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3 Cryptic Parasite Revealed: Improved Prospects for Treatment and Control of Human Cryptosporidiosis Through Advanced Technologies☆ Aaron R. Jex,* Huw V. Smith,† Matthew J. Nolan,* Bronwyn E. Campbell,* Neil D. Young,* Cinzia Cantacessi,* and Robin B. Gasser*

Contents

Abstract

☆

3.1. Introduction 3.2. Cryptosporidium Species and Genotypes Known to Infect Humans 3.3. The Life Cycle of C. Parvum and C. Hominis 3.4. Cryptosporidiosis: Pathogenesis and Immunity 3.5. Genomics and Transcriptomics of Cryptosporidium 3.6. Improved Insights into Cryptosporidium Using In Vitro Techniques 3.7. Concluding Remarks Acknowledgements References

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Cryptosporidium is an important genus of parasitic protozoa of humans and other vertebrates and is a major cause of intestinal disease globally. Unlike many common causes of infectious enteritis,

This review is dedicated to the memory of our colleague and friend, Huw V. Smith.

* Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia {

Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow, United Kingdom

Advances in Parasitology, Volume 77 ISSN 0065-308X, DOI: 10.1016/B978-0-12-391429-3.00007-1

#

2011 Elsevier Ltd. All rights reserved.

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there are no widely available, effective vaccine or drug-based intervention strategies for Cryptosporidium, and control is focused mainly on prevention. This approach is particularly deficient for infections of severely immunocompromised and/or suppressed, the elderly or malnourished people. However, cryptosporidiosis also presents a significant burden on immunocompetent individuals, and can, for example have lasting effects on the physical and mental development of children infected at an early age. In the last few decades, our understanding of Cryptosporidium has expanded significantly in numerous areas, including the parasite life-cycle, the processes of excystation, cellular invasion and reproduction, and the interplay between parasite and host. Nonetheless, despite extensive research, many aspects of the biology of Cryptosporidium remain unknown, and treatment and control are challenging. Here, we review the current state of knowledge of Cryptosporidium, with a focus on major advances arising from the recently completed genome sequences of the two species of greatest relevance in humans, namely Cryptosporidium hominis and Cryptosporidium parvum. In addition, we discuss the potential of next-generation sequencing technologies, new advances in in silico analyses and progress in in vitro culturing systems to bridge these gaps and to lead toward effective treatment and control of cryptosporidiosis.

3.1. INTRODUCTION Cryptosporidium species represent a genus of faecal-orally transmitted parasitic protozoa that infect the epithelial tissues of the alimentary or respiratory tract of vertebrates (Fayer, 2004; Xiao et al., 2004). Infection occurs following the ingestion of viable and resistant oocysts (see Korich et al., 1990; Peeters et al., 1989), through direct host-to-host contact or via food or water (Caccio`, 2005; Caccio` and Pozio, 2006). In humans, cryptosporidial infection may be transmitted via anthroponotic (human-tohuman) or zoonotic (animal-to-human) pathways, depending on the species of parasite (Xiao et al., 2004). Although cryptosporidial infection can be asymptomatic (Checkley et al., 1997; Hellard et al., 2000), common clinical signs of the intestinal disease (cryptosporidiosis) include diarrhoea, abdominal pain, headache, nausea, vomiting, dehydration and/or fever (Kosek et al., 2001; Tzipori and Ward, 2002). Cryptosporidium infections are often short-lived (days to weeks: Chappell et al., 1996; Okhuysen et al., 1999) and eliminated following the stimulation of an effective host immune response (Riggs, 2002). However, in ‘high-risk’ host groups, such as neonatal or young animals, infants, the elderly and immunocompromised or -suppressed patients, an infection can become chronic and sometimes fatal in the absence of supportive and

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chemotherapeutic treatments (Amadi et al., 2001; Colford et al., 1996; Mwachari et al., 1998). Estimating the global ‘disease burden’ for cryptosporidiosis is challenging due to a lack of detailed epidemiological data for many countries ( Jex and Gasser, 2010; Lim et al., 2010; Putignani and Menichella, 2010). In 2004, the World Health Organization (Anonymous, 2004a,b) estimated the global impact of all diarrhoeal diseases to represent approximately 62 million disability adjusted life years (DALYs; second only to HIV among the infectious diseases). Cryptosporidium is considered to be amongst the most common causes of diarrhoea in regions of the world for which substantial epidemiological data are available (Karanis et al., 2007; Leoni et al., 2006; PHLSSG, 1990); thus, although its specific contribution to this burden is difficult to estimate, this genus is a major and potentially underestimated contributor (Guerrant et al., 1999; Ricci et al., 2006). Because effective chemo- or immunotherapeutics for cryptosporidiosis are limited (Armson et al., 2003; Caccio` and Pozio, 2006; Zardi et al., 2005), the control of this infection relies on prevention. Many of these preventative measures are behavioural and include the adherence to appropriate hygienic and sanitation practices (e.g. proper disposal of wastes, frequent hand-washing and suitable water treatment practices; Anonymous, 2002, 2004a,b; HSE, 2000a,b; Kaye, 2001). Although preventative measures aid in limiting the spread of human cryptosporidiosis, there is a pressing need for improved chemotherapeutics or prophylactic vaccines, particularly for use in impoverished nations. In the absence of an effective anti-Cryptosporidium vaccine, there has been a considerable focus on developing chemotherapeutic compounds (Armson et al., 2003; Smith and Corcoran, 2004; Zardi et al., 2005). Specific treatment strategies seem to be improving, and there are case reports describing effective reductions in oocyst excretion levels and the alleviation of clinical signs of cryptosporidiosis in immunocompromised patients upon treatment with paromomycin and/or azithromycin, following effective ‘highly active antiretroviral therapy’ (HAART) intervention (Palmieri et al., 2005; Zardi et al., 2005). Other evidence also suggests that nitazoxanide reduces the duration of diarrhoea associated with cryptosporidiosis in immunocompetent (Rossignol et al., 2001) and malnourished children (Amadi et al., 2002). Although this latter compound is now licensed for the treatment of cryptosporidiosis in immunocompetent children in the USA (Rossignol, 2006), it is not licensed in Europe and is not widely available in developing countries. As a result, in most situations, the treatment of cryptosporidiosis relies solely on maintaining the hydration of the patient and with an expection that the immune response mounted will eliminate the parasite. Further exploration and development of effective anti-cryptosporidial chemotherapeutics and/or vaccines is urgently needed.

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The present chapter reviews key aspects of the biology of the known species of Cryptosporidium, describes the significance of cryptosporidiosis in humans and summarizes recent advances in our knowledge of these parasites. Together with the use of advanced genomic and bioinformatic technologies, an improved understanding of Cryptosporidium should provide better insights into the complexities of the interplay between different genotypes/species and their hosts, with new prospects for the development of improved diagnostic tests, anti-cryptosporidial drugs and vaccines.

3.2. CRYPTOSPORIDIUM SPECIES AND GENOTYPES KNOWN TO INFECT HUMANS Currently, based primarily on molecular data, approximately 20 Cryptosporidium species and more than 40 unclassified genotypes have been recorded among all classes of vertebrates (Xiao and Feng, 2008; Xiao et al., 2004). In humans, the two major Cryptosporidium species recognized as being associated with infection and disease are Cryptosporidium hominis and Cryptosporidium parvum (Caccio`, 2005; Leoni et al., 2006; Xiao and Ryan, 2004). C. hominis (Morgan-Ryan et al., 2002) is considered to be transmitted by anthroponotic pathways only and, with few exceptions (Giles et al., 2001; Morgan et al., 2000; Smith et al., 2005b), is reported to be human-specific. In contrast, C. parvum has a broad reported host range (Fayer et al., 2000; Xiao et al., 2004), including numerous mammalian species which might act as zoonotic reservoirs (Hunter and Thompson, 2005; Smith and Nichols, 2006; Xiao and Feng, 2008). Based on available data, there is strong evidence that cattle represent a major source for zoonotic transmission of C. parvum (see Starkey et al., 2005; Xiao and Feng, 2008). The potential contribution of other animals as reservoirs of zoonotic Cryptosporidium is less certain. Considering their large population numbers in many countries and importance as major livestock animals, small ruminants (e.g. sheep and goats) appear likely candidates (Robertson, 2009). However, the extent to which these animals pose a risk to the public health is not well established, with some research indicating a low-prevalence of C. parvum in sheep (Ryan et al., 2005b) and others (Alves et al., 2001; Caccio` et al., 2000, 2001; Morgan et al., 1998; Santin et al., 2007) identifying this species with significant frequency. Molecular methods have provided evidence of C. parvum also in wild ruminants (Alves et al., 2003; 2006), including deer (Hajdusek et al., 2004; Ryan et al., 2005a; Siefker et al., 2002), and canids (Giangaspero et al., 2006; Matsubayashi et al., 2004). Such findings reinforce that further study of the breadth of the host range for C. parvum is required using molecular tools ( Jex and Gasser, 2009; 2010; Jex et al., 2008). Other species/genotypes

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(e.g. C. meleagridis, C. felis, C. canis, C. muris, C. suis and cervine and monkey genotypes of Cryptosporidium) have been reported to infect humans, but are much less common (Chalmers et al., 2002; Leoni et al., 2006; Xiao et al., 2001) and are likely to be of lesser zoonotic significance. However, the impact of these latter species/genotypes on immunocompromised persons, particularly in developing countries, has not been examined in detail and thus warrants further study. Illness and oocyst excretion patterns can vary significantly among infected individuals due to host factors, including immune status (Goodgame et al., 1993; Lazar and Radulescu, 1989) as well as parasite factors, such as the origin and age of the oocysts, the species/genotype, virulence and/or infective dose (Cama et al., 2007; Okhuysen et al., 1999). Although asymptomatic infections can occur (Biggs et al., 1987; Lacroix et al., 1987), clinical signs of disease usually commence 1–12 days after the ingestion of infective oocysts and usually coincide with the excretion of oocysts in the faeces ( Jokipii and Jokipii, 1986; Jokipii et al., 1983). However, oocyst excretion may continue for up to 2 months after clinical signs disappear ( Jokipii and Jokipii, 1986; Soave and Armstrong, 1986). Conversely, intermittent faecal oocyst excretion has been observed in patients with clinical signs of cryptosporidiosis ( Jokipii and Jokipii, 1986). Infected individuals often defaecate between 2 and > 20 times a day, producing watery, light-coloured, stools containing mucus (Casemore, 1987). Illness usually has a mean duration of approximately 1–3 weeks, with a range of 1–44 days (Elsser et al., 1986; Jokipii and Jokipii, 1986). Although chronic Cryptosporidium infections have been reported to occur in otherwise healthy humans (Lazzari et al., 1991; Rey et al., 2004), they are usually eliminated through an effective immune response. In contrast, infections in immunocompromised patients can develop into chronic disease (Blanshard et al., 1992; Flanigan et al., 1992; Hayward et al., 1997) and spread from the intestine to the hepatobiliary and pancreatic ducts, causing cholangiohepatitis, cholecystitis, choledochitis and/or pancreatitis (Current and Owen, 1989; Current et al., 1983; Soave and Armstrong, 1986). In immunocompromised or -deficient persons, clinical cryptosporidiosis can be fatal (Soave and Armstrong, 1986), causing intractable diarrhoea with severe dehydration, malabsorption, malnutrition and wasting, often in association with infections by other opportunistic pathogens (Huh et al., 2008; Scaglia et al., 1994; Soave and Johnson, 1998).

3.3. THE LIFE CYCLE OF C. PARVUM AND C. HOMINIS Both C. parvum and C. hominis are transmitted via the faecal-oral route and have a direct life-cycle. In brief, an infective dosage of sporulated oocysts (containing four naked, sporozoites) is ingested by the host.

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Upon encountering specific environmental cues, which, for C. parvum and C. hominis, appear to include salt (particularly bile salt) concentration, pH and temperature (Smith et al., 2005a), the oocysts excyst in the small intestine; this process appears to involve the activation of several serine proteases (Rosenthal, 1999). Upon excystation, each sporozoite migrates along the gut epithelium (e.g. microvilli of enterocytes in the small intestine) by gliding motility, ‘powered’ by an intracellular actinomyosin motor (Forney et al., 1998; Wetzel et al., 2005). Cell-surface glycoproteins on the sporozoite, including P23 and the 15-kDa glycoprotein (GP15), appear to be involved in this process (Boulter-Bitzer et al., 2007). Upon finding a suitable site for invasion, the sporozoite forms an attachment zone between its apical complex and the host cell membrane (Valigurova´ et al., 2008). Various proteins associated with the apical complex have been identified to be involved in this process (Boulter-Bitzer et al., 2007; Smith et al., 2005a), including the 40 and 900 kDa cell-surface glycoproteins (Barnes et al., 1998; Bonnin et al., 2001; Cevallos et al., 2000; Strong et al., 2000) as well as the thombrospondin-related attachment proteins (TRAPs; Lally et al., 1992; Spano et al., 1998). Upon attachment, the host cell membrane envelopes the sporozoite, encasing it in an epicellular parasitophorous vacuolar membrane (PVM); (Valigurova´ et al., 2008). The stimuli that initiate this process and the molecular mechanisms by which it proceeds are not well understood, but various cysteine proteases appear to play a critical role (Rosenthal, 1999). Within the PVM, the trophozoite transforms and then undergoes asexual reproduction (called merogony or schizogony; longitudinal binary fission) to produce type 1-meronts (schizonts). Each of these type 1-meronts contains 16 merozoites, which are released from the PVM. Each merozoite infects a new enterocyte (reforming the PVM), then replicates and develops into a new type 1-meront to repeat the cycle, or enters into the reproductive phase to replicate and develop into type 2-meronts, each of which contains four merozoites. After infecting a host cell, each type 2-merozoite initiates the sexual reproductive cycle (gametogony) and develops either into a microgametocyte (containing 12–16 microgametes) or a macrogametocyte (maturing into a macrogamete). Microgametes (male) are released and fertilize macrogametes (female) to form zygotes, which ultimately develop into oocysts. In another asexual reproductive phase (called sporogony), the oocyst sporulates to produce, internally, four naked sporozoites. Two types of oocyst are produced; thin-walled oocysts remain in the alimentary tract and have the ability to sustain an autoinfection, whereas thick-walled oocysts are passed in the faeces. The thin-walled oocysts and/or type 1-meronts are of particular relevance in immunocompromised, -deficient or -suppressed individuals, as they can perpetuate chronic cryptosporidiosis (Arenas-Pinto et al., 2003; Certad et al., 2005; Chhin et al., 2006; Lebbad et al., 2001) due to autoinfection

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within the gut (Sun and Teichberg, 1988). Unlike the motility, attachment and invasion phases, very little is known about the molecules involved in the endogenous phases of the Cryptosporidium life-cycle because of practical limitations in isolating these stages from infected hosts and culturing developmental stages in vitro.

3.4. CRYPTOSPORIDIOSIS: PATHOGENESIS AND IMMUNITY In humans, Cryptosporidium most directly and severely affects the small intestine (Xiao et al., 2004), although, in rare instances, and usually in relation to immunocompromised individuals, gastric cryptosporidiosis has been reported (Lumadue et al., 1998; Ventura et al., 1997). In such infections, combined endoscopic/histopathological examination of the gastric epithelial tissues reveals hyperplasia of the epithelial cells, inflammation in the lamina propria and non-specific lesions and oedema (Rivasi et al., 1999). In contrast, Cryptosporidium infection in the intestine is relatively well characterized. In humans, such infections are initiated when activated ‘zoites’ attach to the vicinal enterocytes and endogenous forms spread to the enterocytes of both the villi and crypts (Current et al., 1983). The infective dose, extent of spread, the sites involved and the immune response induced appear to be involved in determining whether an infection is clinical or subclinical (Tzipori and Ward, 2002). The site of infection within the intestine can be associated with the severity of clinical signs. Infection of the proximal small intestine is mainly related to symptoms of severe and watery diarrhoea, whereas infections confined to the distal ileum and/or the large bowel tend to be associated with intermittent diarrhoea or can be asymptomatic (Tzipori and Ward, 2002). The endogenous stages of Cryptosporidium disrupt the microvillus border, leading to a loss of mature enterocytes, shortening and/or fusion of the villi and lengthening of the crypts due to increased cell division (see Inman and Takeuchi, 1979; Tzipori et al., 1981). These changes lead to the loss of membrane-bound digestive enzymes and diminish the effective surface area of the intestine, leading to the decreased uptake of fluids, electrolytes and nutrients (Adams et al., 1994; Argenzio et al., 1990; Griffiths et al., 1994). Inflammation also occurs to a significant degree and is linked to the host’s immune response against the parasite. Although less commonly reported than gastrointestinal infections, extra-gastrointestinal cryptosporidiosis does occur, and can affect both immunocompetent (Westrope and Acharya, 2001) and, with greater frequency, immunocompromised, -suppressed or -deficient individuals (Bonacini, 1992; Dowsett et al., 1988; Vakil et al., 1996). Such infections can be categorized as pulmonary (Clavel et al., 1996) as well as biliary or pancreatic (Forbes et al., 1993; Goodwin, 1991; Vakil et al., 1996), and often

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appear to result from the systemic spread of an initial infection from the gastrointestinal tract (e.g. Edwards et al., 1990). Ultrasonic examinations of patients with biliary (Dolmatch et al., 1987; Teixidor et al., 1991) or pancreatic (Cappell and Hassan, 1993) infections indicate a notable dilation of the gall bladder and/or bile or pancreatic ducts, respectively, and an increase in pericholecystic fluid and thickening of the epithelial layers. The pathophysiology of diarrhoea is multifactorial and linked, to a significant extent, to a loss of the intestinal surface area due to ‘carpeting’ of the luminal surface by parasites, as well as villus fusion and atrophy (Buret et al., 2003; Inman and Takeuchi, 1979) and enterocytic destruction, following merogony and gametogony. Enterotoxins produced by the parasite have been proposed as playing a possible role in diarrhoeal illness (Guarino et al., 1994, 1995). The recent detection of a neurokinin-1 receptor antagonist, dubbed ‘Substance P’, produced by the parasite supports this hypothesis (Robinson et al., 2003; Sonea et al., 2002). The presence of this substance during an infection with Cryptosporidium appears to correlate with the severity of diarrhoea (Robinson et al., 2003; Sonea et al., 2002). Experimental data have indicated that this receptor antagonist is directly linked to glucose malabsorption and the increased secretion of chloride ions in the host intestinal tract (Hernandez et al., 2007), which has been demonstrated to be an important factor in the inducement of diarrhoeal illness (Kapel et al., 1997). The induction of diarrhoea may also relate to the attachment of C. parvum sporozoites to the apex of enterocytes. This complex process appears to involve multiple parasite ligands and host receptors, and induces a reorganisation of the host cell actin cytoskeleton (Elliott et al., 2001; Smith et al., 2005a; Tzipori and Ward, 2002). Such large changes to the cellular skeletal structure likely impact on host cell function. Indeed, significant negative effects on the integrity and function of enterocytes linked to cryptosporidial infections have been reported (Argenzio et al., 1990), and may, at least in part, be linked to the activation/perturbation of apoptotic pathways in these cells (Chen et al., 1998, 1999). A reduced permeability of the intestinal epithelia, due to a Cryptosporidium-induced disruption, may also play a role in the pathogenesis of diarrhoea. Such changes in permeability have been linked to various intestinal disorders, including inflammatory bowel disease (IBD), Crohn’s disease and ulcerative colitis (Fiocchi, 1998). With respect to cryptosporidiosis, reduced permeability of the intestinal barrier is proposed to relate partly to the disruption of zonula-occludens (ZO)-1, a 220-kDa cytoperipheral protein, which acts as a physical bridge between tight junction occludin and cytoskeletal F-actin (Balda and Anderson, 1993; Fanning et al., 1998). This hypothesis is supported by observations of the affects of Cryptosporidium andersoni on in vitro cultures of human and bovine intestinal epithelial cells in which significant disruptions of the tight junctions and apoptosis of the enterocytes were both noted (Buret et al., 2003). However, when ‘apical

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epidermal growth factor’ (which inhibits the induction of apoptosis and the disruption of ZO-1, but does not kill the parasite) was added to the culture, these effects were reduced (Buret et al., 2003). During the endogenous phases of the Cryptosporidium life-cycle, regardless of the organ being infected (e.g. stomach, intestine, lung, liver or pancreas), a common histopathological manifestation is hyperplasia of the affected epithelial tissues (e.g. Blagburn et al., 1987; Rivasi et al., 1999). This thickening of the epithelia of infected organs might be, in part, the result of ‘scarring’ (or fibrosis) following the infection of the host cells and the effect of Cryptosporidium-induced host cell mitosis (Hatkin et al., 1990; Masuno et al., 2006). Studies of the nuclear genome of C. parvum and C. hominis conducted to date (Abrahamsen et al., 2004; Xu et al., 2004) have indicated that these parasites have greatly reduced metabolic pathways and are heavily dependent on the host for resources (e.g. nucleotides and amino acids) that they cannot produce themselves. One hypothesis is that the parasite’s role in the induction of hyperplasia and mitotic division is to satisfy these resource requirements by eliciting increased production in the host cell. However, the mechanism by which this may occur and the parasite derived signals and/or molecules involved are not yet known. In addition to the major role that the parasite plays in the pathogenesis of disease, various host-related factors, including inflammatory and immunological responses are also of critical importance (Savioli et al., 2006). Although much of the knowledge of the immune responses to cryptosporidial infections relates to studies of mice, key insights have been made also through investigations of humans and cattle (Deng et al., 2004; Gomez Morales and Pozio, 2002; Riggs, 2002) as well as in vitro explorations of cultured monolayers of mammalian cell lines infected with C. parvum (Current and Haynes, 1984). A recent review of the literature ( Jex et al., 2011) reveals that the immunological control of cryptosporidial infection is associated with both innate and adaptive host responses. Epithelial cells and NK cells appear to be central to innate immunity, whereas adaptive immunity required for elimination of the parasite is coordinated by CD4þ T cells. IFN-g expressed by both T cells and NK cells could be central to immunity early in infection (reviewed by Jex et al., 2011).

3.5. GENOMICS AND TRANSCRIPTOMICS OF CRYPTOSPORIDIUM A major advance in our understanding of the molecular biology of Cryptosporidium has arisen from the sequencing of the genomes of C. parvum and C. hominis (Abrahamsen et al., 2004; Xu et al., 2004). The genomes of these closely related species are similar in size ( 9.1–9.2 Mbp), content ( 4000

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genes among 8 chromosomes) and sequence ( 96–97% identity; Abrahamsen et al., 2004; Xu et al., 2004). These genomes are substantially smaller than those reported for other apicomplexans, such as Eimeria tenella ( 60 Mbp; Shirley, 1994, 2000) and Plasmodium falciparum ( 23 Mbp; Gardner et al., 2002). This size difference is consistent with Cryptosporidium having fewer genes (e.g.  4000 vs.  5300 for Plasmodium), fewer introns and shorter non-coding regions (Abrahamsen et al., 2004; Xu et al., 2004). The small genome of C. parvum and C. hominis reveals a reduced complement of genes associated with both anabolic and catabolic phases of metabolism. Energy generation (i.e. ATP) appears to be dependent exclusively upon the degradation of simple sugars via anaerobic glycolysis, with no evidence of a mitochondrial genome or many of the nuclear genes associated with the Krebs cycle or electron transport chain (Abrahamsen et al., 2004; Xu et al., 2004). In addition, there is no evidence for the presence of genes associated with energy production via the digestion of fatty acids or proteins (Abrahamsen et al., 2004; Xu et al., 2004). The substantial reduction in these energy production pathways significantly reduces the ability of both C. parvum and C. hominis to synthesize a variety of essential building blocks (e.g. some nucleotides and amino acids). This deficiency is further accentuated by an absence of the genes encoding the enzymes involved in the urea and nitrogen cycles (i.e. for amino acid synthesis) and the shikimate pathway (Abrahamsen et al., 2004; Xu et al., 2004). The absence and/or substantial depauperacy of enzymes associated with these major metabolic pathways indicates that Cryptosporidium species are highly reliant on the host cell for building blocks (Abrahamsen et al., 2004; Xu et al., 2004). Consistent with this hypothesis is the finding that the nuclear genomes of both C. parvum and C. hominis contain numerous amino acid transporter genes, which are hypothesized to be involved in amino acid salvaging (Abrahamsen et al., 2004; Xu et al., 2004). Complementing these apparent salvage pathways are a variety of enzymes necessary for the conversion of the amino acids and nucleotides (e.g. pyrimidines to purines and purines to pyrimidines; Abrahamsen et al., 2004; Striepen et al., 2004; Xu et al., 2004). Interestingly, there is no evidence of redundancy in these pathways, such that, for example, a single enzyme (inosine 50 monophosphate dehydrogenase) appears to be responsible for the conversion of adenosine monophosphate (AMP) to guanosine monophosphate (GMP). Such metabolic and catabolic ‘bottle-necks’ are likely to represent significant targets for the development of new and specific anticryptosporidial drugs (e.g. Chaudhary and Roos, 2005). In silico drug target prediction, docking and screening represent significant areas of interest in current research of a range of neglected infectious diseases (Fig. 3.1). An emerging array of online resources, such as the Braunschweig Enzyme Database (BRENDA: Chang et al., 2009), CHEMBL (accessible via http://www.ebi.ac.uk/chembl/) and the

Sample collection

In vitro screening Culture purification

NGS

Clinical trials

Bioinformatics

BLAST homology

In silico docking

Structural modelling

Essentiality Lethality Model organisms Chemical inhibitor databases

FIGURE 3.1 An approach to the in silico prediction of novel drug targets and drugs. The diagram outlines the collection of parasite material or production with culturing (in vitro or in a surrogate host), followed by next-generation sequencing (NGS). Following sequencing, bioinformatic analyses allow the rapid assembly and annotation of data. BLAST homology comparisons of sequence data with those from model organisms (such as Drosophila melanogaster, Saccharomyces cerevisiae, Xenopus ranitans, and/or Caenorhabditis elegans—clockwise) allow the prediction of essential genes linked to lethal phenotypes. Peptides encoded by these genes can be screened in silico for potential inhibitors (drugs) in curated chemical databases (e.g. CHEMBL, BRENDA, TDR targets) that bind to them. Structural modelling of the predicted drug targets, supported by crystal structures, and subsequent in silico docking experiments can assist in the prediction of compounds and their analogues. Compounds designed can then be tested in vitro and in vivo for safety and efficacy.

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CHEMBL-Neglected Tropical Disease databases (accessible via http:// www.ebi.ac.uk/chemblntd) as well as the Tropical Disease Research (TDR) targets database (accessible via http://www.tdrtargets.org/; Aguero et al., 2008) provide a tremendous amount of information to facilitate such research. A study by Crowther et al. (2010) represents a recent example of the application of the in silico prediction of drug targets for a range of key parasitic protists, including Leishmania major, Trypanosoma cruzi and P. falciparum. In this study, peptide sequences inferred from genomic data for each species were assessed for their suitability as potential drug targets using a variety of priority-weighted selection criteria, including essentiality (based on lethal RNA interference phenotypes), their absence from the host organism and the availability of a predicted protein and/or a solved (crystallized) structure. Using this approach, these authors (Crowther et al., 2010) inferred 31 high priority ‘druggable’ molecules for P. falciparum. Seventeen of these molecules have homologues in C. parvum and C. hominis (see Table 3.1). Conspicuous among these homologues is a dihydrofolate reductase (DHFR). Dihydrofolate reductase, which plays a critical role in purine synthesis, is a known target of pyrimethamines (Crowther et al., 2010; Sirichaiwat et al., 2004), which have been used historically for the clinical treatment of malaria and toxoplasmosis (Amin, 1992; Roberts et al., 1998; Rosenblatt, 1992; Watkins, 1995). Early in vitro trials of these compounds as anti-cryptosporidial medications were unsuccessful (Lemeteil et al., 1993), suggesting that pyrimethamines were not effective against Cryptosporidium infection. However, such a conclusion could be premature. A more recent study, using recombinant Cryptosporidium DHFR expressed in yeast (Saccharomyces cerevisiae), identified several pyrimethamine structural analogues that had significant inhibitory effect (Lau et al., 2001). Although the rapid emergence in Plasmodium of resistance against pyrimethamine (Mita, 2010; Mita et al., 2009; Talisuna et al., 2007) may limit its appeal as a compound to treat cryptosporidiosis, the ‘blind’ prediction of dhfr as a potential drug target (Crowther et al., 2010) supports the assertion that in silico methodologies do yield genuine targets worthy of pursuit. In addition, the contrasting results reported by Lemeteil et al. (1993) and Lau et al. (2001) highlight that the difference between a successful drug and a failed compound can be small, as subtle changes to the molecular structure of a drug candidate can alter or maximize bioavailability, efficacy and/or safety (Lipinski, 2001; Lipinski et al., 1997; van de Waterbeemd and Gifford, 2003). For novel drug targets whose protein structures have been solved or may be confidently inferred from close homologues (de Beer et al., 2009; Wieman et al., 2004), advances in the predictive modelling of molecular interactions can assist significantly in the design and subsequent synthesis of structural analogues of a particular compound as candidate

TABLE 3.1 Cryptosporidium hominis and C. parvum genes (identification ¼ ID) inferred to encode peptides with high sequence homology to prioritized drug targets in Plasmodium falciparum predicted using the TDR targets database (www.tdrtargets.org; Crowther et al., 2010) C. hominis gene ID

BLASTp homology C. parvum (e-value) gene ID  118

Chro.10305 1.00  10 Chro.10107 2.00  10 41 Chro.70577 3.00  10 44 Chro.30215 Chro.30013 Chro.60090 Chro.70303 Chro.20263 Chro.10337 Chro.10335 Chro.40038 Chro.50038 Chro.20441 Chro.40506

1.00 1.00 1.00 1.00 1.00 3.00 1.00 2.00 4.00 1.00 1.00

          

10 146 10 112 10 118 10 159 10 131 10 65 10 119 10 89 10 56 10 60 10 113

Chro.30017 1.00  10 147 Chro.60435 Chro.70113 Chro.60524 Chro.20464

8.00 1.00 8.00 1.00

   

10 71 10 145 10 71 10 159

BLASTp homology P. falciparum (e-value) gene ID  120

cgd1_2700 1.00  10 cgd1_870 2.00  10 41 cgd7_5170 1.00  10 44           

10 112 10 146 10 121 10 159 10 131 10 64 10 120 10 106 10 60 10 55 10 113

PF10_0150 PF11_0164 PF11_0282

Methionine aminopeptidase, putative Peptidyl-prolyl cis–trans isomerase Deoxyuridine 50 -triphosphate nucleotidohydrolase, putative PF11_0377-b Casein kinase 1, PfCK1

cgd3_40 cgd3_1810 cgd6_690 cgd7_2670 cgd2_2480 cgd1_3040 cgd1_3020 cgd4_240 cgd2_4120 cgd5_3350 cgd4_4460

1.00 1.00 1.00 1.00 1.00 4.00 1.00 1.00 1.00 2.00 1.00

cgd3_80

1.00  10 147

PFE1050w

cgd6_3800 cgd7_910 cgd6_4570 cgd2_4320

5.00 1.00 9.00 1.00

10 69 10 149 10 71 10 159

PFF1155w PFI1105w PFI1110w PFI1170c

   

Gene description

PF14_0053 PF14_0142 PF14_0327 PF14_0378 PF14_0425 PFC0525c PFC0975c

Ribonucleotide reductase small subunit Serine/threonine protein phosphatase Methionine aminopeptidase, type II, putative Triosephosphate isomerase Fructose-bisphosphate aldolase Glycogen synthase kinase 3 Peptidyl-prolyl cis–trans isomerase

PFD0830w

Bifunctional dihydrofolate reductasethymidylate synthase Adenosylhomocysteinase (S-adenosyl-Lhomocystein e hydrolase) Hexokinase Phosphoglycerate kinase Glutamine synthetase, putative Thioredoxin reductase

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inhibitors (Fig. 3.1). Thus, armed with a suite of novel drug targets, for which structural models are available, and having identified classes of inhibitors based on information in current literature or databases (i.e. BRENDA or CHEMBL), in silico prediction and docking can assist in the prioritisation of structural analogues for synthesis, subsequent safety and efficacy testing in vitro (in cultured cells or pathogens) and in vivo (in animals). Some examples of open-source tools available in silico docking include MolDock (Thomsen and Christensen, 2006) and Lidaeus (Taylor et al., 2008) as well as Patchdock and Symmdock (Schneidman-Duhovny et al., 2005). Such an integrated approach to drug design and discovery provides substantial scope to improve the efficiency and reduce the costs associated with the research and development of new drugs (e.g. Campbell et al., 2010; Taylor et al., 2008; Wu et al., 2003; Yang et al., 2007). In the present review, we examined the druggability of the genomes of Cryptosporidium spp. and predicted, on a global scale, selective targets for known chemicals. We selected the sequences for all annotated coding genes common to C. parvum and C. hominis (accessible via http://www. CryptoDB.org), conducted homology searches (BLASTx) against the S. cerevisiae (yeast) genome and discovered > 1400 homologues for Cryptosporidium genes, 536 of which are associated with lethal phenotypes based on gene perturbation experiments (see http://www.yeastgenome. org). Recently, Doyle et al. (2010) demonstrated that functional genomic data for a range of eukaryotic model organisms could be used to assist in the prediction of the essentiality of conserved genes that represented prime targets for anti-parasitic drugs. Thus, genes in Cryptosporidium that are linked to homologues that display lethal phenotypes in S. cerevisiae, if their function/s is/are perturbed, could represent candidate targets for anti-cryptosporidial drugs. The collation of such genes and the corresponding interrogation of publicly available databases for known protein inhibitors (e.g. available via the BRENDA database; Schomburg et al., 2002) revealed 313 molecules in Cryptosporidium that may be inhibited by chemical compounds that are known to have activity against homologues in other organisms and/or in vitro. Conspicuous among these proteins are 61 GTPases, all of which contain a domain consistent with a protein-synthesizing GTPase (EC:3.6.5.3) and 21 of which also contain domains consistent with heterotrimeric G-protein (EC:3.6.5.1), small monomeric (EC:3.6.5.2) and signal-recognition-particle (EC:6.5.3.4) GTPases. In recent years, GTPases have received significant attention as druggable targets for anti-cancer therapies (Saxena et al., 2008; Thomas et al., 2008; Williams et al., 2008). Although, we are not aware of this specific family of GTPases having been suggested or tested as druggable targets in parasites, several GTPases have been proposed as playing an important functional role in key biological pathways in parasitic protozoa, including T. cruzi (Barrias et al., 2010; Yokoyama et al., 2008),

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Toxoplasma gondii (Caldas et al., 2009), Entamoeba histolytica (Welter and Temesvari, 2009) and P. falciparum (Zhou et al., 2009). Furthermore, recent studies have indicated that compounds that actively inhibit these targets may indeed represent new treatments for trypanosomiasis (Barrias et al., 2010) and toxoplasmosis (Caldas et al., 2009), highlighting the potential of GTPase inhibitors to be used specifically against cryptosporidiosis. The link between GTPases and anti-cancer therapies has undoubtedly contributed to an abundance of information for compounds that inhibit/ bind these enzymes. For example, we find that there are currently 37, 9, 7 and 4 known inhibitors of protein-synthesizing, signal-recognitionparticle, small monomeric and heterotrimeric G-protein GTPases in the BRENDA database (Schomburg et al., 2002). Strikingly, many of these inhibitors represent a variety of common, commercially available, antibiotics, including chloramphenicol, fusidic acid, streptogramin and tetracyclin. Notable among these inhibitors are mycins, including dihydrostreptomycin, hygromycin, neomycin, pulvomycin, ribostamycin, sparsomycin and viomycin, all of which are listed as having known activity against protein-synthesizing GTPases. The finding of a significant number of enzymes inhibited/bound by mycins is interesting. Various mycins have been investigated for activity against Cryptosporidium, including spiramycin (Portnoy et al., 1984; Saez-Llorens et al., 1989), salinomycin (Lindsay et al., 1987), clarithromycin (Cama et al., 1994), roxithromycin (Sprinz et al., 1998; Uip et al., 1998) and, most frequently, paromomycin and azrithromycin (Palmieri et al., 2005; Zardi et al., 2005). However, none of them are predicted to have activity against GTPases. Of the mycins that have been evaluated for efficacy against Cryptosporidium, only paromomycin is listed in the BRENDA database as having activity against an ‘essential’ Cryptosporidium gene-product. Furthermore, based on available data, only four enzymes are inhibited/bound by this compound; they are the PAP1P poly A polymerase (encoded by cgd4_930), the Po1 beta superfamily nucleotidyltransferase (cgd2_2730), a putative RNA binding protein (cgd4_3410) and a conserved hypothetical protein of unknown function (cgd3_2820) which is linked to ribonuclease P based on gene ontology (see http://www.Cryptodb.org). Of these molecules, only PAP1P is predicted to be encoded by an essential gene (i.e. has a lethal phenotype in S. cerevisiae). One hypothesis could be that the variable efficacy of paromomycin in field studies (Hewitt et al., 2000; Zardi et al., 2005) is the result of differences in temporal expression of this gene throughout the life-cycle or linked to microenvironmental factors within the lumen of the intestine. Although some aminoglycosides (e.g. mycins) have been reported to be toxic to mammalian cells (Guthrie, 2008; Martinez-Salgado et al., 2007; Rizzi and Hirose, 2007), this can be reduced through careful management (Murakami et al., 2008; Pannu and Nadim, 2008) and/or improved formulations that incorporate, for example, low-molecular

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weight proteins (Tugcu et al., 2006) and/or other compounds ( Jeyanthi and Subramanian, 2009; Nagai and Takano, 2010). Moreover, with current technologies, it should be possible to synthesize analogues with optimum bioavailability and parasite-specificity (supported by chemical, structural and in silico docking studies) but negligible toxicity to host tissues. Given the essentiality of GTPases in Cryptosporidium, there appears to be considerable scope for the design of relatively specific, safe and effective anti-cryptosporidial compounds. An enhanced understanding of the biology of known species and genotypes of Cryptosporidium should support the prediction of a larger and/or better panel of potential drug targets. The genomic sequencing of species of Cryptosporidium other than C. parvum and C. hominis, combined with transcriptomic and proteomic studies, is greatly needed to improve our understanding of these important parasites. In addition to directly revealing potential drug targets, such studies could explore, for example, the genomic characters linked to parasite virulence and pathogenicity as well as host-specificity and infection-site. Furthermore, investigating and understanding the temporal and spatial changes in transcription and expression in these parasites, as they progress through their lifecycle, is of paramount importance. Specific alterations associated with excystation, cellular invasion, development into and existence as the trophozoite, reproduction (asexual and sexual) as well as development into type-1 or -2 merozoites and thin- or thick-walled oocysts are particularly pertinent. To this end, the sequencing and draft assembly of the genome of a distinct species of Cryptosporidium, such as C. muris, which infects the stomach of mice (but not humans; Xiao et al., 2004), appears to be nearing completion (accessible http://www.ncbi.nlm.nih.gov; genome sequencing accession AAZY02000000). This sequence, along with the C. parvum and C. hominis genomes, will provide significant, new insights into the molecular biology of Cryptosporidium, is likely to assist in elucidating the molecular basis of host- and site-specificity of the parasites and provide a wealth of new genetic markers for the development of molecular-diagnostic tools. The sequencing of a range of species and genotypes of Cryptosporidium would greatly assist in both fundamental and applied areas. In particular, C. meleagridis, which is the only species of Cryptosporidium known to infect hosts of multiple taxonomic classes (i.e. birds and mammals; Xiao et al., 2004) and displays a significant plasticity in infection-site (i.e. respiratory tract in birds and intestinal tract in humans; Xiao et al., 2004) would be an interesting candidate. In addition, the exploration of genomic variation within species (e.g. C. parvum and C. hominis) would also be of significant interest. For example, a recent review of the global variation in a key population marker, the 60 kDa glycoprotein gene (¼ gp60), has revealed that, despite the substantial sequence variation recorded for this locus, there are five sequence types (three representing

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C. parvum and two representing C. hominis) that account for approximately 70% of all reports associated with human infections ( Jex and Gasser, 2010). A link between genotypic identity based on gp60 sequence and that based on the complete sequence of the nuclear genome has not been explored. However, there is some evidence of a relationship between identity based on gp60 sequence and the clinical signs associated with infection in some humans (Cama et al., 2007; 2008). Large-scale re-sequencing of the genomes of these and other genetic types of C. parvum and C. hominis would allow the testing of the hypothesis that gp60 sequence type does correlate with overall genomic sequence, which would indicate that, despite the richness and diversity of genetic types of Cryptosporidium reported to date, a small number of sub-specific genotypes are linked to the vast majority of human infections. Insights into the genetics governing the mechanisms (e.g. virulence, infectivity and pathogenicity) that might give rise to such an association would likely have major relevance toward developing new strategies to prevent and control Cryptosporidium and cryptosporidiosis. A major limitation to genomic and genetic research of Cryptosporidium has been access to sufficient quantities of material for next-generation sequencing (NGS). For genomic sequencing, it may be possible to overcome this limitation, to an extent, through the use of whole genomic amplification (WGA) systems (Pinard et al., 2006; Sorensen et al., 2007), which allow the synthesis of microgram quantities of total genomic DNA from minute quantities of starting material (e.g. nanograms). These systems are particularly attractive for re-sequencing projects, wherein the overall structure of the genome is largely established (e.g. for C. parvum and, to a lesser extent, C. hominis; see Abrahamsen et al., 2004; Xu et al., 2004). Although, like any enzymatic amplification, WGA approaches have the potential to introduce artefacts into the genomic DNA, studies directly comparing the sequencing of ‘WGA-amplified’ and ‘non-amplified’ templates have not detected substantial errors (e.g. Pinard et al., 2006; Sorensen et al., 2007), particularly when approaches relying on ‘multiple displacement amplification’ (e.g. using y29 DNA polyermase) are employed (see Burtt, 2011). Although WGA can assist in genomic research, which can take advantage of DNA isolated from purified oocysts, the challenges associated with isolating RNA needed for the study of transcription are not so readily overcome. Although some transcriptomic data are available for oocysts (Ortega-Pierres et al., 2009), little is known about transcription occurring in other life-cycle stages (e.g. trophozoites, merozoites and gametocytes) or linked to important aspects of the parasite biology, including invasion, nutrient uptake and reproduction/development. The acquisition of sufficient amounts of pure parasite material for transcriptomic and genomic studies has been the major obstacle preventing such research. Because the purification of endogenous stages of these parasites from infected animals is challenging, the

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development of in vitro cultivation technologies for any species of Cryptosporidium would represent a major advance.

3.6. IMPROVED INSIGHTS INTO CRYPTOSPORIDIUM USING IN VITRO TECHNIQUES Although some Cryptosporidium species can be maintained in experimental animals, this approach does not allow for the isolation or analysis of the intracellular parasite life-cycle stages. In addition, due to the broad genetic richness and diversity of cryptosporidia infective to humans ( Jex and Gasser, 2010; Leoni et al., 2006; Xiao et al., 2004), the maintenance of experimental infections as ‘reference lines’ for each known variant is impractical and costly. The establishment of a diverse range of Cryptosporidium isolates in in vitro culture would greatly aid a number of areas of research of these important parasites. For example, in vitro culturing has led to the first examination of the genes expressed and/or transcribed during infection of the host cell ( Jakobi and Petry, 2006) and in response to host defences (Zaalouk et al., 2004). Improved culturing techniques may also enable the investigation of changes in transcription during intracellular replication and development, and/or changes in response to external stimuli (e.g. host molecules, immune cells and/or drugs) under wellcontrolled conditions. In any practical sense, such information cannot be obtained using experimental infections in animals. The culturing of Cryptosporidium in vitro has been a challenging prospect and the subject of substantial research. Current and Long (1983) were the first to complete the Cryptosporidium life-cycle in vitro and used oocysts from humans and calves to infect chicken embryos (chorioallantoic membrane). These authors reported the successful completion of the parasite life-cycle and ‘normal’ epicellular development. However, oocyst yields were low, the endogenous stages of the parasite life-cycle were difficult to isolate, and the results could not be reproduced in subsequent investigations (Arrowood, 2002; Hijjawi, 2010). Numerous studies have explored the use of various cell lines for the cultivation of C. parvum from sporozoites (reviewed by Arrowood, 2002). These lines include human rectal tumour (HRT), human foetal lung (HFL), primary chicken (PCK), porcine (PK-10), baby hamster (BHK), Madin-Darby bovine (MDBK) and Madin-Darby canine (MDCK) kidney as well as HT29.74 human colon adenocarcinoma, RL95-2 human endometrial carcinoma and Caco2 human colon adenocarcinoma cells. A major deficiency of many of these cell lines is that the life-cycle of Cryptosporidium does not complete or accurately reflect that in the host animal, and the yield of oocysts is usually low (Arrowood, 2002). More recent publications have reported the successful in vitro cultivation of C. parvum in human ileocaecal

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adenocarcinoma 8 (HCT-8; Hijjawi et al., 2001) and VELI (rabbit chondrocyte; Lacharme et al., 2004) cell lines, resulting in the production of infective oocysts. In particular, the HCT-8 cell line is gaining usage ( Jakobi and Petry, 2006; Sifuentes and Di Giovanni, 2007; Wu et al., 2009). A recent study (Woods and Upton, 2007) has reported that oocyst yields from culture in HCT-8 cells can be enhanced further using serumfree media, with MDCK (Sigma) and PC-1, UltraCHO, UltraCulture and UltraMDCK (BioWhittaker) being most successful. Despite the success of some previous studies, the isolation of purified parasites from cells, particularly specific endogenous stages remains a challenge. Cell-free cultures have been evaluated as an alternative approach to the culturing of Cryptosporidium, greatly facilitating the isolation of parasite stages for subsequent experimentation. One of the first attempts at culturing C. parvum in cell-free medium (Hijjawi et al., 2004) utilized RPMI-1640 (Sigma-Aldrich) containing coagulated new born calf serum (NBCS) inoculated with oocysts. Using this approach, the authors reported the completion of the parasite life-cycle in vitro, resulting in the production of new oocysts. Excitingly, the cultures were maintained successfully for 2 months (after which the experiment was terminated), with the parasites seemingly self-propagating (Hijjawi et al., 2004). The promise of these results, however, were somewhat blunted by subsequent, unsuccessful attempts to replicate the initial findings (e.g. Girouard et al., 2006; Karanis et al., 2008). A publication (Woods and Upton, 2007) has suggested that some of photomicrographs taken of the developing parasitic stages reported in the original study (Hijjawi et al., 2004) appear to have been budding yeasts and/or fungi rather than stages of Cryptosporidium, and the authors questioned the possibility of culturing an ‘obligate, intracellular parasite’ in vitro without host cells. Queries about the identity of the ‘trophozoites’ and ‘merozoites’ described by Hijjawi et al. (2004) and also observed by Karanis et al. (2008) were also raised in a recent study (Petry et al., 2009). In the latter study (Petry et al., 2009), it was suggested that the stages observed by Hijjawi et al. (2004) were aged sporozoites that had become misshapen as a result of nutrient deficiencies due to the lack of a host-cell in cell-free cultures. Unfortunately, it is not possible to confirm the identity of any of the stages from the original study (Hijjawi et al., 2004), as the results were not verified by detailed ultrastructural examinations (including the use of nucleic acid or specific antibody probes). However, a subsequent study (Zhang et al., 2009), attempting to replicate the cell-free culturing of C. parvum, appears to have provided the first independent support of the original findings of Hijjawi et al. (2004). In this study, three distinct Cryptosporidium-specific monoclonal antibodies were used to successfully immunolabel various morphologically distinct cell types detected in host-cell free culture and interpreted them to represent

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distinct phases of the parasite’s life-cycle. Zhang et al. (2009) also used quantitative real-time PCR targeting the glyceraldehyde 3-phosphate dehydrogenase gene employing primers reported to be specific to Cryptosporidium and measured a fivefold increase in genomic DNA over the course of the cell-free culturing. On the basis of these results, the authors’ concluded that C. parvum could indeed be cultured in vitro in cell-free media, albeit with ‘modest’ yields. The validity of this finding appears to be further supported by a recent report describing the successful culturing of C. hominis in cell-free medium (Hijjawi et al., 2010). Here also, fluorescent labelling was utilized to support the morphological identification of each Cryptosporidium life-cycle stage and quantitative PCR was used to estimate the production of new parasite cells. As observed by Zhang et al. (2009), there was a five- to sixfold increase in DNA in a cell-free culture. In order to control for the potential contamination of their cell-cultures with non-cryptosporidial organisms, as suggested previously by Woods and Upton (2007), cell-free cultures were inoculated with heat-deactivated sporozoites also, with no measureable evidence of cellular proliferation, indicating that the culturing of Cryptosporidium cells could be achieved in a cell-free medium. The recent advances in in vitro culturing are intriguing and provide the prospect that problems associated with the inadequate supply of Cryptosporidium stages for molecular, immunological or biochemical investigations might be overcome in the future. Certainly, if the genome of Cryptosporidium were to remain entirely stable in vitro, the culturing of large quantities of parasite material would be a substantial step forward for exploring the genomics of this genus. This has been demonstrated to be a challenge for the culturing of other parasites, such as some genetic types of Giardia (Upcroft and Upcroft, 1994). Despite this, the successful in vitro culturing of Cryptosporidium would allow the exploration of isolates displaying a variety of phenotypes and could facilitate the generation of transgenic lines, as has be achieved for other apicomplexans, including for species of Plasmodium (Fairhurst, 2007; Kocken et al., 2009), and/or well-controlled gene knockout experiments. Further optimization of the proliferation of parasite material through culture and/or the development of an approach to purify specific stages in vitro or in vivo represent some of the last remaining obstacles to broad-scale transcriptomic and proteomic investigation of these parasites. Such research may prove a boon to our understanding of this group. However, extensive experimentation would be required to characterize, and, if possible, account for the impacts of culturing on the parasite (e.g. development, transcription and expression) and to determine the extent to which isolates of Cryptosporidium cultured in vitro reflect their natural phenotype in vivo. Such factors should be considered carefully when interpreting transcriptional or proteomic data derived from cultured parasites.

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3.7. CONCLUDING REMARKS Cryptosporidium derives its name from the small mysterious (hidden or cryptic) ‘spores’ within its resilient and microscopic oocysts and was dubbed so by Edmund Tyzzer in the early years of the last century. In the many years since, and despite substantial research, this has proven a particularly apt moniker, as the species comprising this genus of infectious protozoa have remained, in many respects, cryptic. Expansion of our knowledge of these organisms has progressed with increasing rapidity, from the first description in the early 1900s, to the detailed observation and description of the endogenous stages of the life-cycle, leading, in the early 1980s to the first real recognition of cryptosporidia as parasites rather than commensalists. The onset of the global HIV pandemic led to the first considerations of these organisms as troublesome opportunistic pathogens of immunocompromised or -suppressed people. However, the massive waterborne outbreak of cryptosporidiosis in Milwaukee in the earlier 1990s and the discovery that the resistant, transmissive stage of these parasites are not killed by common water treatment practices led to the revelation of the enormity of the adverse impact that Cryptosporidium/ cryptosporidiosis has on global health. The introduction and application of molecular tools (including the PCR) further accelerated the expansion of the collective knowledge of these organisms, leading to new insights into the mechanisms associated with infection and the disease. This information provided us with an improved understanding of the zoonotic potential, systematics and molecular detection of Cryptosporidium, leading to a range of new species descriptions and the determination that the genus is made up of a number of, in many cases, hostadapted lineages with varying levels of host-specificity and significantly different levels of relevance to human health. These advances have led to substantial expansions in the availability of a range of molecular-diagnostics tools to detect, characterize and identify these parasites in clinical and environmental samples. Within the last decade, we have seen the sequencing of the complete genomes of two key members of this genus (C. parvum and C. hominis) and anticipate the completion of the sequencing of another, systematically and biologically distinct, species (e. g., C. muris) in the very near future. Interestingly, through all of this, Cryptosporidium species have remained enigmatic, their basic biology has remained controversial, and perhaps most significantly, our ability to actively kill these parasites in infected individuals, through drug or vaccine, has remained noticeably absent. Recent advances in drug development have heralded new promise for treating and controlling these pathogens, which cause major human suffering and disease. However, the treatments adopted to date have had limited or ephemeral efficacy or are sometimes toxic. The ‘magic bullet’ has not been found.

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The purpose of the present chapter was to review the present state of our knowledge in the mechanisms behind the biology of these parasites wherein novel forms of treatment may be found. Our knowledge to date reveals parasites that are highly dependent upon specific cues within the host and a cascade of peptides and chemical reactions to successfully conduct the exquisite symphony of their life-cycle. The genomic sequences completed in the early 2000s revealed a genus of parasite with a highly streamlined metabolism, minimal modes of energy production, and a complex, but critically important, armada of transport proteins, allowing it to salvage essential nutrients and building blocks from its host. Much of what was once hidden is now exposed. Herein we see, for example, a variety of molecular mechanisms that are predicted to be essential for the parasites’ survival and could potentially be disrupted by a range of common, commercially available, antimicrobial compounds. These compounds are as yet untried, but the tools with which to test them are readily available. What is more, new and exciting advances in NGS technologies provide real prospects to delve deeper beneath the surface of these cryptic parasites to better understand their biology and further exploit their weaknesses. The advent of these platforms coupled with advances in in vitro culturing provide the means of exploring gene function and critical changes in the cellular biology of the parasite at key moments in its life-cycle, as well as, the prospects to identify, test and optimize novel targets for drug development. Broad-scale genomic, transcriptomic and proteomic research of Cryptosporidium coupled to the ability to test the findings of this research in vitro and in vivo provides the means to ultimately know this enemy and the potential to finally develop efficacious therapies against it.

ACKNOWLEDGEMENTS Support through the Melbourne Water Corporation, the National Health and Medical Research Council (Career Development Award Level 1 Industry Fellowship—ARJ) and the Australian Research Council (LP0989137) is gratefully acknowledged.

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Alves, M., Matos, O., Pereira Da Fonseca, I., Delgado, E., Lourenco, A.M., Antunes, F., 2001. Multilocus genotyping of Cryptosporidium isolates from human HIV-infected and animal hosts. J. Eukaryot. Microbiol. Suppl, 17S–18S. Alves, M., Xiao, L., Sulaiman, I., Lal, A.A., Matos, O., Antunes, F., 2003. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J. Clin. Microbiol. 41, 2744–2747. Alves, M., Xiao, L., Antunes, F., Matos, O., 2006. Distribution of Cryptosporidium subtypes in humans and domestic and wild ruminants in Portugal. Parasitol. Res. 99, 287–292. Amadi, B., Kelly, P., Mwiya, M., Mulwazi, E., Sianongo, S., Changwe, F., et al., 2001. Intestinal and systemic infection, HIV, and mortality in Zambian children with persistent diarrhea and malnutrition. J. Pediatr. Gastroenterol. Nutr. 32, 550–554. Amadi, B., Mwiya, M., Musuku, J., Watuka, A., Sianongo, S., Ayoub, A., et al., 2002. Effect of nitazoxanide on morbidity and mortality in Zambian children with cryptosporidiosis: a randomised controlled trial. Lancet 360, 1375–1380. Amin, N.M., 1992. Prophylaxis for malaria. Helping world travelers come home healthy. Postgrad. Med. 92, 161–168. Anonymous, 2002. United States Public Health Service/Infectious Disease Society of America guidelines for the prevention of opportunistic infections in persons infected with human immunodeficiency virus. Morb. Mortal. Wkly Rep. 51, 1–46. Anonymous, 2004a. World Health Report 2004: Changing History. World Health Organization, Geneva (p. 96). Anonymous, 2004b. Preventing person-to-person spread following gastrointestinal infections: guidelines for public health physicians and environmental health officers. Commun. Dis. Public Health 7, 362–384. Arenas-Pinto, A., Certad, G., Ferrara, G., Castro, J., Bello, M.A., Nunez, L.T., 2003. Association between parasitic intestinal infections and acute or chronic diarrhoea in HIV-infected patients in Caracas, Venezuela. Int. J. STD. AIDS 14, 487–492. Argenzio, R.A., Liacos, J.A., Levy, M.L., Meuten, D.J., Lecce, J.G., Powell, D.W., 1990. Villous atrophy, crypt hyperplasia, cellular infiltration, and impaired glucose-Na absorption in enteric cryptosporidiosis of pigs. Gastroenterol. 98, 1129–1140. Armson, A., Thompson, R.C., Reynoldson, J.A., 2003. A review of chemotherapeutic approaches to the treatment of cryptosporidiosis. Expert Rev. Anti Infect. Ther. 1, 297–305. Arrowood, M.J., 2002. In vitro cultivation of Cryptosporidium species. Clin. Microbiol. Rev. 15, 390–400. Balda, M.S., Anderson, J.M., 1993. Two classes of tight junctions are revealed by Zo-1 isoforms. Am. J. Physiol. 264, C918–C924. Barnes, D.A., Bonnin, A., Huang, J.X., Gousset, L., Wu, J., Gut, J., et al., 1998. A novel multidomain mucin-like glycoprotein of Cryptosporidium parvum mediates invasion. Mol. Biochem. Parasitol. 96, 93–110. Barrias, E.S., Reignault, L.C., De Souza, W., Carvalho, T.M., 2010. Dynasore, a dynamin inhibitor, inhibits Trypanosoma cruzi entry into peritoneal macrophages. PLoS One 5, e7764. Biggs, B.A., Megna, R., Wickremesinghe, S., Dwyer, B., 1987. Human infection with Cryptosporidium spp.: results of a 24-month survey. Med. J. Aust. 147, 175–177. Blagburn, B.L., Lindsay, D.S., Giambrone, J.J., Sundermann, C.A., Hoerr, F.J., 1987. Experimental cryptosporidiosis in broiler chickens. Poult. Sci. 66, 442–449. Blanshard, C., Jackson, A.M., Shanson, D.C., Francis, N., Gazzard, B.G., 1992. Cryptosporidiosis in HIV seropositive patients. Q. J. Med. 85, 813–823. Bonacini, M., 1992. Hepatobiliary complications in patients with human immunodeficiency virus infection. Am. J. Med. 92, 404–411. Bonnin, A., Ojcius, D.M., Souque, P., Barnes, D.A., Doyle, P.S., Gut, J., et al., 2001. Characterization of a monoclonal antibody reacting with antigen-4 domain of gp900 in Cryptosporidium parvum invasive stages. Parasitol. Res. 87, 589–592.

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