Inorganic Metal Nanomaterials Promoting The Formation Of Persister Cells By Inducing Protein Aggregation & Case Report

Bacterial infection is still one of the urgent clinical problems to be solved.Currently,the failure of antibiotics to control infection is mainly attribute to three types of bacterial phenomena:antibiotic resistance,tolerance and persistence.Persisters are a small subgroup of phenotypic drug-tolerant cells in an isogenic bacterial population,which can survive under antibiotic treatment mainly by reducing metabolism.Different from resistant bacteria,persisters cannot grow in an environment with high levels of antibiotics,but can wake up and reproduce when antibiotics are removed.This subgroup of bacteria is thought to be one of the main causes behind the prolonged infection and play important roles in the development of antibiotic resistant.With the development of nanotechnology,a variety of nanoparticles have shown excellent bactericidal properties and have been studied in the treatment of bacterial infections.As nanoparticles do not cause bacterial resistance,they have become one of the candidates for combating bacterial infections.However,most studies have focused on sensitive or resistant bacterial cells,and few studies have elucidated the interaction between nanomaterials and persisters.In this study,common oral inorganic metal nanomaterials and gold nanocages（Au NCs）were used as model nanomaterials to explore the effect of inorganic metal nanomaterials on the persisters and the mechanisms.PART I:The effect and mechanism of oral inorganic metal nanomaterials on persisters.Objective:To investigate the effect and mechanism of common oral inorganic metal nanoparticles on persister cells.Methods:The influence of common oral inorganic nanoparticles on persisters was determined by dilution plating procedure,and the surviving bacteria were determined as persisters by dilution plating procedure,bactericidal curve and antibacterial ring.The interaction between nanoparticles and bacteria was visualized by fluorescence microscope,and the relationship between bacterial behavior and fluorescence intensity was analyzed by Image J software.The intracellular ATP level was detected by ATP kit,and the expression of persistence related genes was detected by q PCR.Scanning electron microscopy,transmission electron microscopy and confocal fluorescence microscopy were used to observe the interaction between the nanomaterials and bacteria.Microscale thermophoresis,protein gel electrophoresis and protein spectrum were used to elucidate the components of the interaction between nanomaterials and cell membranes,and molecular dynamics simulation was used to explore the possible binding forms.RNA-seq results were analyzed to find the possible mechanism of action,and q PCR was used to verify the conjecture.Intracellular water content was measured by thermogravimetry and the change of intracellular potassium ion concentration was detected by a potassium ion dye EPG-2.Molecular dynamics simulation and Zeta potential measurement were used to clarify the possible source of osmotic pressure,and the dilution plate method was used to verify whether high osmotic pressure and nanomaterials have the same effect.The formation of the protein aggregates was analyzed by SDS-PAGE and protein spectrum analysis.Finally,the distribution of Clp A-mcherry was observed by fluorescence microscope to clarify the mechanism of action between common oral inorganic nanoparticles and persisters.Results:Common oral inorganic nanomaterials titanium dioxide（Ti O₂）,silver（Ag）and zinc oxide（Zn O）can all lead to an increase in the number of persisters in the bacterial population.The results of time-lapse imaging showed that the bacteria combined with Ti O₂ nanoparticles survived the antibiotic treatment through growth inhibition,and resumed growth after the removal of antibiotics.After Ti O₂nanoparticles treatment,the intracellular ATP level of bacteria decreased and the expression of genes related to persisters increased.Ti O₂nanoparticles attach to the surface of bacteria mainly by binding to the outer membrane proteins.RNA-seq results showed that the gene expression profile of Ti O₂ nanoparticles treated bacteria was similar to that of hypertonic shock,and this conjecture was verified by q PCR.Intracellular water content and potassium ion level detection confirmed that the addition of Ti O₂ nanoparticles would cause bacteria to suffer from hypertonic shock.Molecular dynamics simulation results show that the presence of Ti O₂ nanoparticles can enrich local ions,and then form ion corona around.The measurement of Zeta potential also confirm this view.SDS-PAGE results showed that the insoluble protein content of bacterial increased after adding Ti O₂ nanoparticles.A variety of proteins related to bacterial life activities and Clp A,a small molecule protein closely related to protein aggregation,were detected in the aggregates.Fluorescence microscopy images showed that fluorescently labeled Clp A would aggregate in the cell under the addition of Ti O₂nanoparticles or hypertonic impact,resulting in the increase of the persisters.Conclusion:The common oral inorganic nanoparticles can induce the formation of protein aggregates by causing local hyperosmosis of bacteria,thus promote the formation of persisters.Part II:The effect and mechanism of photothermal treatment of gold nanocages on persisters.Objective:To investigate the effect and mechanism of photothermal gold nanocages（Au NCs）on persisters.Methods:Au NCs was synthesized by silver cubic displacement method,and transmission electron microscopy was used to observe the morphology and size of the nanomaterial.Hydration particle size and Zeta potential were measured by Zetasizer,and the specific absorption spectrum was measured by ultraviolet spectrophotometer.The temperature rise curve was measured with an 808 nm laser and infrared imager.Dilution plating method was used to determine the bactericidal capacity of Au NCs and the influence on persisters.Other possible factors were excluded by dilution plating method,sterilization curve and bacteriostatic ring.Intracellular ATP levels were measured by ATP kits and expression of persisters-related genes was detected using q PCR.CTC staining detects bacterial respiratory chain activity and Th T staining detect changes in membrane potential.Changes in bacterial morphology were observed by scanning electron microscopy,fluorescence microscopy,and flow cytometry.Protein aggregates content was compared by SDS-PAGE,Th S staining,microscopy observation,and flow cytometry.Finally,the fluorescence distribution of Dna K-e GFP was observed under a fluorescence microscope after Au NCs photothermal and heat treatment.The dilution plating methods with the addition of Dna K inhibitors and agonists to explore the role of Dna K in protein aggregation related persisters formation.Results:The synthesized Au NCs showed a cube morphology with a diameter of 50 nm,a hydrated particle size of 70 nm,and a Zeta potential of-30 m V.The synthesized Au NCs have good photothermal properties and could effectively kill bacteria.On the contrary,Au NCs photothermal treatment leaded to an increase in persisters.After Au NCs photothermal treatment,the ATP level of bacteria decreased and the expression of persisters related genes increased.The respiratory chain on the bacterial membrane was blocked and the membrane potential exhibits depolarization.After the bacteria were irradiated or heat treated,the morphology changed and dark foci appeared.Photothermal or heat treatment can cause protein aggregation,and the effect of photothermal treatment is more violent than that of the heat treatment group.Under Au NCs photothermal treatment,fluorescent-labeled Dna K would aggregates in cells.After the addition of Dna K inhibitor,the number of persisters under photothermal treatment increased.After the addition of Dna K agonist,the number of persisters decreased.These results confirming the important role of Dna K in the formation of persisters under Au NCs photothermal treatment.Conclusion:Photothermal effect of Au NCs can promote the formation of persisters,and the mechanism is that the heat generated by photothermal effect can lead to bacterial protein aggregation,in which Dna K plays a vital role.