Abstract

   Talaromyces marneffei (TM) is an opportunistic pathogenic fungus that
   mainly infects immunocompromised patients. Currently, the global
   prevalence of talaromycosis caused by TM is increasing, leading to an
   increased demand for anti-TM drugs. In our previous study, a novel
   28-membered macrolide compound, antifungalmycin B (ANB), was isolated
   from Streptomyces hiroshimensis GXIMD 06359, exhibiting significant
   antifungal properties. However, its in vivo mechanisms and direct
   antifungal effects warrant further investigation. In this study, we
   employed a mouse model in conjunction with transcriptomic and proteomic
   approaches to explore the antifungal activity of ANB against T.
   marneffei. In an in vivo mouse model infected with T. marneffei
   infection, ANB significantly reduced fungal burdens in the liver,
   spleen, lungs, and kidneys. Additionally, it markedly decreased the
   levels of reactive oxygen species (ROS) and cytokines, including
   interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. Proteomic
   and transcriptomic studies, complemented by parallel reaction
   monitoring (PRM) analysis, revealed that ANB effectively disrupted acid
   biosynthesis and cellular energy metabolism, thereby impairing
   mitochondrial functions in T. marneffei. These effects were exerted
   through multiple pathways. These findings highlight the potential of
   ANB as a versatile inhibitor of polyene macrolide-resistant fungi,
   offering a promising therapeutic avenue for the treatment of
   talaromycosis.

   Keywords: Talaromyces marneffei, antifungal mechanism, proteomics,
   transcriptomics

1. Introduction

   Talaromyces marneffei infection leads to talaromycosis, a severe fungal
   disease predominantly found in the tropical and subtropical areas of
   Southeast Asia [[38]1]. This invasive, systemic infection is especially
   dangerous for immunocompromised individuals who have a significantly
   high mortality rate, thus raising concerns [[39]2]. The disease also
   poses a risk of becoming epidemic in new areas [[40]3]. Since the
   mid-1990s, the prevalence of T. marneffei infection has increased not
   only among people living with human immunodeficiency virus (HIV) but
   also among those who are immunocompromised without HIV infection
   [[41]4,[42]5]. In a review published in 2018, T. marneffei was ranked
   as the third-most common fungal infection in the world. By 2021,
   experts from endemic regions advocated that it be officially recognized
   as a neglected tropical disease, stressing the global impact of this
   infection [[43]6,[44]7].

   With the global rise in fungal infections, existing antifungal
   medications are inadequate, highlighting the critical need for new
   drugs to increase treatment effectiveness. Currently, three main
   classes of agents (polyenes, azoles, and echinocandins) are used to
   treat invasive talaromycosis. Amphotericin B (AmB) is the preferred
   initial treatment for severe cases of talaromycosis [[45]8], but its
   high cost and severe side effects, including renal failure, electrolyte
   imbalances, vasculitis, and bone marrow suppression, limit its use
   [[46]9]. Furthermore, azole antifungals have encountered resistance
   issues with T. marneffei [[47]10,[48]11]. Given these challenges,
   developing novel antifungal agents is an urgent and essential task to
   address the evolving threats of talatomycosis and other fungal
   diseases. Sangkanu et al. (2021) isolated 19 actinomycete extracts from
   marine organisms and sediments that showed antifungal activity against
   T. marneffei [[49]12]. Additionally, Jiang et al. (2024) investigated a
   novel marine-derived compound called bamemacrolactine C, which has
   demonstrated antifungal activity against T. marneffei and is capable of
   disrupting the integrity of cell membranes and inhibiting intracellular
   enzyme activities [[50]13]. Therefore, the search for new and efficient
   antibiotics from the marine sources with fewer side effects and lower
   costs is important for combating T. marneffei.

   Antifungalmycin B (ANB), a novel polyene macrolide derived from the
   mangrove-associated Streptomyces hiroshimensis GXIMD 06359, has shown
   efficacy against T. marneffei [[51]14,[52]15]. Our preliminary study
   revealed that ANB acts on T. marneffei by compromising the integrity of
   its cell membrane and mitochondrial function, leading to apoptosis.
   These findings highlight the potential of ANB as an effective treatment
   for T. marneffei [[53]14]. However, the specific mechanisms by which
   ANB disrupts mitochondrial function and damages cell membranes in T.
   marneffei remain underexplored. Although a few systematic studies
   exist, comprehensive studies on the intracellular responses of T.
   marneffei are lacking. The detailed antifungal mechanism of ANB in T.
   marneffei also remains unclear. Hence, further exploration is needed to
   investigate the potential antifungal effects of ANB in vivo in mouse
   models infected with T. marneffei.

   Therefore, we investigated the antifungal activity of ANB in a mouse
   model of T. marneffei infection. To identify the intracellular targets
   affected by ANB, we employed both transcriptomic and proteomic
   approaches. Additionally, parallel reaction monitoring (PRM) was
   utilized to confirm changes in protein expression. This study helps to
   elucidate the mechanism of action and the in vivo efficacy of ANB
   against T. marneffei and provides valuable insights into the
   multitarget inhibitory mechanisms of polyene macrolides.

2. Results

2.1. Antifungalmycin B Demonstrated Significant Efficacy in an
Immunocompromised Disseminated T. marneffei Model

   To assess the in vivo antifungal effect of ANB, a murine model of
   immunocompromised disseminated T. marneffei was used. The mice were
   administered ANB, AmB, or saline for seven consecutive days. The
   results revealed that the mean survival time (MST) of the mice treated
   with saline was 7 days. In contrast, ANB affected the MST over 14 days.
   As depicted in [54]Figure 1A, both ANB and AmB treatments significantly
   increased survival rates at day 14, with 80% survival at doses of 2
   mg/kg/d or 1 mg/kg/d of ANB or 0.5 mg/kg/d AmB and 90% survival at 0.5
   mg/kg/d) of ANB, while all the saline-treated mice died.

Figure 1.

   [55]Figure 1
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   Therapeutic effect of ANB on talaromycosis in mice. (A) Survival curves
   for mice infected with T. marneffei and treated with ANB (administered
   intraperitoneally). (B–E) Quantitative analysis of fungal burden in
   tissues, expressed as colony-forming units (CFUs) per gram. Data are
   shown as mean ± S.D. with significance levels indicated as ** p < 0.01,
   and *** p < 0.001; statistical analysis performed using Student’s t
   test, n = 3.

   The severity of T. marneffei infection was assessed by determining the
   tissue fungal burden in mice from various experimental groups. As shown
   in [57]Figure 1B–E, the fungal burden in the liver, spleen, lung, and
   kidney decreased after five days of daily intraperitoneal treatment
   with ANB. The quantification of fungal burden in the control group
   revealed that in the control group, the spleen was the most heavily
   affected organ, with a fungal burden of 5.11 ± 0.02 log10 CFU/g,
   followed by the kidney (4.65 ± 0.01 log10 CFU/g), liver (4.27 ± 0.01
   log10 CFU/g), and lungs (3.67 ± 0.04 log10 CFU/g). Notably, a
   significant reduction in fungal burden was observed in the mice treated
   with 2 mg/kg/d, 1 mg/kg/d, and 0.5 mg/kg/d of ANB. The greatest
   decrease was observed in the group receiving 2 mg/kg/d of ANB compared
   with those treated with lower doses.

   The intracellular levels of ROS in the spleen and lung were
   subsequently assessed ([58]Figure 2). ROS are pivotal in regulating
   cell survival, with moderate levels supporting cell proliferation and
   survival. However, excessive ROS can induce oxidative stress, resulting
   in damage such as DNA strand breaks, protein degradation, lipid
   peroxidation, and ultimately cell death [[59]16,[60]17]. In the control
   group, the ROS levels significantly increased from 14.58 to 30.55 in
   the spleen and from 11.57 to 19.16 in the lungs ([61]Figure 2B,C). In
   contrast, the level of ROS was significantly lower in the groups
   treated with ANB at doses of 2 mg/kg/d, 1 mg/kg/d, and 0.5 mg/kg/d. The
   cells in the spleen and lungs of the ANB-treated group presented weak
   green fluorescence, indicating reduced ROS accumulation, whereas the
   cells in the control group presented strong fluorescence, suggesting
   high ROS levels. These findings indicate that ANB can effectively
   reduce the accumulation of intracellular ROS in the spleen and lung.

Figure 2.

   [62]Figure 2
   [63]Open in a new tab

   Measurement of ROS in cells of lung and spleen cells via an ROS
   fluorometric assay. (A) Fluorescence microscopy images of cells. (B,C)
   Quantitative evaluation of fungal burden in tissues, reported as CFUs
   per gram. The data are shown as means ± S.Ds. Control group vs Blank
   group: ^### p < 0.001. Treatment groups vs Control group: ***p < 0.001;
   Student’s t test, n = 3.

   Cytokine levels were measured to assess whether the concentrations of
   inflammatory factors in challenged mice changed in the presence or
   absence of ANB. By day five of treatment, ANB had significantly lowered
   the levels of the proinflammatory cytokines IL-1β, IL-6, and TNF-α
   ([64]Figure 3). These results indicate that ANB effectively controlled
   T. marneffei infection by decreasing the levels of proinflammatory
   IL-1β, IL-6, and TNF-α.

Figure 3.

   [65]Figure 3
   [66]Open in a new tab

   Anti-inflammatory activity of ANB in T. marneffei-infected mice. (A–C)
   Comparison of inflammatory factors in lung tissue. (D–F) Comparison of
   inflammatory factors in spleen tissue. The data are shown as means ±
   S.Ds. Control group vs Blank group: ^### p < 0.001. Treatment groups vs
   Control group: * p < 0.05, ** p < 0.01, and *** p < 0.001; Student’s t
   test, n = 3.

2.2. Transcriptomics of ANB-Treated T. marneffei

   The changes in the transcriptome of T. marneffei in response to ANB
   treatment were analyzed by RNA-Seq to obtain insights into the
   mechanism of action of ANB on T. marneffei. A total of 4973 genes were
   differentially expressed in the ANB-treated group, with 2297 genes
   being upregulated and 2676 genes being downregulated. A volcano plot
   provided a visual representation of the expression levels of each
   differentially expressed gene ([67]Figure 4B). Hierarchical clustering
   analysis of the DEGs revealed consistent patterns within groups and
   distinct differences between groups, demonstrating the high reliability
   of the data ([68]Figure 4A).

Figure 4.

   [69]Figure 4
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   Transcriptomic alterations in T. marneffei following treatment with
   ANB. (A) Heatmap displaying differentially expressed genes (DEGs).
   Hierarchical clustering is shown, with changes in gene expression
   represented by the color scale. (B) Volcano plot illustrating DEGs;
   upregulated genes are highlighted in red, while downregulated genes are
   shown in green. (C) Top 20 cellular component terms from the Gene
   Ontology (GO) classification. (D) Top 20 molecular function terms from
   the GO classification. (E) Top 20 biological process terms from the GO
   classifications. (F) Top 20 pathways from the KEGG analysis.

   The DEPs were annotated in the GO database, resulting in a total of
   1986 DEGs (40.90% of the total) being mapped to three GO categories:
   cellular component (CC), molecular function (MF), and biological
   process (BP). Within the CC category, the significantly enriched terms
   included “proton-transporting V-type ATPase, V1 domain”, “
   proton-transporting V-type ATPase, V0 domain”, and “mitochondrial inner
   membrane” ([71]Figure 4C). The MF category featured significantly
   enriched terms associated with lipid synthesis and biological
   metabolism, such as “fatty acid synthase activity”,
   “enoyl-[acyl-carrier-protein] reductase (NADPH, B-specific) activity”,
   “enoyl-[acyl-carrier-protein] reductase (NADPH, A-specific) activity”,
   and “3-oxoacyl-[acyl-carrier-protein] reductase (NADPH) activity”
   ([72]Figure 4D). In the BP category, terms such as “lipid metabolic
   process”, “lactate metabolic process”, and “ATP hydrolysis coupled
   proton transport” were significantly represented ([73]Figure 4E).

   To gain a deeper understanding of the biological functions of DEGs,
   KEGG enrichment analysis was employed using a public database that
   provides information on various biochemical pathways. The analysis
   revealed that the DEGs were predominantly involved in pathways
   associated with biochemical metabolism and synthesis. Among the genes
   analyzed, 1732 DEGs (35.67%) were successfully mapped via KEGG
   enrichment analysis; the top 20 pathways are illustrated in [74]Figure
   4F. Notably, the pathways for “oxidative phosphorylation” and
   “biosynthesis of unsaturated fatty acids” were significantly enriched
   ([75]Table S1 in the Supplementary Materials). These findings suggest
   that ANB can impact lipid synthesis and the overall biological
   metabolism in T. marneffei.

2.3. Proteomics of ANB-Treated T. marneffei

   Proteomic technology has emerged as a crucial tool for investigating
   the antifungal mechanisms of various compounds [[76]18,[77]19]. A total
   of 779 proteins were identified when T. marneffei was treated with ANB.
   The criteria for screening DEPs required that their expression levels
   be greater than 1.2-fold to be classified as upregulated, or less than
   0.5-fold to be considered downregulated. Consequently, 132 DEPs were
   detected in the ANB-treated samples, with 85 proteins categorized as
   upregulated and 47 as downregulated ([78]Figure 5A).

Figure 5.

   [79]Figure 5
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   Proteome differences induced by ANB in T. marneffei. (A) Volcano plot
   displaying DEPs; proteins with increased expression are marked in red,
   while those with decreased expression are in green. (B) Distribution of
   subcellular localization terms for DEPs. (C) Level 2 statistics from
   the GO annotations. Analysis of correlations between transcriptomics
   and proteomics data.

   Subcellular location classification was used to gain further insight
   into the mechanism of action of ANB on T. marneffei, revealing that the
   DEGs were most enriched in the mitochondria domain ([81]Figure 5B). To
   further dissect the functions of these DEGs, GO functional
   classification was applied, categorizing the DEPs according to BP, CC,
   and MF. As illustrated in [82]Figure 5C, under the BP classification,
   the most represented subcategories of DEPs included cellular processes,
   metabolic processes, single-organism processes, and cellular component
   organization or biogenesis. For the CC classification, the leading
   subcategories were cell part, cell, organelle, and organelle part. In
   the MF classification, the top enriched subcategories were binding,
   catalytic activity, structural molecule activity, and transporter
   activity. During the treatment of T. marneffei with ANB, 20 DEPs were
   associated with the response to stimulation, 89 were associated with
   metabolic processes, 19 acted on the membrane surface, 14 were involved
   membrane parts, and 20 functioned within the membrane-enclosed lumen.
   These proteins may be involved in the mechanism of ANB against T.
   marneffei and play critical roles in fungal growth, development,
   pathogenicity, and metabolism.

   Correlation analysis was employed to elucidate the relationships
   between the proteome and transcriptome. All genes and proteins involved
   in the association study are shown in [83]Figure 6A. The findings,
   displayed in [84]Figure 6B, revealed significant correlations among a
   total of 80 DEGs and DEPs from both groups, including forty-five
   “upregulated-upregulated”, six “upregulated-downregulated”, twenty-four
   “downregulated-upregulated”, and five “downregulated-downregulated”.

Figure 6.

   [85]Figure 6
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   Joint transcriptomic and proteomic analysis of ANB in T. marneffei. (A)
   Venn diagram illustrating the overlap between the transcriptomic and
   proteomic data. (B) Correlation analysis between the transcriptome and
   proteome. (C) Analysis of domains within the transcriptome and
   proteome. (D) PRM analysis of 7 selected differentially expressed
   proteins.

   In a comprehensive examination combining both transcriptome and
   proteome data, domain analysis was prioritized. Genes and proteins were
   selected on the basis of their domains for a thorough combined
   analysis; the results are presented in [87]Figure 6C. This integrated
   domain analysis revealed that DEPs and DEGs primarily characterized as
   “downregulated–downregulated” were significantly enriched in the
   FAD/NAD(P)-binding domain, indicating that ANB can effectively disrupt
   the biological metabolism of T. marneffei.

   Additionally, detailed mappings of two closely associated metabolic
   pathways, the glycolytic pathway and the citrate cycle (TCA cycle),
   were performed. A total of 10 DEPs and 36 DEGs were identified as
   significant for these pathways ([88]Tables S3 and S4 in Supplementary
   Materials). Both pathways exhibited a notable decreasing trend, with
   key enzymes such as pyruvate kinase (PK) and the α-oxoglutarate
   dehydrogenase complex (OGDHC) showing decreased expression levels;
   however, no corresponding genes were identified. Furthermore, seven
   proteins were linked to these pathways (B6Q5P1, B6QD47, B6QDG2, B6QGK4,
   B6QPY8, B6QQB9, and B6QUF8) were selected to test the accuracy of the
   proteomic data ([89]Figure 6D). The results confirmed the proteomic
   findings and indicated that both the glycolytic pathway and the TCA
   cycle are inhibited in T. marneffei following ANB treatment.

3. Discussion

   Owing to the limitations of current antifungal treatments such as
   azoles and AmB, new antifungal drugs for treating T. marneffei
   infection are urgently needed. Our previous study demonstrated that ANB
   significantly inhibits the growth and proliferation of T. marneffei in
   vitro by damaging cell membranes and impairing mitochondrial function
   [[90]14]. Furthermore, in vivo studies confirmed the potent antifugal
   efficacy of ANB in a T. marneffei infection model. This study
   demonstrated that administering high concentrations of ANB
   significantly reduces fungal loads in vital organs of infected mice,
   including the liver, spleen, lungs, and kidneys, thereby aiding
   recovery and confirming the potential of ANB as a viable therapeutic
   option.

   As byproducts of cellular metabolism, the ubiquitous presence of ROS
   indicates their fundamental role in biological systems [[91]20]. While
   ROS play a supportive role in the immune system, they become cytotoxic
   when they accumulate excessively [[92]21]. Excessive ROS can induce
   oxidative damage to DNA, proteins, and lipids within the body [[93]20].
   This study revealed a significant reduction in ROS levels following ANB
   treatment after severe T. marneffei infection, suggesting its potential
   to mitigate excessive ROS production. Importantly, previous studies
   have noted that hyperinflammatory responses lead to mortality in mice
   infected with T. marneffei [[94]22]. Higher levels of proinflammatory
   cytokines were observed in patients with talaromycosis before treatment
   than in both HIV-infected individuals and healthy controls. These
   cytokine levels peaked three days after antifungal therapy and
   gradually declined. Correspondingly, the clinical symptoms of these
   patients also diminished over time [[95]23]. Our findings indicate that
   ANB effectively reduced the concentrations of the proinflammatory
   cytokines IL-1β, IL-6, and TNF-α ([96]Figure 3), likely due to the
   rapid clearance of T. marneffei from infected tissues. The literature
   supports the notion that compounds that reduce proinflammatory
   cytokines through pathogen inhibition can be effective. For example,
   Xiaoyun Liu et al. (2022) reported a significant reduction in TNF-α,
   IL-6, and IL-1β levels following SA-2 treatment in intranasal C.
   neoformans infection, which aligns with our findings [[97]24].
   Additionally, eugenol treatment was shown to decrease the levels of
   proinflammatory cytokines (IL-1β, TNF-α, and iNOS) in Aspergillus
   fumigatus-infected mouse corneas [[98]25]. Collectively, these studies
   suggest that successful antifungal therapy may alleviate inflammatory
   responses.

   Energy metabolism, carbohydrate metabolism, and lipid metabolism serve
   as critical energy sources in fungal cells. DEG annotation via the KEGG
   pathway analysis revealed that ANB treatment substantially affected the
   energy metabolism and lipid synthesis pathways of T. marneffei.
   Furthermore, KEGG pathway enrichment analysis highlighted the
   substantial effects of ANB on oxidative phosphorylation and the
   biosynthesis of unsaturated fatty acids, both of which are essential
   for cellular metabolism. The plasma membrane protein PMA1, a plasma
   membrane H^+-ATPase, plays a pivotal role in proton transport within
   the oxidative phosphorylation pathway. This protein belongs to a family
   of ATPases embedded in the cell membrane and is responsible for ion
   transport across cellular membranes powered by transient ATP
   phosphorylation [[99]26,[100]27]. The significance of PMA1 as a
   membrane protein has been demonstrated through various methods,
   including the disruption of the PMA1 gene, RNA interference studies
   [[101]28], and loss-of-function mutations in yeast [[102]29]. Extensive
   investigations have also explored the potential of targeting PMA1 as an
   antifungal strategy across multiple fungal species. Gene silencing of
   PMA1 leads to reduced cell growth and pathogenicity in Penicillum
   digitatum [[103]30], whereas PGA treatment significantly decreases PMA1
   activity, reducing stress tolerance in Fusarium solani [[104]31]. These
   findings align with the results of our current study, where ANB
   treatment significantly downregulated PMA1 gene expression, thereby
   impairing energy metabolism in T. marneffei. Fatty acid synthase (FAS),
   an enzyme crucial for the biosynthesis of endogenous fatty acids
   [[105]32], has a unique α6β6 configuration in fungi, with its
   seven-component activities distributed across two nonidentical
   polypeptides, α and β [[106]33]. The notable structural differences
   between fungal and mammalian cells render this enzyme a promising
   target for the novel antifungal drugs [[107]34]. The latest evidence
   indicates that the enzyme FAS holds significant potential as a target
   for antifungal drug development. Cerulenin forms a covalent bond with
   the catalytic site of FAS, thereby effectively inhibiting the
   biosynthesis of fatty acids and sterols in yeast [[108]35]. The
   antifungal effects of trans-chalcone and quercetin on Trichophyton
   rubrum have been demonstrated by their ability to reduce ergosterol
   levels and modulate FAS1/2 expression [[109]36]. In this study,
   treatment with ANB resulted in a decreased expression of FAS1/2 in T.
   marneffei ([110]Table S2 in Supplementary Materials). The
   transcriptomic analyses suggest that ANB targets FAS1/2, leading to the
   inhibition of essential fatty acid biosynthesis, findings that are
   consistent with those reported by Kali R. Iyer et al. [[111]37].

   The glycolysis pathway is ubiquitous across organisms and acts as a
   critical energy source for various biological activities; it is one of
   the fundamental metabolic pathways in microorganisms, providing the
   primary energy required for their biological functions [[112]38]. PK
   plays a pivotal role by directly generating ATP and acts as the
   rate-limiting enzyme within the glycolytic pathway [[113]39]. In this
   study, we demonstrated that ANB treatment significantly reduces PK
   activity in T. marneffei, leading to decreased pyruvate accumulation. A
   reduction in pyruvate disrupts the normal function of the TCA cycle, a
   central energy-producing pathway in eukaryotic cells that is critical
   for aerobic respiration in all life forms.

   The OGDHC enzyme complex, which is essential for the oxidative
   decarboxylation of α-oxoglutarate within the TCA cycle, plays a crucial
   role in energy production, energy metabolism, and amino acid metabolism
   [[114]40]; it has been shown to be involved in the energy metabolism of
   Verticillium dahlia and is critical for full virulence because of its
   ability to regulate various fungal developmental processes [[115]41].
   Our findings demonstrate that ANB treatment significantly reduced OGDHC
   activity in T. marneffei, thereby impairing the TCA cycle, as evidenced
   by decreased OGDHC activity and reduced pyruvate levels. Consistent
   with previous studies, ANB has been shown to markedly lower
   intracellular ATP levels and diminish the activities of enzymes such as
   malate dehydrogenase (MDH) and succinate dehydrogenase (SDH), leading
   to mitochondrial dysfunction and energy metabolism disruption in T.
   marneffei [[116]14]. On the basis of these observations, we hypothesize
   that ANB treatment may reduce the activity of key TCA cycle enzymes and
   inhibit pyruvate accumulation by affecting PK activity, thereby
   suppressing energy metabolism in T. marneffei.

   In this study, we constructed a hypothetical model elucidating the
   inhibitory mechanism of ANB against T. marneffei through an integrative
   analysis of proteomics and transcriptomics data from ANB-treated T.
   marneffei in conjunction with our previous investigations ([117]Figure
   7). ANB exerts its inhibitory effect by downregulating the expression
   of genes and proteins involved in fatty acid biosynthesis,
   mitochondrial function, and the respiratory chain. This downregulation
   results in mitochondrial dysfunction and triggers an oxidative stress
   response. The interruption of these critical metabolic processes
   ultimately results in cell death. This model offers valuable insights
   into the potential antifungal mechanism of ANB against T. marneffei.

Figure 7.

   [118]Figure 7
   [119]Open in a new tab

   Hypothetical diagram of the mechanism of action of ANB against T.
   marneffei.

   In summary, we conducted an in vivo evaluation of the antifungal
   activity of ANB against T. marneffei. Our findings demonstrated that
   ANB significantly reduced the fungal burden and effectively mitigated
   the levels of ROS and proinflammatory cytokines in a murine model of T.
   marneffei infection. Through comprehensive proteomic and transcriptomic
   analyses, we revealed that ANB likely impairs the growth and virulence
   of T. marneffei by disrupting pathways associated with fatty acid
   biosynthesis and energy metabolism. Although additional experiments are
   needed to fully elucidate the underlying mechanisms, the extensive
   testing conducted in this research highlights the potential of ANB as a
   promising therapeutic option for T. marneffei. To validate the accuracy
   of ANB for the inhibition of the T. marneffei pathway, we will further
   detect the expression levels of key proteins of the signaling pathway,
   such as pyruvate kinase and α-oxoglutarate dehydrogenase, and changes
   in the concentrations of related metabolites. This research highlights
   not only the direct antifungal activity of ANB but also its role in
   modulating host immune responses, which may be critical for developing
   more effective therapeutic strategies against fungal infections.

4. Materials and Methods

4.1. Experimental Strains and Animals

   The reference strain (Talaromyces marneffei ATCC 58950) was provided by
   Prof. YE Li from Guangxi Key Laboratory of AIDS Prevention and Control,
   Guangxi Medical University. For all experiments, we used a
   seven-day-old pure yeast culture grown on brain–heart infusion (BHI)
   agar. The T. marneffei colonies were thoroughly washed three times with
   phosphate-buffered saline (PBS) and then counted using a hemocytometer.
   The cells were resuspended in PBS and subjected to vigorous vortexing.

   Male Institute of Cancer Research (ICR) mice, aged six to eight weeks
   and weighing between 18 and 22 g, were obtained from Hunan SJA
   Laboratory Animal Co., Ltd (Hunan, China). These mice were accompanied
   by an animal quality certificate that verified their specific pathogen
   free (SPF) status under license number SCXK 2019-0004.

4.2. Drugs

   ANB was isolated from the fermentation broth of the mangrove-derived
   strain Streptomyces hiroshimensis GXIMD 06359 [[120]14,[121]15]. This
   compound was stored at the Institute of Marine Drugs, Guangxi
   University of Chinese Medicine. Cyclophosphamide and AmB were both
   provided by Shanghai Macklin Biochemical Co., Ltd (Shanghai, China).

4.3. Antifungal Activity In Vivo, with the Exception of the Control Group

   Male ICR mice aged 6 to 8 weeks and weighing between 18 and 22 g were
   obtained from SPF Biotechnology. The mice were kept in standard cages
   with free access to water and food and maintained at a temperature of
   21 to 23 °C with proper lighting. The mice were acclimatized for one
   week prior to the start of the experiments. Before T. marneffei
   inoculation, the mice were subjected to immunosuppression by a single
   intraperitoneal (i.p.) injection of cyclophosphamide at a dosage of 200
   mg/kg per day for 4 days. To induce systemic infection, this yeast
   suspension was injected intraperitoneally into each mouse. The mice
   were randomly divided into five groups (n = 20). The experimental
   groups received an intraperitoneal injection of ANB at doses of either
   2 mg/kg, 1 mg/kg, or 0.5 mg/kg. The control group was administered the
   same volume of normal saline or AmB at a concentration of 0.5 mg/kg
   (0.2 mL per mouse). The treatment was initiated 2 h post-inoculation
   and continued for a period of 7 days.

   For survival analysis, 10 mice were assigned to each group and
   monitored daily until day 14 after inoculation. The analysis of the
   tissue fungal load involved sacrificing five mice from each group at 5
   days post-inoculation. The livers, lungs, spleens, and kidneys were
   removed aseptically, weighed, and homogenized in 1 mL of sterile saline
   solution. The homogenates were then serially diluted in tenfold
   increments and plated on Sabouraud dextrose agar (SDA), followed by
   incubation at 37 °C for 72 h. After incubation, the number of
   colony-forming units (CFUs) per gram of tissue was determined. The
   levels of reactive oxygen species (ROS) were measured using a ROS
   fluorometric assay kit (Elabscience, Wuhan, China). Additionally, the
   levels of interleukin (IL)-1β, interleukin (IL)-6, and tumor necrosis
   factor (TNF)-α were quantified using a mouse ELISA kit (Elabscience,
   Wuhan, China) according to the manufacturer’s protocol.

4.4. Transcriptomic Analysis

   The initial inoculum of T. marneffei cells was adjusted to a 1–5 × 10^4
   CFU mL^−1 standard in 20 mL of RPMI-1640 medium and cultured for 72 h
   at 37 °C with shaking at 180 rpm. The treatment group received ANB at a
   concentration of 8 μg/mL. The concentration of 8 μg/mL is equal to the
   ANB 1/2MIC for T. marneffei. The cells were harvested, washed three
   times with sterile PBS, and then rapidly frozen in liquid nitrogen
   before being stored at −80 °C until further use. The cells from the
   untreated T. marneffei group served as the control. Three separate
   cultures were prepared for RNA-Seq analysis. The transcriptomic
   analysis was conducted by MHelix BioTech Co., Ltd. (Shanghai, China),
   involving steps such as total RNA extraction, mRNA enrichment by oligo
   (dT), RNA fragmentation, cDNA synthesis initiated by random hexamers,
   size selection, polymerase chain reaction amplification, and sequencing
   using an Illumina HiSeq 2500 platform.

   The paired-end clean reads were aligned to the T. marneffei strain
   reference genome
   ([122]https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_009556855.1/,
   accessed on 8 January 2024) using HISAT2.2.4. To normalize gene
   expression across samples, the number of fragments per kilobase of
   transcript per million mapped reads value was calculated.
   Differentially expressed genes (DEGs) between the allicin-exposed and
   control groups were identified using DESeq2 on the basis of a false
   discovery rate (FDR) of less than 0.05 and an absolute fold change (FC)
   of greater than 1.

   To explore the biological functions of the DEGs, Gene Ontology (GO)
   enrichment analysis was conducted using the GO database
   ([123]http://geneontology.org/, accessed on 8 January 2024), and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis
   was carried out using the KEGG database
   ([124]https://www.genome.jp/kegg/, accessed on 8 January 2024).

4.5. Proteomics and PRM Analysis

   The initial inoculum of T. marneffei cells was standardized to a
   concentration of 1–5 × 10^4 CFU /mL in 20 mL of RPMI-1640 medium and
   incubated for 72 h at 37 °C with continuous shaking at 180 rpm. The
   treated group was then exposed to ANB at a final concentration of 8
   μg/mL. The cells were then harvested and washed three times with
   sterile PBS. Before being rapidly frozen in liquid nitrogen, the
   samples were stored at −80 °C until analysis. The cells from the
   untreated group served as the control. Three independent cultures were
   prepared for RNA-Seq analysis, and triplicate samples from these
   cultures were submitted to MHelix BioTech Co., Ltd. (Shanghai, China),
   for TMT-based quantitative proteomic analysis.

   The differentially expressed proteins (DEPs) identified in T. marneffei
   mycelia were defined as those showing a minimum 1.20-fold change and a
   p_value less than 0.05 when the ANB-treated samples were compared with
   the control samples. For GO annotation and KEGG enrichment analysis of
   DEPs, the web-based analysis tool Omicsbean
   ([125]http://www.omicsbean.cn, accessed on 25 December 2023) was used.

   The samples employed for omics PRM verification were identical to those
   used in the proteomic analysis. MHelix BioTech Co., Ltd. (Shanghai,
   China), was entrusted with performed PRM verification on 7 target
   proteins.

4.6. Statistical Analysis

   The statistical analyses were carried out using GraphPad Prism 8.3.0
   (GraphPad Software, San Diego, CA, USA), and the results are expressed
   as means and standard deviations (SDs). Each experiment was conducted
   with independent biological replicates. The data were analyzed using
   either one-way ANOVA or an unpaired t_test depending on the
   experimental design. Statistical significance was considered
   probability (p) values of less than 0.05, 0.01, or 0.001.

Supplementary Materials

   The following supporting information can be downloaded at:
   [126]https://www.mdpi.com/article/10.3390/md23020076/s1, Table S1:
   Differential genes related to oxidative phosphorylation pathway. Table
   S2: Differential genes related to fatty acid biosynthesis pathway.
   Table S3: Differentially expressed genes and proteins associated with
   glycolysis. Table S4: Tricarboxylic acid cycle related differentially
   expressed genes and proteins.
   [127]marinedrugs-23-00076-s001.zip^ (535.5KB, zip)

Author Contributions

   Conceptualization, Q.L., Z.W. and C.G.; methodology, Q.L. and Z.W.;
   validation, C.G. and X.Q.; formal analysis, Q.L., Z.W. and C.J.;
   investigation, Q.L. and J.Y.; resources, Q.L.; data curation, Q.L.,
   Z.W. and C.J.; writing—original draft preparation, Q.L., Z.W. and C.J;
   writing—review and editing, C.G., X.Q. and X.Y.; visualization, Q.L.,
   Z.W. and J.Y.; supervision, C.G., X.Q. and X.Y.; project
   administration, C.G., Y.L. and X.Y.; funding acquisition, C.G. and Y.L.
   All authors have read and agreed to the published version of the
   manuscript.

Institutional Review Board Statement

   The animal study protocol was approved by the Animal Ethics Committee
   of the Guangxi University of Traditional Chinese Medicine, as well as
   followed the Guidelines of Animal Care set by the Chinese Ministry of
   Health (GB/T 35892—2018), approval date 30 July 2023.

Data Availability Statement

   The data presented in this study are available upon request from the
   corresponding author.

Conflicts of Interest

   The authors declare no conflicts of interest.

Funding Statement

   This research was funded by National Natural Science Foundation of
   China (82060640), Guangxi Key Research and Development Programme
   (AB24010109), National Natural Science Foundation of China (U20A20101),
   Guangxi Key Laboratory of Marine Drugs (2302603), High-level Talent
   Inheritance, Innovation Team of Guangxi Traditional Chinese Medicine
   (2022A007), Guangxi University of Chinese Medicine “Guipai Traditional
   Chinese Medicine Inheritance and Innovation Team” Project (2022B005),
   and Guangxi University of Chinese Medicine-Guipai Xinglin Top Talent
   Funding Project (2022C008).

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References