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

When organisms are exposed to an increase in temperature, they undergo a heat shock response (HSR) regulated by the transcription factor heat shock factor 1 (HSF-1). The heat shock response includes the rapid changes in gene expression initiated by binding of HSF-1 to response elements in the promoters of heat shock genes. Heat shock proteins function as molecular chaperones to protect proteins during periods of elevated temperature and other stress. During infection, hookworm infective third stage larvae (L3) undergo a temperature shift from ambient to host temperature. This increased temperature is required for the resumption of feeding and activation of L3, but whether this increase initiates a heat shock response is unknown. To investigate the role of the heat shock in hookworm L3 activation and parasitic development, we identified and characterized the expression profile of several components of the heat shock response in the hookworm Ancylostoma caninum. We cloned DNAs encoding an hsp70 family member (Aca-hsp-1) and an hsp90 family member (Aca-daf-21). Exposure to a heat shock of 42°C for one hour caused significant up-regulation of both genes, which slowly returned to near baseline levels following one hour attenuation at 22°C. Neither gene was up-regulated in response to host temperature (37°C). Conversely, levels of hsf-1 remained unchanged during heat shock, but increased in response to incubation at 37°C. During activation, both hsp-1 and daf-21 are down regulated early, although daf-21 levels increase significantly in non-activated control larvae after 12h, and slightly in activated larvae by 24h incubation. The heat shock response modulators celastrol and KNK437 were tested for their effects on gene expression during heat shock and activation. Pre-incubation with celastrol, an HSP90 inhibitor that promotes heat shock gene expression, slightly up-regulated expression of both hsp-1 and daf-21 during heat shock. KNK437, an inhibitor of heat shock protein expression, slightly down regulated both genes under similar conditions. Both modulators inhibited activation-associated feeding, but neither had an effect on hsp-1 levels in activated L3 at 16h. Both celastrol and KNK437 prevent the up-regulation of daf-21 and hsf-1 seen in non-activated control larvae during activation, and significantly down regulated expression of the HSF-1 negative regulator Aca-hsb-1 in activated larvae. Expression levels of heat shock response factors were examined in developing Ancylostoma ceylanicum larvae recovered from infected hosts and found to differ significantly from the expression profile of activated L3, suggesting that feeding during in vitro activation is regulated differently than parasitic development. Our results indicate that a classical heat shock response is not induced at host temperature and is suppressed during larval recovery and parasitic development in the host, but a partial heat shock response is induced after extended incubation at host temperature in the absence of a developmental signal, possibly to protect against heat stress.