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

Abstract

Several species of mycoplasmas, including <i>Mycoplasma fermentans</i>, are associated with certain human cancers. We previously isolated and characterized in our laboratory a strain of human mycoplasma <i>M. fermentans</i> subtype <i>incognitus</i> (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 <i>Mycoplasma</i><i>pneumoniae</i>, <i>Helicobacter</i><i>pylori</i>, <i>Fusobacterium</i><i>nucleatum</i>, <i>Chlamydia</i><i>thrachomatis</i>, and <i>Chlamydia pneumoniae</i>) have closely related DnaK proteins, indicating a potential common mechanism of cellular transformation. Here, we quantify <i>dnaK</i> 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 <i>dnaK</i> mRNA transcripts isolated from intracellular and extracellular MF-I1. Both characteristics were less evident in <i>dnaK</i> 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 <i>dnaK</i> with efficient RNA turnover.