# Spatial organization of mycobacterial cell wall biosynthesis

> **NIH NIH R21** · UNIVERSITY OF MASSACHUSETTS AMHERST · 2020 · $230,935

## Abstract

PROJECT SUMMARY/ABSTRACT
There is a fundamental gap in understanding how peptidoglycan (PG) is biosynthesized in a spatially
coordinated fashion to support the polar growth of Mycobacterium tuberculosis (Mtb). This gap represents an
important problem because the elongation of the cell envelope, an essential process of bacterial growth, is
incomprehensive without understanding the precise mechanism of spatially coordinated PG precursor
biosynthesis, transport and cell wall integration. The intracellular membrane domain (IMD) is a discrete area of
the plasma membrane (PM) particularly enriched in the subpolar region of actively growing mycobacterial cells.
The long-term goal is to understand the role of PM partitioning in mycobacterial physiology and to identify
vulnerabilities in this process. As the next step to achieve this goal, the overall objective of this proposal is to
gain the fundamental insights into the spatial compartmentalization of PG assembly, so that the IMD can be
evaluated as a target for inhibiting the assembly of the PG layer. The central hypothesis is that the IMD is a
region of the PM where polyprenol-linked PG precursors are synthesized. The rationale is that characterizing
the PM partitioning of the PG biosynthesis will lay the foundation for understanding the role of the IMD in the
cell wall elongation, thereby beginning to understand how the robust pathogen Mtb produces and maintains its
highly complex cell wall. Guided by published studies and preliminary data from the applicant’s laboratories,
this hypothesis will be tested by pursuing two specific aims: 1) Determine the subcellular localization of
proteins that synthesize, transport and polymerize PG precursors; 2) Determine the localized
production of polyprenol-linked PG precursors and PG polymer. Under the first aim, the working
hypothesis, that PG biosynthetic enzymes are spatially and biochemically segregated in the PM, will be
addressed by quantitative and super resolution microscopy and by subcellular fractionation. Under the second
aim, the working hypothesis, that precursors and polymerized PG are in distinct PM regions, will be determined
by bioorthogonal metabolic labeling of PG for microscopic detection and in vitro biotinylation of PG precursors
for biochemical detection. The project is innovative because it combines synergistic expertise of two
laboratories to dissect the role of compartmentalized PM in mycobacterial PG synthesis, a substantive
departure from the status quo in both concept and execution. The proposed research is significant because it
reveals whether PM partitioning organizes PG synthesis, an established drug target of high clinical significance.
The key data obtained from this study will form the basis to further investigate the role of the IMD in cell wall
elongation of Mtb, enhancing our evaluation of IMD disruption as a new route for inhibiting PG synthesis and
mycobacterial cell growth.
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## Key facts

- **NIH application ID:** 9884733
- **Project number:** 5R21AI144748-02
- **Recipient organization:** UNIVERSITY OF MASSACHUSETTS AMHERST
- **Principal Investigator:** Yasu S Morita
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $230,935
- **Award type:** 5
- **Project period:** 2019-03-05 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9884733

## Citation

> US National Institutes of Health, RePORTER application 9884733, Spatial organization of mycobacterial cell wall biosynthesis (5R21AI144748-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9884733. Licensed CC0.

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