The Mycobacteria marinum M strain [32], type I human lung epithelial cell line WI-26 VA4 and human monocyte cell line THP-1 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and preserved in our lab [33,34]. Type II human lung epithelial cell line A549 was kindly provided by Dr. Jianying Zhang at The University of Texas at El Paso [35].

The pJSC407, pGOAL17, and pGMCKq1-10M1-sspBopt plasmids were kindly provided by Dr. Hugues Ouellet at The University of Texas at El Paso. The pJSC407 and pGOAL17 plasmids were used to produce the pJSC407-sacB suicide plasmid. The pGMCKq1-10M1-sspBopt was used for inducible expression of adaptor protein SspB for DAS4+ inducible knockout system. The pQE80L-SpyCatcher-ELP-GFP plasmid was purchased from Addgene (#69,835, Watertown, MA, USA) for prokaryotic expression and purification of SpyCatcher-ELP-GFP protein.

Since the ELP linker between SC and GFP was found cytotoxic to human cells [36], we removed it and inserted SpyCather (SC) into pcDNA3-EGFP vector for eukaryotic expression of SpyCatcher-GFP (SC-GFP) in A549. However, we found that transient transfection of pcDNA3-SC-GFP had a very low expression of SC-GFP in several mycobacteria susceptible cell lines, including WI-26, RAW264.7, THP-1, and A549. In order to enhance SC-GFP expression in eukaryotic cells, the coding sequence of SC was optimized for expression in human cell lines and synthesized by ThermoFisher Scientific (Rockford, IL, USA). The codon-optimized SC sequence was inserted into pcDNA3-EGFP (#13,031, Watertown, MA, USA) for eukaryotic expression of SC-GFP. Even after optimization, SC-GFP was efficiently expressed only in A549 cell line.

The primers for site-directed mutagenesis, identification, and RT-qPCR were all designed according to Mm’s genomic DNA sequence (GenBank Accession Number: CP000854.1) and synthesized by Sigma-Aldrich (St. Louis, MO, USA). The primers used in this study are listed in Table 1.

Plasmids and primers used in this study

To fuse ST and DAS4+ to the respective C-terminus of EsxA and EsxB, the homologous arms that flank the genes esxA or esxB were amplified from the genomic DNA of Mm, respectively. Then the amplified fragments were inserted into pJSC407-sacB. Similarly as previously described [21], the recombinant suicide plasmid was electroporated into competent Mm(WT). After selection with 2% sucrose on the 7H10 plates (10% OADC), the colonies were picked to culture until OD600 reached 0.6 to 0.8. Then, the culture pellet was collected, resuspended in ultrapure water, and heated at 95°C for 5 min. The heated pellet was used as PCR template and identified with primers that span the target genes (esxA or esxB) and the tags. The positive colonies were named as Mm(EsxB-DAS4+) and Mm(EsxA-ST), respectively. The plasmid pGMCKq1-10M1-sspBopt was electroporated into Mm(EsxB-DAS4+) to obtain Mm(EsxB-DAS4+)|pGMCKq1 for inducible knockdown of EsxB protein by ATC. All the Mm strains used in this study carry a mCherry-coding plasmid.

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