Hsp70 May Be a Molecular Regulator of Schistosome Host Invasion

Abstract

Schistosomiasis is a debilitating disease that affects over 240 million people worldwide and is considered the most important neglected tropical disease following malaria. Free-swimming freshwater cercariae, one of the six morphologically distinct schistosome life stages, infect humans by directly penetrating through the skin. Cercariae identify and seek the host by sensing chemicals released from human skin. When they reach the host, they burrow into the skin with the help of proteases and other contents released from their acetabular glands and transform into schistosomula, the subsequent larval worm stage upon skin infection. Relative to host invasion, studies have primarily focused on the nature of the acetabular gland secretions, immune response of the host upon exposure to cercariae, and cercaria-schistosomulum transformation methods. However, the molecular signaling pathways involved from host-seeking through the decision to penetrate skin are not well understood. We recently observed that heat shock factor 1 (Hsf1) is localized to the acetabular glands of infectious schistosome cercariae, prompting us to investigate a potential role for heat shock proteins (HSPs) in cercarial invasion. In this study, we report that cercarial invasion behavior, similar to the behavior of cercariae exposed to human skin lipid, is regulated through an Hsp70-dependent process, which we show by using chemical agents that target Hsp70. The observation that biologically active protein activity modulators can elicit a direct and clear behavioral change in parasitic schistosome larvae is itself interesting and has not been previously observed. This finding suggests a novel role for Hsp70 to act as a switch in the cercaria-schistosomulum transformation, and it allows us to begin elucidating the pathways associated with cercarial host invasion. In addition, because the Hsp70 protein and its structure/function is highly conserved, the model that Hsp70 acts as a behavior transitional switch could be relevant to other parasites that also undergo an invasion process and can apply more broadly to other organisms during morphological transitions. Finally, it points to a new function for HSPs in parasite/host interactions.

Fig 1. Visual illustration of cercariae swimming.

During active swimming, most cercariae are found in the water column, while almost no cercariae can be seen at the bottom of the culture well. As cercariae hone, more of them can be seen at the bottom of the well when observed at higher magnification.

Fig 2. Cercariae treated with PES hone and transform more completely than those treated with skin lipid.

Cercariae were treated with filtered water (A-C), 0.5% DMSO (D-F), human skin lipid (G-I), or 250 μM PES (J-L), and observed at various time points. The treatments and observation timings are as follows: (A) water, 8 minutes; (B) water, 56 minutes; (C) water, 1 hour 45 minutes; (D) DMSO, 9 minutes; (E) DMSO, 57 minutes; (F) DMSO, 1 hour 46 minutes; (G) lipid, 0 minutes; (H) lipid, 9 minutes; (I) lipid, 1 hour; (J) PES, 3 minutes; (K) PES, 51 minutes; (L) PES, 1 hour 42 minutes. Each treatment used about 1,000 cercariae in a volume of 1 mL in a 12-well plate well (40× view).

Fig 3. Cercariae treated with other Hsp70 inhibitors do not hone.

Cercariae were treated with filtered water (A), 1% DMSO (B), 250 μM PES (C), 500 μM MKT-077 (D), 400 μM 115-7c (E), or 100 μM VER-155008 (F); observed at 2 hours. Each treatment used about 1,000 cercariae in a volume of 1 mL in a 12-well plate well (10× view).

Fig 4. Cercariae treated with Hsp90 inhibitors do not hone.

Cercariae were treated with 1% DMSO (A), 250 μM PES (B), 100 μM geldanamycin (C), or 50 μM 17-DMAG (D); observed at 2 hours. Each treatment used about 1,000 cercariae in a volume of 1 mL in a 12-well plate well (10× view).

Fig 5. Predictive model of a role for Hsp70 in cercarial honing.

(A) In the absence of strong host signals, Hsp70 binds tightly to its client protein, HHF, inhibiting its activity. (B) Host signals are transmitted through a cercarial signal transduction pathway, releasing Hsp70 inhibition of HHF, which functions in cercarial honing. (C) The inhibitor PES blocks Hsp70 activity by binding to the Hsp70 substrate binding domain and releasing Hsp70 inhibition of HHF, resulting in cercarial honing. (D) Addition of 10 mM ATP leads to release of HHF, possibly by binding to the Hsp70 ATPase domain and reducing its affinity for HHF, resulting in cercarial honing. (E) Addition of a non-hydrolyzable form of ATP leads to release of HHF, possibly by preventing ATP hydrolysis and maintaining the weak affinity state of Hsp70 for binding client proteins, resulting in cercarial honing.

Fig 6. Cercariae treated with ATP, AMP-PNP, and ADP.

Cercariae were treated with filtered water (A), 5 mM ATP (B), 5 mM AMP-PNP (C), or 5 mM ADP (D), and observed 2 hours 30 minutes after treatment. Each treatment used about 500 cercariae in a volume of 0.5 mL in a 24-well plate well (40× view).