Direct reprogramming of fibroblasts to myocytes via bacterial injection of MyoD protein

Abstract

Forced exogenous gene expression has been well characterized as an effective method for directing both cellular differentiation and dedifferentiation. However, transgene expression is not amenable for therapeutic application due to potential insertional mutagenesis. Protein-based techniques provide a safe alternative, but current protein delivery methods are quite limited by labor-intensive purification processes, low protein yield, and inefficient intracellular targeting. Such limitations may be overcome by using a naturally occurring bacterial protein injection system, called the type III secretion system (T3SS), which injects bacterial proteins directly into the eukaryotic cell cytoplasm. Using a genetically attenuated strain of Pseudomonas aeruginosa, we have previously described the ability of this system to easily deliver a high quantity of protein to both differentiated and pluripotent cells. MyoD is a key muscle regulatory factor, the overexpression of which is able to induce transdifferentiation of numerous cell types into functional myocytes. Here we demonstrate transient injection of MyoD protein by P. aeruginosa to be sufficient to induce myogenic conversion of mouse embryonic fibroblasts. In addition to clear morphological changes, muscle-specific gene expression has been observed both at mRNA and protein levels. These studies serve as a foundation for the bacterial delivery of transcription factors to efficiently modulate concentration-dependent and temporal activation of gene expression that directs cell fate without jeopardizing genomic integrity.

FIG. 1.

Bacterial production and injection of ExoS54–MyoD. (A) PAK-JδSTY, PAK-JδpopD, and PAK-JδexsA were electroporated with the vector (V) or a plasmid containing the ExoS54–Cre fusion. (B) Each strain was examined for the ability to produce and secrete the fusion protein by anti-MyoD immunoblot of the bacterial pellet and secretion supernatant. (C) MEFs were infected with each strain at an MOI of 100, unless otherwise noted, lysed and examined for protein injection by anti-MyoD immunoblot.

FIG. 2.

Lentiviral expression of MyoD and ExoS54–MyoD. MEFs were infected with lentiviral particles expressing, MyoD or ExoS54–MyoD. Cells were fixed and stained 6 dpi for expression of desmin. Nuclei were stained with DAPI.

FIG. 3.

ExoS54–MyoD injection halts cell proliferation. CFSE-stained MEFs were infected with PAK-JΔSTY (pExoS54–MyoD) at MOIs of 25, 50, 100, 150, 200, PAK-JΔpopD and vector controls at an MOI of 100. Following a 3-h infection, bacteria were cleared and cells were allowed to proliferate for 5 days. Cells were then collected for CFSE detection by flow cytometry (A). Efficiency of ExoS54–MyoD-mediated growth arrest was compared for each condition as percent of CFSE retaining cells in the total cell population (B). n=3.

FIG. 4.

MEF cell cycle synchronization. MEFs were either left unsynchronized (U) or synchronized through double thymidine blocking and released every 4 h to determine the duration of the cell replication cycle.

FIG. 5.

ExoS54–MyoD injection causes muscle-specific protein expression in MEFs. Fourteen dpi, infected MEFs and uninfected MEFs were fixed and immunostained for expression of the indicated proteins using protein-specific antibodies and DAPI for nuclear staining.

FIG. 6.

ExoS54–MyoD injection activates muscle-specific gene expression in MEFs. MEFs that had been infected (+) or not infected (−) were collected at 14 dpi for RNA extraction and RT-PCR analysis for the expression of the indicated genes, using cDNA from murine tongue muscle cells as a positive control for expression.