Honecker, B. | Search bioRxiv

bioRxiv2024

Entamoeba histolytica extracellular vesicles drive pro-inflammatory monocyte signaling

The parasitic protozoan Entamoeba histolytica secretes extracellular vesicles (EVs), but so far little is known about their function in the interaction with the host immune system. Infection with E. histolytica trophozoites can lead to formation of amebic liver abscesses (ALAs), in which pro-inflammatory immune responses of Ly6Chi monocytes contribute to liver damage. Men exhibit a more severe pathology as the result of higher monocyte recruitment and a stronger immune response. To investigate the role of EVs and pathogenicity in the host immune response, we studied the effect of EVs secreted by low pathogenic EhA1 and highly pathogenic EhB2 amebae on monocytes. Size and quantity of isolated EVs from both clones were similar. However, they differed in their proteome and miRNA cargo, providing insight into factors potentially involved in amebic pathogenicity. In addition, EVs were enriched in proteins with signaling peptides compared with the total protein content of trophozoites. Exposure to EVs from both clones induced monocyte activation and a pro-inflammatory immune response as evidenced by increased surface presentation of the activation marker CD38 and upregulated gene expression of key signaling pathways (including NF-{kappa}B, IL-17 and TNF signaling). The release of pro-inflammatory cytokines was increased in EV-stimulated monocytes and more so in male-than in female-derived cells. While EhA1 EV stimulation caused elevated myeloperoxidase (MPO) release by both monocytes and neutrophils, EhB2 EV stimulation did not, indicating the protective role of MPO during amebiasis. Collectively, our results suggest that parasite-released EVs contribute to the male-biased immunopathology mediated by pro-inflammatory monocytes during ALA formation. Author summaryParasites communicate with their host via small membranous extracellular vesicles (EVs) that can shuttle cargo and thus information between cells. The protozoan parasite Entamoeba histolytica releases EVs but not much is known about their role in the interaction with the host immune system. Infection with E. histolytica can lead to amebic liver abscess (ALA) formation. Innate immune cells, particularly monocytes, contribute to liver damage by releasing microbicidal factors. Men have a more severe ALA pathology as the result of a stronger monocyte immune response. In this study, we analyzed the effect of EVs from differently virulent E. histolytica clones on monocytes to better understand their interaction. EVs of both clones were similar in size and quantity but differed in their cargo, which provides information on factors potentially involved in pathogenicity. Monocytes responded to EVs of both clones in a pro-inflammatory manner that reflected the immune processes occurring during ALA in vivo, including the bias towards the male sex. Only EVs of amebae with low pathogenicity, and not those released by the highly pathogenic clone, elicited secretion of the granular enzyme myeloperoxidase, which plays a protective role during ALA. Overall, our data suggest that EVs may contribute to liver injury.

immunology↗

bioRxiv2023

Proteins differentially expressed between pathogenic and non-pathogenic Entamoeba histolytica clones influence pathogenicity by different mechanisms

Recently, two genes involved in pathogenicity in a mouse model of amoebic liver abscess were identified based on their differential expression between non-pathogenic (A1np) and pathogenic (B2p) clones of the Entamoeba histolytica isolate HM:1-IMSS. While overexpression of a gene encoding the metallopeptidase EhMP8-2 decreases the virulence of the pathogenic clone B2p, overexpression of the gene ehi_127670 (ehhp127), encoding a hypothetical protein, increases the virulence of the non-pathogenic clone A1np, while silencing this gene in B2p decreases virulence. To understand the role of both molecules in determining the pathogenicity of E. histolytica, silencing and overexpression transfectants were characterized in detail. Silencing of ehmp8-2, of the homologous gene ehmp8-1, or of both together in A1np trophozoites significantly altered the transcript levels of 60-350 genes. This strong change in the expression profile caused by the silencing of ehmp8-1 and/or ehmp8-2 implies that these peptidases regulate expression of numerous genes. Consequently, numerous phenotypic characteristics including cytopathic, hemolytic and cysteine peptidase activity were changed in response to their silencing. Silencing of ehhp127 in B2p trophozoites did not affect other genes, whereas overexpression in A1np trophozoites results in an altered expression of approximately 140 genes. EhHP127 appears to be important for trophozoite movement, as silencing negatively affects and overexpression positively affects trophozoite motility. Interestingly, the specific silencing of ehhp127 also impairs cytopathic activity, cysteine peptidase and hemolytic activity. All three molecules of interest, namely EhMP8-1, EhMP8-2, and EhHP127 can be detected in amoeba vesicles. Our results clearly show that the proteins studied here influence the pathogenicity of amoebae in different ways and use entirely different mechanisms to do so. Author summaryThe human pathogen Entamoeba histolytica can live asymptomatically in the intestine or become invasive and cause fatal liver abscesses. Approximately 15,000 people die each year as a result of an amoebic infection. Recently, two clones with different pathogenicity (A1np: non-pathogenic; B2p: pathogenic) derived from the E. histolytica isolate HM:1-IMSS were compared at the transcriptome level. Two highly differentially expressed genes (ehhp127 encoding a hypothetical protein and ehmp8-2 encoding a metallopeptidase) were identified. Analysis of E. histolytica transfectants showed that silencing of ehhp127 and overexpression of ehmp8-2 in B2p trophozoites reduced amoebic liver abscess formation in the mouse model. In this study, we characterized E. histolytica silencing and overexpression transfectants of ehmp8-2, as well as of the homologous gene ehmp8-1 and of ehhp127. It was shown that the altered expression of the metallopeptidase genes has a strong influence on the expression of a large number of genes and that the phenotype is strongly altered as a result. Silencing of ehhp127 does not affect the overall expression profile. However, specific silencing has a negative effect on motility, cysteine peptidase, hemolytic and cytopathic activity. All three molecules were shown to be localized in trophozoite vesicles.

cell biology↗