Thioredoxin peroxidases from Brugia malayi.

Parasite-derived antioxidant proteins have been implicated in playing an important role in protection against the oxygen radicals that are generated during aerobic metabolism and in defense against host immune cell attack. Here we report that filarial nematodes include the thioredoxin peroxidase/thiol-specific antioxidant (TPx/TSA) family of antioxidant proteins as part of their complex defense against radical-mediated damage. At the protein level, the TPx/TSA from Brugia malayi (Bm-TPx-1) was approximately 50% identical and approximately 60% similar to TPx/TSAs from mammals, amphibians and yeast. Bm-TPx-1 was also approximately 60% identical to putative TPx proteins from a related filarial nematode, Onchocerca volvulus, and from the free-living nematode Caenorhabditis elegans. That B. malayi may express multiple forms of molecules with TPx/TSA activity was indicated by the identification of a B. malayi gene encoding a second, distinct member of the TPx/TSA family (Bm-tpx-2). Bm-tpx-1 was found to be transcribed in all stages of the parasite present in the mammalian host and the 25 kDa translation product was present in all of the developmental stages studied. The results of immunohistochemical, immunofluorescent and immunoprecipitation studies showed Bm-TPx-1 to be localized in the cells of the hypodermis/lateral chord in adult parasites and not to be present at the surface or in excretory/secretory products. The distribution in the parasite suggests that Bm-TPx-1 may play its major role in countering radicals produced within cells. A recombinant form of Bm-TPx-1 was biologically active and capable of protecting DNA from oxygen radical-mediated damage. Thioredoxin peroxidases may prove to be a critical component in the parasite's defense against injury caused by oxygen radicals derived from endogenous and exogenous sources.

Muscle actin gene from Aedes aegypti (Diptera:Culicidae).

A recombinant phagemid containing a 1,240-bp insert encoding an actin was isolated from a yellow fever mosquito, Aedes aegypti (L.), complementary DNA library. This insert (pBS-Act35) contained an open reading frame of 822 bp whose deduced amino acid sequence exhibited > 95% homology with the carboxyl terminal 274 amino acids of Drosophila melanogaster Meigen and silkworm, Bombyx mori (L.), actin genes. Reverse transcriptase-polymerase chain reaction was used to clone and determine the sequence of the additional 306 nucleotides that comprise the 5' end of the gene. The coding nucleotide sequence of the whole gene (designated Aeact-1) exhibited between 81 and 89% homology with coding sequences of D. melanogaster and B. mori actin genes, and its deduced amino acid sequence exhibited > 95% homology with those genes. The highest similarity of Aeact-1 gene at the amino acid sequence level was with B. mori and D. melanogaster muscle actins. Southern blot analysis indicated that the Aedes genome contains at least 5 actin-related sequences.

Calgranulin C has filariacidal and filariastatic activity.

The calgranulins are a family of calcium- and zinc-binding proteins produced by neutrophils, monocytes, and other cells. Calgranulins are released during inflammatory responses and have antimicrobial activity. Recently, one of the calgranulins, human calgranulin C (CaGC), has been implicated as an important component of the host responses that limit the parasite burden during filarial nematode infections. The goal of this work was to test the hypothesis that human CaGC has biologic activity against filarial parasites. Brugia malayi microfilariae and adults were exposed in vitro to 0.75 to 100 nM recombinant human CaGC. Recombinant CaGC affected adult and larval parasites in a dose-dependent fashion. Microfilariae were more sensitive to the action of CaGC than were adult parasites. At high levels, CaGC was both macrofilariacidal and microfilariacidal. At lower levels, the percentage of parasites killed was dependent on the level of CaGC in the culture system. The larvae not killed had limited motility. The filariastatic effect of low-level CaGC was reversed when the CaGC was removed from the culture system. Immunohistochemical analysis demonstrated that human CaGC accumulated in the cells of the hypodermis-lateral chord of adult and larval parasites. The antifilarial activity of CaGC was not due to the sequestration of zinc. Thus, the cellular and molecular mechanisms that result in the production and release of CaGC in humans may play a key role in the regulation of filarial parasite numbers.

Filarial nematode parasites secrete a homologue of the human cytokine macrophage migration inhibitory factor.

Filarial nematode parasites establish long-term chronic infections in the context of an antiparasite immunity that is strongly biased toward a Th2 response. The mechanisms that lead to this Th2 bias toward filarial antigens are not clear, but one possibility is that the parasites produce molecules that have the capacity to proactively modify their immunological environment. Here we report that filarial parasites of humans secrete a homologue of the human proinflammatory cytokine macrophage migration inhibitory factor (MIF) that has the capability of modifying the activity of human monocytes/macrophages. A cDNA clone isolated from a Brugia malayi infective-stage larva expression library encoded a 12.5-kDa protein product (Bm-MIF) with 42% identity to human and murine MIF. MIF homologues were also found to be expressed in the related filarial species Wuchereria bancrofti and Onchocerca volvulus. Bm-mif was transcribed by adult and larval parasites, and the protein product was found in somatic extracts and in the parasite's excretory-secretory products. Immunohistocytochemistry revealed that Bm-MIF was localized to cells of the hypodermis/lateral chord, the uterine wall, and larvae developing in utero. Unexpectedly, the activities of recombinant Bm-MIF and human MIF on human monocytes/macrophages were found to be similar. When placed with monocytes/macrophages in a cell migration assay, Bm-MIF inhibited random migration. When placed away from cells, Bm-MIF induced an increase in monocyte/macrophage migration that was specifically inhibited by neutralizing anti-Bm-MIF antibodies. Bm-MIF is the first demonstration that helminth parasites produce cytokine homologues that have the potential to modify host immune responses to promote parasite survival.