Cell Wall Hydrolytic Enzymes Enhance Antimicrobial Drug Activity Against Mycobacterium
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.
- 2.Sotgiu G, Sulis G, Matteelli A (2017) Tuberculosis-a Wold Health Organization perspective. Microbiol Spectr. https://doi.org/10.1128/microbiolspec.TNMI7-0036-2016 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1093/cid/ciw376.CrossRefGoogle Scholar
- 6.WHO (2016) Global tuberculosis report. World Health Organization, GenevaGoogle Scholar
- 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
- 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
- 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
- 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. https://doi.org/10.1128/AAC.00359-18 CrossRefGoogle Scholar