The effect of a Mycoplasma hyorhinis protein (p37) on gene expression in mouse fibroblasts

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Life Sciences, Department of Botany, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

The p37 protein is a lipoprotein associated with the plasma membrane of Mycoplasma hyorhinis. Cell lines treated with the purified p37 protein exhibit reduced heterotypic contact inhibition of locomotion in the Abercrombie confronted explant invasion assay. In addition p37-treated and p37-transformed cells show increased migration through transwell chambers and increased invasivity through a Matrigel matrix. However, the molecular mechanisms underlying these responses are not known. This thesis identifies and characterizes the changes in gene expression of the NIH3T3 (mouse) fibroblast cell line in response to added p37 protein. A microarray analysis of p37-treated and untreated NIH3T3 cells identified many genes that were up or downregulated. The most strongly up-regulated genes encoded cytokines and acute phase proteins which play a major role in the inflammatory response. Quantitative PCR was used to determine the temporal expression pattern of the p37-induced gene expression over 24hr. The cytokine interleukin 6 (Il6) was identified as a primary response gene. Possible explanations for this response are discussed. The Il6 protein, via the Il6 receptor, activates STAT3 transcription of secondary response genes. Unexpectedly, inhibition of the Il6 receptor and of STAT3 activation increased the potency of p37-induced gene expression. Inhibition of the Toll-like receptor 4 inhibited p37-induced gene expression in NIH3T3 cells. NIH3T3 cells were transfected with the p37 gene. The transfected cells lifted readily from the tissue culture plate and were smaller than the wild type NIH3T3 cells. Expression levels of eight of the twenty genes selected based on the microarray data were higher in p37-transfected cells than p37-treated NIH3T3 cells. p37-treated NIH3T3 cells exhibited increased migration rates in wound healing assays and rapid activation of RhoA. Mutating the four amino acids required for thiamine pyrophosphate binding by p37 reduced the capacity of the protein to up-regulate gene expression. Angptl4 was an exception, the mutated p37 protein further stimulated expression of the gene. The removal of the 20 C-terminal amino acids of p37 greatly reduced the proteins capacity for XVI stimulating gene expression. However, the stimulation of Angptl4 was again the exception and induction was only affected when 40 C-terminal amino acids were removed from p37. Finally, the amino acid sequence of p37 was compared with proteins from other mycoplasma species. Several homologues were observed.