| Many financial damages to livestock rearing and public health may result from combined bacterial illnesses.Gram-negative and Gram-positive pathogenic microorganisms that are typical in animal husbandry are E.coli and Streptococcus suis.Bacterial resistance has become more severe recently as a result of the misuse of medications and the horizontal transfer of genes.As a result,bacterial antibiotic resistance has become a global problem.The need to discover novel antibacterial agents and alternative medications has grown.While most compounds are excluded during this step for a variety of reasons,the creation of novel medications is a challenging and complex process that frequently involves several challenges.Hence,increasing precursor yield may be accomplished by employing various screening techniques and enhancing screening throughput.Based on the commonly used whole-bacteria screening method,this study innovatively developed a screening system for virulence proteins,and designed and modified new antimicrobial peptides for bacterial membrane structures.In view of the fact that clinical anti-drug-resistant bacterial infections are often used in combination with different target drugs,multiple screening methods can not only greatly improve the discovery efficiency of active precursors,but also avoid the possibility of crossresistance between the screened active precursors.The final selected drugs all showed good antibacterial and anti-inflammatory activities in vivo and in vitro,and had potential clinical application value.Below are key research findings:1.Whole-bacteria inhibition for antibacterial compound screening By using whole bacterial inhibition,a total of 23,696 small compounds from natural secondary metabolites,FDA-approved medications,NIH clinical medications,natural products,and chemically produced medications were examined.In the end,86 potent antibacterial chemicals were discovered.We discovered that ellipticine had impressive in vitro antibacterial,bactericidal,and antibacterial properties,as well as effective in vivo bactericidal and anti-inflammatory benefits.The outcome of the mechanism study revealed that ellipticine bound to Gram-negative topoisomerase IV to prevent the growth of E.coli.This screening process also discovered the substance auranofin,which significantly inhibits the development of gram-positive Bacteria.Moreover,auranofin demonstrated blatant antibacterial and bactericidal properties in vitro and in vivo against the Gram-positive bacterium Streptococcus.Moreover,animals infected with Streptococcus had a strong inhibitory impact on the inflammation they induced.At the later stages of infection,it also demonstrated superior therapeutic efficacy to the firstline medication ampicillin.The blood biochemistry of mice reveals auranofin had no negative effects,even at greater therapeutic levels.Auranofin was found to have these effects by binding to the thioredoxin Trx B and significantly lowering the number of free thiols in Streptococcus suis,which in turn significantly lowers the organism’s ability to resist oxidative stress.Moreover,eukaryotic cells have an oxygen-sulfur reduction compensation mechanism,which allows them to reduce the negative effects of auranofin.2.Compound screening by suppression of bacteria toxin protein activity Traditional whole-bacteria inhibition screening would miss some inhibition effects that are not immediately apparent but have toxin protein inhibition activity of chemicals.Drugs targeting the structure of bacterial toxin protein and synthesis of substances do not directly threaten bacterial survival,the use of the process is less likely to cause bacteria to produce resistance mutations and can lengthen the use cycle of drugs.The research will greatly expand the range of clinical drug selection,relieve the pressure of antibiotic resistance,and reduce the need for antibiotics.The drug’s use cycle can be prolonged.Streptococcus suis produces hemolysin,a virulence protein that is crucial to the pathogenic process of infecting host cells and piercing host cell membranes.As a result,stopping Streptococcus suis infection by targeting hemolysins is successful.We tested the structural similarities of baicalin and apigenin,two chemicals,for their ability to prevent hemolytic activity.The growth of Streptococcus suis was unaffected by these two substances,but they both significantly inhibited hemolysin-induced inflammation and exhibited strong hemolytic inhibitory action.Both substances had the ability to attach to hemolysin proteins,but apigenin was different in that it produced strong hydrogen bonds with the SLY amino acid residues ASN50,LEU110,ASN112,GLN177,and ASP179 and strong hydrophobic interactions with the residues THR191,PHE193,GLY194,and LYS224.While baicalein and the SLY amino acids SER84 and THR191 formed strong hydrophobic bonds.ASN82,ALA88,GLN177,ASP179,and LYS224 were among the amino acids with which baicalein established hydrogen bonding connections.S.suis was unable to infect cells as a result of this interaction,which altered the hemolysin protein’s structure and interfered with its capacity to puncture cells.In animal tests,we combined the antibacterial activity of ampicillin with the antitoxin impact of herbal medicine in mice with severe sensitization to produce superior therapeutic outcomes than first-line medicines alone.3 Antimicrobial peptide design and modificationThe helical peptide L-10,a positively charged amphipathic-helical peptide with good antibacterial activity,was obtained through design and modification after being discovered in this study to be derived from the chemotactic factor of grass carp.When compared to antibiotics,L-10 did not easily result in the development of resistance.It also had good in vivo and in vitro anti-inflammatory effects and did not affect the blood biochemical indexes of mice.L-10 also showed good therapeutic effects in Galleria mellonella larvae infection,mouse thigh infection,and mouse abdominal infection models.By binding to both LPS and PG,L-10 primarily altered the membrane’s permeability,which influenced medication entrance into the bacteria and,when coupled with other pharmaceuticals,improved the capacity of other drugs to enter the bacterium.According to the RNA-seq findings,L-10 also influences how bacteria transport and metabolize medications,suppresses the production of membrane proteins and restores bacteria’s sensitivity to antibiotics that are previously resistant to it.We resolved the antibacterial mechanism of these small molecules,screened a variety of highly active antibacterial small molecules using three different screening techniques(all bacterial,antibacterial toxic protein,and synthetic peptides),and preliminary assessed the anti-inflammatory activity and in vivo therapeutic effects of these small molecules using animal experiments and toxicity tests,laying the groundwork for further in-depth development and clinical studies. |
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