Cloning and Expression Analysis of Two Mucin-like Genes Encoding Microfilarial Sheath Surface Proteins of the Parasitic Nematodes Brugia and Litomosoides

Abstract

In several filarial genera the first stage larvae (microfilariae) are enclosed by an eggshell-derived sheath that provides a major interface between the parasite and the host immune system. Analysis of the polypeptide constituents of the microfilarial sheath from the cotton rat filaria Litomosoides sigmodontis identified two abundant surface glycoproteins: Shp3a and Shp3. The corresponding genes and the orthologues of the human parasite Brugia malayi and the rodent filaria Brugia pahangi were cloned and sequenced. They encode secreted, mucin-like proteins with N-terminal Ser/Thrrich repeats and a C-terminal anchor domain rich in aromatic amino acids. About 75% of the protein molecular masses result from post-translational modifications. The Ser/Thr-rich motifs are supposed to serve as targets for dimethylaminoethanol-phosphate substitutions. These modifications were detected only on the sheaths of the late developmental stage of stretched microfilariae, corresponding with the expression of the proteins in the epithelium of the distal part of the uterus and the specific transcription of shp3 and shp3a in the anterior female worm segment. Genomic analysis of all three species demonstrated a conserved linkage of the two genes. Their transcripts undergo cis-and transsplicing. The transcription start sites of the primary transcripts were determined for the L. sigmodontis genes. The core promoter regions are remarkably conserved between the paralogue genes Ls-shp3a and Lsshp3 and their orthologues in Brugia, implicating conserved regulatory elements.The parasitic nematodes Brugia malayi, Brugia timori, and Wuchereria bancrofti are the causative agents of human lymphatic filariasis, a disease that afflicts an estimated 128 million people throughout the tropics (1) and in its final form results in severe obstructive alterations (elephantiasis). The adult lymphatic-dwelling parasites survive for years, and the viviparous females release large amounts of first stage larvae, microfilariae, into the blood stream. Completion of the filarial life cycle involves the uptake of the microfilariae by mosquitos, development to third stage larvae and transmission back to the vertebrate host. The microfilariae are completely surrounded by a bag-like, antibody-impermeable structure, the microfilarial sheath, which derives from the embryonic eggshell but is altered by maternal components secreted by the uterus epithelium (2). Parasitaemic hosts develop a parasite stage-specific immunological tolerance and fail to mount an antibody response to the microfilarial sheath surface proteins (3, 4). Therefore, it is of considerable interest to identify and characterize sheath surface components. The cotton rat filaria Litomosoides sigmodontis represents a suitable model organism for the lymphatics dwelling filarial parasites because of, in principle, a corresponding structure and genesis of the sheath (5). Previous studies with this parasite had identified two sheath proteins: Ls-Shp3a and Ls-Shp3 (40 and 120 kDa on SDS-PAGE) that appeared to ...

Stage-specific proteomic expression patterns of the human filarial parasiteBrugia malayiand its endosymbiontWolbachia

“…Some of these immunologically relevant proteins have been described previously (23-33) and relate to those molecules that are highly immunogenic in human infections or that have activity on the mammalian host immune response (through mimicry or other mechanisms). These data corroborate a number of previous studies that demonstrate stage-specific expression and/or serologic reactivity of ALT-2 and the larval allergens (in the L3 stage) (34) and BmR1 (35), BmMIF (36), TGF-β homologue (30), SXP-1 (37), galectins (38), and microfilarial sheath proteins (in the microfilarial stage) (39). Particular sets of ECM-associated proteins were common and highly enriched in the both the L3s (SI Appendix, Fig.…”

Genome-Wide Transcription Profiling of the Early Phase of Biofilm Formation by Candida albicans

“…ORFs with no homology to S. cerevisiae but which encode interesting amino acid motifs are also present in group A: for example, ORF19.5760 encodes a 392-amino-acid protein with a Gly-Ser domain at the carboxy terminal similar to that found in lustrin A, human and mouse cornified envelope loricrin, and the extracellular matrix protein keratin (54); ORF19.7104 encodes a mucin-like protein similar to microfilarial sheath protein Shp3 from the nematode Litomosoides (27).…”

Use of microarray hybridization to identify Brugia genes involved in mosquito infectivity

“…The antigen designated SXP-1 is diagnostic for human infection for several filarial species, and is localized to the hypodermis, where it is possibly secreted to the sheath (Dissanayake et al 1992;Sasisekhar et al 2005). Major microfilarial sheath protein SHP-1 was previously identified as one of six major polypeptide constituents of the sheath (Hirzmann et al 2002), and RNAi phenotypes for SHP-1 in B. malayi consist of reduced mf release by adult females, and shortened sheaths of released mf (Aboobaker and Blaxter 2003). The infectivity phenotype of this mutant is unfortunately unknown.…”

“…Į-tocopherol); it also is associated with alkaline phosphatase and calcium-activated ATPase activities (Araujo et al 1993;Sayers et al 1984;Smith et al 1998). Filarial developmental transitions induce changes in the protein, lipid and carbohydrate composition of the cuticle (Hirzmann 2002;Apfel et al 1992;Ham et al 1988;Lewis et al 1999), and our transcriptome data indicate that mature mf differentially produce transcripts involved in cuticular structure, including two encoding cuticular collagens, one (TC2854) that is highly similar to C. elegans col-34, and the other an uncharacterized B. malayi gene (GenBank accession no.…”

“…The sheath, a remnant of the eggshell membrane, is cast from the worm during or after midgut penetration in a successful infection (Fuhrman et al 1987;Yamamoto et al 1983). The sheath is acellular and insoluble, and is composed of chitin Simpson and Laurence 1974;Paulson et al 1988;Fuhrman and Piessense 1985), matrix proteins (elastin, fibronectin, collagen type IV, laminin), and shp2, an insoluble and cross-linking protein (Hirzmann et al 2002); as well 25 as sulfur-containing proteins, proteoglycans, phospholipids and acidic mucopolysaccharides Simpson and Laurence 1974;Araujo et al 1993;Araujo et al 1994). Enzymatic activities associated with the sheath include acid phosphatase and 5' nucleotidase (Sayers et al 1984).…”