Molecular screening and genetic diversity analysis of anticancer Azurin-encoding and Azurin-like genes in human gut microbiome deduced through cultivation-dependent and cultivation-independent studies

  • Van Duy NguyenEmail author
  • Thanh Tra Nguyen
  • Thu Thuy Pham
  • Michael Packianather
  • Chi Hieu Le
Original Article


Azurin, a bacteriocin produced by a human gut bacterium Pseudomonas aeruginosa, can reveal selectively cytotoxic and induce apoptosis in cancer cells. After overcoming two phase I trials, a functional region of Azurin called p28 has been approved as a drug for the treatment of brain tumor glioma by FDA. The present study aims to improve a screening procedure and assess genetic diversity of Azurin genes in P. aeruginosa and Azurin-like genes in the gut microbiome of a specific population in Vietnam and global populations. Firstly, both cultivation-dependent and cultivation-independent techniques based on genomic and metagenomic DNAs extracted from fecal samples of the healthy specific population were performed and optimized to detect Azurin genes. Secondly, the Azurin gene sequences were analyzed and compared with global populations by using bioinformatics tools. Finally, the screening procedure improved from the first step was applied for screening Azurin-like genes, followed by the protein synthesis and NCI in vitro screening for anticancer activity. As a result, this study has successfully optimized the annealing temperatures to amplify DNAs for screening Azurin genes and applying to Azurin-like genes from human gut microbiota. The novelty of this study is the first of its kind to classify Azurin genes into five different genotypes at a global scale and confirm the potential anticancer activity of three Azurin-like synthetic proteins (Cnazu1, Dlazu11, and Ruazu12). The results contribute to the procedure development applied for screening anticancer proteins from human microbiome and a comprehensive understanding of their therapeutic response at a genetic level.


Anticancer proteins Azurin Cancer therapy Genetic diversity Microbiome NCI in vitro screening 



Authors would like to acknowledge the British Council under the frame of HAPIE and TEAM-SIE projects ( to CHL, MP, and VDN for enhancing the partnership among higher education institutions, companies, and hospitals in Vietnam and the UK. We would also like to give special thanks to Professor Arsénio M. Fialho at the Instituto Superior Técnico, University of Lisbon, Portugal, for his valuable comments and contribution to the manuscript, and Professor Simeon Keates at the Faculty of Engineering and Science, University of Greenwich, UK, for the research administration support.


This work was supported by a grant no. 106-YS.04-2014.40 from the Vietnam National Foundation for Science and Technology Development (NAFOSTED) to VDN.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Research and Education Committee of Nha Trang University (REC-NTU) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants or their parents as minors (less than 16 years old) included in the study.

Supplementary material

10123_2019_70_MOESM1_ESM.pdf (61 kb)
S1 Figure PCR products of Azurin genes amplified from metagenomic DNAs using a pair of primers Azu 677F/R at the annealing temperatures of 54oC. M stands for DNA Marker. Ctr is a Control, which is amplified from genomic DNA of K16P isolate. The name of fecal samples is shown above the lane designations. NOTE: This is a high quality figure for printing and publication. Number of pixels: 1641 x 2625 with Resolution 600 dpi. The original figure can be downloaded at the following link: Original image (TIF), Larger image (PNG) (PDF 60 kb)
10123_2019_70_MOESM2_ESM.pdf (196 kb)
S2 Figure Phylogenetic tree and sequence alignment of 117 Azurin genes in P. aeruginosa in the Vietnamese and global populations. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei 1987). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) (Felsenstein 1985). The evolutionary distances were computed using the Tamura 3-parameter method (Tamura 1992) and cut-off value of consensus tree is 50%. Evolutionary analyses were conducted in Genious program (Kearse et al. 2012). Base substitutions occur popularly in 5 positions at nucleotides 51, 282, 297, 305 and 432. The name of bacterial isolates or samples from the Vietnamese population in the present study was shown along with GenBank Accession number (% pairwise identify to P. aeruginosa strain PAO1) in global populations. NOTE: This is a high quality with number of pixels 2250 × 1984 and Resolution 600 dpi. The original figure can be downloaded at the following link: Original image (TIF), Larger image (PNG) (PDF 195 kb)


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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Institute of Biotechnology and EnvironmentNha Trang UniversityNha TrangVietnam
  2. 2.School of EngineeringCardiff UniversityCardiffUK
  3. 3.Faculty of Engineering and ScienceUniversity of GreenwichChathamUK

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