Novel antibacterial peptides derived from differentiated stem cells

Abstract

The present disclosure provides an antibacterial peptide comprising ENOS or SPARCL1, or a portion of the sequence thereof. The present disclosure also provides a conditioned cell culture medium comprising the antimicrobial peptide, wherein the conditioned cell culture medium is prepared by culturing adipose-derived mesenchymal stem cells. The technology also relates to methods of treating infections using the peptides.

Description

Technical Field

[0001] The present invention relates generally to the field of cell biology. In particular, the present invention relates to antimicrobial peptides. Background Art

[0002] Chronic wound management has become a huge and growing problem due to the aging of the global population and the sharp rise in the incidence of diabetes and obesity. It is estimated that 1%-2% of the world's population will experience chronic wounds in their lifetime. In the United States, chronic wounds affect about 6.5 million patients and are reported to cost $25 billion per year. Chronic wounds are mainly caused by nosocomial infections that cause complications of skin injuries. According to the National Institute for Health and Clinical Excellence (NICE) (2008), an estimated 5% of all surgical operations in the UK will lead to nosocomial infections, that is, one in every seven operations will lead to nosocomial infections. The incidence of chronic wound infections is higher in diseases prone to bacterial infections such as diabetes. Exemplary results of chronic wound infections are diabetic foot ulcers (DFU) and diabetic foot infections (DFI), which affect about 15% of all patients and are the most common risk factors for lower limb amputations. In some cases, pathogens, necrotic tissue, and extracellular matrix (ECM) secreted by the pathogens form a membrane layer called a biofilm, which is particularly difficult to manage due to the presence of a protective matrix barrier.

[0003] To date, antibiotics remain the first-line treatment for wound infections, for example in the form of wound dressings to promote tissue regeneration. However, antibiotics are not able to actively manage chronic infections except with limited existing antibiotics. The vast majority of pathogens causing chronic wounds are multidrug resistant (MDR), which can easily survive standard antibiotic treatment by acquiring antimicrobial resistance (AMR). The World Health Organization (WHO) has declared antimicrobial resistance (AMR) one of the top ten global public health threats, which is expected to cause 10 million deaths per year by 2050, surpassing cancer, and the global economic losses are expected to reach 100 trillion US dollars. The emergence of antibiotic-resistant infections and the lack of new antibiotics have become the biggest challenges.

[0004] To address this problem, attempts such as combination therapies with antimicrobial peptides (AMPs) or polymers and antibiotics have focused specifically on treating infections caused by multidrug-resistant (MDR) bacterial strains. Although combination therapies have shown promising results in in vitro and animal studies, clinical data are conflicting. In addition, adverse reactions have also been reported, such as nephrotoxicity and ototoxicity, which may lead to drug interactions and further toxicity in patients receiving multiple medications. In addition, the use of combined antimicrobial therapies to treat multidrug-resistant (MDR) strains can lead to increased antimicrobial resistance (AMR) of pathogens and impose additional cost burdens on patients. Alternative approaches such as skin grafting are considered the gold standard for chronic wound management. However, skin grafting is limited by the quality and quantity of donor skin and therefore cannot solve the problem of bacterial clearance. Similarly, other new approaches, such as the delivery of angiogenesis-inducing growth factors and cytokines and hyperbaric oxygen therapy, cannot fundamentally solve the problem of infection.

[0005] Therefore, what is needed is a novel antimicrobial agent that is effective in treating chronic infections, and methods of using antimicrobial agents to treat chronic infections.Furthermore, other desirable features and characteristics will become apparent from the subsequent detailed description and the appended claims, taken in conjunction with the drawings and background of the disclosure. Summary of the invention

[0006] In one aspect, the present disclosure provides an antimicrobial peptide consisting of the sequence KVNQIGSVTESIQACKLAQSN (SEQ ID NO: 3).

[0007] In one aspect, the present disclosure provides an antimicrobial peptide consisting of the sequence HHKAEKSSVLKSKEESH (SEQ ID NO: 4).

[0008] In one aspect, the present disclosure provides an antimicrobial peptide consisting of the sequence KQRNKVKKIYLDEKR (SEQ ID NO: 5).

[0009] In one aspect, the present disclosure provides an antimicrobial peptide comprising a partial sequence of ENO3, wherein the peptide comprises at least the sequence KVNQIGSVTESIQACKLAQSN (SEQ ID NO: 3).

[0010] In one aspect, the present disclosure provides an antimicrobial peptide comprising a partial sequence of SPARCL1, wherein the peptide at least comprises the sequence HHKAEKSSVLKSKEESH (SEQ ID NO: 4).

[0011] In one aspect, the present disclosure provides an antimicrobial peptide comprising a partial sequence of SPARCL1, wherein the peptide comprises at least the sequence KQRNKVKKIYLDEKR (SEQ ID NO: 5).

[0012] In one aspect, the present disclosure provides a conditioned cell culture medium (CM) comprising the antimicrobial peptide, full-length ENO3 peptide or full-length SPARCL1 peptide of claims 1-8.

[0013] In one aspect, the present disclosure provides a composition comprising one or more antimicrobial peptides, full-length ENO3 peptides, or full-length SPARCL1 peptides disclosed herein.

[0014] In one aspect, the present disclosure provides a pharmaceutical composition comprising a peptide disclosed herein, a conditioned cell culture medium disclosed herein, or a composition disclosed herein.

[0015] In one aspect, the present disclosure provides a wound dressing comprising a peptide disclosed herein, a conditioned cell culture medium (CM) disclosed herein, a composition disclosed herein, or a pharmaceutical composition disclosed herein.

[0016] In one aspect, the present disclosure provides an in vitro method for inducing an antimicrobial response in tissue-derived stem cells, wherein the method comprises (a) obtaining a tissue sample from a subject; (b) isolating tissue-derived stem cells from the tissue sample of (a) in cell culture; (c) culturing the tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated cells; and (d) incubating the intermediate differentiated cells with a bacterial culture or a bacterial surface antigen to induce an antimicrobial response in the intermediate differentiated cells.

[0017] In one aspect, the present disclosure provides an in vitro method for inducing an antibacterial response in adipose-derived mesenchymalstem cells (ASCs), comprising (a) obtaining an adipose tissue sample from a subject; (b) isolating adipose-derived mesenchymal stem cells (ASCs) from the adipose tissue sample of (a) in cell culture; (c) culturing adipose-derived mesenchymal stem cells (ASCs) in an adipogenic differentiation medium to obtain intermediate differentiated ASCs; and (d) incubating the intermediate differentiated ASCs with a bacterial culture or a bacterial surface antigen to induce an antibacterial response of the intermediate differentiated ASCs.

[0018] In one aspect, the present disclosure provides an in vitro method for producing the conditioned cell culture medium (CM) disclosed herein, comprising: (a) obtaining a tissue sample from a subject; (b) isolating tissue-derived stem cells from the tissue sample of (a); (c) culturing the tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated stem cells; (d) incubating the intermediate differentiated cells with a bacterial culture or a bacterial surface antigen in a cell culture medium; and (e) collecting the conditioned cell culture medium (CM) by removing the bacteria and the intermediate differentiated cells.

[0019] In one aspect, the present disclosure provides a method for preparing the conditioned cell culture medium (CM) disclosed herein, comprising (a) obtaining an adipose tissue sample from a subject; (b) isolating adipose-derived mesenchymal stem cells (ASCs) from the adipose tissue of (a) in cell culture; (c) culturing adipose-derived mesenchymal stem cells (ASCs) in an adipogenic differentiation medium to obtain intermediate-differentiated ASCs; (d) incubating the intermediate-differentiated ASCs in a cell culture medium with a bacterial culture or a bacterial surface antigen; and (e) collecting the conditioned cell culture medium (CM) by removing the bacteria and the intermediate-differentiated ASCs.

[0020] In one aspect, the present disclosure provides a method of treating an infection in a subject, or a method of promoting wound healing and tissue regeneration in a subject, or a method of reducing / preventing biofilm formation in a subject, comprising administering to the subject a pharmaceutically effective amount of a peptide disclosed herein, a conditioned cell culture medium (CM) disclosed herein, a composition disclosed herein, or a pharmaceutical composition disclosed herein.

[0021] In one aspect, the present disclosure provides nucleic acid compositions encoding the peptides disclosed herein.

[0022] In one aspect, the present disclosure provides an expression vector comprising a nucleic acid composition disclosed herein.

[0023] In one aspect, the disclosure provides a host cell comprising one or more nucleic acid compositions disclosed herein or one or more expression vectors disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A provides an overview of the optimization of conditioned medium culture conditions for triggering antimicrobial responses, particularly in adipose-derived mesenchymal stem cells (ASCs), based on the following parameters: (i) Figure 1Adipose-derived mesenchymal stem cells (ASC) are sensitized with heat-killed or live bacteria at a multiplicity of infection (MOI) ratio of 1:2, 1:20, 1:200 or 1:2000 (bacteria: ASC) as indicated by A; (ii) 3, 6, 12 or 24 hours of sensitization period; (iii) adipose-derived mesenchymal stem cells (ASC) undergo a stage of adipogenic differentiation as indicated by the number of days after differentiation (0 days, 6 days or 12 days); (iv) high or low glucose, and culture conditions with or without 10% FBS. Conditions showing >50% bacterial killing are circled compared to the culture medium control. As shown in the overview, exemplary conditions for inducing antibacterial responses in mesenchymal stem cells include live bacteria at a multiplicity of infection (MOI) ratio of 1:200 (bacteria: ASC, as an example) at high or low glucose levels, or in the presence or absence of FBS. The conditions also include co-culturing the bacteria and mesenchymal stem cells, which are intermediate differentiated stem cells, for about 24 hours.

[0025] Figure 1 B summarizes Figure 1 Various combinations of conditions tested in A.

[0026] Figure 1 C shows the viability of an exemplary bacterium, Salmonella typhimurium (ST), treated with conditioned medium obtained by Figure 1 Adipose-derived mesenchymal stem cell lines (ATCC SCRC 4000) from healthy donors were cultured under the conditions summarized in A. The percentage of colony forming unit (CFU) counts of the treated bacteria is shown for each condition stratified based on the stage of stem cell differentiation and the sensitizing bacterial strain used. Figure 1 As demonstrated by C, intermediate differentiated stem cells (D6: 6 days) sensitized with Salmonella Typhimurium (ST) or Pseudomonas aeruginosa (PA) showed less than 50% of the Salmonella Typhimurium (ST) colony forming unit (CFU) count, indicating stronger antibacterial activity against the exemplary bacterium Salmonella Typhimurium (ST) compared with other conditions.

[0027] Figure 1 D shows the viability of exemplary bacteria Pseudomonas aeruginosa (PA) treated with conditioned medium obtained by Figure 1Adipose-derived mesenchymal stem cell lines (ATCC SCRC4000) from healthy donors were cultured under the conditions summarized in A. The percentage of colony forming unit (CFU) counts of the treated bacteria is shown for each condition stratified based on the stage of stem cell differentiation and the sensitizing bacterial strain used. Figure 1 As demonstrated by D, intermediate differentiated stem cells sensitized with Salmonella typhimurium (ST) or Pseudomonas aeruginosa (PA) (D6: 6 days) showed less than 50% of the Pseudomonas aeruginosa (PA) colony forming unit (CFU) count, indicating stronger antibacterial activity against the exemplary bacterium Pseudomonas aeruginosa (PA) compared with other conditions.

[0028] Figure 1 E shows the viability of exemplary Staphylococcus aureus (S. aureus) (SA) treated with conditioned medium obtained by Figure 1 Adipose-derived mesenchymal stem cell lines (ATCC SCRC 4000) from healthy donors were cultured under the conditions summarized in A. The percentage of colony forming unit (CFU) counts of the treated bacteria is shown for each condition stratified based on the stage of stem cell differentiation and the sensitizing bacterial strain used. Figure 1 As demonstrated by E, intermediate differentiated stem cells sensitized with Salmonella typhimurium (ST) or Pseudomonas aeruginosa (PA) (D6: 6 days) showed less than 50% of the colony forming unit (CFU) count of Staphylococcus aureus (SA), indicating stronger antibacterial activity against the exemplary bacterium Staphylococcus aureus (SA) compared with other conditions.

[0029] Figure 1 F shows the viability of an exemplary bacterium, Salmonella typhimurium (ST), treated with conditioned medium obtained by Figure 1 Adipose-derived mesenchymal stem cell lines (S29) from obese patients were cultured under the conditions summarized in A. The percentage of colony forming unit (CFU) counts of the treated microorganisms is shown for each condition stratified based on the stage of stem cell differentiation and the sensitizing bacterial strain used. Figure 1 As demonstrated by F, intermediate differentiated stem cells sensitized with Salmonella typhimurium (ST) or Pseudomonas aeruginosa (PA) (D6: 6 days) showed less than 50% of the colony forming unit (CFU) count of Salmonella typhimurium (ST), indicating stronger antibacterial activity against the exemplary bacterium Salmonella typhimurium (ST) compared with other conditions.

[0030] Figure 1 G shows the viability of exemplary bacteria Pseudomonas aeruginosa (PA) treated with conditioned medium obtained by Figure 1Obtained by culturing an adipose-derived mesenchymal stem cell line (S29) from an obese patient under the conditions summarized in A. The percentage of colony forming unit (CFU) counts of the treated microorganisms is shown for each condition stratified based on the stage of stem cell differentiation and the sensitizing bacterial strain used. Figure 1 As demonstrated in Figure 5, intermediate differentiated stem cells sensitized with Salmonella typhimurium (ST) or Pseudomonas aeruginosa (PA) (D6: 6 days) showed less than 50% of the Pseudomonas aeruginosa (PA) colony forming unit (CFU) count, indicating stronger antibacterial activity against the exemplary bacterium Pseudomonas aeruginosa (PA) compared with other conditions.

[0031] Figure 1 H shows the viability of an exemplary bacterium, Staphylococcus aureus (SA), treated with conditioned medium obtained by Figure 1 Obtained by culturing an adipose-derived mesenchymal stem cell line (S29) from an obese patient under the conditions summarized in A. The percentage of colony forming unit (CFU) counts of the treated microorganisms is shown for each condition stratified based on the stage of stem cell differentiation and the sensitizing bacterial strain used. Figure 1 As demonstrated by H, intermediate differentiated stem cells sensitized with Salmonella typhimurium (ST) or Pseudomonas aeruginosa (PA) (D6: 6 days) showed less than 50% of the Staphylococcus aureus (SA) colony forming unit (CFU) count, indicating stronger antibacterial activity against the exemplary bacterium Staphylococcus aureus (SA) compared with other conditions.

[0032] Figure 1 I shows the viability of an exemplary bacterium, Salmonella typhimurium (ST), treated with conditioned medium obtained by Figure 1 Obtained by culturing an adipose-derived mesenchymal stem cell line (S23) from an obese patient under the conditions summarized in A. The percentage of colony forming unit (CFU) counts of the treated microorganisms is shown for each condition stratified based on the stage of stem cell differentiation and the sensitizing bacterial strain used. Figure 1 As demonstrated by I, intermediate differentiated stem cells (D6: 6 days) sensitized with Salmonella typhimurium (ST) or Pseudomonas aeruginosa (PA) showed less than 50% of the colony forming unit (CFU) count of Salmonella typhimurium (ST), indicating stronger antibacterial activity against the exemplary bacterium Salmonella typhimurium (ST) compared with other conditions.

[0033] Figure 1 J shows the viability of exemplary bacteria Pseudomonas aeruginosa (PA) treated with conditioned medium obtained by Figure 1Obtained by culturing an adipose-derived mesenchymal stem cell line (S23) from an obese patient under the conditions summarized in A. The percentage of colony forming unit (CFU) counts of the treated microorganisms is shown for each condition stratified based on the stage of stem cell differentiation and the sensitizing bacterial strain used. Figure 1 As demonstrated by J, intermediate differentiated stem cells (D6: 6 days) sensitized with Salmonella typhimurium (ST) or Pseudomonas aeruginosa (PA) showed less than 50% of the Pseudomonas aeruginosa (PA) colony forming unit (CFU) count, indicating a stronger antibacterial activity against the exemplary bacterium Pseudomonas aeruginosa (PA) compared with other conditions.

[0034] Figure 1 K shows the viability of exemplary bacteria Staphylococcus aureus (SA) treated with conditioned medium obtained by Figure 1 Obtained by culturing an adipose-derived mesenchymal stem cell line (S23) from an obese patient under the conditions summarized in A. The percentage of colony forming unit (CFU) counts of the treated microorganisms is shown for each condition stratified based on the stage of stem cell differentiation and the sensitizing bacterial strain used. Figure 1 As demonstrated by K, intermediate differentiated stem cells sensitized with Salmonella typhimurium (ST) or Pseudomonas aeruginosa (PA) (D6: 6 days) showed less than 50% of the Staphylococcus aureus (SA) colony forming unit (CFU) count, indicating stronger antibacterial activity against the exemplary bacterium Staphylococcus aureus (SA) compared with other conditions. Figure 1 C to Figure 1 All strains of K were grown in the presence of bacterially sensitized or non-sensitized conditioned medium of adipose-derived mesenchymal stem cell lines from healthy or obese patients according to the procedures of the Clinical and Laboratory Standards Institute (CLSI). Each value is mean ± SD (n = 3). All statistical significance was evaluated using Student's paired t test compared to the medium control normalized to 100%, *p < 0.05.

[0035] Figure 2 A provides crystal violet (CV) staining of 3-day-old Pseudomonas aeruginosa (PA) to quantify the presence of biofilm in response to treatment with conditioned medium (ASC-CM) of different adipose-derived mesenchymal stem cell lines as indicated. Figure 2 As shown in A, conditioned medium from an intermediate adipogenic differentiation stage sensitized with either Salmonella typhimurium (ST) or Pseudomonas aeruginosa (PA) showed significant disruption of the biofilm forming ability of the Pseudomonas aeruginosa (PA) strain.

[0036] Figure 2 B provides crystal violet (CV) staining of 3-day-old Salmonella typhimurium (ST) to quantify the presence of biofilm in response to treatment with conditioned medium (ASC-CM) from different adipose-derived mesenchymal stem cell lines as indicated. Figure 2 The conclusion in A is consistent with that in Figure 2 In B, conditioned media from an intermediate adipogenic differentiation stage sensitized with either S. Typhimurium (ST) or P. aeruginosa (PA) showed a significant disruption of the biofilm forming ability of the S. Typhimurium (ST) strain. Figure 2 A and Figure 2 Each value in B is mean ± SEM (n = 3, biological replicates). All statistical significance was assessed using Student's paired t-test, *p < 0.05.

[0037] Figure 3 A shows the generation of an exemplary resistant microbial strain by serial passage of Salmonella typhimurium (ST) with gentamicin. After 16 days of gentamicin treatment, the minimum inhibitory concentration (MIC) of gentamicin was as high as 1024 μg / ml. The resistant Salmonella typhimurium (ST) bacterial strain was later used to test the conditioned medium (CM) obtained from adipose-derived mesenchymal stem cell line (ASC).

[0038] Figure 3 B provides the viability (colony forming unit (CFU) percentage) of high minimum inhibitory concentration (MIC) (1024 μg / ml) Salmonella typhimurium (ST) strains after treatment with conditioned medium (ASC-CM) of adipose-derived mesenchymal stem cell lines. Conditioned medium obtained from intermediate differentiated mesenchymal stem cell cultures sensitized with Salmonella typhimurium (ST) or Pseudomonas aeruginosa (PA) effectively kills resistant Salmonella typhimurium (ST) strains. Each value is mean ± SD (n = 3). All statistical significances were evaluated using Student's paired t test, *p < 0.05.

[0039] Figure 3 C further demonstrated the long-term antibacterial effect of ATCC SCRC 4000 conditioned medium sensitized with Pseudomonas aeruginosa (PA) against drug-resistant Salmonella Typhimurium (ST) bacterial strains. Based on intracellular ATP levels (quantifying the viability of drug-resistant Salmonella Typhimurium (ST) bacterial strains), the conditioned medium showed a prolonged inhibitory effect for at least 28 days against serially passaged drug-resistant Salmonella Typhimurium (ST) strains without the development of resistance.

[0040] Figure 4A shows the results of the activity test of Salmonella typhimurium (ST) in response to HPLC purification vials, to identify the fractions containing active antimicrobial agents, which are obtained from the conditioned medium (ASC-CM) of the intermediate differentiation adipose-derived mesenchymal stem cell line of obese patient sample S29. The sterile HPLC fractions of the conditioned medium were compared with the culture medium fractions. In all 3 conditioned medium fractions, based on the colony counts of Salmonella typhimurium (ST), the conditioned medium fractions of Salmonella typhimurium (ST) or Pseudomonas aeruginosa (PA) sensitized in No. 3 vials (V3) showed significant antimicrobial effects compared with other vials. Therefore, active antimicrobial agents may be present in V3.

[0041] Figure 4 B shows the results of a Salmonella typhimurium (ST) viability test in response to HPLC purification vials obtained from conditioned medium (ASC-CM) of an intermediate differentiated adipose-derived mesenchymal stem cell line (ATCC SCRC 4000) from a healthy subject sample to identify fractions containing active antimicrobial agents. Figure 4 Consistent with the findings in A, the conditioned media fractions from vial 3 (V3) sensitized with either S. typhimurium (ST) or P. aeruginosa (PA) showed significant antimicrobial effects compared with the other vials based on the colony counts of S. typhimurium (ST). Active antimicrobial agents may be present in V3.

[0042] Figure 4 C shows the results of a viability test of Salmonella typhimurium (ST) in response to HPLC purification vials obtained from conditioned medium (ASC-CM) of an intermediate differentiated adipose-derived mesenchymal stem cell line from obese patient sample S23 compared to culture medium to identify fractions containing active antimicrobial agents. Figure 4 A and Figure 4 The results in B were consistent, with the same vial 3 showing antimicrobial activity based on the percentage of colony forming units (CFU%), while the other vials did not show such antimicrobial activity. Each value is the mean ± SD (n = 3). All statistical significance was assessed using Student's paired t-test, *p < 0.05. Based on Figure 4 A. Figure 4 B and Figure 4 Based on the consistent results of C, the active antimicrobial agent is likely present in the fraction of vial 3, and this fraction can be further separated and identified using mass spectrometry. In fact, new peptides were identified based on mass spectrometry analysis.

[0043] Figure 5 A provides validation of the antimicrobial peptides ENO3 and SPARCL1 identified by mass spectrometry. Figure 5A shows the percentage of colony forming units (CFU%) of Salmonella typhimurium (ST) in response to ATCC SCRC 4000 conditioned medium pretreated with antibodies against ENO3 and SPARCL1 and a control peptide LL-37 known to have antibacterial effects. Conditioned medium treated with anti-ENO3 or anti-SPARCL1 failed to inhibit the growth of Salmonella typhimurium (ST) compared to untreated medium. Conditioned medium treated with anti-LL-37 continued to inhibit the growth of Salmonella typhimurium (ST) bacterial cultures. Therefore, ENO3 and SPARCL1 are shown to be responsible for the antibacterial activity in the conditioned medium (ASC-CM) of the adipose-derived mesenchymal stem cell line.

[0044] Figure 5 B provides validation of the antimicrobial peptides ENO3 and SPARCL1 identified from conditioned medium obtained from obese patient-derived stem cells S29.

[0045] Figure 5 C provides validation of the antimicrobial peptides ENO3 and SPARCL1 identified in conditioned medium obtained from obese patient-derived stem cells S23. Isotype antibodies were used as negative controls. Figure 5 B and Figure 5 C shows the Figure 5 A Consistent results confirming the antimicrobial activities of ENO3 and SPARCL1 identified from conditioned media.

[0046] Figure 6 A tested the antibacterial activity of different concentrations of ENO3 against an exemplary bacterial strain, Salmonella typhimurium (ST). The percentage of colony forming units (CFU%) of Salmonella typhimurium (ST) treated with ENO3 at concentrations ranging from 0 (medium) to 2.04 nM is shown. It can be seen that at a concentration of 2.04 nM, ENO3 exhibited a bacterial reduction of more than 50%.

[0047] Figure 6 B. The antibacterial activity of different concentrations of ENO3 against an exemplary bacterial strain Pseudomonas aeruginosa (PA) was tested. At a low concentration of 0.204 nM, ENO3 exhibited a greater than 50% reduction in Pseudomonas aeruginosa (PA) bacteria.

[0048] Figure 6 C tested the antibacterial activity of ENO3 at different concentrations against an exemplary bacterial strain, Staphylococcus aureus (SA). Even at a lower concentration of 0.102 nM, ENO3 showed a greater than 50% reduction in Pseudomonas aeruginosa (PA) bacteria. Figure 6 A to Figure 6 All bacterial strains used in C. Figure 6 A- Figure 6C, ENO3 exhibited antibacterial activity against a variety of microbial strains at concentrations as low as 0.102 nM.

[0049] Figure 6 D. Different concentrations of SPARCL1 were tested for antibacterial activity against an exemplary bacterial strain, Salmonella typhimurium (ST). At a concentration of 0.0067 nM, SPARCL1 exhibited greater than 50% reduction of Salmonella typhimurium (ST) bacteria.

[0050] Figure 6 E provides the antibacterial activity of SPARCL1 at different concentrations against an exemplary bacterial strain, Pseudomonas aeruginosa (PA). At a concentration of 268 nM, SPARCL1 exhibited greater than 50% reduction of Pseudomonas aeruginosa (PA) bacteria.

[0051] Figure 6 F provides the antibacterial activity of SPARCL1 at different concentrations against an exemplary bacterial strain, Staphylococcus aureus (SA). At a concentration of 1000 nM, SPARCL1 exhibited greater than 50% reduction of Salmonella typhimurium (ST) bacteria. All tested bacterial strains were grown according to CLSI procedures. Each value is mean ± SD (n = 3). All statistical significance was assessed using Student's paired t-test, *p < 0.05. Based on Figure 6 A-6C, SPARCL1 exhibited antibacterial activity against a variety of microbial strains at concentrations as low as 0.0067 nM.

[0052] Figure 6 G shows viable colonies of an exemplary bacterial strain Staphylococcus aureus (SA) after treatment with ENO3. The MIC and MBC values ​​of ENO3 against Staphylococcus aureus (SA) were both 2 μM.

[0053] Figure 6 H shows viable colonies of an exemplary bacterial strain Pseudomonas aeruginosa (PA) after treatment with ENO3. The MIC value of ENO3 against Pseudomonas aeruginosa (PA) is 4 μM, while the MBC value of ENO3 against Pseudomonas aeruginosa (PA) is 6 μM.

[0054] Figure 7 A shows the anti-biofilm effect of different concentrations of ENO3. Crystal violet (CV) staining of 3-day-old Pseudomonas aeruginosa (PA) biofilms in response to ENO3 treatment in the nM range is shown. After treatment with ENO3 at a concentration of about 0.0102 nM, the Pseudomonas aeruginosa (PA) biofilms showed nearly 50% disruption.

[0055] Figure 7B shows the anti-biofilm effect of different concentrations of ENO3. Crystal violet (CV) staining of 3-day-old Salmonella typhimurium (ST) biofilm in response to ENO3 treatment in the nM range. After treatment with ENO3 at a concentration of about 0.0102 nM, the Salmonella typhimurium (ST) biofilm showed more than 50% disruption.

[0056] Figure 7 C shows the anti-biofilm effect of different concentrations of SPARCL1. Figure 7 C shows crystal violet (CV) staining of 3-day-old Pseudomonas aeruginosa (PA) biofilm in response to SPARCL1 treatment in the nM range. After treatment with SPARCL1 at a concentration of approximately 0.00134 nM, the Pseudomonas aeruginosa (PA) biofilm exhibited nearly 50% disruption.

[0057] Figure 7 D shows the anti-biofilm effect of different concentrations of SPARCL1. Figure 7 C shows crystal violet (CV) staining of 3-day-old Salmonella Typhimurium (ST) biofilms in response to SPARCL1 treatment in the nM range. Salmonella Typhimurium (ST) biofilms showed nearly 50% disruption after treatment with SPARCL1 at a concentration of approximately 0.00134 nM. Each value is the mean ± SEM (n = 3, biological replicates). All statistical significance was assessed using Student's paired t-test, *p < 0.05. Figure 7 A to Figure 7 As shown in D, ENO3 and SPARCL1 exhibited strong anti-biofilm activity against a variety of microbial strains.

[0058] Figure 8 A provides exemplary images of injured and uninjured healthy de-epidermised dermis human skin equivalent (DED-HSE) models at day 0. Methylthiazol tetrazolium (MTT) staining of keratinocytes in DED-HSE samples shows the localization of keratinocytes in each sample. As can be seen from the wound images, on day 0, there was a lack of keratinocytes in the wound area.

[0059] Figure 8B shows MTT staining of keratinocytes in a healthy de-epidermized dermal human skin substitute (DED-HSE) model 2 days before and 8 days after injury. The samples were treated with conditioned medium obtained from adipose-derived mesenchymal stem cells (ASCs) undergoing adipogenic differentiation at different stages in the differentiation process (0 days, 6 days, or 12 days). The wounded de-epidermized dermal human skin substitute (DED-HSE) samples treated with conditioned medium obtained from adipose-derived mesenchymal stem cells (ASCs) 6 days after differentiation (intermediate differentiation) showed that keratinocytes were concentrated in the wound area, indicating that keratinocytes proliferated in the wound area.

[0060] Figure 8 C provides quantification of the recovery of wound area based on MTT staining images. Data represent mean ± SD. Figure 8 Consistent with the observations in B, in wounded samples treated with conditioned medium from intermediate differentiated stem cells, the wound area was covered with keratinocytes.

[0061] Figure 8 D shows that the wound of the skin sample treated with the conditioned medium on day 6 was completely closed, indicating accelerated wound healing. The dotted line indicates the wound boundary. The conditioned medium used was from the ATCC healthy subject cell line. Figure 8 D shows that in the sample treated with the conditioned medium obtained from intermediate differentiated mesenchymal stem cells, the wound of the skin model was closed and recovered. Therefore, in addition to the antibacterial effect, the conditioned medium described herein also showed the ability to promote proliferation and wound healing.

[0062] Fig. 9 The cell confluence of human keratinocytes assessed at each time point in the 2D scratch assay by applying the ImageJ plugin PHANTAST on phase contrast time-lapse images is shown. The rate of change of confluence was then calculated by obtaining the best fit line on the confluence versus time plot. Each value is mean ± SD (n = 2). Conditioned medium obtained from intermediate differentiated mesenchymal stem cells and sensitized with Salmonella typhimurium (ST) or Pseudomonas aeruginosa (PA) showed the highest migration speed in the scratch assay, supporting the higher wound healing capacity demonstrated in the skin model.

[0063] Fig.10 The confluence of human keratinocytes assessed at each time point in the 2D scratch assay by applying the ImageJ plugin PHANTAST on phase contrast time-lapse images is shown. The rate of change of confluence was then calculated by obtaining the best fit line on the confluence versus time graph. Each value is mean ± SD (n = 2). Fig. 9The higher keratinocyte migration rate observed in the conditioned medium could be reproduced in isolated ENO3 and SPARCL1. Moreover, no cell migration was observed in the gentamicin-treated group, indicating that the wound healing effects of ENO3 and SPARCL1 are independent of the antimicrobial activity of standard antibiotics.

[0064] Fig.11 In vivo evidence for the antibacterial effect of ENO3 and SPARCL1 in a mouse excisional wound infection model is provided. Mouse excisional wounds were infected with Pseudomonas aeruginosa for 24 hours and then treated with ENO3 or SPARCL1 for 24 hours. Each value is the mean ± SEM (n = 5). Colony forming unit (CFU) counts were assessed in treated and untreated samples, indicating the viability of Pseudomonas aeruginosa bacteria in each sample. It was observed that the bacterial colonies of samples treated with ENO3 and SPARCL1 were reduced, indicating the in vivo antibacterial efficacy of ENO3 and SPARCL1.

[0065] Fig.12 A shows the bacterial viability of Pseudomonas aeruginosa (PA) treated with different concentrations of the short peptide E1A from ENO3. In order to develop clinically useful peptides, short peptide regions within ENO3, such as the E1A region, were identified. At a concentration of 0.39 μM, the short peptide E1A exhibited an antibacterial activity greater than 50%.

[0066] Fig.12 B shows the bacterial viability of Pseudomonas aeruginosa (PA) treated with different concentrations of the short peptide S1A from SPARCL1. At a concentration of 100 μM, the short peptide S1A exhibited an antibacterial activity greater than 50%.

[0067] Fig.12 C shows the bacterial viability of Pseudomonas aeruginosa (PA) treated with different concentrations of the short peptide S1B from SPARCL1. At a concentration of 50 μM, the short peptide S1B showed an antibacterial activity greater than 50%. Fig.12 A- Fig.12 C Growth of Pseudomonas aeruginosa (PA) bacterial strain used. Each value is mean ± SD (n = 3). All statistical significance was assessed using Student's paired t-test, *p < 0.05. Fig.12 A to Fig.12 C provides in vitro experimental data demonstrating the antibacterial activity of short peptides E1A (based on ENO3), S1A (based on SPARCL1) and S1B (based on SPARCL1) against the exemplary bacterial strain Pseudomonas aeruginosa (PA).

[0068] Fig.13A provides crystal violet (CV) staining of 3-day-old Pseudomonas aeruginosa (PA) biofilms in response to μM range of E1A treatment. Within the exemplary range of 0.39 μM-1.56 μM, the short peptide E1A exhibited greater than 50% biofilm disruption activity.

[0069] Fig.13 B provides crystal violet (CV) staining of 3-day-old Pseudomonas aeruginosa (PA) biofilms in response to treatment with S1A in the μM range. Within the exemplary range of 0.78 μM-3.125 μM, the short peptide S1A exhibited greater than 50% biofilm disruption activity.

[0070] Fig.13 C provides crystal violet (CV) staining of 3-day-old Pseudomonas aeruginosa (PA) biofilms in response to S1B treatment in the μM range. In the exemplary concentration range of 6.25 μM-12.5 μM, the short peptide S1B exhibited greater than 50% biofilm disruption activity. Each value is mean ± SEM (n = 3, biological replicates). All statistical significances were evaluated using Student's paired t-test, *p < 0.05. Fig.13 A to Fig.13 C provides in vitro experimental data demonstrating the biofilm disruption activity of short peptides E1A (based on ENO3), S1A (based on SPARCL1) and S1B (based on SPARCL1) against the exemplary bacterial strain Pseudomonas aeruginosa (PA).

[0071] Fig.14 A further provides crystal violet (CV) staining of a 3-day-old Pseudomonas aeruginosa (PA) biofilm in response to a combination of E1A and an exemplary antibiotic ciprofloxacin. Compared to the individual antibiotics, the combined treatment of E1A (concentration: μM) and ciprofloxacin (concentration: μM) showed improved effects in destroying the biofilm formed by the exemplary bacterial strain Pseudomonas aeruginosa (PA). The biofilm staining readings when E1A and ciprofloxacin are combined are also lower than those of E1A alone, which indicates a synergistic effect between the two agents.

[0072] Fig.14 B provides crystal violet (CV) staining of 3-day-old Pseudomonas aeruginosa (PA) biofilms in response to a combination of E1A (concentration: μM) and an exemplary antibiotic colistin (concentration: μM). Each value is mean ± SEM (n = 3, biological replicates). Similar to ciprofloxacin, the combined treatment of E1A and colistin showed improved effects in destroying biofilms formed by the exemplary bacterial strain Pseudomonas aeruginosa (PA) compared to the antibiotic alone. The biofilm staining readings when E1A and colistin were combined were comparable to those of E1A alone, but the E1A concentration was lower, indicating a synergistic effect between the two agents.

[0073] Fig.14 C provides crystal violet (CV) staining of 3-day-old Pseudomonas aeruginosa (PA) biofilms in response to a combination of S1A (concentration: μM) and an exemplary antibiotic ciprofloxacin (concentration: μM). The combined treatment of S1A and ciprofloxacin showed improved effects in destroying biofilms formed by the exemplary bacterial strain Pseudomonas aeruginosa (PA) compared to the antibiotics alone. The biofilm staining readings when S1A and ciprofloxacin were combined were also lower than those of the S1A treatment alone, indicating a synergistic effect between the two agents.

[0074] Fig.14 D provides crystal violet (CV) staining of a 3-day-old Pseudomonas aeruginosa (PA) biofilm in response to a combination of S1B (concentration: μM) and an exemplary antibiotic ciprofloxacin (concentration: μM). Compared to the individual antibiotics, the combined treatment of S1B and ciprofloxacin showed improved effects in destroying the biofilm formed by the exemplary bacterial strain Pseudomonas aeruginosa (PA). The biofilm staining readings when S1B and ciprofloxacin were combined were also lower than those of the individual S1B treatments, which indicated a synergistic effect between the two agents.

[0075] Fig.14 E provides crystal violet (CV) staining of a 3-day-old Pseudomonas aeruginosa (PA) biofilm in response to a combination of ENO3 and an exemplary antibiotic ciprofloxacin. The biofilm control represents "culture medium". All statistical significances were evaluated using Student's paired t-test, *p<0.05. Compared with individual antibiotics, the combined treatment of ENO3 (concentration: nM) and ciprofloxacin (concentration: μM) showed improved effects in destroying the biofilm formed by the exemplary bacterial strain Pseudomonas aeruginosa (PA). The biofilm staining readings when ENO3 and ciprofloxacin were combined were also lower than those treated with ENO3 alone, which indicates a synergistic effect between the two agents.

[0076] Fig.14 F provides crystal violet (CV) staining of 3-day-old Pseudomonas aeruginosa (PA) biofilms in response to a combination of SPARCL1 and the exemplary antibiotic ciprofloxacin (as indicated). The biofilm control represents "medium". All statistical significances were assessed using Student's paired t-test, *p<0.05. The combined treatment of SPARCL1 (concentration: nM) and ciprofloxacin (concentration: μM) showed improved effects in destroying biofilms formed by the exemplary bacterial strain Pseudomonas aeruginosa (PA) compared to the antibiotics alone. The biofilm staining readings when SPARCL1 and ciprofloxacin were combined were also lower than those of SPARCL1 treatment alone, indicating a synergistic effect between the two agents.

[0077] Fig.15The cell confluence of human keratinocytes assessed at each time point in the 2D scratch assay by applying the ImageJ plugin PHANTAST on phase contrast time-lapse images is shown. The rate of change of confluence was calculated by obtaining the best fit line on the confluence versus time plot. Each value is mean ± SD (n = 3). The migration velocity of cells treated with E1A was higher compared to the culture medium, indicating that the wound healing ability of cells treated with E1A was enhanced.

[0078] Fig.16 An overview of exemplary procedures for preparing conditioned media from adipose tissue and identifying novel antimicrobial peptides is provided. The resulting conditioned media exhibited antimicrobial, antibiofilm, and wound healing effects that were effective against antibiotic-resistant pathogens.

[0079] definition

[0080] The term "chronic wound" as used herein refers to a wound that cannot achieve anatomical and functional integrity through a normal, orderly and timely repair process under the influence of various internal or external factors. Wound healing, as a normal biological process of the human body, is achieved through four precise and highly programmed stages: hemostasis, inflammation, proliferation and remodeling. For a wound to heal successfully, all four stages must be carried out in the correct order and time frame. A chronic wound refers to an injury that has not healed and has no healing trend after a long period of treatment (e.g., one month). Examples of chronic wounds include: non-healing or infected surgical or traumatic wounds, venous ulcers, pressure sores, diabetic foot ulcers, and ischemic ulcers.

[0081] As used herein, the term "hospital infection" or "healthcare-associated infection (HAI)" refers to an infection that is not present or latent at the time of hospital admission but occurs during or after treatment. These infections include catheter-associated urinary tract infection, central line-associated bloodstream infection, surgical site infection, ventilator-associated pneumonia, hospital-acquired pneumonia, and Clostridium difficile infection.

[0082] As used herein, the term "diabetes mellitus" or "diabetes" refers to a heterogeneous complex metabolic disorder characterized by hyperglycemia, i.e., elevated blood glucose concentrations secondary to resistance to insulin action, insufficient insulin secretion, or both. Diabetes can generally be divided into two categories, namely type 1 diabetes and type 2 diabetes. Type 1 diabetes is characterized by an absolute lack of insulin secretion. Serological evidence and genetic markers of autoimmune pathological processes in the pancreatic islets can generally identify individuals with an increased risk of developing this type of diabetes. The cause of type 2 diabetes is a combination of resistance to insulin action and insufficient compensatory insulin secretion response. According to statistics, most patients with type 2 diabetes are obese, and obesity itself causes a certain degree of insulin resistance. Diabetic patients are particularly susceptible to infections, which are related to long-term hyperglycemia.

[0083] The term "diabetic foot ulcer (DFU)" or "diabetic foot infection (DFI)" as used herein is one of the most common and challenging complications associated with the onset of diabetes, which reduces the quality of life of patients and causes significant morbidity and mortality. Chronic foot ulcers in patients with diabetes are often caused by poor blood circulation and peripheral neuropathy (i.e., localized nerve damage). The treatment of diabetic foot ulcers (DFU) is particularly challenging due to the high recurrence rate and antibiotic resistance.

[0084] As used herein, the term "antimicrobial resistance (AMR)" or "drug resistance" refers to the ability of a pathogen to evade killing by antimicrobial agents. Pathogens with antimicrobial resistance (AMR) no longer respond to drugs, making infections more difficult to treat and increasing the risk of disease spread, severe illness, and death. Antimicrobials include, but are not limited to, antibiotics, antivirals, antifungals, and antiparasitics. Antimicrobials are drugs used to prevent and treat infections in humans, animals, and plants.

[0085] As used herein, the term "multi-drug resistant (MDR) pathogen" refers to a pathogen that has become resistant to certain antibiotics, which can no longer be used to control or kill the pathogen. Such pathogens include, but are not limited to, fungi, protozoa or bacteria. Exemplary multi-drug resistant fungi include Candida auris, which is resistant to a variety of antifungal drugs commonly used to treat Candida infections. Some strains are resistant to all three types of available antifungal drugs. Multi-drug resistant bacteria are common in medical facilities such as elderly care centers, and include, for example, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), Gram-negative bacteria that produce extended-spectrum β-lactamase (ESBL), and Gram-negative bacteria that produce Klebsiella pneumoniae carbapenemase (KPC).

[0086] The term "ESKAPE pathogens" as used herein is an acronym for six highly virulent and antibiotic-resistant bacterial pathogens, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. These bacterial pathogens can evade or "escape" commonly used antibiotics due to increased multidrug resistance (MDR).

[0087] As used herein, the term "angiogenesis" refers to the growth of blood vessels. The term "angiogenesis-inducing growth factor" refers to a growth factor that induces blood vessel growth, such as vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF).

[0088] The term "mesenchymal stem cells (MSC)" or "mesenchymal stromal cells" as used herein are multipotent stem cells found in the bone marrow that are important for making and repairing skeletal tissues such as cartilage, bone, and fat in the bone marrow. Mesenchymal stem cells are able to differentiate into a variety of cell types, such as osteoblasts, chondrocytes, muscle cells, and adipocytes. With age and the onset of disease, mesenchymal stem cells are primarily transformed into fat cells that accumulate lipids.

[0089] The term "adipose-derived mesenchymal stem cells (ASC)" as used herein refers to mesenchymal stem cells (MSC) obtained from adipose tissue. Although there are two types of adipose tissue (brown and white), white fat produces adipose-derived stem cells (ASC) that are commonly studied. Methods for obtaining adipose-derived mesenchymal stem cells (ASC) are known in the art. The adipose tissue is minced and then enzymatically digested with type II collagenase. After centrifugation, the resulting particles are called stromavascular fraction (stromavascular fraction, SVF). About 2 million to 6 million cells in the stromavascular fraction (SVF) can be obtained from one milliliter of fat extract. The stromavascular fraction (SVF) contains adipose-derived mesenchymal stem cells (ASC), endothelial cells, endothelial progenitor cells, pericytes, smooth muscle cells, leukocytes and erythrocytes. After overnight culture, adipose-derived mesenchymal stem cells (ASC) as a plastic-adherent population are obtained.

[0090] The term "intermediate differentiated cells" or "intermediate differentiated adipose-derived mesenchymal stem cells (ASC)" as used herein refers to tissue-derived stem cells that are in the middle stage of the adipogenic differentiation process in vitro and have not yet completed differentiation into adipocytes (i.e., incompletely differentiated adipocytes), or specifically adipose-derived mesenchymal stem cells (ASC). The timeline for adipogenic differentiation varies from species to species. For example, the adipogenic differentiation of human adipose-derived mesenchymal stem cells (ASC) requires about 12-14 days; the adipogenic differentiation of mice requires about 8-10 days; and the adipogenic differentiation of fish requires about 3-6 days. Therefore, in order to generate intermediate stem cells for mid-term adipogenesis, the timeline should depend on the species being studied. Human intermediate differentiated adipose-derived mesenchymal stem cells (ASC) used herein refer to adipose-derived mesenchymal stem cells (ASC) that undergo adipogenic differentiation for about 4 to 8 days or about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days.

[0091] The term "biofilm" as used herein refers to a membranous tissue formed by pathogens such as bacteria that attach to the chronic wound bed and fuse with the extracellular matrix secreted by the membrane. It is composed of pathogens and their products, extracellular matrix, necrotic tissue, etc. Clinically, biofilm is commonly seen in pressure sores, diabetic foot ulcers, lower extremity arteriovenous ulcers and other chronic wounds.

[0092] As used herein, the term "minimum inhibitory concentration (MIC)" measures the in vitro sensitivity or resistance level of a particular bacterial strain to an antibiotic being used. The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial agent that completely prevents >99% of the visible growth of a test organism strain under strictly controlled in vitro conditions, expressed in mg / L (μg / mL). As used herein, the term "minimum bactericidal concentration (MBC)" measures the lowest concentration of an antimicrobial agent required to kill >99% of a particular bacteria. As used herein, the term "minimum biofilm inhibitory concentration (MBIC)" is defined as the lowest concentration of an antimicrobial agent that inhibits the initial formation of a biofilm. At this concentration, the average number of viable cells in the biofilm does not increase over time.

[0093] As used herein, the expression "microbe" or "microorganism" refers to bacteria, protozoa, algae and fungi.

[0094] As used herein, the expression "antimicrobial response" refers to any host process that is triggered by exposure to bacteria and results in a change in the state, activity or vitality of the microorganism (e.g., motility, secretion, enzyme production or gene expression). For example, the antimicrobial response induced by adipose-derived mesenchymal stem cells (ASCs) described herein after a bacterial sensitization step includes the production of antimicrobial peptides in the culture medium. DETAILED DESCRIPTION

[0095] Chronic wound management is a huge and growing problem in healthcare. Currently, antibiotics are the first-line treatment for wound infections. However, due to the resistance of most pathogens that cause chronic wounds, standard antibiotic treatment has little effect. The emergence of antibiotic-resistant infections and the accompanying lack of new antibiotics are the biggest challenges. Therefore, what is needed is a new antimicrobial agent that is effective in treating chronic infections. In one example, an antimicrobial agent can effectively treat chronic infections, particularly chronic infections caused by antimicrobial-resistant pathogens.

[0096] Mesenchymal stem cells (MSC) have become an attractive alternative cell-based therapy for the treatment of chronic wounds due to their advantages such as ease of isolation, relative abundance, anti-inflammatory effects, angiogenesis regulation and homing ability. Mesenchymal stem cells (MSC) have been studied in a variety of disease indications. It has been demonstrated that mesenchymal stem cells upregulate antimicrobial activity by secreting antimicrobial peptides such as LL-37, cathelicidin, lipocalin-2, β-defensin-2, hepcidin and elastase inhibitors. Superior to existing methods and known peptides, the present disclosure studies the antimicrobial function of adipogenically differentiated mesenchymal stem cells (MSC) based on a bacterial sensitization strategy and isolates novel antimicrobial peptides that can be used to treat chronic infections.

[0097] In one aspect, the present disclosure provides antimicrobial peptides. The antimicrobial peptides disclosed herein are isolated from conditioned medium obtained from intermediately differentiated mesenchymal stem cells sensitized with a bacterial strain. Figure 1 A provides an overview of the parameters for obtaining conditioned medium. Cell viability tests and biofilm staining of exemplary pathogens demonstrated the antibacterial ability of the identified peptides, as detailed in the experimental section. In one example, the antimicrobial peptide has or comprises the amino acid sequence of KVNQIGSVTESIQACKLAQSN (E1A, SEQ ID NO: 3). In another example, the antimicrobial peptide has or comprises the amino acid sequence of HHKAEKSSVLKSKEESH (S1A, SEQ ID NO: 4). In another example, the antimicrobial peptide has or comprises the amino acid sequence of KQRNKVKKIYLDEKR (S1B, SEQ ID No: 5). In some examples, the antimicrobial peptide is at least about 15 amino acids in length.

[0098] In some examples, the antimicrobial peptide comprises one or more partial sequences of ENo3, wherein the antimicrobial peptide comprises at least the sequence KVNQIGSVTESIQACKLAQSN (SEQ ID NO: 3). That is, the peptide sequence may comprise a partial sequence of ENo3 and a peptide sequence of SEQ ID NO: 3. The partial sequence of ENo3 may be from any region of ENo3 and have any length. In some cases, the partial sequence may be located at the N-terminus of SEQ ID NO: 3, or at the C-terminus of SEQ ID NO: 3, or both. Figure 5 A-5C and Figure 6 As demonstrated in Figure 6A-6F, both ENO3 and SPARCL1 showed antibacterial activity against exemplary bacterial strains in the nM range. Figure 7 A to Figure 7D further illustrates the biofilm destruction ability of ENo3 and SPARCL1. In one example, the full-length amino acid sequence of human ENO3 is SEQ ID NO: 1. In some examples, the antimicrobial peptide comprises a partial sequence of SPARCL1, wherein the antimicrobial peptide comprises at least the sequence HHKAEKSSVLKSKEESH (SEQ ID NO: 4). That is, the peptide sequence can comprise a partial sequence of SPARCL1 and a peptide sequence of SEQ ID NO: 4 or 5. The partial sequence of SPARCL1 can be from any region of SPARCL1 and can have any length. In some cases, the partial sequence can be located at the N-terminus of SEQ ID NO: 4 or 5, or at the C-terminus of SEQ ID NO: 4 or 5, or both. In some examples, the antimicrobial peptide comprises a partial sequence of SPARCL1, wherein the antimicrobial peptide comprises at least the sequence KQRNKVKKIYLDEKR (SEQ ID NO: 5). In one example, the full-length amino acid sequence of human SPARCL1 is SEQ ID NO: 2. Table 2 discloses the sequences of full-length human ENO3 and full-length human SPARCL1. In some other examples, the full-length ENO3 or SPARCL1 sequence can be from other species, for example, from a mammal, such as a mouse, monkey, rabbit, dog or hamster. In some other examples, the full-length ENO3 or SPARCL1 sequence can be from a fish.

[0099] In another aspect, the present disclosure provides a conditioned cell culture medium (CM). In one example, the conditioned cell culture medium (CM) comprises an antimicrobial peptide as described herein. In another example, the conditioned cell culture medium (CM) comprises a full-length ENO3 peptide or a full-length SPARCL1 peptide as described herein. In some examples, the culture medium is non-cytotoxic. The conditioned cell culture medium is based on Figure 1 A and Figure 1 B. In one example, the conditioned cell culture medium (CM) is effective against microorganisms with multidrug resistance (MDR). Figure 3 B and Figure 3 As shown in C, after treatment with the conditioned medium described herein, the viability of the resistant bacteria was reduced to less than 50%.

[0100] In another aspect, the present disclosure provides a composition comprising one or more antimicrobial peptides described herein. In one example, the composition comprises a full-length ENO3 peptide or a full-length SPARCL1 peptide as described herein. In another example, the composition further comprises an antibiotic agent. In one example, wherein the composition comprises a full-length ENO3 peptide or a full-length SPARCL1 peptide as described herein, the composition further comprises an antibiotic agent. Fig.14 E and Fig.14F shows the biofilm disruption effect when combined with the exemplary antibiotic ciprofloxacin. Other antibiotics contained in the composition can be, but are not limited to, gentamicin ((3R,4R,5R)-2-{[(1S,2S,3R,4S,6R)-4,6-diamino-3-{[(2R,3R,6S)-3-amino-6-[(1R)-1-(methylamino)ethyl]oxan-2-yl]oxy}-2-hydroxycyclohexyl]oxy}-5-methyl-4-(methylamino)oxane-3,5-diol), colistin (N-(4-amino-1-(1-(4-amino-1-oxo-1-(3,12,23 -tris(2-aminoethyl)-20-(1-hydroxyethyl)-6,9-diisobutyl-2,5,8,11,14,19,22-heptyloxy-1,4,7,10,13,18-hexaazacyclotricosan-15-ylamino)butan-2-ylamino)-3-hydroxybutan-2-ylamino)-1-oxobutan-2-yl)-N,5-dimethylheptylamide), piperacillin ((2S,5R,6R)-6-{[(2R)-2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)amino]-2-phenyl-ethyl =Kanamycin A (2-(aminomethyl)-6-[4,6-diamino-3-[4-amino-3,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-2-hydroxy-cyclohexyloxy]-tetrahydropyran-3,4,5-triol), Kanamycin B ((2S,3R,4S,5S,6R)-4-amino-2-{[(2S,3R,4S,6R)-4,6-diamino- 3-{[(2R,3R,4R,5S,6R)-3-amino-6-(aminomethyl)-4,5-dihydroxyoxan-2-yl]oxy}-2-hydroxycyclohexyl]oxy}-6-(hydroxymethyl)oxane-3,5-diol), nalidixic acid (1-ethyl-7-methyl-4-oxo-[1,8]naphthyridine-3-carboxylic acid), ampicillin ((2S,5R,6R)-6-([(2R)-2-amino-2-phenylacetyl]amino)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid), vancomycin ((1S,2R,18R,19R,22S,25R,28R,40S)-48-{[(2S,3R,4S,5S,6R)-3-{[(2S,4S,5S,6S)-4-amino-5-hydroxy-4,6-dimethyloxan-2-yl]oxy}-4,5-dihydroxy-6-(hydroxymethyl)oxane -2-yl]oxy}-22-(carbamoylmethyl)-5,15-dichloro-2,18,32,35,37-pentahydroxy-19-[(2R)-4-methyl-2-(methylamino)pentanamide]-20,23,26,42,44-pentaoxo-7,13-dioxa-21,24,27,41,43-pentaazaoctane [26.14.2.2. 3,6 .2 14,17 .1 8,12 .1 29,33 .0 10, 25 .0 34,39 ]pentaconta-3,5,8(48),9,11,14,16,29(45),30,32,34,36,38,46,49-pentadecan-40-carboxylic acid), linezolid ((S)-N-({3-[3-fluoro-4-(morpholin-4-yl)phenyl]-2-oxo-1,3-oxazolidin-5-yl}methyl)acetamide) and ciprofloxacin (1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-quinoline-3-carboxylic acid).

[0101] In another aspect, the present disclosure provides a pharmaceutical composition comprising a peptide as described herein. In another example, the present disclosure provides a pharmaceutical composition comprising a conditioned cell culture medium as described herein. In another example, the present disclosure provides a pharmaceutical composition comprising a composition disclosed herein. In some examples, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. Those skilled in the art can provide suitable carriers or excipients for the preparation of the pharmaceutical composition according to the desired dosage form or route of administration.

[0102] In yet another aspect, the present disclosure provides a wound dressing comprising a peptide described herein, a conditioned cell culture medium (CM) described herein, a composition described herein, or a pharmaceutical composition described herein. As used herein, the term "wound dressing" refers to a sterile pad applied to a wound to promote healing and protect the wound from further injury. The dressing is designed to be in direct contact with the wound and may or may not be self-adhesive. In some cases, the wound dressing may be medicated, i.e., contain one or more drugs suitable for wound healing, infection inhibition, pain management, or other purposes. The material of the wound dressing varies depending on the type of wound. For example, the wound dressing may be, but is not limited to, an alginate dressing, a hydrocolloid dressing, or a foam dressing. Those skilled in the art are able to assess the type, shape, and location of the wound to be treated, and select appropriate dressing materials and types for use. The term "conditioned medium" refers to a cell secretory protein group in the form of a cell culture medium, including a collection of proteins shed from the cell surface and intracellular proteins released through atypical secretory pathways or exosomes. These secreted proteins include enzymes, growth factors, cytokines, hormones, and / or other soluble mediators. The term "conditioned medium" as used herein refers to a cell culture medium after culturing the tissue-derived stem cells described herein. The cell culture medium used to prepare the conditioned medium can be a cell culture medium suitable for culturing tissue-derived stem cells. Such cell culture media and methods for preparing them are known in the art, such as mesenchymal stem cell growth medium. The culture medium may not contain xenogeneic components or serum. In one example, the cell culture medium is Essential E6 cell culture medium. Those skilled in the art can select a suitable cell culture medium according to the type of cells to be cultured and add appropriate supplements if necessary.

[0103] In another aspect, the present disclosure provides an in vitro method for inducing an antimicrobial response in tissue-derived stem cells. As used herein, the term "antimicrobial response" refers to a host process that is triggered by exposure to a microorganism and causes a change in the state, activity, or vitality of the microorganism. For example, as used herein, after bacterial sensitization, the antimicrobial response sensitized by the mesenchymal stem cells described herein includes the production of antimicrobial peptides. In one example, the antimicrobial response produces the antimicrobial peptides described herein.

[0104] The term "tissue-derived stem cells" as used herein refers to stem cells derived from somatic tissues that can differentiate into mesenchymal lineages such as bone, cartilage, fat and skin. In one example, the tissue-derived stem cells are mesenchymal stem cells (MSC). In another example, the tissue-derived stem cells can be, but are not limited to: adipose-derived mesenchymal stem cells (ASC), bone marrow-derived MSC (BM-MSC), dental pulp stem cells, and umbilical cord-derived MSC. The term "adipose-derived mesenchymal stem cells (ASC)" as used herein refers to mesenchymal stem cells (MSC) obtained from abundant adipose tissue. Adipose-derived mesenchymal stem cells (ASC) are attached to plastic culture bottles and can be expanded in vitro. Adipose-derived mesenchymal stem cells (ASC) have the ability to differentiate into multiple cell lineages. The exemplary method of inducing antibacterial titer in adipose-derived mesenchymal stem cells (ASC) described in the experimental section can be extended to other types of stem cells commonly recruited at wound sites, such as epidermal stem cells (EPSC), hair follicle stem cells (HFSC), hematopoietic stem cells (HSC) and other multipotent MSCs.

[0105] In one example, the present disclosure provides an in vitro method for inducing an antimicrobial response in tissue-derived stem cells, the method comprising obtaining a tissue sample from a subject. The tissue sample can be, but is not limited to, adipose tissue, bone marrow tissue, dental pulp, or umbilical cord blood. Those skilled in the art can select the appropriate sample type to be obtained from the subject based on the desired tissue-derived stem cell type. In another example, the subject can be a healthy subject. In yet another example, the subject can be an obese subject or a diabetic subject.

[0106] In another example, the present disclosure provides an in vitro method for inducing an antimicrobial response in tissue-derived stem cells, wherein the method includes obtaining a tissue sample from a subject and separating tissue-derived stem cells from the tissue sample in cell culture. Methods for separating tissue-derived stem cells are known in the art. For example, in order to separate adipose-derived mesenchymal stem cells (ASC) from adipose tissue, based on the widely accepted method disclosed in Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001Apr; 7 (2): 211-28), chopped adipose tissue is digested and centrifuged to obtain a stromal vascular fraction (SVF), which contains adipose-derived mesenchymal stem cells (ASC) in a cell group attached to plastic after overnight culture. Those skilled in the art can obtain a suitable separation scheme based on the type of cells and tissue samples used.

[0107] In another example, the present disclosure provides an in vitro method for inducing an antimicrobial response in tissue-derived stem cells. In short, tissue-derived mesenchymal stem cells, such as adipose-derived mesenchymal stem cells, are isolated from a tissue sample of a subject. The isolated cells are cultured in an adipogenic differentiation medium to induce adipogenesis. In the middle stage of adipogenesis, before differentiation is completed, the cells are co-incubated with a live bacterial culture or a bacterial surface antigen to induce an antimicrobial response. The method includes obtaining a tissue sample from a subject, isolating tissue-derived stem cells from the tissue sample in cell culture, and culturing tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated cells. In one example, the differentiation medium is an adipogenic differentiation medium. Various adipogenic methods are known in the art. For example, the adipogenic method is characterized in that tissue-derived stem cells are subjected to treatment with an adipogenic mixture comprising indomethacin, insulin, dexamethasone and IBMX.

[0108] In another example, the present disclosure provides an in vitro method for inducing an antibacterial response in tissue-derived stem cells, wherein the method includes obtaining a tissue sample from a subject, isolating tissue-derived stem cells from the tissue sample in cell culture, culturing tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated cells, and incubating the intermediate differentiated cells with a bacterial culture or a bacterial surface antigen to induce an antibacterial response of the intermediate differentiated cells. "Intermediate differentiated cells" as used herein refer to tissue-derived stem cells undergoing adipogenesis. In one example, "intermediate differentiated cells" refer to tissue-derived stem cells in the middle stage of adipogenesis. The adipogenic differentiation timeline varies from species to species. For example, the adipogenic differentiation of human adipose-derived mesenchymal stem cells (ASC) requires about 12-14 days; the adipogenic differentiation of mice requires about 8-10 days; and the adipogenic differentiation of fish requires about 3-6 days. Therefore, in order to generate intermediate stem cells undergoing mid-term adipogenesis, the timeline should depend on the species being studied. As used herein, the intermediate differentiation adipose-derived mesenchymal stem cells (ASC) from people refer to the adipose-derived mesenchymal stem cells (ASC) that experience about 4-8 days or about 4 days, about 5 days, about 6 days, about 7 days or about 8 days of adipogenic differentiation. In one example, the intermediate differentiation adipose-derived mesenchymal stem cells (ASC) from mice can be obtained between 2-6 days after adipogenic differentiation. In another example, the intermediate differentiation adipose-derived mesenchymal stem cells (ASC) from fish can be obtained between 1-4 days after adipogenic differentiation. In one example, the bacterial culture is the culture of ESKAPE pathogens. In another example, bacterium can be but not limited to Salmonella typhimurium and Pseudomonas aeruginosa. In another example, bacterial surface antigens refer to surface proteins, lipopolysaccharides and peptidoglycans on bacterial cell walls. The bacterial surface antigens for inducing the antibacterial response of intermediate differentiation cells can be but not limited to lipopolysaccharides (LPS), lipoteichoic acid (LTA) and peptidoglycans (PG).

[0109] On the other hand, the present disclosure provides an in vitro method for inducing an antibacterial response in adipose-derived mesenchymal stem cells (ASCs), comprising (a) obtaining an adipose tissue sample from a subject; (b) isolating adipose-derived mesenchymal stem cells (ASCs) from the adipose tissue sample of (a) in cell culture; (c) culturing adipose-derived mesenchymal stem cells (ASCs) in an adipogenic differentiation medium to obtain intermediate-differentiated ASCs; and (d) incubating the intermediate-differentiated ASCs with a bacterial culture or a bacterial surface antigen to induce an antibacterial response in the intermediate-differentiated ASCs. Adipose-derived mesenchymal stem cells (ASCs) are known to interact with pathogenic bacteria and bacterial components at the site of injury, which complements their role in wound regeneration and immune response regulation. However, as Figure 1 C- Figure 1As illustrated in K, intermediately differentiated adipose-derived mesenchymal stem cells (ASCs) elicited an antimicrobial response when sensitized with bacterial culture. In one example of the in vitro method disclosed herein, the antimicrobial response produces the antimicrobial peptides described herein.

[0110] On the other hand, the present disclosure provides a method for producing a conditioned cell culture medium (CM) as described herein, wherein the method comprises: (a) obtaining a tissue sample from a subject; (b) isolating tissue-derived stem cells from the tissue sample of (a); (c) culturing tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated stem cells; (d) incubating intermediate differentiated cells in a cell culture medium with a bacterial culture or a bacterial surface antigen; and (e) collecting conditioned cell culture medium (CM) by removing bacteria and intermediate differentiated cells. Methods for removing cells from cell culture are known in the art, for example, centrifugation at an appropriate speed to separate cells and cell culture medium based on weight difference.

[0111] In yet another aspect, the present disclosure provides a method for preparing the conditioned cell culture medium (CM) described herein, wherein the method comprises: (a) obtaining an adipose tissue sample from a subject; (b) isolating adipose-derived mesenchymal stem cells (ASCs) from the adipose tissue of (a) in cell culture; (c) culturing adipose-derived mesenchymal stem cells (ASCs) in an adipogenic differentiation medium to obtain intermediate differentiated ASCs; (d) incubating the intermediate differentiated ASCs in a cell culture medium with a bacterial culture or a bacterial surface antigen; and (e) collecting the conditioned cell culture medium (CM) by removing the bacteria and the intermediate differentiated ASCs.

[0112] In one example, tissue-derived stem cells / ASC are mammalian cells. In some examples, tissue-derived stem cells / ASC can be human cells, mouse cells or fish cells. In some examples, the incubation time period of tissue-derived stem cells / ASC and bacterial culture or bacterial surface antigens in cell culture medium can be about 14 to 48 hours. In another example, the bacteria to be incubated with intermediate differentiation tissue-derived stem cells / ASC in bacterial culture are live bacteria. In some other examples, the number ratio of bacteria contacting tissue-derived stem cells / ASC can be but is not limited to about 1:50-1:75, about 1:75-1:100, about 1:100-1:200, about 1:200-1:225 and about 1:225-1:250. In some examples, the added glucose concentration is high or low, or whether there is additional FBS in the cell culture medium, it will not affect the results of the methods described herein. Figure 1 B shows the specific conditions of the test. In another example, the subject can be a healthy subject. In yet another example, the subject can be an obese subject or a diabetic subject. Figure 1 C- Figure 1E and Figure 1 Comparison between F-1H showed that the same conditions were consistently effective on cell lines obtained from healthy subjects or diabetic patients.

[0113] In some examples, for the antimicrobial peptides described herein, the conditioned cell culture medium (CM) described herein, or the pharmaceutical compositions described herein, the microorganism can be a bacterium, a fungus, or a protist. In one example, the fungus is Candida albicans. In another example, the protist can be, but is not limited to, Acanthamoeba spp. and Leishmania tropica.

[0114] In one aspect, the present disclosure provides a method for treating an infection in a subject. The method comprises administering to the subject a pharmaceutically effective amount of a peptide described herein, a composition described herein, or a pharmaceutical composition described herein. Figure 6 A-6H demonstrated that ENO3 and SPARCL1 peptides effectively killed exemplary microorganisms. Fig.12 A-12C demonstrates that short peptides such as E1A, S1A and S1B effectively kill exemplary microorganisms. The term "pharmaceutically effective amount" as used herein generally refers to an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or potential causes, prevent the occurrence of symptoms and / or their potential causes, and / or improve or remedy damage caused by or associated with a disease state (e.g., alleviate infection). Those skilled in the art can determine the pharmaceutically effective amount of the peptides, compositions, or pharmaceutical compositions disclosed herein based on considerations such as disease state, body size, frequency of administration, and route. In some instances, the infection is a mixed infection. In one instance, the infection is a bacterial infection. In another instance, the subject may also include a fungal infection, a protist infection, or a combination thereof. In some examples, the infection may be caused by one or more of the following microorganisms: Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Enterobacter cloacae, Serratia marcescens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus sanguinis, or Lactobacillus casei.

[0115] In one example, the present disclosure provides use of a peptide described herein, a composition described herein, or a pharmaceutical composition described herein in the preparation of a medicament for treating an infection in a subject.

[0116] In another example, the infection is a chronic infection, or a nosocomial infection. In yet another example, the infection is an infection caused by a drug-resistant microorganism. In yet another example, the infection is caused by a multidrug-resistant microorganism. In some instances, the infection is resistant to one or more antimicrobial agents, such as but not limited to: vancomycin, ampicillin, gentamicin, kanamycin A, neomycin B, neomycin C, neomycin E, amikacin, tobramycin, dibekacin, sisomicin, netilmicin, streptomycin, plazomicin, ticarcillin, pefloxacin, ceftriaxone and methicillin. Figure 3 B and 3C provide examples of resistant microorganisms that responded to the exemplary conditioned media treatments described herein even after 28 days of long-term treatment.

[0117] In one aspect, the present disclosure provides a method for promoting wound healing and tissue regeneration in a subject. The method comprises administering to the subject a pharmaceutically effective amount of a peptide described herein, a composition described herein, a conditioned medium described herein, or a pharmaceutical composition described herein. Figure 8 A-8D provides examples of healthy exfoliated dermal human skin equivalent (DED-HSE) models with and without wounds. Wounded DED-HSE samples treated with the conditioned medium described herein showed that keratinocytes were concentrated in the wound area, indicating that keratinocytes proliferated in the wound area. Figure 8 D demonstrates wound closure and recovery of a skin model in a sample treated with conditioned medium as described herein. In one example, the wound has not yet shown symptoms of infection. In another example, the wound is an infected wound. In yet another example, the wound undergoes chronic infection. In one example, the wound is infected by one or more microorganisms. In another example, the wound is infected by one or more bacteria. In some examples, the wound is infected by bacteria including but not limited to: Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Enterobacter cloacae, Serratia marcescens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus sanguinis or Lactobacillus casei. In some instances, the bacteria in the wound are resistant to one or more antimicrobial agents, such as, but not limited to, vancomycin, methicillin, gentamicin, kanamycin A, amikacin, tobramycin, dibekacin, sisomicin, netilmicin, neomycin B, neomycin C, neomycin E, streptomycin, prazosin, ticarcillin, pefloxacin, ceftriaxone, and ampicillin.

[0118] In one example, the present disclosure provides use of a peptide described herein, a composition described herein, a conditioned medium described herein, or a pharmaceutical composition described herein in the preparation of a medicament for promoting wound healing and tissue regeneration in a subject.

[0119] In one aspect, the present disclosure provides a method of reducing / preventing biofilm formation in a subject. The method comprises administering to the subject a pharmaceutically effective amount of a peptide described herein, a composition described herein, a conditioned medium disclosed herein, or a pharmaceutical composition described herein. Figure 7 A-7D and Fig.13 A-13C shows that the peptide described herein effectively destroys the biofilm formed by exemplary microorganisms. In one example, the biofilm is formed by one or more microorganisms. In another example, the biofilm comprises one or more bacteria. In yet another example, the bacteria present in the biofilm can be but not limited to: Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Enterobacter cloacae, Serratia marcescens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus sanguis or Lactobacillus casei. In one example, the bacteria present in the biofilm are resistant to one or more antimicrobial agents. In another example, the one or more antibacterial agents can be, but are not limited to, vancomycin, methicillin, gentamicin, kanamycin A, amikacin, tobramycin, dibekacin, sisomicin, netilmicin, neomycin B, neomycin C, neomycin E, streptomycin, prazosin, ticarcillin, pefloxacin, ceftriaxone, and ampicillin.

[0120] In some examples, the effective biofilm disruption concentration range of E1A can be, but is not limited to, at least about 0.1 μM, at least about 0.15 μM, at least about 0.2 μM, at least about 0.25 μM, at least about 0.3 μM, at least about 0.35 μM, or at least about 0.39 μM. In some examples, the effective biofilm disruption concentration range of E1A can be, but is not limited to, less than about 3.125 μM, less than about 3 μM, less than about 2.5 μM, less than about 2 μM, less than about 1.75 μM, less than about 1.25 μM, or less than about 1 μM. In one example, the effective biofilm disruption concentration range of E1A is about 0.39 μM-1.56 μM.

[0121] In some examples, the effective biofilm disruption concentration range of S1A can be, but is not limited to, at least about 0.1 μM, at least about 0.2 μM, at least about 0.3 μM, at least about 0.4 μM, at least about 0.5 μM, at least about 0.6 μM, or at least about 0.78 μM. In some examples, the effective biofilm disruption concentration range of S1A can be, but is not limited to, less than about 6.25 μM, less than about 6 μM, less than about 5 μM, less than about 4 μM, less than about 4.5 μM, less than about 4.25 μM, less than about 3.75 μM, less than about 3.5 μM, or less than about 3.125 μM. In one example, the effective biofilm disruption concentration range of S1A is about 0.1 μM-0.3 μM. In another example, the effective biofilm disruption concentration range of S1A is about 0.78 μM-3.125 μM.

[0122] In some examples, the effective biofilm disruption concentration range of S1B can be, but is not limited to, at least about 3.2 μM, at least about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at least about 5 μM, at least about 5.5 μM, at least about 6 μM, or at least about 6.25 μM. In some examples, the effective biofilm disruption concentration range of S1B can be, but is not limited to, less than about 25 μM, less than about 22 μM, less than about 20 μM, less than about 18 μM, less than about 15 μM, less than about 13 μM, or less than about 12.5 μM. In another example, the effective biofilm disruption concentration range of S1B is about 6.25 μM-12.5 μM.

[0123] In one example, the present disclosure provides use of a peptide described herein, a composition described herein, a conditioned medium described herein, or a pharmaceutical composition described herein in the preparation of a medicament for reducing / preventing biofilm formation in a subject.

[0124] In one aspect, the present disclosure provides a method for treating an infection in a subject, wherein the method comprises administering to the subject a pharmaceutically effective amount of a peptide described herein, a full-length ENO3 peptide, or a full-length SPARCL1 peptide. In one example, the present disclosure provides the use of a peptide described herein, a full-length ENO3 peptide, or a full-length SPARCL1 peptide in the preparation of a medicament for treating an infection in a subject. In one example, the method further comprises administering an antibiotic agent to the subject. In some examples, the infection to be treated is located at a wound, such as a chronic wound. In some examples, the infection to be treated is a situation where there is no wound.

[0125] In another aspect, the present disclosure provides a method for promoting wound healing and tissue regeneration in a subject, wherein the method comprises administering to the subject a pharmaceutically effective amount of a peptide, a full-length ENO3 peptide, or a full-length SPARCL1 peptide as described herein. In one example, the present disclosure provides the use of a peptide, a full-length ENO3 peptide, or a full-length SPARCL1 peptide as described herein in the preparation of a medicament for promoting wound healing and tissue regeneration in a subject.

[0126] In another aspect, the present disclosure provides a method for reducing / preventing biofilm formation in a subject, wherein the method comprises administering to the subject a pharmaceutically effective amount of a peptide, a full-length ENO3 peptide, or a full-length SPARCL1 peptide as described herein. In one example, the present disclosure provides the use of a peptide, a full-length ENO3 peptide, or a full-length SPARCL1 peptide as described herein in the preparation of a medicament for reducing / preventing biofilm formation in a subject. In one example, the method further comprises administering to the subject an antibiotic agent.

[0127] In one example of the methods described herein, the peptide and antibiotic agent are administered together or separately. Administration may be, but is not limited to, intramuscular administration, topical administration, and intravenous administration. In some instances, the peptide, conditioned medium, composition, or pharmaceutical composition described herein to be delivered to the subject is embedded in a hydrogel, polymer, or nanofiber or attached to a nanoparticle. Delivery systems such as hydrogels or poly(lactic-co-glycolic acid) (PLGA) nanoparticles can improve the bioavailability and stability of the peptide. In some instances, the subject is diagnosed with obesity or diabetes, or is diagnosed as being at risk of obesity or diabetes. In some instances, the subject suffers from diabetic foot ulcers and / or diabetic foot infections.

[0128] On the one hand, the present disclosure provides nucleic acid compositions encoding peptides described herein. On the other hand, the present disclosure provides expression vectors comprising nucleic acid compositions described herein. On the other hand, the present disclosure provides host cells comprising one or more nucleic acid compositions described herein or one or more expression vectors described herein. In one example, the host cell is a mammalian cell, a bacterium, a yeast cell, a fungal cell or a plant cell. In another example of the method disclosed herein, the tissue sample is a fat sample. In some other examples, the tissue sample is a subcutaneous fat sample.

[0129] The disclosure illustratively described herein may be appropriately implemented without any one or more elements, any one or more limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. are to be interpreted broadly and without limitation. In addition, the terms and expressions used herein are used as terms of description rather than limitation, and when using these terms and expressions, it is not intended to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications may be made within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been specifically disclosed through preferred embodiments and optional features, those skilled in the art may make modifications and changes to the invention embodied in the present invention disclosed herein, and such modifications and changes are considered to be within the scope of the present invention.

[0130] It should be further appreciated that the exemplary embodiments are merely examples and are not intended to limit the scope, applicability, size or configuration of the present invention in any way. On the contrary, the above detailed description will provide a convenient roadmap for those skilled in the art to implement the exemplary embodiments of the present invention, and it should be understood that various changes can be made to the functions and arrangements of the elements described in the exemplary embodiments and the preparation methods without departing from the scope of the present invention as defined in the appended claims.

[0131] Experimental Section

[0132] Adipose-derived mesenchymal stem cell (ASC) lines

[0133] With the approval of the National Healthcare Group Domain Specific Review Board, white adipose tissue (WAT) was isolated from subcutaneous (abdominal region) fat depots of all human volunteers undergoing bariatric surgery. Adipose-derived mesenchymal stem cells (ASCs) were isolated and enriched by serial subculture of stromal vascular fraction (SVF). Adipose-derived mesenchymal stem cells (ASCs) were obtained from healthy donors (ATCC SCRC 4000) or obese patients (S29, 37M; S23, 34F).

[0134] Bacterial strains

[0135] Salmonella enterica subsp. enterica typhimurium (ATCC 14028s, ST), Pseudomonas aeruginosa (ATCC 14213, PA) and methicillin-resistant Staphylococcus aureus (BAA1717-USA 300, SA) were obtained from the American Type Culture Collection (ATCC). In the priority patent application filed on May 9, 2022 (Singapore Patent Application No.: 10202204848T), the Salmonella typhimurium bacterial strain used in the sensitization step of the disclosed method was inadvertently mistaken for Enterococcus faecalis. The disclosure provided in Singapore Patent Application No. 10202204848T is based on experimental data generated by an exemplary bacterial strain Salmonella typhimurium.

[0136] Peptide synthesis

[0137] The full-length wild-type peptide sequence was obtained from PubMed (http: / / www.ncbi.nlm.nih.gov / ). The recombinant antibodies obtained were anti-ENO3 antibody (Abcam, catalog number ab157474) and anti-SPARCL1 antibody (R&D systems, catalog number CF2728-SL-050). Short antimicrobial peptides were then synthesized with a purity greater than 98%.

[0138] Adipogenesis, bacterial sensitization, and CM collection

[0139] 100,000 adipose-derived mesenchymal stem cells (ASC) were seeded in 6-well plates and grown for 2 days to D0. The method of adipogenic differentiation is known in the art. Generally, an adipogenic mixture of 3-4 components is used to carry out adipogenic induction of adipose-derived mesenchymal stem cells (ASC), and the mixture includes indomethacin, insulin, dexamethasone and IBMX. In a specific example, according to previous reports, two days after adipose-derived mesenchymal stem cells (ASC) reach confluence, an adipogenic mixture containing 1mM dexamethasone, 0.5mM IBMX, 167nM insulin and 100mM indomethacin is used to induce ASC. Adipose-derived mesenchymal stem cells (ASC) are induced with an adipogenic mixture for about 12 days. On the 6th day, cells are transferred to a culture medium with 167nM insulin and 1mM dexamethasone, and maintained until the 12th day. The culture medium is replaced every 3 days until the 12th day.

[0140] Adipose-derived mesenchymal stem cells (ASC) were sensitized with heat-killed (HI, 80°C, 30 minutes) or live Salmonella typhimurium or Pseudomonas aeruginosa at different infection multiplicity (MOI) within 3-24 hours, and the corresponding conditioned medium (CM) was obtained at D0 (undifferentiated stage), D6 (intermediate differentiation) and D12 (complete differentiation). This sensitization was carried out in the presence of high (4.5g / l) or low (1g / l) D-glucose and ±10% FBS. To prevent acidification, 20mM HEPES was added during bacterial sensitization of ASC. The conditioned medium obtained from adipose-derived mesenchymal stem cells (ASC-CM) was filtered with a 0.2μM syringe filter to eliminate any bacterial contamination.

[0141] Antibacterial test

[0142] Conditioned medium and recombinant peptides obtained from adipose-derived mesenchymal stem cells (ASC-CM) were examined for their efficacy against Salmonella typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus strains using the broth microdilution protocol in the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI 2012). Briefly, five freshly plated bacterial colonies were grown in a cation-adjusted MH2 broth in a shaking incubator at 37°C and 220 RPM. Cells were grown to an optical density (OD600) of 0.20 using a microplate spectrophotometer. 100 μL of microbial culture containing approximately 5×10 5 CFU / mL of bacteria in each broth and incubate overnight at 37°C. Next, to check the bactericidal effect, transfer 100 μL from each well and spread on a sterile MH2 agar plate. The plates were incubated overnight at 37°C and the number of colony forming units (CFU) was observed the next day. The readings of bacteria grown in medium without any treatment were normalized to 100% as a medium control. The minimum bactericidal concentration (MBC) is defined as the minimum peptide concentration (μM) required to kill ≥50% of bacterial colonies compared to the medium control.

[0143] ATP detection in bacterial cells

[0144] To determine the antibacterial effect of ASC-CM or antimicrobial peptides, the intracellular ATP level of bacteria was measured by ATP Determination Kit. As indicated in the antibacterial test protocol, after overnight incubation of ASC-CM or peptides with bacteria for 24 hours, the culture was first spun to eliminate extracellular ATP and other metabolic wastes released from dead bacteria. The bacterial pellet was resuspended in an equal amount of water, and the level of intracellular ATP was calculated based on the manufacturer's instructions.

[0145] Estimation of biofilm destruction

[0146] Biofilms were grown in cation-adjusted MH2 broth in 96-well clear-bottom polystyrene plates for 72 h. Overnight bacterial cultures were subcultured for 90 min until OD 600 Reach about 0.3-0.4, prepare the biofilm inoculum. 200 μL of biofilm inoculum was aliquoted into each well of a 96-well plate. After 72 hours of static incubation at 37°C, the spent culture medium was drained and the biofilm was treated with ASC-CM for 24 hours. After treatment, the level of remaining attached biofilm was quantified by crystal violet staining to determine the minimum biofilm inhibition concentration (MBIC) showing a >50% reduction. In brief, all contents in the 96-well plate were removed, and the wells were then washed three times with 1x PBS (phosphate buffered saline) to remove unattached planktonic bacteria. The attached biofilm was fixed with 95% methanol for 5 minutes. The wells were air-dried and stained with 0.1% crystal violet solution for 15 minutes at room temperature. The crystal violet solution was removed and the wells were washed three times with clean water. The plate was air-dried again, and the stained biofilm was dissolved with 30% acetic acid. Micro xMark was used. TM Microplate Absorbance Spectrophotometer(Micro xMark TM Microplate absorbance spectrophotometer) at OD 595 The absorbance of the biofilm was recorded.

[0147] Antimicrobial resistance evolution test

[0148] The development of resistance in Salmonella Typhimurium (ST) strains was evaluated by serial passaging in the presence of subinhibitory concentrations of gentamicin or ASC-CM. Briefly, serial passaging of Salmonella Typhimurium (ST) cells was completed over a 4-week period with full-strength, 1 / 2-strength, and 1 / 4-strength ASC-CM or subinhibitory concentrations of gentamicin. Cells grown in subinhibitory conditioned medium (CM) or gentamicin were diluted to A 600nm (OD 600) was 0.1, and then serial passages were performed at the same concentration every day for 4 weeks at 37°C with agitation every 24 h. A high minimum inhibitory concentration (MIC) strain of Salmonella typhimurium (ST) (1024 μg / ml) was stored and used to evaluate the efficacy of potent ASC-CM.

[0149] Neutralization test

[0150] To verify the bioactive role of ENO3 and SPARCL1, conditioned medium (CM) was pretreated with primary antibodies at a final concentration of 5 μg / ml for 1 hour before the start of the antibacterial assay. Conditioned medium (CM) pre-incubated with 5 μg / ml rabbit IgG (Abcam, catalog number ab172730) was used as an isotype control.

[0151] Protein fractionation of ASC-CM by HPLC

[0152] ASC-CM was fractionated using HPLC using a ZORBAX GF-250 size exclusion (gel filtration) column to identify active fractions. The flow rate was maintained at 0.3 ml / min, and 20 mM Na2HPO4 / NaH2PO4 buffer at pH 7.0 was used as the mobile phase. The chromatograms of the conditioned medium (CM) were superimposed and compared to find the unique peaks present in the potent fractions. Then, at different stages of adipogenesis, conditioned medium (CM) fractions (vials 2, 3, and 4) were collected from unsensitized or sensitized Salmonella typhimurium or Pseudomonas aeruginosa conditioned medium (CM) fractions were stored at -20 ° C until further use. In order to clarify the active fractions that retain antibacterial properties, the collected HPLC fractions were first filtered using a 0.45 μm cellulose acetate syringe filter. The antibacterial effect of the filtered HPLC fractions was checked.

[0153] Mass spectrometry (MS) analysis

[0154] At room temperature, the selected HPLC fractions (100 μl) were first treated with 4 μl 500 mM tris (2-carboxyethyl) phosphine (TCEP) and then 550 mM chloroacetamide (CAA). For digestion, 100 μl of 100 mM triethylammonium bicarbonate (TEAB) and 1 μl of 0.5 μg / μl LysC were added, and the samples were then incubated for 2 h (hours) in a thermomixer set at 37 ° C and 900 rpm. Next, 2 μl of modified trypsin was added and the samples were incubated overnight at room temperature. Each sample was digested with 20 μl of supernatant. The sample was acidified with 1% trifluoroacetic acid (TFA) and then passed through 10 mg reverse phase C18 beads (pore size 10 μm) which had been pre-equilibrated with 50 μl 100% acetonitrile, 75 μl 0.5% (v / v) acetic acid in 100 μl buffer A (0.5% acetic acid in water). The bound sample was washed in 100 μl buffer A to remove residual salts. The sample was eluted with buffer B (0.5% acetic acid in 65% acetonitrile) and dried in a speed vacuum. The dried sample was sent for MS analysis and stored at 4°C until further use. Next, the remaining sample that showed good initial digestion was acidified with 1% TFA. Use Desalting was performed using an HLB 1cc / 10mg column. The reagent or sample was pushed into the column using a syringe. The column was activated with 1ml of 100% acetonitrile. Equilibration was performed with 1ml of 0.5% aqueous acetic acid. The acidified sample was loaded into the column and the flow-through was collected. The column was washed with 1ml of 0.5% aqueous acetic acid. The sample was eluted into a new tube with 1ml of 0.5% acetic acid in 65% acetonitrile. The sample was then dried in a high-speed vacuum. After drying, the sample was resuspended in 30μl of buffer B. The tip was prepared by loading the reverse phase C8 beads into a 200μl pipette tip. The resuspended sample was pushed through the tip into a new tube to remove contaminants. The sample was then dried in a high-speed vacuum and sent for mass spectrometry (MS) analysis.

[0155] Determination of the efficacy of ASC-CM in an ex vivo wound model

[0156] The effect of ASC-CM at different stages of adipogenic differentiation to support human keratinocyte proliferation and migration in 3D superficial wounds was validated in a healthy de-epidermated dermis (DED) human skin equivalent (HSE) model, which closely resembles native human skin. Keratinocytes were transferred to a ring placed on the DED and incubated for 2 days. The ring was removed and the DED-HSE was moved to the air:liquid interface using a stainless steel grid. Keratinocyte proliferation was determined using thiazolyl blue (MTT) and hematoxylin and eosin (H&E) staining was performed to assess wound closure.

[0157] Cell viability assay

[0158] Following the manufacturer's instructions, a cell titer blue assay was performed to determine the safety of the peptides to the mouse ASC lines. Peptides ENO3 and SPARCL1 were incubated with mASC lines at the indicated concentrations for 48 h. Fluorescence intensity was measured using a Cytation 3 Cell Imaging Multi-Mode Reader.

[0159] Mouse excisional wound model

[0160] The mouse wound infection model was performed as described with slight modifications. After treatment with ENO3 and SPARCL1 for 24 hours, the infected skin area was excised, homogenized, and then the viable bacteria were counted by plating the dilutions on BHI plates and antibiotic selection plates (PA 14213 with 12.5 μM vancomycin). Statistical analysis was performed by Student t test compared to the untreated group (PBS). All approved procedures were performed in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) (ARFSBS / NIEA0198Z) of the School of Biological Sciences, Nanyang Technological University, regarding the mouse wound infection model.

[0161] 2D scratch test

[0162] The cell confluence of human keratinocytes at each time point in the presence of peptides in the two-dimensional scratch assay was assessed by applying the ImageJ plugin PHANTAST on phase contrast time-lapse images. The rate of change of keratinocyte confluence was calculated by obtaining the best fit line on the confluence versus time plot as described. The rate of change of confluence was used as a surrogate measure of keratinocyte migration speed.

[0163] Statistical analysis

[0164] Results are presented as mean ± SEM. The differences in the mean values ​​between the two groups were determined using Student's t test. p values ​​were calculated using ANOVA to perform multiple comparisons with correction, and p < 0.05 was considered significant.

[0165] Example

[0166] Bacteria-sensitized intermediate adipogenic ASC-CM exhibits robust antibacterial effects

[0167] Conditioned medium obtained from intermediately differentiated adipose-derived mesenchymal stem cells (ASC-CM) exhibits antimicrobial effects. The conditions that trigger this antimicrobial property of ASC-CM were investigated. ASC-CM obtained from healthy donors (ATCC SCRC 4000) and obese patients (S29 and S23) were compared to ASC growth medium without antibiotics against Salmonella typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus. As shown in the circle ( Figure 1 A) or bold underline ( Figure 1 As highlighted in B), live bacterial primed ASCs at a bacteria:ASC ratio of approximately 1:200 exhibited >50% bacterial killing regardless of culture conditions (high / low glucose or ± FBS). Treatment with bacteria primed intermediate differentiation ASC-CM for 24 hours resulted in significant killing of all bacterial strains of S. typhimurium, P. aeruginosa, and S. aureus based on viable plate counts ( Figure 1 C-1K).

[0168] Intermediate adipogenic differentiation ASC-CM exerts significant biofilm disruption effects

[0169] The intermediate adipogenic differentiation ASC-CM significantly disrupted the biofilm-forming ability of the exemplary bacterial strains Salmonella typhimurium and Pseudomonas aeruginosa. This activated ASC-CM showed >75% disruption of 3-day-old bacterial biofilms grown in Mueller Hinton broth (MHB). Figure 2 ).

[0170] Activated ASC-CM prevents the emergence of antimicrobial resistance (AMR)

[0171] First, an exemplary bacterial strain, Salmonella typhimurium ( Figure 3 A). Potent ASC-CM exhibits robust killing against high MIC S. Typhimurium strains ( Figure 3 B). Most importantly, unlike gentamicin treatment, continuous passage of ASC-CM for 4 weeks showed negligible emergence of S. typhimurium resistance ( Figure 3 C).

[0172] Elucidation of the antibacterial effect of the active fraction obtained by HPLC

[0173] To identify active fractions, the possible antimicrobial properties of sterile HPLC fractions of ASC-CM were compared with each culture medium fraction. In all three adipose-derived mesenchymal stem cell (ASC) samples, the fractions sensitized with Salmonella typhimurium or Pseudomonas aeruginosa from bottle 3 (V3) showed significant antimicrobial effects compared with the other vials based on colony counts ( Figure 4 A- Figure 4 C). Next, the active V3 fractions were subjected to mass spectrometry (MS) analysis to identify novel AMPs.

[0174] Mass spectrometry (MS) analysis of the ASC-CM fraction obtained by HPLC identified two novel antimicrobial peptides, ENO3 and SPARCL1

[0175] MS settings were set to detect peptides with charges of 2-7 and 1. Results of searching the raw data in Proteome Discoverer2.3. Each raw data was searched for the human_uniprot_AMP database using the non-labeled quantitative (Label-free quantification, LFQ) setting. The normalized relative intensity ratio of the bacterial sensitized fraction compared to the non-sensitized fraction was determined by fold change (FC). FC>1 indicates that the abundance of peptides in the sensitized fraction is higher. As shown in Table 2, more abundant peptides ENO3 and SPARCL1 were detected in the sensitized fraction.

[0176] Table 1: FC values ​​of ENO3 and SPARCL1 based on MS results

[0177] ST sensitized, non-sensitized ATCC S29 S23 ENO3(344-358) 15.36 112 23.6 SPARCL1(607-617) 1.64 2.72 ＜1

[0178] PA - sensitized, non-sensitized ATCC S29 S23 ENO3(344-358) 26.2 31.2 11.2 SPARCL1(607-617) 1.43 1.97 ＜1

[0179] Table 2: Sequences of peptides ENO3 and SPARCL1

[0180]

[0181] Pretreatment of CM with anti-ENO3 and anti-SPARCL1 antibodies reversed the antimicrobial properties of potent ASC-CM

[0182] To establish a link between the detected antimicrobial peptides and the observed antimicrobial effects of ASC-CM, antimicrobial assays were performed using conditioned medium (CM) pretreated with anti-ENO3 and SPARCL1 antibodies. In all 3 ASCs, pretreatment with anti-ENO3 antibodies completely reversed the growth inhibitory effects of bacteria-sensitized conditioned medium (CM) compared to untreated ASC-CM or CM pretreated with isotype control antibodies ( Figure 5 A- Figure 5 C). This suggests that ENO3 is the primary cause of the potent CM-mediated growth inhibition induced by bacterial sensitization. Similarly, pretreatment of ATCC SCRC 4000 or S29 ASC-CM with anti-SPARCL1 antibodies also reversed the growth inhibition of S. typhimurium, thus validating that SPARCL1 functions as an antimicrobial peptide that is effective against bacteria.

[0183] Peptides ENO3 and SPARCL1 showed significant bactericidal effects

[0184] To directly identify ENO3 and SPARCL1 as novel AMPs, the antimicrobial efficacy of the recombinant peptides was tested using the broth microdilution protocol from the CLSI guidelines (CLSI 2012). ENO3 significantly killed S. typhimurium at 2.04 nM, while much lower concentrations were required to kill P. aeruginosa (0.204 nM) and S. aureus (0.102 nM) strains, respectively. The minimum bactericidal concentration (MBC) of SPARCL1 was 0.0067 nM (S. typhimurium), 268 nM (P. aeruginosa), and 1000 nM (S. aureus) ( Figure 6 A- Figure 6 F). Overall, both peptides showed significant killing of all three bacterial pathogens in the nM range. For Staphylococcus aureus, 2 μM concentration of ENO3 reduced viable colonies by >99% compared to the growth control (GC). For Pseudomonas aeruginosa, 6 μM concentration of ENO3 reduced viable colonies by >99% compared to the growth control (GC). Figure 6 G and Figure 6 H shows a spot plate image. Table 3 below lists the MIC / MBC of ENO3 against S. aureus and P. aeruginosa. These results validate the bacterial killing potential of ENO3 against both S. aureus and P. aeruginosa.

[0185] Table 3: Killing effect of ENO3 on Staphylococcus aureus and Pseudomonas aeruginosa

[0186]

[0187] Peptides ENO3 and SPARCL1 exhibit bacterial biofilm disruption effects

[0188] Both peptides showed robust antibiofilm effects (>50%) against Pseudomonas aeruginosa (PA) and Salmonella typhimurium (ST) strains in the nM range. Figure 7 A- Figure 7 D).

[0189] ASC-CM accelerates wound healing in an ex vivo wound model

[0190] The safety and efficacy of ASC-CM were examined in a 3D ex vivo de-epidermated dermis (DED) human skin equivalent (HSE) model. ASC-CM showed no toxicity, especially at the intermediate stage, and increased the proliferation of keratinocytes as determined by thiazolyl blue (MTT) and hematoxylin and eosin (H&E) staining. Keratinocytes formed stratified epithelium ( Figure 8 B) to minimize uncovered wound area ( Figure 8 C). In contrast, less keratinocyte invasion at the wound site and a wider uncovered wound area were observed in undifferentiated or fully differentiated CMs ( Figure 8 B. Figure 8 C). Therefore, in addition to its antimicrobial properties, intermediate CM also exhibits properties that promote wound healing ( Figure 8 D).

[0191] Potent ASC-CM accelerates the migration of human keratinocytes

[0192] In a 2D scratch assay, ATCC cells treated with ASC-CM increased the migration of human keratinocytes, highlighting increased wound healing compared to medium control or CM derived from D0 or D12 that did not exhibit antimicrobial properties ( Fig. 9 ).

[0193] AMP, ENO3, and SPARCL all induce keratinocyte migration in a 2D scratch wound model

[0194] Both ENO3 and SPARCL1 peptides showed increased migration of human keratinocytes in a 2D scratch assay, highlighting increased wound healing compared to the media control group or the group significantly damaged by gentamicin, meaning that the antimicrobial peptides disclosed herein are more effective than standard antibiotic treatment ( Fig.10 ).

[0195] Both ENO3 and SPARCL1 exhibit in vivo efficacy

[0196] ENO3 and SPARCL1 biofilm disruption ability in a mouse excisional wound infection model. 3 The excised wounds were infected with CFU of Pseudomonas aeruginosa 14213 and treated for 24 hours to allow biofilm formation. ENO3 (0.2 nM) and SPARCL1 (0.067 nM) were then used for treatment for 24 hours post-infection ( Figure 7 ), ENO3 and SPARCL1 have previously shown significant in vitro biofilm disruption effects. After treatment, the wound area was excised and the number of bacteria was counted.

[0197] Treatment with ENO3 and SPARCL1 both resulted in a decrease in bacterial colonies compared to untreated controls ( Fig.11 ).

[0198] Identification of ultrashort peptide sequences within ENO3 and SPARCL1 with potent antimicrobial activity

[0199] In order to develop antimicrobial peptides as topical drugs for chronic wound infections and reduce production costs, short peptide regions (<22 residues) with potent antimicrobial properties were identified within ENO3 and SPARCL1 for clinical development. To this end, the peptide sequences obtained by mass spectrometry were examined. The predicted secondary structure, cationicity and hydrophobic regions in ENO3 and SPARCL1 that are critical for bactericidal activity were analyzed. Table 4 lists exemplary short peptides with potential antimicrobial functions identified from ENO3 and SPARCL1.

[0200]

[0201] Short peptides E1A, S1A and S1B showed significant bactericidal effects

[0202] In order to directly determine the antibacterial efficacy of the short peptides, the antibacterial effects of the obtained 98% purified E1A, S1A and S1B peptides were tested using the broth microdilution procedure. All three short peptides showed significant killing effects on Pseudomonas aeruginosa 14213, and their minimum bactericidal concentration (MBC) values ​​were different. Compared with S1A (100μM) and S1B (50μM), E1A showed the lowest MBC (0.39μM) ( Fig.12 A- Fig.12 C).

[0203] E1A, S1A and S1B showed destructive effects on PA 14213 biofilm

[0204] All three peptides showed robust antibiofilm effects (>50%) against P. aeruginosa ( Fig.13 A- Fig.13 C).

[0205] E1A, S1A, and S1B significantly enhance the biofilm-disrupting properties of antibiotics

[0206] Combination biofilm disruption assays of E1A and two clinically relevant exemplary antibiotics, ciprofloxacin and colistin, were performed against P. aeruginosa 14213. E1A in combination with the antibiotics significantly reduced P. aeruginosa biofilms compared to the antibiotics alone, implying a synergistic effect ( Fig.14 A and Fig.14B). Combination of S1A and S1B, respectively, with the exemplary antibiotic ciprofloxacin reduced P. aeruginosa biofilm compared to ciprofloxacin alone ( Fig.14 C and Fig.14 D).

[0207] ENO3 and SPARCL1 enhance the biofilm-disrupting properties of antibiotics

[0208] Similarly, a biofilm disruption assay was performed for Pseudomonas aeruginosa 14213 in combination with ENO3 and SPARCL1 and the exemplary antibiotic ciprofloxacin. The combination of ENO3 and SPARCL1, respectively, with the exemplary antibiotic ciprofloxacin reduced Pseudomonas aeruginosa biofilms compared to ciprofloxacin alone ( Fig.14 E and Fig.14 F).

[0209] E1A significantly accelerated the migration of human keratinocytes, demonstrating wound healing efficacy

[0210] In a 2D scratch assay, the exemplary peptide E1A showed increased migration of human keratinocytes compared to the medium control group, highlighting increased wound healing ( Fig.15 ).

[0211] In summary, as described herein, the present disclosure provides conditioned medium derived from intermediate differentiated mesenchymal stem cells triggered by bacterial sensitization. The conditioned medium exhibits robust antibacterial and wound healing properties. The strategy described herein for inducing antibacterial responses with bacterial sensitization can be extended to other stem cell populations that are typically present in the wound environment and are undergoing different differentiation processes. In addition, the present disclosure also provides antimicrobial peptides isolated from conditioned medium as preclinical targets, which combat chronic wound infections through robust antimicrobial efficacy, biofilm disruption efficacy, keratinocyte migration promotion efficacy, and in vivo chronic wound healing efficacy. Antimicrobial peptides also exhibit synergistic biofilm disruption effects when combined with antibiotics such as ciprofloxacin and colistin.

[0212] The above examples are provided in order to fully disclose and describe to those of ordinary skill in the art how to make and use embodiments of the compositions, systems and methods of the present invention, but are not intended to limit the scope of what the inventors regard as their invention. Modifications to the above-described modes of carrying out the present invention will be apparent to those skilled in the art and are intended to fall within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the present invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference were individually incorporated by reference in its entirety.

[0213] Many modifications and variations may be made to the present application without departing from the spirit and scope of the present application, which will be apparent to those skilled in the art. The specific embodiments and examples described herein are provided by way of example only, and the present application is limited only by the terms of the appended claims and the full range of equivalents enjoyed by the claims.

Claims

1. An antimicrobial peptide consisting of the sequence KVNQIGSVTESIQACKLAQSN (SEQ ID NO: 3).

2. An antimicrobial peptide consisting of the sequence HHKAEKSSVLKSEESH (SEQ ID NO: 4).

3. An antimicrobial peptide consisting of the sequence KQRNKVKKIYLDEKR (SEQ ID NO: 5).

4. An antimicrobial peptide comprising a partial sequence of ENO3, wherein the peptide comprises at least the sequence KVNQIGSVTESIQACKLAQSN (SEQ ID NO: 3). The peptide according to claim 4 , wherein the ENO3 has the amino acid sequence of SEQ ID NO:

1.

6. An antimicrobial peptide comprising a partial sequence of SPARCL1, wherein the peptide at least comprises the sequence HHKAEKSSVLKSKEESH (SEQ ID NO: 4).

7. An antimicrobial peptide comprising a partial sequence of SPARCL1, wherein the peptide comprises at least the sequence KQRNKVKKIYLDEKR (SEQ ID NO: 5). The peptide according to claim 6 or 7, wherein the SPARCL1 has the amino acid sequence of SEQ ID NO:

2.

9. A conditioned cell culture medium (CM) comprising the antimicrobial peptide, full-length ENO3 peptide or full-length SPARCL1 peptide according to claims 1 to 8.

10. A composition comprising one or more of the antimicrobial peptides, full-length ENO3 peptides or full-length SPARCL1 peptides according to any one of claims 1 to 8.

11. The composition of claim 10, further comprising an antibiotic agent.

12. The composition of claim 10, wherein the antibiotic agent is selected from the group consisting of gentamicin, colistin, piperacillin, kanamycin, nalidixic acid, ampicillin, vancomycin and linezolid and ciprofloxacin.

13. A pharmaceutical composition comprising the peptide of any one of claims 1 to 8, the conditioned cell culture medium of claim 9, or the composition of any one of claims 10 to 12.

14. A wound dressing comprising the peptide of any one of claims 1 to 8, the conditioned cell culture medium (CM) of claim 9, the composition of any one of claims 10 to 12, or the pharmaceutical composition of claim 13.

15. An in vitro method for inducing an antimicrobial response in tissue-derived stem cells, wherein the method comprises: (a) obtaining a tissue sample from a subject; (b) isolating tissue-derived stem cells from the tissue sample of (a) in cell culture; (c) culturing the tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated cells; and (d) incubating the intermediate differentiated cells with a bacterial culture or a bacterial surface antigen to induce an antibacterial response of the intermediate differentiated cells.

16. The in vitro method according to claim 15, wherein the antimicrobial response produces the antimicrobial peptide according to any one of claims 1 to 8.

17. The in vitro method according to claim 15 or 16, wherein the differentiation medium is an adipogenic differentiation medium.

18. An in vitro method for inducing an antimicrobial response in adipose-derived mesenchymal stem cells (ASCs), comprising: (a) obtaining an adipose tissue sample from a subject; (b) isolating adipose-derived mesenchymal stem cells (ASCs) from the adipose tissue sample of (a) in cell culture; (c) culturing the adipose-derived mesenchymal stem cells (ASCs) in an adipogenic differentiation medium to obtain intermediate differentiated ASCs; and (d) incubating the intermediate differentiated ASCs with bacterial culture or bacterial surface antigens to induce an antibacterial response of the intermediate differentiated ASCs.

19. The in vitro method according to claim 18, wherein the antimicrobial response produces the antimicrobial peptide according to any one of claims 1 to 8.

20. An in vitro method for producing the conditioned cell culture medium (CM) of claim 9, comprising: (a) obtaining a tissue sample from a subject; (b) isolating tissue-derived stem cells from the tissue sample of (a); (c) culturing the tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated stem cells; (d) incubating the intermediate differentiated cells with a bacterial culture or a bacterial surface antigen in a cell culture medium; and (e) collecting the conditioned cell culture medium (CM) by removing the bacteria and the intermediate differentiated cells.

21. A method for preparing the conditioned cell culture medium (CM) of claim 9, comprising: (a) obtaining an adipose tissue sample from a subject; (b) isolating adipose-derived mesenchymal stem cells (ASCs) from the adipose tissue of (a) in cell culture; (c) culturing the adipose-derived mesenchymal stem cells (ASCs) in an adipogenic differentiation medium to obtain intermediate differentiated ASCs; (d) incubating the intermediate differentiated ASCs with bacterial culture or bacterial surface antigens in a cell culture medium; and (e) collecting the conditioned cell culture medium (CM) by removing the bacteria and the intermediate differentiated ASCs.

22. The method according to any one of claims 15 to 21, wherein the bacterium is selected from Salmonella typhimurium or Pseudomonas aeruginosa.

23. The method of any one of claims 15-22, wherein the subject is obese or has diabetes.

24. A method for treating an infection in a subject, or a method for promoting wound healing and tissue regeneration in a subject, or a method for reducing / preventing biofilm formation in a subject, comprising administering to the subject a pharmaceutically effective amount of the peptide of any one of claims 1-8, the conditioned cell culture medium (CM) of claim 9, the composition of any one of claims 10-13, or the pharmaceutical composition of claim 14.

25. The method of claim 24, wherein the infection comprises a bacterial infection.

26. The method of claim 24, wherein the infection is resistant to one or more antimicrobial agents.

27. A nucleic acid composition encoding the peptide of any one of claims 1-8.

28. An expression vector comprising the nucleic acid composition of claim 27.

29. A host cell comprising one or more nucleic acid compositions of claim 27 or one or more expression vectors of claim 28.

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