Expression,Purification And Antimicrobial Activity Of HNP-1 And Its Mechanism

Antibiotic resistance has become a serious problem that endangers global public health.Due to the use of antibiotics in the world continue to increase,almost all of the pathogens are showing antibiotic resistance.Unfortunately,the development of new antibiotics is far behind the growth rate of drug-resistant bacteria,and with the large increase in the use of antibiotics,the emergence of bacterial resistance is even more transient.Antibacterial peptides are important candidates for the development of new antibiotics,which are expressed by the host to eliminate invasive pathogens and enhance the immune response.Among them,human neutrophil protein-1(HNP-1)is the major antibacterial peptide molecule of human neutrophils,which mainly exists in the lysosomal granules of human neutrophils.HNP-1 has a strong antimicrobial activity against gram-positive and gram-negative bacteria as well as adenovirus,influenza virus,and HIV,and may become the new generation of antibacterial drug.Although HNP-1 has broad application prospects,the amount of naturally obtained HNP-1 is extremely limited,and the chemical synthesis of this peptide is a challenge due to the complex pattern of disulfide bonds which stabilize their structure.Therefore,researchers tried to express HNP-1 by genetic engineering through various fusion expression strategies.However,it is difficult to form HNP-1 with a correct structure,or the formed HNP-1 exists in the inclusion bodies.This study intend to investigate the activation process and bactericidal mechanism of HNP-1,which will provide the experimental basis for the efficient expression of HNP-1 in prokaryotic system.The contents and conclusions of this paper mainly include the following three aspects:1.In the process of expression of full-length HNP-1 in E.coli,mature HNP-1 is activated.1)Tricine gel system was used to compare the differences of protein expression before and after IPTG induction,this pattern showed an obvious band near 3.4 kD after induction,and the peptide was identified as mature HNP-1 by bottom-up and top-down mass spectrometry.2)The expression of mature HNP-1 was increased gradually with the prolongation time of IPTG induction,while the number of viable bacteria was significantly decreased.3)The whole protein solution was subjected to centrifugation with 3 kD ultrafiltration tube to obtain the purified mature HNP-1 protein.4)The growth curves were determined by the addition of preproHNP-1 and mature HNP-1,indicating that preproHNP-1 had no antibacterial activity while mature HNP-1 had bacteriostatic effect.2.HNP-1 triggered bacterial programmed cell death.1)Label-free quantitative proteomics were used to compare differentially expressed proteins before and after IPTG induction,then HNP-1 was presumed to trigger bacterial programmed cell death based on functional proteins clustering.2)TUNEL and Annexin V staining showed that DNA and membrane structure were severely damaged in the presence of HNP-1.Moreover,scanning electron microscopy showed that the bacterial surface produced a large number of apoptotic vesicles.These classic phenotypic experiments confirmed that HNP-1 induces bacterial programmed cell death in E.coli.3.HNP-1 directly bind to RecA to exacerbate bacterial programmed cell death.1)The HNP-1 interacting protein was enriched with Ni-NTA beads,then the interacting proteins were detected by mass spectrometry,and the proteins interacting with HNP-1,RecA,was singled out by protein annotation.2)Western blot was used to verify the presence of RecA expression in the HNP-1-enriched interaction protein solution.On the other hand,the RecA protein antibody was used to enrich its interacting proteins,and HNP-1 was also identified,further confirming the interaction between the HNP-1 and RecA.3)With the prolongation of induction time,mature HNP-1 gradually replaced the combination of preproHNP1 and RecA,which further indicated that the combination of mature HNP-1 and RecA was dominant.4)The binding sites of RecA and HNP-1 were predicted by molecular docking software ZDOCK and PyMoL,the results showed that HNP-1 was able to bind to Asp-161 and Ser-162 at RecA to inhibit its recombinant repair activity.5)Adding 10 ?M mature HNP-1 can significantly reduce the binding of RecA and ssDNA by in vitro experiments.Through this study,we found that during the expression of full-length HNP-1 in E.coli,mature HNP-1 was activated.In addition,the purified mature HNP-1 was obtained by ultrafiltration,which provides a simple and cost-effective solution for the production of mature HNP-1.Our study also revealed that mature HNP-1 can inhibit the binding of RecA protein and ssDNA,thereby inhibiting its SOS repair function.Due to its important functions,RecA has become an ideal target for the treatment of drug-resistant bacteria.Because of its important role in inhibiting RecA activity,HNP-1 is expected to become a new drug to combat drug-resistant bacteria.