Characterisation of hookworm heat shock factor binding protein (HSB-1) during heat shock and larval activation

Abstract

When hookworm infective L3s infect their mammalian host, they undergo a temperature shift from that of the ambient environment to that of their endothermic host. Additionally, L3s living in the environment can be exposed to temperature extremes associated with weather fluctuations. The heat shock response (HSR) is a conserved response to heat shock and other stress that involves the expression of protective heat shock proteins (HSPs). The HSR is controlled by heat shock factor 1 (HSF-1), a conserved transcription factor that binds to a heat shock element in the promoter of HSPs, causing their expression. HSF-1 is negatively regulated in part by a HSF binding protein (HSB-1) that binds to and removes HSF-1 trimers bound to HSP gene promoters, resulting in attenuation of the HSR. Herein we describe an HSB-1 ortholog, Ac-HSB-1, from the hookworm Ancylostoma caninum. The Ac-hsb-1 cDNA encodes a 79 amino acid protein that is 71% identical to the Caenorhabditis elegans HSB-1, and is predicted to share the characteristic coiled-coil structural motif comprised of two interacting alpha helices. Recombinant Ac-HSB-1 immunoprecipitated Ce-HSF-1 expressed in mammalian cells that had been heat shocked for 1 h at 42°C, but not from cells incubated at 37°C, indicating that HSB-1 only bound to the active DNA binding form of HSF-1. Expression of Ac-hsb-1 transcripts decreased following 1 h of heat shock, but increased when L3s were incubated at 37°C for 1 h. Activation of hookworm L3s induces an five-to six-fold increase in Ac-hsb-1 expression that peaks at 12 h, coincident with L3 feeding, but that subsequently decreases to two-to three-fold above control at 24 h. Recombinant Ac-HSB-1 immunoprecipitates greater amounts of 70 and 40 kDa proteins from extracts of activated L3s than from non-activated L3s. We propose that an increase in Ac-hsb-1 levels early in activation allows feeding to resume, but that a subsequent decrease in expression permits a HSR that protects nondeveloping L3s at host-like temperatures. Further investigations of the HSR will clarify the role of HSB-1 and HSF-1 in hookworm infection.

Expression profile of heat shock response factors during hookworm larval activation and parasitic development

“…1000655) was maintained and harvested from dog feces as described elsewhere [28]. An Indian strain of A. ceylanicum (USNPC No.…”

“…In vitro activation of iL3 and heat shock experiments were conducted as published [28]. For activation, iL3 were decontaminated in 0.12 N HCl for 30 minutes at 22° C followed by 3 washes in RPMI 1640 tissue culture medium supplemented with 25 mM HEPES (pH 7.0) and antibiotics (RPMI-c).…”

“…For heat shock experiments, 5000 decontaminated iL3 in 0.5 ml of RPMI-c were incubated at 42°C for the desired time, followed by attenuation at 22°C for up to 60 minutes [28]. Control iL3 were incubated at 37°C followed by attenuation.…”

“…RNA isolation and quantitative reverse transcriptase-PCR (qPCR) experiments were performed as described previously [28]. Data are from 2–3 runs in triplicates from 1–3 biological replicates and analysed in GraphPad InStat3 (ANOVA plus Tukey Kramer Test or unpaired t test with Welch’s correction for fewer than 3 groups).…”

“…We recently analysed the function of Aca -HSB-1[28], and herein investigate the expression of other main regulators of the HSR, HSF-1, HSP-1/HSP-70, and DAF-21/HSP-90 during heat shock. We compare the HS expression profile to the profile of in vitro activated L3 as well as developing parasitic larvae.…”