| Objective:Methicillin-resistant Staphylococcus aureus(MRSA)infection is still a major global healthcare problem,causes a wide range of infections commonly involving the skin,soft tissue,bone,joints,and infections associated with indwelling catheters or prosthetic devices.Of concern is pneumonia,which exhibits high rates of morbidity and can cause metastatic or complicated infections.However,the antimicrobial agent recommend treatment for MRSA has limitations.Several issues restrict the utility of the antibiotics,including slow bactericidal activity,low tissue penetration,and increasing reports of resistance and failure.Cell division is extremely important for development,reproduction and death of microorganisms.Binary fission is the primary method of reproduction of bacteria,involves the division of a parent cell into two daughter cells.The essence of antibacterial drugs inhibits the growth and reproduction or kills bacteria,by inhibited the synthesis of cell walls,change the permeability of membranes and hinder the synthesis of cytoplasmic nucleic acids or proteins.With the innovation of science and technology,binary fission has been well studied in spherical bacteria,the mechanisms underlying cell division in bacteria are understood.The latest research about bacterial division proteins and the treadmilling has provided new ideas for the development of antibiotics.Traditional Chinese Medicine mostly utilized in complement therapy with Western Medicine which are filling up the preventive and curative therapy.But they are not acting only in this direction,a major argument had successfully passed the exam of history being in vivo tested for hundreds or thousands of years.In 2000,natural medicine was used by 80%of the population about according to World Health Organization.Traditional Chinese medicine has efficient antimicrobial activities,even revealing the importance of this unexploited resource in antibiotic resistance of pathogen agents.TanReQing(TRQ)injection is the aqueous extract of a formulation from Traditional Chinese Medicine containing five drugs(Scutellaria baicalensis Georgi,Bear Gall powder Goral horn,Lonicera japonica Thunb and Forsythia suspense).Although antimicrobial activities of TRQ have been investigated,but the mechanisms have not revealed.We found that TRQ are different from antibiotics,which could damage the biofilm in view of our early study.In this paper,TRQ was investigated the effect on the cell division of MRSA.The study was elaborated from bacterial division morphology,regulation of bacterial division protein FtsZ,peptidoglycan synthesis and hydrolysis.Method:1.The effect of TRQ on the morphology of MRSA division by Scanning Electron Microscope(SEM)The overnight cultured bacteria were inoculated into the bottom of a 24-well plate with sterile glass slides.Add drugs,and the concentration of bacterial solution was finally OD600=0.05,and cultured at 37? for 24 hours.The bacteria attached to the glass were taken out,and were fixed with glutaraldehyde and citric acid,respectively,subjected to gradient dehydration treatment,and treated with a vacuum freeze dryer to completely dry,and gold was sprayed and photographed by SEM.The length of the bacterial cells was measured using Image J.2.The effect of TRQ on the MRSA cell cycle by Structured Illumination Microscopy(SIM)The bacterial solution was diluted at a ratio of 1:1000.cultured for 5 hours,and repeated three times,ensure that the bacteria were in a logarithmic division phase.The drug was added so that the final concentration of the bacterial solution was OD600=0.05 and cultured at 37? for 30 minutes.Then,the Nile Red and WGA-488 were added for dyeing,and the cells were collected 10 minutes later.After fixing with 1%agarose in PBS solution,it was observed using SIM.Use Imaris for data processing.3.Effect of TRQ on MRSA cleavage protein FtsZ by Western Blot(WB)Bacterial culture and drug treatment were carried out according to the method in the experiment.2.The cells were collected by centrifugation,and the bacterial protein extract was added for extraction.The protein content was measured using a BCA kit The protein were loaded to ensure that the total protein content per well was equal.SDS-PAGE electrophoresis was taken,transferred protein gel to PVDF membrane,blocked using skim milk,incubated primary antibody at 4 ? overnight,cultured secondary antibody after rinsing,added ECL chemiluminescence solution,using ChemiDocTM imaging system for detection.4.Effect of TRQ on the expression of PG synthesis in MRSA by Enzyme-Linked Immunosorbent Assay(ELISA)Bacterial culture and drug treatment were carried out according to the method in the experiment.2.After collecting the cells,added ultrapure water,the cell wall was broken using a cell disrupter to perform peptidoglycan extraction.Follow the protocol in the ELISA kit instructions.Detected with a microplate reader.5.Effects of TRQ on MRSA division-related genes by Real Time PCRDesign and synthesize primers for the interest gene.Bacterial culture and drug treatment were carried out according to the method in the experiment.2.The bacteria were ground by adding liquid nitrogen to extract bacterial total RNA.Concentration and purity were measured by using NanoDrop.Denatured agarose gel electrophoresis was carried out and detected under ultraviolet light transmission and photographed.The RNA was reverse transcribed into cDNA,and Real Time PCR was performed,and the data was analyzed by a method of 2-??CT.Result:1.TRQ inhibit the division of bacteria significantly.The result of bacterial length indicated that the length of the undivided bacteria in the TRQ group was greater than that the control and vancomycin group.Morphologically,single cells can be observed abnormal large,or multi-cell body cannot be divided had been observed with the presentation of the initial bacterial membrane retraction.As well as the position of the old division scar is deviated evidence TRQ inhibiting the division.In addition,the bacteria are suppressed after the treatment of TRQ,and the surface becomes uneven.2.TRQ can destroy the structure of the bacterial dividing membrane,making it impossible to divide.Different from the mature dividing membrane formed by the control and the vancomycin group,a large number of filamentous membranes were observed on and inner of the cell membrane in the TRQ group.and no dividing membrane was formed with an equatorial-like Z-ring.According the stage of the division cycle,the bacterial cell was sorted.TRQ was mostly in the stage of bacterial division phase 2 which the membrane synthesis was being carried out,and the cells did not have obvious elongation.The control and the vancomycin group were mostly in the stage of phase 3,which had formed the mature membrane and the cells were significantly elongated to prepare for dividing into two daughter cells.3.TRQ increase the content of division protein FtsZ,affecting the progression of bacterial division.Compared with the control and the vancomycin group,TRQ increased the expression level of FtsZ protein in MRS A with a dose-dependent manner.When the FtsZ protein excess,the amount of the divsion protein bound to it is diluted,which hinders the formation of the diving complex product.In addition,it can also reduce the space for other division proteins to react with each other,increasing the difficulty of their reaction,thereby inhibiting bacterial division.4.TRQ can block the hydrolysis of peptidoglycan to inhibit bacterial division.TRQ increased the content of peptidoglycan in MRSA compared to the control and the vancomycin group.Peptidoglycan content increased with TRQ concentration.Peptidoglycan can be synthesized and hydrolyzed in bacteria.Since the hydrolysis of peptidoglycan makes the surface of the cell smooth,combined with the uneven surface observed by SEM,we speculated that TRQ can inhibit the hydrolysis of peptidoglycan.Further research is needed to study the synthesis of peptidoglycan.5.TRQ inhibits the transcription of the Z-ring initial stabilization gene GpsB and the anchor protein gene FtsA.FtsZ cannot form a Z-ring anchored to the membrane and form a mature divvied membrane.TRQ can inhibit the transcription of the peptidoglycan hydrolase gene slel,increase the accumulation of peptidoglycan in MRSA.Conclusion:TRQ can increase the content of the division protein FtsZ,and inhibit the anchor protein FtsA and the Z-ring stable protein GpsB,resulting in the inability of MRSA to form a division membrane,and the filamentous membrane is scattered in and on the membrane.Failure to form a mature division membrane has prevented bacterial division.At the same time,TRQ also inhibit the hydrolysis of peptidoglycan,so that even if mature membrane is formed,the bacteria cannot be completely separated,forming a multicellular body,and the position of the division scar is shifted.The bacteria cannot be separated for a long time,resulting in a rough surface and th increase in the length of the cells.In summary,TRQ can synergistically inhibit the division of MRSA from multiple targets. |
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