Abstract
D-alanine (D-Ala) is an essential amino acid that has a key role in bacterial cell wall synthesis. Alanine racemase (Alr) is a unique enzyme that interconverts L-alanine and D-alanine in most bacteria, making this enzyme a potential target for antimicrobial drug development. Streptococcus mutans is a major causative factor of dental caries. The factors involved in the survival, virulence and interspecies interactions of S. mutans could be exploited as potential targets for caries control. The current study aimed to investigate the physiological role of Alr in S. mutans. We constructed alr mutant strain of S. mutans and evaluated its phenotypic traits and interspecies competitiveness compared with the wild-type strain. We found that alr deletion was lethal to S. mutans. A minimal supplement of D-Ala (150 μg·mL−1) was required for the optimal growth of the alr mutant. The depletion of D-alanine in the growth medium resulted in cell wall perforation and cell lysis in the alr mutant strain. We also determined the compromised competitiveness of the alr mutant strain relative to the wild-type S. mutans against other oral streptococci (S. sanguinis or S. gordonii), demonstrated using either conditioned medium assays or dual-species fluorescent in situ hybridization analysis. Given the importance and necessity of alr to the growth and competitiveness of S. mutans, Alr may represent a promising target to modulate the cariogenicity of oral biofilms and to benefit the management of dental caries.
Highlights
D-alanine (D-Ala) is an essential substance for the synthesis of peptidoglycan (PG), an important constituent of the cell wall of both Gram-negative and Gram-positive bacteria.[1,2] The synthesis of the cell wall is initiated with uridine diphosphate (UPD)-N-acetylmuramic acid, L-Ala, D-glutamic acid (D-Glu) and meso-diaminopimelic acid to generate UDP-N-acetylmuramyl-L-Ala-D-Glu-meso-diaminopimelate.[1,3] A D-Ala dipeptide is coupled to this intermediate through UDP-N-acetylmuramyl-tripeptide-D-Ala-D-Ala ligase to generate UDP-N-acetylmuramyl-tripeptide-D-Ala-D-Ala, which is subsequently incorporated into the growing PG peptide chain
Growth of the alr- mutant is strictly dependent on exogenous D-Ala As shown in Figure 2a, alr deletion without the addition of exogenous D-Ala was lethal to S. mutans
alanine racemase (Alr) is responsible for the production of D-Ala, which is used for PG biosynthesis in all bacteria, including harmful pathogens.[1,2,21]
Summary
D-alanine (D-Ala) is an essential substance for the synthesis of peptidoglycan (PG), an important constituent of the cell wall of both Gram-negative and Gram-positive bacteria.[1,2] The synthesis of the cell wall is initiated with uridine diphosphate (UPD)-N-acetylmuramic acid, L-Ala, D-glutamic acid (D-Glu) and meso-diaminopimelic acid to generate UDP-N-acetylmuramyl-L-Ala-D-Glu-meso-diaminopimelate.[1,3] A D-Ala dipeptide is coupled to this intermediate through UDP-N-acetylmuramyl-tripeptide-D-Ala-D-Ala ligase to generate UDP-N-acetylmuramyl-tripeptide-D-Ala-D-Ala, which is subsequently incorporated into the growing PG peptide chain. Alr is a unique enzyme responsible for the racemization of L-Ala to D-Ala.[4] Previous studies have revealed that Alr is essential for the survival of numerous Gram-positive bacteria, including Bacillus, Lactobacillus, Burkholderia pseudomallei, Burkholderia mallei and Mycobacterium tuberculosis. Knockout of the alr gene in these bacteria resulted in a strict exogenous D-Ala-dependent growth phenotype.[5,6,7,8] Similar growth arrest and extensive cell lysis were observed in the alr mutant of Gram-negative Escherichia coli, which underwent rapid and extensive cell lysis when deprived of D-Ala.[5,9] Further in vitro and in vivo studies of M. tuberculosis have shown that Alr is a primary target of D-cycloserine, and the inhibition of Alr alone could reduce the viability and perseverance of this bacterium.[8]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
