Fig 1.
HPX-2 contributes to C. elegans resistance to some pathogens.
(A) Schematic representation of the predicted HPX-2 translated products in N2, hpx-2(dg047), and hpx-2 (gk252521). (B) Survival of N2 and hpx-2 mutants on live Enterococcus faecalis OG1RF. (C) Survival of N2 and hpx-2 mutants on vancomycin inactivated Enterococcus faecalis OG1RF. Survival of N2 and hpx-2 mutants on (D) Candida albicans SC5314, (E) Staphylococcus aureus NCTC8325, (F) Corynebacterium diphtheriae NCTC12129, (G) Pseudomonas aeruginosa PA14, (H) Escherichia coli O157:H7 Sakai (EHEC), and (I) Salmonella enterica SL1344. Results from one representative experiment with an n of approximately 90 worms for each condition are shown. Median survival and P-values are listed in S8 Table as experiment No.1 along with those from additional biological replicates.
Fig 2.
Genomic hpx-2 complementation rescues the susceptibility to E. faecalis.
(A) Survival of N2, hpx-2(dg047) mutant, and its complemented strain on E. faecalis. (B) Survival of N2, hpx-2(gk252521) mutant, and its complemented strain on E. faecalis. The hpx-2 mutants are set as the control comparison. Results from one representative experiment with an n of approximately 90 worms for each condition are shown. Median survival and P-values are listed in S8 Table as experiment No.1 along with those from additional biological replicates.
Fig 3.
hpx-2 is expressed in the hypodermis and pharynx.
(A) Survival of the intestinal RNAi strain on E. faecalis following exposure to hpx-2 or VC control RNAi. (B) Survival of the hypodermal RNAi strain on E. faecalis following exposure to hpx-2 or VC control RNAi. Representative results from single experiments with an n of approximately 90 worms for each condition are shown. Median survival and P-values along with additional biological replicates are listed in S8 Table as experiment No.1. (C) Illustration of upstream region and the first two exons of the hpx-2 gene that are fused to gfp. The expression pattern of hpx-2::gfp under the confocal microscope in young-adult worms (D)and dauer worms (E-G). Red arrow heads indicate the pharyngeal expression of hpx-2 in either the processes (D and E) or the gland cells (F and G). (G) Higher magnification of the terminal bulb showing the pharyngeal expression pattern of hpx-2::gfp. (H) Diagram of the relevant features of the terminal bulb imaged in G. Note that D-G are mergers of images taken using the DIC, FITC and TRITC filters. Background autofluorescence is detected by both FITC and TRITC filters causing the merge to appear yellow, whereas true GFP expression only appears in the FITC filter and is green.
Fig 4.
The cuticle defect of hpx-2 mutants.
(A) Hoechst staining of N2, hpx-2(dg047) and hpx-2(gk252521) worms. Worms with stained nuclei were scored as positive. Images presented are representative of ~300 worms observed. (B) Quantification of Hoechst positive worms in N2, hpx-2(dg047) and hpx-2(gk252521) worms. Compared with N2, both mutants had significantly higher level of Hoechst staining (Phpx-2(dg047) = 0.0022, Phpx-2(gk252521) = 0.033). (C) Hoechst staining at higher magnification of hypodermal cells along body. (D) Quantification of animals with Hoechst staining of body hypodermal nuclei (Phpx-2(dg047) = 0.0023, Phpx-2(gk252521) = 0.0310). (E) Hoechst staining of nuclei in head region, specifically showing a focal plane cutting through the pharynx. (F) Quantification of animals with Hoechst staining of head nuclei (Phpx-2(dg047)<0.0001, Phpx-2(gk252521) = 0.0137). The graphs in B, D and F were generated from 3 independent experiments (n = 100). Error bars represent SEM.
Fig 5.
Intestinal colonization of hpx-2 mutants by pathogens.
Colonization of (A) GFP-labeled E. faecalis OG1RF and (C) dsRed labeled P. aeruginosa PA14 in N2, hpx-2(dg047) and hpx-2(gk252521) worms at day two and day one of infection, respectively. (B) E. faecalis CFU counts per worm (Phpx-2(dg047) = 0.0471, Phpx-2(gk252521) = 0.0275, n = 19 (combined from 3 independent experiments)). (D) E. faecalis CFU counts per worm (Phpx-2(dg047) = 0.1833, Phpx-2(gk252521) = 0.2724, n = 19 (combined from 3 independent experiments)). Error bars represent SEM.
Fig 6.
HPX-2 peroxidase activity is required for pathogen resistance.
(A) Sequence alignment of the peroxidase domain of HPX-2 and HPX-2 site-directed (SD) mutant to other peroxidases and DUOX. hMPO, human myeloperoxidase. (B) The survival of N2, HPX-2(R372A) strain (hpx-2(syb238)), and hpx-2 mutant strain (hpx-2(dg047)) on E. faecalis. Results from one representative experiment with an n of approximately 90 worms for each condition are shown. Median survival and P-values are listed in S8 Table as experiment No.1 along with those from additional biological replicates.
Fig 7.
Transcriptome analysis of hpx-2 mutant.
(A) Scatter plot of genes changed in hpx-2(dg047) animals, as compared to N2 animals, when either exposed to E. coli (x-axis) or E. faecalis (y-axis). Values of 10 or -10 indicate expression level under one condition was 0 RPKM. (B) Gene Ontology (GO) term of Biological Process (BP) analysis of genes that changed upon loss of hpx-2(dg047) compared to N2, when exposed to E. faecalis. (C) qRT-PCR validation of 10 selected genes differentially expressed in the RNA-seq analysis of hpx-2(dg047) to N2 on E. faecalis. Mean log2 (fold change) determined in three independent qRT-PCR were plotted against the log2 (fold change) from the RNAseq experiments. (D) Scatter plot of genes changed when exposed to E. faecalis, as compared to E. coli, in N2 (x-axis) or hpx-2(dg047) (y-axis) animals. Genes that are significantly changed under at least one condition are presented. Values of 10 or -10 indicate expression level under one condition was 0 RPKM.
Fig 8.
Model for HPX-2 function in hypodermis and pharynx.
Diagram of head of the worm. The green structure is the pharynx. Brown lines on the outside of the worm as well as in the pharyngeal lumen indicate the cuticle material that makes up the hypodermal cuticle and pharyngeal cuticle, respectively. The blue globular structures in the terminal bulb of the pharynx are the gland cells, and the blue line extending from them represent the processes through which vesicles travel for export of material to the lumen. We postulate that HPX-2 is released from the gland cells for building of the pharyngeal cuticle and released from the underlying hypodermal cells for building of the hypodermal cuticle. See main text for details.