# Role of DdIR-regulated D-Ala-D-Ala biosynthesis in peptidoglycan synthesis and virulence

> **NIH NIH R21** · TUFTS UNIVERSITY BOSTON · 2020 · $206,250

## Abstract

ABSTRACT
 D-alanine-D-alanine is an essential precursor for biosynthesis of peptidoglycan and bacterial cell wall.
This dipeptide is synthesized by D-Ala-D-Ala ligase, an enzyme usually encoded by one or two ddl gene(s).
Mutants lacking ddl genes are not viable in the absence of D-Ala-D-Ala; it is assumed that such mutants
cannot grow in animals due to D-Ala-D-Ala limitation.
 DdlR, an unusual transcriptional regulator containing an aminotransferase domain, is a positive
regulator of the ddl gene in Clostridioides (formerly Clostridium) difficile, an important pathogen that can cause
severe disease, including antibiotic-associated diarrhea and pseudomembranous colitis, in humans. A C.
difficile ddlR null mutant retains some activity of D-Ala-D-Ala ligase, but this residual activity is not sufficient for
growth in the absence of the dipeptide.
 We hypothesize that C. difficile strains that lack DdlR will be unable to grow in animals. We propose to
explore the potential of DdlR as a novel target for antibacterial therapy and to illuminate prospective drug
design by identifying molecules that interact with DdlR and affect its activity. DdlR may be especially well
suited as a potential drug target due to its essentiality and expected ability to interact with two different types of
small molecules, pyridoxal 5’-phosphate and D-Ala-D-Ala. Moreover, simultaneous targeting of DdlR and D-
Ala-D-Ala ligase is a viable possibility. To achieve our goal, we will study in detail the mechanism of DdlR-
mediated regulation of D-Ala-D-Ala synthesis in C. difficile using genetic and molecular biological approaches
and test the role of DdlR and D-Ala-D-Ala synthesis in cell growth and virulence using a mouse model. Though
a very important pathogen itself, C. difficile will also serve as a model for other DdlR-containing pathogenic
Gram-positive bacteria, including Bacillus anthracis, Clostridium tetani, Clostridium botulinum, and Clostridium
perfringens.

## Key facts

- **NIH application ID:** 9822148
- **Project number:** 5R21AI137641-02
- **Recipient organization:** TUFTS UNIVERSITY BOSTON
- **Principal Investigator:** BORIS R BELITSKY
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $206,250
- **Award type:** 5
- **Project period:** 2018-11-13 → 2021-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9822148

## Citation

> US National Institutes of Health, RePORTER application 9822148, Role of DdIR-regulated D-Ala-D-Ala biosynthesis in peptidoglycan synthesis and virulence (5R21AI137641-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9822148. Licensed CC0.

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