Skip to main content
SpringerLink
Log in

DNA aptamers specific for Legionella pneumophila: systematic evolution of ligands by exponential enrichment in whole bacterial cells

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

Legionella pneumophila is the major causative agent of Legionnaires’ disease and Pontiac fever, which pose major public health problems. Rapid detection of L. pneumophila is important for global control of these diseases. Aptamers, short oligonucleotides that bind to targets with high affinity and specificity, have great potential for use in pathogenic bacterium detection, diagnostics, and therapy. Here, we used a whole-cell SELEX (systematic evolution of ligands by exponential enrichment) method to isolate and characterize single-stranded DNA (ssDNA) aptamers against L. pneumophila. A total of 60 ssDNA sequences were identified after 17 rounds of selection. Other bacterial species (Escherichia coli, Bacillus subtilis, Pseudomonas syringae, Staphylococcus aureus, Legionella quateirensis, and Legionella adelaidensis) were used for counterselection to enhance the specificity of ssDNA aptamers against L. pneumophila. Four ssDNA aptamers showed strong affinity and high selectivity for L. pneumophila, with Kd values in the nanomolar range. Bioinformatic analysis of the most specific aptamers revealed predicted conserved secondary structures that might bind to L. pneumophila cell walls. In addition, the binding of these four fluorescently labeled aptamers to the surface of L. pneumophila was observed directly by fluorescence microscopy. These aptamers identified in this study could be used in the future to develop medical diagnostic tools and public environmental detection assays for L. pneumophila.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Data availability

All data generated or analyzed during this study are included in this published article, and are available from the corresponding author (sunyunhaoscope@163.com) on reasonable request.

Abbreviations

FAM:

6-Carboxyfluorescein

SELEX:

Systematic evolution of ligands by exponential enrichment

ELISA:

Enzyme linked immunosorbent assay

CFU:

Colony forming units

References

  • Bruno JG (2015) Predicting the uncertain future of aptamer-based diagnostics and therapeutics. Molecules 20:6866–6887

    Article  CAS  Google Scholar 

  • Cao X, Li S, Chen L, Ding H, Xu H, Huang Y, Li J, Liu N, Cao W, Zhu Y, Shen B, Shao N (2009) Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus. Nucleic Acids Res 37:4621–4628

    Article  CAS  Google Scholar 

  • Casini B, Aquino F, Totaro M, Miccoli M, Galli I, Manfredini L, Giustarini C, Costa AL, Tuvo B, Valentini P, Privitera G, Baggiani A (2017) Application of hydrogen peroxide as an innovative method of treatment for legionella control in a hospital water network. Pathogens 6(2):556–565

    Article  Google Scholar 

  • Chen F, Zhou J, Luo F, Mohammed AB, Zhang XL (2007) Aptamer from whole-bacterium SELEX as new therapeutic reagent against virulent Mycobacterium tuberculosis. Biochem Biophys Res Commun 357:743–748

    Article  CAS  Google Scholar 

  • Cloutman-Green E, Barbosa VL, Jimenez D, Wong D, Dunn H, Needham B, Ciric L, Hartley JC (2019) Controlling Legionella pneumophila in water systems at reduced hot water temperatures with copper and silver ionization. Am J Infect Control 47(7):761–766

    Article  Google Scholar 

  • Dua P, Ren S, Lee SW, Kim JK, Shin HS, Jeong OC, Kim S, Lee DK (2016) Cell-SELEX based identification of an RNA aptamer for Escherichia coli and its use in various detection formats. Mol Cells 39:807–813

    Article  CAS  Google Scholar 

  • Duan N, Wu S, Chen X, Huang Y, Wang Z (2012) Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus. J Agric Food Chem 60:4034–4038

    Article  CAS  Google Scholar 

  • Duan N, Ding X, Wu S, Xia Y, Ma X, Wang Z, Chen J (2013a) In vitro selection of a DNA aptamer targeted against Shigella dysenteriae. J Microbiol Methods 94:170–174

    Article  CAS  Google Scholar 

  • Duan N, Wu S, Chen X, Huang Y, Xia Y, Ma X, Wang Z (2013b) Selection and characterization of aptamers against Salmonella typhimurium using whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX). J Agric Food Chem 61:3229–3234

    Article  CAS  Google Scholar 

  • Dwivedi HP, Smiley RD, Jaykus LA (2010) Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX. Appl Microbiol Biotechnol 87:2323–2334

    Article  CAS  Google Scholar 

  • Dwivedi HP, Smiley RD, Jaykus LA (2013) Selection of DNA aptamers for capture and detection of Salmonella Typhimurium using a whole-cell SELEX approach in conjunction with cell sorting. Appl Microbiol Biotechnol 97:3677–3686

    Article  CAS  Google Scholar 

  • Gopinath SC, Tang TH, Chen Y, Citartan M, Lakshmipriya T (2014) Bacterial detection: from microscope to smartphone. Biosens Bioelectron 60:332–342

    Article  CAS  Google Scholar 

  • Keefe AD, Pai S, Ellington A (2010) Aptamers as therapeutics. Nat Rev Drug Discov 9:537–550

    Article  CAS  Google Scholar 

  • Kolovskaya OS, Savitskaya AG, Zamay TN, Reshetneva IT, Zamay GS, Erkaev EN, Wang X, Wehbe M, Salmina AB, Perianova OV, Zubkova OA, Spivak EA, Mezko VS, Glazyrin YE, Titova NM, Berezovski MV, Zamay AS (2013) Development of bacteriostatic DNA aptamers for salmonella. J Med Chem 56:1564–1572

    Article  CAS  Google Scholar 

  • Maita C, Matushita M, Okubo T, Matsuo J, Miyake M, Nagai H, Yamaguchi H (2016) Draft genome sequences of Legionella pneumophila JR32 and Lp01 laboratory strains domesticated in Japan. Genome Announc 4(4):1–2

    Article  Google Scholar 

  • Moon J, Kim G, Lee S, Park S (2013) Identification of Salmonella Typhimurium-specific DNA aptamers developed using whole-cell SELEX and FACS analysis. J Microbiol Methods 95:162–166

    Article  CAS  Google Scholar 

  • Park HC, Baig IA, Lee SC, Moon JY, Yoon MY (2014) Development of ssDNA aptamers for the sensitive detection of Salmonella typhimurium and Salmonella enteritidis. Appl Biochem Biotechnol 174:793–802

    Article  CAS  Google Scholar 

  • Peng Z, Ling M, Ning Y, Deng L (2014) Rapid fluorescent detection of Escherichia coli K88 based on DNA aptamer library as direct and specific reporter combined with immuno-magnetic separation. J Fluoresc 24:1159–1168

    Article  CAS  Google Scholar 

  • Rajendhran J, Gunasekaran P (2011) Microbial phylogeny and diversity: small subunit ribosomal RNA sequence analysis and beyond. Microbiol Res 166:99–110

    Article  CAS  Google Scholar 

  • Saad M, Chinerman D, Tabrizian M, Faucher P, S. (2020) Identification of two aptamers binding to Legionella pneumophila with high affinity and specificity. Sci Rep 10:9145

    Article  CAS  Google Scholar 

  • Savory N, Lednor D, Tsukakoshi K, Abe K, Yoshida W, Ferri S, Jones BV, Ikebukuro K (2013) In silico maturation of binding-specificity of DNA aptamers against Proteus mirabilis. Biotechnol Bioeng 110:2573–2580

    Article  CAS  Google Scholar 

  • Schmolders J, Manske C, Otto A, Hoffmann C, Steiner B, Welin A, Becher D, Hilbi H (2017) Comparative proteomics of purified pathogen vacuoles correlates intracellular replication of Legionella pneumophila with the small GTPase Ras-related protein 1 (Rap1). Mol Cell Proteomics 16:622–641

    Article  Google Scholar 

  • Suh SH, Jaykus LA (2013) Nucleic acid aptamers for capture and detection of Listeria spp. J Biotechnol 167:454–461

    Article  CAS  Google Scholar 

  • Suh SH, Dwivedi HP, Choi SJ, Jaykus LA (2014) Selection and characterization of DNA aptamers specific for Listeria species. Anal Biochem 459:39–45

    Article  CAS  Google Scholar 

  • Syed MA, Jamil B (2018) Aptamers and aptasensors as novel approach for microbial detection and identification: an appraisal. Curr Drug Targets 19:1560–1572

    Article  CAS  Google Scholar 

  • Sypabekova M, Bekmurzayeva A, Wang R, Li Y, Nogues C, Kanayeva D (2017) Selection, characterization, and application of DNA aptamers for detection of Mycobacterium tuberculosis secreted protein MPT64. Tuberculosis (edinb) 104:70–78

    Article  CAS  Google Scholar 

  • Tang X, Zheng J, Yan Q, Li Z, Li Y (2013) Selection of aptamers against inactive Vibrio alginolyticus and application in a qualitative detection assay. Biotechnol Lett 35:909–914

    Article  CAS  Google Scholar 

  • Torres-Chavolla E, Alocilja EC (2009) Aptasensors for detection of microbial and viral pathogens. Biosens Bioelectron 24:3175–3182

    Article  CAS  Google Scholar 

  • Wang KY, Zeng YL, Yang XY, Li WB, Lan XP (2011) Utility of aptamer-fluorescence in situ hybridization for rapid detection of Pseudomonas aeruginosa. Eur J Clin Microbiol Infect Dis 30:273–278

    Article  Google Scholar 

  • Woo PC, Lau SK, Teng JL, Tse H, Yuen KY (2008) Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect 14:908–934

    Article  CAS  Google Scholar 

  • Xiong L, Zhao H, Mo Z, Shi L (2015) Prevalence of 7 virulence genes of Legionella strains isolated from environmental water sources of public facilities and sequence types diversity of L. pneumopila strains in Macau. Biosci Trends 9:214–220

    Article  CAS  Google Scholar 

  • Xiong L, Yan H, Shi L, Mo Z (2016) Antibiotic susceptibility of Legionella strains isolated from public water sources in Macau and Guangzhou. J Water Health 14:1041–1046

    Article  Google Scholar 

  • Xiong L, Yamasaki S, Chen H, Shi L, Mo Z (2017) Intracellular growth and morphological characteristics of Legionella pneumophila during invasion and proliferation in different cells. Biol Pharm Bull 40:1035–1042

    Article  CAS  Google Scholar 

  • Yang M, Peng Z, Ning Y, Chen Y, Zhou Q, Deng L (2013) Highly specific and cost-efficient detection of Salmonella Paratyphi A combining aptamers with single-walled carbon nanotubes. Sensors (basel) 13:6865–6881

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. Zhenhuang Ge at Sun Yat-sen University for technical assistance in culture of L. pneumophila. We also thank Guangzhou Saite Testing Co., LTD, Guangzhou, China for technical assistance in detecting the characteristics of aptamers.

Funding

This work was supported by Funds for the Key Scientific and Technological Innovation Projects of Guangdong Forestry Bureau (Grant No. 2020KJCX009 to Yunhao Sun, for the collection of data), the National Natural Science Foundation of China (Grant No. 32000086 to Yunhao Sun, for the analysis of the data and writing of the manuscript), the Applied Basic Research Programs of Science and Technology Commission Foundation of Guangdong Province (Grant No. 2019 A1515110593 to Yunhao Sun, for the analysis of the data and writing of the manuscript), the Research Project of Innovative Institute for Plant Health from Zhongkai University of Agriculture and Engineering (Grant No. KA21031H103 to Yunhao Sun, for the collection of data), and the Starting Research Fund from Zhongkai University of Agriculture and Engineering (Grant No. KA200540844 to Yunhao Sun, for the collection of data).

Author information

Authors and Affiliations

  1. School of Life Sciences, Sun Yat-Sen University, Guangzhou, China

    Lina Xiong, Qinglin Wang & Yongjun Lu

  2. Guangzhou Saite Testing Co., LTD, Guangzhou, China

    Mingchen Xia

  3. Innovative Institute for Plant Health, Zhongkai University of Agriculture and Engineering, No. 24, Dongsha Street, Guangzhou, China

    Zhen Meng, Jie Zhang, Guohui Yu, Zhangyong Dong & Yunhao Sun

  4. College of Agriculture and Biology, Zhongkai University of Agriculture and Engineering, No. 24, Dongsha Street, Guangzhou, China

    Zhen Meng, Guohui Yu, Zhangyong Dong & Yunhao Sun

  5. College of Resources and Environment, Zhongkai University of Agriculture and Engineering, Guangzhou, China

    Jie Zhang

Authors
  1. Lina Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mingchen Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qinglin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhen Meng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jie Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Guohui Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhangyong Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yongjun Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yunhao Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

LX, MX and YS: conceived and designed the study. LX, QW, ZM, JZ, GY and ZD: performed the experiments. LX, YL and YS: wrote the paper. GY and ZD: reviewed and edited the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Yunhao Sun.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xiong, L., Xia, M., Wang, Q. et al. DNA aptamers specific for Legionella pneumophila: systematic evolution of ligands by exponential enrichment in whole bacterial cells. Biotechnol Lett 44, 777–786 (2022). https://doi.org/10.1007/s10529-022-03252-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-022-03252-z

Keywords

Search

Navigation