INTRODUCTION
Parasitic protozoans of the genus Cryptosporidium continue to cause outbreaks of gastrointestinal disease throughout the world (Schuster et al. Reference Schuster, Ellis, Robertson, Charron, Aramini, Marshall and Medeiros2005; Black and McAnulty, Reference Black and McAnulty2006; Smith et al. Reference Smith, Reacher, Smerdon, Adak, Nichols and Chalmers2006; Semenza and Nichols, Reference Semenza and Nichols2007). The infectious form is the oocyst, which contains 4 sporozoites. Following ingestion by a susceptible host, exposure to stomach acid, bile salts and host metabolic temperature, sporozoites can excyst and infect the epithelial cells lining the luminal surfaces of the digestive tract (O'Donoghue, Reference O'Donoghue1995; Chen et al. Reference Chen, O'Hara, Huang, Nelson, Lin, Zhu, Ward and Larusso2004). While Cryptosporidium excystation and host cell invasion have been characterized in detail ultrastructurally (Harris and Petry, Reference Harris and Petry1999; Huang et al. Reference Huang, Chen and Larusso2004; O'Hara et al. Reference O'Hara, Huang, Chen, Nelson and Larusso2005; Valigurova et al. Reference Valigurova, Jirku, Koudela, Gelnar, Modry and Slapeta2008), until recently our knowledge of the biochemical and physiological changes taking place within sporozoites during these processes has been limited (see recent review by Borowski et al. Reference Borowski, Clode and Thompson2008).
Upon excystation, sporozoites must find and attach to a susceptible target cell. Like other apicomplexans, Cryptosporidium parvum sporozoites have been shown to advance upon target host cells for invasion using a unique active process termed gliding motility, consisting of circular and helical gliding (Wetzel et al. Reference Wetzel, Schmidt, Kuhlenschmidt, Dubey and Sibley2005). During active motility, numerous proteins, which have recently been indicated to mediate attachment and invasion of the host cell, are secreted by sporozoites onto their cell surface (Barnes et al. Reference Barnes, Bonnin, Huang, Gousset, Wu, Gut, Doyle, Dubremetz, Ward and Petersen1998; Cevallos et al. Reference Cevallos, Zhang, Waldor, Jaison, Zhou, Tzipori, Neutra and Ward2000; Tosini et al. Reference Tosini, Agnoli, Mele, Gomez Morales and Pozio2004). After location of a host epithelial cell, an infective sporozoite can attach to the host cell by its apical end (Tzipori and Griffiths, Reference Tzipori and Griffiths1998; Chen et al. Reference Chen, O'Hara, Huang, Nelson, Lin, Zhu, Ward and Larusso2004). It is within the apical region that a number of vesicular secretory organelles, including micronemes, dense granules and a single rhoptry reside (Tetley et al. Reference Tetley, Brown, Mcdonald and Coombs1998; Harris et al. Reference Harris, Adrian and Petry2003). These secretory organelles have been identified as essential in sporozoite motility, secretion, cell attachment and penetration for a number of apicomplexans including C. parvum (Huang et al. Reference Huang, Chen and Larusso2004; Mercier et al. Reference Mercier, Adjogble, Daubener and Delauw2005; Smith et al. Reference Smith, Nichols and Grimason2005). The apical discharge of these secretory organelles is essential for cellular invasion and this discharge has been demonstrated to be temperature, cytoskeleton and intracellular calcium dependent and capable of proceeding in the absence of host cells (Chen et al. Reference Chen, O'Hara, Huang, Nelson, Lin, Zhu, Ward and Larusso2004).
To date, there is no reported methodology for the rapid assessment of biochemical and physiological processes taking place within Cryptosporidium sporozoites once excysted from the oocyst. Assessment has relied on differential interference contrast (DIC), immunofluorescence, electron microscopy and immunochemical hybridization techniques to further our understanding of the processes taking place within sporozoites (Carruthers et al. Reference Carruthers, Giddings and Sibley1999; Carruthers and Sibley, Reference Carruthers and Sibley1999; Chaturvedi et al. Reference Chaturvedi, Qi, Coleman, Rodriguez, Hanson, Striepen, Roos and Joiner1999; Chen et al. Reference Chen, O'Hara, Huang, Nelson, Lin, Zhu, Ward and Larusso2004). Flow cytometry (FCM), in combination with an ever increasing array of fluorescent dyes for the quantification of cellular and metabolic processes, offers the ability to precisely and rapidly examine biochemical and physiological changes taking place within sporozoites post-excystation.
This is the first study to report the use of the fluorescent dyes SYTO9/PI, DiBAC4 (3), Fluo-4 AM and FM1-43 for the assessment of sporozoite membrane integrity, membrane depolarization, intracellular calcium and exocytosis respectively in combination with FCM analysis. In addition, we determine if forward angle light scatter (FSC) and side angle light scatter (SSC) are capable of measuring changes in the size, shape and internal granularity of sporozoites post-excystation, where we relate these observations to changes in sporozoite ATP content and infectivity. We conclude that by using multi-parametric flow cytometry, we are able to identify distinct physiological and biochemical events occurring within the sporozoite post-excystation, indicative of apical organelle discharge in the absence of host cells.
MATERIALS AND METHODS
Parasites
C. parvum cattle isolate oocysts (Swiss cattle C26), originally obtained from the Institute of Parasitology, Zurich, were purchased from the Department of Veterinary and Biomedical Sciences, Murdoch University, Perth, Australia. Oocysts were genotyped as previously described by direct PCR analysis and sequencing of the 18S rRNA gene (Morgan et al. Reference Morgan, Constantine, Forbes and Thompson1997). Oocysts were passaged through mice and purified as previously described (Hijjawi et al. Reference Hijjawi, Meloni, Morgan and Thompson2001). Oocysts were stored in sterile phosphate-buffered saline (PBS) supplemented with antibiotic solution (15 μl/ml) containing ampicillin (10 mg/ml) and lincomycin (4 mg/ml). On receipt, infectivity of each oocyst batch was determined using a cell culture-Taqman PCR assay (Keegan et al. Reference Keegan, Fanok, Monis and Saint2003), and all experiments using oocysts were carried out within 9 weeks of purification.
Oocyst excystation
C. parvum oocysts were pre-treated to promote excystation using a standard excystation procedure. Oocysts were incubated in 1 ml of acidified water (pH 2·7)-trypsin (0·025% (w/v)) for 20 min at 37°C with mixing by inversion (5 times) every 5 min, centrifugation (10 min at 1800 g) and re-suspension in 1 ml of supplemented RPMI 1640 medium (Sigma) (Keegan et al. Reference Keegan, Fanok, Monis and Saint2003) ready for subsequent experimentation. Excystation rates were determined for each oocyst batch using flow cytometry (Vesey et al. Reference Vesey, Griffiths, Gauci, Deere, Williams and Veal1997). Only oocyst batches that achieved excystation rates of greater than 80% after excystation pre-treatment and 10 min incubation at 37°C in supplemented RPMI were used for subsequent experiments.
Fluorescent dye labelling of excysted sporozoites
After pre-treatment to promote excystation, oocysts suspended in 1·5 ml centrifuge tubes were incubated within RPMI medium in heating blocks for time-course and temperature incubation experiments. Following incubation, excysted sporozoites were centrifuged for 10 min at 1800 g, the majority of the supernatant removed and sporozoites resuspended in Isoton II and stained with either SYTO9/PI, DiBAC4(3), or Fluo-4 AM (Molecular probes, Eugene, Oregon, USA). For FM1-43 (Molecular Probes, Eugene, Oregon, USA) staining, sporozoites were pre-stained in RPMI before incubation time-course experiments. At the end of FM1-43 incubation experiments stained sporozoites were washed with RPMI medium before centrifugation for 10 min at 1800 g and subsequent analysis. Dye characteristics and fluorescent dye labelling of excysted sporozoites are summarized in Table 1. For all time-course and temperature incubation experiments oocysts were used at a concentration of 20 000 oocysts/ml.
Dye characteristics and labelling of excysted sporozoites
For evaluation of the suitability of SYTO9/PI for assessing membrane integrity of sporozoites, excysted sporozoites were heat treated at 37°C, 45°C, 48°C and 50°C for 20 min before staining with SYTO9/PI and analysis by flow cytometry. A minimum of 3 independent experiments were conducted at each temperature regime. To evaluate the suitability of DiBAC4(3) for assessing membrane depolarization, excysted sporozoites were heat treated at 37°C, 45°C and 48°C for 20 min before staining with DiBAC4(3) and analysis by flow cytometry. A minimum of 3 independent experiments were conducted for each temperature regime. Potassium chloride (50 mm), a classical depolarizing agent and inducer of exocytosis, was also used to depolarize sporozoites and to promote sporozoite exocytosis (Meffert et al. Reference Meffert, Premack and Schulman1994; de Castro Junior et al. Reference De Castro Junior, Pinheiro, Guatimosim, Cordeiro, Souza, Richardson, Romano-Silva, Prado and Gomez2008).
Fluo-4 AM was loaded into sporozoites at the end of the incubation period/treatment before analysis by flow cytometry. Sporozoite intracellular calcium was also manipulated by the use of BAPTA-AM, a highly specific chelator of intracellular calcium, and A23187, a calcium ionophore. Sporozoites were incubated with either BAPTA-AM (20 μm) or A23187 (25 μm) (Molecular Probes, Eugene, Oregon, USA) at 18°C for 20 min after the excystation pre-treatment but prior to time-course experiments. Excysted sporozoites were stained with FM1-43 before time-course experiments and washed at the end of each time-period in dye-free medium to allow the cell surface to de-stain as the dye molecules leave the plasma membrane, allowing the selective labelling of endosomes which form in the presence of the dye (Brumback et al. Reference Brumback, Lieber, Angleson and Betz2004).
Time-course incubation experiments
Time-course experiments were conducted after an initial excystation treatment. Excysted sporozoites were subsequently incubated at 37°C in supplemented RPMI medium for 0 min, 30 min, 60 min, 90 min, 120 min, 3 h, 4 h, and 5 h post-excystation treatment before being stained with SYTO9/PI, DiBAC4(3), or Fluo-4 AM ester. To monitor exocytosis, sporozoites were pre-stained with FM1-43 before time-course experiments commenced and washed at the conclusion of each time-point. Stained excysted sporozoites were subsequently analysed by flow cytometry. All time-course experiments were independently replicated a minimum of 3 times for each individual dye.
Microscopy
Excysted sporozoites from time-course experiments were also analysed by DIC and fluorescence microscopy. An Olympus BX60 microscope fitted with a 10×eyepiece and either a 40×or 100×objectives were used for examination of samples.
Flow cytometric analysis
Flow cytometry was performed using a Becton Dickinson (San Jose, USA) FACS Calibur flow cytometer. Logarithmic signals were used for all parameters and the forward light scatter detector was set at E00 for all assays. For SYTO9/PI and DiBAC4(3) stained particles, the side scatter detector was set to 335 V and the green fluorescent detector (FL1) set to 500 V. The fluorescence detector FL1 was used as the threshold and set to a value of 289. For Fluo-4 AM stained particles, the side scatter detector was set to 335 V and the green fluorescent detector was set to 700 V. The forward scatter detector was used as the threshold and set to a value of 111. For FM1-43, the side scatter detector was set to 335 V and orange fluorescence detector (FL2) was set to 800 V. The forward scatter detector was used as the threshold and set to a value of 160. A minimum of 10 000 events were analysed for SYTO9/PI stained particles, while a minimum of 20 000 events were analysed for DiBAC4(3), Fluo-4 AM and FM1-43 stained particles due to increased numbers of events detected that were of a smaller size (lower FSC) and of less complexity (lower SSC) than that of the sporozoites.
Data were examined on scatter-plots of Log FSC vs Log SSC using WinMDI software (version 2.8, http://facs.scripps.edu/software.html). Regions of interest were defined on scatter-plots by enclosing populations (gating) and displaying these events as histograms of green fluorescence (FL-1 channel), or orange fluorescence (FL-2 channel) for FM1-43 stained particles. For flow cytometric sorting, a minimum of 10 000 gated events within regions defined on a scatter-plot of forward angle light scatter (FSC) and side angle light scatter (SCC) were sorted by exclusion mode into 50 ml polystyrene tubes, transferred to 40 ml tubes and centrifuged in a Sorvall RC-5B centrifuge with a fixed angle rotor (SS-34) at 4000 g for 10 min at 4°C. The majority of supernatant was removed leaving approximately 3 ml, and the remaining supernatant was transferred to 1·5 ml tubes before centrifugation on a bench-top centrifuge for 10 min at 4000 g. The majority of the supernatant was removed leaving approximately 100 μl which was re-stained with SYTO9 and re-analysed on the flow cytometer to confirm the correct population was captured. Subsequently, 10 μl of the remaining sample was analysed on a glass slide under a sealed cover-slip by DIC and fluorescence microscopy. Isoton II (Beckman Coulter) sheath fluid was used in the cytometer.
Determination of sporozoite infectivity
Sporozoites were incubated in cell-free supplemented medium as described above for defined time-periods before application to HCT-8 (ATCC CCL244 [American Type Culture Collection]; human ileocecal adenocarcinoma) cell monolayers. Sporozoite infectivity was determined for time-course incubation periods using a cell culture-Taqman PCR assay previously described (Keegan et al. Reference Keegan, Fanok, Monis and Saint2003).
Quantification of sporozoite ATP levels
Sporozoite ATP levels were determined for time-course incubation periods using a method previously described for the determination of oocyst ATP concentration (King et al. Reference King, Keegan, Monis and Saint2005).
Quantification of sporozoite total soluble secreted protein
Sporozoites were excysted as described above and incubated for either 30 min or 3 h in Hank's Balanced Salt Solution. At the end of each incubation period excysted sporozoites were spun on a bench-top centrifuge for 10 min at 18 000 g and the supernatant removed and analysed for total soluble protein using the Bio-Rad Quick Start™ Bradford Protein Assay (Bradford, Reference Bradford1976). Each time-point was replicated 4 times and each individual replicate contained sporozoites from a total of 2×107 excysted oocysts.
RESULTS
Flow cytometric identification of sporozoites stained with SYTO9/PI, DiBAC4(3), Fluo-4 AM and FM1-43
To identify Cryptosporidium sporozoites by flow cytometric analysis, oocysts were treated using a standard excystation procedure, stained with the nucleic acid-specific dyes SYTO9/PI and analysed by flow cytometry. Comparison of flow cytometric scatter plots for unexcysted and excysted oocysts (Fig. 1 plots A1, A2) identified 3 distinct populations on the scatter plot for excysted oocysts. The region representing the sporozoite population was discriminated from intact oocysts (R3), empty oocyst shells (R2) and associated residual body components (crystalline proteins, lipid bodies and amylopectin granules) by gating these different populations and plotting a histogram of events against green fluorescence intensity (FL1) (Fig. 1. plots B1–B3). The sporozoite region (R1) exhibited higher fluorescence on the FL1 channel in comparison to the other regions analysed. Untreated oocysts and intact oocysts (containing sporozoites) exhibited reduced fluorescence on the FL1 channel as expected due to the impermeability of the oocyst wall to the SYTO9/PI dyes. This region was further confirmed to predominately represent sporozoites by sorting the populations on the flow cytometer and examining samples by DIC and fluorescence microscopy (data not shown).
Scatter plots representing the flow cytometric analysis of unexcysted Cryptosporidium oocysts (A1) and excysted oocysts (A2) stained with SYTO9/PI. Region 1 (R1) was identified as the region representing Cryptosporidium sporozoites on the scatter plot (A2) and could be discriminated from intact oocysts (region 3 (R3)), empty oocyst shells and associated oocyst contents (region 2 (R2)) by increased green fluorescence (FL1 channel). (B1) Histogram of fluorescence intensity plotted for the gated population R3 from the unexcysted oocysts. (B2) Histogram of fluorescence intensity plotted for the gated population R2 from the excysted oocysts. (B3) Histogram of fluorescence intensity plotted for the gated population R1 from the excysted oocysts.
Excysted sporozoites were heat treated at 37°C, 45°C, 48°C and 50°C for 20 min and stained with SYTO9/PI to assess the suitability of the dyes for assessing sporozoite membrane integrity. Heat treatments at 50°C showed significant decreases in FL1 signal of the gated region when compared to the 37°C control (t-test, P=0·015) (a near log decrease in FL1, data not shown), identifying the suitability of SYTO9/PI for the assessment of sporozoite membrane integrity. Scatter plot profiles for DiBAC4(3), Fluo-4 AM and FM1-43 stained sporozoites and associated oocyst contents were similar to that of the SYTO9/PI dyes; however, these profiles had substantially more noise demonstrated by the detection of more events below the size range of the gated sporozoite region. Therefore the number of total events analysed was increased for excysted oocysts stained with these dyes for subsequent experiments.
Excysted sporozoites were heat treated at 37°C, 45°C, and 48°C for 20 min or treated with 50 mm KCl for 30 min and stained with DiBAC4(3) to assess the suitability of this dye to measure membrane depolarization. Significant increases in FL1 of the gated region were evident at 45°C compared to 37°C (t-test, P=0·003), with greater than a log increase in FL1 signal at 48°C (t-test, P<0·001) demonstrating the sensitivity of this dye to measuring membrane depolarization. Potassium chloride treatment significantly depolarized the membrane (t-test, P<0·001) with an accompanying reduction in the SSC of the gated region (t-test, P<0·001). The assessment of Fluo-4 AM for the quantification of intracellular calcium levels within sporozoites was performed using a calcium chelator BAPTA-AM and the calcium ionophore A23187, and is described in the following section. FM1-43 was assessed by fluorescence microscopy and determination of its localization to the sporozoite's surface membrane.
Flow cytometric time-course studies of excysted sporozoites stained with Fluo-4 AM SYTO9/PI, DiBAC4(3) and FM1-43
Time-course studies were conducted on excysted sporozoites incubated at 37°C in RPMI supplemented medium in the absence of a host cell line. At defined time-intervals sporozoites were stained using the fluorescent dyes Fluo-4 AM, SYTO9/PI DiBAC4(3) and FM1-43 in order to investigate physiological and biochemical changes taking place within the sporozoite, post-excystation. Sporozoites incubated at 37°C and stained with Fluo-4 AM at defined time-intervals showed an increase in intracellular calcium (Fig. 2) (Single factor ANOVA, P=0·01) over a time-course of 2 h. A significant decrease in SSC (Single factor ANOVA, P<0·001) of the sporozoites was also evident over this time-period.
A representative time-course experiment identifying changes in sporozoite intracellular calcium. Scatter plots represent the flow cytometric analysis of Cryptosporidium oocysts incubated at 37°C in RPMI supplemented medium for (A1) 30 min, (A2) 60 min, (A3) 90 min and (A4) 120 min post-excystation treatment and sporozoites subsequently loaded with Fluo-4 AM. The sporozoite region R1 was gated and histograms of fluorescence intensity (FL1 channel) plotted for the population for each time-period, (B1) 30 min, (B2) 60 min, (B3) 90 min and (B4) 120 min post-excystation treatment.
Excysted sporozoites stained with SYTO9/PI at defined time-intervals revealed small but significant changes in the sporozoite membrane permeability over the time-course (Single factor ANOVA, P=0·002). The sporozoite plasma membrane gradually became more permeable over this time-period as exhibited by a decrease in FL1 signal (Fig. 3). A gradual and significant decrease in SSC (Single factor ANOVA, P<0·001) of the sporozoites also paralleled this decrease in plasma membrane permeability (correlation coefficient of determination, r 2=0·72).
A representative time-course experiment identifying changes in sporozoite plasma membrane permeability. Scatter plots represent the flow cytometric analysis of Cryptosporidium oocysts incubated at 37°C in RPMI supplemented medium for (A1) 30 min, (A2) 60 min, (A3) 90 min and (A4) 120 min post-excystation treatment and subsequently stained with SYTO9/PI. The sporozoite region R1 was gated and histograms of fluorescence intensity (FL1 channel) plotted for the population for each time-period, (B1) 30 min, (B2) 60 min, (B3) 90 min and (B4) 120 min post-excystation treatment.
Excysted sporozoites stained with DiBAC4(3) at the same defined time-intervals showed rapid and significant membrane depolarization as indicated by an increase in FL1 signal (Single factor ANOVA, P<0·001). Greater than one log increase in FL1 signal was evident by the end of the 2 h time-period, indicating a large depolarization of the plasma membrane of the sporozoites (Fig. 4). A gradual and significant decrease in SSC (Single factor ANOVA, P=0·007) of the sporozoites accompanied this increase in membrane depolarization (correlation coefficient of determination r 2=0·79). Time-course studies using DIC and fluorescence microscopy did not detect any changes in the gross appearance of sporozoites over this time-period except for the detection of a few smaller swollen ellipsoid-shaped sporozoites.
A representative time-course experiment identifying changes in sporozoite membrane depolarization. Scatter plots represent the flow cytometric analysis of Cryptosporidium oocysts incubated at 37°C in RPMI supplemented medium for (A1) 30 min, (A2) 60 min, (A3) 90 min and (A4) 120 min post-excystation treatment and subsequently stained with DiBAC4(3). The sporozoite region R1 was gated and histograms of fluorescence intensity (FL1 channel) plotted for the population for each time-period, (B1) 30 min, (B2) 60 min, (B3) 90 min and (B4) 120 min post-excystation treatment.
Excysted sporozoites stained with FM1-43 showed a gradual and significant decrease in SSC over the 2 h time-period (Single factor ANOVA, P<0·001). However, there was no significant increase in FL-2 signal until 3 h post-excystation (Single factor ANOVA, P<0·001) (Fig. 5). Fluorescence microscopy of sporozoites stained with FM1-43 at 3 h post-excystation identified sporozoites of a typical slender aubergine morphology with light lateral staining and intense posterior staining of the sporozoite (Fig. 6).
Representative scatter plots and histograms from a time-course experiment identifying changes in sporozoite exocytosis. Scatter plots represent the flow cytometric analysis of Cryptosporidium oocysts stained with FM1-43 and incubated at 37°C in RPMI supplemented medium for (A1) 30 min, (A2) 180 min post-excystation treatment. The sporozoite region R1 was gated and histograms of fluorescence intensity (FL2 channel) plotted for the population for each time-period, (B1) 30 min, (B2) 180 min post-excystation treatment.
Representative FM-143 staining of typical Cryptosporidium sporozoites incubated at 37°C in RPMI supplemented medium for 3 h post-excystation treatment; composite image, DIC above, blue-light-excited fluorescence below. Staining is most intense around the posterior of excysted sporozoites. Note staining of some bacteria (b), but not vacant oocyst walls (o). Scale bar=5 μm.
Beyond 2 h incubation at 37°C, the number of swollen sporozoites increased as determined by DIC microscopy, and sporozoites of the normal slender aubergine-shaped morphology depicted in Fig. 7 (A1) decreased. Large numbers of smaller swollen sporozoites were evident by 5 h (Fig. 7 A2). The appearance of smaller swollen sporozoites coincided with flow cytometric detection of another distinct population (Fig. 7 plots B1, B2). In contrast to the region already defined to represent sporozoites (R1), the second population (R2) was smaller in size (decreased FSC) with a reduced internal complexity (decreased SSC). This was consistent for all scatter-plots of excysted sporozoites stained using all 4 fluorescent dyes Fluo-4 AM, SYTO9/PI, DiBAC4(3) and FM1-43. This population (R2) was further confirmed to represent smaller swollen sporozoites by flow cytometric sorting and DIC and fluorescence microscopy. It was also noted that many of the sporozoites recovered from this region lysed after the sorting and centrifugation process indicating the fragile nature of the swollen sporozoites.
Light microscope images of Cryptosporidium sporozoites viewed with DIC optics. (A1) Represents the typical slender aubergine-shaped sporozoite cell, and the arrow denotes the clearly visible apical complex. (A2) Represents a sporozoite cell exhibiting a smaller swollen shape. Scale bars=5 μm. Scatter plots representing the flow cytometric analysis of Cryptosporidium oocysts incubated at 37°C in RPMI supplemented medium for 30 min (B1) and 5 h (B2) post-excystation treatment. Sporozoites were subsequently stained with SYTO9/PI. The region R1 represents the typical slender aubergine-shaped sporozoite cell, while the region R2 represents sporozoites of a smaller and swollen morphology.
Intracellular calcium levels in the smaller swollen sporozoites returned to the baseline levels identified in freshly excysted sporozoites. A significantly large decrease in FL1 signal of SYTO9/PI stained sporozoites was apparent for the R2 population (t-test, P<0·001), indicating further increase in the sporozoite plasma membrane permeability. However, in contrast, smaller swollen sporozoites stained with DiBAC4(3) exhibited a significant decrease in FL1 fluorescence (t-test, P=0·008), initially indicating the opposite, a re-establishment of membrane potential.
To further confirm that decreases in sporozoite internal granularity (SSC) and the appearance of smaller swollen sporozoites were representative of apical organelle discharge, a calcium chelator, BAPTA-AM, previously identified to block apical organelle discharge in a number of apicomplexans including Cryptosporidium, and the calcium ionophore A23187, shown to induce apical organelle discharge, were loaded into sporozoites prior to incubation studies. BAPTA-AM delayed the decrease in sporozoite internal granularity (t-test, P=0·002) by chelating sporozoite intracellular calcium when compared to the BAPTA-AM minus treatment (t-test, P<0·001) as well as delaying the appearance of the region R2 representing the smaller swollen sporozoites. The calcium ionophore A23187 rapidly induced a calcium burst (t-test, P<0·003) and an associated decrease in internal granularity (t-test, P<0·001). Changes in sporozoite size, internal granularity, membrane integrity, membrane potential and intracellular calcium were all able to be completely arrested if excysted sporozoites were incubated at 4°C instead of 37°C over the 5-h time-period.
Sporozoite infectivity and ATP content were examined at defined time-intervals on excysted sporozoites incubated at 37°C in RPMI supplemented medium, in order to relate changes observed in multi-parametric flow cytometry of sporozoites to changes in the above parameters. A rapid reduction in sporozoite infectivity was evident within the first 2 h of incubation at 37°C (Fig. 8A). Total sporozoite ATP content began to decrease after 30 min incubation at 37°C to 18% of the initial ATP levels, and then remained at approximately 15% for the remaining incubation period (Fig. 8B).
Cryptosporidium oocysts were excysted and incubated at 37°C in supplemented RPMI medium for defined time-periods before application to HCT-8 cell monolayers for infectivity studies (A1) or processing for quantification of ATP levels (B1). A minimum of 3 independent time-course experiments were conducted for each time-point, and each time-point replicated 3 times within each individual experiment. Infectivity was calculated using a cell-culture-Taqman PCR assay and expressed as a percentage of the starting infectivity. ATP levels were expressed as a percentage of the levels of untreated oocysts incubated at 4°C. Each data point is the mean value from the independent experiments and the error bars indicate the standard deviation between experiments.
Total sporozoite soluble secreted protein was also quantified at 30 min and 3 h after incubation at 37°C in Hank's Balanced Salt Solution (HSB), used in place of RPMI supplemented medium due to the high levels of endogenous protein in the latter. However, flow cytometric analysis in combination with DiBAC4(3) staining identified changes in sporozoite internal granularity and membrane depolarization that were similar in HSB buffer to those incubated in RPMI supplemented medium. Total soluble secreted protein was found to have increased by more than 3-fold for sporozoites incubated for 3 h at 37°C in comparison to those incubated for only 30 min (t-test, P<0·001).
DISCUSSION
One of the main goals of this work was to evaluate if flow cytometry, in combination with fluorescent dye staining, was capable of identifying distinct physiological events occurring within C. parvum sporozoites post-excystation. Like other Apicomplexa, C. parvum sporozoites contain a number of vesicular secretory organelles including a single rhoptry, numerous micronemes and several dense granules predominantly localized at the apical region of the sporozoite (Tetley et al. Reference Tetley, Brown, Mcdonald and Coombs1998; Harris et al. Reference Harris, Adrian and Petry2003). Within the Apicomplexa, these apical organelles and their associated proteins have been either hypothesized or shown to be involved in directional gliding motility, host cell adhesion and parasitophorous vacuole formation, physiological processes essential for the successful establishment of infection (for review see Smith et al. Reference Smith, Nichols and Grimason2005). However, assessment of the physiological processes described above has relied on the use of techniques such as DIC, immunofluorescence and electron microscopy and immunochemistry hybridization techniques. During this study we demonstrated for the first time that flow cytometry in combination with the fluorescent dyes SYTO9/PI, DiBAC4(3), Fluo-4 AM and FM1-43 was a suitable and rapid methodology for the assessment of sporozoite membrane integrity, membrane depolarization, intracellular calcium and exocytosis respectively.
Chen et al. (Reference Chen, O'Hara, Huang, Nelson, Lin, Zhu, Ward and Larusso2004) reported that C. parvum sporozoites were able to discharge their apical contents after 2 h incubation at 37°C in the absence of host cells. They established that this apical organelle discharge was temperature, intracellular calcium and cytoskeleton dependent, and required for successful host cell invasion and infection. Based on these observations, we decided to examine freshly excysted C. parvum sporozoites incubated at 37°C in a cell-free medium using multi-parametric flow cytometry in an endeavour to see if we could detect such changes.
Flow cytometric time-course studies identified rapid reductions in the side scatter (SSC) of sporozoites stained with all 4 fluorescent dyes over a 2 h incubation period. SSC is a function of the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). As described above, sporozoites have numerous secretory organelles contributing to a complex internal granularity. Our results identified a rapid reduction in sporozoite internal complexity within 2 h, suggestive of a rapid mobilization and utilization of proteins within these organelles. This is in agreement with transmission electron micrograph studies of sporozoites showing a significant reduction in both the numbers of dense granules and micronemes within sporozoites after incubation at 37°C for 2 h (Chen et al. Reference Chen, O'Hara, Huang, Nelson, Lin, Zhu, Ward and Larusso2004). Furthermore, it has previously been demonstrated that sporozoites after excystation secrete protein during helical gliding (Chen et al. Reference Chen, O'Hara, Huang, Nelson, Lin, Zhu, Ward and Larusso2004), indicating active organelle discharge. Comparably, we were able to measure large increases in total soluble secreted protein between the 30 min and 3 h incubation periods.
After excystation, C. parvum sporozoites undergo circular and helical gliding as they advance upon a target host cell (Wetzel et al. Reference Wetzel, Schmidt, Kuhlenschmidt, Dubey and Sibley2005). This motility appears to be driven by coupling the translocation of surface adhesions to an actin-myosin motor beneath the parasite plasma membrane (Sibley, Reference Sibley2004). It is postulated that during gliding motility, microneme and vesicular contents are secreted from the anterior conoid of the sporozoite before translocation down the lateral membrane to the posterior, and subsequent shedding onto a substrate (Smith et al. Reference Smith, Nichols and Grimason2005). The use of the styryl dye FM1-43 with flow cytometric analysis identified large increases in exocytosed membrane after incubation for 2 h, and fluorescence microscopy localized its distribution predominantly to the posterior end of the sporozoite. This is confirmatory of the proposed model of surface molecule adhesion translocation, and further evidence for extensive sporozoite exocytosis having occurred during this time-course.
It is noteworthy that during the 2 h incubation period, while large changes in internal granularity were evident, no exocytosed membrane could be visualized by fluorescence microscopy or quantified by flow cytometry until shortly after this period. However, previous work by others (Chen et al. Reference Chen, O'Hara, Huang, Nelson, Lin, Zhu, Ward and Larusso2004) and ourselves has identified active protein secretion within this period. We can think of 3 possible explanations for this observation. Firstly, not enough exocytosed membrane may have been present until this time for either flow cytometric quantification or microscopic visualization. Secondly, the exocytosed membrane may have been actively shed within the 2 h incubation period along with adhesion molecules by helical gliding (Chen et al. Reference Chen, O'Hara, Huang, Nelson, Lin, Zhu, Ward and Larusso2004), and until active motility decreased, as indicated by lowered levels of ATP, this could not be either quantified or visualized. Finally the exocytosed membrane may have been recycled once translocated to the posterior end of the sporozoite and not shed, and this may have discontinued once ATP levels dropped below a threshold level. However, we think that this final explanation is unlikely as we did not observe internalization of the styryl dye.
After incubation for 2 h at 37°C both sporozoite infectivity and ATP levels had dramatically dropped. The large reductions in ATP are not surprising considering the energy intensive nature of helical gliding and apical organelle discharge. The reductions in infectivity over this time-course re-enforce the importance of these processes taking place in near or intimate contact with a compatible host cell. Beyond 2 h incubation at 37°C, we observed both the appearance and increase in the number of sporozoites exhibiting a smaller swollen shape instead of the characteristic slender aubergine-shaped sporozoite cell, possibly suggestive of a conclusion of apical organelle discharge and/or sporozoites becoming necrotic. This is analogous to previous work identifying the appearance of non-motile smaller swollen sporozoites after increased incubation time following excystation in RPMI medium at room temperature for longer incubation times (Harris et al. Reference Harris, Adrian and Petry2003). After 2 h sporozoite ATP levels had fallen below 20% of initial sporozoite levels, low enough for events leading to cellular death to occur (Lipton, Reference Lipton1999).
Large decreases observed in the green fluorescence signal for SYTO9/PI stained smaller swollen sporozoites when compared to aubergine-shaped sporozoite cells is strong evidence suggesting that smaller swollen sporozoites may have become necrotic. This is further emphasized by the fragility of this stage observed in flow cytometric sorting. However, the large decreases observed in the green fluorescence signal for DiBAC4(3) stained smaller swollen sporozoites when compared to aubergine-shaped sporozoite cells appear at first to be contradictory. The decrease in DiBAC4(3) green fluorescence would infer the opposite, a re-establishment of ionic potential. However, this can be explained in the terms of apical organelle discharge. Since apical organelles consist of both lipid and protein, which DiBAC4(3) would stain in depolarized cells, further discharge could account for the observed reduction in fluorescence. By the end of the 5 h time-period a large number of sporozoites exhibited the smaller swollen morphology. It is unknown whether these sporozoites are destined to perish, or whether they can undertake the extracellular life cycle as described by Hijjawi et al. (Reference Hijjawi, Meloni, Ng'anzo, Ryan, Olson, Cox, Monis and Thompson2004). Longer time-course studies using the techniques described in this paper may help resolve this question.
The mechanisms by which secretory organelles such as micronemes discharge their contents are not clear or readily observed. However, there is an increasing body of evidence to suggest that this process may occur via regulated exocytosis (Matthiesen et al. Reference Matthiesen, Shenoy, Kim, Singer and Satir2003; Chen et al. Reference Chen, O'Hara, Huang, Nelson, Lin, Zhu, Ward and Larusso2004; Dowse and Soldati, Reference Dowse and Soldati2004), in which a cell directs secretory vesicles to the cell membrane. Once vesicles are transported to the cell surface they fuse with the plasma membrane, and their contents are extruded from the cell (Sollner, Reference Sollner2003). Calcium is a major signalling trigger in a diverse range of eukaryotic cells and critical for host cell invasion by intracellular parasites (Tsien and Tsien, Reference Tsien and Tsien1990; Clapham, Reference Clapham1995; Moreno and Docampo, Reference Moreno and Docampo2003; Nagamune and Sibley, Reference Nagamune and Sibley2006). The rapid release or influx of Ca2+ into the cytosol of the cell has been coupled to a number of key physiological processes including regulated exocytosis, with a rise in intracellular calcium generally triggering the final fusion event with the plasma membrane (Holz et al. Reference Holz, Senyshyn and Bittner1991).
Our data identified a rapid increase in sporozoite cytosolic free Ca2+ coincident with reductions in sporozoite internal granularity shortly after excystation. Chelation of this intracellular calcium delayed decreases in sporozoite internal granularity and the appearance of smaller swollen sporozoites, indicating inhibition of apical organelle discharge. These changes could be more rapidly induced by use of the calcium ionophore A23187, as previously shown in Toxoplasma (Carruthers and Sibley, Reference Carruthers and Sibley1999). This is in agreement with results of Chen et al. (Reference Chen, O'Hara, Huang, Nelson, Lin, Zhu, Ward and Larusso2004), identifying that chelation of intracellular calcium inhibited apical organelle discharge and sporozoite gliding motility. Monitoring of calcium in Toxoplasma gondii using Fluo-4-loaded cells revealed that cytosolic Ca2+ undergoes oscillations during sporozoite motility. Concurrent with cell invasion, T. gondii cytosolic Ca2+ levels were dramatically dampened down (Lovett and Sibley, Reference Lovett and Sibley2003). Comparably, this study identified, after an initial rise in cytoplasmic Ca2+, a subsequent decrease back to basal levels within smaller swollen-shaped sporozoites. In concert with an increasing body of evidence in the literature, this suggests a conserved role for the calcium-dependent secretion pathway within the Apicomplexa. However, it must also be considered that prolonged and large rises in cytosolic calcium are cytotoxic and can result in cell death (Lipton, Reference Lipton1999). Therefore, the reduction in cytosolic calcium in smaller swollen sporozoites may just be a result of inactivated or depleted esterases unable to cleave the Fluo-4-AM ester due to cellular necrosis.
In protozoan parasites, cytosolic Ca2+ concentration is regulated by the concerted operation of several transporters in the plasma membrane, endoplasmic reticulum, mitochondria (absent in C. parvum) and acidocalcisomes (Moreno and Docampo, Reference Moreno and Docampo2003). However, the systems that control calcium responses in parasites are incompletely understood. Depolarization-dependent rises in cytoplasmic Ca2+ have been shown to trigger exocytosis and the release of proteins in a number of eukaryotic systems (Koga et al. Reference Koga, Kozaki and Takahashi2002; Sadakata et al. Reference Sadakata, Mizoguchi, Sato, Katoh-Semba, Fukuda, Mikoshiba and Furuichi2004; Cohen et al. Reference Cohen, Schmitt and Atlas2005). Interestingly, coincident with reductions in sporozoite internal granularity and increased cytosolic free Ca2+, we also observed coordinated reductions in plasma membrane permeability and large and sustained increases in sporozoite membrane depolarization. Potassium chloride, which has been widely used as a depolarizing agent and inducer of exocytosis in secretory cells (Meffert et al. Reference Meffert, Premack and Schulman1994; de Castro Junior et al. Reference De Castro Junior, Pinheiro, Guatimosim, Cordeiro, Souza, Richardson, Romano-Silva, Prado and Gomez2008), was shown to induce both membrane depolarization and associated reductions in internal granularity, indicative of sporozoite exocytosis.
While regulation of cytoskeleton filaments has been implicated in the machinery of secretory vesicle-plasma membrane fusion resulting in exocytic discharge of vesicle contents from cells (Burgoyne and Morgan, Reference Burgoyne and Morgan1993), this is the first study providing evidence to suggest a role for plasma-membrane depolarization in apical organelle discharge for an apicomplexan. Until this work, it has been unclear what the initial triggers are that are required for apical organelle discharge. Discharge can begin shortly after excystation and occur in the absence of host cells at 37°C (Chen et al. Reference Chen, O'Hara, Huang, Nelson, Lin, Zhu, Ward and Larusso2004; Feng et al. Reference Feng, Nie, Sheoran, Zhang and Tzipori2006). We propose that in the absence of host cells, extensive apical organelle discharge is a result of a rundown in the metabolic currency of the cell, ATP. This run down is most likely a result of prolonged helical gliding and limited internal energy reserves available to the sporozoite. Limiting ATP would reduce the activity of ATP-dependent pumps in the plasma membrane and endoplasmic reticulum, resulting in membrane depolarization, an inability to clear cytosolic calcium and calcium release from ATP-dependent stores. The resultant calcium rise would in turn activate calcium-dependent kinases, which would phosphorylate SNARE (soluble NSF attachment receptor) proteins activating the sporozoite secretory pathway.
When a sporozoite attaches to a host cell via specific receptor-ligand interactions (Nesterenko et al. Reference Nesterenko, Woods and Upton1999), vacuolated structures thought to arise from the apical organelles are secreted from the sporozoite at the host cell interface during the internalization stage (Huang et al. Reference Huang, Chen and Larusso2004). While calcium has already been shown to be essential for this process to proceed, it is a secondary messenger and the exact trigger(s) prompting this discharge in the presence of a compatible host cell are unknown. Whether an ATP depletion induced discharge has any role to play in the parasite-cell interaction has not been resolved. However, Silverman et al. (Reference Silverman, Qi, Riehl, Beckers, Nakaar and Joiner1998) established that in Toxoplasma-infected cells a collapse in host cell ATP pools induced by DTT activation of a potent apyrase triggered a calcium flux, signalling parasite egress. Interestingly it has been suggested that the same calcium release regulatory pathway involved in egress may have a role in invasion (Arrizabalaga and Boothroyd, Reference Arrizabalaga and Boothroyd2004).
In light of a recent review by Borowski et al. (Reference Borowski, Clode and Thompson2008) emphasizing the role that host cell membrane protrusion has in encapsulating the parasite, as well as re-evaluating the question of whether Cryptosporidium actively invades cells, it is tempting to speculate that sporozoites may be primed to infect regardless of host cell contact. That is, successful cell infection may be a matter of being in right place at the right time; the right place being a compatible cell expressing the appropriate receptor/s, and the right time denoted by a sporozoite finding such a cell, but still having enough energy in reserve after helical gliding to attach to the target cell before extensive apical organelle discharge ensues. If so, of special interest is what happens to sporozoites in vivo which do not find a compatible cell in a limited timeframe. Does this lead to cell necrosis, or are some sporozoites capable of developing an extracellular life cycle that co-exists with intracellular stages in vivo (Hijjawi et al. Reference Hijjawi, Meloni, Ng'anzo, Ryan, Olson, Cox, Monis and Thompson2004; Borowski et al. Reference Borowski, Clode and Thompson2008)?
We undertook this study in order to examine if flow cytometry in combination with vital dye staining could be used to identify physiological events occurring within sporozoites post-excystation. The work described here should prove useful for further investigation into biochemical and physiological processes taking place within C. parvum sporozoites once excysted. In particular, these techniques may help in the elucidation of sporozoite receptor-ligand mediated exocytosis and/or gastrointestinal cues involved in the activation of the sporozoite secretory pathway. It also has the potential to be applied to other parasitic protozoa. Finally, this methodology also lends itself to examination of the effect of stresses (e.g. abiotic, pharmacological) on sporozoite-specific physiological processes involving apical organelle discharge, membrane potential, intracellular cytosolic Ca2+ and exocyotsis.
We are grateful to Suzanne Froscio and Richard Bright for comments and suggestions on the manuscript. We thank the South Australian Water Corporation, the Cooperative Research Centre for Water Quality and Treatment, and the Water Research Foundation (WRF) for financial, technical, and administrative assistance in funding the project through which this information was discovered. Mention of trade names or commercial products does not constitute WRF endorsement or recommendations for use. Similarly, omission of products or trade names indicates nothing concerning WRF's position regarding product effectiveness or applicability. The comments and views detailed herein may not necessarily reflect the views of the WRF, its officers, directors, affiliates or agents.
