Fig 1.
The aggregation of fimbriated and non-fimbriated bacteria to the polar plugs of Trichuris muris eggs precedes hatching.
Representative SEM images from T. muris eggs co-cultured for 60–90 min at 37°C with A, E and F. Escherichia coli, B. Salmonella typhimurium, C. Pseudomonas aeruginosa, and D. Staphylococcus aureus. Fimbriated bacteria (A, B and C) attach to a zone of surface material (sm) covering the polar plugs of T. muris eggs and extending to the egg collars immediately adjacent to the plugs (zoomed into insets II and III, asterisks) via fimbriae/pili (arrows in insets III). Bacteria attached to the eggshell surface in the nonpolar region between the two plugs (black border insets IV) have no fimbriae. Non-fimbriated S. aureus (D) is in intimate contact with the sm in the collar (asterisk in inset III) and making a cup-like indentation at the surface of the T. muris egg polar plug. E. The tilted axis view of the polar plug and collar of a T. muris egg shows the extent of the sm zone (dashed circle) to which E. coli binds. F. The sm zone is present at each pole of the eggs, and E. coli is visibly binding to both (zoomed insets I and II).
Fig 2.
Escherichia coli anchors to the Pellicula Ovi of the polar plugs of Trichuris muris eggs via fimbriae during the induction phase of the hatching cascade.
Representative SEM and TEM images from T. muris eggs untreated (A and B) or co-cultured with Escherichia coli (C and D) for 90 min at 37°C. Polar plugs and collars of untreated eggs are covered by surface material (sm) detected by SEM (A) and display an intact Pellicula Ovi (PO) extending across the plug and eggshell (B). E. coli in contact with the polar plugs and collars of the eggs, express fimbriae (arrows in insets in C and D) that anchors the bacteria to the PO directly below. Chitin Layer (CL), Electron-dense Parietal Coating (EdPC), Inner Layer of the Polar Plug (ILPP) and Spatio Externum (SpEx) are visible in D.
Fig 3.
Interactions of fimbriated and non-fimbriated bacteria with the Pellicula Ovi of Trichuris muris eggs polar plugs during the induction phase of the hatching cascade.
Representative TEM images from T. muris eggs co-cultured for 90 min at 37°C with: A. Salmonella typhimurium, B. Pseudomonas aeruginosa, and C. Staphylococcus aureus, showing bacteria in contact with Pellicula Ovi (PO) of the T. muris egg polar plug bordering the eggshell chitin layer (CL). Insets show the detail of bacterial attachment to the PO via fimbriae and pili (arrows in insets of A and B) or direct contact of the S. aureus cell wall to a shallow cup-like indentation zone on the PO surface (arrow in inset C).
Fig 4.
Temporal and physical changes leading to the asymmetric disintegration of the polar plugs during the hatching of Trichuris muris eggs.
A-C. Representative TEM and Toluidine Blue-stained images showing progressive degradation of the polar plug of T. muris eggs during hatching induced by Escherichia coli at 75–90 min of co-culture. A. Intact plug of an embryonated egg showing Pellicula Ovi (PO) continuous across the chitin layer (CL) and plug, Electron-dense Parietal Coating (EdPC) separating the plug from the Spatio Externum (SpEx) and first-stage larva (L1). B. Degradation proceeds with visible loss of electron density within the Inner Layer of the Polar Plug (ILPP), which leads to the EdPC and SpEx expanding upwards. L1 larva is enclosed by the Permeability Barrier Membrane (PBM, asterisks) and the Spatio Internum (SpIn) is visible. C. Finally, one of the two polar plugs is destroyed following degradation and larval eclosion (inset I), while the contralateral polar plug remains unbreached (inset II). D. Representative SEM image showing S. typhimurium binding both poles of a T. muris egg undergoing eclosion.
Fig 5.
Bacteria-mediated hatching of Trichuris muris eggs is protease-dependent.
Trichuris muris eggs were co-cultured with Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa and Staphylococcus aureus in the absence (-) or presence (+) of a protease inhibitor cocktail (1x) for A. 2 h and B. 4 h at 37°C. The number of total embryonated eggs and hatched larvae were counted, from which the hatching percentage was calculated. Hatching was completed in triplicate across three independent experiments (n = 9), except for S. typhimurium in A, where data comes from two independent experiments (n = 6). Median and interquartile range are shown, and statistical differences between the absence (-) or presence (+) of a protease inhibitor cocktail for each bacterial species were evaluated using the Mann-Whitney test (**p<0.005, ****p<0.0001). C. T. muris eggs were co-cultured with E. coli, S. typhimurium, P. aeruginosa and S. aureus in the absence (-) or presence (1x or 2x) of a protease inhibitor cocktail for 24 h at 37°C. The number of total embryonated eggs and hatched larvae were counted, from which the hatching percentage was calculated. Hatching was completed in triplicate across three independent experiments (n = 9). Median and interquartile range are shown, and statistical differences between the absence (-) or presence (1x or 2x) of a protease inhibitor cocktail for each bacterial species were evaluated using Kruskal Wallis with Dunn’s multiple comparison tests performed (*p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001). D. T. muris eggs were co-cultured with E. coli in the absence (-) or presence (+) of a protease inhibitor cocktail (1x) for 24 h at 37°C. The number of total embryonated eggs and hatched larvae were counted, from which the hatching percentage was calculated. Next, T. muris eggs co-cultured in the presence of the inhibitor were washed to remove it and re-exposed to fresh E. coli cultures for 24 h at 37°C; the percentage hatching was recorded 24 h later (Washed). Hatching was completed in triplicate in the absence and presence of the protease inhibitor cocktail and sextuplicate for the washed condition across three independent experiments (n = 9 and n = 18, respectively). Median and interquartile range are shown, and statistical differences between groups were evaluated using Kruskal Wallis with Dunn’s multiple comparison tests performed (*p<0.05, **p<0.005, ****p<0.0001).
Fig 6.
Complete Trichuris muris egg embryonation leads to the development of ‘hatching-ready’ L1 larvae expressing S01A family serine endopeptidases and inhibitors.
A-B. T. muris un-embryonated eggs were placed in deionised water in the dark at room temperature. Embryonation was monitored over time at 0, 1, 5, 6, 7, 8 and 12 weeks by A. brightfield imaging (scale bars = 10 μm) and B. percentage of egg hatching was determined upon co-culture with Escherichia coli for 24 h at 37°C (n = 6). C-K. Differential gene expression by DESeq2 between T. muris eggs embryonated for six (n = 5) and eight weeks (n = 6). C. Principal Component Analysis (PCA) of normalised transcripts (log2, variance stabilised transcripts (VST)), showing the relationship between 6 and 8 week samples and replicates. D. Volcano plot of differentially expressed transcripts from T. muris eggs embryonated for six (left side) and eight weeks (right side). Dashed lines represent thresholds for fold-change (<-1 and >1) and adjusted p-value (<0.01); transcripts that are significantly differentially expressed are indicated by red points. E. Gene set enrichment analysis of transcripts significantly differentially expressed and upregulated at six or eight weeks of embryonation. Gene Ontology (GO) terms were analysed in gProfiler; shown are -log10(Padj) (colour scale) and term size (size) for enriched GO terms in each time point, categorised by biological process (BP), cellular component (CC), and molecular function (MF). F-K. Volcano plots focused on transcripts for specific GO terms. Each plot is assembled as in D; however, transcripts associated with a specific GO term are highlighted, with significantly differentially expressed transcripts (dashed lines indicated thresholds, as in panel D) shown in red, whereas non-significant transcripts are marked in orange. F. Chitinase activity (GO:0004568). G. Metallopeptidase activity (GO:0008237). H. Serine-type endopeptidase activity (GO:0004252). I. Peptidase inhibitor activity (GO:0030414). J. Lipase activity (GO:0016298). K. Cysteine-type peptidase activity (GO:0008234).
Fig 7.
Bacterial-mediated hatching of Trichuris muris eggs is serine-protease dependent.
A. T. muris eggs were co-cultured with Escherichia coli in the absence (-) or presence of a protease inhibitor cocktail, the dual-class protease inhibitors antipain and leupeptin, the serine protease inhibitors Pefabloc, chymostatin and aprotinin, and the cysteine protease inhibitor E64. Percentage hatching was calculated after 24 h of co-culture at 37°C. Hatching was completed in triplicate across three independent experiments (n = 9). The median and interquartile range are shown. Kruskal Wallis with Dunn’s multiple comparison tests were performed (****p<0.0001). B. T. muris eggs were co-cultured with E. coli, Salmonella typhimurium, Pseudomonas aeruginosa and Staphylococcus aureus in the absence (-) or presence (1 mg/mL or 2 mg/mL) of Pefabloc for 24 h at 37°C. The number of total embryonated eggs and hatched larvae were counted, from which the hatching percentage was calculated. Hatching was completed in triplicate across three independent experiments (n = 9). Median and interquartile range are shown, and statistical differences between absence (-) or presence (1 mg/mL or 2 mg/mL) of Pefabloc for each bacterial species were evaluated using Kruskal Wallis with Dunn’s multiple comparison tests were performed (*p<0.05, ****p<0.0001).