Feed-Forward Regulation of Phagocytosis by Entamoeba histolytica

Abstract

The parasitic protozoan Entamoeba histolytica is aptly named for its capacity to destroy host tissue. When E. histolytica trophozoites invade the lamina propria of a host colon, extracellular matrices are degraded while host cells are killed and phagocytosed. The ability of E. histolytica to phagocytose host cells correlates with virulence in vivo. In order to better understand the mechanism of phagocytosis, we used an E. histolytica Affymetrix microarray chip to measure the total gene expression of phagocytic and nonphagocytic subpopulations. Using paramagnetic beads coated with a known host ligand that stimulates phagocytosis, phagocytic and nonphagocytic amoebae from a single culture were purified. Microarray analysis of the subpopulations identified 121 genes with >2-fold higher expression in phagocytic than in nonphagocytic amoebae. Functional annotation identified genes encoding proteins involved in actin binding and cytoskeletal organization as highly enriched gene clusters. Post hoc analyses of selected genes showed that the gene expression profile identified in the microarray experiment did not exist prior to cell sorting but rather was stimulated through phagocytosis. Further, these expression profiles correlated with an increase in phagocytic ability, as E. histolytica cultures exposed to an initial stimulus of phagocytosis showed increased phagocytic ability upon a second stimulus. To our knowledge, this is the first description of such feed-forward regulation of gene expression and phagocytic ability in a phagocyte.

Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells

“…HCT-8 cell nuclei were stained using cell-permeant Hoechst 33342 (blue), and SNAP fusion proteins were labeled using a cell-permeant TMR-Star substrate (red). Bars ϭ 10 m. ment of apoptotic cells and the engulfment of apparently whole cells seen in vivo (26)(27)(28), they provide further evidence that amebic cell killing and phagocytosis occur largely as a sequence of events, and (ii) they further validate previously published microarray data demonstrating the upregulated gene expression following phagocytosis that correlates with an increased phagocytic ability (33).…”

“…E. histolytica contains 55 putative tyrosine kinases, 44 of which are putative receptor tyrosine kinases (51). In our earlier microarray analysis comparing gene regulation between phagocytic and nonphagocytic populations of E. histolytica trophozoites, the SH3 domain was found to be enriched in genes upregulated in phagocytic trophozoites (33).…”

“…Trophozoites in which EHI_150430 was silenced had the greatest defect at a 50% phagocytic index compared to that for the empty vector-transfected control. Despite this redundancy, said pathways appear to share a program of feed-forward regulation in response to phagocytosis, in which phagocytosis stimulates the expression of genes that enhance the phagocytic ability (33).…”

“…By modeling of phagocytosis of apoptotic cells through ingestion of C1q-coated particles, our laboratory previously showed that E. histolytica trophozoites have a program of gene expression changes that is initiated through the act of phagocytosis and that actually increases their phagocytic ability (33). Stimulation with host cells also enhances trogocytosis (32), likely through the same changes in gene expression.…”

“…Stimulation with host cells also enhances trogocytosis (32), likely through the same changes in gene expression. Microarray analysis comparing gene expression in phagocytic and nonphagocytic populations of trophozoites identified 121 genes that were upregulated in the phagocytic population (33). Gene clustering based on functional annotation identified strong clusters of genes encoding proteins involved in actin binding and cytoskeletal organization.…”