Coordination of apicoplast transcription in a malaria parasite by internal and host cues.

The malaria parasite Plasmodium falciparum has a nonphotosynthetic plastid called the apicoplast, which contains its own genome. Regulatory mechanisms for apicoplast gene expression remain poorly understood, despite this organelle being crucial for the parasite life cycle. Here, we identify a nuclear-encoded apicoplast RNA polymerase σ subunit (sigma factor) which, along with the α subunit, appears to mediate apicoplast transcript accumulation. This has a periodicity reminiscent of parasite circadian or developmental control. Expression of the apicoplast subunit gene, apSig, together with apicoplast transcripts, increased in the presence of the blood circadian signaling hormone melatonin. Our data suggest that the host circadian rhythm is integrated with intrinsic parasite cues to coordinate apicoplast genome transcription. This evolutionarily conserved regulatory system might be a future target for malaria treatment.

PTEN differentially regulates endocytosis, migration, and proliferation in the enteric protozoan parasite Entamoeba histolytica.

PTEN is a lipid phosphatase that is highly conserved and involved in a broad range of biological processes including cytoskeletal reorganization, endocytosis, signal transduction, and cell migration in all eukaryotes. Although regulation of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] signaling via PTEN has been well established in model organisms and mammals, it remains elusive in the parasitic protist E. histolytica, which heavily relies on PtdIns phosphate(s)-dependent membrane traffic, migration, and phago- and trogocytosis for its pathogenesis. In this study, we characterized the major PTEN from E. histolytica, EhPTEN1, which shows the highest expression at the transcript level in the trophozoite stage among 6 possible PTENs, to understand the significance of PtdIns(3,4,5)P3 signaling in this parasite. Live imaging of GFP-EhPTEN1 expressing amebic trophozoites showed localization mainly in the cytosol with a higher concentration at pseudopods and the extending edge of the phago- and trogocytic cups. Furthermore, quantitative analysis of phago- and trogocytosis using a confocal image cytometer showed that overexpression of EhPTEN1 caused reduction in trogo- and phagocytosis while transcriptional gene silencing of EhPTEN1 gene caused opposite phenotypes. These data suggest that EhPTEN1 has an inhibitory role in these biological processes. Conversely, EhPTEN1 acts as a positive regulator for fluid-phase and receptor-mediated endocytosis in E. histolytica trophozoites. Moreover, we showed that EhPTEN1 was required for optimal growth and migration of this parasite. Finally, the phosphatase activity of EhPTEN1 towards PtdIns(3,4,5)P3 was demonstrated, suggesting that the biological roles of EhPTEN1 are likely linked to its catalytic function. Taken together, these results indicate that EhPTEN1 differentially regulates multiple cellular activities essential for proliferation and pathogenesis of the organism, via PtdIns(3,4,5)P3 signaling. Elucidation of biological roles of PTEN and PtdIns(3,4,5)P3 signaling at the molecular levels promotes our understanding of the pathogenesis of this parasite.

Gene expression of axenically-isolated clinical Entamoeba histolytica strains and its impact on disease severity of amebiasis.

The severity of Entamoeba histolytica infection is determined by host immunology, pathogen virulence, and the intestinal environment. Conventional research for assessing pathogen virulence has been mainly performed using laboratory strains, such as a virulent HM-1: IMSS (HM-1) and an avirulent Rahman, under various artificial environmental conditions because of the difficulties of axenic isolation of the clinical strains. However, it is still unclear whether scientific knowledge based on laboratory strains are universally applicable to the true pathogenesis. Hereby, we performed transcriptomic analysis of clinical strains from patients with different degrees of disease severity, as well as HM-1 under different conditions. Even after several months of axenization, Clinical strains show the distinct profile in gene expression during in vitro passage, moreover, difference between any 2 of these strains was much greater than the changes on the liver challenge. Interestingly, 26 DEGs, which were closely related to the biological functions, were oppositely up- or down regulated between virulent Ax 19 (liver abscess) and avirulent Ax 11 (asymptomatic carrier). Additionally, RNAseq using laboratory strain (HM1) showed more than half of genes were differently expressed between continuously in vitro passaged HM1 (in vitro HM1) and periodically liver passaged HM1 (virulent HM1), which was much greater than the changes on the liver passage of virulent HM1. Also, transcriptomic analysis of a laboratory strain revealed that continuous environmental stress enhances its virulence via a shift in its gene expression profile. Changes in gene expression patterns on liver abscess formation were not consistent between clinical and laboratory strains.

The kinesin KIF4 mediates HBV/HDV entry through the regulation of surface NTCP localization and can be targeted by RXR agonists in vitro.

Intracellular transport via microtubule-based dynein and kinesin family motors plays a key role in viral reproduction and transmission. We show here that Kinesin Family Member 4 (KIF4) plays an important role in HBV/HDV infection. We intended to explore host factors impacting the HBV life cycle that can be therapeutically addressed using siRNA library transfection and HBV/NLuc (HBV/NL) reporter virus infection in HepG2-hNTCP cells. KIF4 silencing resulted in a 3-fold reduction in luciferase activity following HBV/NL infection. KIF4 knockdown suppressed both HBV and HDV infection. Transient KIF4 depletion reduced surface and raised intracellular NTCP (HBV/HDV entry receptor) levels, according to both cellular fractionation and immunofluorescence analysis (IF). Overexpression of wild-type KIF4 but not ATPase-null KIF4 mutant regained the surface localization of NTCP and significantly restored HBV permissiveness in these cells. IF revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study revealed that KIF4 interacts with NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1-mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and subsequent HBV infection in both HepG2-hNTCP and primary human hepatocyte (PXB) (Bexarotene, IC50 1.89 ± 0.98 µM) cultures. Overall, our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress or alleviate KIF4 expression would be potential antiviral candidates targeting HBV and HDV entry.

Genetic characterization of the Entamoeba moshkovskii population based on different potential genetic markers.

Entamoeba moshkovskii, according to recent studies, appears to exert a more significant impact on diarrhoeal infections than previously believed. The efficient identification and genetic characterization of E. moshkovskii isolates from endemic areas worldwide are crucial for understanding the impact of parasite genomes on amoebic infections. In this study, we employed a multilocus sequence typing system to characterize E. moshkovskii isolates, with the aim of assessing the role of genetic variation in the pathogenic potential of E. moshkovskii. We incorporated 3 potential genetic markers: KERP1, a protein rich in lysine and glutamic acid; amoebapore C (apc) and chitinase. Sequencing was attempted for all target loci in 68 positive E. moshkovskii samples, and successfully sequenced a total of 33 samples for all 3 loci. The analysis revealed 17 distinct genotypes, labelled M1­M17, across the tested samples when combining all loci. Notably, genotype M1 demonstrated a statistically significant association with diarrhoeal incidence within E. moshkovskii infection (P = 0.0394). This suggests that M1 may represent a pathogenic strain with the highest potential for causing diarrhoeal symptoms. Additionally, we have identified a few single-nucleotide polymorphisms in the studied loci that can be utilized as genetic markers for recognizing the most potentially pathogenic E. moshkovskii isolates. In our genetic diversity study, the apc locus demonstrated the highest Hd value and π value, indicating its pivotal role in reflecting the evolutionary history and adaptation of the E. moshkovskii population. Furthermore, analyses of linkage disequilibrium and recombination within the E. moshkovskii population suggested that the apc locus could play a crucial role in determining the virulence of E. moshkovskii.

Kagimminols A and B, Cembrene-Type Diterpenes from an Okeania sp. Marine Cyanobacterium.

Kagimminols A (1) and B (2), new cembrene-type diterpenoids, were isolated from an Okeania sp. marine cyanobacterium. By combining DP4 analysis with an efficient NMR chemical shift calculation protocol, we clarified the relative configurations of 1 and 2 without consuming precious natural products. We determined the absolute configurations by a comparison of theoretical electronic circular dichroism (ECD) spectra with experimental spectra, and the absolute configuration of 1 was verified experimentally. Finally, we found that 1 and 2 showed selective growth-inhibitory activity against the causative agent of human African trypanosomiasis. This study exemplifies that computational chemistry is an efficient tool for clarifying the configurations of natural products possessing tautomers in equilibrium.

Andrographolide induced cytotoxicity and cell cycle arrest in Giardia trophozoites.

Giardiasis is a prevalent parasitic diarrheal disease caused by Giardia lamblia, affecting people worldwide. Recently, the availability of several drugs for its treatment has highlighted issues such as multidrug resistance, limited effectiveness and undesirable side effects. Therefore, it is necessary to develop alternative new drugs and treatment strategies that can enhance therapeutic outcomes and effectively treat giardiasis. Natural compounds show promise in the search for more potent anti-giardial agents. Our investigation focused on the effect of Andrographolide (ADG), an active compound of the Andrographis paniculata plant, on Giardia lamblia, assessing trophozoite growth, morphological changes, cell cycle arrest, DNA damage and inhibition of gene expression associated with pathogenic factors. ADG demonstrated anti-Giardia activity almost equivalent to the reference drug metronidazole, with an IC50 value of 4.99 µM after 24 h of incubation. In cytotoxicity assessments and morphological examinations, it showed significant alterations in trophozoite shape and size and effectively hindered the adhesion of trophozoites. It also caused excessive ROS generation, DNA damage, cell cycle arrest and inhibited the gene expression related to pathogenesis. Our findings have revealed the anti-giardial efficacy of ADG, suggesting its potential as an agent against Giardia infections. This could offer a natural and low-risk treatment option for giardiasis, reducing the risk of side effects and drug resistance.

Isolation and structure determination of a new analog of polycavernosides from marine Okeania sp. cyanobacterium.

Polycavernoside E (1), a new polycavernoside analog, was isolated from a marine Okeania sp. cyanobacterium. The relative configuration was elucidated primarily by analyzing the two dimensional nuclear magnetism resonance (2D NMR) data. The absolute configuration was clarified by comparing the electronic circular dichroism (ECD) data of 1 with those of known analogs. Polycavernoside E (1) exhibited moderate antitrypanosomal activity against Trypanosoma brucei rhodesiense. Furthermore, the isolation of polycavernoside E (1) from marine cyanobacteria provides additional evidence that marine cyanobacteria, and not red algae, are responsible for the biosynthesis of polycavernosides.

Fumagillin inhibits growth of the enteric protozoan parasite Entamoeba histolytica by covalently binding to and selectively inhibiting methionine aminopeptidase 2.

Amebiasis is an important cause of morbidity and mortality worldwide, and caused by infection with the protozoan parasite Entamoeba histolytica. Metronidazole is currently the first-line drug despite adverse effects and concerns on the emergence of drug resistance. Fumagillin, a fungal metabolite from Aspergillus fumigatus, and its structurally related natural and synthetic compounds have been previously explored as potential anti-angiogenesis inhibitors for cancers, anti-microbial, and anti-obese compounds. Although fumagillin was used for human amebiasis in clinical trials in 1950s, the mode of action of fumagillin remains elusive until now. In this report, we showed that fumagillin covalently binds to methionine aminopeptidase 2 (MetAP2) and non-covalently but abundantly binds to patatin family phospholipase A (PLA). Susceptibility against fumagillin of the amebic strains in which expression of E. histolytica MetAP2 (EhMetAP2) gene was silenced increased compared to control strain. Conversely, overexpression of EhMetAP2 mutants that harbors amino acid substitutions responsible for resistance to ovalicin, a fumagillin analog, in human MetAP2, also resulted in decrease in fumagillin susceptibility. In contrast, neither gene silencing nor overexpression of E. histolytica PLA (EhPLA) affected fumagillin susceptibility. These data suggest that EhPLA is not essential and not the target of fumagillin for its amebicidal activity. Taken together, our data have demonstrated that EhMetAP2 is the primary target for amebicidal activity of fumagillin, and EhMetAP2 represents a rational explorable target for the development of alternative therapeutic agents against amebiasis.

Eukaryotic translation initiation factor 5A and its posttranslational modifications play an important role in proliferation and potentially in differentiation of the human enteric protozoan parasite Entamoeba histolytica.

The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein and is essential in all eukaryotes. However, the specific roles of eIF5A in translation and in other biological processes remain elusive. In the present study, we described the role of eIF5A, its posttranslational modifications (PTM), and the biosynthetic pathway needed for the PTM in Entamoeba histolytica, the protozoan parasite responsible for amoebic dysentery and liver abscess in humans. E. histolytica encodes two isotypes of eIF5A and two isotypes of enzymes, deoxyhypusine synthase (DHS), responsible for their PTM. Both of the two eIF5A isotypes are functional, whereas only one DHS (EhDHS1, but not EhDHS2), is catalytically active. The DHS activity increased ~2000-fold when EhDHS1 was co-expressed with EhDHS2 in Escherichia coli, suggesting that the formation of a heteromeric complex is needed for full enzymatic activity. Both EhDHS1 and 2 genes were required for in vitro growth of E. histolytica trophozoites, indicated by small antisense RNA-mediated gene silencing. In trophozoites, only eIF5A2, but not eIF5A1, gene was actively transcribed. Gene silencing of eIF5A2 caused compensatory induction of expression of eIF5A1 gene, suggesting interchangeable role of the two eIF5A isotypes and also reinforcing the importance of eIF5As for parasite proliferation and survival. Furthermore, using a sibling species, Entamoeba invadens, we found that eIF5A1 gene was upregulated during excystation, while eIF5A2 was downregulated, suggesting that eIF5A1 gene plays an important role during differentiation. Taken together, these results have underscored the essentiality of eIF5A and DHS, for proliferation and potentially in the differentiation of this parasite, and suggest that the hypusination associated pathway represents a novel rational target for drug development against amebiasis.

Two StAR-related lipid transfer proteins play specific roles in endocytosis, exocytosis, and motility in the parasitic protist Entamoeba histolytica.

Lipid transfer proteins (LTPs) are the key contributor of organelle-specific lipid distribution and cellular lipid homeostasis. Here, we report a novel implication of LTPs in phagocytosis, trogocytosis, pinocytosis, biosynthetic secretion, recycling of pinosomes, and motility of the parasitic protist E. histolytica, the etiological agent of human amoebiasis. We show that two StAR-related lipid transfer (START) domain-containing LTPs (named as EhLTP1 and 3) are involved in these biological pathways in an LTP-specific manner. Our findings provide novel implications of LTPs, which are relevant to the elucidation of pathophysiology of the diseases caused by parasitic protists.

An atypical EhGEF regulates phagocytosis in Entamoeba histolytica through EhRho1.

The parasite Entamoeba histolytica is the etiological agent of amoebiasis, a major cause of morbidity and mortality due to parasitic diseases in developing countries. Phagocytosis is an essential mode of obtaining nutrition and has been associated with the virulence behaviour of E. histolytica. Signalling pathways involved in activation of cytoskeletal dynamics required for phagocytosis remains to be elucidated in this parasite. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica and have described some of the molecules that play key roles in the process. Here we showed the involvement of non-Dbl Rho Guanine Nucleotide Exchange Factor, EhGEF in regulation of amoebic phagocytosis by regulating activation of EhRho1. EhGEF was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. Our observation from imaging, pull down experiments and down regulating expression of different molecules suggest that EhGEF interacts with EhRho1 and it is required during initiation of phagocytosis and phagosome formation. Also, biophysical, and computational analysis reveals that EhGEF mediates GTP exchange on EhRho1 via an unconventional pathway. In conclusion, we describe a non-Dbl EhGEF of EhRho1 which is involved in endocytic processes of E. histolytica.

Isolation and Structure Determination of Akunolides, Macrolide Glycosides from a Marine Okeania sp. Cyanobacterium.

Akunolides A (1), B (2), C (3), and D (4), new macrolide glycosides, were isolated from a marine Okeania sp. cyanobacterium. Their structures were elucidated by spectroscopic analyses and derivatization reactions. Akunolides A-D (1-4) are classified as 16-membered macrolide glycosides, which are relatively rare structures for marine cyanobacterium-derived natural products. Akunolides A-D (1-4) showed moderate antitrypanosomal activities against Trypanosoma brucei rhodesiense, with IC50 values ranging from 11 to 14 µM. Furthermore, akunolides A (1) and C (3) exhibited no cytotoxicity against normal human WI-38 cells even at a concentration of 150 µM.

Functional characterization of phospholipase B enzyme from Giardia lamblia.

The microaerotolarent amitochondriate protozoan Giardia lamblia causes Giardiasis and produces a unique enzyme called Phospholipase B (PLB) in contrast to higher eukaryotes. The enzyme is produced upon induction with oxidative (H2O2) stress, thus leading to prostaglandin E2 (PGE2) production. It exists in dimeric form, and its molecular weight is 56 kDa. This PLB was extracellularly cloned in the pET21d vector. The ORF is 1620 bp (Genbank accession no. -OM939681) long and codes for a protein 539 amino acid long, with a 15 amino acid long amino-terminal signal peptide. The highest enzyme activity of PLB was identified at pH 7.5 and 35 °C. This specific enzyme was also active at 50 °C pH 10, but activity was low. We also analyzed the expression of PLB protein in G. lamblia, which was significantly induced under increased oxidative stress.

Development of a simple PCR-RFLP technique for detection and differentiation of E. histolytica, E. dispar and E. moshkovskii.

Epidemiological studies on amoebic infections are complicated by morphological overlap between the pathogenic E. histolytica, the commensal E. dispar and the amphizoic E. moshkovskii, necessitating molecular identification. The present study developed a simple and economical 18S PCR-RFLP method for the simultaneous detection and differentiation of the three species. PCR products were differentiated by Tat1 restriction digestion generating three different RFLP patterns. Validation was conducted by screening 382 faecal samples from human patients from Kolkata, India, hospitalized for diarrhoea. Analysis indicated that the PCR-RFLP could successfully differentiate between the three species and was confirmed by sequence analysis. This method could prove useful for clinical and epidemiological studies of amoebiasis.

Investigating genetic polymorphism in E. histolytica isolates with distinct clinical phenotypes.

Amoebiasis is an infection caused by enteric protozoa, most commonly Entamoeba histolytica, and is globally considered a potentially severe and life-threatening condition. To understand the impact of the parasite genome on disease outcomes, it is important to study the genomes of infecting strains in areas with high disease prevalence. These studies aim to establish correlations between parasite genotypes and the clinical presentation of amoebiasis. We employ a strain typing approach that utilizes multiple loci, including SREHP and three polymorphic non-coding loci (tRNA-linked array N-K2 and loci 1-2 and 5-6), for high-resolution analysis. Distinct clinical phenotype isolates underwent amplification and sequencing of studied loci. The nucleotide sequences were analysed using Tandem Repeats Finder to detect short tandem repeats (STRs). These patterns were combined to assign a genotype, and the correlation between clinical phenotypes and repetitive patterns was statistically evaluated. This study found significant polymorphism in the size and number of PCR fragments at SREHP and 5-6 locus, while the 1-2 locus and NK2 locus showed variations in PCR product sizes. Out of 41 genotypes, two (I6 and I41) were significantly associated with their respective disease outcomes and were found in multiple isolates. We observed that I6 was linked with a symptomatic outcome, with a statistically significant p-value of 0.0183. Additionally, we found that I41 was associated with ALA disease outcome, with a p-value of 0.0089. Our study revealed new repeat units not previously reported, unveiling the genetic composition of E. histolytica strains in India, associated with distinct disease manifestations.

Genotyping and epidemiological distribution of diarrhea-causing isolates of Giardia duodenalis in southeastern part of West Bengal, India.

The prevalence and genetic diversity of the protozoan pathogen Giardia duodenalis have been extensively studied worldwide. There is currently a lack of data regarding the genetic variability of the organism in eastern India. Understanding the circulating genotypes and associated risk factors is crucial for effective planning and implementing control measures. Therefore, the objective of the study was to conduct an epidemiological study to determine the prevalence and identify the various genotypes present. This survey adds to our knowledge on the occurrence and distribution of Giardia genotypes in the studied region. The overall prevalence was found to be 6.8%. This parasitic infection was significantly associated with two age groups, i.e., >0-5 years and >5-12 years. Using a multilocus genotyping method, we genotyped 52 human Giardia isolates that were obtained from diarrheal patients. Two distinct assemblages were found in the population-30.8% belonged to assemblage A; 63.5% belonged to assemblage B, prevalent in the population; and 5.7% belonged to a combined assemblage A+B. Sub-assemblage AII was found in 17.3% of the cases, followed by sub-assemblage AI (13.5%). High levels of genetic diversity were found within the population of assemblage B undergoing balancing selection. Overall, the high prevalence of the parasite observed, particularly among children, raises a major concern and necessitates implementation of robust control measures. Furthermore, we report the presence of numerous unique genotypes, circulating in this limited geographical boundary, which can be useful dataset for future studies.

Non-Vesicular Lipid Transport Machinery in Leishmania donovani: Functional Implications in Host-Parasite Interaction.

Eukaryotic cells have distinct membrane-enclosed organelles, each with a unique biochemical signature and specialized function. The unique identity of each organelle is greatly governed by the asymmetric distribution and regulated intracellular movement of two important biomolecules, lipids, and proteins. Non-vesicular lipid transport mediated by lipid-transfer proteins (LTPs) plays essential roles in intra-cellular lipid trafficking and cellular lipid homeostasis, while vesicular transport regulates protein trafficking. A comparative analysis of non-vesicular lipid transport machinery in protists could enhance our understanding of parasitism and basis of eukaryotic evolution. Leishmania donovani, the trypanosomatid parasite, greatly depends on receptor-ligand mediated signalling pathways for cellular differentiation, nutrient uptake, secretion of virulence factors, and pathogenesis. Lipids, despite being important signalling molecules, have intracellular transport mechanisms that are largely unexplored in L. donovani. We have identified a repertoire of sixteen (16) potential lipid transfer protein (LTP) homologs based on a domain-based search on TriTrypDB coupled with bioinformatics analyses, which signifies the presence of well-organized lipid transport machinery in this parasite. We emphasized here their evolutionary uniqueness and conservation and discussed their potential implications for parasite biology with regards to future therapeutic targets against visceral leishmaniasis.

Dephospho-Coenzyme A Kinase Is an Exploitable Drug Target against Plasmodium falciparum: Identification of Selective Inhibitors by High-Throughput Screening of a Large Chemical Compound Library.

Malaria is a mosquito-borne fatal infectious disease that affects humans and is caused by Plasmodium parasites, primarily Plasmodium falciparum. Widespread drug resistance compels us to discover novel compounds and alternative drug discovery targets. The coenzyme A (CoA) biosynthesis pathway is essential for the malaria parasite P. falciparum. The last enzyme in CoA biosynthesis, dephospho-CoA kinase (DPCK), is essential to the major life cycle development stages but has not yet been exploited as a drug target in antimalarial drug discovery. We performed a high-throughput screen of a 210,000-compound library using recombinant P. falciparum DPCK (PfDPCK). A high-throughput enzymatic assay using a 1,536-well platform was developed to identify potential PfDPCK inhibitors. PfDPCK inhibitors also inhibited parasite growth in a P. falciparum whole-cell asexual blood-stage assay in both drug-sensitive and drug-resistant strains. Hit compounds were selected based on their potency in cell-free (PfDPCK) and whole-cell (Pf3D7 and PfDd2) assays, selectivity over the human orthologue (HsCOASY) and no cytotoxicity (HepG2). The compounds were ranked using a multiparameter optimization (MPO) scoring model, and the specific binding and the mechanism of inhibition were investigated for the most promising compounds.

Rab7D small GTPase is involved in phago-, trogocytosis and cytoskeletal reorganization in the enteric protozoan Entamoeba histolytica.

Rab small GTPases regulate membrane traffic between distinct cellular compartments of all eukaryotes in a tempo-spatially specific fashion. Rab small GTPases are also involved in the regulation of cytoskeleton and signalling. Membrane traffic and cytoskeletal regulation play pivotal role in the pathogenesis of Entamoeba histolytica, which is a protozoan parasite responsible for human amebiasis. E. histolytica is unique in that its genome encodes over 100 Rab proteins, containing multiple isotypes of conserved members (e.g., Rab7) and Entamoeba-specific subgroups (e.g., RabA, B, and X). Among them, E. histolytica Rab7 is the most diversified group consisting of nine isotypes. While it was previously demonstrated that EhRab7A and EhRab7B are involved in lysosome and phagosome biogenesis, the individual roles of other Rab7 members and their coordination remain elusive. In this study, we characterised the third member of Rab7, Rab7D, to better understand the significance of the multiplicity of Rab7 isotypes in E. histolytica. Overexpression of EhRab7D caused reduction in phagocytosis of erythrocytes, trogocytosis (meaning nibbling or chewing of a portion) of live mammalian cells, and phagosome acidification and maturation. Conversely, transcriptional gene silencing of EhRab7D gene caused opposite phenotypes in phago/trogocytosis and phagosome maturation. Furthermore, EhRab7D gene silencing caused reduction in the attachment to and the motility on the collagen-coated surface. Image analysis showed that EhRab7D was occasionally associated with lysosomes and prephagosomal vacuoles, but not with mature phagosomes and trogosomes. Finally, in silico prediction of structural organisation of EhRab7 isotypes identified unique amino acid changes on the effector binding surface of EhRab7D. Taken together, our data suggest that EhRab7D plays coordinated counteracting roles: a inhibitory role in phago/trogocytosis and lyso/phago/trogosome biogenesis, and an stimulatory role in adherence and motility, presumably via interaction with unique effectors. Finally, we propose the model in which three EhRab7 isotypes are sequentially involved in phago/trogocytosis.