Characterizing the Transcriptional Effects of Endolysin Treatment on Established Biofilms of Staphylococcus aureus
鉴定内溶素处理对已建立的金黄色葡萄球菌生物膜转录的影响   

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Antimicrobial Agents and Chemotherapy
May 2017

 

Abstract

Biofilms are the most common lifestyle of bacteria in both natural and human environments. The organized structure of these multicellular communities generally protects bacterial cells from external challenges, thereby enhancing their ability to survive treatment with antibiotics or disinfectants. For this reason, the search for new antibiofilm strategies is an active field of study. In this context, bacteriophages (viruses that infect bacteria) and their derived proteins have been proposed as promising alternatives for eliminating biofilms. For instance, endolysins can degrade peptidoglycan and, ultimately, lyse the target bacterial cells. However, it is important to characterize the responses of bacterial cells exposed to these compounds in order to improve the design of phage-based antimicrobial strategies.

This protocol was developed to examine the transcriptional responses of Staphylococcus aureus biofilm cells exposed to endolysin treatment, as previously described in Fernández et al. (2017). However, it may be subsequently adapted to analyze the response of other microorganisms to different antimicrobials.

Keywords: Biofilms (生物膜), Endolysins (内溶素), Staphylococcus aureus (金黄色葡萄球菌), RNA-seq (RNA-seq), Responses to antimicrobials (对抗菌剂的反应)

Background

It is becoming increasingly clear that subinhibitory doses of antimicrobials may have a regulatory effect on different phenotypes of the target microbes, including biofilm formation, metabolism or virulence. Therefore, studying the potential impact of a novel compound on the target cells at low-level concentrations should be a part of the development process. Indeed, a very effective antibacterial agent that triggers production of virulence factors or antibiotic resistance determinants may not be a good candidate for therapeutic application. On the other hand, considering the physiological differences between biofilm and planktonic cells, it seems logical that the effect of new antibiofilm agents should be analyzed on biofilm-forming cells. Here, we describe a protocol for the analysis of transcriptional responses of biofilm cells upon exposure to subinhibitory concentrations of endolysins, phage-derived proteins that show great promise as biofilm removal agents. Thus, the transcriptome of endolysin-treated cells was compared to control cells by RNA-seq and differential expression of selected genes was later confirmed by RT-qPCR.

Materials and Reagents

  1. Standard Petri dishes (Labbox, catalog number: PDIP-09N-500 )
  2. Sterile 10 ml polystyrene culture tubes (Deltalab, catalog number: 300903 )
  3. Cuvettes for OD600 reading (Deltalab, catalog number: 303103 )
  4. 1.5 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.690.001 )
  5. 12-well microtiter plates with Nunclon Delta surface (Thermo Fisher Scientific, Nunc, catalog number: 150628 )
  6. Sterile plastic loops (1 μl) (VWR, catalog number: 612-9351 )
  7. MicroAmp® Fast optical 96-well reaction plate with barcode (Thermo Fisher Scientific, Applied Biosystems, catalog number: 4346906 )
  8. MicroAmp® optical adhesive film (Thermo Fisher Scientific, Applied Biosystems, catalog number: 4311971 )
  9. Frozen stock of Staphylococcus aureus (for example, S. aureus IPLA1 from our laboratory collection) stored in glycerol at -80 °C
  10. Filtered LysH5 endolysin stock stored in NaPi buffer with 30% glycerol at -80 °C (~350 μg/ml = 5.8 μM) purified as described previously (Gutiérrez et al., 2014)
  11. Agarose for electrophoresis (Conda, catalog number: 8008 )
  12. Glass beads, acid washed (≤ 106 µm, sterile) (Sigma-Aldrich, catalog number: G4649 )
  13. RNA protect® Bacteria Reagent (QIAGEN, catalog number: 76560 )
  14. IllustraTM RNAspin Mini Kit (GE Healthcare, catalog number: 25050071 )
  15. Chloroform (Merck, catalog number: 1024451000 )
  16. Ethanol (Fisher Scientific, catalog number: BP28184 )
  17. SUPERase-InTM RNase Inhibitor (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2694 )
  18. Turbo DNA-free kitTM (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1907 )
  19. DL-Dithiothreitol (Sigma-Aldrich, catalog number: D0632-5G )
  20. Phenol, Molecular Biology Grade (Merck, Calbiochem, catalog number: 516724-100GM )
  21. iScriptTM Reverse Transcription Supermix for RT-qPCR (Bio-Rad Laboratories, catalog number: 1708841 )
  22. Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4367659 )
  23. Bacteriological agar (ROKO S.A.)
  24. D(+)-Glucose (Merck, catalog number: 1.08337.1000 )
  25. Sodium chloride (NaCl) (Merck, catalog number: 1.06404.1000 )
  26. Potassium chloride (KCl) (VWR, BDH, catalog number: 437025H )
  27. Sodium phosphate dibasic (Na2HPO4) (VWR, AnalaR NORMAPUR, catalog number: 102495D )
  28. Potassium phosphate monobasic (KH2PO4) (Merck, catalog number: 1048731000 )
  29. Sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O) (ITW Reagents Division, AppliChem, catalog number: 131965.1211 )
  30. UltraPureTM Tris Buffer (Thermo Fisher Scientific, catalog number: 15504020 )
  31. Glacial acetic acid (Merck, catalog number: 1.00063.2500 )
  32. 0.5 M EDTA (pH 8.0) (Alfa Aesar, USB, catalog number: J15701 )
  33. TSB medium (tryptic soy broth, Scharlab, catalog number: 02-200-500 ) (see Recipes)
  34. TSA agar plates (see Recipes)
  35. TSB medium supplemented with glucose (TSBG) (see Recipes)
  36. Phosphate buffered saline (PBS) solution (see Recipes)
  37. Sodium phosphate (NaPi) buffer (see Recipes)
  38. Tris-acetate-EDTA (TAE) buffer (see Recipes)

Equipment

  1. Pipettes (volume ranges: 1 μl-10 μl, 2 μl-20 μl, 20 μl-200 μl, 200 μl-1,000 μl)
  2. Shaking (250 rpm) and static incubators at 25 °C and 37 °C
  3. Spectrophotometer
    Note: It is used to measure optical density (OD600) of cell culture.
  4. Epoch microplate spectrophotometer (BioTek Instruments, model: Epoch )
  5. Refrigerated centrifuge (Eppendorf, model: 5415 R )
  6. FastPrep®-24 (MP Biomedicals, catalog number: 116004500 )
  7. Gel electrophoresis apparatus (Bio-Rad Laboratories, Mini-Sub® Cell GT Cell)
  8. Vortex
  9. 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Applied Biosystems, catalog number: 4351107 )
  10. Illumina HiSeq2000 platform
  11. Computer equipped with four Intel Xeon E5-4650 v2 2.4GHz 25M 8GT/s 10-core processors, 256 GB RAM, and running CentOS Linux release 7.3.1611
    Note: The computer is for carrying out computation.

Software

  1. FastQC (http://www.bioinformatics.babraham.ac.uk/projects/download.html#fastqc)
  2. BowTie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) (Langmead and Salzberg, 2012)
  3. EDGE-Pro (http://ccb.jhu.edu/software/EDGE-pro/) (Magoc et al., 2013)
  4. DEseq2 (http://bioconductor.org/packages/release/bioc/html/DESeq2.html) (Love et al., 2014)

Procedure

  1. Biofilm formation and treatment (Figure 1)
    1. Streak out S. aureus strain (IPLA1) from the frozen stock onto a TSB agar plate and incubate statically overnight at 37 °C.
    2. To obtain three biological replicates, pick 3 isolated colonies of S. aureus from the agar plate with a sterile plastic loop and inoculate into three 10-ml polystyrene tubes containing 2 ml of TSB medium.
    3. Grow bacterial cultures overnight at 37 °C, with shaking at 250 rpm.
    4. Dilute the overnight cultures to an OD600 of 0.1 in TSBG medium (TSB supplemented with glucose), containing approximately 107 CFU/ml, and then make a 1:20 dilution in the same medium to prepare the inoculum for the biofilm assays.
    5. Inoculate 2 ml from this cell suspension (approximately 5 x 105 CFU/ml) into each well of a 12-well microtiter plate (four wells per biological replicate).
    6. Incubate the microtiter plate in static for 24 h at 25 °C.
      Note: In this case, the temperature used for biofilm formation and treatment was 25 °C, which represents treatment/disinfection at “room temperature”. Nonetheless, the experiment could have also been performed at different temperatures; for instance, at 37 °C to represent treatment of human infection.
    7. Remove the planktonic phase from the wells and wash the biofilms twice each with 2 ml of PBS.
    8. For each replicate, add 1 ml of NaPi buffer alone to two wells and 1 ml of NaPi containing 10.94 μg/ml (0.18 μM) of LysH5 to the other two wells.
    9. Incubate in static for 30 min at 25 °C.
    10. Remove supernatant.
    11. Wash twice with PBS.
    12. Harvest cells corresponding to the same biological replicate and treatment in 1 ml of RNA protect® and 500 μl PBS by scraping with a pipette tip and transfer to a clean Eppendorf tube.
    13. Process the samples according to the RNA protect® manufacturer’s instructions.
    14. Store at -80 °C or proceed to RNA purification.


      Figure 1. Schematic representation of the protocol. The different steps of this method include biofilm formation and treatment (purple), subsequent RNA purification and RNA-sequencing (pink) and, finally, computer analysis of the generated data (light green).

  2. RNA purification and sequencing (Figure 1)
    1. To achieve cell lysis, perform mechanical disruption of the cells with glass beads and phenol using FastPrep equipment.
    2. After lysis, samples were centrifuged at 9,000 x g for 10 min at 4 °C.
    3. Transfer the upper phase to a clean tube and add 500 μl chloroform and then centrifuge for 5 min at 9,000 x g and 4 °C.
    4. Transfer the upper phase to a clean tube and mix with 250 μl of ethanol by pipetting.
    5. Transfer the samples mixed with ethanol to the columns provided with the illustraTM RNAspin Mini kit and perform the rest of RNA purification steps following the instructions provided by the manufacturer.
    6. Elute in 50 μl of nuclease-free water and add 1 μl of SUPERase-InTM.
    7. Add 5 μl of Turbo DNase buffer and 1 μl of Turbo DNase per 50 μl sample and incubate for 30 min at 37 °C.
    8. Add 1 μl of Turbo DNase per sample and incubate for another 30 min at 37 °C.
    9. Remove DNase from sample with inactivation reagent as indicated by the manufacturer.
    10. Add 1 μl of SUPERase inhibitor per 50 μl sample.
    11. Check RNA quality and concentration by agarose gel electrophoresis (1% agarose) in TAE buffer and the Epoch microplate spectrophotometer (Figure 2). RNA concentrations obtained with this protocol usually range between 200 and 700 ng/μl.


      Figure 2. Agarose gel electrophoresis of total RNA from S. aureus biofilm samples. Aliquots (1-2 μl) from different RNA samples were run in a 1% agarose gel. Two bands corresponding to the 23S and 16S rRNAs should be visible and preferably in a proportion of 2:1 (23S:16S) indicating RNA integrity. Sometimes a lower band corresponding to 5S rRNA can also be observed.

    12. Samples with A260/A280 ratios ≥ 1.8 can be considered adequate for RNA-seq analysis. Otherwise, clean up the samples with the illustraTM RNAspin Mini kit following the protocol recommended by the manufacturer.
    13. Take 8 μg of RNA from each sample and proceed with sequencing steps according to the protocols recommended by the manufacturer of the selected platform. For example, in this study samples were sent to an external service provider (Macrogen Inc., South Korea) for sequencing with an Illumina HiSeq2000 platform according to the protocols recommended by Illumina, generating 100-bp paired-end reads.

  3. Computer analysis of the generated data (Figure 1)
    1. Check the quality of the reads in FASTQ format with FastQC.
    2. Download the reference genome in FASTA format (.fa or .fna), the protein table file (.ptt) and the RNA table (.rnt) from the NCBI archive (ftp://ftp.ncbi.nlm.nih.gov/genomes/archive/old_genbank/Bacteria/).
    3. Run script “edge.pl” with arguments indicating the FASTQ files containing paired-end reads for each sample (-u and –v) as well as the three files mentioned above (-g, -p and –r) and the prefix for the output files names (-o). In a first step, EDGE-Pro will map the reads to the reference genome using program BowTie2 and create an alignment file as output (this file will be in sequence alignment map or SAM format). BowTie2 also indicates the percentage of alignment to the reference genome. Once completed this step, EDGE-Pro performs transcript quantification into Reads Per Kilobase of transcript per Million mapped reads (RPKMs). The output files containing the RPKM counts will end in “.rpkm_0”.
      Example: /edge.pl -g SAreference.fna -p SAreference.ptt -r SAreference.rnt -u Lys_1-1.fastq -o Lys1 -v Lys_1-2.fastq
    4. Run script “edgeToDeseq.perl” indicating the .rpkm_0 files to be analyzed in order to generate a table gathering the raw counts for each gene and each sample. This table will be saved in the output “deseqFile”.
      Example: /edgeToDeseq.perl NaPi1.rpkm_0 NaPi2.rpkm_0 NaPi3.rpkm_0 Lys1.rpkm_0 Lys2.rpkm_0 Lys3.rpkm_0
      Note: This step is necessary because DESeq2 requires information on raw counts and not RPKMs.
    5. Perform differential expression analysis between treated and untreated samples with DESeq2 by using the “deseqFile” from the previous step as an input. Select genes with an adjusted P-value < 0.05 for further analysis and save the table of differentially-expressed genes in .csv format.

  4. Confirmation of RNA-seq results by RT-qPCR (Figure 1)
    1. Convert 0.5 μg RNA from each sample into cDNA with iScriptTM Reverse Transcription Supermix for RT-qPCR as indicated by the manufacturer.
    2. Dilute cDNA samples 1:25 in nuclease-free water and use them as a template for qPCR.
    3. To perform qPCR, add 2.5 μl aliquots from the different samples to each well of a MicroAmp® Fast optical 96-well reaction plate together with 3.25 μl of nuclease-free water, 0.25 μl of each primer from a 10 μM stock, and 6.25 μl of Power SYBR® Green PCR Master Mix.
    4. Analyze each biological replicate in duplicate.
    5. Determine changes in gene expression by using a reference gene (in this case rplD) according to the 2-ΔΔCT method ( Livak and Schmittgen, 2001), in which ΔCT = CT(target gene) - CT(reference gene) and ΔΔCT = ΔCT(target sample) - ΔCT(reference sample).

Data analysis

For reproducibility, it is recommended to analyze three independent biological replicates (BR). Statistical analysis of RNAseq data was performed as part of the differential gene expression analysis with the DESeq2 package, and only those genes with adjusted P-values < 0.05 were selected for further analysis. Regarding fold-change, we normally set the cut-off at 2-fold change (log2 fold-change = 1). However, in this case all genes displaying significant changes based on the adjusted P-values were analyzed further. The small changes are probably due to the fact that only part of the biofilm population was exposed to the antimicrobial. In addition to confirming the genes under the conditions described here, changes were further evaluated in a liquid culture exposed to endolysin LysH5. This analysis showed more evident changes in some of the genes identified by RNA-seq, which reinforced the idea that the transcriptional changes observed in the biofilm were indeed a result of endolysin exposure.

Recipes

  1. Tryptic soy broth (TSB)
    30 g TSB medium
    Dissolve in 1 L ddH2O and autoclave
  2. TSA agar plates
    TSB medium with 2% agar
    Dissolve in ddH2O and autoclave
  3. TSBG medium
    TSB medium with 0.25% glucose
    Dissolve in ddH2O and autoclave
  4. Phosphate buffered saline (PBS) solution
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    2 mM KH2PO4
    Adjust pH to 7.4
    Dissolve in ddH2O and autoclave
  5. NaPi buffer
    50 mM sodium phosphate
    Adjust pH to 7.4
    Dissolve in ddH2O and autoclave
  6. TAE buffer (50x stock solution)
    242 g of Tris
    57.1 ml of glacial acetic acid
    100 ml of 0.5 M EDTA (pH 8.0)
    Add deionized water to 1 L

Acknowledgments

The development of this protocol was funded by grant AGL2012-40194-C02-01 (Ministry of Science and Innovation, Spain), AGL2015-65673-R (Program of Science, Technology and Innovation 2013-2017), Proyecto Intramural CSIC 201770E016, EU ANIWHA ERA-NET (BLAAT ID: 67), and GRUPIN14-139 (FEDER EU funds, Principado de Asturias, Spain). L.F. was awarded a Marie Curie Clarin-Cofund postdoctoral fellowship. P.G. and A.R. are members of the FWO Vlaanderen-funded PhageBiotics Research community (WO.016.14) and the bacteriophage network FAGOMA. This protocol was adapted from the previously published article Fernández et al. (2017).
The authors declare that they have no conflict of interest.

References

  1. Fernández, L., González, S., Campelo, A. B., Martínez, B., Rodríguez, A. and García, P. (2017). Downregulation of autolysin-encoding genes by phage-derived lytic proteins inhibits biofilm formation in Staphylococcus aureus. Antimicrob Agents Chemother 61(5).
  2. Gutiérrez, D., Ruas-Madiedo, P., Martínez, B., Rodríguez, A. and García, P. (2014). Effective removal of staphylococcal biofilms by the endolysin LysH5. PLoS One 9(9): e107307.
  3. Langmead, B. and Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4): 357-359.
  4. Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25(4): 402-408.
  5. Love, M. I., Huber, W. and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12): 550.
  6. Magoc, T., Wood, D. and Salzberg, S. L. (2013). EDGE-pro: Estimated degree of gene expression in prokaryotic genomes. Evol Bioinform Online 9: 127-136.

简介

生物膜是自然和人类环境中最常见的细菌生活方式。这些多细胞社区的有组织结构通常保护细菌细胞免受外部挑战,从而增强其抗生素或消毒剂治疗的生存能力。为此,寻找新的抗菌膜策略是一个积极的研究领域。在这种情况下,已提出噬菌体(感染细菌的病毒)及其衍生蛋白作为消除生物膜的有希望的替代物。例如,内溶素可降解肽聚糖,并最终裂解靶细菌细胞。然而,表征暴露于这些化合物的细菌细胞的反应以改进基于噬菌体的抗微生物策略的设计是重要的。

如以前在Fernández等人(2017)中所描述的,开发该协议以检查暴露于内溶素处理的金黄色葡萄球菌生物膜细胞的转录反应。然而,它可能随后适用于分析其他微生物对不同抗菌剂的反应。

【背景】越来越清楚的是,亚抑制剂量的抗菌剂可能对目标微生物的不同表型具有调节作用,包括生物膜形成,代谢或毒力。因此,研究新化合物对低浓度靶细胞的潜在影响应该是发展过程的一部分。事实上,引发毒力因子或抗生素耐药决定簇产生的非常有效的抗菌剂可能不是治疗应用的良好候选者。另一方面,考虑到生物膜和浮游细胞之间的生理差异,应该对生物膜形成细胞分析新抗生物膜剂的作用似乎是合乎逻辑的。在这里,我们描述了一种协议,用于分析生物膜细胞在亚抑制浓度的内抑素浓度下的转录反应,噬菌体来源的蛋白质作为生物膜去除剂展现出巨大的前景。因此,通过RNA-seq将内溶素处理的细胞的转录组与对照细胞进行比较,并且后来通过RT-qPCR证实了所选基因的差异表达。

关键字:生物膜, 内溶素, 金黄色葡萄球菌, RNA-seq, 对抗菌剂的反应

材料和试剂

  1. 标准培养皿(Labbox,目录号:PDIP-09N-500)
  2. 无菌10毫升聚苯乙烯培养管(Deltalab,目录号:300903)
  3. OD 600读数的比色皿(Deltalab,目录号:303103)

  4. 1.5 ml微量离心管(SARSTEDT,目录号:72.690.001)
  5. 具有Nunclon Delta表面的12孔微量滴定板(Thermo Fisher Scientific,Nunc,目录号:150628)
  6. 无菌塑料环(1μl)(VWR,目录号:612-9351)
  7. MicroAmp快速光学96孔反应板(Thermo Fisher Scientific,Applied Biosystems,产品目录号:4346906)
  8. MicroAmp®光学粘合剂薄膜(Thermo Fisher Scientific,Applied Biosystems,产品目录号:4311971)
  9. 在-80°C储存在甘油中的 Staphylococcus aureus (例如,来自我们实验室收集的金黄色葡萄球菌IPLA1)的冷冻库存
  10. 如前所述(Gutiérrez等人,2014)纯化,在-80℃(〜350μg/ ml =5.8μM)中用30%甘油储存在NaPi缓冲液中的经过滤的LysH5细胞内溶素储备液。
  11. 琼脂糖电泳(Conda,目录号:8008)

  12. 酸洗玻璃珠(≤106μm,无菌)(Sigma-Aldrich,目录号:G4649)
  13. RNA保护细菌试剂(QIAGEN,目录号:76560)
  14. Illustra TM RNAspin Mini Kit(GE Healthcare,目录号:25050071)
  15. 氯仿(Merck,目录号:1024451000)
  16. 乙醇(Fisher Scientific,目录号:BP28184)
  17. SUPERase-In TM RNase抑制剂(Thermo Fisher Scientific,Invitrogen TM,目录号:AM2694)
  18. 不含Turbo DNA的试剂盒TM(Thermo Fisher Scientific,Invitrogen TM,目录号:AM1907)
  19. DL-二硫苏糖醇(Sigma-Aldrich,目录号:D0632-5G)
  20. 苯酚,分子生物学级(Merck,Calbiochem,目录号:516724-100GM)
  21. 用于RT-qPCR的iScript TM逆转录超混合物(Bio-Rad Laboratories,目录号:1708841)
  22. Power SYBR Green Green Master Mix(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4367659)
  23. 细菌琼脂(ROKO S.A.)
  24. D(+) - 葡萄糖(Merck,目录号:1.08337.1000)
  25. 氯化钠(NaCl)(Merck,目录号:1.06404.1000)
  26. 氯化钾(KCl)(VWR,BDH,目录号:437025H)
  27. 磷酸二氢钠(Na 2 HPO 4)(VWR,AnalaR NORMAPUR,目录号:102495D)
  28. 磷酸二氢钾(KH 2 PO 4)(Merck,目录号:1048731000)
  29. 磷酸二氢钠一水合物(NaH 2 PO 4·2H 2 O)(ITW Reagents Division,AppliChem,目录号:131965.1211) >
  30. UltraPure TM Tris缓冲液(Thermo Fisher Scientific,目录号:15504020)
  31. 冰醋酸(Merck,目录号:1.00063.2500)
  32. 0.5M EDTA(pH8.0)(Alfa Aesar,USB,目录号:J15701)
  33. TSB培养基(胰蛋白酶大豆肉汤,Scharlab,目录号:02-200-500)(见食谱)
  34. TSA琼脂平板(见食谱)
  35. 补充葡萄糖的TSB培养基(TSBG)(见食谱)
  36. 磷酸盐缓冲盐水(PBS)溶液(见食谱)
  37. 磷酸钠(NaPi)缓冲液(见食谱)
  38. Tris-acetate-EDTA(TAE)缓冲液(见食谱)

设备

  1. 移液器(体积范围:1μl-10μl,2μl-20μl,20μl-200μl,200μl-1,000μl)

  2. 在25°C和37°C摇动(250 rpm)和静态培养箱
  3. 分光光度计
    注:它用于测量细胞培养的光密度(OD 600 )。
  4. Epoch微孔板分光光度计(BioTek Instruments,型号:Epoch)
  5. 冷冻离心机(Eppendorf,型号:5415 R)
  6. FastPrep®/ -24(MP Biomedicals,产品目录号:116004500)
  7. 凝胶电泳装置(Bio-Rad Laboratories,Mini-Sub Cell GT Cell)
  8. 涡流
  9. 7500 Fast Real-Time PCR System(Thermo Fisher Scientific,Applied Biosystems,产品目录号:4351107)
  10. Illumina HiSeq2000平台
  11. 计算机配备四个Intel Xeon E5-4650 v2 2.4GHz 25M 8GT / s 10核处理器,256 GB RAM,并运行CentOS Linux 7.3.1611版本。
    注:计算机用于执行计算。

软件

  1. FastQC( http://www.bioinformatics.babraham.ac.uk/projects /download.html#fastqc
  2. BowTie2( http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)(Langmead和Salzberg,2012)
  3. EDGE-Pro( http://ccb.jhu.edu/software/EDGE-pro/)(Magoc et al。,2013)
  4. DEseq2( http://bioconductor.org/packages/release/bioc/html/DESeq2 .html )(Love et。,2014)

程序

  1. 生物膜的形成和处理(图1)
    1. Streak out S。将来自冷冻原料的金黄色葡萄球菌菌株(IPLA1)置于TSB琼脂平板上,并在37℃静置培养过夜。
    2. 为了获得三个生物学复制品,挑选3个分离的菌落。用无菌塑料环从琼脂平板上接种金黄色葡萄球菌,接种到含有2毫升TSB培养基的三个10毫升聚苯乙烯管中。

    3. 在37°C过夜培养细菌培养物,摇动速度为250 rpm。
    4. 在含有约10 7 CFU / ml的TSBG培养基(补充有葡萄糖的TSB)中将过夜培养物稀释至OD 600的0.1,然后进行1:20稀释在相同的培养基中制备用于生物膜测定的接种物。

    5. 在12孔微量滴定板的每个孔中接种2ml(约5×10 5 CFU / ml)(每个生物学复制品4个孔)。

    6. 在25℃静置微量滴定板24h。
      注:在这种情况下,用于生物膜形成和处理的温度为25℃,这代表“室温”下的处理/消毒。尽管如此,该实验也可以在不同的温度下进行;例如,在37°C下代表人类感染的治疗。

    7. 除去孔中的浮游生物相,每次用2 ml PBS洗生物膜两次
    8. 对于每个重复,将1ml NaPi缓冲液单独添加到两个孔中,并向另外两个孔中添加1ml含有10.94μg/ ml(0.18μM)LysH5的NaPi。

    9. 在25°C静态孵育30分钟
    10. 去除上清液。
    11. 用PBS洗两次。
    12. 在1ml RNA中收获对应于相同生物学重复和处理的细胞,通过用移液枪尖端刮取500μlPBS并转移到干净的Eppendorf管中。
    13. 根据RNA保护®制造商的说明处理样品。
    14. 储存在-80°C或进行RNA纯化。


      图1.该方案的示意图。该方法的不同步骤包括生物膜形成和处理(紫色),随后的RNA纯化和RNA测序(粉红色)以及最后生成的计算机分析数据(浅绿色)。

  2. RNA纯化和测序(图1)
    1. 为了实现细胞裂解,使用FastPrep设备对玻璃珠和酚进行机械破碎。
    2. 裂解后,将样品在4℃以9,000×g离心10分钟。
    3. 将上层相转移到干净的试管中并加入500μl氯仿,然后在9,000gxg和4℃下离心5分钟。
    4. 将上层相转移到干净的管中,并通过移液将其与250μl乙醇混合。
    5. 将混有乙醇的样品转移到illustra™RNAspin Mini试剂盒提供的色谱柱中,按照制造商提供的说明执行RNA纯化步骤的其余部分。
    6. 用50μl不含核酸酶的水洗脱并加入1μlSUPERase-In TM 。
    7. 每50μl样品加5μlTurbo DNase缓冲液和1μlTurbo DNase,37℃孵育30分钟。

    8. 每个样品加1μlTurbo DNase,37℃孵育30分钟。

    9. 按照制造商的说明使用灭活剂从样本中去除DNA酶

    10. 每50μl样品加1μlSUPERase抑制剂
    11. 在TAE缓冲液和Epoch微孔板分光光度计(图2)中通过琼脂糖凝胶电泳(1%琼脂糖)检查RNA质量和浓度。使用该方案获得的RNA浓度通常介于200和700 ng /μl之间。


      图2.来自 S的总RNA的琼脂糖凝胶电泳。生物膜样品 不同RNA样品的等分试样(1-2μl)在1%琼脂糖凝胶中运行。对应于23S和16S rRNA的两个条带应该是可见的并且优选地以2:1(23S:16S)的比例指示RNA完整性。有时也可以观察到对应于5S rRNA的较低带。

    12. 具有A 260 / A 280比率≥1.8的样品可被认为足以用于RNA-seq分析。否则,请按照制造商推荐的方案使用illustra™RNAspin Mini试剂盒清洁样品。
    13. 从每个样品取8μgRNA,按照所选平台制造商推荐的方案进行测序步骤。例如,在本研究中,根据Illumina推荐的方案,将样品送至外部服务提供商(Macrogen Inc.,South Korea),用Illumina HiSeq2000平台进行测序,产生100-bp配对末端读数。

  3. 计算机分析生成的数据(图1)
    1. 使用FastQC检查FASTQ格式的读取质量。
    2. 从NCBI档案库( ftp://ftp.ncbi.nlm.nih.gov/genomes/archive/old_genbank/Bacteria/ )。
    3. 运行带有参数的脚本“edge.pl”,该参数指示包含每个样本(-u和-v)以及上述三个文件(-g,-p和-r)的配对结尾读取的FASTQ文件以及输出文件名称(-o)。在第一步中,EDGE-Pro将使用BowTie2程序将读数映射到参考基因组,并创建一个比对文件作为输出(该文件将以序列比对图或SAM格式)。 BowTie2也显示与参照基因组的比对百分比。完成此步骤后,EDGE-Pro将转录本定量转换为每百万次映射读取(RPKM)转录本的读数。包含RPKM计数的输出文件将以“.rpkm_0”结尾。
      示例:
      /edge.pl -g SAreference.fna -p SAreference.ptt -r SAreference.rnt -u Lys_1-1.fastq -o Lys1 -v Lys_1-2.fastq
    4. 运行指示要分析的.rpkm_0文件的脚本“edgeToDeseq.perl”,以生成收集每个基因和每个样本的原始计数的表格。该表格将保存在输出“deseqFile”中。
      示例:
      /edgeToDeseq.perl NaPi1.rpkm_0 NaPi2.rpkm_0 NaPi3.rpkm_0 Lys1.rpkm_0 Lys2.rpkm_0 Lys3.rpkm_0
      注意:这一步是必需的,因为DESeq2需要关于原始计数而不是RPKM的信息。
    5. 使用上一步中的“deseqFile”作为输入,使用DESeq2对已处理和未处理样本进行差异表达分析。选择具有调整的 P 值的基因 0.05进行进一步分析并保存.csv格式的差异表达基因表。

  4. 通过RT-qPCR确认RNA-seq结果(图1)
    1. 如制造商所示,将来自每个样品的0.5μgRNA转化成具有用于RT-qPCR的iScript TM逆转录超混合物的cDNA。

    2. 在无核酸酶的水中稀释cDNA样品1:25,并将它们用作qPCR的模板。
    3. 为了进行qPCR,将来自不同样品的2.5μl等分试样与3.25μl不含核酸酶的水一起加入到MicroAmp Fast Fiber 96孔反应板的每个孔中,将0.25μl来自10μM储备液和6.25μlPowerSYBR®Green Green Master Mix。

    4. 分析每个生物学重复
    5. 根据2-ΔΔCT方法(Livak和Schmittgen,2001),通过使用参考基因(在这种情况下为rplD)确定基因表达的变化,其中ΔCT= CT (目标基因)-CT(参考基因)和ΔΔCT=ΔCT(目标样品)-ΔCT(参考样品)。

数据分析

为了重现性,建议分析三个独立的生物学重复(BR)。 RNAseq数据的统计分析是用DESeq2软件包作为差异基因表达分析的一部分进行的,并且只有那些具有调整后的值的基因选择0.05作进一步分析。关于倍数变化,我们通常将截止值设定为2倍变化(log <2> fold-change = 1)。然而,在这种情况下,进一步分析所有基于调整的P 值显示显着变化的基因。这些小的变化可能是由于只有部分生物膜群体暴露于抗微生物剂的事实。除了在此处描述的条件下确认基因之外,在暴露于细胞内溶素LysH5的液体培养物中进一步评估了变化。该分析显示由RNA-seq鉴定的一些基因中的更明显的变化,其强化了观察到的生物膜中的转录变化确实是内溶素暴露的结果的观点。

食谱

  1. 胰蛋白酶大豆肉汤(TSB)
    30克TSB培养基
    溶于1升ddH 2 O和高压灭菌器中
  2. TSA琼脂平板
    含2%琼脂的TSB培养基
    溶于ddH 2 O和高压灭菌器
  3. TSBG中等
    含0.25%葡萄糖的TSB培养基
    溶于ddH 2 O和高压灭菌器
  4. 磷酸盐缓冲盐水(PBS)溶液
    137mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4 4/2 2mM KH 2 PO 4 4/2 调整pH值到7.4
    溶于ddH 2 O和高压灭菌器
  5. NaPi缓冲液
    50 mM磷酸钠
    调整pH值到7.4
    溶于ddH 2 O和高压灭菌器
  6. TAE缓冲液(50x储备液)
    242克Tris

    57.1毫升冰醋酸 100毫升0.5M EDTA(pH8.0)
    将去离子水加入1升

致谢

该协议的制定由AGL2012-40194-C02-01(科学和创新部,西班牙),AGL2015-65673-R(科学,技术和创新计划2013-2017),Proyecto Intramural CSIC 201770E016,欧盟ANIWHA ERA-NET(BLAAT ID:67)和GRUPIN14-139(FEDER EU基金,西班牙Principado de Asturias)。 L.F.被授予Marie Curie Clarin-Cofund博士后奖学金。 P.G。和A.R.是FWO Vlaanderen资助的噬菌体生物研究社区(WO.016.14)和噬菌体网络FAGOMA的成员。该协议改编自以前发表的文章Fernández et。(2017)。
作者声明他们没有利益冲突。

参考

  1. Fernández,L.,González,S.,Campelo,A. B.,Martínez,B.,Rodríguez,A.和García,P.(2017年)。 噬菌体衍生溶解蛋白下调自溶素编码基因抑制金黄色葡萄球菌中的生物膜形成 Antimicrob Agents Chemother 61(5)。
  2. Gutiérrez,D.,Ruas-Madiedo,P.,Martínez,B.,Rodríguez,A.和García,P.(2014年)。 通过内溶素LysH5有效去除葡萄球菌生物膜 PLoS一个9(9):e107307。
  3. Langmead,B。和Salzberg,S.L。(2012)。 快速阅读与Bowtie 2对齐。 Nat Methods 9(4):357-359。
  4. Livak,K.J。和Schmittgen,T.D。(2001)。 使用实时定量PCR分析相对基因表达数据和2 -ΔΔCT方法。方法 25(4):402-408。
  5. Love,M. I.,Huber,W.和Anders,S。(2014)。 使用DESeq2对RNA-seq数据的倍数变化和扩散进行适度估计 基因组生物学 15(12):550。
  6. Magoc,T.,Wood,D。和Salzberg,S.L。(2013)。 EDGE-pro:原核生物基因组中的基因表达的估计程度。 Evol Bioinform Online 9:127-136。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fernández, L., González, S., Gutiérrez, D., Campelo, A. B., Martínez, B., Rodríguez, A. and García, P. (2018). Characterizing the Transcriptional Effects of Endolysin Treatment on Established Biofilms of Staphylococcus aureus. Bio-protocol 8(12): e2891. DOI: 10.21769/BioProtoc.2891.
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