| Staphylococcus aureus is an important pathogen that can to cause human infectious diseases.According to the reports of CHINET,the clinical isolation rate of S.aureus always ranks the first among Gram-positive bacteria isolated in China.The pathogenic S.aureus can produce hundreds of virulence factors to cause skin and soft tissue infections,pneumonia,sepsis,and other diseases,which raise great concerns to human health.Moreover,the increase of superbug infections casued by methicillin-resistant S.aureus(MRSA)and vancomycin-intermediate S.aureus(VISA)complicates the control of infectious diseases and often results in high morbidity and mortality.Preclinical animal models are crucial in studying bacterial pathogenesis and assessing the antimicrobial efficacy of new drugs and therapeutic regimens,however,a noninvasive and effective strategy allowing the accurate and real-time track of S.aureus in vivo is lack.Bioluminescence imaging(BLI)relying on the catalysis of luciferase on its substrate is a new technology to track target cells or report biological events in living models.BLI can provide dynamic monitoring and noninvasive evaluation of labeled active organisms in vivo.The most applicable bacterial BLI system is constructed with lux CDABE operon derived from the insect pathogen Photorhabdus luminescens.Lux system in metabolically active bacteria can naturally emit blue-green light with a maximum wavelength of 490 nm,which has limited tissue penetration in vivo.In this study,bioluminescence reporter strains of S.aureus were constructed by using plasmid expression and chromosome recombination methods.The luminescence properties of S.aureus reporters on different substrates were analyzed and compared in vitro and in vivo,and a stable and efficient bioluminescence tracking system was screened out.In addition,the preliminary application of the selected luminescence system in S.aureus colonization and response to antibiotic treatment was performed in animal models.The main research contents and results are as following:1.Construction and stability detection of S.aureus bioluminescence reporter strains based on plasmid expression.The promoter of S.aureus fhu D2 gene that encodes a protein involved in iron metabolism was amplified and cloned into the Escherichia coli-S.aureus shuttle plasmid pLI50 to achieve pFH plasmid,and then the lux CDABE operon from P.luminescens and nluc gene derived from deep sea shrimp Oplophorus gracilirostris were colned into pFH to generate pFH-lux and pFH-nluc,respectively.S.aureus bioluminescence reporter strains were constructed by transformation of pFH-lux or pFH-nluc into strains RN4220,Newman and USA300.In vitro BLI analysis showed that Nluc-catalyzed hydrofurimazine(HFZ)substrate produced 2～10~6-fold higher bioluminescence signals than its Lux counterpart at all growth phases and exhibited better luminous stability.However,about 0.15%～47.4%of S.aureus reporter strains retained the pFH-nluc plasmid after 48 h of culture in vitro,and only 0.0018%of S.aureus USA300/pFH-nluc existed in the kidneys of mice 5 days postinfection.Therefore,S.aureus bioluminescence reporters based on plasmid expression were not stable.2.Construction of chromosome recombination-based bioluminescence S.aureus reporters and their luminescence performance analysis in vitro.Staphylococcal enolase(Eno)was screened out as an appropriate fusion candidate for the delivery of exogenous Nluc-based luciferases.Genes that encode Nluc-based luciferases,including Nluc,Teluc,and Antare2,were chemically synthesized and genetically in-frame fused with the 3’-terminal of eno in the chromosome of S.aureus USA300 to achieve USA300/Eno-Nluc,USA300/Eno-Teluc,and USA300/Eno-Antares2 reporter strains.Bioluminescence detection demonstrated that all three luciferase reporter strains could catalyze HFZ to generate stable signals within 24 h of testing.The varied luminescence performance was observed during the catalysis of S.aureus reporter on different substrates,such as HFZ,furimazine(FUR),and diphenylterazine(DTZ).Among all reporter/substrate combinations,USA300/Eno-Teluc/HFZ pair generated the highest total luminescence emission,while the USA300/Eno-Antares2/HFZ pair emitted>2.5-fold more photons of orange-red light than other pairs.Approximately 50 colony forming unit(CFU)of S.aureus could produce detectable bioluminescence signals in the three reporters by using HFZ as the substrate.3.Preliminary applications of bioluminescence system in S.aureus colonization and antibiotic treatment evaluation.The engineered USA300/Eno-Teluc and USA300/Eno-Antares2 reporter strains were used to infect BALB/c mice intravenously through tail-vein injection,and different substrates were individually administered to evaluate the performance of reporter strain/substrate pairs for S.aureus deep-tissue BLI analysis.The results showed that USA300/Eno-Antares2/HFZ pair presented better performance than other reporter/substrate combinations.Bacterial loads in right and left kidneys of mice 6 h postinfection were determined,and an satisfactory correlation between BLI signals of the USA300/Eno-Antares2/HFZ pair and viable bacterial numbers within one day of infection was observed(R~2=0.8437).A sensitivity of approximately 750 CFU from deep-tissue(kidney)with USA300/Eno-Antares2/HFZ was detected via BLI analysis,and this value was 40-fold higher than that of the S.aureus luc reporter system.In vitro bacterial CFU counts from kidneys and livers of the infected mice dynamically changed over time,and abscess formed in kidneys of mice 6 days after infection.The formation of abscess may impair BLI signals by impeding the entrance of Nluc substrate.Therefore,the reporter system of USA300/Eno-Antares2/HFZ may provide a useful tool for monitoring deep-seated bacterial dissemination during the early stages of bacterial sepsis and mainly within 24 h postinfection.A mouse skin infection model was constructed by injecting the USA300/Eno-Antares2reporter strain on the skin of the dorsal back.BLI detection revealed that a sensitivity of about100 CFU from mouse skins was achieved with S.aureus USA300/Eno-Antares2/HFZ system.A linear correlation between BLI signals and the number of USA300/Eno-Antares2 loads in murine skins was presented between 2 and 6 days postinfection(R2=0.9181).The longitudinal evaluation of antibiotic efficacy against S.aureus was performed with USA300/Eno-Antares2/HFZ bioluminescence system in a mouse skin infection model.The administration of HFZ could track the location of S.aureus USA300/Eno-Antares2 reporter in mouse skin for 11 days,and BLI signals were remarkably lower in mice treated with vancomycin for 11 days than those in mice administrated with erythromycin and normal saline.However,comparable skin abscess sizes and body weight variations were observed among the infected mice,indicating a poor marker for S.aureus skin colonization with skin abscess size or mouse weight.Overall,the optimized S.aureus BLI system based on the USA300/Eno-Antares2/HFZ pair provides a technological advancement for investigating the preclinical efficacy of anti-S.aureus agents.In conclusion,several bioluminescent reporter S.aureus strains were constructed in the present study.An optimized USA300/Eno-Antares2/HFZ BLI system with improved stability and brightness was screened out with different luciferase substrates in vitro and in vivo.The preliminary applications of USA300/Eno-Antares2/HFZ BLI system in the colonization and dissemination of S.aureus in vivo and the evaluation of antibiotic treatment efficacy were investigated by using mouse sepsis and skin infection models.The carrier molecule-based optimizing strategy for BLI systems may provide a model for generating other bacterial reporters to establish noninvasive,stable,and accurate animal models for focal and systemic studies. |
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