• Functional analysis of Paramecium genes responsiveness to Holospora infection

      Bright, Lydia; Weiler, Jared (2021-05)
      Paramecium caudatum is a single-celled ciliate that sweeps food into its oral groove to acquire nutrients. The cells become infected through this feeding apparatus by Holospora undulata, an obligate endosymbiont that occupies the micronucleus of P. caudatum in two morphologically distinct forms, the infectious and reproductive forms. Once the P. caudatum cells begin to starve, the reproductive forms of the parasitic bacteria consume most of the food in the micronuclei and differentiate into infectious forms. The bacteria eventually break out of the cell and seek other hosts to infect. To better understand the mechanism by which H. undulata successfully infiltrates their host’s defense systems, P. caudatum strains were infected and varying levels of susceptibility to infection were identified. Whole genome RNA sequencing found that certain proteins are significantly upregulated upon uptake of the H. undulata. By cloning the significantly up-regulated genes into E. coli, feeding this silencing media to naïve stocks of P. caudatum, and analyzing the infection phenotype of each, we hoped to characterize the genes that function in the infiltration of H. undulata. Varying levels of infection prevalence were seen amongst the gene knockdowns and across replicates of each infection. However, five knockdowns show significant resistance compared to their respective controls, revealing a possible involvement of 5 genes in H. undulata susceptibility. After finding which genes are capable of inducing resistance, we plan to identify the associated proteins and track the gene’s evolutionary divergence in P. caudatum strains with varying infectability.
    • Measuring gene expression of MORN, SANT, and Sig 1, “ ” 4, in Paramecium caudatum over the course of Holospora infection

      Vislocka, Karin (2021-05)
      Paramecia are single-celled organisms that live in ponds and feed on other single celled organisms like bacteria or algae. They are typically oblong and are covered with short structures called cilia. They have interactions with bacteria as they are infected intranuclearly by Holospora. Paramecium caudatum is known to express genes such as MORN, SANT, and Sig 1, “ ”, 4. Paramecium shows a response upon infection by Holospora as it navigates through the various stages of infection. The Holospora bacteria may cause varying degrees of expression of genes in the single celled organism. The genes were chosen due to their upregulation in Paramecium in a previous RNA sequencing study (Kagemann et al, in prep). Through the use of microscopy, imaging and qPCR, gene expression was tracked in the control and in the samples in which MORN, SANT, and Sig 1, “ ”, 4 genes were knocked down. The amount of gene expression was measured in each stage of the infection using qPCR. A comparison was made between the results of the control samples, which have no knocked down genes, to the samples which have knocked down genes. The amplitude of expression between Sig 1, Sig 2, Sig 3, and Sig 4 was compared as they function as protein kinases in Paramecium caudatum.