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Cloning, Characterization, and Antibacterial Properties of Endolysin LysE Against Planktonic Cells and Biofilms of Aeromonas hydrophila

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Abstract

Multidrug-resistant bacteria are emerging as a major global threat to public health. Bacteriophages are an important source of antimicrobial enzymes and could be developed as an alternative antibiotic candidate. This study investigates the antibacterial capacity of the endolysin LysE against Aeromonas hydrophila. The endolysin LysE gene was cloned and expressed in Escherichia coli BL21 (DE3) cells. Purified recombinant LysE protein was tested for its antimicrobial activity against A. hydrophila. The study reveals that recombinant LysE protein was highly effective against Gram-negative bacteria when combined with antimicrobials that alter the permeability of the outer membrane. Specifically, the enzyme had the highest muralytic activity at pH 4, and maintained over 50% of the activity at pH 10. Moreover, endolysin displayed more than 50% activity even after 30 min of incubation at 100 °C. Also, endolysin LysE resulted in one log reduction in CFU/mL in 30 min and demonstrated antibiofilm capabilities when combined with EDTA. Interestingly, checkerboard assay showed its synergistic effects in combination with lower concentrations of colistin against A. hydrophila. Additionally, in vitro tests with Channa striatus kidney (CSK) cell lines do not show cytotoxic effects. Taken together, these findings suggest that LysE can be employed with outer membrane permeabilizers to expand the arsenal repertoire against Gram-negative bacteria in the aquaculture, food, and medical industries.

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All the data generated or analyzed during this study are included in this published article.

References

  1. Rasmussen-Ivey CR, Figueras MJ, McGarey D, Liles MR (2016) Virulence factors of Aeromonas hydrophila: in the wake of reclassification. Front Microbiol 7:1337. https://doi.org/10.3389/fmicb.2016.01337

    Article  PubMed  PubMed Central  Google Scholar 

  2. Yu HB, Rao PS, Lee HC et al (2004) A type III secretion system is required for Aeromonas hydrophila AH-1 pathogenesis. Infect Immun 72:1248–1256. https://doi.org/10.1128/iai.72.3.1248-1256.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Skwor T, Shinko J, Augustyniak A et al (2014) Aeromonas hydrophila and Aeromonas veronii predominate among potentially pathogenic ciprofloxacin and tetracycline-resistant Aeromonas isolates from Lake Erie. Appl Environ Microbiol 80:841–848. https://doi.org/10.1128/AEM.03645-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Algammal AM, Mohamed MF, Tawfiek BA et al (2020) Molecular typing, antibiogram and PCR-RFLP based detection of Aeromonas hydrophila complex isolated from Oreochromis niloticus. Pathogens 9:238. https://doi.org/10.3390/pathogens9030238

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Matamp N, Bhat SG (2019) Phage endolysins as potential antimicrobials against multidrug resistant Vibrio alginolyticus and Vibrio parahaemolyticus: current status of research and challenges ahead. Microorganisms 7:84. https://doi.org/10.3390/microorganisms7030084

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Schmelcher M, Donovan DM, Loessner MJ (2012) Bacteriophage endolysins as novel antimicrobials. Future Microbiol 7:1147–1171. https://doi.org/10.2217/fmb.12.97

    Article  CAS  PubMed  Google Scholar 

  7. Guo M, Feng C, Ren J et al (2017) A novel antimicrobial endolysin, LysPA26, against Pseudomonas aeruginosa. Front Microbiol 8:293. https://doi.org/10.3389/fmicb.2017.00293

    Article  PubMed  PubMed Central  Google Scholar 

  8. Chang Y (2020) Bacteriophage-derived endolysins applied as potent biocontrol agents to enhance food safety. Microorganisms 8:724. https://doi.org/10.3390/microorganisms8050724

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pallavi B, Puneeth T, Shekar M, Girisha S (2021) Isolation, characterization and genomic analysis of vB-AhyM-AP1, a lytic bacteriophage infecting Aeromonas hydrophila. J Appl Microbiol 131:695–705. https://doi.org/10.1111/jam.14997

    Article  CAS  PubMed  Google Scholar 

  10. Tamura K, Peterson D, Peterson N et al (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. https://doi.org/10.1093/molbev/msr121

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lowry OH, Rosebrough NJ, Farr AL et al (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    Article  CAS  PubMed  Google Scholar 

  12. Oliveira H, Boas VD, Mesnage S et al (2016) Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-Gram-negative bacteria activity. Front Microbiol 7:208. https://doi.org/10.3389/fmicb.2016.00208

    Article  PubMed  PubMed Central  Google Scholar 

  13. Khakhum N, Yordpratum U, Boonmee A et al (2016) Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79. AMB Express 6:77. https://doi.org/10.1186/s13568-016-0251-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Oliveira H, Thiagarajan V, Walmagh M et al (2014) A thermostable Salmonella phage endolysin, Lys68, with broad bactericidal properties against Gram-negative pathogens in presence of weak acids. PLoS One 9:e108376. https://doi.org/10.1371/journal.pone.0108376

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Blasco L, Ambroa A, Trastoy R et al (2020) In vitro and in vivo efficacy of combinations of colistin and different endolysins against clinical strains of multi-drug resistant pathogens. Sci Rep 10:7163. https://doi.org/10.1038/s41598-020-64145-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Majeed AS, Nambi KS, Taju G, Sahul Hameed AS (2013) Development, characterization and application of a new fibroblastic-like cell line from kidney of a freshwater air breathing fish Channa striatus (Bloch, 1793). Acta Trop 127:25–32. https://doi.org/10.1016/j.actatropica.2013.03.013

    Article  CAS  PubMed  Google Scholar 

  17. Stratev D, Odeyemi OA (2016) Antimicrobial resistance of Aeromonas hydrophila isolated from different food sources: a mini-review. J Infect Public Health 9:535–544. https://doi.org/10.1016/j.jiph.2015.10.006

    Article  PubMed  Google Scholar 

  18. Kutter EM (2013) Lysozyme. In: Stanley Maloy, Kelly Hughes (eds) Brenner's Encyclopedia of Genetics, 2nd edn. Academic Press, pp 292–293 https://doi.org/10.1016/B978-0-12-374984-0.00893-7

  19. Shang X, Nelson DC (2019) Contributions of net charge on the PlyC Endolysin CHAP Domain. Antibiotics 8:70. https://doi.org/10.3390/antibiotics8020070

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tsugita A, Inouye M (1968) Purification of bacteriophage T4 lysozyme. J Biol Chem 243:391–397

    Article  CAS  PubMed  Google Scholar 

  21. Khondker A, Rheinstädter MC (2020) How do bacterial membranes resist polymyxin antibiotics? Commun Biol 3:77. https://doi.org/10.1038/s42003-020-0803-x

    Article  PubMed  PubMed Central  Google Scholar 

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Funding

This work is supported by the Science and Engineering Research Board (SERB), India (Grant Number: PDF/2018/000811).

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Authors and Affiliations

  1. Department of Aquatic Animal Health Management, College of Fisheries, Karnataka Veterinary, Animal and Fisheries Sciences University, Mangalore, 575 002, India

    Pallavi Baliga, Puneeth Thadooru Goolappa, Malathi Shekar & Girisha Shivani Kallappa

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  1. Pallavi Baliga
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  2. Puneeth Thadooru Goolappa
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  3. Malathi Shekar
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  4. Girisha Shivani Kallappa
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Contributions

PB and PTG conceived and designed the study and experiments involved. PB and PTG performed the experiments. PB, PTG, and GSK analyzed the data. GSK supervised the study. PB wrote the manuscript. PB, MS, PTG, and GSK reviewed and validated the result. PB acquired the funding for the study. All the authors read, commented, and approved the final manuscript.

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Baliga, P., Goolappa, P.T., Shekar, M. et al. Cloning, Characterization, and Antibacterial Properties of Endolysin LysE Against Planktonic Cells and Biofilms of Aeromonas hydrophila. Probiotics & Antimicro. Prot. 15, 646–654 (2023). https://doi.org/10.1007/s12602-021-09880-7

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  • DOI: https://doi.org/10.1007/s12602-021-09880-7

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