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Infection and Immunity, April 2005, p. 1995-2004, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.1995-2004.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Pyrophosphatase of the Roundworm Ascaris suum Plays an Essential Role in the Worm's Molting and Development

M. Khyrul Islam,1 Takeharu Miyoshi,1 Manabu Yamada,2 and Naotoshi Tsuji1*

Laboratory of Parasitic Diseases,1 Laboratory of Chronic Disease Pathology, National Institute of Animal Health, National Agricultural Research Organization, Tsukuba, Ibaraki, Japan2

Received 31 August 2004/ Returned for modification 25 October 2004/ Accepted 15 December 2004

Previous studies indicated that inorganic pyrophosphatase of Ascaris suum (AsPPase) plays an important role in larval survival in the host. Here we describe a precise role for AsPPase in larval molting and development and also describe the potential role of recombinant AsPPase (rAsPPase) in protective immunity to A. suum infection. Using reverse transcriptase PCR analysis, we found that disruption of AsPPase gene function by RNA interference resulted in suppression of AsPPase mRNA levels. RNA interference also caused inhibition of molting of third-stage larvae (31%) and suppression of native protein expression, as demonstrated by a 56% reduction in enzyme activity and quantified by immunoblot and immunofluorescence analyses, suggesting that AsPPase has a role in the molting process. The anatomic location of the AsPPase native enzyme in the hypodermis of larvae along with its elevated expression prior to and during the molting process supports such a role. Anti-rAsPPase immunoglobulin G (IgG) also resulted in 57% inhibition of molting of A. suum lung-stage third-stage larvae to fourth-stage larvae in vitro with developmental arrest. Antigenic epitopes of AsPPase overlapped the enzyme active sites. Mice immunized with rAsPPase exhibited high antigen-specific IgG antibody responses and were protected (>70%) against a challenge A. suum migratory-phase infection. Splenic T cells from rAsPPase-immunized mice produced low levels of T helper 1-type cytokines (gamma interferon and interleukin-2) in vitro but exhibited an elevated interleukin-10 response. A significantly high level of IgG1 subclass antibodies was found in immunized mice. Our results establish that AsPPase has a critical role in the molting and development of Ascaris roundworms and suggest the potential of AsPPase for use as a candidate vaccine against ascariasis.

* Corresponding author. Mailing address: Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural Research Organization, 3-1-5, Kannondai, Tsukuba, Ibaraki 305-0856, Japan. Phone: 81-29-838-7749. Fax: 81-29-838-7880. E-mail: tsujin{at}affrc.go.jp.

Editor: W. A. Petri, Jr.

Infection and Immunity, April 2005, p. 1995-2004, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.1995-2004.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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