Modulation of the Host Nuclear Compartment by Trypanosoma cruzi Uncovers Effects on Host Transcription and Splicing Machinery

Host manipulation is a common strategy for invading pathogens. Trypanosoma cruzi, the causative agent of Chagas Disease, lives intracellularly within host cells. During infection, parasite-associated modifications occur to the host cell metabolism and morphology. However, little is known about the effect of T. cruzi infection on the host cell nucleus and nuclear functionality. Here, we show that T. cruzi can modulate host transcription and splicing machinery in non-professional phagocytic cells during infection. We found that T. cruzi regulates host RNA polymerase II (RNAPII) in a time-dependent manner, resulting in a drastic decrease in RNAPII activity. Furthermore, host cell ribonucleoproteins associated with mRNA transcription (hnRNPA1 and AB2) are downregulated concurrently. We reasoned that T. cruzi may hijack the host U2AF35 auxiliary factor, a key regulator for RNA processing, as a strategy to affect the splicing machinery activities directly. In support of our hypothesis, we carried out in vivo splicing assays using an adenovirus E1A pre-mRNA splicing reporter, showing that intracellular T. cruzi directly modulates the host cells by appropriating U2AF35. For the first time, our results provide evidence of a complex and intimate molecular relationship between T. cruzi and the host cell nucleus during infection

Myosin Va phosphorylated on ser(1650) is found in nuclear speckles and redistributes to nucleoli upon inhibition of transcription

Nuclear actin and nuclear myosins have been implicated in the regulation of geneexpression in vertebrate cells. Myosin V is a class of actin-based motor proteins involved in cytoplasmic vesicle transport and anchorage, spindle-pole alignment and mRNA translocation. In this study, myosin-Va, phosphorylated on a conserved serine in the tail domain (phospho-ser(1650) MVa), was localized to subnuclear compartments. A monoclonal antibody, 9E6, raised against a peptide corresponding to phosphoserine(1650) and flanking regions of the murine myosin Va sequence, was immunoreactive to myosin Va heavy chain in cellular and nuclear extracts of HeLa cells, PC12 cells and B16-F10 melanocytes. Immunofluorescence microscopy with this antibody revealed discrete irregular spots within the nucleoplasm that colocalized with SC35, a splicing factor that earmarks nuclear speckles. Phospho-ser(1650) MVa was not detected in other nuclear compartments, such as condensed chromatin, Cajal bodies, gems and perinucleolar caps. Although nucleoli also were not labeled by 9E6 under normal conditions, inhibition of transcription in HeLa cells by actinomycin D caused the redistribution of phospho-ser(1650) MVa to nucleoli, as well as separating a fraction of phosphoser(1650) MVa from SC35 into near-neighboring particles. These observations indicate a novel role for myosin Va in nuclear compartmentalization and offer a new lead towards the understanding of actomyosin-based gene regulation