Deficiency of D‐alanyl‐D‐alanine ligase A attenuated cell division and greatly altered the proteome of Mycobacterium smegmatis
Abstract 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...
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doaj-e68f6bd2b88341dc96e7f7d972b426002020-11-25T02:39:33ZengWileyMicrobiologyOpen2045-88272019-09-0189n/an/a10.1002/mbo3.819Deficiency of D‐alanyl‐D‐alanine ligase A attenuated cell division and greatly altered the proteome of Mycobacterium smegmatisYingfei Chen0Yuefei Xu1Shufeng Yang2Sheng Li3Wenyong Ding4Wenli Zhang5Dalian Yuming Senior High School Liaoning ChinaBiochemistry and Molecular Biology Department of College of Basic Medical Sciences Dalian Medical University Dalian ChinaDepartment of Microbiology Dalian Medical University Dalian ChinaBiochemistry and Molecular Biology Department of College of Basic Medical Sciences Dalian Medical University Dalian ChinaBiochemistry and Molecular Biology Department of College of Basic Medical Sciences Dalian Medical University Dalian ChinaBiochemistry and Molecular Biology Department of College of Basic Medical Sciences Dalian Medical University Dalian ChinaAbstract 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.https://doi.org/10.1002/mbo3.819cell divisionD‐alanyl‐D‐alanine ligase A (DdlA)DdlA deficiencyMycobacterium smegmatisproteome |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yingfei Chen Yuefei Xu Shufeng Yang Sheng Li Wenyong Ding Wenli Zhang |
spellingShingle |
Yingfei Chen Yuefei Xu Shufeng Yang Sheng Li Wenyong Ding Wenli Zhang Deficiency of D‐alanyl‐D‐alanine ligase A attenuated cell division and greatly altered the proteome of Mycobacterium smegmatis MicrobiologyOpen cell division D‐alanyl‐D‐alanine ligase A (DdlA) DdlA deficiency Mycobacterium smegmatis proteome |
author_facet |
Yingfei Chen Yuefei Xu Shufeng Yang Sheng Li Wenyong Ding Wenli Zhang |
author_sort |
Yingfei Chen |
title |
Deficiency of D‐alanyl‐D‐alanine ligase A attenuated cell division and greatly altered the proteome of Mycobacterium smegmatis |
title_short |
Deficiency of D‐alanyl‐D‐alanine ligase A attenuated cell division and greatly altered the proteome of Mycobacterium smegmatis |
title_full |
Deficiency of D‐alanyl‐D‐alanine ligase A attenuated cell division and greatly altered the proteome of Mycobacterium smegmatis |
title_fullStr |
Deficiency of D‐alanyl‐D‐alanine ligase A attenuated cell division and greatly altered the proteome of Mycobacterium smegmatis |
title_full_unstemmed |
Deficiency of D‐alanyl‐D‐alanine ligase A attenuated cell division and greatly altered the proteome of Mycobacterium smegmatis |
title_sort |
deficiency of d‐alanyl‐d‐alanine ligase a attenuated cell division and greatly altered the proteome of mycobacterium smegmatis |
publisher |
Wiley |
series |
MicrobiologyOpen |
issn |
2045-8827 |
publishDate |
2019-09-01 |
description |
Abstract 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. |
topic |
cell division D‐alanyl‐D‐alanine ligase A (DdlA) DdlA deficiency Mycobacterium smegmatis proteome |
url |
https://doi.org/10.1002/mbo3.819 |
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