Heterologous production of the D-cycloserine intermediate O-acetyl-L-serine in a human type II pulmonary cell model
Laurel Robbins1, Ariane Balaram1, Stefanie Dejneka1
1Department of Chemistry and Biochemistry and Molecular Biology Program, Lake Forest College, Lake Forest, USA.
Abstract:
Tuberculosis (TB) is the second leading cause of death by a single infectious disease behind COVID-19. Despite a century of effort, the current TB vaccine does not effectively prevent pulmonary TB, promote herd immunity, or prevent transmission. Therefore, alternative approaches are needed. We seek to develop a cell therapy that produces an effective antibiotic in response to TB infection. D-cycloserine (D-CS) is a second-line antibiotic for TB that inhibits bacterial cell wall synthesis. We have determined D-CS to be the optimal candidate for anti-TB cell therapy due to its effectiveness against TB, relatively short biosynthetic pathway, and its low-resistance incidence. The first committed step towards D-CS synthesis is catalyzed by the L-serine-O-acetyltransferase (DcsE) which converts L-serine and acetyl-CoA to O-acetyl-L-serine (L-OAS). To test if the D-CS pathway could be an effective prophylaxis for TB, we endeavored to express functional DcsE in A549 cells as a human pulmonary model. We observed DcsE-FLAG-GFP expression using fluorescence microscopy. DcsE purified from A549 cells catalyzed the synthesis of L-OAS as observed by HPLC-MS. Therefore, human cells synthesize functional DcsE capable of converting L-serine and acetyl-CoA to L-OAS demonstrating the first step towards D-CS production in human cells.
Insights
Developing a novel cell therapy for tuberculosis (TB) involves engineering human cells to produce the antibiotic D-cycloserine (D-CS). This study demonstrates the first step by successfully expressing functional DcsE in lung cells.
Area of Science:
- Biotechnology
- Infectious Disease Research
- Cell Therapy Development
Background:
- Tuberculosis (TB) remains a leading infectious cause of death, with current vaccines lacking efficacy in preventing pulmonary TB and transmission.
- Existing treatments face challenges, necessitating innovative therapeutic strategies like cell-based antibiotic production.
- D-cycloserine (D-CS) is a crucial second-line TB antibiotic, and its biosynthetic pathway offers a target for cell therapy.
Purpose of the Study:
- To engineer human pulmonary cells to synthesize D-cycloserine (D-CS), a key antibiotic for tuberculosis (TB) treatment.
- To establish a proof-of-concept for cell therapy as an alternative approach to combat TB.
- To investigate the feasibility of expressing functional DcsE, the first enzyme in the D-CS pathway, within human cells.
Main Methods:
- The L-serine-O-acetyltransferase (DcsE) gene was expressed in A549 human lung cells, serving as a model for pulmonary TB.
- Successful expression of DcsE fused with FLAG and GFP tags was confirmed using fluorescence microscopy.
- The enzymatic activity of purified DcsE from A549 cells was analyzed using High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS).
Main Results:
- Functional DcsE enzyme was successfully expressed in A549 human lung cells, visualized via fluorescence microscopy.
- Purified DcsE from these engineered cells demonstrated catalytic activity, synthesizing O-acetyl-L-serine (L-OAS) from L-serine and acetyl-CoA.
- This confirms the capability of human cells to perform the initial step in D-CS biosynthesis.
Conclusions:
- Human cells can be engineered to express functional DcsE, the enzyme initiating D-cycloserine (D-CS) synthesis.
- This represents a significant first step towards developing a cell therapy for tuberculosis (TB) leveraging D-CS production.
- The successful synthesis of L-OAS in human cells validates the potential of this approach for novel TB prophylaxis and treatment.
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