RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade - PubMed (original) (raw)

RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade

Maria E Morales et al. Mol Biochem Parasitol. 2008 Feb.

Abstract

The aspartic protease cathepsin D (Clan AA, Family A1) is expressed in the schistosome gut where it plays an apical role in the digestion of hemoglobin released from ingested erythrocytes. In this report, RNA interference approaches were employed to investigate the effects of knockdown of schistosome cathepsin D. Cultured schistosomules of Schistosoma mansoni were exposed by square wave electroporation to double stranded RNA (dsRNA) specific for cDNA encoding S. mansoni cathepsin D. RNAi-mediated reductions in transcript levels led to phenotypic changes including significant growth retardation in vitro and suppression of aspartic protease enzyme activity. In addition, black-pigmented heme, the end point by-product of normal hemoglobin proteolysis that accumulates in the schistosome gut, was not apparent within the guts of the treated schistosomules. Their guts appeared to be red in color, rather than black, apparently indicating the presence of intact rather than digested host hemoglobin. These phenotypic effects were apparent when either of two forms of dsRNA, a long form spanning the entire target transcript or a short form specific for the 3'-region was employed. Off-target effects were not apparent in transcript levels of the gut-localized cysteine protease cathepsin B1. Finally, cathepsin D may be an essential enzyme in the mammal-parasitic stages of schistosomes because schistosomules treated with dsRNA did not survive to maturity after transfer into Balb/c mice. These and earlier findings suggest that, given its essential function in parasite nutrition, schistosome cathepsin D could be developed as a target for novel anti-schistosomal interventions.

PubMed Disclaimer

Figures

Figure 1

Schematic representation of the Schistosoma mansoni cathepsin D (_Sm_CD) transcript and open reading frames (ORFs) illustrating the regions from which the double-stranded RNAs (red and blue) used in this study were synthesized. Solid black arrows indicate the relative positions of the primers used in the RT-PCRs.

Figure 2

_Sm_CD is essential for normal worm growth. (A) Examples of schistosomes at day 14 following electroporation with either 10 μg of _Sm_CD 1.2 kb-dsRNA (left) or Luc-dsRNA (right). (Scale bars, 100 μm). (B) Population-wide quantitation of parasite size (as measured by the area occupied by parasites on a 2D image) on day 14 following dsRNA treatment; (1) _Sm_CD 1.2 kb-dsRNA, (2) Luc-dsRNA; (p ≤ 0.0001).

Figure 3

Induction of specific RNAi following treatment with cathepsin D dsRNA. (A) _Sm_CD enzyme activity in extracts of schistosomula. Three hour old schistosomula were electroporated with 10 μg of _Sm_CD 1.2 kb-dsRNA or Luc-dsRNA, and cultured for 14 days post-electroporation in the presence of human erythrocytes. (B) Shows relative cathepsin D gene expression of the same treatment group. RNA was 10 fold serially diluted, and assayed by RT-PCR for _Sm_CD and _Sm_CB1. The dilution series reflects input of 50.0, 5.0, 0.05 ng of total RNA into the RT-PCRs. In all cases, statistically significant differences between experimental and control groups were observed (p<0.05).

Figure 4

Cathepsin D may be required for normal hemoglobin proteolysis. Seven, nine and 11 day old schistosomules were transformed by electroporation with 30 μg of _Sm_CD 1.2kb-dsRNA or Luc-dsRNA, after which they were cultured for seven days in Basch’s medium supplemented with human erythrocytes. Examples of the 11 day old schistosomes at seven days after electroporation are presented: panel A, _Sm_CD-dsRNA; panel B, Luc-dsRNA; panel C, higher magnifications of three worms from the _Sm_CD-dsRNA-treated population, with arrows pointing to the worm’s gut; panel D, higher magnifications of three worms from the Luc-dsRNA-treated population. Scale bars = 100 μm. Similar findings were apparent in the seven- and nine-day-old schistosomules (not shown).

Figure 5

Gene suppression of S. mansoni cathepsin D (_Sm_CD) in schistosomules. Seven, nine and 11day old schistosomes were transformed by electroporation with 30 μg of _Sm_CD 0.5 kb-dsRNA or Luc-dsRNA, and then cultured in vitro. Forty eight hours and seven days later, batches of schistosomules were harvested for analysis of (A) total RNA by RT-PCR and (B) aspartic protease activity. At 48 h following electroporation, knockdown of both _Sm_CD transcripts and aspartic protease activity was apparent (not shown). By seven days, _Sm_CD transcription was completely silenced (A) and protease activity profoundly suppressed (B). Panel A: RNA was 10-fold serially diluted for analysis by RT-PCR. The dilution series reflects input of 50.0, 5.0, 0.05 ng of total parasite RNAs. Two other genes, _Sm_CB1 and GAPDH, were quantified by RT-PCR to monitor for possible off-target effects of dsRNA exposure. Panel B: _Sm_CD protease activity was measured at pH 3.5 in soluble extracts of schistosomules using the cathepsin D-specific substrate MCA-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-Arg. Extracts of _Sm_CD-dsRNA treated schistosomules exhibited markedly less protease activity than the Luc-dsRNA treated controls. For all three age groups of the schistosomules, significant suppression of _Sm_CD was evident (P ≤ 0.05 in each case), although there was a trend for diminished silencing with increasing age of the schistosomules for seven to nine to 11 days of age.

References

1. Brindley PJ, Kalinna BH, Dalton JP, Day SR, Wong JY, Smythe ML, McManus DP. Proteolytic degradation of host hemoglobin by schistosomes. Mol Biochem Parasitol. 1997;1:1–9. - PubMed
1. Tort J, Brindley PJ, Knox D, Wolfe KH, Dalton JP. Proteinases and associated genes of parasitic helminths. Adv Parasitol. 1999:161–266. - PubMed
1. Wasilewski MM, Lim KC, Phillips J, McKerrow JH. Cysteine protease inhibitors block schistosome hemoglobin degradation in vitro and decrease worm burden and egg production in vivo. Mol Biochem Parasitol. 1996;2:179–89. - PubMed
1. Bogitsh BJ, Kirschner KF, Rotmans JP. Schistosoma japonicum: immunoinhibitory studies on hemoglobin digestion using heterologous antiserum to bovine cathepsin D. J Parasitol. 1992;3:454–9. - PubMed
1. Williamson AL, Brindley PJ, Knox DP, Hotez PJ, Loukas A. Digestive proteases of blood-feeding nematodes. Trends Parasitol. 2003;9:417–23. - PubMed

RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade - PubMed (original) (raw)