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| Localization of MEDLE-2 protein expression on Cryptosporidium parvum sporozoites (Top) and developmental stages of C. parvum in HCT-8 cell cultures at 24 and 48 h (Middle, Bottom, respectively). Images were taken by differential interference contrast microscopy (DIC), fluorescence microscopy using nuclear stain 4, 6-diamidino-2-phenylindole (DAPI), fluorescence microscopy using polyclonal MEDLE-2 antibodies (MEDLE-2), and superimposition of the three (Merge).
Source publication
Cryptosporidium spp. are important causes of diarrhea in humans, ruminants, and other mammals. Comparative genomic analysis indicated that genetically related and host-adapted Cryptosporidium species have different numbers of subtelomeric genes encoding the Cryptosporidium-specific MEDLE family of secreted proteins, which could contribute to differ...
Contexts in source publication
Context 1
... staining of sporozoites with anti-MEDLE-2 polyclonal antibodies showed a diffused expression of MEDLE-2 within sporozoites, including the anterior end. The highest MEDLE-2 expression was in the area just above the nucleus, which was located at the 1/3 posterior end of sporozoites by DAPI staining (Figure 3, Top). The immunofluorescence staining of developmental stages in 24 and 48 h HCT-8 cultures showed the expression of MEDLE-2 proteins on both the parasitophorus vacuoles of meronts and merozoites within them (Figure 3, Middle, Bottom). ...
Context 2
... highest MEDLE-2 expression was in the area just above the nucleus, which was located at the 1/3 posterior end of sporozoites by DAPI staining (Figure 3, Top). The immunofluorescence staining of developmental stages in 24 and 48 h HCT-8 cultures showed the expression of MEDLE-2 proteins on both the parasitophorus vacuoles of meronts and merozoites within them (Figure 3, Middle, Bottom). ...
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Citations
... Serine protease Excystation [115] Arginine aminopeptidase Excystation [116] CSL Attachment, invasion [117] gp900 Invasion [118,119] gp40/15 Invasion, motility [21,120,121] TRAP-C1 Invasion, motility [122] CP47 Attachment [123] CpMIC1 Attachment, invasion, motility [124] P23 Attachment, motility [125,126] P30 Attachment, invasion [127] CPS-500 Attachment, motility [128] CpClec Attachment, infection [129] INS-15 Invasion [130] Cp2 Attachment, motility [131] Cpa135 Attachment, motility [132] CpMuc Invasion [133] CpMEDLE-1/2 Invasion [134,135] CpABC Nutrient transport [136] CpATPase1 ...
... hominis Ia subtype family) were evolutionarily close to those of the C. parvum MLG1 � (IIc subtype family), which has been described in the literature [45]. Cryptosporidium parvum IIc subtype has been associated with an anthroponotic transmission [45,49,50], and results from a comparative genomic study carried out by Nader et al. demonstrated that it shares loci with C. hominis, with high mutation levels in sub telomeric genes, where MEDLE proteins and insulin-like proteases are located [51,52]. ...
Multilocus Sequence Typing has become a useful tool for the study of the genetic diversity and population structure of different organisms. In this study, a MLST approach with seven loci (CP47, MS5, MS9, MSC6-7, TP14, and gp60) was used to analyze the genetic diversity of Cryptosporidium hominis and Cryptosporidium parvum isolated from 28 Colombian patients. Five Cryptosporidium species were identified: C. hominis, C. parvum, Cryptosporidium felis, Cryptosporidium meleagridis, and Cryptosporidium suis. Unilocus gp60 analysis identified four allelic families for C. hominis (Ia, Ib, Id, and Ie) and two for C. parvum (IIa and IIc). There was polymorphic behavior of all markers evaluated for both C. hominis and C. parvum, particularly with the CP47, MS5, and gp60 markers. Phylogenetic analysis with consensus sequences (CS) of the markers showed a taxonomic agreement with the results obtained with the 18S rRNA and gp60 gene. Additionally, two monophyletic clades that clustered the species C. hominis and C. parvum were detected, with a higher number of subclades within the monophyletic groups compared to those with the gp60 gene. Thirteen MLG were identified for C. hominis and eight for C. parvum. Haplotypic and nucleotide diversity were detected, but only the latter was affected by the gp60 exclusion from the CS analysis. The gene fixation index showed an evolutionary closeness between the C. hominis samples and a less evolutionary closeness and greater sequence divergence in the C. parvum samples. Data obtained in this work support the implementation of MLST analysis in the study of the genetic diversity of Cryptosporidium, considering the more detailed information that it provides, which may explain some genetic events that with an unilocus approach could not be established. This is the first multilocus analysis of the intra-specific variability of Cryptosporidium from humans in South America.
... These proteins can modulate signalling pathways for intracellular development of the parasite. Further, antisera raised against recombinant MEDLE1 or MEDLE2 have been show to diminish the efficiency of sporozoite infection by 40% (Fei et al., 2018;Li et al., 2017), stressing the important role played by these proteins. ...
Cryptosporidium parvum is a globally distributed zoonotic pathogen and a major cause of diarrhoeal disease in humans and ruminants. The parasite's life cycle comprises an obligatory sexual phase, during which genetic exchanges can occur between previously isolated lineages. Here, we compare 32 whole genome sequences from human- and ruminant-derived parasite isolates collected across Europe, Egypt and China. We identify three strongly supported clusters that comprise a mix of isolates from different host species, geographic origins, and subtypes. We show that: (1) recombination occurs between ruminant isolates into human isolates; (2) these recombinant regions can be passed on to other human subtypes through gene flow and population admixture; (3) there have been multiple genetic exchanges, and most are likely recent; (4) putative virulence genes are significantly enriched within these genetic exchanges, and (5) this results in an increase in their nucleotide diversity. We carefully dissect the phylogenetic sequence of two genetic exchanges, illustrating the long-term evolutionary consequences of these events. Our results suggest that increased globalisation and close human-animal contacts increase the opportunity for genetic exchanges between previously isolated parasite lineages, resulting in spillover and spillback events. We discuss how this can provide a novel substrate for natural selection at genes involved in host-parasite interactions, thereby potentially altering the dynamic coevolutionary equilibrium in the Red Queens arms race.
... The quantity of polyclonal antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot. The reactivity of anti-CDPK antibodies to native proteins in C. parvum sporozoites was assessed by Western blot as described [18], with polyclonal antibodies being used as primary antibodies and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Yeasen, Shanghai, China) as secondary antibodies. ...
As the invasion, egress, and growth of Cryptosporidium spp. are regulated by the calcium ion, calcium-dependent protein kinases (CDPKs) are considered potential drug targets against these pathogens. In this study, we expressed CpCDPK1 of Cryptosporidium parvum encoded by the cgd3_920 gene and CpCDPK9 encoded by the the cgd7_1260 gene in Escherichia coli, and we conducted some comparative studies with quantitative PCR, immunofluorescence staining, and in vitro neutralization assays. By immunofluorescence microscopy, CpCDPK1 was expressed over the entirety of the sporozoites, while CpCDPK9 was mainly expressed in the apical region. The expression of the cgd3_920 gene was the highest at 12 h of the in vitro culture, whereas the expression of the cgd7_1260 gene peaked between 2 h and 6 h. Polyclonal antibodies against these two CpCDPK proteins had similar neutralization efficiency on C. parvum growth, reaching approximately 40%. Of the 50 candidate compounds from the molecular docking of CpCDPK1, 10 had significant in vitro anti-cryptosporidial effects, but only one inhibited enzyme activity. For CpCDPK9, five of the forty-five candidate compounds showed significant in vitro anti-cryptosporidial effects. Results obtained from this study suggest that CpCDPK1 and CpCDPK9 might function differently in C. parvum infection.
... They also observed that the two branches had undergone genetic recombination, as evidenced by genetic exchanges between both branches and some incorporation of C. hominis related regions, mainly in C. p. anthroponosum. The majority of the detected recombination events are located in subtelomeric regions, which are a common hotspot for genes associated with host interactions and virulence in other apicomplexan parasites [54][55][56] and seem to play an important role in Cryptosporidium. ...
Cryptosporidiosis is ranked sixth in the list of the most important food-borne parasites globally, and it is an important contributor to mortality in infants and the immunosuppressed. Recently, the number of genome sequences available for this parasite has increased drastically. The majority of the sequences are derived from population studies of Cryptosporidium parvum and Cryptosporidium hominis, the most important species causing disease in humans. Work with this parasite is challenging since it lacks an optimal, prolonged, in vitro culture system, which accurately reproduces the in vivo life cycle. This obstacle makes the cloning of isolates nearly impossible. Thus, patient isolates that are sequenced represent a population or, at times, mixed infections. Oocysts, the lifecycle stage currently used for sequencing, must be considered a population even if the sequence is derived from single-cell sequencing of a single oocyst because each oocyst contains four haploid meiotic progeny (sporozoites). Additionally, the community does not yet have a set of universal markers for strain typing that are distributed across all chromosomes. These variables pose challenges for population studies and require careful analyses to avoid biased interpretation. This review presents an overview of existing population studies, challenges, and potential solutions to facilitate future population analyses.
... The copyright holder has placed this preprint this version posted June 4, 2021. authors to consider that MEDLE proteins may play a role in invasion and that host specificity is 297 rooted in receptor specificity (Fei et al., 2018;Li et al., 2017). In this study, we screened 298 polymorphic genes for the localization of the proteins they encode by epitope tagging the 299 endogenous loci. ...
The parasite Cryptosporidium is responsible for diarrheal disease in young children causing death, malnutrition, and growth delay. Cryptosporidium invades enterocytes where it develops in a unique intracellular niche. Infected cells exhibit profound changes in morphology, physiology and transcriptional activity. How the parasite effects these changes is poorly understood. We explored the localization of highly polymorphic proteins and found members of the C. parvum MEDLE protein family to be translocated into the cytoplasm of infected cells. All intracellular life stages engage in this export, which occurs after completion of invasion. Mutational studies defined an N-terminal host-targeting motif and demonstrated proteolytic processing at a specific leucine residue. Direct expression of MEDLE2 in mammalian cells triggered an ER stress response that was also observed during infection. Taken together, our studies reveal the presence of a Cryptosporidium secretion system capable of delivering pathogenesis factors into the infected enterocyte.
... They are known to be involved in host cell invasion but their presence and expression varies between Cryptosporidium species implying that they may have a role in defining host range. Although the precise function of this protein is still unclear, it gives an insight into how different Cryptosporidium species are able to infect different host species [112][113][114] . Interestingly, with the establishment of species-specific enteroid models, and methods for creating transgenic parasites, it may be possible to determine how the MEDLE proteins determine host range. ...
Toxoplasma gondii and Cryptosporidium spp. can cause devastating pathological effects in humans and livestock, and in particular to young or immunocompromised individuals. The current treatment plans for these enteric parasites are limited due to long drug courses, severe side effects, or simply a lack of efficacy. The study of the early interactions between the parasites and the site of infection in the small intestinal epithelium has been thwarted by the lack of accessible, physiologically relevant, and species‐specific models. Increasingly, 3D stem cell‐derived enteroid models are being refined and developed into sophisticated models of infectious disease. In this review we shall illustrate the use of enteroids to spearhead research into enteric parasitic infections, bridging the gap between cell line cultures and in vivo experiments.
... Cyclic-GMP dependent protein kinase G (PKG) has been identified in related organisms as a key mediator of the signal transduction cascade leading to merozoite egress [6][7][8][9][10]. In Toxoplasma gondii, PKG is upstream of microneme secretion and a cofactor in subsequent egress [9][10][11]. In Plasmodium falciparum, inhibition of PKG activity has been found to block egress, leading to clearance of infection and decreased transmission to mosquitoes [7,12]. ...
Cryptosporidiosis is an obligate intracellular pathogen causing diarrhea. Merozoite egress is essential for infection to spread between host cells. However, the mechanisms of egress have yet to be defined. We hypothesized that Cyclic GMP-Dependent Protein Kinase G (PKG) maybe involved in Cryptosporidium egress. In this study, Cryptosporidium parvum PKG was silenced by using antisense RNA sequences. PKG-silencing significantly inhibited egress of merozoites from infected HCT-8 cells into the supernatant and led to retention of intracellular forms within the host cells. This data identifies PKG as a key mediator of merozoite egress, a key step in the parasite lifecycle.
... In terms of host range, the latter species is unique for its broad host range which includes birds (Slavin, 1955), humans and other mammals (Chappell et al., 2011). Putative genetic determinants of host range have been described (Li et al., 2017), but due to a lack of suitable research tools, Cryptosporidium "host specificity genes" remain to be discovered, assuming they exist. ...
Parasites in the genus Cryptosporidium, phylum Apicomplexa, are found worldwide in the intestinal tract of many vertebrate species and in the environment. Driven by sensitive PCR methods, and the availability of abundant sequence data and reference genomes, the taxonomic complexity of the genus has steadily increased; 38 species have been named to date. Due to its public health importance, Cryptosporidium hominis has long attracted the interest of the research community. This species was initially described as infectious to humans only. This perception has persisted in spite of an increasing number of observations of natural and experimental infections of animals with this species. Here we summarize and discuss this literature published since 2000 and conclude that the host range of C. hominis is broader than originally described. The evolving definition of the C. hominis host range raises interesting questions about host specificity and the evolution of Cryptosporidium parasites.
... It was suggested that these proteins could be responsible for host expansion in C. parvum (Guo et al. 2015c). This has been recently supported by biological characterizations of MEDLE proteins, which appear to have developmentally regulated expression and are potentially involved in the invasion of host cells (Fei et al. 2018;Li et al. 2017;Xu et al. 2019). In addition, a recent study has shown that cgd6_5510 and cgd6_5520 in C. parvum are actually fragments of one gene (cgd6_5520-5510) encoding a full insulinase-like protease (INS20-19), and provided some preliminary data on the potential involvement of the protein in the invasion or early developmental process of C. parvum (Zhang et al. 2019). ...
Whole genomic sequencing (WGS) and comparative genomics are increasingly used in the characterization of Cryptosporidium spp. They are facilitated by the establishment of procedures for WGS analysis of clinical specimens without laboratory propagation of pathogens. Results of recent comparative genomics analysis suggest that gene duplication might be associated with broad host ranges of some zoonotic Cryptosporidium species and subtypes, while genetic recombination could be involved in the emergence of virulent subtypes. The availability of WGS data has further facilitated the development of advanced molecular typing tools. The use of these tools together with comparative genomics analyses has begun to improve the investigations of outbreaks in industrialized nations. More WGS data, however, are needed from both industrialized nations and developing countries before we can have in-depth understanding of the population genetics and evolution of Cryptosporidium spp. and genetic determinants of various phenotypic traits in human-pathogenic subtypes.
