Heat shock and developmental expression of hsp83 in the filarial nematode Brugia pahangi

Abstract

hsp83 was cloned from the filarial nematode Brugia pahangi. The mRNA was constitutively expressed at 37 8C in life cycle stages that live in the mammalian host (microfilariae and adult worms). Heat shock resulted in only a minimal increase in levels of transcription. A genomic copy of hsp83 was isolated and was shown to contain 11 introns while sequencing of the 5 0 upstream region revealed several heat shock elements. Using a chloramphenicol acetyltransferase (CAT) reporter gene construct the expression of hsp83 from B. pahangi (Bp-hsp83 ) was studied by transfection of COS-7 cells. Similar to the expression pattern in the parasite, CAT activity was detected at 37 8C and was not influenced by heat shock. When the free-living nematode Caenorhabditis elegans was transfected with the same construct, CAT activity was not observed at normal growth temperatures (21 8C) or under moderate heat shock conditions (28 8C). However exposure to more severe heat shock (35 8C) resulted in an increase in CAT activity. These results suggest that Bp-hsp83 has a temperature threshold $ 35 8C for expression.Keywords: Caenorhabditis elegans; CAT assay; heat shock elements; temperature threshold; transient transfection.Proteins of the heat shock protein (Hsp)90 family have been characterized extensively in yeast and higher eukaryotes, where they function both as molecular chaperones and as stress proteins [1,2]. In many cell types Hsp90 specifically associates with a range of regulatory proteins including steroid hormone receptors [3,4] and cyclin-dependant kinases [5]. Rutherford & Lindquist [6] proposed that the ability of Hsp90 to associate with signal transduction proteins, together with its capacity to act as a stress protein, provides a link between various developmental pathways and environmental change. Indeed they provided experimental evidence that Hsp90 is able to 'buffer' various mutant phenotypes in Drosophila and demonstrated that inhibition of Hsp90 allowed expression of otherwise silent mutations.The association of Hsp90 with components of the cell cycle provides a mechanism by which Hsp90 may be involved in developmental switching. In the free-living nematode Caenorhabditis elegans, hsp90 transcripts were shown to be 10 to15-fold enriched in the dauer larvae [7], while the transcription of other mRNAs tested was significantly reduced. The dauer larva is a developmentally arrested stage arising in response to adverse environmental conditions such as over-crowding or food shortages [8]. When worms were stimulated to emerge from dauer, hsp90 levels showed a marked decline within 2 h of recovery [7]. hsp90 in C. elegans has more recently been shown to be a member of the daf (dauer formation) gene family, daf-21. A gain of function mutant in daf-21 confers a dauerconstitutive phenotype suggesting that Hsp90 has a function in the dauer pathway, whereas the daf-21 null phenotype displays a growth arrest at the L2 to L3 stage [9].In this study, we report the cloning and characterization of the hsp90 homologue from the paras...

Characterization of the heat shock protein 90 gene in the plant parasitic nematode Meloidogyne artiellia and its expression as related to different developmental stages and temperature

“…A little decrease of Mthsp90 level was determined in J2s, compared to that of mature egg masses. The increase of Mt-hsp90 transcript levels observed in J3s-J4s and in males suggests that Mt-hsp90 transcripts are essential for both basal and developmental functions and for coping with environmental changes (Krishna et al, 1995;Martinez et al, 2001;Thompson et al, 2001;Wiesgigl and Clos, 2001;Zilka et al, 2001;Echeverria et al, 2005).…”

“…The comparison of the complete sequences of hsp90 genes from two free-living nematodes C. briggsae, C. elegans (Birnby et al, 2000) and two parasitic nematodes Brugia pahangi (Thompson et al, 2001) and M. artiellia revealed that the number of introns is variable: from 3 introns in the free-living nematodes up to 9 in M. artiellia and 11 in B. pahangi ( Fig. 2A).…”

Nematode Hsp90: highly conserved but functionally diverse

“…Using 35 S methionine labelling, an increase in expression of a range of heat shock proteins, including Hsp90, was observed as L3 were shifted from 28 to 37°C (Jecock and Devaney, 1992). However, there is no evidence to suggest that the expression of Bp-hsp90 (either mRNA or protein) is significantly increased following exposure of adult worms to heat shock conditions (Thompson et al 2001;Devaney et al 2005), suggesting that most Bp-hsp90 is constitutively expressed. Parasitic nematodes secrete a range of molecules when cultured in serum-free medium (the so called excretory-secretory products or ES), and analysis of the ES of adult B. malayi demonstrated that B. malayi Hsp90 (Bm-Hsp90) was present (Kumari et al 1994), a finding which has been confirmed in this laboratory using B. pahangi (Devaney et al unpublished observations).…”

“…While studies from this laboratory have failed to demonstrate a significant induction of DAF-21 in C. elegans following heat shock (as quantified by immuno-blotting with a specific antibody) (Thompson et al 2001;Devaney et al 2005), other studies using in situ hybridization with daf-21 specific probes have revealed differences in both the staining patterns and abundance of daf-21 mRNA in C. elegans upon heat shock (Inoue et al 2003). At normal growth temperatures, DAF-21 is predominantly localized to the germline of C. elegans, as detected by antibody staining as well as in situ hybridization (Inoue et al 2003).…”

Hsp90 Inhibitors in Parasitic Nematodes: Prospects and Challenges

“…In common with other eukaryotes, filarial worms express relatively high levels of Hsp90, but in contrast to other heat shock proteins, Hsp90 is not significantly upregulated, at either the mRNA or protein level, upon temperature shift from 28 o C to 37 o C (mimicking the vector to mammalian transition) or upon heat shock [34,35]. What then might be the function of Hsp90 in these organisms?…”