EhRho6‐mediated actin degradation in Entamoeba histolytica is associated with compromised pathogenicity

Abstract

Entamoeba histolytica causes amoebiasis which is a major health concern in developing countries. E. histolytica pathogenicity has been implicated to a large repertoire of small GTPases which switch between the inactive GDP bound state and the active GTP bound state with the help of guanine nucleotide exchange factors (GEFs) and GTPase activating protein (GAPs). Rho family of small GTPases are well known to modulate the actin cytoskeletal dynamics which plays a major role in E. histolytica pathogenicity. Here, we report an atypical amoebic RhoGEF, and its preferred substrate EhRho6, which, upon overexpression abrogated the pathogenic behavior of the amoeba such as adhesion to host cell, monolayer destruction, erythrophagocytosis, and formation of actin dots. A causative immunoblot analysis revealed actin degradation in the EhRho6 overexpressing trophozoites that could be inhibited by blocking the amoebic proteasomal pathway. A careful analysis of the results from a previously published transcriptomics study, in conjunction with our observations, led to the identification of a clade of Rho GTPases in this pathogenic amoeba which we hypothesize to have implications during the amoebic encystation.

EhRacM differentially regulates macropinocytosis and motility in the enteric protozoan parasiteEntamoeba histolytica

“…To identify and characterize Rhos (or Racs) that are involved in macropinocytosis in E. histolytica trophozoites, we established gene silenced strains for the ten most highly expressed Ehrho/Ehrac genes (23) using antisense small RNA-mediated transcriptional gene silencing (24,25): EHI_070730 [( EhracC (19) or Ehrho7 (26)], EHI_029020 ( Ehrho1B ) (19), XP_651936 [in the current database, EHI_013650 ( EhracR2 ) and EHI_068240 ( EhracR1 ), two identical copies present in two independent loci (19), EHI_129750 [ EhracG (19) or Ehrho2 (26)], EHI_012240 [ EhracD1 (19) or Ehrho5 (26)], EHI_197840 [ EhracA2 (19) or Ehrho6 (26)], EHI_135450 [ EhracM (19) or Ehrho13 (26)], EHI_146180 [ EhracQ (19) or Ehrho16 (26)], EHI_194390 [ EhracJ (19) or Ehrho15 (26)], and EHI_192450 ( EhracP (19) or Ehrho14 (26))]. We examined the repression of the target gene expression by reverse transcriptase (RT)-PCR (Fig 1A) and quantitative real- time (qRT)-PCR (Fig 1B).…”

“…For antisense small RNA-mediated transcriptional silencing of ten highly expressed Ehrho genes [EHI_070730 ( EhracC (19) or Ehrho7 (26)), EHI_029020 ( Ehrho1B (19)), XP_651936 (in the current database, EHI_013650 ( EhRacR2 (19)) and EHI_068240 ( EhracR1 (19))), EHI_129750 ( EhracG (19) or EhRho2 (26)), EHI_012240 ( EhracD1 (19) or Ehrho5 (26)), EHI_197840 ( EhracA2 (19) or Ehrho6 (26)), EHI_135450 ( EhracM (19) or Ehrho13 (26)), EHI_146180 ( EhracQ (19) or Ehrho16 (26)), EHI_194390 ( EhracJ (19) or Ehrho15 (26)), and EHI_192450 ( EhracP (19) or Ehrho14 (26))] (S1 Table), around 420 bp fragments of the protein-coding region of each gene were amplified by PCR from cDNA with sense and antisense oligonucleotides containing StuI and Sac1 restriction sites. The amplified product was digested by StuI and Sac1 and ligated into the compatible sites of the double-digested psAP2-Gunma plasmid (24) to synthesize a gene silencing plasmid designated as psAP2-EhRacC, psAP2-EhRho1B, psAP2-XP_651936, psAP2-EhRacG, psAP2-EhRacD1, psAP2- EhRacA2, psAP2-EhRacM, psAP2-EhRacQ, psAP2-EhRacJ, and psAP2-EhRacP.…”