Julien Santi-Rocca

The lysine‐ and glutamic acid‐rich protein KERP1 plays a role in Entamoeba histolytica liver abscess pathogenesis

The parasite Entamoeba histolytica colonizes the large bowel where it may persist as an asymptomatic luminal gut infection, which changes to virulence. Parasite invasion of the intestine leads to dysentery and spreads to the liver, where amoebae form abscesses. We took advantage of changes in virulence that occurs after long‐term in vitro culture of E. histolytica strains. Using microarrays, we concluded that virulence correlates with upregulation of key genes involved in stress response, including molecular chaperones, ssp1 and peroxiredoxin; as well as the induction of unknown genes encoding lysine‐rich proteins. Seven of these were retained with respect to their lysine content higher than 25%. Among them, we found KERP1, formerly identified as associated to parasite surface and involved in the parasite adherence to host cells. Experimentally induced liver abscesses, using molecular beacons and protein analysis, allowed us to draw a parallel between the intricate upregulation of kerp1 gene expression during abscess development and the increased abundance of KERP1 in virulent trophozoites. Following its characterization as a marker for the progression of infection, KERP1 was also seen to be a virulence marker as trophozoites affected in kerp1 expression by an antisense strategy were unable to form liver abscesses.

Proteomic Analysis of Intact Flagella of Procyclic Trypanosoma brucei Cells Identifies Novel Flagellar Proteins with Unique Sub-localization and Dynamics

Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity.

Host-Microbe Interactions and Defense Mechanisms in the Development of Amoebic Liver Abscesses

SUMMARY Amoebiasis by Entamoeba histolytica is a major public health problem in developing countries and leads to several thousand deaths per year. The parasite invades the intestine (provoking diarrhea and dysentery) and the liver, where it forms abscesses (amoebic liver abscesses [ALAs]). The liver is the organ responsible for filtering blood coming from the intestinal tract, a task that implies a particular structure and immune features. Amoebae use the portal route and break through the sinusoidal endothelial barrier to reach the hepatic parenchyma. When faced with systemic and cell-mediated defenses, trophozoites adapt to their new environment and modulate host responses, leading to parasite survival and the formation of inflammatory foci. Cytopathogenic effects and the onset of inflammation may be caused by diffusible products originating from parasites and/or immune cells either by their secretion or by their release after cell death. Liver infection thus results from the interplay between E. histolytica and hepatic cells. Despite its importance in terms of public health burden, the lack of integrated data on ALA genesis means that we have only an incomplete description of the initiation and development of hepatic amoebiasis. Here, we review the main steps of ALA development as well as the responses triggered in both the host and the parasite. Transcriptome studies highlighted parasite factors involved in adherence to human cells, cytopathogenic effects, and adaptative and stress responses. An understanding of their role in ALA development will help to unravel the host-pathogen interactions and their evolution throughout the infection.

Calcium‐binding protein 1 of Entamoeba histolytica transiently associates with phagocytic cups in a calcium‐independent manner

EhCaBP1, a calcium‐binding protein of the parasite Entamoeba histolytica, is known to participate in cellular processes involving actin filaments. This may be due to its direct interaction with actin. In order to understand the kinetics of EhCaBP1 in such processes, its movement was studied in living cells expressing GFP‐EhCaBP1. The results showed that EhCaBP1 accumulated at phagocytic cups and pseudopods transiently. The time taken for appearance and disappearance of EhCaBP1 was found to be around 12 s. Site‐directed mutagenesis was used to generate an EhCaBP1 mutant with reduced Ca2+‐ and G‐actin binding ability without any defect in its ability to bind F‐actin. The overexpression of this mutant EhCaBP1 in the E. histolytica trophozoites resulted in the impairment of erythrophagocytosis, uptake of bacterial cells, killing of target cells but not fluid‐phase pinocytosis. However, the mutant protein was still found to transiently localize with F‐actin at the phagocytic cups and pseudopods. The mutant protein displayed reduced ability to activate endogenous kinase(s) suggesting that phagosome formation may require Ca2+‐EhCaBP1 transducing downstream signalling but initiation of phagocytosis may be independent of its intrinsic ability to bind Ca2+. The results suggest a dynamic association of EhCaBP1 with F‐actin‐mediated processes.

Endoplasmic Reticulum Stress-Sensing Mechanism Is Activated in Entamoeba histolytica upon Treatment with Nitric Oxide

The Endoplasmic Reticulum stores calcium and is a site of protein synthesis and modification. Changes in ER homeostasis lead to stress responses with an activation of the unfolded protein response (UPR). The Entamoeba histolytica endomembrane system is simple compared to those of higher eukaryotes, as a canonical ER is not observed. During amoebiasis, an infection of the human intestine and liver by E. histolytica, nitric oxide (NO) triggers an apoptotic-like event preceded by an impairment of energy production and a loss of important parasite pathogenic features. We address the question of how this ancient eukaryote responds to stress induced by immune components (i.e. NO) and whether stress leads to ER changes and subsequently to an UPR. Gene expression analysis suggested that NO triggers stress responses marked by (i) dramatic up-regulation of hsp genes although a bona fide UPR is absent; (ii) induction of DNA repair and redox gene expression and iii) up-regulation of glycolysis-related gene expression. Enzymology approaches demonstrate that NO directly inhibits glycolysis and enhance cysteine synthase activity. Using live imaging and confocal microscopy we found that NO dramatically provokes extensive ER fragmentation. ER fission in E. histolytica appears as a protective response against stress, as it has been recently proposed for neuron self-defense during neurologic disorders. Chronic ER stress is also involved in metabolic diseases including diabetes, where NO production reduces ER calcium levels and activates cell death. Our data highlighted unique cellular responses of interest to understand the mechanisms of parasite death during amoebiasis.

Use of Bacterially Expressed dsRNA to Downregulate Entamoeba histolytica Gene Expression

Background Modern RNA interference (RNAi) methodologies using small interfering RNA (siRNA) oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules. Principal Findings Here we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA) targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica β-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite. Conclusions Our results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets.

Generation of a Nanobody Targeting the Paraflagellar Rod Protein of Trypanosomes

Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa), costly to produce and require cold chain maintenance, a condition that is difficult to achieve in trypanosomiasis endemic regions, which are mostly rural. Nanobodies are recombinant, heat-stable, small-sized (15 kDa), antigen-specific, single-domain, variable fragments derived from heavy chain-only antibodies in camelids. Because of numerous advantages over classical antibodies, we investigated the use of nanobodies for the targeting of trypanosome-specific antigens and diagnostic potential. An alpaca was immunized using lysates of Trypanosoma evansi. Using phage display and bio-panning techniques, a cross-reactive nanobody (Nb392) targeting all trypanosome species and isolates tested was selected. Imunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry assays were combined to identify the target recognized. Nb392 targets paraflagellar rod protein (PFR1) of T. evansi, T. brucei, T. congolense and T. vivax. Two different RNAi mutants with defective PFR assembly (PFR2RNAi and KIF9BRNAi) were used to confirm its specificity. In conclusion, using a complex protein mixture for alpaca immunization, we generated a highly specific nanobody (Nb392) that targets a conserved trypanosome protein, i.e., PFR1 in the flagella of trypanosomes. Nb392 is an excellent marker for the PFR and can be useful in the diagnosis of trypanosomiasis. In addition, as demonstrated, Nb392 can be a useful research or PFR protein isolation tool.

New insights into host-pathogen interactions during Entamoeba histolytica liver infection

Amoebiasis is the third worldwide disease due to a parasite. The causative agent of this disease, the unicellular eukaryote Entamoeba histolytica, causes dysentery and liver abscesses associated with inflammation and human cell death. During liver invasion, before entering the parenchyma, E. histolytica trophozoites are in contact with liver sinusoidal endothelial cells (LSEC). We present data characterizing human LSEC responses to interaction with E. histolytica and identifying amoebic factors involved in the process of cell death in this cell culture model potentially relevant for early steps of hepatic amoebiasis. E. histolytica interferes with host cell adhesion signalling and leads to diminished adhesion and target cell death. Contact with parasites induces disruption of actin stress fibers and focal adhesion complexes. We conclude that interference with LSEC signalling may result from amoeba-triggered changes in the mechanical forces in the vicinity of cells in contact with parasites, sensed and transmitted by focal adhesion complexes. The study highlights for the first time the potential role in the onset of hepatic amoebiasis of the loss of liver endothelium integrity by disturbance of focal adhesion function and adhesion signalling. Among the amoebic factors required for changed LSEC adherence properties we identified the Gal/GalNAC lectin, cysteine proteases and KERP1.

Uncoupling flagellum formation and maintenance

Objective The role of Intraflagellar transport (IFT) in the construction of cilia and flagella is well established in numerous species. In contrast, its involvement in flagellum maintenance has only been shown in Chlamydomonas. Here, the protist Trypanosoma brucei was used as a model to investigate the role of IFT in flagellum maintenance. This organism has the advantage to maintain the existing flagellum whilst assembling the new one during the cell cycle. Knocking down the expression of any IFT protein inhibits flagellum formation in trypanosomes. Nevertheless, 25% of the cells still possess a flagellum of variable length. It was initially proposed that this was a consequence of RNAi that targets mRNA and not protein, hence IFT would still be active in these flagella.

Mechanisms of flagellum construction and maintenance

Cilia and flagella are cylindrical organelles that protrude at the surface of numerous eukaryotes including most human cells. They are composed, from base to tip, of the basal body (9 triplet microtubules), the transition zone (TZ, 9 doublet microtubules) and the axoneme (9 doublet microtubules ± 2 central microtubules). New flagellar sub-units are added at the distal tip by intraflagellar transport (IFT), a dynamic process where IFT motors drag IFT particles in both anterograde and retrograde directions. IFTs concentrate in a pool at the base of the flagellum, whose localization depends on the organism. In Trypanosomatidae, immunofluorescence assays reveal the association of this pool to the TZ, split apart from the cytoplasm by the transitional fibres. In Trypanosoma brucei, we undertook to investigate about the role of the TZ in flagellum formation and maintenance of IFT in mature flagella, by deciphering the role of RP2, a candidate protein located at the TFs [Stephan et al., Traffic 2007]. We constructed a strain impaired in RP2 production by tetracycline-inducible RNAi and obtained the expected phenotype: parasite growth was affected from the third day of induction. In parallel, diminution of IFT concentration at the flagellum base suggests that RP2 is involved in IFT recruitment during flagellum formation and/or RP2 takes part in a ciliary gate that hinders IFT leaking towards the cytoplasm. We are currently studying these hypotheses by live microscopy using fluorescent fusions of IFT proteins, RP2, and other ciliary gate candidates, dissecting the molecular structure and mechanisms involved in flagellum construction and homeostasis.