scholarly journals Exploration of synergistic action of cell wall-degrading enzymes against Mycobacterium tuberculosis

2021 ◽  
Author(s):  
Loes van Schie ◽  
Katlyn Borgers ◽  
Gitte Michielsen ◽  
Evelyn Plets ◽  
Marnik Vuylsteke ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Growth Inhibition ◽  
Cell Envelope ◽  
Mycolic Acid ◽  
Wall Layer ◽  
Cell Wall Degrading Enzymes ◽  
Growth Inhibition Assay ◽  
Degrading Enzymes ◽  
Hydrophobic Barrier

Background The major global health threat tuberculosis is caused by Mycobacterium tuberculosis (Mtb). Mtb has a complex cell envelope – a partially covalently linked composite of polysaccharides, peptidoglycan and lipids, including a mycolic acid layer – which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating gram-positive and -negative infections with cell wall degrading enzymes, we investigated such approach for Mtb. Objectives (i) Development of an Mtb microtiter growth inhibition assay that allows undisturbed cell envelope formation, to overcome the invalidation of results by typical clumped Mtb-growth in surfactant-free assays. (ii) Exploring anti-Mtb potency of cell wall layer-degrading enzymes. (iii) Investigation of the concerted action of several such enzymes. Methods We inserted a bacterial luciferase-operon in an auxotrophic Mtb strain to develop a microtiter assay that allows proper evaluation of cell wall degrading anti-Mtb enzymes. We assessed growth-inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase (LysB), fungal α-amylase and human and chicken egg white lysozymes) and combinations thereof, in presence or absence of biopharmaceutically acceptable surfactant. Results Our biosafety level-2 assay identified both LysB and lysozymes as potent Mtb-inhibitors, but only in presence of surfactant. Moreover, most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, α-amylase and polysorbate 80. Conclusions Synergistically acting cell wall degrading enzymes are potently inhibiting Mtb – which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.

scholarly journals Exploration of synergistic action of cell wall-degrading enzymes against Mycobacterium tuberculosis

2021 ◽  
Author(s):  
Loes van Schie ◽  
Katlyn Borgers ◽  
Gitte Michielsen ◽  
Evelyn Plets ◽  
Marnik Vuylsteke ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Growth Inhibition ◽  
Cell Envelope ◽  
Mycolic Acid ◽  
Wall Layer ◽  
Cell Wall Degrading Enzymes ◽  
Growth Inhibition Assay ◽  
Degrading Enzymes ◽  
Hydrophobic Barrier

Background: The major global health threat tuberculosis is caused by Mycobacterium tuberculosis (Mtb). Mtb has a complex cell envelope – a partially covalently linked composite of polysaccharides, peptidoglycan and lipids, including a mycolic acid layer – which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating gram-positive and -negative infections with cell wall degrading enzymes, we investigated such approach for Mtb. Objectives: (i) Development of an Mtb microtiter growth inhibition assay that allows undisturbed cell envelope formation, to overcome the invalidation of results by typical clumped Mtb-growth in surfactant-free assays. (ii) Exploring anti-Mtb potency of cell wall layer-degrading enzymes. (iii) Investigation of the concerted action of several such enzymes. Methods: We inserted a bacterial luciferase-operon in an auxotrophic Mtb strain to develop a microtiter assay that allows proper evaluation of cell wall degrading anti-Mtb enzymes. We assessed growth-inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase (LysB), fungal α-amylase and human and chicken egg white lysozymes) and combinations thereof, in presence or absence of biopharmaceutically acceptable surfactant. Results: Our biosafety level-2 assay identified both LysB and lysozymes as potent Mtb-inhibitors, but only in presence of surfactant. Moreover, most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, α-amylase and polysorbate 80.

scholarly journals Two Accessory Proteins Govern MmpL3 Mycolic Acid Transport in Mycobacteria

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Allison Fay ◽  
Nadine Czudnochowski ◽  
Jeremy M. Rock ◽  
Jeffrey R. Johnson ◽  
Nevan J. Krogan ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Cell Envelope ◽  
Complex Structure ◽  
Mycolic Acid ◽  
Accessory Proteins ◽  
Acid Transport ◽  
Content Type ◽  
Mycolic Acids ◽  
Antibiotic Target

ABSTRACT Mycolic acids are the signature lipid of mycobacteria and constitute an important physical component of the cell wall, a target of mycobacterium-specific antibiotics and a mediator of Mycobacterium tuberculosis pathogenesis. Mycolic acids are synthesized in the cytoplasm and are thought to be transported to the cell wall as a trehalose ester by the MmpL3 transporter, an antibiotic target for M. tuberculosis. However, the mechanism by which mycolate synthesis is coupled to transport, and the full MmpL3 transport machinery, is unknown. Here, we identify two new components of the MmpL3 transport machinery in mycobacteria. The protein encoded by MSMEG_0736/Rv0383c is essential for growth of Mycobacterium smegmatis and M. tuberculosis and is anchored to the cytoplasmic membrane, physically interacts with and colocalizes with MmpL3 in growing cells, and is required for trehalose monomycolate (TMM) transport to the cell wall. In light of these findings, we propose MSMEG_0736/Rv0383c be named “TMM transport factor A”, TtfA. The protein encoded by MSMEG_5308 also interacts with the MmpL3 complex but is nonessential for growth or TMM transport. However, MSMEG_5308 accumulates with inhibition of MmpL3-mediated TMM transport and stabilizes the MmpL3/TtfA complex, indicating that it may stabilize the transport system during stress. These studies identify two new components of the mycobacterial mycolate transport machinery, an emerging antibiotic target in M. tuberculosis. IMPORTANCE The cell envelope of Mycobacterium tuberculosis, the bacterium that causes the disease tuberculosis, is a complex structure composed of abundant lipids and glycolipids, including the signature lipid of these bacteria, mycolic acids. In this study, we identified two new components of the transport machinery that constructs this complex cell wall. These two accessory proteins are in a complex with the MmpL3 transporter. One of these proteins, TtfA, is required for mycolic acid transport and cell viability, whereas the other stabilizes the MmpL3 complex. These studies identify two new components of the essential cell envelope biosynthetic machinery in mycobacteria.

Use of cell wall degrading enzymes to improve the recovery of lipids from Chlorella sorokiniana

2019 ◽  
Vol 377 ◽  
pp. 120325 ◽  
Author(s):  
Antonio Zuorro ◽  
Veronica Malavasi ◽  
Giacomo Cao ◽  
Roberto Lavecchia
Keyword(s):  
Cell Wall ◽  
Chlorella Sorokiniana ◽  
Cell Wall Degrading Enzymes ◽  
Degrading Enzymes

scholarly journals The thick waxy coat of mycobacteria, a protective layer against antibiotics and the host's immune system

2020 ◽  
Vol 477 (10) ◽  
pp. 1983-2006 ◽  
Author(s):  
Sarah M. Batt ◽  
David E. Minnikin ◽  
Gurdyal S. Besra
Keyword(s):  
Infectious Disease ◽  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Immune System ◽  
Mortality Rate ◽  
Cell Envelope ◽  
Protective Layer ◽  
Structure And Function ◽  
Complex Lipids ◽  
And Function

Tuberculosis, caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease, with a mortality rate of over a million people per year. This pathogen's remarkable resilience and infectivity is largely due to its unique waxy cell envelope, 40% of which comprises complex lipids. Therefore, an understanding of the structure and function of the cell wall lipids is of huge indirect clinical significance. This review provides a synopsis of the cell envelope and the major lipids contained within, including structure, biosynthesis and roles in pathogenesis.

Sign in / Sign up

Export Citation Format

Share Document