Fatty Acid Modification Of Antimicrobial Peptide CGA-N9 And The Anti-candida Activity

CGA-N9（NH₂-RILSILRHQ）is an antifungal peptide found in our lab.It is a Candida selective antibacterial peptide that preferentially kills Candida,but has low activity against Candida albicans.In order to enhance the anti-Candida activity and improve the bioavailability of the antimicrobial peptide CGA-N9,the analogues of CGA-N9 modified with different fatty acids were synthesized.The structures were characterized by high resolution mass spectrometry（HRMS）,nuclear magnetic resonance（NMR）and circular dichroism.The antimicrobial activity,stability,anti-biofilm activity,hemolysis activity and human hepatocyte toxicity of the antimicrobial peptide CGA-N9 and its fatty acid modified analogues were measured.After selecting the best peptide,further in vitro biological activity and in vivo inflammatory inhibition effect were studied,and the following results were obtained:（1）Structural characterization of antimicrobial peptide CGA-N9 and its fatty acid modified analogues:CGA-N9-C2、CGA-N9-C4、CGA-N9-C6、CGA-N9-C8 and CGA-N9-C10 were obtained by modifying CGA-N9 with fatty acids of different carbon chain lengths.Circular dichroism showed that the secondary structure of CGA-N9、CGA-N9-C2、CGA-N9-C4 and CGA-N9-C6 consisted mainly ofα-helix and random coil.The secondary structure of CGA-N9-C8 is dominated byα-helix.The results above indicate that with the increase of fatty acid chain length,theα-helix content of CGA-N9 and its fatty acid modified analogues increases,and the water solubility of CGA-N9 analogues decreases.Finally,CGA-N9-C10 is insoluble in PBS.（2）Studies on the activity of antimicrobial peptide CGA-N9 and its fatty acid modified analogues:With the extension of the adipose chain,the anti-Candida albicans activity increased.CGA-N9-C8 is the highest antimicrobial peptide among CGA-N9 and its analogues,and the activity of CGA-N9-C8（MIC:7.8±0.03μg/mL）increased by 31.6 times compared with CGA-N9（MIC:246.4±2.3μg/mL）.The antimicrobial peptide CGA-N9 and its fatty acid modified analogies CGA-N9 are less affected by serum.The serum stability of CGA-N9 and its fatty acid modified analogies CGA-N9 is maintained in the range of 72.6%to 78.9%by HPLC.The peptide CGA-N9-C8 has the highest inhibition or elimination ability of C.albicans biofilm,compared with CGA-N9 MBIC₅₀（105.2μg/mL）,the MBIC₅₀of CGA-N9-C8 was 50.5μg/mL.Meanwhile,270.9μg/mL CGA-N9-C8 eliminated half of the biofilm.The safety of CGA-N9-C8 is the highest in mammatic cells,and the therapeutic index of CGA-N9-C8 is up to 93.6 in human erythrocytes and 84.1 in human hepatocytes.By comparison of biological activity,CGA-N9-C8 is the best fatty acid modified analogue of CGA-N9.（3）Under 2×MIC treatment for 3 h,CGA-N9-C8 can kill C.albicans.Increase the concentration,CGA-N9-C8 can inhibit（31.3μg/mL）and kill（62.5μg/mL）more than 99%of the C.albicans persister cells.CGA-N9-C8 has low resistance to C.albicans.The antimicrobial peptide CGA-N9-C8 induces active oxygen production and cell membrane depolarization,which may be the main causes of C.albicans cell apoptosis.These results indicate that CGA-N9-C8 is an effective choice to solve the problems of C.albicans infection and drug resistance.（4）Therapeutic effect of CGA-N9-C8 on mice with C.albicans deep infection:After treatment with 0.94 mg/kg CGA-N9-C8,the survival rate of mice increased from 40%to90%.After treatment with 0.94 mg/kg CGA-N9-C8,the body weight increased by about6.77-6.92 g,the health index of 4-5 points（out of 5）.After treatment with 0.94 mg/kg CGA-N9-C8,the amount of bacteria in the kidneys of mice infected with C.albicans decreased by 52.61%on average.The change trend of immune organ index showed that CGA-N9-C8 could stimulate the enhancement of immune function of mice immune organs（spleen and thymus）.After 7 days of treatment,the spleen index of mice reached 10.33 mg/g,4.29 mg/g higher than that of infection group（6.04 mg/g）.After 14 days of treatment,the thymus index of mice was as high as 4.78 mg/g,which was 2.04 mg/g higher than that of the infection group（2.74 mg/g）.The pathological changes of the internal tissue sections of mice before and after the treatment of the peptide CGA-N9-C8 of 0.94 mg/kg were found that the liver lobule structure was normalized,the pulmonary interstitial congestion was reduced,the alveolar cavity was enlarged,the germinal center of the spleen was clearly visible,and the renal interstitial tissue was not bleeding after the treatment of CGA-N9-C8.It’s indicating that the peptide CGA-N9-C8 had the therapeutic effect on the deep infection of C.albicans.Results show that CGA-N9-C8 is the best CGA-N9 analogues,and with the highest resistant C.albicans activity.Compared with CGA-N9,CGA-N9-C8 inhibit or kill C.albicans,and inhibit or kill C.albicans persister cells activity,and show the biofilm inhibition activity and ability of biofilm eradication.CGA-N9-C8 showed low tendency of drug resistance in C.albicans.CGA-N9-C8 reduces the pathological effect of C.albicans infection in a mouse model of deep Candida infection.In conclusion,CGA-N9-C8 is the best candidate that to resist C.albicans infection and solve the problem of C.albicans resistance.