The effect of epetraborole on the transcriptome and proteome profiles of an Escherichia coli strain overexpressing leuS, Leucyl-tRNA Synthetase

Abstract

Epetraborole (EP) is a boron-containing antibiotic known for its effectiveness against gram-negative enteric bacteria and Mycobacterium species. It is designed to bind and inhibit the LeuS enzyme (Leucyl-tRNA Synthetase), which is encoded by the essential gene leuS in Escherichia coli. EP inhibits protein translation, impeding bacterial growth. However, when leuS is overexpressed in a recombinant plasmid, the amount of EP required for growth inhibition needs to be increased. This study explored the impact of EP on the transcriptome and proteome of E. coli overexpressing leuS, aiming to reveal additional gene and pathway insights beyond LeuS, shedding light on the biochemical players orchestrating the bacterium’s molecular response. 2D-PAGE Proteomics analysis identified four differentially regulated proteins influenced by EP in the leuS overexpression strain. Notably, LeuA and DeoA emerged as identified proteins. EP may affect LeuA in the cells overexpressing LeuS, which could result in truncated LeuA protein variants. Transcriptomics analyses, based on microarray data, revealed 23 up-regulated and 9 down-regulated genes responding to EP in the overexpression strain (p<0.05, fold change; FC>2). Based on the statistical analyses, the first five up-and down-regulated genes showing the highest fold differences in their mRNA levels are yiaW, mglB, narH, ybiO, flgB and yhdY, deoR, recX, yobB, potF, respectively. Analyses using the Omics Dashboard pathway and String indicate that the EP effect on the leuS overexpressing strain mainly induces alterations in the expression of genes related to the cell exterior, regulation, and response to stimuli. It is suggested that EP and higher levels of LeuS may interfere with the translational and transcriptional regulation of the expression of the leuA gene, which encodes the first enzyme, 2-isopropylmalate synthase, in L-leucine biosynthesis. This study offers new insights into the effects of EP on the bacterium, specifically when the level of the aminoacyl-tRNA synthetase LeuS is increased.

Keywords

• Epetraborole
• Escherichia coli
• LeuS
• Leucyl-tRNA Synthetase
• proteomics
• transcriptomics

References

1. Alméciga-Díaz, C. J., Tolosa-Díaz, A. D., Pimentel, L. N., Bonilla, Y. A., Rodríguez-López, A., Espejo-Mojica, A. J., ... & Gonzalez-Santos, J. (2017). Anaerobic sulfatase maturase AslB from Escherichia coli activates human recombinant iduronate-2-sulfate sulfatase (IDS) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Gene, 634, 53-61.
2. Arai, M., Koizumi, Y., Sato, H., Kawabe, T., Suganuma, M., Kobayashi, H., ... & Omura, S. (2004). Boromycin abrogates bleomycin-induced G2 checkpoint. The Journal of Antibiotics, 57(10), 662-668.
3. Benjdia, A., Dehò, G., Rabot, S., & Berteau, O. (2007). First evidences for a third sulfatase maturation system in prokaryotes from E. coli aslB and ydeM deletion mutants. FEBS Letters, 581(5), 1009-1014.
4. Bie, L., Zhang, M., Wang, J., Fang, M., Li, L., Xu, H., & Wang, M. (2023). Comparative analysis of transcriptomic response of Escherichia coli K-12 MG1655 to nine representative classes of antibiotics. Microbiology Spectrum, 11(2), e00317-23.
5. BioCyc, (2024). BioCyc Pathway/Genome Database Collection, https://biocyc.org/dashboard/dashboard-intro.shtml, Last accessed on March 30, 2024.
6. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2), 248-254.
7. Cardenas, C. A. (2023). Review of boron-based compounds: Advancing cancer therapy and beyond. Clinical Oncology: Case Reports 6, 8, 2.
8. Cole, S. T., Eiglmeier, K., Ahmed, S., Honore, N., Elmes, L., Anderson, W. F., & Weiner, J. H. (1988). Nucleotide sequence and gene-polypeptide relationships of the glpABC operon encoding the anaerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12. Journal of Bacteriology, 170(6), 2448-2456.

9. Cummings, J. E., Lunde, C. S., Alley, M. R. K., & Slayden, R. A. (2023). Epetraborole, a leucyl-tRNA synthetase inhibitor, demonstrates murine efficacy, enhancing the in vivo activity of ceftazidime against Burkholderia pseudomallei, the causative agent of melioidosis. PLOS Neglected Tropical Diseases, 17(11), e0011795.
10. Daley, D. O., Rapp, M., Granseth, E., Melén, K., Drew, D., & Von Heijne, G. (2005). Global topology analysis of the Escherichia coli inner membrane proteome. Science, 308(5726), 1321-1323.
11. Dibek, E., Babayeva, A., Kürkçü, M. S., Çöl, N. A., & Çöl, B. (2020). Bor içeren bazı biyoaktif bileşikler. Journal of Boron, 5(1), 29-39.
12. dos Santos, K. V., Diniz, C. G., de Castro Veloso, L., de Andrade, H. M., da Silva Giusta, M., da Fonseca Pires, S., ... & de Macêdo Farias, L. (2010). Proteomic analysis of Escherichia coli with experimentally induced resistance to piperacillin/tazobactam. Research in Microbiology, 161(4), 268-275.
13. Egan, A. J., Errington, J., & Vollmer, W. (2020). Regulation of peptidoglycan synthesis and remodeling. Nature Reviews Microbiology, 18(8), 446-460.
14. Ganapathy, U. S., Gengenbacher, M., & Dick, T. (2021). Epetraborole is active against Mycobacterium abscessus. Antimicrobial Agents and Chemotherapy, 65(10), 10-1128.
15. Garoff, L., Huseby, D. L., Praski Alzrigat, L., & Hughes, D. (2018). Effect of aminoacyl-tRNA synthetase mutations on susceptibility to ciprofloxacin in Escherichia coli. Journal of Antimicrobial Chemotherapy, 73(12), 3285-3292.
16. Gemmill, R. M., Wessler, S. R., Keller, E. B., & Calvo, J. M. (1979). leu operon of Salmonella typhimurium is controlled by an attenuation mechanism. Proceedings of the National Academy of Sciences, 76(10), 4941-4945.
17. Goswami, M., & Narayana Rao, A. V. S. S. (2018). Transcriptome profiling reveals interplay of multifaceted stress response in Escherichia coli on exposure to glutathione and ciprofloxacin. Msystems, 3(1), 10-1128.
18. Harayama, S., Bollinger, J., Iino, T., & Hazelbauer, G. L. (1983). Characterization of the mgl operon of Escherichia coli by transposon mutagenesis and molecular cloning. Journal of Bacteriology, 153(1), 408-415.
19. Hatch, N. D., & Ouellette, S. P. (2020). Inhibition of tRNA synthetases induces persistence in Chlamydia. Infection and Immunity, 88(4), 10-1128.
20. Hernandez, V., Crépin, T., Palencia, A., Cusack, S., Akama, T., Baker, S. J., ... & Plattner, J. J. (2013). Discovery of a novel class of boron-based antibacterials with activity against gram-negative bacteria. Antimicrobial Agents and Chemotherapy, 57(3), 1394-1403.
21. Hurdle, J. G., O’Neill, A. J., & Chopra, I. (2005). Prospects for aminoacyl-tRNA synthetase inhibitors as new antimicrobial agents. Antimicrobial Agents and Chemotherapy, 49(12), 4821-4833.
22. Irschik, H., Schummer, D., Gerth, K., Höfle, G., & Reichenbach, H. (1995). The tartrolons, new boron-containing antibiotics from a myxobacterium, Sorangium cellulosum. The Journal of Antibiotics, 48(1), 26-30.
23. Ito, A., May, T., Kawata, K., & Okabe, S. (2008). Significance of rpoS during maturation of Escherichia coli biofilms. Biotechnology and Bioengineering, 99(6), 1462-1471.
24. Jiang, H., Shang, L., Yoon, S. H., Lee, S. Y., & Yu, Z. (2006). Optimal production of poly-γ-glutamic acid by metabolically engineered Escherichia coli. Biotechnology Letters, 28, 1241-1246.
25. Keseler, I. M., Collado-Vides, J., Gama-Castro, S., Ingraham, J., Paley, S., Paulsen, I. T., ... & Karp, P. D. (2005). EcoCyc: a comprehensive database resource for Escherichia coli. Nucleic Acids Research, 33(suppl_1), D334-D337.
26. Kim, T., Hanh, B. T. B., Heo, B., Quang, N., Park, Y., Shin, J., ... & Jang, J. (2021). A screening of the mmv pandemic response box reveals epetraborole as a new potent inhibitor against mycobacterium abscessus. International Journal of Molecular Sciences, 22(11), 5936.
27. Kitagawa, M., Ara, T., Arifuzzaman, M., Ioka-Nakamichi, T., Inamoto, E., Toyonaga, H., & Mori, H. (2005). Complete set of ORF clones of Escherichia coli ASKA library (A Complete Set of E. coli K-12 ORF Archive): Unique resources for biological research. DNA Research, 12(5), 291-299.
28. Kohno, J., Kawahata, T., Otake, T., Morimoto, M., Mori, H., Ueba, N., ... & Kawashima, K. (1996). Boromycin, an anti-HIV antibiotic. Bioscience, Biotechnology and Biochemistry, 60(6), 1036-1037.
29. Linton, K. J., & Higgins, C. F. (1998). The Escherichia coli ATP‐binding cassette (ABC) proteins. Molecular Microbiology, 28(1), 5-13.
30. Mendes, R. E., Alley, M. R. K., Sader, H. S., Biedenbach, D. J., & Jones, R. N. (2013). Potency and spectrum of activity of AN3365, a novel boron-containing protein synthesis inhibitor, tested against clinical isolates of Enterobacteriaceae and nonfermentative Gram-negative bacilli. Antimicrobial Agents and Chemotherapy, 57(6), 2849-2857.
31. Mering, C. V., Huynen, M., Jaeggi, D., Schmidt, S., Bork, P., & Snel, B. (2003). STRING: a database of predicted functional associations between proteins. Nucleic Acids Research, 31(1), 258-261.
32. Metcalf, W. W., Steed, P. M., & Wanner, B. L. (1990). Identification of phosphate starvation-inducible genes in Escherichia coli K-12 by DNA sequence analysis of psi:: lacZ (Mu d1) transcriptional fusions. Journal of Bacteriology, 172(6), 3191-3200.
33. Milija, J., Lilic, M., Janjusevic, R., Jovanovic, G., & Savic, D. J. (1999). tRNA synthetase mutants of Escherichia coli K-12 are resistant to the gyrase inhibitor novobiocin. Journal of Bacteriology, 181(9), 2979-2983.
34. Monteferrante, C. G., Jirgensons, A., Varik, V., Hauryliuk, V., Goessens, W. H. F., & Hays, J. P. (2016). Evaluation of the characteristics of leucyl-tRNA synthetase (LeuRS) inhibitor AN3365 in combination with different antibiotic classes. European Journal of Clinical Microbiology & Infectious Diseases, 35, 1857-1864.
35. Nakamura, H., Iitaka, Y., Kitahara, T., Okazaki, T., & Okami, Y. (1977). Structure of aplasmomycin. The Journal of Antibiotics, 30(9), 714-719.
36. Nguyen, T. Q., Heo, B. E., Hanh, B. T. B., Jeon, S., Park, Y., Choudhary, A., ... & Jang, J. (2023). DS86760016, a Leucyl-tRNA Synthetase Inhibitor, Is Active against Mycobacterium abscessus. Antimicrobial Agents and Chemotherapy, 67(6), e01567-22.
37. NIH, (2024). Official Website of National Institutes of Health, https://david.ncifcrf.gov/tools.jsp, Last accessed on March 30, 2024.
38. Novikova, M., Metlitskaya, A., Datsenko, K., Kazakov, T., Kazakov, A., Wanner, B., & Severinov, K. (2007). The Escherichia coli Yej transporter is required for the uptake of translation inhibitor microcin C. Journal of Bacteriology, 189(22), 8361-8365.
39. Paley, S., Parker, K., Spaulding, A., Tomb, J. F., O’Maille, P., & Karp, P. D. (2017). The Omics Dashboard for interactive exploration of gene-expression data. Nucleic Acids Research, 45(21), 12113-12124.
40. Pavoncello, V., Barras, F., & Bouveret, E. (2022). Degradation of exogenous fatty acids in Escherichia coli. Biomolecules, 12(8), 1019.
41. Pistocchi, R., Kashiwagi, K., Miyamoto, S., Nukui, E., Sadakata, Y., Kobayashi, H., & Igarashi, K. (1993). Characteristics of the operon for a putrescine transport system that maps at 19 minutes on the Escherichia coli chromosome. Journal of Biological Chemistry, 268(1), 146-152.
42. Saier Jr, M. H., Reddy, V. S., Tsu, B. V., Ahmed, M. S., Li, C., & Moreno-Hagelsieb, G. (2016). The transporter classification database (TCDB): recent advances. Nucleic Acids Research, 44(D1), D372-D379.
43. Schirmer, A., & Kolter, R. (1998). Computational analysis of bacterial sulfatases and their modifying enzymes. Chemistry & Biology, 5(8), R181-R186.
44. Scholle, A., Vreemann, J., Blank, V., Nold, A., Boos, W., & Manson, M. D. (1987). Sequence of the mglB gene from Escherichia coli K12: comparison of wild-type and mutant galactose chemoreceptors. Molecular and General Genetics MGG, 208, 247-253.
45. Seong, W., Han, G. H., Lim, H. S., Baek, J. I., Kim, S. J., Kim, D., ... & Lee, D. H. (2020). Adaptive laboratory evolution of Escherichia coli lacking cellular by product formation for enhanced acetate utilization through compensatory ATP consumption. Metabolic Engineering, 62, 249-259.
46. Shafiee, A., & Chanda, S. (2024). In vitro evaluation of drug–drug interaction potential of epetraborole, a novel bacterial leucyl-trna synthetase inhibitor. Pharmaceuticals, 17(1), 120.
47. Sivasankar, S., Premnath, M. A., Boppe, A., Grobusch, M. P., & Jeyaraj, S. (2023). Screening of MMV pandemic response and pathogen box compounds against pan-drug-resistant Klebsiella pneumoniae to identify potent inhibitory compounds. New Microbes and New Infections, 55, 101193.
48. Soo, V. W., Hanson-Manful, P., & Patrick, W. M. (2011). Artificial gene amplification reveals an abundance of promiscuous resistance determinants in Escherichia coli. Proceedings of the National Academy of Sciences, 108(4), 1484-1489.
49. Spoering, A. L., Vulic, M., & Lewis, K. (2006). GlpD and PlsB participate in persister cell formation in Escherichia coli. Journal of Bacteriology, 188(14), 5136-5144.
50. Stieglitz, B. I., & Calvo, J. M. (1974). Distribution of the isopropylmalate pathway to leucine among diverse bacteria. Journal of Bacteriology, 118(3), 935-941.
51. STRING, (2024). Official Website of String, https://string-db.org/, Last accessed on March 30, 2024. Snel, B., Lehmann, G., Bork, P., & Huynen, M. A. (2000). STRING: a web-server to retrieve and display the repeatedly occurring neighbourhood of a gene. Nucleic Acids Research, 28(18), 3442-3444.
52. Sullivan, J. R., Lupien, A., Kalthoff, E., Hamela, C., Taylor, L., Munro, K. A., .. & Behr, M. A. (2021). Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline. PLoS Pathogens, 17(10), e1009965.
53. Sun, H., Wang, M., Liu, Y., Wu, P., Yao, T., Yang, W., ... & Yang, B. (2022). Regulation of flagellar motility and biosynthesis in enterohemorrhagic Escherichia coli O157: H7. Gut Microbes, 14(1), 2110822.
54. Vinella, D., Joseleau-Petit, D., Thévenet, D., Bouloc, P., & D’Ari, R. (1993). Penicillin-binding protein 2 inactivation in Escherichia coli results in cell division inhibition, which is relieved by FtsZ overexpression. Journal of Bacteriology, 175(20), 6704-6710.
55. Wessler, S. R., & Calvo, J. M. (1981). Control of leu operon expression in Escherichia coli by a transcription attenuation mechanism. Journal of Molecular Biology, 149(4), 579-597.
56. Xu, C., Lin, X., Ren, H., Zhang, Y., Wang, S., & Peng, X. (2006). Analysis of outer membrane proteome of Escherichia coli related to resistance to ampicillin and tetracycline. Proteomics, 6(2), 462-473.
57. Zhao, Q., Wu, Y., Li, B., He, L., Sun, J., Pang, X., ... & Li, X. (2023). OmpA is involved in the early response of Escherichia coli to antibiotics as a Hub outer membrane protein. Applied Biochemistry and Microbiology, 59(5), 608-621.