Deficiency of D‐alanyl‐D‐alanine ligase A attenuated cell division and greatly altered the proteome of Mycobacterium smegmatis

• Published in: MicrobiologyOpen (Weinheim) Vol. 8; no. 9; pp. e00819 - n/a
• Main Authors: Chen, Yingfei, Xu, Yuefei, Yang, Shufeng, Li, Sheng, Ding, Wenyong, Zhang, Wenli
• Format: Journal Article
• Language: English
• Published: England John Wiley & Sons, Inc 01.09.2019; John Wiley and Sons Inc; Wiley
• Subjects: Alanine; Antibiotics; Antibodies; Antisense RNA; Biodegradation; Biosynthesis; Carbohydrates; Catabolism; Cell division; Cell survival; Cloning; DdlA deficiency; Deoxyribonucleic acid; Dimerization; DNA; D‐alanyl‐D‐alanine ligase A (DdlA); E coli; Electron microscopy; Electrophoresis; Electrophoresis, Gel, Two-Dimensional; Fatty acids; Gel electrophoresis; Gene expression; Gene Knockdown Techniques; Mass Spectrometry; Mass spectroscopy; Methanol dehydrogenase; Microscopy, Electrochemical, Scanning; Microscopy, Electron, Transmission; Morphology; Mycobacterium smegmatis; Mycobacterium smegmatis - chemistry; Mycobacterium smegmatis - enzymology; Mycobacterium smegmatis - growth & development; Mycobacterium smegmatis - ultrastructure; N-Nitrosodimethylamine; Original; Peptide Synthases - deficiency; Peptide Synthases - metabolism; Peptidoglycans; Plasmids; Protein biosynthesis; Proteins; proteome; Proteome - analysis; Proteomes; Ribonucleic acid; RNA; Scanning electron microscopy; Studies; Transmission electron microscopy; Tuberculosis; Virulence; China; Mycobacterium smegmatis; cell division; DdlA deficiency; D-alanyl-D-alanine ligase A (DdlA); proteome
• ISSN: 2045-8827; 2045-8827
• DOI: 10.1002/mbo3.819

D‐Alanyl‐D‐alanine ligase A (DdlA) catalyses the dimerization of two D‐alanines yielding D‐alanyl‐D‐alanine required for mycobacterial peptidoglycan biosynthesis, and is a promising antimycobacterial drug target. To better understand the roles of DdlA in mycobacteria in vivo, we established a cell model in which DdlA expression was specifically downregulated by ddlA antisense RNA by introducing a 380 bp ddlA fragment into pMind followed by transforming the construct into nonpathogenic Mycobacterium smegmatis. The M. smegmatis cell model was verified by plotting the growth inhibition curves and quantifying endogenous DdlA expression using a polyclonal anti‐DdlA antibody produced from the expressed DdlA. Scanning electron microscopy and transmission electron microscopy were used to investigate mycobacterial morphology. Bidimensional gel electrophoresis and mass spectrometry were used to analyze differentially expressed proteins. Consequently, the successful construction of the M. smegmatis cell model was verified. The morphological investigation of the model indicated that DdlA deficiency led to an increased number of Z rings and a rearrangement of intracellular content, including a clear nucleoid and visible filamentous DNA. Proteomic techniques identified six upregulated and 14 downregulated proteins that interacted with each other to permit cell survival by forming a regulatory network under DdlA deficiency. Finally, our data revealed that DdlA deficiency inhibited cell division in mycobacteria and attenuated the process of carbohydrate catabolism and the pathway of fatty acid anabolism, while maintaining active protein degradation and synthesis. N‐Nitrosodimethylamine (NDMA)‐dependent methanol dehydrogenase (MSMEG_6242) and fumonisin (MSMEG_1419) were identified as potential antimycobacterial drug targets. Mycobacterium smegmatis cell model, which was downregulated by Sm‐ddlA antisense RNA was constructed. The ultrastructure analysis showed that DdlA deficiency inhibited mycobacterial cell divisions. Proteomic analysis showed that DdlA deficiency attenuated catabolic process of carbohydrate and anabolic pathway of fatty acid, maintained active degradation and synthesis of proteins. NDMA‐dependent methanol dehydrogenase and fumonisin were identified as greatly differentially expressed proteins by proteomic analysis and displayed a great potential to be developed into anti‐TB drug targets.