. The ISGF3
complex is composed of IRF9, STAT1, and STAT2 28. Our results revealed that PSMα3 treatment
significantly upregulated IRF9 expression in THP-1-derived macrophages, as confirmed by both Western
blot and high-content fluorescence assays (Figure 4a–d, Supplementary Figure 5). This finding was
consistent with our proteomic profiling data. Additionally, the phosphorylation levels of STAT1 and
STAT2 were also markedly increased (Figure 4a–d, Supplementary Figure 5). For the necroptosis
pathway, RIP1, RIP3, and MLKL are essential components of the necrosome complex 30,31, while
SQSTM1/p62 plays a significant role in regulating its formation. Our results showed that the expression
of SQSTM1/p62 was significantly upregulated in THP-1-derived macrophages treated with PSMα3
(Figure 4e–h, Supplementary Figure 5). Moreover, the phosphorylation levels of RIP1, RIP3, and MLKL
were notably increased (Figure 4e–h, Supplementary Figure 5), indicating the activation of necroptosis
signaling. To further validate the formation of these complexes, co-immunoprecipitation (Co-IP)
experiments were also performed. The results confirmed that IRF9 directly interacted with phosphorylated
STAT1 and STAT2 following 3 μM PSMα3 treatment (Figure 4i, Supplementary Figure 6a), thereby
facilitating the formation of the ISGF3 complex. Moreover, SQSTM1/p62 was found to directly interact
with p-RIP1, p-RIP3, and p-MLKL (Figure 4j, Supplementary Figure 6b), supporting its role in the
regulation of the necrosome complex formation. Collectively, these results indicate that PSMα3 activates
the IRF9-related ISGF3 complex and modulates the formation of the necrosome complex through
SQSTM1/p62 in macrophages.

ISGF3 complex modulates the M1 macrophage polarization and necroptosis
To investigate the role of the ISGF3 complex in modulating M1 macrophage polarization and necroptosis,
we employed siRNAs targeting the components of ISGF3 complex, IRF9, STAT1, and STAT2, to knock
down their expression. Western blot analysis confirmed significant knockdown of these proteins
(Supplementary Figure 7). Then, we assessed the secretion of M1 macrophage polarization-related
cytokines (TNF-α, IL-1β, IL-8) and chemokines (CCL-2, CCL-3, CCL-5) in the supernatants of PSMα3-
treated THP-1-derived macrophages using Luminex multiplex cytokine analysis. The results showed that
the increased secretion of these cytokines and chemokines induced by PSMα3 was significantly reduced
following the knockdown of STAT1 (Figure 5a). These findings indicate that the ISGF3 complex is
essential for PSMα3-induced M1 macrophage polarization. Additionally, the cytotoxic effects of PSMα3
were significantly inhibited by siRNA-mediated knockdown of STAT1, STAT2, or IRF9 (Figure 5b), as
well as by the STAT1 inhibitor fludarabine (Supplementary Figure 8). These results indicate that the
ISGF3 complex is a key regulator of PSMα3-driven necroptosis. To further investigate this, we performed
Western blot analysis and found that the increased phosphorylation levels of RIP1, RIP3, and MLKL
induced by PSMα3 were significantly reduced following the knockdown of IRF9, STAT1, and STAT2
(Figure 5c, d, Supplementary Figure 9, 10). Consistent results were also observed in high-content
fluorescence assays (Figure 5e, Supplementary Figure 11). In summary, these findings demonstrate that
the ISGF3 complex plays a dual regulatory role in modulating both M1 macrophage polarization and
necroptosis following PSMα3 treatment.

FPR2 receptor modulates the M1 macrophage polarization and necroptosis
Previous studies have reported that PSMα3 could activate the FPR2 receptor and cause the inflammatory
response 32,33. However, the role of FPR2 in modulating PSMα3-induced M1 macrophage polarization
and necroptosis remains unexplored. To investigate this, we knocked down FPR2 using siRNA, and the
silencing efficiency was confirmed by Western blot analysis (Supplementary Figure 7). With FPR2
effectively silenced, we firstly examined its impact on PSMα3-induced M1 macrophage polarization.
Luminex multiplex cytokine analysis revealed that increased secretion of M1 macrophage polarization-
related cytokines (TNF-α, IL-1β) and chemokines (CCL-2, CCL-3, CXCL-10) induced by PSMα3 was
significantly reduced following FPR2 knockdown (Figure 6a). This finding suggests that FPR2 is a critical
receptor of PSMα3-induced M1 macrophage polarization. Given that the ISGF3 complex is essential for
PSMα3-induced M1 macrophage polarization, as mentioned above, we evaluated the effect of FPR2
knockdown on the activation of STAT1, which is critical for the formation of the ISGF3 complex. Both
Western blot and high-content fluorescence assays revealed that FPR2 knockdown markedly reduced
PSMα3-induced STAT1 phosphorylation (Figure 6b, c, e, f, Supplementary Figure 10). These findings
suggested that the ISGF3 signaling pathway was involved in FPR2-mediated PSMα3-induced M1
macrophage polarization. Moreover, we found that FPR2 also modulated PSMα3-induced necroptosis.
FPR2 knockdown significantly inhibited the cytotoxic effects of PSMα3 (Figure 6d). High-content
fluorescence assays and Western blot further confirmed that the increased phosphorylation levels of RIP1,
RIP3, and MLKL induced by PSMα3 were significantly reduced following knockdown of FPR2 (Figure
6e–h, Supplementary Figure 10). Taken together, these findings demonstrate that the FPR2 receptor plays
a significant role in modulating both M1 macrophage polarization and necroptosis following PSMα3
treatment.

PSMα3 modulates macrophage polarization and necroptosis in human primary macrophages
To substantiate our findings in a more physiologically relevant model, we investigated the effects of
PSMα3 on primary human macrophages. The results showed that PSMα3 exhibited concentration-
dependent cytotoxicity against primary human macrophages, with an IC50 value of 1.593 ± 0.206 μM
(Figure 7a). Consistent with previous results in THP-1-derived macrophages, PSMα3 treatment induced
a concentration-dependent increase in the secretion of M1 macrophage polarization-related pro-
inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the supernatant of primary human macrophages
(Figure 7b–d). Fluorescence analysis revealed a significant increase in the surface marker CD86 following
PSMα3 exposure (Figure 7e, f). These results demonstrate that PSMα3 can induce M1 macrophage
polarization in primary human macrophages. Furthermore, PSMα3 treatment significantly upregulated
IRF9 expression and enhanced the phosphorylation of STAT1 and STAT2 (Figure 7e–h, Supplementary
Figure 12). In the necroptosis pathway, we observed increased expression of SQSTM1/p62 and elevated
phosphorylation levels of RIP1, RIP3, and MLKL in PSMα3-treated primary macrophages (Figure 7e, f,
i, j, Supplementary Figure 12). Co-IP assays verified a direct interaction between IRF9 and
phosphorylated STAT1 and STAT2 (Figure 7k, Supplementary Figure 13a), and revealed that
SQSTM1/p62 directly associates with p-RIP1, p-RIP3, and p-MLKL (Figure 7l, Supplementary Figure
13b). Together, these data indicate that PSMα3 activates the ISGF3 complex and facilitates the assembly
of the necrosome complex in primary human macrophages.
To further define the role of FPR2 in PSMα3-induced M1 polarization and necroptosis in primary
human macrophages, we employed the FPR2 inhibitor WRW4. The results showed that WRW4
significantly attenuated the PSMα3-triggered secretion of TNF-α, IL-1β, and IL-6 in primary human
macrophages (Figure 8a). Western blot and high-content fluorescence assays revealed that WRW4
significantly reduced the PSMα3-induced upregulation of CD86 and phosphorylated STAT1 (Figure 8b–
d, Supplementary Figure 14). Additionally, WRW4 significantly alleviated the cytotoxic effects of
PSMα3 (Figure 8e). Further high-content fluorescence and Western blot analyses demonstrated that
WRW4 also reduced the increased phosphorylation of RIP1, RIP3, and MLKL induced by PSMα3 (Figure
8f–i, Supplementary Figure 14). Collectively, these results demonstrate that FPR2 is a key upstream
receptor mediating PSMα3-induced M1 macrophage polarization and necroptosis in primary human
macrophages. These findings are consistent with the observations in THP-1-derived macrophages.

Fludarabine mitigates MRSA infection
Recent studies have highlighted the potential of targeting bacterial virulence factors and their associated
pathways as a novel strategy to combat antibiotic-resistant infections 8. Thus, targeting the key proteins
and pathways involved in PSMα3-induced M1 macrophage polarization and necroptosis may represent
an effective approach to control MRSA infection. To verify this strategy, we reviewed the literatures and
summarized several inhibitors targeting these key proteins (Supplementary Table 1) 34, 35, 36, 37, 38, 39, 40, 41,
42. Among these, fludarabine, as a clinically approved drug for leukemia 43, offers distinct advantages
over other candidate compounds due to its established safety profiles, regulatory approval, and well-
documented pharmacokinetic properties, thereby facilitating clinical translation. However, the therapeutic
effects of fludarabine on MRSA infections have not yet been reported.
To evaluate the antibacterial activity of fludarabine against MRSA in vivo, we established a MRSA-
infected sepsis model using the wild-type LAC strain. In this model, all septic mice died within 2 d post
infection, whereas treatment with 50 mg/kg fludarabine markedly increased the survival rate from 0% to
80% (Figure 9a). Additionally, the colony forming units (CFUs) of LAC significantly decreased in the
blood, lung, liver, spleen, and kidney of septic mice after fludarabine treatment (Figure 9b). Consistently,
compared to the tissue damage induced by LAC strain, tissue damage was effectively alleviated after
treatment with 50 mg/kg fludarabine (Figure 9c). To explore the underlying mechanism, we first ruled
out direct antibacterial activity, as the minimum inhibitory concentration (MIC) of fludarabine exceeded
512 μg/mL against both the LAC and its Δpsmα3 mutant strains (Supplementary Figure 15). We therefore
investigated whether fludarabine exerts its therapeutic effect by targeting PSMα3-driven pathogenesis.
To test this, we evaluated its activity in a sepsis model induced by the Δpsmα3 mutant. The results showed
that infection with the Δpsmα3 mutant alone led to a 50% survival rate, while virulence was fully restored
by psmα3 complementation (0% survival rate) (Figure 9d). However, the therapeutic benefit of
fludarabine in the Δpsmα3 infection model was significantly reduced, with fludarabine only providing a
statistically non-significant increase in survival rate (from 50% to 60%) (Figure 9d). These findings
demonstrate that the potent therapeutic effect of fludarabine is mainly mediated through suppressing
PSMα3-driven pathogenesis.
We also assessed the therapeutic efficacy of fludarabine in a murine model of MRSA pneumonia.
Consistent with the results from the sepsis model, fludarabine treatment significantly increased the
survival rate from 0% to 60% and reduced bacterial counts in the lungs (Figure 9e, f). These results
demonstrate that fludarabine effectively mitigates MRSA infections in different mouse models. Given the
multiple reported mechanisms of fludarabine, we sought to assess the contribution of STAT1 inhibition
to its protective effect. We therefore used STAT1/3-IN-1 36, a distinct STAT1 inhibitor with minimal
reported in vivo toxicity, for further validation. Consistent with the effects of fludarabine, treatment with
STAT1/3-IN-1 provided significant protection against MRSA infection, resulting in a 50% survival rate
and a marked reduction in pulmonary bacterial loads (Figure 9e, f). These collective findings suggest that
STAT1 inhibition is a key mechanism underlying the efficacy of fludarabine against MRSA infection.

Fludarabine attenuates cytokine storm and necroptosis in MRSA infection
Excessive activation of M1 macrophages and the subsequent secretion of pro-inflammatory cytokines are
a major cause of cytokine storm 44. To determine whether fludarabine could mitigate cytokine storm in
vivo, we examined its effects in a murine sepsis model. Immunohistochemical analysis of lung and liver
tissues from septic mice infected with the LAC strain revealed increased levels of IL-1β expression, which
was effectively reduced by fludarabine treatment (Figure 10a). Consistently, the levels of M1 macrophage
polarization-related cytokines (TNF-α, IL-1β, and IL-6) were significantly increased in the plasma of the
model group, but fludarabine treatment effectively attenuated these increases (Figure 10b–d). These
results demonstrate that fludarabine suppresses the excessive release of pro-inflammatory cytokines
during MRSA infection.
To further explore the effects of fludarabine on macrophage necroptosis during MRSA infection, we
conducted immunofluorescence analysis. The results showed a notable infiltration of macrophages
(F4/80-positive cells) in lung tissues of LAC-infected septic mice, indicating macrophage recruitment to
infection sites (Figure 10e). However, fludarabine treatment effectively reduced macrophage recruitment,
a phenotype similar to that observed in Δpsmα3 mutant-infected mice (Figure 10e). Additionally,
fludarabine treatment effectively decreased the phosphorylation levels of MLKL in the lung macrophages
of septic mice, indicating an inhibition of necroptosis (Figure 10e). Taken together, these results confirmed
that fludarabine not only reduces the excessive production of pro-inflammatory cytokine but also protects
host macrophages by suppressing necroptosis in vivo. This dual mechanism enables fludarabine to inhibit
the bacterial immune evasion strategy, thereby restoring the host’s immune response against MRSA. A
schematic diagram summarizing the mechanisms of this study is presented in Figure 10f.

Methods
Ethical Statement
All experiments in this study were conducted in compliance with relevant ethical regulations and
approved by the Institutional Animal Care and Use Committee (IACUC) of the Academy of Military
Medical Sciences. Female C57BL/6 and male BALB/c mice, aged 8–10 weeks, were purchased from SPF
Biotechnology Co. Ltd. (Beijing, China). The mice were housed under controlled ambient conditions (12
h light/dark cycle, temperature of 20 ± 2°C, and 50% relative humidity) and were allowed free access to
a standard rodent diet and distilled water. All animals were acclimatized for at least 3 days prior to
experimental procedures.

Reagents and antibodies
RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), β-
mercaptoethanol (1000 ×, 55 mM), and penicillin-streptomycin (10000 U/mL) were purchased from
Gibco. Phorbol 12-myristate 13-acetate (PMA), WRW4, and STAT1/3-IN-1 were obtained from
MedChemExpress (MCE). Z-VAD-FMK, Z-IETD-FMK, Ac-DEVD-CHO, belnacasan (VX-765),
necrostatin-1 (Nec-1), necrosulfonamide (NSA), GSK’872, wortmannin, 3-Methyladenine (3-MA),
chloroquine (CQ), ferrostatin-1 (Fer-1), and fludarabine were purchased from Selleck. Antibodies against
STAT1 (Cat No. 14994), Phospho-STAT1 (Cat No. 7649), STAT2 (Cat No. 72604), Phospho-STAT2
(Cat No. 88410), IRF9 (Cat No. 76684), RIP1 (Cat No. 3493), Phospho-RIP1 (Cat No. 65746), RIP3 (Cat
No. 13526), Phospho-RIP3 (Cat No. 93654), MLKL (Cat No. 14993), Phospho-MLKL (Cat No. 91689
and 37333), SQSTM1/p62 (Cat No. 8025), GAPDH (Cat No. 5174), horseradish peroxidase (HRP)-linked
anti-rabbit IgG (Cat No. 7074), Alexa Fluor 488-conjugated F4/80 (Cat No. 27076) were purchased from
Cell Signaling Technology (CST). Anti-FPR2 antibody (Cat No. GTX130627) was obtained from
GeneTex. Anti-IL-1β antibody (Cat No. ab283818) and Alexa Fluor 488-conjugated anti-CD86 antibody
(Cat No. ab290990) were purchased from Abcam. Propidium iodide (PI), Hoechst 33342, CellMask Green
plasma membrane stain, Alexa Fluor 488-labeled, Alexa Fluor 546-labeled, and Alexa Fluor 647-labeled
secondary antibodies (Cat No. A21206; A10040; A31573) were obtained from Thermo Fisher Scientific.
Tryptic soy broth (TSB) and Mueller–Hinton broth (MHB) were obtained from Solarbio. All chemicals
from commercial sources were of analytical grade.

Synthesis of PSM peptides
PSMα1, PSMα2, PSMα3, PSMα4, PSMβ1, PSMβ2, and δ-toxin were synthesized using standard Fmoc
(9-fluorenylmethoxycarbonyl) solid-phase peptide synthesis protocols. Crude peptides were purified by
reverse-phase high-performance liquid chromatography (RP-HPLC) to >98% purity, and their identities
were confirmed by mass spectrometry (MS). Peptide sequences are listed in Supplementary Table 2.

Construction of Δpsmα3 mutant and its complemented strain
To construct the Δpsmα3 mutant strain from MRSA USA300 strain, the upstream and downstream
homologous arm fragments of psmα3 were amplified from MRSA USA300 genomic DNA using the
psmα3-up-F/psmα3-up-R, psmα3-down-F/psmα3-down-R sets of primers and ligated by overlap
extension PCR (Supplementary Table 3). The obtained fragment was cloned into the pBTs plasmid
digested with EcoRI and HindIII. The recombinant plasmid was introduced to E. coli DH5α, and correct
clones were selected on ampicillin plates. The resulting plasmid pBTs-psmα3 was then electroporated into
RN4220 for modification and transformed into MRSA USA300 for allelic replacement. Successful
deletion of psmα3 was confirmed by PCR and DNA sequencing.
To construct the complemented mutant strain, the pEX-psmα3 plasmid was constructed based on the
pEX expression vector (Wengene Biosciences, China). The psmα3 gene, along with its native promoter
and terminator sequences, was amplified from the genomic DNA of USA300 using the psmα3-F/ psmα3-
R primer pair as detailed in Supplementary Table 3. Subsequently, the amplified fragment was cloned to
the pEX vector following the instructions of the ClonExpress II One Step Cloning Kit (Vazyme Biotech,
China). Then, the resulting plasmid pEX-psmα3 was electroporated into USA300 Δpsmα3 mutant strain.
PCR was used to verify the mutation.

Cell culture
Human primary macrophages (ICHX-C104) were purchased from Hycyte and cultured in the
manufacturer’s specialized medium (ICHX-G104, Hycyte). Human leukemia monocytic THP-1 cells
(TIB-202, ATCC) were cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin-
streptomycin, and 50 μM β-mercaptoethanol at 37°C with 5% CO2. Differentiation of THP-1 monocytes
to macrophage was induced by treatment with 100 ng/mL PMA for 24 h. MLKL knockout THP-1 cells
(YKO-HT0390, Ubigene) were cultured and confirmed by Western blot analysis (Supplementary Figure
16).
Mouse bone marrow-derived macrophages (BMDMs) were isolated from the femurs and tibias of
female C57BL/6 mice following an established protocol 53. This protocol indicated no significant
differences in the viability or yield of harvested cells between male and female C57BL/6 mice aged 6–12
weeks. Freshly isolated cells were suspended in DMEM supplemented with 10% FBS, 2 mM L-glutamine
(Invitrogen, USA), 1% penicillin/streptomycin, and 20 ng/mL recombinant mouse macrophage colony-
stimulating factor (M-CSF) (Peprotech, USA). After induction for 7 days, mature BMDMs were collected
and used for subsequent assays.

Quantitative real-time polymerase chain reaction (qRT-PCR) assay
The qRT-PCR was employed to evaluate the expression levels of macrophage polarization markers in
THP-1-derived macrophages and BMDMs following treatment with PSMα3. For THP-1-derived
macrophages, the M1 macrophage polarization markers analyzed included TNF-α, IL-1β, IL-6, IL-12,
and CXCL-10, while the M2 markers included CD206, IL-1RA, and FN-1. For BMDMs, iNOS, TNF-α,
IL-1β, IL-6, and IL-12 were analyzed as M1 markers, while CD206, Arg-1, and FN-1 were considered
M2 markers. THP-1 cells were seeded in 6-well plates (1 × 106 cells/well) and treated with 100 ng/mL
PMA for 24 h to induce differentiation. Mature BMDMs were seeded in 6-well plates (1 × 106 cells/well)
and cultured for 24 h. Then, the cells were exposed to PSMα3 for 24 h, with the addition of 100 ng/mL
LPS and 20 ng/mL IFN-γ serving as a positive control for M1 macrophage polarization, and 20 ng/mL
IL-4 as a positive control for M2 macrophage polarization. After incubation, total RNA was extracted
from cells using Trizol Reagent (Takara, Japan), and cDNA was synthesized using a PrimeScript RT
Reagent Kit (Takara, Japan). Subsequently, qRT-PCR was performed using a SYBR Premix Ex Taq II
Kit (Takara, Japan) on a 7500 Real-Time PCR System (Applied Biosystems, USA), Quantification was
performed using GAPDH as the internal control. The relative expression level of each gene was calculated
using the 2–ΔΔCT method. Values were expressed as fold changes. All primer sequences are listed in
Supplementary Table 3.

Cytokine and chemokine detection
Following treatment with PSMα3 or other PSMs for 24 h, the concentrations of M1 macrophage
polarization markers, including cytokines (TNF-α, IL-1β, IL-6, IL-8, IL-12p70, IFN-α, IFN-β, IFN-γ) and
chemokines (CCL-2, CCL-3, CCL-5, CXCL-10), in the culture supernatants of THP-1-derived
macrophages and human primary macrophages were quantified using a multiplex cytokine Luminex kit
(R&D system, USA), according to the manufacturer’s protocol. The cytokines (TNF-α, IL-1β, IL-6) in
the culture supernatants of BMDMs were quantified using mouse ELISA kits (R&D Systems, USA) 
according to the manufacturer’s protocol.

Cytotoxicity assay
Wild-type and MLKL-knockout THP-1 cells were seeded in 96-well plates (1 × 104 cells/well) and treated
with 100 ng/mL PMA for 24 h to induce differentiation. Human primary macrophages were seeded in 96-
well plates (1 × 104 cells/well). After removing the culture medium, 200 µL of fresh culture medium
containing PSMα3 or other PSMs was added to each well. After incubation for 24 h at 37°C, the culture
medium was replaced with fresh medium containing 10% CCK-8 reagent, and the cells were incubated
for 4 h at 37°C. The optical density was measured by a microplate reader (Infinite M1000 pro, TECAN,
Switzerland) at 450 nm. The cell survival rate was calculated.

Membrane permeability assay
To assess plasma membrane permeability and cell death in response to PSMα3 treatment, THP-1-derived
macrophages were stained with propidium iodide (PI), Hoechst 33342, and CellMask Green plasma
membrane stain. Cell fluorescence images were captured using a PerkinElmer Opera Phenix™ high-
content screening (HCS) platform with a 20 × objective.

Lactate dehydrogenase (LDH) release assay
The LDH release assay was conducted to assess the cytotoxic effects of the USA300 strain, its Δpsmα3
mutant and complemented mutant on THP-1-derived macrophages. The USA300 strain and its Δpsmα3
mutant were grown overnight in TSB broth at 37°C with shaking at 250 rpm. The complemented mutant
was grown overnight in TSB broth supplemented with 50 µg/mL apramycin at 37°C with shaking at 250
rpm. The overnight culture supernatants were collected by centrifugation at 12,000 × g, 4°C for 20 min
and filtered through 0.22 μm filters (Millipore, USA) to eliminate bacteria. The precipitates were then
harvested, washed twice, and resuspended in THP-1 cell culture medium. Subsequently, the THP-1-
derived macrophages were infected with the USA300 strain, Δpsmα3 mutant, or complemented mutant at
a multiplicity of infection (MOI) of 10:1. In parallel, the filtered bacterial culture supernatants were
diluted 1:1 with cell culture medium and co-incubated with macrophages. After challenge for 2 h, the cell
culture supernatants were collected by centrifugation and filtered through 0.22 μm filters. The LDH
released into the culture supernatants was quantified using a cytotoxicity detection kit (Roche, USA),
following the manufacturer’s protocol. The lysis rate of the infected macrophages was calculated using
the following formula: [(experimental release – spontaneous release) / (maximum release – spontaneous
release)] × 100%, where spontaneous release refers to LDH release from uninfected macrophages, and
maximum release is the LDH released from macrophages lysed with 0.1% Triton X-100. Additionally,
the cytokines (TNF-α, IL-1β, IL-6) in the culture supernatants were quantified using human ELISA kits
(R&D Systems, USA).

Cell death inhibitor assay
To determine which cell death pathways are activated, pharmacological inhibitors were used to block
specific cell death pathways. THP-1 cells were seeded in 96-well plates (1 × 104 cells/well) and treated
with 100 ng/mL PMA for 24 h. After induction, the cell culture medium was removed and replaced with
fresh medium. Subsequently, the specific cell death inhibitors were added to the wells. For the inhibition
of apoptosis, 50 µM Z-VAD-FMK, 50 µM Z-IETD-FMK or 50 µM Ac-DEVD-CHO were utilized. To
inhibit pyroptosis, 50 µM VX-765 was applied. For the prevention of necroptosis, 10 µM Nec-1, 2 µM
NSA, or 5 µM GSK'872 were used. To inhibit autophagy, 2 mM 3-MA, 100 nM wortmannin, or 50 µM CQ
were administered. For the inhibition of ferroptosis, 1 µM Fer-1 was employed. The cells were pre-
incubated with inhibitors for 1 h at 37°C, after which they were treated with 3 μM PSMα3 for 24 h. Cell
viability was then evaluated using the CCK-8 assay.
To determine whether STAT1 and FPR2 regulate PSMα3-induced cell death, THP-1-derived
macrophages and human primary macrophages were pretreated with 10 µM fludarabine (a STAT1
inhibitor) or 5 µM WRW4 (an FPR2 inhibitor) for 1 h. The cells were then exposed to PSMα3 for 24 h.
Cell viability was subsequently assessed using the CCK-8 assay.

Proteomics analysis
THP-1 monocytes were differentiated with 100 ng/mL PMA stimulation and treated with 0, 1, or 3 μM
PSMα3 for 24 h (n = 3 biological replicates per condition). The cells were lysed by sonication three times
on ice using a high intensity ultrasonic processor (Scientz, China) in lysis buffer (8 M urea, 1% protease
inhibitor cocktail). The supernatant was collected after centrifugation at 12 000 × g at 4°C for 10 min, and
the protein concentration was determined using a BCA assay kit. For digestion, the protein solution was
reduced with 5 mM dithiothreitol for 30 min at 56°C and alkylated with 11 mM iodoacetamide for 15 min
at room temperature in darkness. The protein sample was then diluted with 200 mM TEAB
(triethylammonium bicarbonate). Finally, trypsin was added at a 1:50 trypsin-to-protein mass ratio for
overnight digestion. Each channel of peptide was labeled by its respective TMT reagent, and incubated
for 2 h at room temperature. The pooled samples were desalted using a Strata X C18 SPE column
(Phenomenex, USA) and dried by vacuum centrifugation.
For LC-MS/MS analysis, peptides were dissolved in solvent A (0.1% formic acid, 2% acetonitrile in
water) and separated on a homemade reversed-phase analytical column (25-cm length, 100 μm i.d.) using
an EASY-nLC 1200 UPLC system (Thermo Fisher Scientific, USA). The mobile phase consisted of
solvent A and solvent B (0.1% formic acid, 90% acetonitrile in water). Peptides were separated with the
following gradient: 0–4 min, 7–10% B; 4–53 min, 10–32% B; 53–57 min, 32–80% B; 57–60 min, 80%
B, at a constant flow rate of 500 nL/min. The separated peptides were analyzed using an Orbitrap Exploris
480 (Thermo Fisher Scientific, USA) with a nano-electrospray ion source. The electrospray voltage was
set to 2.3 kV. Full MS scans (400–1200 m/z) were acquired at a resolution of 60,000. MS/MS scans were
acquired with a fixed first mass of 110 m/z at a resolution of 15,000, with the TurboTMT function enabled.
Up to 25 most abundant precursors were selected for further MS/MS analyses with 30 s dynamic exclusion.
Fragmentation was performed via higher-energy collisional dissociation (HCD) at a normalized collision
energy (NCE) of 35%. The automatic gain control (AGC) target was set to 100%, with an intensity
threshold of 50,000 ions/s and a maximum injection time of Auto.
The resulting MS/MS data were processed using the Thermo Proteome Discoverer software (version
2.4.1.15). Tandem mass spectra were searched against the human Swiss-Prot database (20,376 entries)
concatenated with reverse decoy databases. Trypsin (Full) was specified as cleavage enzyme allowing up
to 2 missing cleavages. The minimum peptide length was set to 6 amino acid residues, and a maximum
of 3 modifications per peptide was allowed. The mass tolerance for precursor ions was set as 10 ppm, and
the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on cysteine and TMT6plex on
lysine and peptide N-termini were set as fixed modifications, and oxidation on methionine, acetylation on
protein N-terminal, Met-loss, and Met-loss+Acetyl on methionine were set as variable modifications. The
quantitative method is set as TMT6-plex. False discovery rate (FDR) was adjusted to < 1%. Proteins with
a fold change >1.3 and P < 0.05 (two-sided t-test) between PSMα3-treated and control groups were
defined as differentially expressed. These proteins were subjected to further functional analysis based on
Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and STRING
protein-protein interaction databases.

Transfection of siRNAs
THP-1 cells were differentiated with PMA stimulation and transfected with the negative control siRNA
(NC siRNA), FPR2 siRNAs (1–3), STAT2 siRNAs (1–3), IRF9 siRNAs (1–3) (Sangon Biotech, China),
and STAT1 siRNA (6544S, CST) at a final concentration of 100 nM using jetPRIME transfection reagent
(Polyplus-transfection, France), following the manufacturer’s instructions. Knockdown efficiency was
assessed 72 h post-transfection by Western blot analysis of the target proteins (Supplementary Figure 7).
Based on the validation results, STAT1 siRNA, STAT2 siRNA 3, IRF9 siRNA 3, and FPR2 siRNA 3
were selected for subsequent experiments. All siRNA sequences are listed in Supplementary Table 4.

Western blot analysis
Total protein was extracted from cells using RIPA lysis buffer (Beyotime, China) supplemented with
protease and phosphatase inhibitors (Roche, Germany) on ice for 30 min. Cell debris was removed by
centrifugation at 12,000 × g and 4°C for 20 min. Protein concentration was determined using the Pierce
BCA Protein Assay Kit (Thermo Fisher Scientific, USA) and normalized. Equal amounts (30 µg) of
protein were loaded onto 4–15% gradient SDS-PAGE gels (Beyotime, China) and then transferred to
PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk (Solarbio,
China) in TBST (Tris-buffered saline with 0.1% Tween-20) for 2 h at room temperature, and then
incubated overnight at 4°C with the primary antibodies against FPR2, STAT1, Phospho-STAT1, STAT2,
Phospho-STAT2, IRF9, RIP, Phospho-RIP, RIP3, Phospho-RIP3, MLKL, Phospho-MLKL,
SQSTM1/p62, and GAPDH. The primary antibodies were diluted at 1:1000 in TBST. After three washes
with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (1:3000 dilution
in TBST) for 1 h at room temperature. The protein bands were visualized using an ECL enhanced kit
(Abclonal, China) and imaged with a FluorChem R system (ProteinSimple, USA). Band intensities on
Western blot were quantified using ImageJ software (version 1.52p).

Cell immunofluorescence
THP-1 cells were cultured in 96-well plates (black plate, clear bottom) at a density of 1 × 104 cells per
well and differentiated with 100 ng/mL of PMA for 24 h. Following differentiation, the cells were gently
washed twice with serum-free culture medium and then treated with PSMα3 or other PSMs for an
additional 24 h. For the knockdown assay, THP-1 cells were first transfected with siRNAs for 72 h and
then treated with PSMα3 for 24 h. Human primary macrophages were seeded in 96-well plates (1 × 104
cells/well), pre-incubated with or without WRW4 for 1 h, and then treated with PSMα3 for 24 h. After
treatment, the cells were fixed with 4% formaldehyde for 30 min at room temperature, permeabilized with
0.3% Triton X-100 for 30 min, washed twice with PBS, and blocked with 10% bovine serum albumin.
The cells were then incubated with the indicated primary antibody overnight at 4°C. Following three
washes with PBS, the cells were incubated with appropriate Alexa Fluor 488-, Alexa Fluor 546-, or Alexa
Fluor 647-labeled secondary antibodies (1:500 dilution) for 1 h at 37°C. The nuclei were stained with 1
μM Hoechst 33342 for 30 min at 37°C. Finally, fluorescence images of cells were captured using a
PerkinElmer Opera Phenix™ HCS platform. For quantitative analysis, at least 9 fields and more than
1000 cells per well were analyzed using the Harmony software (version 4.3). Excitation and emission
filters were set according to the specific wavelengths of each fluorescent dye.

Co-immunoprecipitation (Co-IP)
Following treatment with PSMα3 for 24 h, THP-1-derived macrophages or human primary macrophages
(1 × 107 cells) were harvested and lysed in 1 mL of IP lysis buffer (Thermo Fisher Scientific, USA) on
ice for 30 min. After lysis, cell debris was removed by centrifugation at 12,000 × g for 15 min at 4°C. To
minimize non-specific binding, the supernatant was pre-cleared with protein A/G magnetic beads (MCE,
USA) for 4 h. The cell lysates were then incubated with primary antibodies against IRF9 (1:50 dilution)
or SQSTM1/p62 (1:100 dilution) overnight at 4°C, followed by incubation with protein A/G magnetic
beads for 3 h at 4°C. The complexes were washed 7 times with IP wash buffer (50 mM Tris-HCl, 150
mM NaCl, 0.5% Triton X-100, and 1 mM EDTA, pH 7.4). The presence of interacting proteins was
subsequently analyzed by Western blot.

Minimum inhibitory concentration (MIC)
The USA300 strain and its Δpsmα3 mutant were grown overnight in TSB broth at 37°C with shaking at
250 rpm. The bacterial cultures were then adjusted to a concentration of 5 × 105 CFU/mL in MHB. Two-
fold serial dilutions of fludarabine were prepared in 96-well microtiter plates, and 100 μL of the adjusted
bacterial suspension was added to each well. After a 16 h incubation at 37°C, the MIC was defined as the
lowest drug concentration that completely inhibited visible bacterial growth.

Sepsis model
The sepsis model was established using male BALB/c mice. This strain was selected due to its higher
susceptibility to Staphylococcus aureus infection compared to C57BL/6 mice and to align with clinical
epidemiological evidence indicating that males are more prone to MRSA infection 54, 55. The USA300
strain, its Δpsmα3 mutant, and complemented mutant were grown overnight in TSB broth at 37°C. The
overnight cultures were harvested, washed, and diluted in PBS. Then, the male BALB/c mice were
intraperitoneally infected with 3 × 108 CFU of USA300 strain, the Δpsmα3 mutant or the complemented
mutant. After infection, mice infected with the USA300 strain were treated with fludarabine (dissolved in
5% DMSO, 40% PEG300, 5% Tween 80, and 50% double-distilled water) at doses of 10, 20, and 50
mg/kg for durations of 1 and 6 h. Mice infected with the Δpsmα3 mutant were treated with 50 mg/kg
fludarabine for durations of 1 and 6 h. After bacterial challenge for 24 h, six mice from each group were
anesthetized with 60 mg/kg of pentobarbital. Blood samples were collected from the retro-orbital sinus,
and the lungs, livers, spleens, and kidneys were aseptically harvested. These organs were weighed,
homogenized in sterile saline, and plated on agar plates at appropriate dilution. Colonies were counted
after an 18-h incubation at 37°C. Sections of the lung, liver, spleens, and kidneys were washed with sterile
PBS, fixed in 10% formaldehyde for 24 h, and processed for hematoxylin and eosin (HE) staining. The
survival of an additional 10 mice from each group was monitored for 7 days after infection, and the
cumulative survival rate was calculated.

Pneumonia model
An MRSA pneumonia model was established in male BALB/c mice. Mice were anesthetized by
intraperitoneal injection of pentobarbital (60 mg/kg) and then intranasally infected with 30 μL of PBS
containing the USA300 strain or its Δpsmα3 mutant ( 3 × 107 CFU). After infection, mice were treated
with 50 mg/kg fludarabine or STAT1/3-IN-1 for durations of 1 and 6 h. CFUs in the lungs were recorded
at 24 h post infection. The survival of 10 mice per group was monitored for 7 d post-infection, and the
cumulative survival rate was calculated.

Immunohistochemistry and immunofluorescence
Paraffin-embedded lungs and livers from the mouse sepsis model were sectioned at a thickness of 3 μm.
The tissue sections were deparaffinized with xylene and rehydrated a graded ethanol series. Then, the
slides were subjected to antigen retrieval by heating at 94°C in 10 mM citrate buffer (pH 6.0). Peroxidase
activity was subsequently quenched by treating the sections with 3% hydrogen peroxide for 15 min. To
minimize nonspecific staining, the sections were blocked with normal goat serum. For
immunohistochemistry, sections were incubated overnight at 4°C with anti-IL-1β primary antibody (1:100
dilution). After washing three times with PBS, sections were incubated with a secondary antibody for 30
min at 37°C. Following additional PBS washes, the sections were stained with 3, 3′-diaminobenzidine
(DAB) for visualization, and nuclear was counterstained with hematoxylin. For immunofluorescence, the
tissue sections were incubated overnight at 4°C with an Alexa Fluor 488-conjugated F4/80 antibody (1:50
dilution). After three PBS washes, sections were incubated with a Phospho-MLKL antibody (1:500
dilution). After washing, the sections were then incubated with Alexa Fluor 647-conjugated secondary
antibody (1:500 dilution) for 30 min at 37°C. The nuclear were stained with Hoechst 33342. Finally, all
stained sections were scanned and digitized using a Pannoramic 250 Flash III scanner (3DHISTECH,
Hungary). The resulting images were viewed and analyzed with SlideViewer (version 2.5.0.143918)

Plasma cytokines detection
Blood samples from the mouse sepsis model were collected in heparinized tubes and centrifuged at 1000
× g for 15 min to isolate the plasma. The concentrations of TNF-α, IL-1β, and IL-6 in the plasma were
quantified using an Ella device (ProteinSimple, CIA, USA), according to the manufacturer’s instructions.

Statistics and reproducibility
Statistical analysis was performed using GraphPad Prism 8.0 and Microsoft Excel 2016. Results are
expressed as means ± standard error of the mean (SEM). The unpaired two-tailed t-test, one-way analysis
of variance (ANOVA), two-way ANOVA, Fisher’s exact test, or the log-rank test were used for statistical
comparisons, as appropriate. *P < 0.05, **P < 0.01, ***P <0.001. All assays were performed in at least
three independent experiments.

Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the
PRIDE partner repository with the dataset identifier PXD073725
[https://www.ebi.ac.uk/pride/archive/projects/PXD073725]. All identified proteins and the differentially
expressed proteins from proteomic analysis are provided in Supplementary Data 1 and 2 (Excel files).
Full, uncropped images of all Western blots are included in the Supplementary Information. All data that
support the findings of this study are available within the Article, Supplementary Information, or Source
Data file. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "1. Please include the cat no of the antibodies.\n2. The author chooses female C57BL/6 mice for bone marrow cell isolation and male BALB/c mice for sepsis model. What is\nthe relevance of use two types of mice for a single study. Though some epidemiological studies found that males are more\nprone to MRSA BSI infection than female, why the author choose female mice for bone marrow cell isolation. Please justify\nit.\n3. In the figure 4 (e, f) the author must include the loading control and histogram graph of western blot analysis and fluoresce\nactivity for all others.\n4. In the figure 7 HE stains the author must point the suitable area with a proper scale bar.\n5. The author must include the cell viability in treatment condition (especially after adding of fludarabine) in this manuscript.", "reviewer_2": "The authors describe in the manuscript with the title “Targeting phenol-soluble modulin α3-driven macrophage M1\npolarization and necroptosis mitigates MRSA infection by blocking the STAT1 activation with fludarabine “that PSMa3\npromotes both macrophage M1 polarization and necroptosis through the interaction between the interferon-stimulated gene\nfactor 3 (ISGF3) and necrosome complexes. They suggest that formyl peptide receptor 2 (FPR2) was the key regulator of\nPSMα3-induced M1 polarization and necroptosis. The inhibition of STAT1, part of the ISGF3 complex with clinically\napproved cancer drug fludarabine mitigated MRSA infection in a mouse sepsis model and increased survival of treated\nmice. These are interesting data. However, I have some concerns:\nMajor questions/comments:\n1.) The focus of this study is on the polarization of macrophages toward M1. However, only cytokine expression is examined,\nand surface markers in differentiated THP1s are not analysed . Furthermore, it is unclear to what extent differentiated THP1s\nrepresent a good model for human macrophages. Therefore, primary human macrophages should be used to demonstrate\nthat stimulation with PSM 3 or other FPR2 ligands leads to M1 polarization.\n2.) To demonstrate that the cytotoxic effects of PSM 3 are not due to membrane effects but to FPR2 activation, non-toxic\nFPR2 ligands should be used.\n3.) The authors stated that FPR2 is responsible for M1 polarization and necroptosis. (Figure 1c, Fig 6) however the effects\nare small. Only for CC2L a strong effect was observed. To be sure that FPR2 is involved the same experiment should be\ndone with WRW4, an inhibitor for FPR2.\n4.) The authors use different assays to analyse viability (LDH and CCK8). Why?\n5.) The proteomic analysis was performed using differentiated THPs. Why were primary human macrophages not used\nhere?\n6.) The font in Figure 3 c is too small and in Figure 3 d all the prominent points should be labelled, not just 5\n7.) Figure 2d-g was not mentioned in the text. What happens when instead of a PSMa3 mutant a d-toxin mutant was used?\ndelta-toxin is stronger expressed and as PSM 3 a ligand for FPR2. What happens when instead of PSM 3 a  -PSM was used\nregarding necroptosis and M1 polarization?\n8.) Would the effect of PSMa2 be comparable to PSMa3\n9.) Is the drug fludarabine itself antimicrobial. Please add MIC Assays to analyze this -with USA300 and with the USA300\n PSM 3.\n10.) Fig 7: Only the mice infected with WT USA300 were treated with fludarabine-what happens with the USA300  PSM 3\ninfected mice. To demonstrate that the effect is specific, the mice infected with USA300  PSM 3 must also be treated with the\ndrug.\n11.) To be sure that PSMa3 is responsible for the observed effects a complemented mutant should be used\nMinor comments:\nIntroduction: tone done the first section sounds sensational, exaggerated (line 38-53)\nPSMs are toxic because of their  -helical structure, not because of their  - amyloids. Please change this accordingly (line 71-\n72) e.g. Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA. PMID:\n17994102\nIt is not new that PSMs can induce unspecific lysis of various cells\nPage numbering is missing", "reviewer_3": "This study investigates how the MRSA virulence factor phenol-soluble modulin α3 (PSMα3) drives macrophage M1\npolarization and necroptosis through crosstalk between the ISGF3 and necrosome complexes, with FPR2 as a key\nregulator. The authors further demonstrate that targeting STAT1 with the clinically approved drug fludarabine effectively\nmitigates MRSA infection in a murine sepsis model. These findings highlight a promising anti-virulence therapeutic strategy\nby repurposing existing drugs to combat antibiotic-resistant pathogens.\nMinor Comments\n1. The work relies heavily on THP-1 macrophages and murine BMDMs. While informative, conclusions about therapeutic\nrelevance would be strengthened by confirming key findings (M1 markers, necroptosis readouts) in primary human\nmacrophages.\n2. Fludarabine has broad immunosuppressive and anti-proliferative effects. The authors attribute its benefit solely to STAT1\ninhibition, but alternative mechanisms (e.g., general immune suppression) should be considered. A control using a different\nSTAT1 inhibitor or CRISPR/siRNA knockdown of STAT1 in vivo would make the therapeutic claim stronger.\n3. Only a sepsis model is used. Consider testing fludarabine in an additional infection model (e.g., pneumonia or skin\ninfection), as MRSA causes diverse disease manifestations. This would improve translational significance.\n4. The authors convincingly show necroptosis involvement with inhibitors. However, they should provide additional\nvalidation using genetic knockouts (e.g., RIPK3−/− or MLKL−/− macrophages). This would strengthen the conclusion that\nnecroptosis, rather than overlapping cell death pathways, drives the observed effects.\n5. Some figure legends (based on text) lack sufficient detail about replicates, statistical tests, and controls. Ensure all\nexperiments report n values, replicates, error bars, and significance tests clearly.\nMinor Comments\n1. Ensure consistent tense (past tense for results, present tense for conclusions).\n2. Abstract: “increased mice survival rate from 0% to 80%” → should read “increased the survival rate of mice from 0% to\n80%.”\n3. Introduction: “Macrophages play a central in host defense” → should be “central role.”\n4. Results section: “Previous study has reported” → should be “Previous studies have reported.”\n5.\n6. Standardize use of “M1 polarization” vs. “M1 macrophage polarization.”\n7. Define all abbreviations at first use (e.g., CQ, NSA, Fer-1 in methods).\n8. Explain why STAT1 was chosen as the therapeutic target rather than STAT2 or IRF9, which are also part of ISGF3.\n9. Clarify whether fludarabine treatment affected bacterial clearance directly, or if effects were entirely immune-mediated.\n10. The discussion could benefit from a balanced perspective, while fludarabine shows promise, potential risks of\nimmunosuppression should be addressed more critically.\n11. Add a section comparing anti-virulence strategies to conventional antibiotics and explain how repurposed drugs may\ncomplement rather than replace antibiotics.", "reviewer_4": "Comments to the Authors:\nMajor Points\n1. Generalizability of in vitro results: Most experiments are performed in THP-1 cells and mouse BMDMs. Consider including\ndata from primary human macrophages or discussing potential limitations for translational relevance.\n2. Fludarabine specificity and dosing: While the in vivo results are promising, discuss possible off-target or pleiotropic effects\nand provide comparison with other JAK-STAT inhibitors. Clarify dose rationale and safety considerations for future clinical\ntranslation.\n3. FPR2 mechanism: Strengthen mechanistic understanding beyond siRNA knockdown. Consider complementary\napproaches such as agonists or antagonists to confirm the role of FPR2.\n4. Causality in vivo: While fludarabine effectively modulates M1 polarization and necroptosis, acknowledge limitations in\ndirectly attributing effects to specific molecular interactions observed in vitro.\nMinor Points\n- Define technical terms (e.g., ISGF3, necrosome, LD10) at first mention for broader readability.\n- Ensure consistency between main and supplementary figures (cytokine quantification, in vivo survival data).\n- Consider splitting long sentences in Discussion to improve readability.\n- Figure 1i: why IFN-α, ϐ, Ƴ, and chemokines CCL2, 3, and 5 were not quantified by qRT-PCR but only through Luminex\nmultiplex cytokine analysis?\n- Figure 2c: a non-treated control (no PSMa3) should be included in the test.\n- Figure 7: Did the authors try to test what happens to IL-6, TNF-a and IL-1b when treating mice infected with △psmα3 mutant\nstrains with fludarabine?\nDiscussion-specific points\n- Explicitly discuss limitations of translating in vitro THP-1 and BMDM results to primary human macrophages.\n- Briefly address potential off-target effects or pleiotropic effects of fludarabine in vivo.\n- Include short comparison with other inhibitors listed in Supplementary Table 3 regarding translational potential.\n- Consider discussing possible upstream or downstream modulators of ISGF3-necrosome crosstalk to highlight mechanistic\ndepth.\nLine 42: bacteria -> bacterium\nLine 46: its -> bacterial\nLine 60: combating\nLine 68: action of PSM\nLine 134: indicated\nLine 188: et al.\nLine 250: active -> activate\nLine 275: factors\nLine 281: offers\nLine 286: in vivo\nLine 362: concluded doesn’t seem the appropriate term\nLine 383: a previous study have reported\nLine 414: were purchased from Abcam\nLine 498: cultures were grown in static or shaking conditions?\nLine 534: with\nLine 535: respectively.\nLine 542: The meaning of TEAB should be explicated.\nLine 543: Each channel of peptide was labeled by their its respective\nLine 578: in what reagent did you dilute the antibodies?\nLine 912: were assessed\nLine 933: vs control\nLine 968: accessed -> assessed", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "1 RESPONSE TO REVIEWERS’ COMMENTS\n2\n6 for all antibodies in the Reagents and antibodies section of the Methods in the revised\n7 manuscript (lines 537–551).\n8\n10 and male BALB/c mice for sepsis model. What is the relevance of use two types of\n11 mice for a single study. Though some epidemiological studies found that males are more\n12 prone to MRSA BSI infection than female, why the author choose female mice for bone\n13 marrow cell isolation. Please justify it.\n15 different mouse types and sexes in our study. Our decision to use female C57BL/6 mice\n16 for bone marrow-derived macrophages (BMDMs) isolation was based on an established\n17 protocol [Ref: Preparation and culture of bone marrow-derived macrophages from mice\n18 for functional analysis. STAR Protoc. 2020;2(1):100246. PMID: 33458708]. This\n19 protocol indicates that there are no marked differences in the viability or yield of\n20 harvested cells among mice aged 6–12 weeks or between male and female C57BL/6\n21 mice. Moreover, isolating bone marrow cells from female C57BL/6 mice is a reliable\n22 and commonly used practice, and we opted for this approach to ensure consistency with\n23 previous studies.\n25 documented susceptibility to infections, particularly Staphylococcus aureus. In contrast,\n27 mice survive after an intravenous injection of 107 Staphylococcus aureus, all BALB/c\n28 mice die within a week [Ref: Immunological mechanisms underlying the genetic\n29 predisposition to severe Staphylococcus aureus infection in the mouse model. Am J\n1\n32 epidemiological data indicate that males are more prone to MRSA BSI infection than\n33 female; therefore, male BALB/c mice were used in the sepsis model.\n34\n36 histogram graph of western blot analysis and fluoresce activity for all others.\n38 we have repeated the Co-IP experiments and included the loading control GAPDH. The\n39 revised figure is now labeled as Figure 4 (i, j). Additionally, we have provided the\n40 histogram graph of Western blot analysis and the fluoresce activity in the revised\n41 manuscript.\n42\n44 proper scale bar.\n46 with a proper scale bar for the HE stains. The revised figure is now labeled as Figure\n47 9c.\n48\n50 (especially after adding of fludarabine) in this manuscript.\n52 in the fludarabine-treated group following PSMα3 exposure, using the CCK-8 assay.\n55\n58 phenol-soluble modulin α3-driven macrophage M1 polarization and necroptosis\n2\n59 mitigates MRSA infection by blocking the STAT1 activation with fludarabine “that\n61 interaction between the interferon-stimulated gene factor 3 (ISGF3) and necrosome\n62 complexes. They suggest that formyl peptide receptor 2 (FPR2) was the key regulator\n63 of PSMα3-induced M1 polarization and necroptosis. The inhibition of STAT1, part of\n64 the ISGF3 complex with clinically approved cancer drug fludarabine mitigated MRSA\n65 infection in a mouse sepsis model and increased survival of treated mice. These are\n66 interesting data. However, I have some concerns.\n68 helpful suggestions, which have enabled us to strengthen our study. Below, we provide\n69 a point-by-point response to each of the concerns you raised.\n70\n75 represent a good model for human macrophages. Therefore, primary human\n76 macrophages should be used to demonstrate that stimulation with PSMα3 or other FPR2\n77 ligands leads to M1 polarization.\n79 analysis of the M1 polarization surface marker CD86 in differentiated THP-1 cells. Our\n80 results demonstrate that PSMα3 significantly upregulates CD86 expression, supporting\n82 response to your suggestion, we have conducted additional experiments using primary\n83 human macrophages to validate our findings. Consistent with the findings in THP-1\n84 cells, stimulation with PSMα3 induced M1 polarization in primary human macrophages,\n85 as evidenced by increased CD86 expression and elevated levels of pro-inflammatory\n86 cytokines (revised Figure 7b–e).\n87\n3\n89 membrane effects but to FPR2 activation, non-toxic FPR2 ligands should be used.\n91 of PSMα3 is mediated through FPR2 activation rather than non-specific membrane\n92 disruption, we employed the FPR2 inhibitor WRW4. As shown in the revised Figure 8e,\n93 pre-treatment of primary human macrophages with WRW4 significantly inhibited\n95 dependent on FPR2 activation, rather than being a consequence of direct membrane\n96 damage.\n97\n99 necroptosis. (Figure 1c, Fig 6) however the effects are small. Only for CC2L a strong\n100 effect was observed. To be sure that FPR2 is involved the same experiment should be\n101 don with WRW4, an inhibitor for FPR2.\n103 knockdown of FPR2 in THP-1 cells significantly attenuated the production of multiple\n104 pro-inflammatory cytokines/chemokines induced by PSMα3 stimulation, not just CCL-\n105 2 (Figure 6a). Specifically, the downregulation of CCL-2, CCL-3, and TNF-α, as well\n106 as CXCL-10, was highly significant (***P < 0.001), and the decrease in IL-1β was also\n107 significant (**P < 0.01).\n109 confirm the role of FPR2. Our results demonstrated that pre-treatment with WRW4\n110 effectively suppressed the PSMα3-induced upregulation of TNF-α, IL-1β, and IL-6 in\n111 primary human macrophages (Figure 8a). Additionally, Western blot and high-content\n112 fluorescence analyses revealed that WRW4 markedly reduced the PSMα3-induced\n113 upregulation of CD86 and phosphorylation of STAT1 (Figure 8b–d). WRW4 also\n114 significantly alleviated the cytotoxic effects of PSMα3 (Figure 8e). Further high-\n115 content fluorescence and Western blot analyses demonstrated that WRW4 also reduced\n116 the increased phosphorylation of RIP1, RIP3, and MLKL induced by PSMα3 (Figure\n4\n117 8f–i). These findings consistently support the crucial role of FPR2 in mediating\n119\n123 established methods for assessing cell viability. In this study, the CCK-8 assay was used\n124 for experiments involving treated cells alone, while the LDH assay was applied to cell-\n125 bacteria co-culture experiments. This distinction is critical, as the CCK-8 assay has\n126 been reported to also detect bacterial viability [Ref: A simple colorimetric method for\n127 viable bacteria detection based on cell counting Kit-8. Anal Methods.\n128 2021;13(43):5211-5215. PMID: 34694314]. To avoid potential interference from\n129 bacteria in cell viability measurements, we specifically selected the LDH assay. By\n130 collecting cell supernatants and filtering out bacteria prior to performing the LDH assay,\n131 the accuracy and specificity of cell viability assessments were significantly enhanced.\n132\n134 were primary human macrophages not used here?\n136 we utilized differentiated THP-1 cells for the proteomic analysis based on several\n137 practical considerations. The THP-1 model offers significant advantages in terms of\n138 accessibility, cost-effectiveness, ease of propagation, and a homogeneous genetic\n139 background, which are crucial for large-scale, reproducible profiling experiments. This\n140 model is also widely used in macrophage-related proteomic analysis [PMID: 27796300;\n141 41315768; 34731634]. In contrast, primary human macrophages cannot be expanded\n142 in vitro, making it logistically and financially challenging to obtain the large cell\n143 numbers required for deep proteomic coverage (typically >2×10⁶ cells per replicate).\n146 validation experiments using primary human macrophages to confirm key findings\n5\n147 from the differentiated THP-1 cells. These new results demonstrate that PSMα3\n148 similarly induces M1 polarization and necroptosis in primary human macrophages\n149 (revised Figure 7). The remarkable consistency between the data from THP-1-derived\n150 macrophages and primary human macrophages strongly confirms the reliability and\n151 biological relevance of our initial findings.\n152\n154 points should be labelled, not just 5.\n156 improve readability. Regarding Figure 3d, we generated a volcano plot in which all the\n157 prominent points are labelled, but the graphic became overcrowded. Therefore, we\n158 labelled some prominent points in revised Figure 3d and supplied the fully labelled\n159 version as Supplementary Figure 4. The complete list of all the differentially expressed\n160 proteins have been provided in Supplementary Table 2.\n161\n163 a PSMa3 mutant a d-toxin mutant was used? delta-toxin is stronger expressed and as\n165 regarding necroptosis and M1 polarization?\n168 manuscript (revised Figure 2g–j, lines192–200).\n170 mutants are not currently available, we have performed additional experiments using\n171 synthetic and purified PSMα1, PSMα2, PSMα3, PSMα4, PSMβ1, PSMβ2, and δ-toxin\n172 to evaluate their effects. Our results demonstrate that among all the tested PSMs,\n174 whereas PSMα4, PSMβ1, and PSMβ2 showed minimal cytotoxicity. Furthermore,\n6\n176 necroptosis, as evidenced by elevated levels of TNF-α, IL-6, and IL-1β, along with the\n177 strongest upregulation of the necroptosis marker p-MLKL. These findings have been\n178 incorporated into the revised manuscript (revised Figure 2e, f; Supplementary Figure\n179 2).\n180\n183 indicate that although both PSMα2 and PSMα3 exhibit cytotoxic activity and promote\n185 effects across all evaluated parameters—including cell death, secretion of M1-\n186 associated cytokines, and upregulation of the necroptosis marker p-MLKL—when\n187 compared to PSMα2 (revised Figure 2e, f, and Supplementary Figure 2).\n188\n190 analyze this with USA300 and with the USA300 ΔPSMα3.\n192 as requested. The results indicate that the MIC of fludarabine against both USA300 and\n194 high MIC value suggests that fludarabine does not exhibit antibacterial activity against\n196 375).\n197\n199 fludarabine-what happens with the USA300 ΔPSMα3 infected mice. To demonstrate\n200 that the effect is specific, the mice infected with USA300 ΔPSMα3 must also be treated\n201 with the drug.\n203 effect of fludarabine is specific to PSMα3-driven pathogenesis, we had performed the\n204 additional experiment. The △psmα3 mutant-infected mice were treated with 50 mg/kg\n7\n205 fludarabine. As now showed in the revised Figure 9d, the results showed that infection\n206 with the Δpsmα3 mutant alone led to a 50% survival rate. Fludarabine treatment\n207 provided a statistically non-significant improvement, increasing the survival rate from\n208 50% to 60%. These findings demonstrate that the therapeutic effect of fludarabine is\n209 mainly mediated through targeting PSMα3-driven pathogenesis.\n210\n212 complemented mutant should be used.\n214 strain Δpsmα3 (pEX-psmα3) and evaluated its impact both in vitro and in vivo. As\n215 shown in revised Figure 2g, infection of THP-1 macrophages with the Δpsmα3 mutant\n216 led to a significant reduction in cytotoxicity compared to the wild-type LAC strain,\n217 while the complemented strain fully restored cytotoxicity to wild-type levels. A similar\n218 trend was observed when macrophages were treated with bacterial supernatants (Figure\n219 2h). Furthermore, cytokine analysis revealed that the Δpsmα3 mutant induced\n220 significantly lower secretion of TNF-α, IL-1β, and IL-6, and this defect was also\n221 rescued in the complemented strain (Figure 1k–m).\n223 survival rate, mice infected with the complemented Δpsmα3 (pEX-psmα3) strain\n224 exhibited 0% survival rate, confirming the essential role of PSMα3 (Figure 9d).\n226 observed effects.\n227\n230 (line 38-53)\n232 tone is essential in academic writing and have revised the introduction accordingly.\n8\n234 \"alarming situation\") with more measured alternatives (e.g., \"significant,\" \"serious\n235 challenge\") to ensure the language remains factual and balanced.\n237 the MRSA threat are directly supported by the cited literatures: the 127% mortality\n238 increase is from The Lancet (2024), the \"Priority 1: Critical\" classification is from the\n240 reported by Kullar et al. (2011). Our intention was to accurately summarize these\n241 published findings, which themselves underscore the significant public health concern.\n243 sources (lines 37-52).\n244\n246 β-amyloids. Please change this accordingly (line 71-72) e.g. Identification of novel\n247 cytolytic peptides as key virulence determinants for community-associated MRSA.\n250 manuscript (lines 71-72) to: “Among PSMs, PSMα3 stands out for its potent cytotoxic\n251 and lytic activity, which is attributed to its α-helical structure.” We have also cited the\n252 reference (PMID: 17994102).\n253\n256 in the literature. The focus of our current study is to investigate the unreported\n257 mechanisms by which PSMs induce macrophage M1 polarization and regulate\n258 necroptosis. By understanding these mechanisms, we uncovered potential new\n259 strategies and drugs for combating bacterial infections.\n260\n9\n263 page numbers to the manuscript in the revised version.\n264\n267 phenol-soluble modulin α3 (PSMα3) drives macrophage M1 polarization and\n268 necroptosis through crosstalk between the ISGF3 and necrosome complexes, with\n270 clinically approved drug fludarabine effectively mitigates MRSA infection in a murine\n271 sepsis model. These findings highlight a promising anti-virulence therapeutic strategy\n272 by repurposing existing drugs to combat antibiotic-resistant pathogens.\n274 appreciate your insightful comments, which have greatly helped us improve our work.\n276\n279 confirming key findings (M1 markers, necroptosis readouts) in primary human\n280 macrophages.\n282 have performed additional experiments using primary human macrophages. Consistent\n283 with the results from THP-1 and BMDMs, we found that PSMα3 similarly upregulated\n284 the expression of M1 polarization markers and the necroptosis markers in primary\n285 human macrophages (revised Figure 7). Furthermore, we confirmed that PSMα3\n286 activates the ISGF3 complex and promotes the assembly of the necrosome complex in\n287 primary human macrophages (revised Figure 7).\n288\n291 (e.g., general immune suppression) should be considered. A control using a different\n10\n293 therapeutic claim stronger.\n295 regarding the specificity of fludarabine's action, we sought to validate the role of STAT1\n296 inhibition using an independent approach. As STAT1 knockout mice are not readily\n297 available, we used STAT1/3-IN-1, a distinct STAT1 inhibitor with minimal reported in\n298 vivo toxicity, for further validation. Consistent with the effects of fludarabine, treatment\n299 with STAT1/3-IN-1 provided significant protection against MRSA infection, resulting\n300 in a 50% survival rate and a marked reduction in pulmonary bacterial loads (Figure 9e,\n301 f). These findings suggest that STAT1 inhibition is a key mechanism underlying the\n302 efficacy of fludarabine against MRSA infection.\n303\n305 infection model (e.g., pneumonia or skin infection), as MRSA causes diverse disease\n306 manifestations. This would improve translational significance.\n308 we have now evaluated fludarabine in a MRSA-induced pneumonia model. In this\n309 model, fludarabine treatment significantly improved survival and markedly reduced\n310 bacterial burdens in the lungs (revised Figure 9e, f). These results demonstrate that\n311 fludarabine effectively mitigates MRSA infection in different mouse models.\n312\n316 necroptosis, rather than overlapping cell death pathways, drives the observed effects.\n319 cytotoxicity. The result showed that PSMα3-induced cytotoxicity was significantly\n11\n320 attenuated in MLKL-/- macrophages compared to wild-type controls (revised Figure\n321 2d), indicating that PSMα3 induces macrophage death primarily through the\n322 necroptosis pathway.\n323\n325 statistical tests, and controls. Ensure all experiments report n values, replicates, error\n326 bars, and significance tests clearly.\n328 legends to explicitly include the n values, replicates, error bars, the statistical tests used,\n329 and a clear description of the controls for each experiment.\n330\n333 conclusions).\n335 ensure consistent use of verb tenses. We have used the past tense to describe the results\n336 of our experiments and the present tense to present our conclusions and discuss the\n337 implications of our findings.\n338\n340 “increased the survival rate of mice from 0% to 80%.”\n342 rate from 0% to 80%” to “increased the survival rate of mice from 0% to 80%” (line\n343 28)\n344\n346 “central role.”\n349 role in host defense.” (line 78)\n12\n350\n352 studies have reported.”\n354 to read “Previous studies have reported”. (line 290)\n355\n358 throughout the manuscript to consistently use “M1 macrophage polarization” to avoid\n359 confusion and ensure clarity.\n360\n363 including CQ, NSA, and Fer-1, are defined at their first use in the revised manuscript.\n364\n369 necroptosis (RIP1, RIP3, and MLKL), as potential therapeutic targets. Accordingly, we\n370 reviewed literature and summarized several inhibitors targeting these key proteins\n371 (Supplementary Table 3). While many of these inhibitors remain research tools with\n372 unvalidated safety and efficacy profiles in humans, fludarabine, a STAT1 inhibitor,\n373 stands out as an already clinically approved agent for the treatment of chronic\n374 lymphocytic leukemia. The use of an approved drug provides significant advantages\n375 for translational research, including established safety, regulatory acceptance, and well-\n376 characterized pharmacokinetics, thereby facilitating faster clinical application. For\n377 these reasons, we focused on evaluating the therapeutic effect of the fludarabine on\n13\n379\n381 directly, or if effects were entirely immune-mediated.\n383 that the MIC value of fludarabine against MRSA > 512 μg/mL, indicating that\n384 fludarabine does not exhibit direct antibacterial activity in vitro. However, in vivo,\n385 fludarabine treatment significantly increased the survival rate of MRSA-infected mice\n386 and reduced tissue bacterial load. These findings suggest that fludarabine does not\n387 directly kill or clear bacteria. Instead, its protective effect is largely mediated through\n388 the suppression of PSMα3-driven virulence. This conclusion is supported by the\n389 markedly diminished therapeutic benefit of fludarabine in the Δpsmα3 infection model,\n390 where fludarabine only provided a statistically non-significant increase in survival rate\n391 (from 50% to 60%) (Figure 9d). And we also confirm that STAT1 inhibition is a key\n392 mechanism underlying the efficacy of fludarabine against MRSA infection (Figure 9e).\n394 vivo (Figure 10). This immune-mediated mechanism of action is crucial in enhancing\n395 the host's defense against bacterial infections.\n396\n398 fludarabine shows promise, potential risks of immunosuppression should be addressed\n399 more critically.\n401 statement to the Discussion section: “Nevertheless, given the potential for off-target\n402 and pleiotropic effects associated with fludarabine, future investigations should\n403 carefully optimize dose selection and thoroughly evaluate the potential risks,\n404 particularly immunosuppression.” (Lines 507–509)\n405\n407 antibiotics and explain how repurposed drugs may complement rather than replace\n14\n408 antibiotics.\n410 to the Discussion section: “Our study provides an anti-virulence strategy, which differs\n411 fundamentally from conventional antibiotics. While antibiotics aim to kill bacteria or\n412 halt their growth, thereby imposing strong selective pressure for resistance, our\n413 approach targets the PSMα3-associated pathway to disarm the pathogen and mitigate\n414 host damage. This anti-virulence strategy, particularly when employing repurposed\n415 drugs, could also act as a complementary force to conventional antibiotic. By inhibiting\n416 virulence-mediated host damage, this strategy can buy critical time for antibiotics to\n417 effectively clear the bacteria.” (Lines 510–517)\n418\n423 macrophages or discussing potential limitations for translational relevance.\n425 experiments using primary human macrophages to validate our findings (revised Figure\n426 7). Specifically, we have demonstrated that PSMα3 induces M1 macrophage\n427 polarization and necroptosis in primary human macrophages, consistent with the results\n428 obtained in THP-1 macrophages and murine BMDMs.\n429\n431 discuss possible off-target or pleiotropic effects and provide comparison with other\n433 clinical translation.\n435 pleiotropic effects and to evaluate the specificity of the STAT1 inhibition in our\n15\n436 observed protection, we compared fludarabine with a distinct STAT1 inhibitor,\n438 core therapeutic benefits of fludarabine, significantly increasing survival in the MRSA\n439 pneumonia model. The concordant efficacy of these two structurally distinct inhibitors\n440 suggests that the protective effect is primarily mediated through the inhibition of STAT1.\n442 and safety. (Lines 499–509)\n443\n445 knockdown. Consider complementary approaches such as agonists or antagonists to\n446 confirm the role of FPR2.\n448 to further confirm the role of FPR2. Our results demonstrated that pre-treatment with\n451 content fluorescence analyses revealed that WRW4 markedly reduced the PSMα3-\n452 induced upregulation of CD86 and phosphorylation of STAT1 (Figure 8b–d). WRW4\n453 also significantly alleviated the cytotoxic effects of PSMα3 (Figure 8e). Further high-\n454 content fluorescence and western blot analyses demonstrated that WRW4 also reduced\n455 the increased phosphorylation of RIP1, RIP3, and MLKL induced by PSMα3 (Figure\n456 8f–i). These findings consistently support the crucial role of FPR2 in mediating\n458\n460 ion and necroptosis, acknowledge limitations in directly attributing effects to specific\n461 molecular interactions observed in vitro.\n463 address it, we have revised the manuscript to focus on describing the observed\n464 phenomena in vivo, avoiding directly attributing effects of fludarabine to specific\n16\n465 molecular interactions observed in vitro. For example, the sentence “In a sepsis model,\n466 50 mg/kg fludarabine significantly increased mice survival rate from 0% to 80% and\n467 alleviated tissue damage, which were attributed to its ability to reduce the cytokine\n468 storm and enhance host immune defense by inhibiting macrophage M1 polarization and\n469 necroptosis.” has been revised to “In a sepsis model, 50 mg/kg fludarabine significantly\n470 increased the survival rate of mice from 0% to 80% and alleviated tissue damage. It\n471 also reduced the cytokine storm and enhanced host immune defense by inhibiting M1\n472 macrophage polarization and necroptosis in vivo.” (Lines 27–30)\n473\n476 for broader readability.\n478 definitions for technical terms at their first appearance in the revised manuscript. “The\n480 factor complex involved in the regulation of macrophage polarization and plays a key\n481 role in the immune response. The necrosome complex, consisting of RIP1, RIP3, and\n483 (10 % lethal dose; the dose causing 10 % mortality)” (lines 501–502).\n484\n486 quantification, in vivo survival data).\n488 figure (Figure 1i) presents the Luminex multiplex analysis of a panel of cytokines and\n489 chemokines in human THP-1-derived macrophages following PSMα3 treatment. We\n490 performed this comprehensive analysis because our study focuses primarily on human\n491 cells. The data in Supplementary Figure 1i–k validate three representative cytokines\n492 (TNF‑α, IL‑6, IL‑1β) in murine BMDMs. These results align with the corresponding\n493 observations in human cells, demonstrating the conserved inflammatory response\n494 across species and thereby supporting our subsequent in vivo modeling. Regarding in\n17\n495 vivo survival data, all results are consistently presented in the main figures, with no\n496 survival data included in supplementary materials.\n497\n501\n503 quantified by qRT-PCR but only through Luminex multiplex cytokine analysis?\n505 manner to thoroughly investigate the role of PSMα3 in M1 macrophage polarization.\n508 mRNA level prompted us to proceed with validation at the protein level.\n510 available Luminex multiplex cytokine panel. The composition of this panel was fixed\n511 and included not only the protein counterparts of the mRNAs we initially assessed but\n512 also additional analytes such as IFN-α, β, γ, and the chemokines CCL-2, CCL-3, and\n514 and not by qRT-PCR.\n516 of a functional inflammatory response, the measurement of secreted cytokines and\n517 chemokines offers a more direct and physiologically relevant readout of macrophage\n518 activation. We believe that the protein-level data presented strongly corroborate our\n519 conclusion that PSMα3 promotes M1 macrophage polarization.\n520\n523 included in our original experiment. The cell survival rate for each group was calculated\n18\n524 with the non-treated control group set as 100%. To better illustrate this, we have now\n525 added the non-treated control group in revised Figure 2c.\n526\n528 1b when treating mice infected with △psmα3 mutant strains with fludarabine?\n530 performed the experiment that includes a group of mice infected with the Δpsmα3\n531 mutant strain and treated with fludarabine (50 mg/kg), thereby enabling a direct and\n532 rigorous comparison. The plasma concentrations of TNF-α, IL-1β, and IL-6 were\n533 subsequently quantified using the Ella system.The results showed that there was no\n534 statistically significant difference in the levels of these pro-inflammatory cytokines\n535 (TNF-α, IL-1β, and IL-6) between the fludarabine-treated and untreated Δpsmα3\n536 mutant-infected mice (revised Figure 10b–d).\n537\n540 results to primary human macrophages.\n542 relevance, we have conducted additional experiments using primary human\n543 macrophages to validate our findings. Consistent with the results observed in THP-1\n544 macrophages and murine BMDMs, PSMα3 upregulated the expression of M1\n545 polarization markers and the necroptosis markers in primary human macrophages\n546 (revised Figure 7). Furthermore, we confirmed that PSMα3 activates the ISGF3\n547 complex and promotes the assembly of the necrosome complex in this primary human\n548 cell model (revised Figure 7).\n549\n551 fludarabine in vivo.\n19\n553 pleiotropic effects of fludarabine in vivo, we investigated whether its protective effect\n554 was attributable to STAT1 inhibition. To this end, a distinct STAT1 inhibitor STAT1/3-\n556 fludarabine, increasing the survival rate from 0% to 50% in the MRSA pneumonia\n557 model (Figure 9e). These results confirm that STAT1 inhibition is a key mechanism\n558 underlying the efficacy of fludarabine against MRSA infection.\n560 potential for off-target and pleiotropic effects associated with fludarabine, future\n561 investigations should carefully optimize dose selection and thoroughly evaluate the\n562 potential risks, particularly immunosuppression.” (lines 507–509).\n563\n567 inhibitors, as listed in Supplementary Table 3, in the Discussion section: “According to\n568 this strategy, we have summarized several inhibitors targeting key proteins involved in\n571 model induced by cecal ligation and puncture, highlighting its translational potential.\n573 yet been developed into clinically approved drugs, with their safety and efficacy in\n574 humans remaining unclear. In contrast, fludarabine, which is clinically approved for\n575 treating certain cancers primarily due to its ability to inhibit DNA synthesis and repair,\n576 also functions as a STAT1 inhibitor.” (lines 470–478)\n577\n581 downstream modulators of ISGF3-necrosome crosstalk in the Discussion section:\n20\n582 “Previous studies have indicated that FPR2 modulates ERK1/2 46, a kinase known to\n583 regulate STAT1 activation, raising the possibility that FPR2 may influence ISGF3\n584 activation via an ERK1/2-dependent mechanism. Additionally, our proteomics data\n585 revealed the differential expression of CHMP6, a subunit of the endosomal protein\n586 complex required for transport-III (ESCRT-III) complex, following PSMα3 stimulation.\n588 this finding suggests its potential involvement in the PSMα3-induced signaling\n589 pathway.” (lines 459–466)\n590\n21\n615 inaccuracies. All of the listed issues have been corrected in the revised manuscript:\n626 revised to “the antibacterial activity of fludarabine against MRSA in vivo”\n629 reported”\n632 rpm\n634 to “treated with 0, 1, or 3 μM PSMα3 for 24 h”\n637 bicarbonate)\n639 to “Each channel of peptide was labeled by its respective”\n22\n641 containing 0.1% Tween-20 (TBST).\n645\n648 provided the listed reports. This is part of the Nature Communications initiative to\n649 facilitate training in peer review and to provide appropriate recognition for Early Career\n652 manuscript. Your insights are incredibly helpful and have significantly contributed to\n653 the improvement of our work.\nReviewer #1 (Remarks to the Author):\nComments: The authors have addressed all the comments satisfactorily\nAnswer: We sincerely thank you for your time and effort in reviewing our manuscript\nand for your positive feedback on our revisions.\nReviewer #2 (Remarks to the Author):\nComments: Thank you for the revised manuscript. The points of criticism have been\naddressed satisfactorily. I have no further questions.\nAnswer: Thank you very much for your positive comments. We appreciate that you\nhave provided many constructive suggestions to help us improve our study.\nReviewer #3 (Remarks to the Author):\nComments: The authors have adequately addressed all the comments and suggestions\nraised during the review process. The revised manuscript has improved substantially.\nIn its present form, the manuscript is suitable for publication.\nAnswer: We are grateful for your valuable comments and suggestions throughout the\nreview process, which have greatly helped us strengthen the manuscript. We appreciate\nyour recommendation for publication.\nReviewer #4 (Remarks to the Author):\nComments: I have carefully evaluated the revised manuscript and the authors’ detailed\nresponses to all reviewer comments. I would like to commend the authors for the\nsubstantial effort invested in this revision, which has significantly strengthened the\nmanuscript both conceptually and experimentally.\nAll major concerns raised during the first round of review have been thoroughly and\nconvincingly addressed. In particular:\n- The authors have extended their key findings to primary human macrophages,\nsubstantially improving translational relevance.\n- The involvement of necroptosis is now robustly supported by genetic validation using\nMLKL-deficient macrophages, in addition to pharmacological inhibition.\n- The role of FPR2 has been reinforced using complementary approaches, including\nsiRNA knockdown and the pharmacological inhibitor WRW4, across both cell lines and\nprimary cells.\n- The specificity of fludarabine has been carefully examined through MIC assays,\nexperiments with the Δpsmα3 mutant, use of a complemented strain, an independent\nSTAT1 inhibitor, and validation in an additional in vivo infection model.\n- The authors have appropriately revised the manuscript to avoid over-interpretation of\nin vivo causality and have improved clarity, tone, and methodological transparency\nthroughout.\nWhile some aspects of the proposed signaling cascade (e.g., the relative contribution of\ndirect membrane-disruptive effects of PSMα3 versus receptor-mediated signaling) are\ninherently complex and difficult to disentangle fully, the authors now provide a\nbalanced and appropriately cautious discussion of these points. Importantly, no major\nconceptual or experimental gaps remain that would warrant further rounds of revision.\nOverall, the revised manuscript presents a coherent, well-supported, and biologically\nmeaningful study, offering novel insights into MRSA virulence mechanisms and a\ncompelling proof-of-concept for an anti-virulence therapeutic strategy.\nI therefore recommend acceptance of the manuscript, in its current form or after minor\neditorial adjustments at the discretion of the editor.\nAnswer: We thank you for your thorough evaluation and positive recommendation. We\nappreciate your acknowledgment of the revisions and are grateful for your constructive\nfeedback, which has significantly strengthened our study.\nReviewer #5 (Remarks to the Author):\nComment: I co-reviewed this manuscript with one of the reviewers who provided the\nlisted reports. This is part of the Nature Communications initiative to facilitate training\nin peer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nAnswer: We appreciate that you have provided many constructive suggestions to help\nus improve our study."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all the comments satisfactorily", "reviewer_2": "Thank you for the revised manuscript. The points of criticism have been addressed satisfactorily. I have no further questions.", "reviewer_3": "The authors have adequately addressed all the comments and suggestions raised during the review process. The revised\nmanuscript has improved substantially. In its present form, the manuscript is suitable for publication.", "reviewer_4": "I have carefully evaluated the revised manuscript and the authors’ detailed responses to all reviewer comments. I would like\nto commend the authors for the substantial effort invested in this revision, which has significantly strengthened the\nmanuscript both conceptually and experimentally.\n\nAll major concerns raised during the first round of review have been thoroughly and convincingly addressed. In particular:\n- The authors have extended their key findings to primary human macrophages, substantially improving translational\nrelevance.\n- The involvement of necroptosis is now robustly supported by genetic validation using MLKL-deficient macrophages,\nin addition to pharmacological inhibition.\n- The role of FPR2 has been reinforced using complementary approaches, including siRNA knockdown and the\npharmacological inhibitor WRW4, across both cell lines and primary cells.\n- The specificity of fludarabine has been carefully examined through MIC assays, experiments with the Δpsmα3 mutant, use\nof a complemented strain, an independent STAT1 inhibitor, and validation in an additional in vivo infection model.\n- The authors have appropriately revised the manuscript to avoid over-interpretation of in vivo causality and have improved\nclarity, tone, and methodological transparency throughout.\n\nWhile some aspects of the proposed signaling cascade (e.g., the relative contribution of direct membrane-disruptive effects\nof PSMα3 versus receptor-mediated signaling) are inherently complex and difficult to disentangle fully, the authors now\nprovide a balanced and appropriately cautious discussion of these points. Importantly, no major conceptual or experimental\ngaps remain that would warrant further rounds of revision.\n\nOverall, the revised manuscript presents a coherent, well-supported, and biologically meaningful study, offering novel\ninsights into MRSA virulence mechanisms and a compelling proof-of-concept for an anti-virulence therapeutic strategy.\n\nI therefore recommend acceptance of the manuscript, in its current form or after minor\neditorial adjustments at the discretion of the editor.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "RESPONSE TO REVIEWERS’ COMMENTS\nReviewer #1 (Remarks to the Author):\nComments: The authors have addressed all the comments satisfactorily\nAnswer: We sincerely thank you for your time and effort in reviewing our manuscript\nand for your positive feedback on our revisions.\nReviewer #2 (Remarks to the Author):\nComments: Thank you for the revised manuscript. The points of criticism have been\naddressed satisfactorily. I have no further questions.\nAnswer: Thank you very much for your positive comments. We appreciate that you\nhave provided many constructive suggestions to help us improve our study.\nReviewer #3 (Remarks to the Author):\nComments: The authors have adequately addressed all the comments and suggestions\nraised during the review process. The revised manuscript has improved substantially.\nIn its present form, the manuscript is suitable for publication.\nAnswer: We are grateful for your valuable comments and suggestions throughout the\nreview process, which have greatly helped us strengthen the manuscript. We appreciate\nyour recommendation for publication.\nReviewer #4 (Remarks to the Author):\nComments: I have carefully evaluated the revised manuscript and the authors’ detailed\nresponses to all reviewer comments. I would like to commend the authors for the\nsubstantial effort invested in this revision, which has significantly strengthened the\nmanuscript both conceptually and experimentally.\nAll major concerns raised during the first round of review have been thoroughly and\nconvincingly addressed. In particular:\n- The authors have extended their key findings to primary human macrophages,\nsubstantially improving translational relevance.\n- The involvement of necroptosis is now robustly supported by genetic validation using\nMLKL-deficient macrophages, in addition to pharmacological inhibition.\n- The role of FPR2 has been reinforced using complementary approaches, including\nsiRNA knockdown and the pharmacological inhibitor WRW4, across both cell lines and\nprimary cells.\n- The specificity of fludarabine has been carefully examined through MIC assays,\nexperiments with the Δpsmα3 mutant, use of a complemented strain, an independent\nSTAT1 inhibitor, and validation in an additional in vivo infection model.\n- The authors have appropriately revised the manuscript to avoid over-interpretation of\nin vivo causality and have improved clarity, tone, and methodological transparency\nthroughout.\nWhile some aspects of the proposed signaling cascade (e.g., the relative contribution of\ndirect membrane-disruptive effects of PSMα3 versus receptor-mediated signaling) are\ninherently complex and difficult to disentangle fully, the authors now provide a\nbalanced and appropriately cautious discussion of these points. Importantly, no major\nconceptual or experimental gaps remain that would warrant further rounds of revision.\nOverall, the revised manuscript presents a coherent, well-supported, and biologically\nmeaningful study, offering novel insights into MRSA virulence mechanisms and a\ncompelling proof-of-concept for an anti-virulence therapeutic strategy.\nI therefore recommend acceptance of the manuscript, in its current form or after minor\neditorial adjustments at the discretion of the editor.\nAnswer: We thank you for your thorough evaluation and positive recommendation. We\nappreciate your acknowledgment of the revisions and are grateful for your constructive\nfeedback, which has significantly strengthened our study.\nReviewer #5 (Remarks to the Author):\nComment: I co-reviewed this manuscript with one of the reviewers who provided the\nlisted reports. This is part of the Nature Communications initiative to facilitate training\nin peer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nAnswer: We appreciate that you have provided many constructive suggestions to help\nus improve our study."}]

Decision letter