Research On Therapeutic Potential Of TP11A(Tachyplesin Analog Peptide)against C.albicans-S.aureus Mixed Biofilm Formation And Infection

Among infectious diseases,microbial biofilm infections pose a great therapeutic challenge.With the discovery of fungal-bacterial mixed biofilms on wounds and medical devices,antimicrobial spectrum range should be emphasized during the development of novel antibacterial agents.Candida albicans and Staphylococcus aureus are common symbiotic strains in fungal-bacterial mixed biofilms.These two species could exhibit synergistically enhanced resistance to antibiotics when they co-form the biofilms.As the raising problem of antimicrobial resistance has burdened the difficulty of clinical therapies of microbial infections,antimicrobial peptides（AMPs）have attracted increasing attentions as an alternative of novel antimicrobials.AMPs,basic components of innate immunity,are considered to be less susceptible to serious drug resistance with a broad-spectrum antimicrobial activity against bacteria,fungi,viruses,and parasites.Tachyplesin I（TP1）,a broad-spectrum AMP,is derived from the hemolymph of Tri-spine horseshoe crab（Tachypleus tridentatus）.Limited by the high mammalian cytotoxicity,few researches have exploited TP1’s efficient antimicrobial activities for therapeutic uses.By changing the amino acid sequence of TP1 at the 11th position,its analog TP11A（KWCFRVCYRGACYRRCR）were obtained to reduce the cytotoxicity by 64 times while retaining the advantageous antimicrobial properties,showing great development prospects.According to previous studies,both TP1 and TP11A can effectively eradicate a variety of bacteria and fungi,but there is a lack of relevant data on their antibiofilm activity,and meanwhile,the research on their in vivo efficacy and application to infectious diseases is quite scarce.Thus,the present study further explored TP1’s and TP11A’s antibiofilm activities and mechanisms,and subsequently investigated the appropriate delivery strategy of TP11A and its potential as a polymicrobial wound infection therapeutic agent.（1）Comparison of TP1’s and TP11A’s in vitro antimicrobial activities against Candida albicans and Staphylococcus aureus,antibiofilm activities,and cytotoxicity to skin cells.The MIC₉₀ of TP11A（the lowest drug concentration that inhibits more than90%microbial growth）determined by the micro-double dilution method was 24μg/m L against C.albicans SC5314 and 2μg/m L against S.aureus ATCC 6538 respectively,which basically retained TP1’s antimicrobial activity.The results of crystal violet staining and colony forming units（CFU）counting showed that TP1 and TP11A had similar antibiofilm activities——both had significant eradicating and inhibitory abilities against S.aureus monomicrobial biofilms,and can strongly inhibit the growth of C.albicans monomicrobial biofilms and C.albicans-S.aureus polymicrobial biofilms.The cytotoxicity of TP1 and TP11A to human keratinocyte Ha Ca T and mouse fibroblast NIH3T3 was detected using cck-8 kits,and the results confirmed that TP11A had a higher skin cell cytocompatibility than TP1.All the above results suggest that TP11A has a more promising potential for therapeutic development and application for biofilm infection.（2）Investigation of TP11A’s antimicrobial and antibiofilm mechanisms:reactive oxygen species（ROS）generation,morphology observation of C.albicans-S.aureus mixed biofilms,q RT-PCR.On the basis of inhibitory effect of TP11A against C.albicans-S.aureus polymicrobial biofilms,we further explore their antimicrobial and antibiofilm mechanisms.Firstly,the results of DCFH-DA fluorescence intensity detected by confocal laser scanning microscope showed that TP11A induced ROS production when killing C.albicans and S.aureus.Then,field emission scanning electron microscopy（FE-SEM）was used to observe the structural changes of C.albicans-S.aureus polymicrobial biofilm induced by TP11A,in which TP11A significantly inhibited the adhesion of S.aureus and the maturation of the biofilm.In addition,we performed q RT-PCR to investigate the expression of biofilm-and virulence-related genes in TP11A-induced C.albicans-S.aureus polymicrobial biofilm.It was found that key genes for hyphal production and adhesion of C.albicans were significantly changed by TP11A,in which hwp1 was significantly down-regulated,while als1 and als3 was significantly up-regulated.Several studies have confirmed that the deletion of hwp 1 led to biofilm deletion and immaturity.In the present study,immature development of TP11A-induced biofilms may be related to hwp 1 down-regulation,which might also cause negative feedback regulation to up-regulate the expression of als1 and als3.Concerning S.aureus,TP11A globally down-regulated the expression of biofilm-and virulence-related genes,involving adhesion-related genes ica A and fnb B,QS system regulator agr A,major virulence gene hla,drug resistance efflux pump gene nor A,and global stress regulator Sig B,which showed the molecular basis of TP11A’s robust antimicrobial ability to eradicate and inhibit S.aureus in the polymicrobial biofilm.（3）In vivo efficacy of TP11A against C.albicans-S.aureus mixed polymicrobial infection:suitable delivery strategies for TP11A,curative effects on mice in vivo model of polymicrobial infected wounds.Proteolysis,hydrolysis,oxidation,and photolysis of AMPs are known to easily occur as a result of wound-related and environmental variables.Thus,TP11A was embedded in biomimetic nanovesicles and poly（D,L-acrylonitrile）ester)-poly（ethylene glycol）-poly（D,L-lactide）（PLEL）gel,aiming to improve application and compatibility in in vivo anti-infection investigations.The results showed that nanovesicles were not suitable for TP11A delivery as the AMP destroyed the integrity of macrophage biomimetic nanovesicles;while in situ thermosensitive gel system PLEL@TP11A created by simply adding TP11A into PLEL solution can achieve topical fixation at wound sites and sustained release of loaded TP11A,which is a suitable delivery strategy for TP11A.In order to investigate therapeutic efficacy in vivo,C.albicans-S.aureus polymicrobial infected wounds of BALB/c mice were constructed and administered TP11A and PLEL@TP11A.Both TP11A and PLEL@TP11A effectively reduced bacterial and fungal loads in polymicrobial wound infections,accelerated the wound healing process,and promoted epithelialization and angiogenesis.To sum up,the present study demonstrated the potential of Tachyplesin analog peptide TP11A to combat biofilms and polymicrobial infections,and revealed the molecular basis for TP11A-induced inhibition of C.albicans-S.aureus polymicrobial biofilms.These findings lay a foundation for further therapeutic development of TP11A to treat polymicrobial biofilms and infections.