Infection by the Helminth Parasite Fasciola hepatica Requires Rapid Regulation of Metabolic, Virulence, and Invasive Factors to Adjust to Its Mammalian Host - PubMed (original) (raw)

Infection by the Helminth Parasite Fasciola hepatica Requires Rapid Regulation of Metabolic, Virulence, and Invasive Factors to Adjust to Its Mammalian Host

Krystyna Cwiklinski et al. Mol Cell Proteomics. 2018 Apr.

Abstract

The parasite Fasciola hepatica infects a broad range of mammals with impunity. Following ingestion of parasites (metacercariae) by the host, newly excysted juveniles (NEJ) emerge from their cysts, rapidly penetrate the duodenal wall and migrate to the liver. Successful infection takes just a few hours and involves negotiating hurdles presented by host macromolecules, tissues and micro-environments, as well as the immune system. Here, transcriptome and proteome analysis of ex vivo F. hepatica metacercariae and NEJ reveal the rapidity and multitude of metabolic and developmental alterations that take place in order for the parasite to establish infection. We found that metacercariae despite being encased in a cyst are metabolically active, and primed for infection. Following excystment, NEJ expend vital energy stores and rapidly adjust their metabolic pathways to cope with their new and increasingly anaerobic environment. Temperature increases induce neoblast proliferation and the remarkable up-regulation of genes associated with growth and development. Cysteine proteases synthesized by gastrodermal cells are secreted to facilitate invasion and tissue degradation, and tegumental transporters, such as aquaporins, are varied to deal with osmotic/salinity changes. Major proteins of the total NEJ secretome include proteases, protease inhibitors and anti-oxidants, and an array of immunomodulators that likely disarm host innate immune effector cells. Thus, the challenges of infection by F. hepatica parasites are met by rapid metabolic and physiological adjustments that expedite tissue invasion and immune evasion; these changes facilitate parasite growth, development and maturation. Our molecular analysis of the critical processes involved in host invasion has identified key targets for future drug and vaccine strategies directed at preventing parasite infection.

© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

Fig. 1.

Fig. 1.

Network graph of the 17901 genes expressed within the first 24 h. A, 3D layout graph represented by 13,559 nodes connected by 765,001 edges at a Pearson correlation threshold of r ≥ 0.97.B–E, Temporal gene expression by the_F. hepatica_ life-cycle stages: metacercariae (met), NEJ 1 h, 3 h, and 24 h post-excystment, respectively. Low levels of gene transcription are depicted by the small node size and the yellow/green node color. Increased gene transcription is represented by an increase in node size and the node color change from yellow/green to blue/purple/red. The number of genes with increased gene transcription at each time point and the number of unique Gene Ontology (GO) terms represented by these genes are shown.

Fig. 2.

Fig. 2.

Differential gene expression during the first 24 h. Genes expressed by biological replicates of metacercariae and NEJ 1 h, 3 h and 24 h post-excystment with a baseline cut-off of 2 TPM were grouped by hierarchical clustering, represented by a heatmap (Up-regulation represented in red; down-regulation represented in blue). The 6009 gene models broadly clustered into two groups: (1) Genes up-regulated during the metacercariae and NEJ 1 h stages; (2) Genes showing an up-regulation during the NEJ 3 h and 24 h stages. Gene ontology terms reflecting biological processes associated with each group are shown.

Fig. 3.

Fig. 3.

Graphical representation of transcript expression for the TCA and glycolysis/gluconeogenesis KEGG pathways represented as heatmaps for the metacercariae and newly excysted juveniles (NEJ) 1 h, 3 h, and 24 h post-excystment. Relative expression is shown by a blue to red scale depicting low to high levels of expression, respectively.

Fig. 4.

Fig. 4.

F. hepatica metabolism. A, Graphical representation of the transcription of genes associated with the metabolic KEGG modules (ko00001) across the_F. hepatica_ lifecycle within the first 24 h, normalizing the global patterns of expression at the KEGG module level. Relative expression is shown by a blue to red scale depicting low to high levels of expression, respectively. B, Graphical representation of the somatic protein abundance corresponding to the proteins associated with the metabolic KEGG modules (ko00001) within the infective stage, metacercariae and the NEJ up to 48 h post-excystment. The global patterns of expression were normalized at the KEGG module level. Relative protein abundance is shown by light to dark red scale, depicting low to high protein abundance, respectively.

Fig. 5.

Fig. 5.

Analysis of NEJ-specific F. hepatica cathepsin L (FhCL3) and B (FhCLB) cysteine proteases. A, Immunolocalization of FhCL3 in NEJ by CSLM, over a time-course of 48 h, represented by green fluorescence (FITC staining) within the NEJ gut. B, Immunolocalization of FhCB in NEJ by CSLM, over a time-course of 48 h, represented by green fluorescence within the NEJ gut (FITC staining). Time-course: (i) at excystment, (ii) 1 h post-excystment, (iii) 6 h post-excystment, (iv) 10 h post-excystment, (v) 24 h post-excystment. All specimens were counter-stained with phalloidin- TRITC to stain muscle tissue (red fluorescence) and provide structure. Scale bars = 20 μ

m

.C, Relative fold expression of cathepsin L (FhCL3) and B (FhCB1/CB3 and FhCB2) genes over a time-course of 48 h normalized to expression at NEJ excystment relative to a GAPDH reference, with S.E. Statistical analysis was carried out using One Way ANOVA with Tukey's post hoc test (p < 0.05: *; p < 0.01: **; p < 0.001: ***). D, Graphical representation of gene transcription (i), protein abundance within the somatic proteome (ii) and protein abundance within the secretome (iii). Relative expression/abundance is shown by a blue to red scale, depicting low to high levels of expression/abundance, respectively.

Fig. 6.

Fig. 6.

Proliferation of neoblast-like cells during the first 48 h post excystment. A, Incorporation of 5-ethynyl-2-deoxyuridine (EdU) by the proliferative neoblast-like cells highlighted by green fluorescence; (i) NEJ following 24 h culture with Edu at 4 °C, (ii) NEJ following 24 h culture at 4 °C followed by 24 h culture with Edu at 37 °C, (iii) NEJ following 48 h culture with Edu at 37 °C. Scale bars = 20 μ

m

. B, Graphical representation of the expression of genes associated with neoblast-like cells in transcripts per million (TPM) across the _F. hepatica_lifecycle, displayed on a log2 scale. C, Number of neoblast cells identified after (a) NEJ incubated for 48 h with 5-ethynyl-2-deoxyuridine (EdU) at 37 °C, (b) NEJ cultured for 48 h at 37 °C, with the addition of Edu for the last 24 h of culture, (c) NEJ cultured for 48 h, the first 24 h at 4 °C, following by the remaining 24 h culture at 37 °C with the addition of Edu. The differences between these groups were statistically significant (p < 0.001: ***). D, Relative fold expression of genes associated with neoblast-like cells normalized to expression at 24 h relative to a GAPDH reference, performed in duplicate, with S.E. Statistical analysis was carried out using One Way ANOVA with Tukey's post hoc test (p < 0.01: **;p < 0.001: ***).

Fig. 7.

Fig. 7.

Proteins identified by NEJ secretome analysis. A, Venn diagram representing the mean value of proteins identified within biological replicates of NEJ secretomes (1 h, 3 h, and 24 h post-excystment; 4, 3, and 3 biological replicates, respectively).B, Venn diagram representing those proteins across all three NEJ secretomes with a cutoff of at least 2 unique peptides (biological replicates as above). Those proteins that were uncharacterised are included in brackets. C, Graphical representation of the composition of the NEJ secretomes, based on the emPAI abundance of the different proteins types as a proportion of the total protein secreted.

Fig. 8.

Fig. 8.

Comparison of protein abundance for the NEJ 1 h, 3 h, and 24 h secretomes. A, Each NEJ secretome (1 h, 3 h, and 24 h) is represented as a heatmap ranked by emPAI score for each sample separately, indicated by the boxed sample name. Up-regulation: red; Down-regulation: blue. The top 10 proteins in terms of abundance are indicated within the brackets. B, Highlighted section depicting the top 10 proteins from each heatmap including protein annotation.

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