The molecular mechanism by which novel antibacterial peptides inhibit Cryptococcus neoformans

The molecular mechanism by which novel antibacterial peptides inhibit Cryptococcus neoformans | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The molecular mechanism by which novel antibacterial peptides inhibit Cryptococcus neoformans Na Zhao, Mengtao Wang, Die Zhang, Yuxuan Luo, Can Liu, Dongxu Song, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8150680/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Cryptococcosis, caused by Cryptococcus neoformans , is a major global public health concern. The infection primarily begins in the lungs. Cryptococcosis is characterized by significant consequences and low cure rates in immunocompromised individuals. Pulmonary cryptococcosis has become more common in recent years. Amphotericin B (AmB) and fluconazole (FLC) are commonly used as first-line medications in clinical treatment. However, the increasing incidence of azole resistance has led to an increase in clinical treatment failure rates. Antimicrobial peptides (AMPs) are considered attractive substitutes for conventional antibiotics for resolving this urgent problem because of their distinct processes and low risk of resistance development. Our earlier research revealed antimicrobial peptide-17 (AMP-17), which has a molecular weight of 17 kDa, as a possible antifungal agent from the transcriptome database of Musca domestica generated by Candida albicans . Nevertheless, the use of quantitative structure-activity relationship (QSAR) techniques to modify AMP-17 has not been used to investigate the antifungal mechanisms of AMP-17 derivatives. Results This study examined the potential potency of the peptide AMP-17-6 against clinically drug-resistant C. neoformans 314. The results revealed that AMP-17-6 possesses high bacteriostatic activity and bactericidal efficiency against C. neoformans 314, with a minimum inhibitory concentration (MIC) of 4 μg/mL. Additionally, AMP-17-6 is sensitive to trypsin and pepsin but is stable in a range of serum conditions and temperatures. Mechanistic studies revealed that AMP-17-6 induces membrane damage by targeting fungal-specific membrane components and dissipating the proton motive force (PMF), leading to metabolic disturbances and the accumulation of toxic metabolic byproducts. In a mouse model of pneumonia induced by C. neoformans 314 infection, AMP reduced the fungal burden and inflammatory reactivity in lung tissue. Conclusion These data demonstrate that AMP exhibits significant antibacterial efficacy against C. neoformans 314 by decreasing fungal viability and displaying antivirulence effects, suggesting its potential as a novel antibacterial drug for the treatment of resistant C. neoformans infections. Cryptococcus neoformans 314 Antimicrobial peptide AMP-17 derivatives Membrane mechanism In vivo Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction As a fungal pathogen that poses a threat to human life and health, Cryptococcus neoformans frequently causes pneumonia and potentially fatal cryptococcal meningitis in immunocompromised individuals, including those with HIV/AIDS, organ transplant recipients, and cancer patients undergoing chemotherapy [ 1 ]. Typically, cryptococcosis is acquired when the host inhales fungal spores or yeast cells from the air through the respiratory tract, resulting in long-term latent colonization in the lungs. When the host's immune function is impaired, dormant C. neoformans in the lungs rapidly multiply, causing primary pulmonary infection. Severe cryptococcal meningitis may ensue if the infection spreads to the central nervous system [ 2 ]. Therefore, the development of safe and effective novel antimicrobial agents is critically important in the treatment of infections caused by C. neoformans . As prospective alternatives to traditional antibiotics, antimicrobial peptides (AMPs) have gained extensive attention from researchers because of their in vivo safety and efficacy. Drug-resistant bacteria may be treated with antimicrobial peptides (AMPs), which are tiny proteins with broad-spectrum antibacterial action [ 3 ]. According to previous studies, amphibian peptides have been successfully employed to treat local infections caused by a variety of drug-resistant strains, including C. neoformans infections [ 4 ]. Natural AMPs have broad-spectrum antibacterial activity and a quick death time, effectively eliminating viruses, fungi, and bacteria [ 5 ][ 6 ][ 7 ]. Additionally, the clinical use of peptides as therapeutic options for treating diseases is expanding due to new types and technologies of AMPs, such as cell-penetrating peptides, artificially generated multifunctional peptides, and peptide-drug conjugates [ 8 ]. Because of these benefits, they have potential as substitutes for traditional antibiotics. However, their hemolytic characteristics and high toxicity restrict their usefulness [ 9 ]-[ 10 ]. Owing to the diversity of antimicrobial processes, the mechanism of antimicrobial peptides is still unclear. Our earlier research revealed antimicrobial peptide-17 (AMP-17), which has a molecular weight of 17 kDa, as a possible antifungal agent from the transcriptome database of Musca domestica generated by Candida albicans [ 11 ]-[ 12 ]. The whole ORF of AMP-17 is 495 bp long and contains 164 amino acids [ 12 ]. The protein polypeptide chain generated by the AMP-17 gene is hydrophilic, and the protein has a transmembrane region, which is similar to a typical membrane-acting antimicrobial peptide. The PBIL LYON-GERLAND database study revealed three primary secondary structures of AMP-17, namely, α-helices, random coils and extended strands [ 12 ]. Furthermore, AMP-17 exhibits broad-spectrum antibacterial action against both gram-positive and gram-negative bacteria, heat resistance, freeze-thaw resistance, and nontoxicity to human red blood cells, which results in the flow of cell contents [ 13 ]. However, by altering AMP-17 via quantitative structure-activity relationship (QSAR) techniques, the antifungal mechanisms of AMP-17 derivatives have not been investigated. AMP-17-6 demonstrated strong antibacterial activity against drug-resistant C. neoformans in an antibacterial spectrum screening. We also evaluated a few clinical isolates of C. neoformans to assess their antibacterial activity, and we discovered that AMP-17-6 had the best antimicrobial activity against C. neoformans 314. The antibacterial mechanism of AMP-17-6 against C. neoformans 314 was examined physicochemically and morphologically, and animal studies were used to assess its in vivo effectiveness. The results of this investigation offer a theoretical foundation for the development of new antimicrobial medications. Materials and methods Synthesis and validation of antimicrobial peptides GL Biochem (Shanghai, China) used solid-phase synthesis to create AMP-17-6 (TKFAVCFGLLGAKCKILAG-NH2). Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to purify AMP-17-6 to more than 95% purity (Supplementary Fig. 1A). High-performance liquid chromatography tandem mass spectrometry (HPLC/MS) was used to measure the molecular weight (Supplementary Fig. 1B). A final concentration of 1 mg/mL AMP-17-6 was produced in water. The online program HeliQuest ( https://heliquest.ipmc.cnrs.fr/ ) was used to assess the hydrophobicity of AMP-17-6. The 3D spatial structure of AMP-17-6 was predicted with I-TASSER ( https://zhanggroup.org/I-TASSER/ ) [ 14 ][ 15 ][ 16 ]. Growth media and fungal strains A tertiary healthcare center in Guiyang, a city in Guizhou Province, China, provided the clinical strains (311, 313, 181, 182, 183, 314, 178, 185, and 194) used in this investigation. Cryptococcus neoformans H99 and C. albicans were obtained from the Key and Characteristic Laboratory of Modern Pathogen Biology at Guizhou Medical University in Guiyang, China. Every strain was regularly cultivated in YPD media at 30°C and stored at -80°C in LB media. Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) The Clinical and Laboratory Standards Institute (CLSI) criteria were followed to determine the peptide's minimum inhibitory concentration (MIC) via the broth microdilution method. The microdilution method in RPMI 1640 medium was used to determine the MICs. The fungal suspension in the media had a final concentration of 1.0×103 CFU/mL. In a 96-well plate, broth was incubated at 30°C for 16–18 h, and the final AMP content ranged from 1-256 µg/mL. RPMI 1640 was utilized as a blank control, AmB was used as a positive control, and bacteria cultured untreated were used as a negative control. The OD 630 was measured to determine the MIC. One hundred microliter samples from holes that had never undergone fungal growth were placed on YPD agar boards. The MBC was defined as the concentration of AMPs without any fungal growth. Growth curve assay The determination of the growth curve of C. neoformans in reaction to AMP-17-6 was accomplished via a method similar to that described previously [ 17 ]. The fungal suspension of C. neoformans 314 was resuspended to 1.0×10 6 CFU/mL with various concentrations of AMP-17-6 added (final peptide concentrations of 1, 2, or 4 ×MIC) or in PBS in 96-well plates and grown at 30°C. A microplate reader was used to measure the absorbance at 630 nm every two h for a full d. Time-kill assays The time-kill kinetics of AMP-17-6 on C. neoformans 314 were determined following a previously reported technique [ 18 ]. The fungal suspensions (1.0×10 6 CFU/mL) were combined with different doses of AMP-17-6 and subsequently cultivated at 30°C. The final concentrations of AMP-17-6 were 1, 2, or 4 ×MIC. Samples in 10 µL aliquots were diluted in 90 µL of PBS at 0, 1, 2, 4, 6, 12, and 24 h. The dilutions were then disseminated onto YPD agar plates in 5 µL aliquots. The colony counts were calculated, and a time-kill curve was drawn following a 24-h incubation period at 30°C. Assay for cytotoxicity The number of viable cells in a sample can be ascertained via the readily accessible, premixed Cell Counting Kit-8 (CCK-8) test. The cytotoxicity of AMP-17-6 on A549 and HeLa cells was assessed via previously published techniques with a few adjustments [ 19 ]. In brief, HeLa or A549 cells were grown at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. AMP-17-6 (1-256 µg/mL) was administered for 24 h after all the cells were injected into 96-well plates at a density of 1×10 6 cells/well. Cells cultivated without AMP-17-6 in DMEM were utilized as a negative control, and DMEM alone was used as a blank control. After incubation, 10 µL of CCK-8 solution was added to each well and incubated for 2 h. A microplate reader was used to measure the absorbance at 450 nm. $$\:\text{T}\text{h}\text{e}\:\text{r}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{g}\text{r}\text{o}\text{w}\text{t}\text{h}\:\text{r}\text{a}\text{t}\text{e}\:\left(\text{%}\right)=\frac{\left({\text{A}}_{450\:\text{n}\text{m}}\:\text{o}\text{f}\:\text{t}\text{e}\text{s}\text{t}\:\text{w}\text{e}\text{l}\text{l}-\:{\text{A}}_{450\:\text{n}\text{m}}\:\text{o}\text{f}\:\text{b}\text{l}\text{a}\text{n}\text{k}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}{\left({\text{A}}_{450\:\text{n}\text{m}}\:\text{o}\text{f}\:\text{n}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\:{\text{A}}_{450\:\text{n}\text{m}}\:\text{o}\text{f}\:\text{b}\text{l}\text{a}\text{n}\text{k}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}\times\:100\text{%}$$ Hemolytic activity assay The hemolytic activity of AMP-17-6 on human red blood cells was tested according to a previous study [ 20 ]. Centrifugation at 1000 × g for 10 min was used to collect human blood cells, which were subsequently washed three times with 1× PBS. Red blood cells were collected and suspended in 1× PBS to 4%. Next, 96-well plates containing varying doses of AMP-17-6 (1 ~ 256 µg/mL) were incubated for one h at 37°C with 100 µL of 4% red blood cell mixture. The absorbance at 540 nm was measured with a microplate reader. While PBS served as the negative control, a blood cell suspension treated with 1% Triton X-100 was employed as a positive control for 100% hemolysis. $$\:\text{T}\text{h}\text{e}\:\text{h}\text{e}\text{m}\text{o}\text{l}\text{y}\text{s}\text{i}\text{s}\:\text{r}\text{a}\text{t}\text{e}\:\left(\text{%}\right)=\frac{\left({\text{A}}_{540\:\text{n}\text{m}}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{t}\text{e}\text{s}\text{t}\:\text{w}\text{e}\text{l}\text{l}-{\:\text{A}}_{540\:\text{n}\text{m}}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{n}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}{\left({\text{A}}_{540\:\text{n}\text{m}}\:\text{o}\text{f}\:\text{p}\text{o}\text{s}\text{i}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:-{\:\text{A}}_{540\:\text{n}\text{m}}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{n}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}\times\:100%$$ Antimicrobial efficacy in the presence of salts, serum, temperature and trypsin Mouse serum, various temperatures, trypsin, and MICs were examined once more to investigate the action of AMP-17-6 at high salt concentrations. In 96-well plates, C. neoformans 314 was grown at 37°C for 16 h with various doses of AMP-17-6 in YPD supplemented with 150 mM NaCl, 2 mM CaCl 2 , 10% mouse serum, and 10% FBS. The resulting solutions were subjected to MIC testing to evaluate any modifications in the antibacterial activity of AMP-17-6. AMP-17-6 were exposed to different temperatures (40°C, 60°C, 80°C, and 100°C) for 30 min to evaluate its thermal stability. The MIC was then calculated after the temperature was lowered to room temperature. After AMP-17-6 was combined with a 1 mg/ml trypsin solution, the mixture was incubated for one h at 37°C. Then, trypsin inactivation was carried out at 60°C for 30 min. After the samples were treated with trypsin, the antibacterial activity of AMP-17-6 and its analogs was evaluated. For comparative analysis, untreated AMP-17-6 was used as the control. Scanning electron microscopy Fungal suspensions (1.0×10 6 CFU/mL) were treated with AMP-17-6 at a final dose of 1 × MIC or in PBS for two h to examine the morphological alterations caused by AMP-17-6 in C. neoformans 314. After two washes with PBS, the bacteria were fixed with 2.5% glutaraldehyde at 4°C overnight. After that, the bacteria were dehydrated in a range of ethanol solutions. A fungal suspension without AMP-17-6 was utilized as a control for comparison purposes. A Hitachi Regulus SU8100 (Tokyo, Japan) was used to acquire the images. Fungal viability The percentage of viable and dead bacteria was visualized via confocal laser scanning microscopy (CLSM; Olympus SpinSR10, Japan) and the fluorescence probe staining method [ 21 ]. The bacteria were collected after the fungal suspension (1.0 × 10 6 CFU/ml) and AMP-17-6 at various concentrations were incubated for one h at 37°C and centrifuged at 2,000 × g for five min. After two rounds of washing, the bacteria were again suspended in PBS. After adding final amounts of 10 µM SYTO 9 and 10 µM PI to each group, the mixture was incubated for 15 min at 37°C in the dark. Following the incubation period, PBS was used to remove the unbound fluorescent dye. CLSM was used to evaluate the samples' fluorescence. Membrane permeability test Fungal suspensions were diluted with PBS to a concentration of 1.0×10 6 CFU/mL in compliance with previously reported procedures [ 22 ]. The fungal suspensions were grown at 30°C for two h in Eppendorf tubes containing varying doses of AMP-17-6 or PBS (final peptide concentrations of 1, 2, or 4 ×MIC). Next, 10 µM propidium iodide (PI; Sigma, US) was added to each group, and the mixture was incubated at 37°C for 15 min in the dark. The samples were evaluated with a microplate reader (BioTek SYNERGY-H, Agilent, USA). Reactive oxygen species measurements The intracellular production of ROS was measured via the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA (final concentration of 10 µM) was combined with the fungal suspension (1.0 × 10 6 CFU/mL) and incubated at 37°C for 30 min in the dark. After incubation, the suspensions were treated with AMP-17-6 (1, 2, or 4 times the MIC) at the indicated doses for 1 h at 30°C. The fluorescence intensity was assessed via the use of a multifunctional fluorescent enzyme marker with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Effects of AMP-17-6 on fungal cell membranes Antimicrobial peptides work primarily by influencing the permeability, fluidity, and charge of cell membranes. Thus, how AMP-17-6 affects C. neoformans 314 cell membranes was identified via fluorescence intensity tests. The overnight fungal culture mixture was rinsed and resuspended in PBS to a concentration of 1 × 10 6 CFU/mL. The fluorescent dye PI was used to measure the permeability of the fungal inner membrane [ 23 ]. The final concentration of 5 µM PI was incubated with the fungal suspension for 10 min, followed by treatment with AMP-17-6 (0–32 µg/mL) at 30°C for 1 h. The bacterial fluorescence value was then determined via a microplate reader (BioTek SYNERGY-H, Agilent, USA) with excitation and emission wavelengths of 535 nm and 615 nm, respectively. The fluorescent dye Luardan (Sigma, USA) at a concentration of 10 µM was used to measure the fluidity of the fungal membrane. Luardan was found at 350 nm excitation and 435 and 490 nm absorption wavelengths. The computation of the generalized polarization (GP) was conducted via the following formula: GP = (I435 - I490)/(I435 + I490). DiSC3(5) (Sigma, USA) was then used to evaluate the effect of AMP on the potential energy of the cell [ 23 ]. The bacteria were cultured at a final concentration of 0.5 µM DiSC3(5), and the fluorescence values were measured at an excitation wavelength of 622 nm and an emission wavelength of 670 nm. Additionally, the proton motive force of fungi treated with AMP-17-6 was measured with BCECF-AM (UElandy, Suzhou) [ 23 ]. The fungal suspensions were incubated with 1 µM BCECF-AM after being treated with different doses of AMP-17-6. The fluorescence value was calculated using excitation/emission wavelengths of 488 nm and 535 nm. The impact of AMP-17-6 on fungal respiration was examined via the use of the oxygen-sensitive dye resazurin (Solarbio, Beijing) [ 24 ]. Briefly, the fungal cultures were treated with various concentrations of 0–32 µg/mL AMP-17-6 and a constant concentration of 0.1 µg/mL resazurin. A microplate reader with excitation and emission wavelengths of 550 nm and 590 nm, respectively, was used to observe the fluorescence change constantly for one h. Model of mouse infection The female BALB/c mice used in this investigation were purchased from SPF Biotechnology Co., Ltd. (Beijing, China), were 6–8 weeks old and weighed 18–20 g. A typical 12-h light/12-h dark cycle was employed to keep the animals. The Institutional Animal Care and Use Committee (Approval No: 2400247) approved all the animal experiments, which were carried out in compliance with Guizhou Medical University's Ethical Principles in Animal Research. To test the in vivo effectiveness of AMP-17-6, we employed a mouse pneumonia model. A total of 60 mice were divided into four groups: the treatment group (10 mg/kg AMP-17-6, intraperitoneal injection), the positive control group (10 mg/kg AmB, intraperitoneal injection), the blank control group (PBS, intraperitoneal injection), and the model group (fungal injection, PBS, intraperitoneal injection). To create a Cryptococcus-mouse lung infection model, a 100 µL suspension of 1.0 × 10 8 CFU C. neoformans 314 was gradually injected into the mouse lungs via tracheal intubation after each mouse was rendered unconscious by isoflurane inhalation via a small animal anesthetic unit [ 25 ]. To monitor the in vivo therapeutic effects in each mouse treatment group, once-daily medication treatment was started via intraperitoneal injection as soon as possible after 48 h of infection, and three doses were given consecutively. Every day, the survival status of the mice in each group was tracked and documented. Moreover, throughout the 23-d experimental observation period, body weight measurements were performed daily for all groups to analyze changes in mouse weight following medication treatment [ 25 ]. Seven d after the medicine was administered to examine the therapeutic effects of AMP-17-6, ten mice from each group exposed to isoflurane anesthesia in a gas-tight chamber and stayed in the chamber and were then sacrificed by cervical dislocation. And infected lung tissues were collected aseptically. While the tissues were frozen in 4% paraformaldehyde and stained with hematoxylin and eosin (H&E) to examine their morphology, the number of colonies in the lung was ascertained by cultivation on YPD agar. Statistical analysis Every experiment was conducted at least three times. Student's t -test was used to evaluate group differences. The means ± standard deviations (means ± SDs) are used to express the data. P < 0.05 was considered statistically significant. Results Characterization of antimicrobial peptides. The synthesized peptide and its sequences, and the biological characteristics of AMP-17-6 are listed in Table 1 . The cationic peptide AMP-17-6 is more than 95% pure. The precise synthesis of the peptide was demonstrated by the agreement between the calculated and observed molecular weights. Additionally, AMP-17-6 is a cationic antimicrobial peptide whose structure is primarily composed of coil and helical conformations (Fig. 1 ). Table 1 Physicochemical parameters of AMP-17-6. Peptide Sequence(N→C) Formula Charge a pI a Calcd MW a Obsd MW b Purity(%) AMP-17-6 TKFAVCFGLLGAKCKILAG C 100 H 163 N 27 O 24 S 2 + 3 9.39 1940.68 1939.46 95.44 a Charge, isoelectric point (pI) and molecular weight (MW) were calculated online at https://web.expasy.org/protparam/ . b The observed molecular weight (MW) was determined via liquid chromatography-mass spectrometry (LC-MS). (A) The chemical structure of AMP-17-6. (B) AMP-17-6 helical wheel projection diagrams generated via HeliQuest analysis ( https://heliquest.ipmc.cnrs.fr/ ). Hydrophilic amino acids are indicated in blue and are positively charged. The amino acids displayed in yellow and gray are hydrophobic. (C) I-TASSER ( https://zhanggroup.org/I-TASSER/ ) was used to estimate the peptide's three-dimensional structure. The secondary structure is represented by various hues. The helical structure is represented in magenta, whereas the coil structure is represented in white. Antimicrobial action of AMP-17 derivatives. Through alteration of the functional domain of AMP-17, we effectively created six variants of AMP-17. C. neoformans C and H99. Candida albicans was employed as a test strain to investigate the antibacterial activity of the six derivatives of AMP-17. We discovered that AMP-17-6 had the strongest antibacterial action against C among all the peptides that were examined. H99 neoformans (Table 2 ). Table 2 Antimicrobial activity screening of six AMP-17 derivatives. MIC(µg/mL) C. albicans C. neoformans H99 AMP-17-1 > 64 64 AMP-17-2 64 64 AMP-17-3 > 64 32 AMP-17-4 > 64 > 64 AMP-17-5 32 32 AMP-17-6 > 64 16 We chose other clinically drug-resistant isolates of C. neoformans and C. neoformans H99 for testing to examine further the antibacterial activity of AMP-17-6 (Table 3 ). The AMP-17-6 MICs ranged from 4–32 µg/mL. Strain 314 was selected to gain a better understanding of how AMP-17-6 affects clinical drug-resistant isolates. Table 3 Antimicrobial activity of AMP-17-6 against clinical strains of C. neoformans. Bacteria MIC (µg/mL) C. neoformans H99 16 C. neoformans 311 8 C. neoformans 312 8 C. neoformans 314 4 C. neoformans 181 32 C. neoformans 182 8 C. neoformans 183 8 C. neoformans 178 32 C. neoformans 185 16 C. neoformans 194 8 AMP-17-6 growth assay and quick bactericidal effectiveness against 314. The growth curves of C. neoformans 314 in the presence of AMP-17-6 are displayed in Fig. 2 A. AMP-17-6 suppressed C. neoformans 314 growth in a dose-dependent manner at doses ranging from 1 × MIC to 2 × MIC. The kinetic killing effects of AMP-17-6 on C. neoformans 314 were further assessed (Fig. 2 B). At concentrations of 1 × MIC and 2 × MIC, the C. neoformans 314 treatment tended to eradicate the germs within 2 h. These results indicated that AMP-17-6 displayed fast bactericidal activity against fungal infections. Effects of salt, heat and trypsin on AMP-17-6 activity. The effects of heat, salt, and trypsin on AMP-17-6 activity were measured via MIC tests. Table 4 displays the outcomes. In the absence of physiological CaCl 2 , NaCl, or KCl, the MICs of AMP-17-6 did not differ from those of the control. The impact of heat on the antibacterial activity of AMP-17-6 was examined via a heating test. Nevertheless, the antibacterial activity of AMP-17-6 did not decrease following this treatment, and its antimicrobial activity was heat stable. Unfortunately, trypsin and pepsin removed the antibacterial action of AMP-17-6. Table 4 Effects of salt, temperature and trypsin on AMP-17-6 activity in C. neoformans 314. Treatments AMP-17-6 (MIC, µg/mL) Control 4 150 mM Na + 4 4.5 mM K + 4 2 mM Ca 2+ 4 1 mM Mg 2+ 4 1 mg/mL Trypsin > 64 1 mg/mL Pepsin > 32 4°C 30 min 4 37°C 30 min 4 70°C 30 min 4 100°C 30 min 4 AMP-17-6 hemolysis and cytotoxicity. AMP-17-6 was tested for its ability to induce hemolysis and cytotoxic action against human red blood cells, specifically HeLa and A549 (human non-small cell lung cancer) cells, at doses ranging from 2 to 256 µg/mL. The viability of HeLa cells treated with different concentrations of AMP-17-6 was consistently greater than 100%, according to the cytotoxicity data (Fig. 3 A). However, following treatment with 128 µg/mL AMP-17-6, the viability of A549 cells decreased to 75.60%. The majority of the cells remained intact in the hemolysis results (Fig. 3 B), indicating that AMP-17-6 had minimal hemolytic action against human erythrocytes. These findings imply that AMP-17-6 has positive safety characteristics. Antibacterial mechanism. Scanning electron microscopy (SEM) images revealed that normal C. neoformans 314 was consistently rod-shaped with a smooth surface. Furthermore, AMP-17-6 treatment at the appropriate MICs for two h caused significant damage and rupture (Fig. 4 ). The effects of AMP-17-6 on the integrity of the fungal cell membrane were assessed via CLSM and PI and SYTO9 nucleic acid stains to investigate the antibacterial activity of AMP-17-6. STOY9 is a green fluorescent nucleic acid stain that can stain both living and dead bacteria, whereas PI is a red nucleic acid-binding dye that only enters cells with broken membranes. Figure 5 shows that after one h, the untreated C. neoformans 314 cells were stained with STOY9 but not PI, suggesting that most of the cells were viable. On the other hand, after treatment with AMP-17-6 at doses of 1, 2, and 4 times the MIC, the quantity and intensity of red fluorescent dots increased in a concentration-dependent manner. These studies indicated that AMP-17-6 was able to disrupt fungal cell membranes and that rupture increased with increasing peptide content. Additionally, PI has been employed as a probe to investigate cell membrane integrity (Sautrey et al., 2016). PI can cross the membrane and intercalate with DNA in injured cells. On the other hand, PI cannot enter undamaged cells (Thulshan Jayathilaka et al., 2021). As shown in Fig. 6 A, the untreated C. neoformans 314 group presented a low percentage of PI fluorescence signals. In contrast, cells treated with AMP-17-6 at 1, 2, or 4 times the MIC for 1 h presented PI fluorescence signals. These findings suggest that AMP-17-6 increases the permeability of fungal membranes while compromising their integrity. Fungal mortality is significantly influenced by reactive oxygen species (ROS). Following AMP-17-6 treatment, ROS accumulation in bacteria dramatically increased, as shown in Fig. 6 B, exacerbating membrane damage, further impairing fungal homeostasis. These results demonstrate that ROS are essential for the sterilization of antimicrobial peptides. Functions of membranes and energy metabolism. When examined, AMP-17-6 can not only impair the integrity of the inner membrane but also affect membrane malfunction and intracellular metabolic alterations. We also observed changes in C. neoformans 314 respiration levels after treatment with AMP-17-6 and resazurin. The findings showed that AMP-17-6 considerably increased C's respiratory level. neoformans 314 (Fig. 7 A). Laurdan is a fluorescent probe used to research cell membrane characteristics and biomembrane lipid dynamics, and can be applied to identify alterations in membrane fluidity. The membrane fluidity of C. neoformans 314 was improved by AMP-17-6 (Fig. 7 B). The proton motive force (PMF) is known to be impacted by membrane rupture. The transmembrane proton gradient (ΔpH) and Δψ are the two main components of the PMF and are crucial for material movement, energy production, and antibiotic resistance. Δψ was measured with DisC3(5). The fluorescence intensity of DisC3(5) dramatically increased in C. neoformans 314 after treatment with AMP-17-6 in a dose-dependent manner (Fig. 7 C), indicating that AMP-17-6 disrupts the Δψ of C. neoformans 314 and causes membrane potential depolarization. Additionally, we chose BCECF-AM, another fluorescent probe, to assess how AMP-17-6 affects the ΔpH. The fluorescence intensity of C. neoformans 314 was marginally increased by AMP-17-6, suggesting that AMP-17-6 increased the ΔpH (Fig. 7 D). The rapid bacterial disintegration caused by AMP-17-6 may be connected to this increase in fluorescence intensity. Antimicrobial activity in vivo. We evaluated the effectiveness of AMP-17-6 in a mouse model since it can inhibit C. neoformans 314 (Fig. 8 A). We monitored the body weight fluctuations of the mice in each group. All the treatment groups presented a decrease in body weight, although the model group presented the fastest rate of decrease (Fig. 8 B). We assessed the fungal burden in the mouse lung on the fifth d after infection to further assess the effects of AMP-17-6. The fungal content in the positive control group treated with AmB decreased to 4.20 ± 0.25 log 10 CFU/mL (P < 0.0001), whereas the fungal count in the AMP-17-6-treated group decreased to 7.23 ± 0.36 log10 CFU/g (P < 0.05), and that in the model group was 8.73 ± 0.38 log10 CFU/g. At 15 d, the survival rate of the model group was 0%, whereas that of the control group was 100%, and that of the AMP-17-6 group was 40%. Therefore, treatment with AMP-17-6 at a dose of 10 mg/kg increased the survival rate of the mice (Fig. 8 D). The histological images of the lung tissue sections are presented in Fig. 8 E. The mice in the model group had more Cryptococcus in their alveoli and pulmonary interstitium as the infection period progressed, and the lung tissue showed diffuse necrosis. The pulmonary interstitium, interlobular septa, and alveolar walls were greatly enlarged, accompanied by dispersed or focal infiltration of numerous inflammatory cells, such as neutrophils and lymphocytes. In certain places, nodular lesions develop, causing the destruction and consolidation of the lung tissue structure. Occasionally, this was followed by the formation of an abscess. Compared with those in the control group, the lung tissue in the model group contained fewer fungi and fewer severe lesions. Additionally, HE-stained lung tissue sections revealed that mice treated with AMP-17-6 presented relatively few inflammatory lung tissue symptoms. Discussion Hospital-acquired illnesses are now frequently caused by drug-resistant bacteria. These bacteria greatly impair the efficacy of antibiotics, whereas the discovery of novel therapeutic medications has become an important concern [ 26 ]-[ 27 ]. The use of AMPs as antibiotic preparations is a new method for the treatment of drug-resistant bacteria. In this investigation, we explored the robust in vitro antibacterial activity of AMP-17-6 against C. neoformans 314 (Table 3 ). AMP-17-6 suppressed C. neoformans 314 in a dose-dependent manner, according to the MIC data. Effective antimicrobial peptides require rapid bactericidal action, stressing the importance of understanding the time-dependent variations in their antimicrobial activity. According to the growth curve, AMP-17-6 considerably slowed the growth of C. neoformans 314 (Fig. 2 A), and the bactericidal kinetics (Fig. 2 B) demonstrated that AMP-17-6 was dose dependent. These findings demonstrate that AMP-17-6 is a powerful bactericidal agent with a persistent impact over 24 h, offering vital insights into the creation of new antimicrobial peptides. Because the cellular membrane is the major target of action for most antimicrobial peptides, their cytotoxicity, particularly their hemolytic activity and cytotoxicity, is not unexpected [ 28 ]. However, Dong et al. reported a favorable correlation between the hydrophobicity of the peptide and both hemolytic activity and cytotoxicity [ 29 ]. Interestingly, our results indicated that AMP-17-6 was less harmful to HeLa cells than to human non-small cell lung cancer cells (A549) (Fig. 3 A) at a dose of 256 µg/mL and had essentially no hemolytic effects (Fig. 3 B). The high concentration of hydrophilic amino acids in AMP-17-6, which have been demonstrated to lower their hemolytic potential, may be the cause of these outcomes. These results serve as a foundation for the local clinical use of AMP-17-6. Most AMPs are susceptible to salts and enzymes [ 30 ]-[ 32 ]. The stability of antimicrobial peptides under high-salt conditions restricts their use as novel treatment alternatives [ 33 ]. The presence of salt in the environment did not affect the MIC values of AMP-17-6 in this study. When exposed to trypsin, the activity of AMP-17-6 decreases, presumably due to the high selectivity of trypsin for arginine and lysine residues [ 34 ]. Similarly, Seo et al. reported a considerable decrease in the activity of SJGAP following trypsin therapy [ 35 ]. To date, the specific mechanism underlying the antibacterial effect of AMPs remains obscure, with the prevailing view positing their interaction with the membrane[ 36 ]. AMP-17-6 destroys the fungal membrane structure and disrupts membrane fluidity (Figs. 4 – 7 ). The accumulation of ROS in a cell kills different biological components, such as proteins and lipids. ROS dramatically increased in fungi treated with AMP-17-6 in the present study (Fig. 6 ), indicating that the oxidative stress caused by ROS eventually damages C. neoformans 314. This behavior accords with recent observations revealing the crucial involvement of endogenous ROS in the bactericidal activity of antimicrobial drugs [ 37 ]. Therefore, AMP-17-6 triggers fungal mortality by disrupting the cell membrane, increasing the quantity of ROS in C. neoformans 314. A number of physiological alterations, such as increased membrane permeability and fluidity, decreased membrane potential, disrupted PMF, bacterial metabolic disorders, and leakage of cytoplasmic contents, accompany the main mechanism by which AMPs exert their antibacterial effects on the biophysical integrity of bacteria [ 38 ][ 39 ]. For example, NP-6 can enhance membrane permeability and compromise the integrity of the bacterial cell membrane [ 40 ]. Bacterial membranes are believed to be appealing primary targets for the development of novel antimicrobial medicines and are particularly critical for bacterial growth and survival [ 41 ]. In this investigation, we showed that AMP-17-6 can lead to the breakdown of fungal membranes by interfering with membrane permeability and increasing membrane fluidity in C. neoformans 314, resulting in the release of intracellular substances. These results imply that AMP-17-6 interacts with different bacterial membrane components. Moreover, these data show that AMP-17-6 selectively attaches to the components of both the outer and inner fungal membranes, leading to cell membrane destruction. Moreover, PMF dissipation plays a vital role in the lethality of AMPs by affecting essential fungal processes and increasing fungal death [ 40 ]-[ 42 ]. The ΔΨ component of PMF in C. neoformans 314 was dispersed by AMP-17-6 in our investigation. Furthermore, AMP-17-6 results in metabolic disturbances and the accumulation of hazardous metabolites. These results highlight the possibility of using fungal PMF as a target for the creation of new antimicrobial drugs. Mice with a peritoneal infection caused by C. neoformans 314 were utilized in experiments to examine the in vivo therapeutic effects of AMP-17-6. The results of the in vivo experiments revealed that AMP-17-6 successfully decreased the fungal burden in the lungs of the mice, which in turn decreased the death rate. Additionally, AMP-17-6 has a good protective effect on the pathophysiology of damaged organs, highlighting its potential as a promising therapeutic agent for abdominal infections in mice. In conclusion, this study highlights the bactericidal mechanism of AMP-17-6 (Fig. 8 ). Our findings demonstrate that in drug-resistant C. neoformans 314, AMP-17-6 induces ROS, which compromises membrane integrity. These findings show that AMP-17-6 has considerable inhibitory effects on C. neoformans 314. Thus, this work offers a theoretical foundation for the antibacterial activity of antimicrobial peptides as well as potential targets for AMP-17-6 against C. neoformans . Declarations Authors’ Contributions Na Zhao and Guo Guo conceived and designed the experiments. Na Zhao performed the experiments. Mengtao Wang, Die Zhang, Yuxuan Luo, and Can Liu analyzed the data. Dongxu Song contributed materials and analysis tools. Chaoqin Sun revised the article.Na Zhao and Guo Guo wrote the paper. All authors participated in the data analysis, made substantive contributions to the manuscript, granted their final approval of the version intended for publication, and accepted responsibility for all facets of the research. Funding This research was supported by the National Natural Science Foundation of China (No. 82460406); Guizhou Provincial Natural Science Foundation (ZK [2024] General Program 111); and High-level Talents of Guizhou Medical University Scientific Research Start-up Fund Project (Xiao Bo He J Zi [2022] No. 038); National Natural Science Foundation of China Cultivation Project (25NSFCP06 and 22NSFCP12); The local science foundation of Guizhou Province guided by the Central Committee of China ( No. Qiankehe〔2025〕024); Doctor Start-up Fund of Affiliated Hospital of Guizhou Medical University (gyfybsky-2024-34); College Students' Innovation and Entrepreneurship Program (26252030818). The funders had no role in the study design, data collection or analysis, preparation of the manuscript, or the decision to publish it. Data availability The original contributions presented in this study are included in the article; further inquiries can be directed to the corresponding authors. Ethics and consent to participate The animal study was approved by the Institutional Animal Care and Use Committee of Guizhou Medical University, and laboratory animal usage license number is 2400247. All participants in this study have provided written informed consent for the publication of any potentially identifiable data, including images and case details. All authors agree to the publication of this paper. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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16:58:31","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":72704,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8150680/v1/7bcd2724764354e9281d9a0a.png"},{"id":98453567,"identity":"b57999fe-2d8e-498c-b7b5-64e2b6bbccf8","added_by":"auto","created_at":"2025-12-17 17:58:00","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":136190,"visible":true,"origin":"","legend":"","description":"","filename":"83b24d2dffcf451784889587730b64201structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8150680/v1/b625fa38322aa67e1371b4dc.xml"},{"id":98453572,"identity":"7459aee2-7d67-41d1-b74b-bf4bc94636d6","added_by":"auto","created_at":"2025-12-17 17:58:00","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154054,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8150680/v1/b01a9b21e8bc2c97488d435e.html"},{"id":98453552,"identity":"4d8463e5-46cd-44f3-ad90-ca0bfb909b96","added_by":"auto","created_at":"2025-12-17 17:58:00","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82272,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNovel antimicrobial peptide characterization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The chemical structure of AMP-17-6. (B) AMP-17-6 helical wheel projection diagrams generated via HeliQuest analysis (https://heliquest.ipmc.cnrs.fr/). Hydrophilic amino acids are indicated in blue and are positively charged. The amino acids displayed in yellow and gray are hydrophobic. (C) I-TASSER (https://zhanggroup.org/I-TASSER/) was used to estimate the peptide's three-dimensional structure. The secondary structure is represented by various hues. The helical structure is represented in magenta, whereas the coil structure is represented in white.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150680/v1/43e6822949035c09cc7852e1.jpeg"},{"id":98453554,"identity":"d1603156-a5f5-4b33-afbd-2e8b87895672","added_by":"auto","created_at":"2025-12-17 17:58:00","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":105636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe antibacterial efficacy of AMP-17-6 against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. neoformans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e314.\u003c/strong\u003e (A) AMP-17-6 growth curves of \u003cem\u003eC. neoformans\u003c/em\u003e 314. (B) Killing kinetics of \u003cem\u003eC. neoformans\u003c/em\u003e 314. AMP-17-6 at various doses was incubated with an inoculum of approximately 1´10\u003csup\u003e6\u003c/sup\u003e CFU/mL. Samples were obtained at different time intervals and plated on YPD media, and CFUs were counted in triplicate.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150680/v1/1e1e0ccfc95909df12006c41.jpeg"},{"id":98623523,"identity":"9c3d43b7-bf38-4ee5-8785-1bd6edb78f04","added_by":"auto","created_at":"2025-12-19 17:06:49","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":64340,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vitro safety investigation of AMP-17-6.\u003c/strong\u003e (A) The cytotoxicity of AMP-17-6 in HeLa cells and A549 cells was assessed via a CCK-8 test. (B) AMP-17-6 hemolytic activity at various concentrations following a one-h incubation period with 4% human red blood cells. The data are presented as the means ± standard deviations of three independent experiments. ****P\u0026lt;0.0001 compared with the PC (positive control) group. The means ± standard deviations (n = 3) are shown by the error bars.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150680/v1/e5f3ad0ac5eb33dda4ffafee.jpeg"},{"id":98622784,"identity":"aab0971a-8605-4c69-b914-2747e7c4576a","added_by":"auto","created_at":"2025-12-19 17:02:14","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":567893,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScanning electron microscopy images of AMP-17-6-exposed and unexposed \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. neoformans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e314.\u003c/strong\u003e (A) \u003cem\u003eC. neoformans\u003c/em\u003e 314 treated without AMP-17-6. (B) \u003cem\u003eC. neoformans\u003c/em\u003e 314 treated with peptide AMP-17-6 for 1 h.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150680/v1/d70733ee4711c6732fd3669d.jpeg"},{"id":98622959,"identity":"63ff34fa-b32f-45cc-902f-e25a205391a8","added_by":"auto","created_at":"2025-12-19 17:03:45","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":480695,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConfocal laser scanning microscopy images of AMP-17-6-exposed and unexposed \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. neoformans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e 314.\u003c/strong\u003e AMP-17-6 was not used to treat the CK. After \u003cem\u003eC. neoformans \u003c/em\u003e314 was treated with AMP-17-6 for one h, live/dead bacteria stained with SYTO9 (green; green fluorescence indicates live bacteria) and PI (red; red fluorescence indicates dead bacteria) probes were examined via confocal microscopy.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150680/v1/2b5f4c7ab62c557c3ad60a30.jpeg"},{"id":98623388,"identity":"e68f3fab-320b-4ce9-b5d9-c60a88b44329","added_by":"auto","created_at":"2025-12-19 17:06:04","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":86786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAMP-17-6 caused membrane permeabilization in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. neoformans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e314. \u003c/strong\u003e(A) Fluorescence intensity of propidium iodide (PI). (B) AMP-17-6 increased intracellular ROS levels.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150680/v1/4da9835c9d444a94c88676f3.jpeg"},{"id":98623230,"identity":"9e498cfb-7a00-4213-8d95-90e4c53db686","added_by":"auto","created_at":"2025-12-19 17:05:24","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":261240,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAMP-17-6 administration results in membrane disruption in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. neoformans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e314.\u003c/strong\u003e (A) Effects of AMP-17-6 on the fungal electron respiratory chain. (B) Laurdan GP plots revealing changes in membrane fluidity upon interaction with AMP-17-6. (C) Disc 35 Dissipated the membrane potential (Δψ) in \u003cem\u003eC. neoformans\u003c/em\u003e 314 treated with increasing doses of AMP-17-6. (D) Dissipation pH in \u003cem\u003eC. neoformans\u003c/em\u003e 314 treated with increasing AMP-17-6 concentrations.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150680/v1/7dd4625e394fdda47ab4ac84.jpeg"},{"id":98453559,"identity":"e159bbf3-2be2-4364-9283-052f4dc3709a","added_by":"auto","created_at":"2025-12-17 17:58:00","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":97654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo effectiveness of AMP-17-6 against 314 infections in a mouse pneumonia model. \u003c/strong\u003e(A) Scheme of the experimental protocol for generating the mouse pneumonia model. (B) Survival curves of mice treated with AMP-17-6 for pneumonia caused by 314. (C) Fungal burdens in the peritoneal fluid of mice treated with AMP-17-6 were lower than those in the infection group; * indicates \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ** indicates \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, *** indicates P\u0026lt;0.005, and **** indicates\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001. D. Mouse livers and kidneys undergo pathological alterations following fungal challenge. Original magnification, × 20.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8150680/v1/11e735f47d35a1312e0d00be.jpeg"},{"id":99215828,"identity":"95f1db8d-6320-44ac-8430-42b27faf1b65","added_by":"auto","created_at":"2025-12-30 08:56:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3058738,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8150680/v1/34be635d-e7d3-4932-869e-3e85099843f9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The molecular mechanism by which novel antibacterial peptides inhibit Cryptococcus neoformans","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs a fungal pathogen that poses a threat to human life and health, \u003cem\u003eCryptococcus neoformans\u003c/em\u003e frequently causes pneumonia and potentially fatal cryptococcal meningitis in immunocompromised individuals, including those with HIV/AIDS, organ transplant recipients, and cancer patients undergoing chemotherapy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Typically, cryptococcosis is acquired when the host inhales fungal spores or yeast cells from the air through the respiratory tract, resulting in long-term latent colonization in the lungs. When the host's immune function is impaired, dormant \u003cem\u003eC. neoformans\u003c/em\u003e in the lungs rapidly multiply, causing primary pulmonary infection. Severe cryptococcal meningitis may ensue if the infection spreads to the central nervous system [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, the development of safe and effective novel antimicrobial agents is critically important in the treatment of infections caused by \u003cem\u003eC. neoformans\u003c/em\u003e. As prospective alternatives to traditional antibiotics, antimicrobial peptides (AMPs) have gained extensive attention from researchers because of their in vivo safety and efficacy.\u003c/p\u003e \u003cp\u003eDrug-resistant bacteria may be treated with antimicrobial peptides (AMPs), which are tiny proteins with broad-spectrum antibacterial action [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. According to previous studies, amphibian peptides have been successfully employed to treat local infections caused by a variety of drug-resistant strains, including \u003cem\u003eC. neoformans\u003c/em\u003e infections [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Natural AMPs have broad-spectrum antibacterial activity and a quick death time, effectively eliminating viruses, fungi, and bacteria [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e][\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e][\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, the clinical use of peptides as therapeutic options for treating diseases is expanding due to new types and technologies of AMPs, such as cell-penetrating peptides, artificially generated multifunctional peptides, and peptide-drug conjugates [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Because of these benefits, they have potential as substitutes for traditional antibiotics. However, their hemolytic characteristics and high toxicity restrict their usefulness [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]-[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Owing to the diversity of antimicrobial processes, the mechanism of antimicrobial peptides is still unclear.\u003c/p\u003e \u003cp\u003eOur earlier research revealed antimicrobial peptide-17 (AMP-17), which has a molecular weight of 17 kDa, as a possible antifungal agent from the transcriptome database of \u003cem\u003eMusca domestica\u003c/em\u003e generated by \u003cem\u003eCandida albicans\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]-[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The whole ORF of AMP-17 is 495 bp long and contains 164 amino acids [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The protein polypeptide chain generated by the AMP-17 gene is hydrophilic, and the protein has a transmembrane region, which is similar to a typical membrane-acting antimicrobial peptide. The PBIL LYON-GERLAND database study revealed three primary secondary structures of AMP-17, namely, α-helices, random coils and extended strands [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, AMP-17 exhibits broad-spectrum antibacterial action against both gram-positive and gram-negative bacteria, heat resistance, freeze-thaw resistance, and nontoxicity to human red blood cells, which results in the flow of cell contents [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, by altering AMP-17 via quantitative structure-activity relationship (QSAR) techniques, the antifungal mechanisms of AMP-17 derivatives have not been investigated.\u003c/p\u003e \u003cp\u003eAMP-17-6 demonstrated strong antibacterial activity against drug-resistant \u003cem\u003eC. neoformans\u003c/em\u003e in an antibacterial spectrum screening. We also evaluated a few clinical isolates of \u003cem\u003eC. neoformans\u003c/em\u003e to assess their antibacterial activity, and we discovered that AMP-17-6 had the best antimicrobial activity against \u003cem\u003eC. neoformans\u003c/em\u003e 314. The antibacterial mechanism of AMP-17-6 against \u003cem\u003eC. neoformans\u003c/em\u003e 314 was examined physicochemically and morphologically, and animal studies were used to assess its in vivo effectiveness. The results of this investigation offer a theoretical foundation for the development of new antimicrobial medications.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis and validation of antimicrobial peptides\u003c/h2\u003e \u003cp\u003eGL Biochem (Shanghai, China) used solid-phase synthesis to create AMP-17-6 (TKFAVCFGLLGAKCKILAG-NH2). Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to purify AMP-17-6 to more than 95% purity (Supplementary Fig.\u0026nbsp;1A). High-performance liquid chromatography tandem mass spectrometry (HPLC/MS) was used to measure the molecular weight (Supplementary Fig.\u0026nbsp;1B). A final concentration of 1 mg/mL AMP-17-6 was produced in water. The online program HeliQuest (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://heliquest.ipmc.cnrs.fr/\u003c/span\u003e\u003cspan address=\"https://heliquest.ipmc.cnrs.fr/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to assess the hydrophobicity of AMP-17-6. The 3D spatial structure of AMP-17-6 was predicted with I-TASSER (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zhanggroup.org/I-TASSER/\u003c/span\u003e\u003cspan address=\"https://zhanggroup.org/I-TASSER/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e][\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e][\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGrowth media and fungal strains\u003c/h3\u003e\n\u003cp\u003eA tertiary healthcare center in Guiyang, a city in Guizhou Province, China, provided the clinical strains (311, 313, 181, 182, 183, 314, 178, 185, and 194) used in this investigation. \u003cem\u003eCryptococcus neoformans\u003c/em\u003e H99 and \u003cem\u003eC. albicans\u003c/em\u003e were obtained from the Key and Characteristic Laboratory of Modern Pathogen Biology at Guizhou Medical University in Guiyang, China. Every strain was regularly cultivated in YPD media at 30\u0026deg;C and stored at -80\u0026deg;C in LB media.\u003c/p\u003e\n\u003ch3\u003eDetermination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)\u003c/h3\u003e\n\u003cp\u003e The Clinical and Laboratory Standards Institute (CLSI) criteria were followed to determine the peptide's minimum inhibitory concentration (MIC) via the broth microdilution method. The microdilution method in RPMI 1640 medium was used to determine the MICs. The fungal suspension in the media had a final concentration of 1.0\u0026times;103 CFU/mL. In a 96-well plate, broth was incubated at 30\u0026deg;C for 16\u0026ndash;18 h, and the final AMP content ranged from 1-256 \u0026micro;g/mL. RPMI 1640 was utilized as a blank control, AmB was used as a positive control, and bacteria cultured untreated were used as a negative control. The OD\u003csub\u003e630\u003c/sub\u003e was measured to determine the MIC. One hundred microliter samples from holes that had never undergone fungal growth were placed on YPD agar boards. The MBC was defined as the concentration of AMPs without any fungal growth.\u003c/p\u003e\n\u003ch3\u003eGrowth curve assay\u003c/h3\u003e\n\u003cp\u003eThe determination of the growth curve of \u003cem\u003eC. neoformans\u003c/em\u003e in reaction to AMP-17-6 was accomplished via a method similar to that described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The fungal suspension of \u003cem\u003eC. neoformans\u003c/em\u003e 314 was resuspended to 1.0\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL with various concentrations of AMP-17-6 added (final peptide concentrations of 1, 2, or 4 \u0026times;MIC) or in PBS in 96-well plates and grown at 30\u0026deg;C. A microplate reader was used to measure the absorbance at 630 nm every two h for a full d.\u003c/p\u003e\n\u003ch3\u003eTime-kill assays\u003c/h3\u003e\n\u003cp\u003eThe time-kill kinetics of AMP-17-6 on \u003cem\u003eC. neoformans\u003c/em\u003e 314 were determined following a previously reported technique [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The fungal suspensions (1.0\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) were combined with different doses of AMP-17-6 and subsequently cultivated at 30\u0026deg;C. The final concentrations of AMP-17-6 were 1, 2, or 4 \u0026times;MIC. Samples in 10 \u0026micro;L aliquots were diluted in 90 \u0026micro;L of PBS at 0, 1, 2, 4, 6, 12, and 24 h. The dilutions were then disseminated onto YPD agar plates in 5 \u0026micro;L aliquots. The colony counts were calculated, and a time-kill curve was drawn following a 24-h incubation period at 30\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssay for cytotoxicity\u003c/h2\u003e \u003cp\u003eThe number of viable cells in a sample can be ascertained via the readily accessible, premixed Cell Counting Kit-8 (CCK-8) test. The cytotoxicity of AMP-17-6 on A549 and HeLa cells was assessed via previously published techniques with a few adjustments [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In brief, HeLa or A549 cells were grown at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. AMP-17-6 (1-256 \u0026micro;g/mL) was administered for 24 h after all the cells were injected into 96-well plates at a density of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/well. Cells cultivated without AMP-17-6 in DMEM were utilized as a negative control, and DMEM alone was used as a blank control. After incubation, 10 \u0026micro;L of CCK-8 solution was added to each well and incubated for 2 h. A microplate reader was used to measure the absorbance at 450 nm.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{T}\\text{h}\\text{e}\\:\\text{r}\\text{e}\\text{l}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{g}\\text{r}\\text{o}\\text{w}\\text{t}\\text{h}\\:\\text{r}\\text{a}\\text{t}\\text{e}\\:\\left(\\text{%}\\right)=\\frac{\\left({\\text{A}}_{450\\:\\text{n}\\text{m}}\\:\\text{o}\\text{f}\\:\\text{t}\\text{e}\\text{s}\\text{t}\\:\\text{w}\\text{e}\\text{l}\\text{l}-\\:{\\text{A}}_{450\\:\\text{n}\\text{m}}\\:\\text{o}\\text{f}\\:\\text{b}\\text{l}\\text{a}\\text{n}\\text{k}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)}{\\left({\\text{A}}_{450\\:\\text{n}\\text{m}}\\:\\text{o}\\text{f}\\:\\text{n}\\text{e}\\text{g}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}-\\:{\\text{A}}_{450\\:\\text{n}\\text{m}}\\:\\text{o}\\text{f}\\:\\text{b}\\text{l}\\text{a}\\text{n}\\text{k}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)}\\times\\:100\\text{%}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHemolytic activity assay\u003c/h3\u003e\n\u003cp\u003eThe hemolytic activity of AMP-17-6 on human red blood cells was tested according to a previous study [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Centrifugation at 1000 \u0026times; g for 10 min was used to collect human blood cells, which were subsequently washed three times with 1\u0026times; PBS. Red blood cells were collected and suspended in 1\u0026times; PBS to 4%. Next, 96-well plates containing varying doses of AMP-17-6 (1\u0026thinsp;~\u0026thinsp;256 \u0026micro;g/mL) were incubated for one h at 37\u0026deg;C with 100 \u0026micro;L of 4% red blood cell mixture. The absorbance at 540 nm was measured with a microplate reader. While PBS served as the negative control, a blood cell suspension treated with 1% Triton X-100 was employed as a positive control for 100% hemolysis.\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{T}\\text{h}\\text{e}\\:\\text{h}\\text{e}\\text{m}\\text{o}\\text{l}\\text{y}\\text{s}\\text{i}\\text{s}\\:\\text{r}\\text{a}\\text{t}\\text{e}\\:\\left(\\text{%}\\right)=\\frac{\\left({\\text{A}}_{540\\:\\text{n}\\text{m}}\\:\\text{o}\\text{f}\\:\\text{t}\\text{h}\\text{e}\\:\\text{t}\\text{e}\\text{s}\\text{t}\\:\\text{w}\\text{e}\\text{l}\\text{l}-{\\:\\text{A}}_{540\\:\\text{n}\\text{m}}\\:\\text{o}\\text{f}\\:\\text{t}\\text{h}\\text{e}\\:\\text{n}\\text{e}\\text{g}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)}{\\left({\\text{A}}_{540\\:\\text{n}\\text{m}}\\:\\text{o}\\text{f}\\:\\text{p}\\text{o}\\text{s}\\text{i}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\:-{\\:\\text{A}}_{540\\:\\text{n}\\text{m}}\\:\\text{o}\\text{f}\\:\\text{t}\\text{h}\\text{e}\\:\\text{n}\\text{e}\\text{g}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)}\\times\\:100%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eAntimicrobial efficacy in the presence of salts, serum, temperature and trypsin\u003c/h3\u003e\n\u003cp\u003eMouse serum, various temperatures, trypsin, and MICs were examined once more to investigate the action of AMP-17-6 at high salt concentrations. In 96-well plates, \u003cem\u003eC. neoformans\u003c/em\u003e 314 was grown at 37\u0026deg;C for 16 h with various doses of AMP-17-6 in YPD supplemented with 150 mM NaCl, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 10% mouse serum, and 10% FBS. The resulting solutions were subjected to MIC testing to evaluate any modifications in the antibacterial activity of AMP-17-6. AMP-17-6 were exposed to different temperatures (40\u0026deg;C, 60\u0026deg;C, 80\u0026deg;C, and 100\u0026deg;C) for 30 min to evaluate its thermal stability. The MIC was then calculated after the temperature was lowered to room temperature. After AMP-17-6 was combined with a 1 mg/ml trypsin solution, the mixture was incubated for one h at 37\u0026deg;C. Then, trypsin inactivation was carried out at 60\u0026deg;C for 30 min. After the samples were treated with trypsin, the antibacterial activity of AMP-17-6 and its analogs was evaluated. For comparative analysis, untreated AMP-17-6 was used as the control.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy\u003c/h2\u003e \u003cp\u003eFungal suspensions (1.0\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) were treated with AMP-17-6 at a final dose of 1 \u0026times; MIC or in PBS for two h to examine the morphological alterations caused by AMP-17-6 in \u003cem\u003eC. neoformans\u003c/em\u003e 314. After two washes with PBS, the bacteria were fixed with 2.5% glutaraldehyde at 4\u0026deg;C overnight. After that, the bacteria were dehydrated in a range of ethanol solutions. A fungal suspension without AMP-17-6 was utilized as a control for comparison purposes. A Hitachi Regulus SU8100 (Tokyo, Japan) was used to acquire the images.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFungal viability\u003c/h2\u003e \u003cp\u003eThe percentage of viable and dead bacteria was visualized via confocal laser scanning microscopy (CLSM; Olympus SpinSR10, Japan) and the fluorescence probe staining method [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The bacteria were collected after the fungal suspension (1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/ml) and AMP-17-6 at various concentrations were incubated for one h at 37\u0026deg;C and centrifuged at 2,000 \u0026times; g for five min. After two rounds of washing, the bacteria were again suspended in PBS. After adding final amounts of 10 \u0026micro;M SYTO 9 and 10 \u0026micro;M PI to each group, the mixture was incubated for 15 min at 37\u0026deg;C in the dark. Following the incubation period, PBS was used to remove the unbound fluorescent dye. CLSM was used to evaluate the samples' fluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMembrane permeability test\u003c/h2\u003e \u003cp\u003eFungal suspensions were diluted with PBS to a concentration of 1.0\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL in compliance with previously reported procedures [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The fungal suspensions were grown at 30\u0026deg;C for two h in Eppendorf tubes containing varying doses of AMP-17-6 or PBS (final peptide concentrations of 1, 2, or 4 \u0026times;MIC). Next, 10 \u0026micro;M propidium iodide (PI; Sigma, US) was added to each group, and the mixture was incubated at 37\u0026deg;C for 15 min in the dark. The samples were evaluated with a microplate reader (BioTek SYNERGY-H, Agilent, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eReactive oxygen species measurements\u003c/h2\u003e \u003cp\u003eThe intracellular production of ROS was measured via the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA (final concentration of 10 \u0026micro;M) was combined with the fungal suspension (1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL) and incubated at 37\u0026deg;C for 30 min in the dark. After incubation, the suspensions were treated with AMP-17-6 (1, 2, or 4 times the MIC) at the indicated doses for 1 h at 30\u0026deg;C. The fluorescence intensity was assessed via the use of a multifunctional fluorescent enzyme marker with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffects of AMP-17-6 on fungal cell membranes\u003c/h2\u003e \u003cp\u003eAntimicrobial peptides work primarily by influencing the permeability, fluidity, and charge of cell membranes. Thus, how AMP-17-6 affects \u003cem\u003eC. neoformans\u003c/em\u003e 314 cell membranes was identified via fluorescence intensity tests. The overnight fungal culture mixture was rinsed and resuspended in PBS to a concentration of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL. The fluorescent dye PI was used to measure the permeability of the fungal inner membrane [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The final concentration of 5 \u0026micro;M PI was incubated with the fungal suspension for 10 min, followed by treatment with AMP-17-6 (0\u0026ndash;32 \u0026micro;g/mL) at 30\u0026deg;C for 1 h. The bacterial fluorescence value was then determined via a microplate reader (BioTek SYNERGY-H, Agilent, USA) with excitation and emission wavelengths of 535 nm and 615 nm, respectively.\u003c/p\u003e \u003cp\u003eThe fluorescent dye Luardan (Sigma, USA) at a concentration of 10 \u0026micro;M was used to measure the fluidity of the fungal membrane. Luardan was found at 350 nm excitation and 435 and 490 nm absorption wavelengths. The computation of the generalized polarization (GP) was conducted via the following formula: GP = (I435 - I490)/(I435\u0026thinsp;+\u0026thinsp;I490).\u003c/p\u003e \u003cp\u003eDiSC3(5) (Sigma, USA) was then used to evaluate the effect of AMP on the potential energy of the cell [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The bacteria were cultured at a final concentration of 0.5 \u0026micro;M DiSC3(5), and the fluorescence values were measured at an excitation wavelength of 622 nm and an emission wavelength of 670 nm.\u003c/p\u003e \u003cp\u003eAdditionally, the proton motive force of fungi treated with AMP-17-6 was measured with BCECF-AM (UElandy, Suzhou) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The fungal suspensions were incubated with 1 \u0026micro;M BCECF-AM after being treated with different doses of AMP-17-6. The fluorescence value was calculated using excitation/emission wavelengths of 488 nm and 535 nm.\u003c/p\u003e \u003cp\u003eThe impact of AMP-17-6 on fungal respiration was examined via the use of the oxygen-sensitive dye resazurin (Solarbio, Beijing) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, the fungal cultures were treated with various concentrations of 0\u0026ndash;32 \u0026micro;g/mL AMP-17-6 and a constant concentration of 0.1 \u0026micro;g/mL resazurin. A microplate reader with excitation and emission wavelengths of 550 nm and 590 nm, respectively, was used to observe the fluorescence change constantly for one h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eModel of mouse infection\u003c/h2\u003e \u003cp\u003eThe female BALB/c mice used in this investigation were purchased from SPF Biotechnology Co., Ltd. (Beijing, China), were 6\u0026ndash;8 weeks old and weighed 18\u0026ndash;20 g. A typical 12-h light/12-h dark cycle was employed to keep the animals. The Institutional Animal Care and Use Committee (Approval No: 2400247) approved all the animal experiments, which were carried out in compliance with Guizhou Medical University's Ethical Principles in Animal Research.\u003c/p\u003e \u003cp\u003eTo test the in vivo effectiveness of AMP-17-6, we employed a mouse pneumonia model. A total of 60 mice were divided into four groups: the treatment group (10 mg/kg AMP-17-6, intraperitoneal injection), the positive control group (10 mg/kg AmB, intraperitoneal injection), the blank control group (PBS, intraperitoneal injection), and the model group (fungal injection, PBS, intraperitoneal injection). To create a Cryptococcus-mouse lung infection model, a 100 \u0026micro;L suspension of 1.0 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU \u003cem\u003eC. neoformans\u003c/em\u003e 314 was gradually injected into the mouse lungs via tracheal intubation after each mouse was rendered unconscious by isoflurane inhalation via a small animal anesthetic unit [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To monitor the in vivo therapeutic effects in each mouse treatment group, once-daily medication treatment was started via intraperitoneal injection as soon as possible after 48 h of infection, and three doses were given consecutively. Every day, the survival status of the mice in each group was tracked and documented. Moreover, throughout the 23-d experimental observation period, body weight measurements were performed daily for all groups to analyze changes in mouse weight following medication treatment [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeven d after the medicine was administered to examine the therapeutic effects of AMP-17-6, ten mice from each group exposed to isoflurane anesthesia in a gas-tight chamber and stayed in the chamber and were then sacrificed by cervical dislocation. And infected lung tissues were collected aseptically. While the tissues were frozen in 4% paraformaldehyde and stained with hematoxylin and eosin (H\u0026amp;E) to examine their morphology, the number of colonies in the lung was ascertained by cultivation on YPD agar.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eEvery experiment was conducted at least three times. Student's \u003cem\u003et\u003c/em\u003e-test was used to evaluate group differences. The means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs) are used to express the data. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCharacterization of antimicrobial peptides.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe synthesized peptide and its sequences, and the biological characteristics of AMP-17-6 are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The cationic peptide AMP-17-6 is more than 95% pure. The precise synthesis of the peptide was demonstrated by the agreement between the calculated and observed molecular weights. Additionally, AMP-17-6 is a cationic antimicrobial peptide whose structure is primarily composed of coil and helical conformations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical parameters of AMP-17-6.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeptide\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence(N\u0026rarr;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFormula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCharge\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003epI\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCalcd MW\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eObsd MW\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePurity(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMP-17-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTKFAVCFGLLGAKCKILAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e100\u003c/sub\u003eH\u003csub\u003e163\u003c/sub\u003eN\u003csub\u003e27\u003c/sub\u003eO\u003csub\u003e24\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1940.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1939.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e95.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e Charge, isoelectric point (pI) and molecular weight (MW) were calculated online at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003csup\u003eb\u003c/sup\u003e The observed molecular weight (MW) was determined via liquid chromatography-mass spectrometry (LC-MS).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) The chemical structure of AMP-17-6. (B) AMP-17-6 helical wheel projection diagrams generated via HeliQuest analysis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://heliquest.ipmc.cnrs.fr/\u003c/span\u003e\u003cspan address=\"https://heliquest.ipmc.cnrs.fr/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Hydrophilic amino acids are indicated in blue and are positively charged. The amino acids displayed in yellow and gray are hydrophobic. (C) I-TASSER (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zhanggroup.org/I-TASSER/\u003c/span\u003e\u003cspan address=\"https://zhanggroup.org/I-TASSER/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to estimate the peptide's three-dimensional structure. The secondary structure is represented by various hues. The helical structure is represented in magenta, whereas the coil structure is represented in white.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntimicrobial action of AMP-17 derivatives.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThrough alteration of the functional domain of AMP-17, we effectively created six variants of AMP-17. \u003cem\u003eC. neoformans\u003c/em\u003e C and H99. Candida albicans was employed as a test strain to investigate the antibacterial activity of the six derivatives of AMP-17. We discovered that AMP-17-6 had the strongest antibacterial action against C among all the peptides that were examined. H99 neoformans (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial activity screening of six AMP-17 derivatives.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMIC(\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC. neoformans\u003c/em\u003e H99\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMP-17-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMP-17-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMP-17-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMP-17-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMP-17-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMP-17-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe chose other clinically drug-resistant isolates of \u003cem\u003eC. neoformans\u003c/em\u003e and \u003cem\u003eC. neoformans\u003c/em\u003e H99 for testing to examine further the antibacterial activity of AMP-17-6 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The AMP-17-6 MICs ranged from 4\u0026ndash;32 \u0026micro;g/mL. Strain 314 was selected to gain a better understanding of how AMP-17-6 affects clinical drug-resistant isolates.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial activity of AMP-17-6 against clinical strains of \u003cem\u003eC. neoformans.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBacteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. neoformans\u003c/em\u003e H99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. neoformans\u003c/em\u003e 311\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. neoformans\u003c/em\u003e 312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. neoformans\u003c/em\u003e 314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. neoformans\u003c/em\u003e 181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. neoformans\u003c/em\u003e 182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. neoformans\u003c/em\u003e 183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. neoformans\u003c/em\u003e 178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. neoformans\u003c/em\u003e 185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. neoformans\u003c/em\u003e 194\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAMP-17-6 growth assay and quick bactericidal effectiveness against 314.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe growth curves of \u003cem\u003eC. neoformans\u003c/em\u003e 314 in the presence of AMP-17-6 are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. AMP-17-6 suppressed \u003cem\u003eC. neoformans\u003c/em\u003e 314 growth in a dose-dependent manner at doses ranging from 1 \u0026times; MIC to 2 \u0026times; MIC. The kinetic killing effects of AMP-17-6 on \u003cem\u003eC. neoformans\u003c/em\u003e 314 were further assessed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). At concentrations of 1 \u0026times; MIC and 2 \u0026times; MIC, the \u003cem\u003eC. neoformans\u003c/em\u003e 314 treatment tended to eradicate the germs within 2 h. These results indicated that AMP-17-6 displayed fast bactericidal activity against fungal infections.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of salt, heat and trypsin on AMP-17-6 activity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe effects of heat, salt, and trypsin on AMP-17-6 activity were measured via MIC tests. Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e displays the outcomes. In the absence of physiological CaCl\u003csub\u003e2\u003c/sub\u003e, NaCl, or KCl, the MICs of AMP-17-6 did not differ from those of the control. The impact of heat on the antibacterial activity of AMP-17-6 was examined via a heating test. Nevertheless, the antibacterial activity of AMP-17-6 did not decrease following this treatment, and its antimicrobial activity was heat stable. Unfortunately, trypsin and pepsin removed the antibacterial action of AMP-17-6.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of salt, temperature and trypsin on AMP-17-6 activity in \u003cem\u003eC. neoformans\u003c/em\u003e 314.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAMP-17-6 (MIC, \u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150 mM Na\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4.5 mM K\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 mM Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 mM Mg\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 mg/mL Trypsin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 mg/mL Pepsin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u0026deg;C 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e37\u0026deg;C 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e70\u0026deg;C 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u0026deg;C 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAMP-17-6 hemolysis and cytotoxicity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAMP-17-6 was tested for its ability to induce hemolysis and cytotoxic action against human red blood cells, specifically HeLa and A549 (human non-small cell lung cancer) cells, at doses ranging from 2 to 256 \u0026micro;g/mL. The viability of HeLa cells treated with different concentrations of AMP-17-6 was consistently greater than 100%, according to the cytotoxicity data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, following treatment with 128 \u0026micro;g/mL AMP-17-6, the viability of A549 cells decreased to 75.60%. The majority of the cells remained intact in the hemolysis results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicating that AMP-17-6 had minimal hemolytic action against human erythrocytes. These findings imply that AMP-17-6 has positive safety characteristics.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibacterial mechanism.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eScanning electron microscopy (SEM) images revealed that normal \u003cem\u003eC. neoformans\u003c/em\u003e 314 was consistently rod-shaped with a smooth surface. Furthermore, AMP-17-6 treatment at the appropriate MICs for two h caused significant damage and rupture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The effects of AMP-17-6 on the integrity of the fungal cell membrane were assessed via CLSM and PI and SYTO9 nucleic acid stains to investigate the antibacterial activity of AMP-17-6. STOY9 is a green fluorescent nucleic acid stain that can stain both living and dead bacteria, whereas PI is a red nucleic acid-binding dye that only enters cells with broken membranes. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows that after one h, the untreated \u003cem\u003eC. neoformans\u003c/em\u003e 314 cells were stained with STOY9 but not PI, suggesting that most of the cells were viable. On the other hand, after treatment with AMP-17-6 at doses of 1, 2, and 4 times the MIC, the quantity and intensity of red fluorescent dots increased in a concentration-dependent manner. These studies indicated that AMP-17-6 was able to disrupt fungal cell membranes and that rupture increased with increasing peptide content.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, PI has been employed as a probe to investigate cell membrane integrity (Sautrey et al., 2016). PI can cross the membrane and intercalate with DNA in injured cells. On the other hand, PI cannot enter undamaged cells (Thulshan Jayathilaka et al., 2021). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, the untreated \u003cem\u003eC. neoformans\u003c/em\u003e 314 group presented a low percentage of PI fluorescence signals. In contrast, cells treated with AMP-17-6 at 1, 2, or 4 times the MIC for 1 h presented PI fluorescence signals. These findings suggest that AMP-17-6 increases the permeability of fungal membranes while compromising their integrity.\u003c/p\u003e \u003cp\u003eFungal mortality is significantly influenced by reactive oxygen species (ROS). Following AMP-17-6 treatment, ROS accumulation in bacteria dramatically increased, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, exacerbating membrane damage, further impairing fungal homeostasis. These results demonstrate that ROS are essential for the sterilization of antimicrobial peptides.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFunctions of membranes and energy metabolism.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWhen examined, AMP-17-6 can not only impair the integrity of the inner membrane but also affect membrane malfunction and intracellular metabolic alterations. We also observed changes in \u003cem\u003eC. neoformans\u003c/em\u003e 314 respiration levels after treatment with AMP-17-6 and resazurin. The findings showed that AMP-17-6 considerably increased C's respiratory level. neoformans 314 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Laurdan is a fluorescent probe used to research cell membrane characteristics and biomembrane lipid dynamics, and can be applied to identify alterations in membrane fluidity. \u003cem\u003eThe membrane fluidity of C. neoformans\u003c/em\u003e 314 was improved by AMP-17-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The proton motive force (PMF) is known to be impacted by membrane rupture. The transmembrane proton gradient (ΔpH) and Δψ are the two main components of the PMF and are crucial for material movement, energy production, and antibiotic resistance. Δψ was measured with DisC3(5). The fluorescence intensity of DisC3(5) dramatically increased in \u003cem\u003eC. neoformans\u003c/em\u003e 314 after treatment with AMP-17-6 in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), indicating that AMP-17-6 disrupts the Δψ of \u003cem\u003eC. neoformans\u003c/em\u003e 314 and causes membrane potential depolarization. Additionally, we chose BCECF-AM, another fluorescent probe, to assess how AMP-17-6 affects the ΔpH. The fluorescence intensity of \u003cem\u003eC. neoformans\u003c/em\u003e 314 was marginally increased by AMP-17-6, suggesting that AMP-17-6 increased the ΔpH (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The rapid bacterial disintegration caused by AMP-17-6 may be connected to this increase in fluorescence intensity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAntimicrobial activity in vivo.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe evaluated the effectiveness of AMP-17-6 in a mouse model since it can inhibit \u003cem\u003eC. neoformans\u003c/em\u003e 314 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). We monitored the body weight fluctuations of the mice in each group. All the treatment groups presented a decrease in body weight, although the model group presented the fastest rate of decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). We assessed the fungal burden in the mouse lung on the fifth d after infection to further assess the effects of AMP-17-6. The fungal content in the positive control group treated with AmB decreased to 4.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 log\u003csub\u003e10\u003c/sub\u003e CFU/mL (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), whereas the fungal count in the AMP-17-6-treated group decreased to 7.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 log10 CFU/g (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and that in the model group was 8.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 log10 CFU/g. At 15 d, the survival rate of the model group was 0%, whereas that of the control group was 100%, and that of the AMP-17-6 group was 40%. Therefore, treatment with AMP-17-6 at a dose of 10 mg/kg increased the survival rate of the mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe histological images of the lung tissue sections are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE. The mice in the model group had more Cryptococcus in their alveoli and pulmonary interstitium as the infection period progressed, and the lung tissue showed diffuse necrosis. The pulmonary interstitium, interlobular septa, and alveolar walls were greatly enlarged, accompanied by dispersed or focal infiltration of numerous inflammatory cells, such as neutrophils and lymphocytes. In certain places, nodular lesions develop, causing the destruction and consolidation of the lung tissue structure. Occasionally, this was followed by the formation of an abscess. Compared with those in the control group, the lung tissue in the model group contained fewer fungi and fewer severe lesions. Additionally, HE-stained lung tissue sections revealed that mice treated with AMP-17-6 presented relatively few inflammatory lung tissue symptoms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHospital-acquired illnesses are now frequently caused by drug-resistant bacteria. These bacteria greatly impair the efficacy of antibiotics, whereas the discovery of novel therapeutic medications has become an important concern [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]-[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The use of AMPs as antibiotic preparations is a new method for the treatment of drug-resistant bacteria.\u003c/p\u003e \u003cp\u003eIn this investigation, we explored the robust in vitro antibacterial activity of AMP-17-6 against \u003cem\u003eC. neoformans\u003c/em\u003e 314 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). AMP-17-6 suppressed \u003cem\u003eC. neoformans\u003c/em\u003e 314 in a dose-dependent manner, according to the MIC data. Effective antimicrobial peptides require rapid bactericidal action, stressing the importance of understanding the time-dependent variations in their antimicrobial activity. According to the growth curve, AMP-17-6 considerably slowed the growth of \u003cem\u003eC. neoformans\u003c/em\u003e 314 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and the bactericidal kinetics (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) demonstrated that AMP-17-6 was dose dependent. These findings demonstrate that AMP-17-6 is a powerful bactericidal agent with a persistent impact over 24 h, offering vital insights into the creation of new antimicrobial peptides.\u003c/p\u003e \u003cp\u003eBecause the cellular membrane is the major target of action for most antimicrobial peptides, their cytotoxicity, particularly their hemolytic activity and cytotoxicity, is not unexpected [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, Dong et al. reported a favorable correlation between the hydrophobicity of the peptide and both hemolytic activity and cytotoxicity [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Interestingly, our results indicated that AMP-17-6 was less harmful to HeLa cells than to human non-small cell lung cancer cells (A549) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) at a dose of 256 \u0026micro;g/mL and had essentially no hemolytic effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The high concentration of hydrophilic amino acids in AMP-17-6, which have been demonstrated to lower their hemolytic potential, may be the cause of these outcomes. These results serve as a foundation for the local clinical use of AMP-17-6.\u003c/p\u003e \u003cp\u003eMost AMPs are susceptible to salts and enzymes [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]-[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The stability of antimicrobial peptides under high-salt conditions restricts their use as novel treatment alternatives [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The presence of salt in the environment did not affect the MIC values of AMP-17-6 in this study. When exposed to trypsin, the activity of AMP-17-6 decreases, presumably due to the high selectivity of trypsin for arginine and lysine residues [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Similarly, Seo et al. reported a considerable decrease in the activity of SJGAP following trypsin therapy [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo date, the specific mechanism underlying the antibacterial effect of AMPs remains obscure, with the prevailing view positing their interaction with the membrane[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. AMP-17-6 destroys the fungal membrane structure and disrupts membrane fluidity (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe accumulation of ROS in a cell kills different biological components, such as proteins and lipids. ROS dramatically increased in fungi treated with AMP-17-6 in the present study (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), indicating that the oxidative stress caused by ROS eventually damages \u003cem\u003eC. neoformans\u003c/em\u003e 314. This behavior accords with recent observations revealing the crucial involvement of endogenous ROS in the bactericidal activity of antimicrobial drugs [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Therefore, AMP-17-6 triggers fungal mortality by disrupting the cell membrane, increasing the quantity of ROS in \u003cem\u003eC. neoformans\u003c/em\u003e 314.\u003c/p\u003e \u003cp\u003eA number of physiological alterations, such as increased membrane permeability and fluidity, decreased membrane potential, disrupted PMF, bacterial metabolic disorders, and leakage of cytoplasmic contents, accompany the main mechanism by which AMPs exert their antibacterial effects on the biophysical integrity of bacteria [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e][\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. For example, NP-6 can enhance membrane permeability and compromise the integrity of the bacterial cell membrane [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Bacterial membranes are believed to be appealing primary targets for the development of novel antimicrobial medicines and are particularly critical for bacterial growth and survival [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In this investigation, we showed that AMP-17-6 can lead to the breakdown of fungal membranes by interfering with membrane permeability and increasing membrane fluidity in \u003cem\u003eC. neoformans\u003c/em\u003e 314, resulting in the release of intracellular substances. These results imply that AMP-17-6 interacts with different bacterial membrane components. Moreover, these data show that AMP-17-6 selectively attaches to the components of both the outer and inner fungal membranes, leading to cell membrane destruction. Moreover, PMF dissipation plays a vital role in the lethality of AMPs by affecting essential fungal processes and increasing fungal death [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]-[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The ΔΨ component of PMF in \u003cem\u003eC. neoformans\u003c/em\u003e 314 was dispersed by AMP-17-6 in our investigation. Furthermore, AMP-17-6 results in metabolic disturbances and the accumulation of hazardous metabolites. These results highlight the possibility of using fungal PMF as a target for the creation of new antimicrobial drugs.\u003c/p\u003e \u003cp\u003eMice with a peritoneal infection caused by \u003cem\u003eC. neoformans\u003c/em\u003e 314 were utilized in experiments to examine the in vivo therapeutic effects of AMP-17-6. The results of the in vivo experiments revealed that AMP-17-6 successfully decreased the fungal burden in the lungs of the mice, which in turn decreased the death rate. Additionally, AMP-17-6 has a good protective effect on the pathophysiology of damaged organs, highlighting its potential as a promising therapeutic agent for abdominal infections in mice.\u003c/p\u003e \u003cp\u003eIn conclusion, this study highlights the bactericidal mechanism of AMP-17-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Our findings demonstrate that in drug-resistant \u003cem\u003eC. neoformans\u003c/em\u003e 314, AMP-17-6 induces ROS, which compromises membrane integrity. These findings show that AMP-17-6 has considerable inhibitory effects on \u003cem\u003eC. neoformans\u003c/em\u003e 314. Thus, this work offers a theoretical foundation for the antibacterial activity of antimicrobial peptides as well as potential targets for AMP-17-6 against \u003cem\u003eC. neoformans\u003c/em\u003e.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNa Zhao and Guo Guo conceived and designed the experiments. Na Zhao performed the experiments. Mengtao Wang, Die Zhang, Yuxuan Luo, and Can Liu analyzed the data. Dongxu Song contributed materials and analysis tools. Chaoqin Sun revised the article.Na Zhao and Guo Guo wrote the paper. All authors participated in the data analysis, made substantive contributions to the manuscript, granted their final approval of the version intended for publication, and accepted responsibility for all facets of the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (No. 82460406); Guizhou Provincial Natural Science Foundation (ZK [2024] General Program 111); and High-level Talents of Guizhou Medical University Scientific Research Start-up Fund Project (Xiao Bo He J Zi [2022] No. 038); National Natural Science Foundation of China Cultivation Project (25NSFCP06 and 22NSFCP12); The local science foundation of Guizhou Province guided by the Central Committee of China ( No. Qiankehe〔2025〕024); Doctor Start-up Fund of Affiliated Hospital of Guizhou Medical University (gyfybsky-2024-34); College Students\u0026apos; Innovation and Entrepreneurship Program (26252030818). The funders had no role in the study design, data collection or analysis, preparation of the manuscript, or the decision to publish it.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in this study are included in the article; further inquiries can be directed to the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study was approved by the Institutional Animal Care and Use Committee of Guizhou Medical University, and laboratory animal usage license number is 2400247.\u0026nbsp;All participants in this study have provided written informed consent for the publication of any potentially identifiable data, including images and case details. All authors agree to the publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026rsquo;s Note\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGifford A, Jayawardena N, Carlesse F, Lizarazo J, McMullan B, Groll AH, Warris A. Pediatric cryptococcosis. 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Protein Pept Lett. 2022;29(8):641\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2174/0929866529666220613102145\u003c/span\u003e\u003cspan address=\"10.2174/0929866529666220613102145\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cryptococcus neoformans 314, Antimicrobial peptide, AMP-17 derivatives, Membrane mechanism, In vivo","lastPublishedDoi":"10.21203/rs.3.rs-8150680/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8150680/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCryptococcosis, caused by \u003cem\u003eCryptococcus neoformans\u003c/em\u003e, is a major global public health concern. The infection primarily begins in the lungs. Cryptococcosis is characterized by significant consequences and low cure rates in immunocompromised individuals. Pulmonary cryptococcosis has become more common in recent years. Amphotericin B (AmB) and fluconazole (FLC) are commonly used as first-line medications in clinical treatment.\u003c/p\u003e\n\u003cp\u003eHowever, the increasing incidence of azole resistance has led to an increase in clinical treatment failure rates. Antimicrobial peptides (AMPs) are considered attractive substitutes for conventional antibiotics for resolving this urgent problem because of their distinct processes and low risk of resistance development. Our earlier research revealed antimicrobial peptide-17 (AMP-17), which has a molecular weight of 17 kDa, as a possible antifungal agent from the transcriptome database of \u003cem\u003eMusca domestica\u003c/em\u003e generated by \u003cem\u003eCandida albicans\u003c/em\u003e. Nevertheless, the use of quantitative structure-activity relationship (QSAR) techniques to modify AMP-17 has not been used to investigate the antifungal mechanisms of AMP-17 derivatives.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study examined the potential potency of the peptide AMP-17-6 against clinically drug-resistant \u003cem\u003eC. neoformans\u003c/em\u003e 314. The results revealed that AMP-17-6 possesses high bacteriostatic activity and bactericidal efficiency against\u003cem\u003eC. neoformans\u003c/em\u003e 314, with a minimum inhibitory concentration (MIC) of 4 μg/mL. Additionally, AMP-17-6 is sensitive to trypsin and pepsin but is stable in a range of serum conditions and temperatures. Mechanistic studies revealed that AMP-17-6 induces membrane damage by targeting fungal-specific membrane components and dissipating the proton motive force (PMF), leading to metabolic disturbances and the accumulation of toxic metabolic byproducts. In a mouse model of pneumonia induced by\u003cem\u003e C. neoformans\u003c/em\u003e 314 infection, AMP reduced the fungal burden and inflammatory reactivity in lung tissue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese data demonstrate that AMP exhibits significant antibacterial efficacy against \u003cem\u003eC. neoformans\u003c/em\u003e 314 by decreasing fungal viability and displaying antivirulence effects, suggesting its potential as a novel antibacterial drug for the treatment of resistant \u003cem\u003eC. neoformans\u003c/em\u003e infections.\u003c/p\u003e","manuscriptTitle":"The molecular mechanism by which novel antibacterial peptides inhibit Cryptococcus neoformans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-17 17:57:55","doi":"10.21203/rs.3.rs-8150680/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2d75cd6b-d6d5-47f7-9eff-a1ec225e0717","owner":[],"postedDate":"December 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-30T08:54:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-17 17:57:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8150680","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8150680","identity":"rs-8150680","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}