Analysis of DnaK Expression from a Strain of Mycoplasma fermentans in Infected HCT116 Human Colon Carcinoma Cells

Abstract

Several species of mycoplasmas, including Mycoplasma fermentans, are associated with certain human cancers. We previously isolated and characterized in our laboratory a strain of human mycoplasma M. fermentans subtype incognitus (MF-I1) able to induce lymphoma in a Severe Combined Immuno-Deficient (SCID) mouse model, and we demonstrated that its chaperone protein, DnaK, binds and reduces functions of human poly-ADP ribose polymerase-1 (PARP1) and ubiquitin carboxyl-terminal hydrolase protein-10 (USP10), which are required for efficient DNA repair and proper p53 activities, respectively. We also showed that other bacteria associated with human cancers (including Mycoplasmapneumoniae, Helicobacterpylori, Fusobacteriumnucleatum, Chlamydiathrachomatis, and Chlamydia pneumoniae) have closely related DnaK proteins, indicating a potential common mechanism of cellular transformation. Here, we quantify dnaK mRNA copy number by RT-qPCR analysis in different cellular compartments following intracellular MF-I1 infection of HCT116 human colon carcinoma cells. DnaK protein expression in infected cells was also detected and quantified by Western blot. The amount of viable intracellular mycoplasma reached a steady state after an initial phase of growth and was mostly localized in the cytoplasm of the invaded cells, while we detected a logarithmically increased number of viable extracellular bacteria. Our data indicate that, after invasion, MF-I1 is able to establish a chronic intracellular infection. Extracellular replication was more efficient while MF-I1 cultured in cell-free axenic medium showed a markedly reduced growth rate. We also identified modifications of important regulatory regions and heterogeneous lengths of dnaK mRNA transcripts isolated from intracellular and extracellular MF-I1. Both characteristics were less evident in dnaK mRNA transcripts isolated from MF-I1 grown in cell-free axenic media. Taken together, our data indicate that MF-I1, after establishing a chronic infection in eukaryotic cells, accumulates different forms of dnaK with efficient RNA turnover.