Cell Wall Hydrolytic Enzymes Enhance Antimicrobial Drug Activity Against Mycobacterium

  • Joshua N. Gustine
  • Matthew B. Au
  • John R. Haserick
  • Erik C. Hett
  • Eric J. Rubin
  • Frank C. GibsonIII
  • Lingyi L. Deng


Cell wall hydrolases are enzymes that cleave bacterial cell walls by hydrolyzing specific bonds within peptidoglycan and other portions of the envelope. Two major sources of hydrolases in nature are from hosts and microbes. This study specifically investigated whether cell wall hydrolytic enzymes could be employed as exogenous reagents to augment the efficacy of antimicrobial agents against mycobacteria. Mycobacterium smegmatis cultures were treated with ten conventional antibiotics and six anti-tuberculosis drugs—alone or in combination with cell wall hydrolases. Culture turbidity, colony-forming units (CFUs), vital staining, and oxygen consumption were all monitored. The majority of antimicrobial agents tested alone only had minimal inhibitory effects on bacterial growth. However, the combination of cell wall hydrolases and most of the antimicrobial agents tested, revealed a synergistic effect that resulted in significant enhancement of bactericidal activity. Vital staining showed increased cellular damage when M. smegmatis and Mycobacterium bovis bacillus Calmette–Guérin (M. bovis BCG) were treated with both drug and lysozyme. Respiration analysis revealed stress responses when cells were treated with lysozyme and drugs individually, and an acute increase in oxygen consumption when treated with both drug and lysozyme. Similar trends were also observed for the other three enzymes (hydrolase-30, RipA-His6 and RpfE-His6) evaluated. These findings demonstrated that cell wall hydrolytic enzymes, as a group of biological agents, have the capability to improve the potency of many current antimicrobial drugs and render ineffective antibiotics effective in killing mycobacteria. This combinatorial approach may represent an important strategy to eliminate drug-resistant bacteria.



The authors thank Drs. Igor Kramnik, Michael Kirber (BU Cellular Imager Core) and Raman Sahadevan for their technical advice and guidance. We thank Linda L. Deng for her graphic design assistance needed to improve some of the figures. We would also like to thank Lisa Siswanto, Albert Jones, Mario V. Lopez, Sonia Benzor, Keri LaBelle, Anjali Taneja and Mike M. Liu for their technical assistance. This research was supported by Public Health Service Grant AI-45617 from the National Institute of Allergy and Infectious Diseases (NIAID), a research Grant RG-107N from American Lung Association, a pilot Grant 9500300388 from the BU CTSI and Evans Foundation, and a grant through the BU Undergraduate Research Opportunities Program (UROP). The studies were conducted in the Analytical Instrumentation Core (AIC) at BUMC.

Supplementary material

284_2018_1620_MOESM1_ESM.docx (185 kb)
Supplementary material 1 (DOCX 184 KB)


  1. 1.
    Keren I, Minami S, Rubin E, Lewis K (2011) Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. mBio 2:e00100–e00111CrossRefGoogle Scholar
  2. 2.
    Sotgiu G, Sulis G, Matteelli A (2017) Tuberculosis-a Wold Health Organization perspective. Microbiol Spectr. CrossRefPubMedGoogle Scholar
  3. 3.
    Floyd K, Glaziou P, Zumla A, Raviglione M (2018) The global tuberculosis epidemic and progress in care, prevention, and research: an overview in year 3 of the End TB era. Lancet Respir Med 6(4):299–314. CrossRefPubMedGoogle Scholar
  4. 4.
    Nahid P, Dorman SE, Alipanah N, Barry PM, Brozek JL, Cattamanchi A et al (2016) Official American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America Clinical Practice Guidelines: treatment of drug-susceptible tuberculosis. Clin Infect Dis 63:147–195. Scholar
  5. 5.
    Munro SA, Lewin SA, Smith HJ, Engel ME, Fretheim A, Volmink J (2007) Patient adherence to tuberculosis treatment: a systematic review of qualitative research. PLoS Med 4:1230–1245. CrossRefGoogle Scholar
  6. 6.
    WHO (2016) Global tuberculosis report. World Health Organization, GenevaGoogle Scholar
  7. 7.
    Brennan PJ, Nikaido H (1995) The envelope of mycobacteria. Annu Rev Biochem 64:29–63CrossRefGoogle Scholar
  8. 8.
    Draper P (1998) The outer parts of the mycobacterial envelope as permeability barriers. Front Biosci 3:1253–1261CrossRefGoogle Scholar
  9. 9.
    McNeil M, Daffé M, Brennan PJ (1990) Evidence for the nature of the link between the arabinogalactan and peptidoglycan of mycobacterial cell walls. J Biol Chem 265:18200–18206PubMedGoogle Scholar
  10. 10.
    Rosen BP, Mobashery S (1998) Resolving the antibiotic paradox progress in understanding drug resistance and development of new antibiotics. Plenum Press, New YorkCrossRefGoogle Scholar
  11. 11.
    Niederweis M (2003) Mycobacterial porins - new channel proteins in unique outer membranes. Mol Microbiol 49:1167–1177. CrossRefPubMedGoogle Scholar
  12. 12.
    Deng LL, Humphries DE, Arbeit RD, Carlton LE, Smole SC, Carroll JD (2005) Identification of a novel peptidoglycan hydrolase CwlM in Mycobacterium tuberculosis. Biochim Biophys Acta 1747:57–66CrossRefGoogle Scholar
  13. 13.
    Shockman GD, Höltje JV (1994) Microbial peptidoglycan (murein) hydrolases. In: Ghuysen JM, Hakenbeck R (eds) New comprehensive biochemistry. Elsevier, Amsterdam, pp 131–166Google Scholar
  14. 14.
    Callewaert L, Michiels CW (2010) Lysozymes in the animal kingdom. J Biosci 35:127–160CrossRefGoogle Scholar
  15. 15.
    Ercan D, Demirci A (2015) Recent advances for the production and recovery methods of lysozyme. Crit Rev Biotechnol 36:1078–1088CrossRefGoogle Scholar
  16. 16.
    Catalão MJCA, Gil F, Moniz-Pereira J, Pimentel M (2010) The mycobacteriophage Ms6 encodes a chaperone-like protein involved in the endolysin delivery to the peptidoglycan. Mol Microbiol 77:672–686. CrossRefPubMedGoogle Scholar
  17. 17.
    Mahapatra S, Piechota C, Gil F, Ma Y, Huang H, Scherman MS et al (2012) Mycobacteriophage Ms6 LysA: a peptidoglycan amidase and a useful analytical tool. Appl Environ Microbiol 79:768–773CrossRefGoogle Scholar
  18. 18.
    Grover N, Paskaleva EE, Mehta KK, Dordick JS, Kane RS (2014) Growth inhibition of Mycobacterium smegmatis by mycobacteriophage-derived enzymes. Enzyme Microb Technol 63:1–6CrossRefGoogle Scholar
  19. 19.
    Piuri M, Hatfull GF (2006) A peptidoglycan hydrolase motif within the mycobacteriophage TM4 tape measure protein promotes efficient infection of stationary phase cells. Mol Microbiol 62:1569–1585CrossRefGoogle Scholar
  20. 20.
    Connolly LE, Edelstein PH, Ramakrishnan L (2007) Why is long-term therapy required to cure tuberculosis? PLoS Med 4:120. Scholar
  21. 21.
    Djurkovic S, Loeffler JM, Fischetti VA (2005) Synergistic killing of Streptococcus pneumoniae with the bacteriophage lytic enzyme cpl-1 and penicillin or gentamicin depends on the level of penicillin resistance. Antimicrob Agents Chemother 49:1225–1228CrossRefGoogle Scholar
  22. 22.
    Hett EC, Rubin EJ (2008) Bacterial growth and cell division: a mycobacterial perspective. Microbiol Mol Biol Rev 72:126–156CrossRefGoogle Scholar
  23. 23.
    Dwyer DJ, Belenky PA, Yang JH, MacDonald IC, Martell JD, Takahashi N et al (2014) Antibiotics induce redox-related physiological alterations as part of their lethality. Proc Natl Acad Sci USA 111:2100–2109. CrossRefGoogle Scholar
  24. 24.
    Asensi V, Fierer J (1991) Synergistic effect of human lysozyme plus ampicillin or beta-lysin on the killing of Listeria monocytogenes. J Infect Dis 163:574–578CrossRefGoogle Scholar
  25. 25.
    Hett EC, Chao MC, Steyn AJ, Fortune SM, Deng LL, Rubin EJ (2007) A partner for the resuscitation-promoting factors of Mycobacterium tuberculosis. Mol Microbiol 66:658–668CrossRefGoogle Scholar
  26. 26.
    Sassetti CM, Boyd DH, Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48(1):77–84CrossRefGoogle Scholar
  27. 27.
    Smith I (2003) Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin Microbiol Rev 16:463–496. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Daniel A, Euler C, Collin M, Chahales P, Gorelick KJ, Fischetti VA (2010) Synergism between a novel chimeric lysin and oxacillin protects against infection by methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 54:1603–1612CrossRefGoogle Scholar
  29. 29.
    Wallace RJ, Nash DR, Tsukamura M, Blacklock ZM, Silcox VA (1988) Human disease due to Mycobacterium smegmatis. J Infect Dis 158(1):52–59CrossRefGoogle Scholar
  30. 30.
    Lu T, Zhao X, Li X, Hansen G, Blondeau J, Drlica K (2003) Effect of chloramphenicol, erythromycin, moxifloxacin, penicillin and tetracycline concentration on the recovery of resistant mutants of Mycobacterium smegmatis and Staphylococcus aureus. J Antimicrob Chemother 52(1):61–64CrossRefGoogle Scholar
  31. 31.
    Deng L, Mikusova K, Robuck KG, Scherman M, Brennan PJ, Mcneil MR (1995) Recognition of multiple effects of ethambutol on metabolism of mycobacterial cell envelope. Antimicrob Agents Chemother 39:694–701CrossRefGoogle Scholar
  32. 32.
    Au MB, Siswando L, Hall J, Weinberg JS, Deng LL (2017) Identification of native Mycobacterium Smegmatis cell wall degradative enzymes using electrophoretic fluorescent assays. Int Educ Res J 3:31–34Google Scholar
  33. 33.
    Mukamolova GV, Kaprelyants AS, Young DI, Young M, Kell DB (1998) A bacterial cytokine. Proc Natl Acad Sci USA 95:8916–8921CrossRefGoogle Scholar
  34. 34.
    Aguilar-Pérez C, Gracia B, Rodrigues L, Vitoria A, Cebrián R, Deboosère N, Song OR, Brodin P, Maqueda M, Aínsa JA (2018) Synergy between Circular Bacteriocin AS-48 and Ethambutol against Mycobacterium tuberculosis. Antimicrob Agents Chemother 62(9):1–13. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Joshua N. Gustine
    • 1
  • Matthew B. Au
    • 1
  • John R. Haserick
    • 1
  • Erik C. Hett
    • 2
    • 4
  • Eric J. Rubin
    • 2
  • Frank C. GibsonIII
    • 3
  • Lingyi L. Deng
    • 1
  1. 1.Department of MedicineBoston University School of MedicineBostonUSA
  2. 2.Department of Immunology and Infectious DiseasesHarvard School of Public HealthBostonUSA
  3. 3.Department of Oral Biology, College of DentistryUniversity of FloridaGainesvilleUSA
  4. 4.Merck, Exploratory Science Center, Chemical BiologyCambridgeUSA

Personalised recommendations