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 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.

Role of microtubule-associated protein 6 glycosylated with Gal-(β-1,3)-GalNAc in Parkinson's disease

“…Colorimetric detection of protein bands was performed using a silver staining kit (Sigma). Silver-stained bands were excised and identified using tandem mass spectrometric analysis as described previously [34].…”

Identification of Secreted O-Mannosylated Proteins From BCG and Characterization of Immunodominant Antigens BCG_0470 and BCG_0980

“…Gels were silver-stained according to the manufacturer's instructions. In addition, bidimensional gel electrophoresis (2D-DIGE) was performed as described previously (Chen et al, 2019). Briefly, samples were processed using a Bio-Rad clean-up kit as per the manufacturer's instructions (Hercules, CA, United States).…”

“…In addition, the recombinant mannosylated proteins were treated with α-mannosidase (Sigma-Aldrich, St. Louis, MO, United States) as per the manufacturer's instructions. Mannosidase-treated proteins were collected using magnetic agarose beads as described previously (Chen et al, 2019).…”

Proteomic and Morphologic Evidence for Taurine-5-Bromosalicylaldehyde Schiff Base as an Efficient Anti-Mycobacterial Drug

“…Fifty micrograms of total soluble protein was subjected to bidimensional (2D) electrophoresis analysis (Protein IEF Cell, Bio-Rad). The subsequent 2D electrophoresis analysis and the image analyses were performed as described previously [10]. Proteins were separated in the first dimension using Immobiline DryStrips (11 cm, pH 4-7, Bio-Rad), and in the second dimension using 12% SDS-PAGE gels.…”