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Selective activation of stress proteins in the muscle of the parasitic worm Ascaris suum from pig intestine.

Abstract. -- The major tissues of Ascaris suum contain the stress proteins (HSP) corresponding to the HSP70 and HSP90 family. Western blot analysis determined that in muscle, intestine and reproductive tract, the HSP70 family was the most abundant stress protein. Two forms of immunoreactive HSP70 proteins were detected. The concentrations of HSP72 protein were 1.9, 2.3, and 0.76 [micro]g/mg protein in intestine, muscle and reproductive tract, respectively. Constitutive levels of the HSP52 protein, which was also detected with the HSP70 antibody, were 0.80, 5.8, and 0.34 [micro]g/mg in intestine, muscle and reproductive tract, respectively. Further studies on the HSP70 proteins in muscle revealed that the HSP72 protein is a cytoplasmic protein whereas the HSP52 was equally distributed in the soluble and particulate fraction of the muscle. In contrast, both proteins appeared to be cytoplasmic in intestine and reproductive tract. Parasites maintained ex vivo at 37[degrees]C or 40[degrees]C showed a 2-fold increase in both HSP72 and HSP52, consistent with results in other organisms which suggest that parasites mount only a modest heat stress response. By contrast, maintenance of Ascaris suum, an essentially anaerobic organism, in an oxygen atmosphere resulted in a 4-fold increase in HSP52, but no change in HSP72 levels. Collectively, the data suggest that the parasite Ascaris suum responds to selective stresses which may occur in its micro environment.

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The stress protein family (HSP) was first observed in response to heat shock of Drosophila, but numerous studies since that time have established the universality of this response (Lindquist 1986). The major HSP protein families are highly conserved in prokaryotes and eukaryotes with respect to both structures and functions (Schlesinger 1990; Ang et al. 1991; Hendrick & Hartl 1993). The major HSP proteins, HSP70, occur in both cytoplasm and organelles where they bind to nascent or damaged unfolded proteins. Although these proteins are induced by a variety of environmental stresses, they also play an essential role in normal protein folding and cellular trafficking. By contrast, the HSP60 or groEl family of stress proteins are found only in bacteria or bacteria-derived organelles (mitochondria and chloroplasts). These proteins direct folding and oligomerization of proteins transported into these organelles although additional "housekeeping" functions have been proposed for these proteins. The functions of the HSP90/htpG and HSP27 families are less well understood. Deletion of the htpG gene in bacteria produces no detectable phenotypes, whereas deletion of the genes in yeast is lethal. In mammals, the HSP90 proteins are abundant in normal cells and mediate a variety of regulatory events, of which steroid receptor activation is the best documented. HSP27 occurs in most cells, albeit at low concentrations, and is induced by heat shock or toxic chemicals. Other functions which have been associated with this protein include growth, differentiation and neoplastic transformation (Zhou et al. 1993).

In contrast to the acute heat shock response, some organisms which are persistently exposed to thermal variations develop thermal tolerance (Subject & Shyy 1986). In organisms with acquired thermotolerance, the HSP proteins are expressed at high constitutive level which in some cases is not further elevated after acute thermal stress. Studies of parasites which have developmental stages in both poikilotherm and homeotherms have suggested that thermal tolerance may be a common property of many parasitic organisms. Brandau et al. (1995) demonstrated that in two Leishmania strains (L. major and L. donovani) high constitutive levels of HSP70 and HSP83 were observed and neither of these proteins were induced by acute heat stress. In Tritrichomonas mobilensis, a mammalian parasite, and Tritrichomonas augusta, an amphibian trichomonad, considerably different thermotolerance was observed (Bozner 1996). Thermotolerance for HSP70 induction exhibited only a 4[degrees]C range in T. mobilensis, whereas T. augusta tolerates a 13[degrees]C cultivation range. De Carvalho et al. (1994) failed to detect a classical heat treatment response in Trypanosoma cruzi maintained in a 48 hr culture. Studies of HSP70 mRNA turnover in this study, as well as in the work of Brandau et al. (1995) suggest that regulation of HSP induction may occur primarily at post-transcriptional level in these parasites.

Members of the nematode genus Ascaris are predominantly parasitic. Since Ascaris species are adapted to a spectrum of hosts, the public health and agricultural impact of this intestinal parasite is world wide. Ascaris lumbricoides is the most prevalent human parasite world wide, ranging from semi-tropical climates in the southern United States to most of Asia, Latin America and Africa (Bundy 1994; Long-Qi et al. 1995). Ascaris suum infects virtually all domestic swine, at significant expense to the agricultural industry. In the free living larval state of A. suum development, oxygen is required and a mitochondrial cytochrome system similar to that of the mammalian host has been described (Saruta et al. 1995). However, oxygen is toxic to the adult parasite which resides in the mainly anaerobic environment of the host small intestine. There is no functional tricarboxylic acid cycle in these organisms and unsaturated organic acids are used as terminal electron acceptors (Saruta et al. 1995). Since Ascaris is biochemically more complex than the parasites in which the stress response has been studied, this study investigated the HSP family in Ascaris suum with particular emphasis on the response of the organism to oxygen toxicity.

MATERIALS AND METHODS

Specimen aquisition. -- Specimens of Ascaris suum were collected within 30 min after slaughter in a local abattoir. For some experiments animals were removed immediately from the pig small intestine and the parasite intestine, muscle and reproductive tract were dissected out and frozen in liquid nitrogen. For ex vivo (organisms removed from the host and maintained in a synthetic laboratory environment) experiments, intestine were transported to the laboratory before animals were removed. Adult female parasites that measured 20-30 cm in length were used in all experiments.

Ex vivo maintenance. -- Ascaris suum were maintained in A. suum saline at 37[degrees]C in the presence of 95% [N.sub.2] - 5% C[O.sub.2] (Donahue et al. 1982). Three to five worms were suspended in 500 ml media for the designated time intervals. In experiments designed to test the effects of oxygen, the media were gassed with 80% [N.sub.2] - 20% [O.sub.2]. Under anaerobic conditions the worms can be maintained 3 - 4 days ex vivo without detectable loss of viability (Donahue et al. 1981a).

Tissue preparation. -- Specimens were dissected longitudinally and intestine, muscle and reproductive tract were immediately frozen in liquid nitrogen and stored at -80[degrees]C until analyzed. The frozen tissues were weighed and immersed in 3 vol of ice-cold 20 mM Tris-Cl, pH 7.5, 5 mM EDTA, 5 mM EGTA, 2 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 6 mM benzamidine, and 2 [micro]M leupeptin. All subsequent tissue preparation was conducted at 4[degrees]C. The tissues were homogenized in a precooled polytron for 30 sec and the resulting homogenate was centrifuged at 3000 g for 20 min. The resulting supernatant (LSS) was removed from the pellet (LSP) and centrifuged at 103,000 g for 30 min. The resulting supernatant (HSS) was frozen in 30 [micro]l aliquots at -80[degrees]C. Protein concentrations were determined by the Coomassie dye binding method. The protein concentration of the LSP was estimated by subtracting the total protein concentration of the HSS from the total protein concentration of the LSS.

HSP analysis. -- Frozen HSS was thawed in SDS PAGE sample buffer and analyzed by SDS PAGE on 12% polyacrylamide gels. Resolved proteins were transferred to PVDF membranes and stored in phosphate buffered saline, pH 7.3 (PBS). Membranes were blocked with 3% dry skim milk containing 0.1% Tween 20 in PBS. Membranes were probed with anti-HSP antibodies at a dilution of 1:2000. Anti-HSP70, anti-HSP60, and anti-HSP90 were obtained from StressGen (Victoria, B.C., Canada). Each of these antibodies is directed towards a universally conserved sequence in the respective HSP. In addition, the HSP70 reacts with both cognate and induced HSP70. Anti-HSP27 was generously provided by Dr. John Strahler of the University of Michigan (Bitar et al. 1991). Washed blots were probed with appropriate secondary antibodies (1:5000) conjugated with alkaline phosphatase (BioRad, Richmond, CA), and immunocomplexes were detected with chemiluminescent substrate (CSPD) according to the manufacturers instructions (Tropix, New Bedford, MA). The developed films were quantitated by video densitometry. Authentic HSP70, HSP60 and HSP90 were purchased from StressGen (Victoria, B.C., Canada). Authentic HSP27 was generously provided by Dr. Jacques Landry of the Universite Laval (Zhou et al. 1993). Prestained molecular weight standards were purchased from BioRad (Richmond, CA).

RESULTS

Occurrence of Ascaris suum HSP. -- HSP70 and HSP90 were readily detected by their respective antibodies in Western blots of HSS obtained from A. suum muscle and intestine (Figure 1). In both tissues, two forms of the HSP70 were detected. The heavier isoform appeared as a 72 kD protein and the smaller HSP70 isoform was 52 kD. These proteins will be referred to as HSP72 and HSP52 although both are homologous with the HSP70 as shown by the antibody reactivity. The HSP90 antibody detected a 90 kD protein in both tissues although twice as much sample was required to reliably detect this HSP. Several smaller proteins at lesser concentrations were also detected with the anti-HSP90 antibody; these proteins were not further investigated. HSP72, HSP52, and HSP90 could also be detected in reproductive tract although the immunoreactivity observed in these tissues were less than that in muscle and intestine.

Extract from intestine, muscle and reproductive tract was also analyzed with HSP60 and HSP27 antibodies. HSP60 could not be reliably detected in any tissue examined even when the sample concentration was increased to ~0.5 mg/gel lane. HSP27 was detected in muscle and intestine with amounts of sample comparable to that required for detection of HSP90.

Authentic HSP70 was analyzed as described for the tissue samples using concentrations of protein ranging from 50 - 150 ng/lane. Immunoreactive bands were quantitated by video densitometry and a standard curve was constructed. Extract was diluted so that the densitometry tracings of the immunoreactive bands fell within the standard curve and the amount of HSP72 and HSP52 was estimated. By this method, the amount of HSP72 and HSP52 in muscle was estimated to be 2.3 [micro]g/mg soluble protein and 5.8 [micro]g/mg soluble protein, respectively. In contrast, in intestine the amount of HSP72 and HSP52 was estimated to be 1.9 [micro]g/mg and 0.80 [micro]g/ml, respectively. In reproductive tissue, the levels of HSP72 and HSP52 were 0.76 [micro]g/ml and 0.34 [micro]g/mg, respectively.

[FIGURE 1 OMITTED]

HSP occur as both soluble proteins and proteins associated with particulate fractions. Since the HSP70 was the most prevalent HSP among those investigated, the distribution of HSP70 isoforms in the insoluble fraction obtained from low speed centrifugation (LSP) and the soluble fraction obtained from high speed centrifugation (HSS) was compared. The LSP was resuspended in SDS sample buffer to give a protein concentration approximately twice that determined for the HSS. Samples from both fractions of intestine, muscle and reproductive tract were analyzed for HSP70 as previously described. Results are shown in Figure 2. Virtually all of the HSP72 and HSP52 in intestine and reproductive tract was found in the soluble fraction. However, in muscle the distribution of HSP70 isoforms was significantly different. Similar to intestine and reproductive tract, the HSP72 was found primarily in the soluble protein fraction, whereas HSP52 clearly occurred in both the soluble and particulate fractions. When the results are corrected for volume differences, the data suggest that ~20% of the muscle HSP52 is associated with the particulate fraction.

[FIGURE 2 OMITTED]

Heat Shock HSP70 induction in muscle. -- Since the most abundant parasite stress protein is the HSP70 isoforms in muscle, the induction of these proteins was further investigated. A major concern was that removal of the parasite from the host might be sufficient to induce stress protein synthesis. The pigs are killed in a controlled temperature environment of ~20[degrees]C and intestines are obtained within 30 min after slaughter and within 5 min after removal from the animal. No difference in the relative distribution of HSP72 and HSP52 or the total amounts of these proteins could be detected when tissues which were dissected immediately from the worms at the slaughter house were compared to tissues obtained after transport to the laboratory in the intestine (Figure 3). In both samples, the amount of HSP72 was approximately twice the concentration of HSP52 in intestine, and the amount HSP72 was approximately half the amount of HSP52 in muscle. The relative amounts of HSP70 isoforms in the two tissues was unchanged in tissues obtained at the slaughter house as compared to those obtained after transport. On the basis of these results, no significant induction of HSP70 isoforms appears to occur in the time interval between removal of the parasite from the host and transport to the laboratory.

Induction of HSP70 isoforms in the ex vivo incubations was investigated at 37[degrees]C and 40[degrees]C. Worms were dissected at the beginning of the incubation (0 hr) and at 2 hr intervals up to 6 hr. Analysis of muscle extracts prepared from parasites incubated at 40[degrees]C (Figure 4) or 37[degrees]C (Figure 5) showed an increase in both HSP72 and HSP52 with time. At 40[degrees]C, the amount of HSP72 increased 1.3 fold and HSP52 increased 1.7 fold in the first 2 hr. After 4 hr, both HSP72 and HSP52 values were increased by ~2 fold and no further increase was observed after a total incubation time of 6 hr.

[FIGURE 3 OMITTED]

At 37[degrees]C, induction of HSP72 followed a comparable time course and after 6 hr the stress protein level was increased 2 fold. At 37[degrees]C, no induction was observed until the 4 hr time point at which time the increase was 1.2 fold. After 6 hr HSP52 was increased 2.4 fold. This is in contrast to some mammalian and fruitfly systems in which a 100-fold increase in HSP70 is observed in a comparable time frame (Lindquist 1986). Since the increase in HSP72 and HSP52 was not different at 37[degrees]C and 40[degrees]C, the induction may be the result of ex vivo incubation as opposed to temperature shock.

Oxygen-induced induction of HSP70 in muscle. -- Adult parasites normally inhabit the small intestine where the oxygen content is than 0.5%. Consistent with this microaerophilic environment, in the ex vivo incubation the animals survive less than 24 hr in the presence of atmospheric oxygen. To test the hypothesis that oxygen might induce a stress protein response before apoptosis is initiated, parasites were incubated at 37[degrees]C in the presence of an oxygen-free atmosphere and in a 20% oxygen atmosphere. HSP70 in muscle extracts was analyzed and results are shown in Figure 5. HSP52 was increased 3.8 fold in the presence of oxygen as opposed to 2.4 fold in the presence of nitrogen. In the first 4 hr of ex vivo incubation, a modest change in HSP52 was observed in the presence of nitrogen (<1.2 fold) or oxygen (1.5 fold). However, in the final 2 hr incubation, the amount of HSP52 increased 2 fold in the presence of nitrogen and 2.6 fold in the presence of oxygen (Figure 6). The total increase in HSP52 was 3.8 fold in the presence of oxygen and 2.4 fold in the presence of nitrogen. These data suggest that the time frame required for HSP52 induction is 4 - 6 hr. In contrast, no difference in the amount in HSP72 was observed in the presence or absence of oxygen in the time frame investigated.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

DISCUSSION

Similar to other studies with parasitic organisms, the HSP70 isoforms are constitutively expressed at a relatively high level in adult Ascaris suum tissues. The proteins were most abundant in muscle and least abundant in reproductive tract with intestine containing an intermediate concentration. No HSP70 could be detected in the cuticle/hypodermis, the fourth major tissue of the organism, although the possibility that HSP70 was present, but below levels of detection, cannot be excluded. In muscle, the major A. suum tissue, the HSP70 isoforms constitute approximately 0.8% of the soluble protein and 0.16% of the particulate protein. These estimates are obtained from a standard curve constructed with authentic bovine HSP70. Since the antibody used to detect the protein is a monoclonal antibody directed toward a unique conserved domain of the HSP70 protein, the reactivity of both HSP72 and HSP52 with this antibody identifies the parasite proteins as HSP70 proteins. In addition, since the antibody is specific for this domain, it is likely that the reactivity of the A. suum proteins and other HSP70 proteins with the monoclonal antibody would be comparable. Therefore, it is concluded that this is a reasonably reliable estimate of HSP70 expression in the parasite.

[FIGURE 6 OMITTED]

Since two other studies on parasitic organisms have suggested that induction of stress proteins in these species may occur by a post-translational mechanism which could be very rapid (De Carvalho et al. 1994; Brandau et al. 1995), the induction of the HSP70 proteins in the time interval between killing of the host and dissection of the tissues was investigated. During the 30 min interval in question, the host intestines do not change temperature significantly since they are held in the carcass of the host. After removal of the host intestine, the parasites were obtained and dissected within 10 min. Analysis of HSP70 in these tissues and those obtained after transport to the laboratory showed no significant difference in HSP72 or HSP52 levels. Since induction would be predicted to continue through this time interval, the data suggest that the high level of HSP70 proteins is indeed constitutive synthesis, and not induction.

The occurrence of two forms of the HSP70 proteins was unexpected. Even though the tissues were homogenized in a cocktail of protease inhibitors, the possibility that the lower molecular weight form was a product of HSP72 proteolysis was considered. Several observations suggest that HSP52 is not a product of HSP72. First, the distribution of HSP72 and HSP52 are not comparable in the various tissues. HSP52 is the predominant HSP70 isoform in muscle, but in intestine and reproductive tract, HSP72 is the predominant isoform. This argues against proteolysis since the intestine contains higher protease activity than the other two tissues. Second, induction of HSP70 synthesis in the presence of oxygen occurred selectively in the HSP52 isoform whereas both HSP72 and HSP52 were induced by ex vivo incubation. Finally, the ratio of HSP72/HSP52 observed in all tissues studied was remarkably constant and independent of the time between host sacrifice and tissue dissection. This suggests that anomalous degradation of the proteins was not occurring during this time. Collectively, the data suggest that both HSP72 and HSP52 are distinct isoforms of the HSP70 family.

The presence of particularly abundant HSP70 isoforms in A. suum muscle is consistent with the central role this tissue plays in the organism's metabolism. Since the parasite is free swimming and does not attach to the intestinal wall, muscle activity is continually required to counteract the flushing effect of host peristalsis. Muscle ATP is primarily derived from glucose since the tissue lacks a functional tricarboxylic acid cycle (Saruta et al. 1995). The source of glucose in host nonfeeding intervals is glycogen and the relatively sophisticated regulation of glycogen synthesis and mobilization has been studied extensively in this laboratory (Donahue et al. 1981a; 1981b; 1981c; 1982; Ghosh et al. 1989). In the laboratory, worms survive stresses, including starvation up to 60 hr, without noticeably compromises in vitality. The single exception to this apparent adaptation to stress is the toxicity of oxygen.

In other organisms multiple forms of the HSP70 proteins are observed (Hendrick & Hartl 1993). These isoforms correspond to genes which are constitutively expressed and genes which are selectively induced by unique stimuli. The selective induction of HSP52 with oxygen suggests that the HSP72 and HSP52 genes are independently regulated in this comparatively simple organism. Since elevated temperature failed to induce HSP70, but oxygen stress did selectively increase one HSP70 isoform, A. suum may be another example of an organism which is thermal tolerant, but retains the potential to respond to other environmental stresses. Further studies are in progress to define the stress response in this parasite more extensively.

ACKNOWLEDGMENTS

This work was supported in part by a grant to RAM from the University of North Texas Organized Research Fund. We gratefully acknowledge the technical assistance of Ms. Donna Ritchey.

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Sheng-Hao Chao, Manus J. Donahue and Ruthann A. Masaracchia

Department of Biological Sciences, University of North Texas

Denton, Texas 76203-5220

RAM at: ruthannm@facstaff.cas.unt.edu
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Author:Chao, Sheng-Hao; Donahue, Manus J.; Masaracchia, Ruthann A.
Publication:The Texas Journal of Science
Date:Nov 1, 1997
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