Live imaging of the Cryptosporidium parvum life cycle reveals direct development of male and female gametes from type I meronts

Abstract

Cryptosporidium is a leading infectious cause of diarrhea around the world associated with waterborne outbreaks, community spread, or zoonotic transmission. The parasite has significant impact on early childhood mortality, and infection is both a consequence and cause of malnutrition and stunting. There is currently no vaccine, and treatment options are very limited. Cryptosporidium is a member of the Apicomplexa, and, as typical for this, protist phylum relies on asexual and sexual reproduction. In contrast to other Apicomplexa, including the malaria parasite Plasmodium, the entire Cryptosporidium life cycle unfolds in a single host in less than 3 days. Here, we establish a model to image life cycle progression in living cells and observe, track, and compare nuclear division of asexual and sexual stage parasites. We establish the length and sequence of the cell cycles of all stages and map the developmental fate of parasites across multiple rounds of invasion and egress. We propose that the parasite executes an intrinsic program of 3 generations of asexual replication, followed by a single generation of sexual stages that is independent of environmental stimuli. We find no evidence for a morphologically distinct intermediate stage (the tetraploid type II meront) but demonstrate direct development of gametes from 8N type I meronts. The progeny of each meront is collectively committed to either asexual or sexual fate, but, importantly, meronts committed to sexual fate give rise to both males and females. We define a Cryptosporidium life cycle matching Tyzzer's original description and inconsistent with the coccidian life cycle now shown in many textbooks.

Fig 1. Sexual differentiation of C. parvum is not dependent upon a secreted factor.

(A) Schematic representation of both the induction and program hypotheses of C. parvum sexual differentiation. (B) Schematic representation of the conditioned media experiment. Briefly, media was conditioned for 48 hours in the presence of HCT-8 host cells with or without C. parvum infection. Conditioned media was passed through a 0.45-μm filter and then used for new infections that were scored for growth (C) and sexual differentiation (D). (C) Growth assay by luminescence for C. parvum growth in conditioned media. (D) Stage scoring of C. parvum at 24, 48, and 72 hours postinfection when grown in conditioned media. (E) Schematic representation of super-infection experiment. Briefly, HCT-8 cells were infected with H2BmNeon parasites that were previously infected for 24 hours with WT parasites or not previously infected. Both the primary infection (WT) and super-infection (H2BmNeon) were scored for parasite life stage at indicated time points postinfection. (F) Stage scoring and life cycle progression of C. parvum super-infection experiment. Underlying data are provided in the Supporting information as S1 Data. WT, wild type.

Fig 2. The intracellular development of all stages can be observed in real time in living cells.

(A) Images taken from time-lapse microscopy depicting asexual growth and nuclear division, 2 representative meronts. Scale bar: 5 μm; insets represent hours from time of invasion. (B) Images taken from time-lapse microscopy depicting male parasite growth and nuclear division, 2 representative gamonts. Scale bar: 5 μm; insets represent hours from time of invasion. (C) Duration of phases of asexual growth. Parasites remain as a single nucleus for an average of 7.92 hours, followed by 0.83 hours as 2 nuclei, 1.11 hours as 4 nuclei, and 2.77 hours as 8 nuclei prior to egress (n = 366 to 380). (D) Asexual parasites remain intracellular (invasion to egress) for an average of 12.57 hours (n = 3 80). (E) Graphical representation of the timing of nuclear divisions during asexual development. (F) Duration of phases of male growth. Parasites remain as a single nucleus for an average of 6.28 hours, followed by 0.92 hours as 2 nuclei, 1.07 hours as 4 nuclei, and 1.69 hours as 8 nuclei, and 2.05 hours as 16 nuclei prior to egress (n = 58 to 60). (G) Male parasites remain intracellular (invasion to egress) for an average of 12.08 hours (n = 60). (H) Graphical representation of the timing of nuclear divisions during male development. (I) Images taken from time-lapse microscopy depicting female growth, 2 representative gametes. Scale bar: 5 μm; insets represent hours from time of invasion. (J) Total area of female parasites over time. Gray lines represent individual female parasites, and red line indicates the average area of female parasites over time (n = 29). (K) Total fluorescent intensity of cytoplasmic mScarlet for the entire area of female parasites over time. Gray lines represent individual female parasites, and red line indicates the average of female parasite over time (n = 29). Underlying data are provided in the Supporting information as S2 Data.

Fig 3. Extended time-lapse imaging reveals 3 cycles of asexual schizogony followed by differentiation into gametes.

(A) Individually tracked parasites over time. Each horizontal line represents an individual parasite from invasion to egress (green = asexual, blue = male, and pink = female). Lines for females cutoff at 72 hours, as we do not observe egress of these parasites. Parasites with observed egress, but which invaded prior to the start of the experiment are included with lines beginning at 11 hours (n = 729). (B) Individually plotted nuclear division events for asexual parasites over time. The time point at which we observe 2, 4, or 8 nuclei for the first time for parasites designated as asexual are plotted as individual points over time (n = 869 to 973). (C) Individually plotted nuclear division events for male parasites over time. The time point at which we observe 2, 4, 8, or 16 nuclei for the first time for parasites designated as male are plotted as individual points over time (n = 161 to 170). (D) Individually plotted egress events for both asexual (green) and male (blue) parasites over time. The time at which we no longer observe a parasite is designated the time of egress. Each point represents a single asexual meront or male gamont. There are 3 distinct clusters of asexual egress events and a single cluster of male egress events (asexual n = 629, male n = 79). (E) Individually plotted invasion events for asexual (green), male (blue), and female (pink) parasites over time. The time at which a parasite is first observed is considered the time of invasion. There are 2 observed clusters of invasion events leading to asexual parasites and only 1 cluster leading to male or female cells, respectively (asexual n = 600, male n = 133, and female n = 222). Underlying data are provided in the Supporting information as S2 Data.

Fig 4. Meronts commit their merozoites to either asexual or sexual fate, but when committed to sex give rise to both males and females.

(A) Images taken from time-lapse microscopy depicting multiple generations of parasite replication, including both asexual and sexual stage growth and development. Stills take from video (see S4 Movie) over 27 hours of observation; insets represent hours from time of infection of the culture. Green arrows indicate final fate of asexual meronts, and white arrows indicate newly invaded parasites immediately following an egress event. Blue arrows indicate parasites designated with a male fate, and pink arrows indicate parasites designated with a female fate. Scale bar: 5 μm. (B) Representative trees depicting the mapped fate of parasites for which the fate of progeny of a single parental meront could be tracked. The tree in the center represents the images seen in (A). Times are shown in hours postinfection. Crosses indicate cells that were lost or died before fate could be assigned. (C) Fate of progeny of single 8N meronts, ordered by time of egress. For each parental meront, the fate of all observable progeny was tracked and is indicated (asexual = green, male = blue, female = pink, and lost to observation or ambiguous = gray). Individual parental meronts are ordered by the time at which the parental meront was last observed. Underlying data are provided in the Supporting information as S3 Data.

Fig 5. Development to gametes occurs directly from type I meronts, and there is no evidence to support a 4N type II meront as the committed stage.

(A) Schematic depicting hypotheses of life cycle progression. Parasites might undergo direct development in which type I meronts producing 8 progeny develop into sexual stages or indirect development in which an intermediary type II meront producing only 4 progeny gives rise to sexual stages. (B) Images depicting the possible fates of parasites that progress through a stage with 4 nuclei. Parasites were assigned 3 fates: (1) those that progress from 4, to 8, and eventually 16 nuclei are male; (2) those that progress from 4 to 8 nuclei are asexual type I meronts; and (3) those that appear to egress as 4 nuclei are asexual type II meronts. Parasites that remained as 4 nuclei until the end of the experiment were excluded from this analysis. (C) Graph of the fate of 4N parasites over time. Prior to 16 hours of infection, nearly 100% of the parasites that pass through a 4N stage become 8N prior to egress. This decrease after 40.5 hours of infection, and this decrease is proportional to the increase in parasites that become 16N (male) at these time points (n = 1,095). (D and E) Immunofluorescence of HCT-8 cells infected with transgenic C. parvum expressing ROP3-HA or CDPK1-HA, respectively. Representative images of 4N or 8N meronts at 20 and 34 hours postinfection are shown. Only parasites with 8 nuclei express ROP3 (D) or CDPK1 (E) at either time point. Scale bar: 1 μm. (F) Ifnγ^−/− mice were infected with CDPK1-HA parasites and immunofluorescence staining was conducted on frozen sections of the small intestine. Top, low magnification micrograph of a highly infected segment of the intestinal tissue. Scale bar: 15 μm. All parasite stages are labeled with an antibody to LDH (green) and mature meronts ready to egress are labeled with CDPK1 (red). Bottom, higher magnification images of 4N or 8N parasite. Parasites with 8 nuclei but not 4 nuclei express CDPK1 in vivo. Scale bar: 1 μm. (G) Quantification of the ROP3 positive meronts for the entire 4N or 8N population at 20 and 34 hours postinfection. (H) Quantification of the CDPK1 positive meronts at 20 and 34 hours postinfection. (I) Quantification of the number of nuclei of a total of 159 CDPK1 positive parasites observed in 26 independent fields of view of intestinal sections. Only young 1N trophozoite and meronts 8 nuclei were positive for CDPK1, and in matching our studies in culture, we did not observe a single positive tetraploid parasite. Underlying data are provided in the Supporting information as S4 Data. LDH, lactate dehydrogenase.

Fig 6. Model of Cryptosporidium life cycle and sexual commitment.

(A) Schematic representation of the life cycle for Cryptosporidium summarizing the findings of this study. Infection begins with the oocyst that releases 4 sporozoites that invade intestinal epithelial cells. The parasites replicate asexually (green) by synchronous schizogony for 3 cycles and invariantly produce 8 merozoites. Merozoites emerging from the third round upon reinvasion give rise to sexual stages, both males (blue) and female (pink). The male gamont undergoes 4 rounds of synchronous nuclear division producing 16 gametes, while the female gamete is cell cycle arrested and remains haploid while expanding in size and stockpiling proteins, lipids, and carbohydrates for the future oocyst. Male gametes egress and fertilize intracellular female gametes. Following fertilization, meiosis and sporulation oocysts are released from the host cell and are immediately infectious. Oocysts can be shed with the feces resulting in transmission or excyst and reinfect the same host. (B) We propose a developmental commitment model of life cycle progression for Cryptosporidium. Merozoites emerging a merogony cycle are collectively committed to an asexual or sexual fate and, when sexually committed, give rise to both male and females (commitment is represented symbolically here by coloring the nuclei forestalling future fate; however, we note that the mechanism is unknown and may be independent of the nucleus).