Cryptosporidium virulence determinants – are we there yet?

International Journal for Parasitology 32 (2002) 517–525 www.parasitology-online.com

Invited Review

Cryptosporidium virulence determinants – are we there yet? q Pablo C. Okhuysen*, Cynthia L. Chappell Department of Medicine, Division of Infectious Diseases and The School of Public Health, The University of Texas Health Science Center at Houston Medical School, 6431 Fannin, 1.728 JFB, Houston, TX 77030, USA Received 12 July 2001; received in revised form 31 August 2001; accepted 10 September 2001

Abstract Exposure to Cryptosporidium parvum in healthy individuals results in transient infection that may be asymptomatic or can result in selflimited diarrhoea. In contrast, acquired immune deficiency syndrome patients with cryptosporidiosis can experience severe manifestations of disease. Volunteer studies have demonstrated that as few as 10 oocysts can cause infection in otherwise healthy adults and that isolates from geographically diverse regions differ in infectivity and, perhaps, virulence. Variability in isolate pathogenicity and infectivity has also been seen in bovine and murine models, respectively. Furthermore, isolate specific differences in protein composition and in host immunoreactivity have been observed. The molecular basis for differences in pathogenicity is not understood. Determining which factors are responsible for host selectivity and for the initiation, establishment, and perpetuation of infection with Cryptosporidium is key to rational drug design and vaccine development. To date, no specific virulence factors have been unequivocally shown to individually cause direct or indirect damage to host tissues nor have mutant strains been produced that could prove that particular deletions result in less virulent strains. Nevertheless, a number of candidate molecules have been identified by immunological and molecular methods. Here, we review the salient characteristics of some of these putative virulence determinants, including molecules that are involved in adhesion, protein degradation and the modulation of the host responses. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Cryptosporidium; Virulence; Diarrhoea

1. Cryptosporidium biology Cryptosporidium is a protozoan parasite that is resistant to disinfectants commonly used for the treatment of drinking water and is highly infectious to adults and children (Guerrant et al., 1990). After ingesting Cryptosporidium parvum, immunocompetent humans can experience asymptomatic infection or self-limited diarrhoea (DuPont et al., 1995). However, those with defects in innate (Summerfield et al., 1995; Kelly et al., 2000), humoral (Levy et al., 1997) or cellular immunity (Gomez Morales et al., 1996) can experience severe or prolonged illness. For instance, patients with advanced human immunodeficiency virus (HIV) infection and acquired immune deficiency syndrome (AIDS) can experience chronic diarrhoea that frequently leads to wasting and eventually death (Navin et al., 1999). Cryptosporidium survives prolonged periods of time in the environment as an oocyst (Robertson et al., 1992). Once q Informed consent was obtained from all participating volunteers. This study was approved by The University of Texas, Houston Health Science Center Committee for the Protection of Human Subjects. * Corresponding author. Tel.: 11-713-500-6736; fax: 11-713-500-5495. E-mail address: [email protected] (P.C. Okhuysen).

ingested by a susceptible host, the oocysts undergo excystation and release infective sporozoites. Cryptosporidium sporozoites possess an apical complex composed of micronemes, a single rhoptry (Tetley et al., 1998) and dense granules. These secretory organelles are also present in closely related parasites such as Toxoplasma, Plasmodium and Eimeria. Typically, four sporozoites are released from an oocyst and, as in the case of other Apicomplexa, glide over the intestinal epithelial cell releasing material from the apical complex, which presumably aids in attaching to and fusing with the host cell membrane. After penetration and fusion, a parasitophorous vacuole is formed within the host membrane. This unique niche places the parasite in an intracellular, but extracytoplasmic space where zoites undergo further development. This specialised vacoule provides an environment that protects the parasite from direct damage by the host immune response and limits the penetration of anti-parasitic agents. An electron dense region known as the feeding organelle is located at the basal margin of this vacuole. As parasites mature in the vacuole, merozoites are formed and then released to infect new cells. After subsequent asexual replication cycles, a small proportion of parasites differentiate into sexual stages, resulting in

0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(01)00356-3

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the formation of micro- and macrogametes that eventually fuse and form zygotes and new oocysts. Traditionally, Cryptosporidium species were classified by their ability to cause infection in their preferred hosts. For instance, natural infection and cross-infectivity studies showed that C. parvum infected only humans and other mammals; whereas, Cryptosporidium serpentis preferentially infected snakes, and Cryptosporidium muris preferentially infected mice. Such limitations in host range provided a phenotypic classification that is now being redefined based on population genetics research. The application of molecular genetic analysis tools, such as restriction fragment length polymorphisms, genotype specific polymerase chain reaction (PCR), and microsatellite analysis has resulted not only in the confirmation of these known species, but also in the description of distinct genotypes within a species (Xiao et al., 1998). For example, C. parvum is now recognised to be composed of a zoonotic genotype that can infect humans and other animals (also known as type 2) and a separate genotype apparently restricted to causing infection in humans (also known as type 1) (Peng et al., 1997; Morgan et al., 1998; Xiao et al., 1998). Detailed analysis of individual isolates has demonstrated a wide diversity of polymorphisms within these last two genotypes (Feng et al., 2000; Strong et al., 2000). Additionally, species not previously recognised as pathogenic for humans are now being recognised as causative agents of diarrhoea in children (Xiao et al., 2001) and in patients who are immunocompromised (Pieniazek et al., 1999). This is evidenced by a study recently conducted in Peru where children were studied for the presence of C. parvum species and genotypes by PCR. Analysis of 85 infection episodes in 80 children demonstrated that five types of Cryptosporidium accounted for the majority of the infections. Cryptosporidium parvum type 1 (human) accounted for 79% of the episodes, while type 2 (bovine or canine) accounted for 12% of episodes. A minority of episodes were due to Cryptosporidium meleagridis (8%), and Cryptosporidium felis (1%). Of particular interest, only 29% of the 85 infection episodes were associated with diarrhoea. There was no significant difference in age, antecedent stunting, percentage with diarrhoea, or duration of diarrhoea for episodes between type 1 and type 2 Cryptosporidium. However, the duration of oocyst shedding was longer for type 1 versus type 2 infections (mean, 13.9 days and 6.4 days, respectively; P ¼ 0:004) (Xiao et al., 2001).

It has long been known that animals and humans with cryptosporidiosis have variable expression of disease severity and immune responses. While some of these variations in the expression of disease may be due to differences in host susceptibility to infection, these variations may also be due to intrinsic diversity in isolate pathogenicity. Observations supporting differences in isolate pathogenicity have been made in otherwise healthy volunteers and in patients with AIDS. In the latter, cryptosporidiosis results in diverse manifestations (Hashmey et al., 1997) and fulminant infections occur that often end in death. Previous studies also demonstrated that diverse isolates differ in their ability to cause infection in calves (Pozio et al., 1992) and in mice. Furthermore, others have observed isolate specific differences in protein composition (Mead et al., 1990), and in host immunoreactivity (Lumb et al., 1988; Ditrich et al., 1993). It is unknown if these differences are associated with genotypic differences, since these studies were done prior to the availability of tools that allowed for the identification of the different genotypes. The infectivity of C. parvum has been studied in healthy adults in order to develop risk assessment models for drinking water standards. Initial studies were conducted in healthy volunteers using the C. parvum Iowa isolate (an isolate that belongs to type 2) at challenge doses ranging from 30 to 10 6 oocysts (DuPont et al., 1995). These subjects were negative for C. parvum antibodies as determined by an enzyme linked immunosorbent assay (ELISA) to a whole oocyst extract. The estimated ID50 for that particular isolate was approximately 132 oocysts as calculated by a simple linear regression model. This initial data confirmed the epidemiological observation that a low inoculum was sufficient to cause infection in humans. As a continuation of that challenge study, groups of healthy volunteers were exposed to three geographically distinct isolates. The isolates included oocysts that were originally collected in Ames, Iowa from a calf (Iowa), Maine from a calf (UCP), and Texas from a horse (TAMU) (Okhuysen et al., 1999). In this study, the ID50 for humans was determined using the method of Reed and Muench (1938) and for statistical comparisons the data were analysed by linear regression fit on the basis of least-squares estimation. The ID50 differed among isolates (Table 1), as did the attack rate. These clinical observations suggested that isolates belonging to the same genotype and obtained from different geographical sites differed in their infectivity and perhaps in pathogeni-

Table 1 Clinical outcomes of volunteers exposed to four distinct isolates of Cryptosporidium parvum (type 2) a Isolate

Type

Animal source

ID50 in volunteers

Illness attack rate (%)

Mean duration of diarrhoea, h ^ SD (range)

UCP Iowa TAMU

2 2 2

Bovine Bovine Equine

1042 87 9

59 52 86*

81.6 ^ 59.68 (6–193) 64.2 ^ 67.43 (6–223) 94.5 ^ 66.7 (6–195)

a

Modified from Okhuysen et al. (1999). *P , 0:05 TAMU vs. UCP or IOWA.

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city. Interestingly, in volunteer studies, the duration and severity of diarrhoea was milder compared with what has been seen in natural infections that occur sporadically in the community or from outbreaks. The reasons for a milder expression of disease are unclear. It is possible that this selected group of healthy volunteers controlled cryptosporidiosis better than the population in general or that repeated passage in calves decreased the virulence of a given isolate. Thus, a number of factors are likely to contribute to the pathogenicity of Cryptosporidium and virulence determinants should be evaluated in the context of the different Cryptosporidium species, genotypes, and, potentially, polymorphisms within a genotype as well as the immune status of the host. 2. Cryptosporidium virulence determinants Virulence determinants are the factors present in a microorganism that are responsible for the relative capacity of a parasite to cause damage in a host. As with any other infectious agent, it is important to determine which factors are responsible for the initiation, establishment and perpetuation of C. parvum infection. As previously mentioned, C. parvum is a relatively non-invasive parasite that establishes itself in a membrane-bound compartment on the apical surface of the intestinal epithelium. Nevertheless, this parasite causes significant abnormalities in the absorptive and secretory functions of the gut. This epithelial damage likely results from direct injury to the host epithelial cell or indirectly through an effect that parasite derived molecules can

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have on recruiting inflammatory cells and cytokines to the site of infection. Inflammation and associated cytokines may, in fact, be important in controlling or eradicating the parasite (Laurent et al., 1997). Direct tissue damage by the parasite and the influx of inflammatory cells is likely responsible for morphologic and biochemical/functional abnormalities that result in diarrhoea. To date Cryptosporidium specific virulence factors have not been characterised to the point of unequivocally establishing specific roles in causing damage or proving that deletions result in less virulent strains. Nevertheless, a number of candidate molecules have been identified by immunological and molecular methods (Table 2). The identification and characterisation of such virulence determinants will allow advances in the understanding of the pathogenesis and contribute to the development of pharmacological and immunological therapeutic approaches. In this review, we present the salient characteristics of some of these putative virulence determinants. 3. Adherence factors A critical initial step in establishing infection is parasite attachment to host cells. As with other parasites, lectins are necessary for initiation of Cryptosporidium attachment, and competition with soluble lectins can partially block infection (reviewed in Gut and Nelson, 1999). Recently, several molecules thought to mediate adhesion have been characterised, including CSL (for circumsporozoite-like) (Riggs et al., 1997), gp900 (Barnes et al., 1998; Petersen et al., 1997),

Table 2 Summary of the putative virulence factors discussed in this review Virulence factors

Putative or defined role

References

CSL

Adhesion

Riggs et al., 1997; Langer et al., 2001

gp900

Adhesion

Gp60/40/15

Adhesion

p23

Adhesion, locomotion

TRAP C-1 cp47 CPS-500 Haemolysin H4

Adhesion? locomotion? Adhesion Adhesion, locomotion Membrane disruption?

ATP binding cassette

Transport

Cysteine protease Serine protease Aminopeptidase HSP70

Unknown Excystation Excystation Heat shock protein

HSP90

Heat shock protein

Comments

Specific antibodies to result in the formation of a circumsporozoite-like reaction and block infection. Affinity for an 85 kDa epithelial cell receptor (CSL-R) Petersen et al., 1992 Heavy glycosylation accounts for high MW(r), an unusual feature in Apicomplexa. mAbs block infection Cevallos et al., 2000a,b; Strong et al., 2000 Shares epitopes with gp900. Undergoes proteolytic cleavage presumably on the surface of sporozoites. Shed in trails during locomotion Arrowood et al., 1991; Perryman et al., 1996 Deposited in trails. Specific humoral and cellular responses to p23 detectable after natural infection Spano et al., 1998 Orthologs in Toxoplasma, Plasmodium, Eimeria Nesterenko et al., 1999 Affinity for a 57 kDa membrane receptor Riggs et al., 1989; Bjorneby et al., 1990 Polar glycolipid Steele et al., 1995 Homology to the haemolysin of enterohemorrhagic Escherichia coli O157 H7 Perkins et al., 1999 Homology to MRP 1 and the cystic fibrosis conductance regulator Nesterenko et al., 1995 Localised on the surface of sporozoites Forney et al., 1996a,b,c Specific inhibitors block excystation and infection in vitro Okhuysen et al., 1994 Specific inhibitors block excystation Khramtsov et al., 1995 Considerable polymorphisms. In the closely related parasite Toxoplasma, quantitative and qualitative differences in expression of HSP correlate with virulence Woods and co-workers, 1999

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the gp15/40/60 complex (Cevallos et al., 2000a,b; Strong et al., 2000), TRAPC-1 (for thrombospondin related adhesion protein) (Spano et al., 1998), cp47 (Nesterenko et al., 1999) and Cps 500 (Riggs et al., 1999). When incubated in the presence of hyperimmune bovine calostral antibodies, sporozoites undergo morphological changes characterised by the formation and eventual release of membranous sporozoite surface antigen–antibody complexes similar to the malaria circumsporozoite precipitate reaction (Riggs et al., 1994). A monoclonal antiboby (mAb) (3E2) also produces a CSL reaction in vitro. mAb 3E2 recognises a carbohydrate epitope on a protein denominated CSL of approximately 1300 kDa (Riggs et al., 1997). CSL is found associated with the apical complex of sporozoites and merozoites (Langer and Riggs, 1999; Schaefer et al., 2000). In vitro, mAb 3E2 prevents infection of intestinal cells in culture and can passively protect mice against infection. In an effort to characterise a receptor for this protein, the permissiveness of a battery of cell lines for infection with C. parvum and the cells ability to bind CSL was investigated by Langer et al. Epithelial cell lines demonstrate a higher permissiveness to infection and bind more CSL than mesenchymal cell lines. Using affinity chromatography and radio-immunoprecipitation, an 85 kDa epithelial cell receptor (CSL-R) has been identified. Infection of Caco 2 intestinal epithelial cells is inhibited when sporozoites were preincubated with soluble CSL-R or when intestinal cells were pre-treated with CSL. In addition to cultured intestinal epithelial cells, CSL-R is present in the intestinal tract of calves (Langer et al., 2001). Further characterisation of the nature of CSL-R should assist in determining the molecular basis for host susceptibility, the preferential selection for infection of the distal ileum over other segments of the gastrointestinal tract as well to provide insights on how the parasite is internalised. A large glycoprotein termed gp900 was identified by immunoprecipitation of sporozoite extracts with hyperimmune bovine colostrum (Petersen et al., 1992). This large mucin-like glycoprotein is composed of a large proximal core region with type N-linked oligosaccharides, a distal region with cysteine-rich domains that in turn are separated by polythreonine domains. gp900 is stored in the parasite’s micronemes and is shed to the surface of the sporozoite during the infection process. Specific antibodies to gp900 can competitively inhibit infection in vitro (Petersen et al., 1997; Barnes et al., 1998). In enterocyte tissue culture systems, native gp900 can competitively inhibit infection in vitro in micromolar concentrations; whereas, recombinant polypeptides containing the cysteine-rich domains can also competitively inhibit infection in the nanomolar range. Studies have shown that an mAb to gp900 termed 4E9 recognises antigenic epitopes present in the sporozoite as well as in intracellular stages. This mAb has aided in demonstrating that gp900 shares an alpha-N-galactosamine containing epitope with a separate glycoprotein called gp40. The genes for both of these proteins contain sequences for

mucin-like domains. gp40 in turn is thought to be the proteolytic product of a larger molecule called gp60 that when cleaved releases gp40 and gp15. gp40 has also been found to be of importance in cell attachment (Cevallos et al., 2000a,b; Strong et al., 2000) and shares features in common with glycoproteins from other Apicomplexa, including expression during the invasive stages of the parasite, localisation on the surface or the apical complex, and shedding from the parasite in trails as the parasites glide over the surface of the enterocyte. Furthermore, specific inhibition of infection has been achieved with the use of mAbs or with competing lectins, making this protein another candidate for prevention and or treatment strategies. Cryptosporidium p23 is a 23 kDa sporozoite surface protein that is antigenically conserved across geographically diverse isolates (Perryman et al., 1996) and is also deposited in trails during the initial stages of infection (Arrowood et al., 1991). p23 contains two distinct neutralisation sensitive epitopes defined by mAbs C6B6 and 7D10, which react with a linear, repeated epitope and a non-linear epitope, respectively. Both antibodies reduce the infection of mice by C. parvum. Murine IgA mAbs derived from payer patches of mice with cryptosporidiosis also react with p23 and have been shown to be protective (Enriquez and Riggs, 1998). Inoculation with recombinant p23 results in the generation of protective calostral antibodies (Perryman et al., 1999). Humoral and cellular immune responses to p23 can be detected during natural infection in animals and in humans (Smith et al., 2001; Wyatt et al., 2001) and in the case of calves elicits a TH1 type immune response (Wyatt et al., 2001). A Cryptosporidium micronemal protein named TRAP-C1 has recently been cloned. TRAP-C1 contains type I repeats that are characteristic of the human thrombospondin (TSP) gene (Spano et al., 1998). These TSP type I repeats have also been described in F-spondin, properdin and several protein members of the complement cascade. TRAP-C1 orthologues have been identified in Toxoplasma (MIC2), Plasmodium falciparum (pfTRAP) and Eimeria tenella (EtMIC-1 and EtMIC-4). Toxoplasma’s MIC 2 is secreted from the micronemes in bursts and in concert with other micronemal proteins is of importance for locomotion and cell attachment/invasion. This has been the subject of a recent review (Soldati et al., 2001). A second protein, the sporozoite cysteine-rich protein (SCRP) also has TSP type I motifs as well as an epidermal growth factor domain and is thought to mediate adhesion (Spano, personal communication). Eimeria tenella secretes an additional cysteine-rich micronemal protein (EtMIC5) that shows homology to the adhesive Apple (A-) domains of coagulation factor XI and plasma pre-kallikrein (Brown et al., 2000; Ryan et al., 2000). Homologues have also been identified in Sarcocystis and Toxoplasma gondii, which suggests that this cysteinerich class of proteins may be found in all Apicomplexans. Another molecule that is localised in the apical complex of importance in attachment to host cells is cp47. This novel

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protein with affinity to ileal cells of animal and human origin was purified by anion-exchange chromatography on FPLC and immunoaffinity chromatography. Purified cp47 has been shown to bind to a 57 kDa host cell membrane protein and to competitively inhibit the binding of sporozoites to ileal cells. Of interest the binding between cp47 and the 57 kDa ileal protein was abrogated in the presence of Mn 21 (Nesterenko et al., 1999). An additional pellicle antigen called CPS-500 is included in this review because it is also involved in cell attachment, is localised to the pellicle, is shed during gliding motility and has been identified in sporozoites and merozoites (Riggs et al., 1989; Bjorneby et al., 1990) of human and bovine origin (Uhl et al., 1992). CPS-500 was originally identified by mAb 18.44 and contains one or more neutralisation sensitive epitopes (Riggs et al., 1999). mAb 18.44 can inhibit C. parvum infection in vitro in a time-dependent fashion as well as in mice when co-administered with other monoclonal neutralising antibodies (Perryman et al., 1990). Biochemical characterisation studies have revealed that CPS-500 is of non-proteinaceous nature but is rather a polar glycolipid. The neutralisation epitope recognised by mAb 18.44 is dependent on terminal b-d-mannopyranosyl residues. Glycosyl analysis identified mannose and inositol as the predominant residues. Two observations make this molecule of particular importance. First is the fact that mammals lack terminal b-d-mannopyranosyl residues, making it a parasite specific therapeutic target and second is the fact that humans recognise multiple mannose residues and N-acetyl glucosamine as important components of innate immunity via a lectin known as mannose binding protein (MBP). Individuals with mutations in the MBP gene are susceptible to prolonged episodes of intestinal infections including cryptosporidiosis (Summerfield et al., 1995). A recent report demonstrated that MBP can be found in duodenal aspirates of AIDS patients and that purified MBP binds to C. parvum sporozoites and not to oocysts via a mannose residue resulting in the activation of complement (Kelly et al., 2000). In this same study, 72 patients with AIDS and diarrhoea were studied for MBP mutations. Seventeen (24%) had infection with Cryptosporidium, 21 (29%) with Isospora belli, and 23 (32%) had microsporidia identified in their stools. Four of six patients with homozygote or compound heterozygote mutations were found to have cryptosporidiosis, whereas only 13 of 66 patients who were heterozygotes or had the wild type gene were found to have cryptosporidiosis (odds ratio of 8.2, 95% confidence intervals, 1.5–42; P ¼ 0:02). No associations were noted for patients infected with I. belli or microsporidia. From the number of molecules described above, it can be concluded that as is the case with other Apicomplexa, Cryptosporidium has a number of distinct molecules that are used during, attachment and fusion with the host cell. The interaction with specific sugar moieties on the host cells membrane may assist in countering peristaltic motion and

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preserving adherence. The shedding of membrane components may be of use in locomotion and evasion of host immunity. It is likely that by using a large number of seemingly redundant adhesive molecules the parasite can maximise the opportunity for cell attachment across a broad range of potential hosts. Conversely, it is also possible that quantitative or qualitative differences in these glycoproteins may confer selectivity for host attachment. The relative contribution that the individual molecules have in these processes remains to be determined. 4. Toxins Diarrhoea is the prominent symptom of cryptosporidiosis, but the specific mechanism by which C. parvum induces diarrhoea has not been identified. It has long been postulated that C. parvum produces an enterotoxin because of the profuse secretory diarrhoea that some patients experience even in the absence of oral intake. Several observations support this notion. In vitro models have shown that there is altered glucose-stimulated Na 1 and H20 absorption as well as increased Cl 2 secretion in tissues taken from animal models of infection (Argenzio et al., 1990,1993; Moore et al., 1995). The decrease in anion flux in the presence of cyclo-oxigenase inhibitors has led to the hypothesis that a potential mechanism of action of the enterotoxigenic activity of Cryptosporidium may actually involve the secretion of prostaglandins by the infected intestinal epithelial cells. The production of secretagogenic prostaglandins in turn could be a consequence of production of cytokines by the epithelial cells themselves, or the inflammatory cells they recruit, potential toxic effects of phospholipid-like proteins or an enterotoxic molecule that has yet to be identified. It is unlikely that the diarrhoea associated with cryptosporidiosis is solely due to an enterotoxigenic effect since structural villous atrophy and other structural abnormalities result in the malabsorption of fluids and nutrients leading to diarrhoea (Goodgame et al., 1993, 1995). 5. Cellular damage Using enterocyte monolayer cultures, cell damage has been documented by the disruption of tight cell junctions, loss of barrier function, the release of intracellular lactate dehydrogenase, and increased cell death (Adams et al., 1994). The mechanisms involved in the disruption of membranes during Cryptosporidium invasion remain unknown. Phospholipases, proteases, or haemolysins are potential molecules that can cause direct tissue damage. Two specific proteins are of particular interest as candidates that mediate invasion and loss of barrier function. The first is a haemolytic polypeptide (haemolysin H4) encoded by the hemA gene. This haemolysin was identified by screening a C. parvum expression library on sheep blood agar and selecting clones surrounded by a haemolytic halo

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(Steele et al., 1995). The function of Cryptosporidium haemolysin H4 is unknown but its ability to disrupt membranes would suggest roles in cellular invasion, or disruption of vacuolar membranes that would allow intracellular survival or for merozoites to exit the parasitophorous vacuole and spread to adjacent cells. Of interest, this protein contains sequence similarity to the plasmid-encoded haemolysin of enterohemorrhagic (EHEC) Escherichia coli 0157 H7. This E. coli haemolysin is a member of the pore forming repeat in toxin family. In bacterial systems, such as in Vibrio cholera, haemolysins belonging to this family are found in gene clusters that encode genes physically linked to the cholera toxin element. These gene clusters include activators and ATP binding cassette transporter genes (ABC). The second protein of interest is an ABC transporter gene that has also been described in C. parvum. This transporter, the C. parvum ATP binding cassette (CpABC) is localised in the electron dense feeding organelle of the parasitophorous vacuole and shares conserved protein structure features with multidrug resistance protein 1 (MRP1) and the cystic fibrosis conductance regulator (Perkins et al., 1999). The function of CpABC is unknown but its location would suggest it mediates the efflux of ions or other molecules. It is unknown if the CpABC is linked to the C. parvum hemA. The presence of these genetic elements that share structural similarity with genes that in bacterial systems are critical in producing secretory diarrhoea warrants further investigation. Analysis of upstream and downstream regions to hemA is in progress. Proteases have a number of important functions in a parasite’s life cycle. Examples of some of these functions are the degradation of nutrient proteins (Shenai et al., 2000), invasion of host tissues (Que and Reed, 2000), and evasion of host immunity (Shin et al., 2001). Thus, proteases are attractive targets for anti-parasitic agents (Rosenthal, 1998). At least three distinct types of protease activities have been identified in Cryptosporidium sporozoites, including a cysteine (Nesterenko et al., 1995) and a serine endopeptidase (Forney et al., 1996c) as well as aminopeptidase activity. The proteins responsible for the latter two activities have not been fully characterised, but have been implicated in the excystation process (Okhuysen et al., 1996; Forney et al., 1997b). A metallo-dependent proteinase originally identified by Nesterenko et al. (1995) is a 24-kDa protein that can hydrolyse azocasein, casein, bovine serum albumin (BSA), and gelatin. Biochemical studies suggest that this enzyme is a metallo-dependent cysteine proteinase. Using monospecific antibodies, the enzymatic activity has been localised to the surface of sporozoites, but is not associated with oocyst walls, the rhoptry, or micronemes. Using azocasein as a substrate, a different proteolytic activity was demonstrated by Forney in Cryptosporidium extracts obtained from partially excysted oocysts. Inhibition of proteolytic activity by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, aprotinin, and alpha-1-anti-

trypsin suggested that the measured activity belonged to a serine protease and that optimal detection peaked during excystation. Further studies demonstrated that specific inhibitors of serine proteases can indeed block excystation and also infection in vitro (Forney et al., 1996a,b,c, 1997a,b). Aminopeptidase activity has been identified in oocyst extracts and preferentially cleaves substrates containing alanine, arginine and phenylalanine in the N-terminus (Okhuysen et al., 1994). Under fluorescent microscopy, arginine aminopeptidase activity is localised on the surface of the parasite and at the apical complex region. Detergent fractionation with Triton X-114 treated sporozoite membrane extracts identified maximal activity in the insoluble phase, suggesting that the enzyme(s) responsible for this activity is(are) integral membrane protein(s). Subsequent work demonstrated that the aminopeptidase activity correlated with excystation and that traditional metalloprotease inhibitors, as well as boronic acid-based reversible inhibitors of aminopeptidases, blocked excystation (Okhuysen et al., 1996). The sequence of C. parvum aminopeptidase demonstrates striking homology to the aminopeptidase of Plasmodium and contains an RGD motif characteristic of adhesive molecules (Padda et al., submitted for publication). The identification of pre-formed functional proteases in infectious sporozoites during excystation, the prevention of infection in the presence of selected protease inhibitors and the fact that attachment molecules such as the GP60/40/ 15 undergo proteolytic processing on the surface of the sporozoite suggest that proteases are important in the initial stages of infection. 6. Apoptosis Several groups have suggested that C. parvum is cytopathic to epithelial cells directly infected but also uninfected bystander cells. Using biliary epithelium cell lines, Chen et al. (1999) have demonstrated that this cytopathic effect is mediated by apoptosis in infected cells and that noninfected cells in co-culture also experience accelerated apoptosis via a Fas–Fas ligand-dependent mechanism. This same group (Chen et al., 2001) has also demonstrated that C. parvum activates nuclear factor kB preventing epithelial cell apoptosis of infected cells. This in addition to observations by others (McCole et al., 2000) suggests that C. parvum is able to modulate cell apoptosis in order to facilitate its growth in infected cells and induce programmed cell death in surrounding uninfected cells. Widmer et al. (2000) have noted that cell apoptosis is decreased by growing cells on a base that is composed of laminin. The parasite molecules responsible for programmed cell death signalling have not been described. 7. Heat shock proteins Heat shock proteins (HSPs) are a large, ubiquitous family

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of polypeptides that have been well conserved in evolution. HSPs serve many functions including being chaperones to other proteins, binding to steroid receptors, and facilitating the transport, folding, assembly, biosynthesis and secretion of newly formed proteins. HSPs also assist in the refolding of damaged proteins. In the setting of environmental stressors (such as sudden shifts in temperature, decreased availability of certain nutrients or during immune attack), the synthesis of HSPs increases substantially. In the closely related apicomplexan T. gondii, there is evidence to suggest that quantitative and qualitative differences in the expression of HSP are directly related to parasite virulence (Lyons and Johnson, 1998; Miller et al., 2000). It has long been recognised that infection with avirulent strains of T. gondii (defined as those considered to have an LD100 (lethal dose required to kill all animals) in outbred Swiss mice of .1000 tachyzoites) results in asymptomatic brain infections and chronic asymptomatic toxoplasmosis. In contrast, a virulent strain has an LD100 of less than 10 tachyzoites and can result in the death of the mice within 7–10 days. Virulent strains of T. gondii produce less HSP70 than virulent isolates, and certain polymorphisms of this protein are directly correlated with virulence. While some of the qualitative differences are parasite derived, studies have also demonstrated that the amount of HSP produced varies according to the stress imparted by the immune competence of the host. Thus, it has been proposed that the interactions between the immune system and HSPs influence parasite virulence. Two Cryptosporidium HSPs, HSP70 and HSP90 have been identified and sequenced (Khramtsov et al., 1995; Woods and coworkers, 1999). Considerable polymorphism has been identified and has assisted in genotyping Cryptosporidium at the species level in human infections (Morgan et al., 2000). To date it is unknown if quantitative or qualitative differences in the expression of Cryptosporidium HSPs correlate with virulence.

8. Conclusions Until recently, the inability to propagate this C. parvum in vitro has limited the identification of putative virulence determinants to only a few molecules, the characterisation of which is in its most early stages. The variability in pathogenicity that the different species and types have for different hosts suggests that determinants of virulence differ among isolates in semiquantitative and/or qualitative ways. The recent report of the successful long-term maintenance of C. parvum in culture (Hijjawi et al., 2001) may be of use in the development of clonal isolates and transfectants in which the deletion of these individual components can be accomplished. This will assist in determining the relative importance that these and other factors have in parasite viability and pathogenicity. The identification of isolates or laboratory strains with impaired virulence but not with decreased viability could be exploited in the devel-

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opment of attenuated strains that could be useful as vaccines. In the mean time, genome based surveys of different Cryptosporidium genotypes and the analysis of the host responses to them will assist in further understanding the pathogenesis of cryptosporidiosis.

Acknowledgements Grant Support: US Environmental Protection Agency (#CR-819814 and #CR-824759), National Institutes of Health R01 AI 41735-01 and RR-02558. ImmuCell Corporation and The Food and Drug Administration (FDU-001621-01).

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