Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages

Abstract

Cytidine deaminase (MtCDA), encoded by cdd gene (Rv3315c), is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.

EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis

“…Approximately 30 μg of M. smegmatis total protein (crude extract) from detergent fraction was denatured at 70°C for 10 min, subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes (Merck Millipore) in buffer Tris 25 mM, glycine 192 mM (pH 8.8), and methanol 20% for 4 h at 70 V. After transfer, the membrane was blocked with 5% nonfat dried milk (Santa Cruz Biotechnology) and 0.05% Tween 20 (Sigma-Aldrich) in Tris-buffered saline (T-TBS) (2 h, 4°C) and probed with anti- Mt EPSPS polyclonal mouse antibody in a 1:500 dilution (overnight at 4°C). Membranes were washed three times with T-TBS, and alkaline phosphatase-conjugated anti-mouse secondary antibody (Invitrogen) was used at a dilution of 1:5,000 ( 45 ). Chemiluminescent substrate (Novex by Life Technologies, USA) was used for detection with ChemiDoc (Bio-Rad).…”

EvaluatingaroAgene essentiality and EPSP synthase vulnerability inMycobacterium smegmatisunder different nutritional conditions

“…Approximately 30 µg of M. smegmatis proteins from detergent fraction were boiled at 70°C for 10 min, loaded on 12% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), and transferred to nitrocellulose membranes (Merck Millipore, Ltd-Ireland) in Buffer Tris 25 mM, glycine 192 mM pH 8.8 and methanol 20% for 4h at 70 v. After transfer, the membrane was blocked with 5% non-fat dried milk (Santa Cruz Biotechnology, USA), 0.05% tween-20 (Sigma-Aldrich, USA) in TBS (T-TBS) (2h, 4°C) and probed with anti- Mt EPSPS polyclonal mouse antibody in a 1:500 dilution (overnight at 4°C). Membranes were washed three times with T-TBS, and alkaline phosphatase-conjugated anti-mouse secondary antibody (Invitrogen, USA) was used at a dilution of 1:5,000 [27]. Chemiluminescent substrate (Novex by Life Technologies, USA) was used for detection with ChemiDoc (Bio-Rad, USA).…”