- Email: [email protected]
Invasion of skin by schistosome cercariae: some neglected facts
Update
TRENDS in Parasitology
on a scale needed for comprehensive evaluation of field trials requires considerable laboratory resources. But, DNA microarrays and other high-throughput techniques for analyzing polymorphisms will reduce hands-on time and costs, and will enable molecular monitoring to become a routine component of future malaria field trials. References 1 Barker, R.H. et al. (1994) Plasmodium falciparum and P. vivax: factors affecting sensitivity and specificity of PCR-based diagnosis of malaria. Exp. Parasitol. 79, 41 – 49 2 Snounou, G. et al. (1993) High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol. Biochem. Parasitol. 61, 315– 320 3 Brockman, A. et al. (1999) Application of genetic markers to the identification of recrudescent Plasmodium falciparum infections on the northwestern border of Thailand. Am. J. Trop. Med. Hyg. 60, 14–21 4 Genton, B. et al. (2002) A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts selective pressure on parasite populations in a Phase I/IIb trial in Papua New Guinea. J. Infect. Dis. 185, 820 – 827 5 Cheng, Q. et al. (1997) Measurement of Plasmodium falciparum growth rates in vivo: a test of malaria vaccines. Am. J. Trop. Med. Hyg. 57, 495 – 500 6 Lawrence, G. et al. (2000) Effect of vaccination with 3 recombinant asexual-stage malaria antigens on initial growth rates of Plasmodium falciparum in non-immune volunteers. Vaccine 18, 1925 – 1931 7 Hermsen, C.C. et al. (2001) Detection of Plasmodium falciparum malaria parasites in vivo by real-time quantitative PCR. Mol. Biochem. Parasitol. 118, 247 – 251 8 Witney, A.A. et al. (2001) Determining liver stage parasite burden by real time quantitative PCR as a method for evaluating pre-erythrocytic malaria vaccine efficacy. Mol. Biochem. Parasitol. 118, 233 – 245 9 Smith, T. et al. (1999) Effect of insecticide-treated bed nets on the dynamics of multiple Plasmodium falciparum infections. Trans. R. Soc. Trop. Med. Hyg. 93 (Suppl 1), 53 – 57 10 Smith, T. and Vounatsou, P. Estimation of infection and recovery rates for highly polymorphic parasites when detectability is imperfect, using hidden Markov models. Stat. Med. (in press) 11 Fraser-Hurt, N. et al. (1999) Effect of insecticide-treated bed nets on haemoglobin values, prevalence and multiplicity of infection with Plasmodium falciparum in a randomized controlled trial in Tanzania. Trans. R. Soc. Trop. Med. Hyg. 93 (Suppl 1), 47 – 51 12 Coulibaly, D. et al. Impact of pre-season treatment on incidence of falciparum malaria and parasite density at a site for testing malaria vaccines in Bandiagara. Mali. Am. J. Trop. Med. Hyg. (in press) 13 Owusu-Agyei, S. et al. (2001) Incidence of symptomatic and asymptomatic Plasmodium falciparum infection following curative
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therapy in adult residents of northern Ghana. Am. J. Trop. Med. Hyg. 65, 197 – 203 Arnot, D. (1998) Unstable malaria in Sudan: the influence of the dry season. Clone multiplicity of Plasmodium falciparum infections in individuals exposed to variable levels of disease transmission. Trans. R. Soc. Trop. Med. Hyg. 92, 580– 585 Alonso, P.L. et al. (1994) Randomised trial of efficacy of SPf66 vaccine against Plasmodium falciparum malaria in children in southern Tanzania. Lancet 344, 1175– 1181 Beck, H-P. et al. (1997) Analysis of multiple Plasmodium falciparum infections in Tanzanian children during the phase III trial of the malaria vaccine SPf66. J. Infect. Dis. 175, 921 – 926 Haywood, M. et al. (1999) Reduction in the mean number of Plasmodium falciparum genotypes in Gambian children immunized with the malaria vaccine SPf66. Trans. R. Soc. Trop. Med. Hyg. 93 (Suppl 1), 65– 68 Doumbo, O.K. et al. (2000) Rapid selection of Plasmodium falciparum dihydrofolate reductase mutants by pyrimethamine prophylaxis. J. Infect. Dis. 182, 993– 996 Bojang, K.A. et al. (2001) Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomised trial. Lancet 358, 1927– 1934 Gandon, S. et al. (2001) Imperfect vaccines and the evolution of pathogen virulence. Nature 414, 751 – 756 Babiker, H. et al. (1994) Genetic evidence that RI chloroquine resistance of Plasmodium falciparum is caused by recrudescence of resistant parasites. Trans. R. Soc. Trop. Med. Hyg. 88, 328 – 331 Al Yaman, F. et al. (1997) Evidence that recurrent Plasmodium falciparum infection is caused by recrudescence of resistant parasites. Am. J. Trop. Med. Hyg. 56, 436– 439 Ranford-Cartwright, L.C. et al. (1997) Molecular analysis of recrudescent parasites in a Plasmodium falciparum drug efficacy trial in Gabon. Trans. R. Soc. Trop. Med. Hyg. 91, 719 – 724 von Seidlein, L. et al. (1997) Treatment of African children with uncomplicated falciparum malaria with a new antimalarial drug, CGP 56697. J. Infect. Dis. 176, 1113 – 1116 von Seidlein, L. et al. (1998) A randomized controlled trial of artemether/benflumetol, a new antimalarial and pyrimethamine/ sulfadoxine in the treatment of uncomplicated falciparum malaria in African children. Am. J. Trop. Med. Hyg. 58, 638 – 644 Irion, A. et al. (1998) Distinction of recrudescences from new infections by PCR-RFLP analysis in a comparative trial of CGP 56 697 and chloroquine in Tanzanian children. Trop. Med. Int. Health 3, 490 – 497 Snounou, G. and Beck, H-P. (1998) The use of PCR genotyping in the assessment of recrudescence or reinfection after antimalarial drug treatment. Parasitol. Today 14, 462 – 467
1471-4922/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(02)00066-1
Invasion of skin by schistosome cercariae: some neglected facts Rachel S. Curwen and R. Alan Wilson Department of Biology, Area 5, PO Box 373, University of York, York, UK, YO10 5YW
The process of skin invasion by Schistosoma cercariae was reviewed in a recent Trends Research Update, accompanied by a computer animation. Some aspects of that article were misleading and perpetuated Corresponding author: Rachel S. Curwen ([email protected]). http://trepar.trends.com
misconceptions about parasite migration through the skin that should by now have been dispelled. This article sets out a different interpretation of events, taking account of the extensive data on migration and larval structural changes that have been documented for Schistosoma mansoni over the past 20 years.
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Schistosome cercariae, newly emerged from the snail host, swim tail-first with alternate bursts of active upward movement and passive downward sinking in which the furcae on the tail behaves like a parachute (see the animation associated with Ref. [1] at http://archive.bmn. com/supp/part/part0502.html). This behaviour takes the cercariae to the water surface, where their rhythmic sinking and swimming persists for some hours until glycogen reserves have been depleted. It appears to be a ‘sit and wait’ strategy to conserve energy reserves against the unpredictable appearance of the host [2]. Host location The environmental stimuli to which the cercaria responds are precisely those that a human wading through water would generate, namely turbulence, shadows and skin chemicals (N.P. Carter, DPhil. thesis, University of York, 1978). Turbulence and shadows increase the length of the swim period, with a shadow as brief as three seconds resulting in the cercaria swimming parallel to the water surface, but still tail-first. Skin chemicals have the most potent effect on cercarial behaviour, causing continuous swimming with frequent reversals of direction, which is achieved by altering the angle at which the tail furcae are held. In all the above, there is no indication of a directed movement (taxis) towards the host. Rather, the increased swimming activity (kinesis) with a strong, random lateral component enhances the probability of contact with host skin. If the stimuli cease without host contact, the behaviour of the cercaria reverts to the normal up and down rhythm. Obstacles to invasion To infect the host, the cercaria must traverse the stratified epithelium of the epidermis, then the dermis, to exit the skin via a blood or lymphatic vessel. It is not clear whether the cercaria forces its way through the epidermal layers by lysing cells, or insinuates itself between cells by digesting the extracellular matrix (ECM). There is some debris in penetration tunnels, but cells in proximity to the parasite are compressed, implying that insinuation predominates over cytolysis [3]. If this is the case, the multi-lamellar lipid complex that fills the intercellular spaces between the squames of the stratum corneum is the first barrier. The larva then negotiates the living keratinocyte layer which is held together primarily by the adherens junctions and desmosomes between adjacent cells [4]. The principal macromolecules of this ECM are various members of the cadherin family, while elastin is absent. The final barrier to dermal entry is the epidermal basement membrane, the lamina densa of which contains two forms of collagen IV and a third form, the more unusual collagen VII [5]. Anchoring filaments, which comprise laminin 5 trimers, form the link between basal keratinocytes and collagen VII in the lamina densa. The principal obstacles in the dermis itself are the collagen, elastin and reticulin fibres, originating in fibroblasts. The timing of events in skin migration Skin excision experiments have provided a quantitative definition of the kinetics of migration [6]. They revealed http://trepar.trends.com
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that virtually no parasites leave the skin before 48 h in mouse, hamster or rat, with the time for half to exit estimated as 88 h, 65 h and 70 h, respectively [6]. Autoradiographic tracking of 75Se-labelled parasites [2], which confirmed the data for duration of stay in the skin and the time of first arrival in the lungs of mice and rats, and provided similar information for baboons [7], suggesting that the estimates derived from rodents are relevant to primate hosts. Balance sheets, drawn up for the total numbers of parasites in all mouse organs over time, revealed that invading parasites suffer little mortality in the skin, with the vast majority (. 90%) of penetrating larvae travelling to the lungs [8]. The precise compartments in which the parasites reside at any given time have been identified by histology and electron microscopy of, for example, the infected hamster cheek pouch [3,9]. The epidermal basement membrane is reached within 30 min by the majority of penetrating cercariae, but they are arrested there, with some deflected along the basement membrane for a short distance, emphasizing its status as a barrier. The larvae seem to have no problem in disrupting the keratinocyte tethers, so that the epidermis lifts away to form ‘blisters’ in which they lie, but they are stopped by the lamina densa [3]. The early and prolonged larval retardation, external to the dermis, suggests that either a molecular signal is perceived to suspend migration, or one or more components of the laminin and collagen networks in the basement membrane cannot be hydrolyzed by acetabular gland secretions. Data from cheek pouch migration [9] indicate a minimum stay in the epidermis of 40 h and a mean time of entry to the dermis of 52.5 h. Exceptionally, some parasites can reach the dermis in shorter times following exposure to biologically unrealistic numbers of cercariae (. 1000 per cm2 [10]). Once in the dermis, a further 10 h are required to locate a dermal venule and 8.5 h to penetrate its wall to exit the skin. Secretory sources The cercaria has three types of gland cell which contain pre-formed secretory vesicles: (1) the acetabular glands; (2) the head gland; and (3) the sub-tegumental cell bodies [11], each of which plays a role in host invasion or parasite transformation. Several proteases with broad substrate specificities have been reported in the pre- and postacetabular gland secretions [12]. Of these, a serine protease, conventionally referred to as cercarial elastase [13], is considered the most important [14]. The acetabular gland contents are observed to be exhausted as soon as 1 – 2 h after penetration commences [15,16], and are certainly absent at 40 h [3] (Fig. 1a,b). Given the timing of passage through the skin layers described above, degeneration of the acetabular glands is complete before entry into the dermis occurs, and long before vascular entry (c.f. [1]). Their contents could not therefore be involved in digestion of the basement membrane ‘barrier’ or any subsequent migratory event (c.f. [1]). So, how does the parasite breach the basement membrane, cross the dermis and penetrate a venule wall? Strong morphological evidence points to the head gland as the source of any hydrolytic activity used [3]. It has been suggested that the head gland is also empty
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schistosomula (Fig. 1c,d). Its dense granular secretions are released from the schistosomular apical region when pressed against the basement membrane and venule wall. In addition, the muscular head capsule remains until the larva is at least five days old, potentially assisting the spined apical region to act as a ‘battering ram’ to cause mechanical disruption. It should also be noted that elastase-like protease activity has been eluted from schistosomula surface membranes [19], and could potentially participate in dermal migration. When is an elastase not an elastase? It is paradoxical that the supply of a protease seemingly capable of degrading dermal elastin is exhausted before penetration into the dermis. Is the readily demonstrable elastinolytic activity of acetabular gland secretions [18] a relic of former function? Alternatively, because there is evidence that the cleavage sites of the cercarial protease differ from those of other elastases [20], are its intended substrates the cells or ECM of the epidermis, and its classification as an elastase an unfortunate misnomer? Nevertheless, schistosomula lacking acetabular glands can disrupt the basement membrane and venule wall, and another, as yet uncharacterized, protease potentially emanating from the head gland could be responsible. Metalloprotease activity has already been noted in secretions from maturing schistosomula after the first 24 h, but was not tested for its ability to cleave all basement membrane components [21]. Characterization of head gland secretions should resolve these issues. Acknowledgements We thank Patricia Coulson for critical reading of the article. Our work received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. R.S.C. is a BBSRC CASE graduate student with the Natural History Museum, London, UK.
References
Fig. 1. Secretory sources in Schistosoma. Parasites stained with cell-tracker green (Molecular Probes) to reveal the presence of the acetabular glands in a schistosome cercaria (a) and their absence from an ex vivo schistosomulum 24 h after cercarial penetration (b). Electron micrographs of: (c) a 40 h schistosomulum in the epidermis (E), lying external to the basement membrane (BM), with the head gland (HG) containing abundant secretory vesicles; (d) a 72 h schistosomulum with its apical area (A) pressed against a small venule (VN). The muscular head capsule (HC) is evident. Arrows point to fibrin and platelets in the dermis. The inset shows a secretory vesicle (OG) fusing with a tegumental pit. Electron micrographs courtesy of Jean Crabtree (St James University Hospital, Leeds, UK). Scale bars ¼ 20 mm (a,b); 5 mm (c,d); 0.5 mm [inset in (d)].
one hour after skin penetration commences and that its contents are used for repair of the tegument [17]. However, the head gland is clearly visible both in vitro (R.S. Curwen, unpublished) and in vivo [3] in three-day-old, and older, http://trepar.trends.com
1 McKerrow, J.H. and Salter, J. (2002) Invasion of skin by Schistosoma cercariae. Trends Parasitol. 18, 193– 195 2 Wilson, R.A. (1987) Cercariae to liver worms: development and migration in the mammalian host. The Biology of Schistosomes: from Genes to Latrines (Rollinson, D., Simpson, A. eds), pp. 115 – 146, Academic Press Ltd 3 Crabtree, J.E. and Wilson, R.A. (1985) Schistosoma mansoni: an ultrastructural examination of skin migration in the hamster cheek pouch. Parasitology 91, 111 – 120 4 Cozzani, E. et al. (2001) Adhesion molecules in keratinocyte. Clin. Dermatol. 19, 544 – 550 5 Ghohestani, R.F. et al. (2001) Molecular organization of the cutaneous basement membrane zone. Clin. Dermatol. 19, 551– 562 6 Miller, P. and Wilson, R. (1978) Migration of the schistosomula of Schistosoma mansoni from skin to lungs. Parasitology 77, 281 – 302 7 Wilson, R.A. et al. (1990) Schistosome migration in primates: a study in the olive baboon (Papio anubis ). Trans. R. Soc. Trop. Med. Hyg. 84, 80 – 83 8 Wilson, R.A. et al. (1986) Migration of the schistosomula of Schistosoma mansoni in mice vaccinated with radiation-attenuated cercariae, and normal mice: an attempt to identify the timing and site of parasite death. Parasitology 92, 101 – 116 9 Wilson, R.A. and Lawson, J.R. (1980) An examination of the skin phase of schistosome migration using a hamster cheek pouch preparation. Parasitology 80, 257 – 266 10 Wheater, P.R. and Wilson, R.A. (1979) Schistosoma mansoni: a
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histological study of migration in the laboratory mouse. Parasitology 79, 49 – 62 Dorsey, C.H. et al. (2002) Ultrastructure of the Schistosoma mansoni cercaria. Micron 33, 279 – 323 McKerrow, J. and Doenhoff, M. (1988) Schistosome proteases. Parasitol. Today 4, 334 – 340 Newport, G.R. et al. (1988) Cloning of the proteinase that facilitates infection by schistosome parasites. J. Biol. Chem. 263, 13179 – 13184 Salter, J.P. et al. (2000) Schistosome invasion of human skin and degradation of dermal elastin are mediated by a single serine protease. J. Biol. Chem. 275, 38667 – 38673 Brink, L.H. et al. (1977) Schistosoma mansoni: a comparative study of artificially transformed schistosomula and schistosomula recovered after cercarial penetration of isolated skin. Parasitology 74, 73 – 86 Cousin, C.E. et al. (1981) Schistosoma mansoni: ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules. Exp. Parasitol. 51, 341 – 365
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17 Dorsey, C.H. (1976) Schistosoma mansoni: description of the head gland of cercariae and schistosomules at the ultrastructural level. Exp. Parasitol. 39, 444– 459 18 McKerrow, J.H. et al. (1983) Degradation of extracellular matrix by larvae of Schistosoma mansoni. I. Degradation by cercariae as a model for initial parasite invasion of host. Lab. Invest. 49, 195– 200 19 Ghendler, Y. et al. (1996) Schistosoma mansoni: evidence for a 28-kDa membrane-anchored protease on schistosomula. Exp. Parasitol. 83, 73 – 82 20 Salter, J.P. et al. (2002) Cercarial elastase is encoded by a functionally conserved gene family across multiple species of schistosomes. J. Biol. Chem. 277, 24618 – 24624 21 Auriault, C. et al. (1981) Proteolytic cleavage of IgG bound to the Fc receptor of Schistosoma mansoni schistosomula. Parasite Immunol. 3, 33 – 44 1471-4922/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(02)00019-3
Invasion of skin by schistosome cercariae: some neglected facts Response from James J. McKerrow
James J. McKerrow Sandler Center for Basic Research in Parasitic Diseases, University of California, Box 0511 HSW 511, San Francisco, CA 94148, USA
In response to the Research Focus article by Rachel Curwen and Alan Wilson, some of the issues concerning invasion of skin by schistosome cercariae are addressed or clarified. The conclusions drawn by Curwen and Wilson concerning the kinetics of invasion and the exhaustion of schistosome acetabular glands are incorrect because they are based on rodent models and not on direct assessment of cercariae of human parasites that invade human skin. It is reassuring that despite the dwindling number of laboratories studying schistosome biology and the decreasing funding support for this field, enough interest remains to engender debate. That said, I must address some of the misunderstandings that are referred to as ‘perpetuated misconceptions’ by Rachel Curwen and Alan Wilson. Host location In our article [1] and animation (http://archive.bmn.com/ supp/part/part0502.html), we could not detail each step in cercarial invasion. Indeed, Curwen and Wilson are correct in providing more detail on the tail-first movement of cercariae, and the active upward and passive downward movements of cercariae in the absence of hosts. We noted in our article that: ‘Specific host signals recognized by schistosomes vary with specific host– parasite interactions’; however, Curwen and Wilson have fallen into a Corresponding author: James J. McKerrow ([email protected]). http://trepar.trends.com
trap of lumping turbulence, shadows and skin chemicals as the key stimuli for all schistosome cercariae. Haas et al. have provided the most detailed analysis of swimming and tactic stimuli [2]. In their article, Table 1 outlines how shadows are important for Trichobilharzia cercariae to find ducks, water turbulence is particularly important for Diplostonum to find fish and chemical stimuli help Isthmiophora find amphibia [2]. However, not all of these stimuli translate to human Schistosoma. There are even significant differences in the responses of Schistosoma mansoni and Schistosoma haematobium to stimuli. Stirewalt [3] reviewed simple biological experiments showing that S. mansoni cercariae follow a thermal gradient (thermotaxis), and we have confirmed those results. Haas has also indicated the importance of a thermal stimulus to cercarial attachment. However, like Haas, we were unable to demonstrate chemotactic behavior in S. mansoni cercariae. Curwen and Wilson are right to point out that continuous swimming of cercariae will increase the probability of finding a host, but thermotactic behavior in both host finding and host attachment has not been ruled out. To date, no intrepid parasitologist has followed cercariae with an underwater camera to ensure that they do not move towards a warm, immersed limb. Obstacles to invasion We agree that cercarial penetration through the epidermis involves degradation of extracellular adhesion molecules such as the cadherin family of proteins.
TRENDS in Parasitology
on a scale needed for comprehensive evaluation of field trials requires considerable laboratory resources. But, DNA microarrays and other high-throughput techniques for analyzing polymorphisms will reduce hands-on time and costs, and will enable molecular monitoring to become a routine component of future malaria field trials. References 1 Barker, R.H. et al. (1994) Plasmodium falciparum and P. vivax: factors affecting sensitivity and specificity of PCR-based diagnosis of malaria. Exp. Parasitol. 79, 41 – 49 2 Snounou, G. et al. (1993) High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol. Biochem. Parasitol. 61, 315– 320 3 Brockman, A. et al. (1999) Application of genetic markers to the identification of recrudescent Plasmodium falciparum infections on the northwestern border of Thailand. Am. J. Trop. Med. Hyg. 60, 14–21 4 Genton, B. et al. (2002) A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts selective pressure on parasite populations in a Phase I/IIb trial in Papua New Guinea. J. Infect. Dis. 185, 820 – 827 5 Cheng, Q. et al. (1997) Measurement of Plasmodium falciparum growth rates in vivo: a test of malaria vaccines. Am. J. Trop. Med. Hyg. 57, 495 – 500 6 Lawrence, G. et al. (2000) Effect of vaccination with 3 recombinant asexual-stage malaria antigens on initial growth rates of Plasmodium falciparum in non-immune volunteers. Vaccine 18, 1925 – 1931 7 Hermsen, C.C. et al. (2001) Detection of Plasmodium falciparum malaria parasites in vivo by real-time quantitative PCR. Mol. Biochem. Parasitol. 118, 247 – 251 8 Witney, A.A. et al. (2001) Determining liver stage parasite burden by real time quantitative PCR as a method for evaluating pre-erythrocytic malaria vaccine efficacy. Mol. Biochem. Parasitol. 118, 233 – 245 9 Smith, T. et al. (1999) Effect of insecticide-treated bed nets on the dynamics of multiple Plasmodium falciparum infections. Trans. R. Soc. Trop. Med. Hyg. 93 (Suppl 1), 53 – 57 10 Smith, T. and Vounatsou, P. Estimation of infection and recovery rates for highly polymorphic parasites when detectability is imperfect, using hidden Markov models. Stat. Med. (in press) 11 Fraser-Hurt, N. et al. (1999) Effect of insecticide-treated bed nets on haemoglobin values, prevalence and multiplicity of infection with Plasmodium falciparum in a randomized controlled trial in Tanzania. Trans. R. Soc. Trop. Med. Hyg. 93 (Suppl 1), 47 – 51 12 Coulibaly, D. et al. Impact of pre-season treatment on incidence of falciparum malaria and parasite density at a site for testing malaria vaccines in Bandiagara. Mali. Am. J. Trop. Med. Hyg. (in press) 13 Owusu-Agyei, S. et al. (2001) Incidence of symptomatic and asymptomatic Plasmodium falciparum infection following curative
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therapy in adult residents of northern Ghana. Am. J. Trop. Med. Hyg. 65, 197 – 203 Arnot, D. (1998) Unstable malaria in Sudan: the influence of the dry season. Clone multiplicity of Plasmodium falciparum infections in individuals exposed to variable levels of disease transmission. Trans. R. Soc. Trop. Med. Hyg. 92, 580– 585 Alonso, P.L. et al. (1994) Randomised trial of efficacy of SPf66 vaccine against Plasmodium falciparum malaria in children in southern Tanzania. Lancet 344, 1175– 1181 Beck, H-P. et al. (1997) Analysis of multiple Plasmodium falciparum infections in Tanzanian children during the phase III trial of the malaria vaccine SPf66. J. Infect. Dis. 175, 921 – 926 Haywood, M. et al. (1999) Reduction in the mean number of Plasmodium falciparum genotypes in Gambian children immunized with the malaria vaccine SPf66. Trans. R. Soc. Trop. Med. Hyg. 93 (Suppl 1), 65– 68 Doumbo, O.K. et al. (2000) Rapid selection of Plasmodium falciparum dihydrofolate reductase mutants by pyrimethamine prophylaxis. J. Infect. Dis. 182, 993– 996 Bojang, K.A. et al. (2001) Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomised trial. Lancet 358, 1927– 1934 Gandon, S. et al. (2001) Imperfect vaccines and the evolution of pathogen virulence. Nature 414, 751 – 756 Babiker, H. et al. (1994) Genetic evidence that RI chloroquine resistance of Plasmodium falciparum is caused by recrudescence of resistant parasites. Trans. R. Soc. Trop. Med. Hyg. 88, 328 – 331 Al Yaman, F. et al. (1997) Evidence that recurrent Plasmodium falciparum infection is caused by recrudescence of resistant parasites. Am. J. Trop. Med. Hyg. 56, 436– 439 Ranford-Cartwright, L.C. et al. (1997) Molecular analysis of recrudescent parasites in a Plasmodium falciparum drug efficacy trial in Gabon. Trans. R. Soc. Trop. Med. Hyg. 91, 719 – 724 von Seidlein, L. et al. (1997) Treatment of African children with uncomplicated falciparum malaria with a new antimalarial drug, CGP 56697. J. Infect. Dis. 176, 1113 – 1116 von Seidlein, L. et al. (1998) A randomized controlled trial of artemether/benflumetol, a new antimalarial and pyrimethamine/ sulfadoxine in the treatment of uncomplicated falciparum malaria in African children. Am. J. Trop. Med. Hyg. 58, 638 – 644 Irion, A. et al. (1998) Distinction of recrudescences from new infections by PCR-RFLP analysis in a comparative trial of CGP 56 697 and chloroquine in Tanzanian children. Trop. Med. Int. Health 3, 490 – 497 Snounou, G. and Beck, H-P. (1998) The use of PCR genotyping in the assessment of recrudescence or reinfection after antimalarial drug treatment. Parasitol. Today 14, 462 – 467
1471-4922/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(02)00066-1
Invasion of skin by schistosome cercariae: some neglected facts Rachel S. Curwen and R. Alan Wilson Department of Biology, Area 5, PO Box 373, University of York, York, UK, YO10 5YW
The process of skin invasion by Schistosoma cercariae was reviewed in a recent Trends Research Update, accompanied by a computer animation. Some aspects of that article were misleading and perpetuated Corresponding author: Rachel S. Curwen ([email protected]). http://trepar.trends.com
misconceptions about parasite migration through the skin that should by now have been dispelled. This article sets out a different interpretation of events, taking account of the extensive data on migration and larval structural changes that have been documented for Schistosoma mansoni over the past 20 years.
64
Update
TRENDS in Parasitology
Schistosome cercariae, newly emerged from the snail host, swim tail-first with alternate bursts of active upward movement and passive downward sinking in which the furcae on the tail behaves like a parachute (see the animation associated with Ref. [1] at http://archive.bmn. com/supp/part/part0502.html). This behaviour takes the cercariae to the water surface, where their rhythmic sinking and swimming persists for some hours until glycogen reserves have been depleted. It appears to be a ‘sit and wait’ strategy to conserve energy reserves against the unpredictable appearance of the host [2]. Host location The environmental stimuli to which the cercaria responds are precisely those that a human wading through water would generate, namely turbulence, shadows and skin chemicals (N.P. Carter, DPhil. thesis, University of York, 1978). Turbulence and shadows increase the length of the swim period, with a shadow as brief as three seconds resulting in the cercaria swimming parallel to the water surface, but still tail-first. Skin chemicals have the most potent effect on cercarial behaviour, causing continuous swimming with frequent reversals of direction, which is achieved by altering the angle at which the tail furcae are held. In all the above, there is no indication of a directed movement (taxis) towards the host. Rather, the increased swimming activity (kinesis) with a strong, random lateral component enhances the probability of contact with host skin. If the stimuli cease without host contact, the behaviour of the cercaria reverts to the normal up and down rhythm. Obstacles to invasion To infect the host, the cercaria must traverse the stratified epithelium of the epidermis, then the dermis, to exit the skin via a blood or lymphatic vessel. It is not clear whether the cercaria forces its way through the epidermal layers by lysing cells, or insinuates itself between cells by digesting the extracellular matrix (ECM). There is some debris in penetration tunnels, but cells in proximity to the parasite are compressed, implying that insinuation predominates over cytolysis [3]. If this is the case, the multi-lamellar lipid complex that fills the intercellular spaces between the squames of the stratum corneum is the first barrier. The larva then negotiates the living keratinocyte layer which is held together primarily by the adherens junctions and desmosomes between adjacent cells [4]. The principal macromolecules of this ECM are various members of the cadherin family, while elastin is absent. The final barrier to dermal entry is the epidermal basement membrane, the lamina densa of which contains two forms of collagen IV and a third form, the more unusual collagen VII [5]. Anchoring filaments, which comprise laminin 5 trimers, form the link between basal keratinocytes and collagen VII in the lamina densa. The principal obstacles in the dermis itself are the collagen, elastin and reticulin fibres, originating in fibroblasts. The timing of events in skin migration Skin excision experiments have provided a quantitative definition of the kinetics of migration [6]. They revealed http://trepar.trends.com
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that virtually no parasites leave the skin before 48 h in mouse, hamster or rat, with the time for half to exit estimated as 88 h, 65 h and 70 h, respectively [6]. Autoradiographic tracking of 75Se-labelled parasites [2], which confirmed the data for duration of stay in the skin and the time of first arrival in the lungs of mice and rats, and provided similar information for baboons [7], suggesting that the estimates derived from rodents are relevant to primate hosts. Balance sheets, drawn up for the total numbers of parasites in all mouse organs over time, revealed that invading parasites suffer little mortality in the skin, with the vast majority (. 90%) of penetrating larvae travelling to the lungs [8]. The precise compartments in which the parasites reside at any given time have been identified by histology and electron microscopy of, for example, the infected hamster cheek pouch [3,9]. The epidermal basement membrane is reached within 30 min by the majority of penetrating cercariae, but they are arrested there, with some deflected along the basement membrane for a short distance, emphasizing its status as a barrier. The larvae seem to have no problem in disrupting the keratinocyte tethers, so that the epidermis lifts away to form ‘blisters’ in which they lie, but they are stopped by the lamina densa [3]. The early and prolonged larval retardation, external to the dermis, suggests that either a molecular signal is perceived to suspend migration, or one or more components of the laminin and collagen networks in the basement membrane cannot be hydrolyzed by acetabular gland secretions. Data from cheek pouch migration [9] indicate a minimum stay in the epidermis of 40 h and a mean time of entry to the dermis of 52.5 h. Exceptionally, some parasites can reach the dermis in shorter times following exposure to biologically unrealistic numbers of cercariae (. 1000 per cm2 [10]). Once in the dermis, a further 10 h are required to locate a dermal venule and 8.5 h to penetrate its wall to exit the skin. Secretory sources The cercaria has three types of gland cell which contain pre-formed secretory vesicles: (1) the acetabular glands; (2) the head gland; and (3) the sub-tegumental cell bodies [11], each of which plays a role in host invasion or parasite transformation. Several proteases with broad substrate specificities have been reported in the pre- and postacetabular gland secretions [12]. Of these, a serine protease, conventionally referred to as cercarial elastase [13], is considered the most important [14]. The acetabular gland contents are observed to be exhausted as soon as 1 – 2 h after penetration commences [15,16], and are certainly absent at 40 h [3] (Fig. 1a,b). Given the timing of passage through the skin layers described above, degeneration of the acetabular glands is complete before entry into the dermis occurs, and long before vascular entry (c.f. [1]). Their contents could not therefore be involved in digestion of the basement membrane ‘barrier’ or any subsequent migratory event (c.f. [1]). So, how does the parasite breach the basement membrane, cross the dermis and penetrate a venule wall? Strong morphological evidence points to the head gland as the source of any hydrolytic activity used [3]. It has been suggested that the head gland is also empty
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schistosomula (Fig. 1c,d). Its dense granular secretions are released from the schistosomular apical region when pressed against the basement membrane and venule wall. In addition, the muscular head capsule remains until the larva is at least five days old, potentially assisting the spined apical region to act as a ‘battering ram’ to cause mechanical disruption. It should also be noted that elastase-like protease activity has been eluted from schistosomula surface membranes [19], and could potentially participate in dermal migration. When is an elastase not an elastase? It is paradoxical that the supply of a protease seemingly capable of degrading dermal elastin is exhausted before penetration into the dermis. Is the readily demonstrable elastinolytic activity of acetabular gland secretions [18] a relic of former function? Alternatively, because there is evidence that the cleavage sites of the cercarial protease differ from those of other elastases [20], are its intended substrates the cells or ECM of the epidermis, and its classification as an elastase an unfortunate misnomer? Nevertheless, schistosomula lacking acetabular glands can disrupt the basement membrane and venule wall, and another, as yet uncharacterized, protease potentially emanating from the head gland could be responsible. Metalloprotease activity has already been noted in secretions from maturing schistosomula after the first 24 h, but was not tested for its ability to cleave all basement membrane components [21]. Characterization of head gland secretions should resolve these issues. Acknowledgements We thank Patricia Coulson for critical reading of the article. Our work received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. R.S.C. is a BBSRC CASE graduate student with the Natural History Museum, London, UK.
References
Fig. 1. Secretory sources in Schistosoma. Parasites stained with cell-tracker green (Molecular Probes) to reveal the presence of the acetabular glands in a schistosome cercaria (a) and their absence from an ex vivo schistosomulum 24 h after cercarial penetration (b). Electron micrographs of: (c) a 40 h schistosomulum in the epidermis (E), lying external to the basement membrane (BM), with the head gland (HG) containing abundant secretory vesicles; (d) a 72 h schistosomulum with its apical area (A) pressed against a small venule (VN). The muscular head capsule (HC) is evident. Arrows point to fibrin and platelets in the dermis. The inset shows a secretory vesicle (OG) fusing with a tegumental pit. Electron micrographs courtesy of Jean Crabtree (St James University Hospital, Leeds, UK). Scale bars ¼ 20 mm (a,b); 5 mm (c,d); 0.5 mm [inset in (d)].
one hour after skin penetration commences and that its contents are used for repair of the tegument [17]. However, the head gland is clearly visible both in vitro (R.S. Curwen, unpublished) and in vivo [3] in three-day-old, and older, http://trepar.trends.com
1 McKerrow, J.H. and Salter, J. (2002) Invasion of skin by Schistosoma cercariae. Trends Parasitol. 18, 193– 195 2 Wilson, R.A. (1987) Cercariae to liver worms: development and migration in the mammalian host. The Biology of Schistosomes: from Genes to Latrines (Rollinson, D., Simpson, A. eds), pp. 115 – 146, Academic Press Ltd 3 Crabtree, J.E. and Wilson, R.A. (1985) Schistosoma mansoni: an ultrastructural examination of skin migration in the hamster cheek pouch. Parasitology 91, 111 – 120 4 Cozzani, E. et al. (2001) Adhesion molecules in keratinocyte. Clin. Dermatol. 19, 544 – 550 5 Ghohestani, R.F. et al. (2001) Molecular organization of the cutaneous basement membrane zone. Clin. Dermatol. 19, 551– 562 6 Miller, P. and Wilson, R. (1978) Migration of the schistosomula of Schistosoma mansoni from skin to lungs. Parasitology 77, 281 – 302 7 Wilson, R.A. et al. (1990) Schistosome migration in primates: a study in the olive baboon (Papio anubis ). Trans. R. Soc. Trop. Med. Hyg. 84, 80 – 83 8 Wilson, R.A. et al. (1986) Migration of the schistosomula of Schistosoma mansoni in mice vaccinated with radiation-attenuated cercariae, and normal mice: an attempt to identify the timing and site of parasite death. Parasitology 92, 101 – 116 9 Wilson, R.A. and Lawson, J.R. (1980) An examination of the skin phase of schistosome migration using a hamster cheek pouch preparation. Parasitology 80, 257 – 266 10 Wheater, P.R. and Wilson, R.A. (1979) Schistosoma mansoni: a
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histological study of migration in the laboratory mouse. Parasitology 79, 49 – 62 Dorsey, C.H. et al. (2002) Ultrastructure of the Schistosoma mansoni cercaria. Micron 33, 279 – 323 McKerrow, J. and Doenhoff, M. (1988) Schistosome proteases. Parasitol. Today 4, 334 – 340 Newport, G.R. et al. (1988) Cloning of the proteinase that facilitates infection by schistosome parasites. J. Biol. Chem. 263, 13179 – 13184 Salter, J.P. et al. (2000) Schistosome invasion of human skin and degradation of dermal elastin are mediated by a single serine protease. J. Biol. Chem. 275, 38667 – 38673 Brink, L.H. et al. (1977) Schistosoma mansoni: a comparative study of artificially transformed schistosomula and schistosomula recovered after cercarial penetration of isolated skin. Parasitology 74, 73 – 86 Cousin, C.E. et al. (1981) Schistosoma mansoni: ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules. Exp. Parasitol. 51, 341 – 365
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17 Dorsey, C.H. (1976) Schistosoma mansoni: description of the head gland of cercariae and schistosomules at the ultrastructural level. Exp. Parasitol. 39, 444– 459 18 McKerrow, J.H. et al. (1983) Degradation of extracellular matrix by larvae of Schistosoma mansoni. I. Degradation by cercariae as a model for initial parasite invasion of host. Lab. Invest. 49, 195– 200 19 Ghendler, Y. et al. (1996) Schistosoma mansoni: evidence for a 28-kDa membrane-anchored protease on schistosomula. Exp. Parasitol. 83, 73 – 82 20 Salter, J.P. et al. (2002) Cercarial elastase is encoded by a functionally conserved gene family across multiple species of schistosomes. J. Biol. Chem. 277, 24618 – 24624 21 Auriault, C. et al. (1981) Proteolytic cleavage of IgG bound to the Fc receptor of Schistosoma mansoni schistosomula. Parasite Immunol. 3, 33 – 44 1471-4922/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(02)00019-3
Invasion of skin by schistosome cercariae: some neglected facts Response from James J. McKerrow
James J. McKerrow Sandler Center for Basic Research in Parasitic Diseases, University of California, Box 0511 HSW 511, San Francisco, CA 94148, USA
In response to the Research Focus article by Rachel Curwen and Alan Wilson, some of the issues concerning invasion of skin by schistosome cercariae are addressed or clarified. The conclusions drawn by Curwen and Wilson concerning the kinetics of invasion and the exhaustion of schistosome acetabular glands are incorrect because they are based on rodent models and not on direct assessment of cercariae of human parasites that invade human skin. It is reassuring that despite the dwindling number of laboratories studying schistosome biology and the decreasing funding support for this field, enough interest remains to engender debate. That said, I must address some of the misunderstandings that are referred to as ‘perpetuated misconceptions’ by Rachel Curwen and Alan Wilson. Host location In our article [1] and animation (http://archive.bmn.com/ supp/part/part0502.html), we could not detail each step in cercarial invasion. Indeed, Curwen and Wilson are correct in providing more detail on the tail-first movement of cercariae, and the active upward and passive downward movements of cercariae in the absence of hosts. We noted in our article that: ‘Specific host signals recognized by schistosomes vary with specific host– parasite interactions’; however, Curwen and Wilson have fallen into a Corresponding author: James J. McKerrow ([email protected]). http://trepar.trends.com
trap of lumping turbulence, shadows and skin chemicals as the key stimuli for all schistosome cercariae. Haas et al. have provided the most detailed analysis of swimming and tactic stimuli [2]. In their article, Table 1 outlines how shadows are important for Trichobilharzia cercariae to find ducks, water turbulence is particularly important for Diplostonum to find fish and chemical stimuli help Isthmiophora find amphibia [2]. However, not all of these stimuli translate to human Schistosoma. There are even significant differences in the responses of Schistosoma mansoni and Schistosoma haematobium to stimuli. Stirewalt [3] reviewed simple biological experiments showing that S. mansoni cercariae follow a thermal gradient (thermotaxis), and we have confirmed those results. Haas has also indicated the importance of a thermal stimulus to cercarial attachment. However, like Haas, we were unable to demonstrate chemotactic behavior in S. mansoni cercariae. Curwen and Wilson are right to point out that continuous swimming of cercariae will increase the probability of finding a host, but thermotactic behavior in both host finding and host attachment has not been ruled out. To date, no intrepid parasitologist has followed cercariae with an underwater camera to ensure that they do not move towards a warm, immersed limb. Obstacles to invasion We agree that cercarial penetration through the epidermis involves degradation of extracellular adhesion molecules such as the cadherin family of proteins.