Characterization of INS-15, A Metalloprotease Potentially Involved in the Invasion of Cryptosporidium parvum

Xu, Rui; Guo, Yaqiong; Li, Na; Zhang, Qiang; Wu, Haizhen; Ryan, Una; Feng, Yaoyu; Xiao, Lihua

doi:10.3390/microorganisms7100452

Cited by 20 publications

(26 citation statements)

References 31 publications

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“…INS genes in C. parvum are expressed at various levels and with different patterns (Mauzy et al, 2012), implying that these INS proteins play different biological functions at various life cycle stages. This has been supported by recent studies of , which have different expression patterns (Xu et al, 2019;Zhang et al, 2019). Another INS that has four domains of classic INS, INS-4 encoded by the cgd2_930 gene, has been identified as a putative rhoptry protein of sporozoites (Sanderson et al, 2008).…”

Section: Discussionmentioning

confidence: 65%

“…In this study, we have conducted one of the first studies of INS proteins in Cryptosporidium spp. Previously, only two INS proteins of Cryptosporidium spp., , had been characterized biologically (Xu et al, 2019;Zhang et al, 2019). We selected INS-5 in this study because it has homologs in most other Cryptosporidium species such as Cryptosporidium hominis, Cryptosporidium muris, and Cryptosporidium ubiquitum (Liu et al, 2016), indicating that it has conserved functions among Cryptosporidium spp.…”

Section: Discussionmentioning

confidence: 99%

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Expression and Functional Studies of INS-5, an Insulinase-Like Protein in Cryptosporidium parvum

Jia

Guo

et al. 2020

Front. Microbiol.

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The small Cryptosporidium genome (∼9 Mb) has over 20 copies of genes encoding insulinase-like proteases (INS), suggesting that these enzymes may have important biological functions in the pathogen and could be developmentally regulated. In this study, INS-5, a unique member of the INS family in Cryptosporidium parvum, was cloned and expressed in Escherichia coli BL21 (DE3). In addition to the predicted INS-5 of ∼78 kDa, smaller fragments of ∼70, ∼55, and ∼30 kDa were simultaneously generated. After purification through a nickel-nitrilotriacetic acid affinity column, the full recombinant protein obtained was used to prepare polyclonal antibodies. Antibodies raised against INS-5 recognized the recombinant protein and native protein in sporozoite extracts. Further characterization of INS-5 included qRT-PCR assessment of gene expression; immunofluorescence localization of the protein expression in sporozoites, merozoites, and other developmental stages; and neutralization of invasion of C. parvum in vitro. The results obtained indicated that although INS-5 was expressed in sporozoites and merozoites, the high gene expression was from 36 to 48 h of the in vitro culture after invasion. Anti-INS-5 antibodies partially neutralized the invasion (inhibition rate = 38.5%). Results of this study suggest that INS-5 plays some role in the invasion and growth of C. parvum.

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Section: Discussionmentioning

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Expression and Functional Studies of INS-5, an Insulinase-Like Protein in Cryptosporidium parvum

Jia

Guo

et al. 2020

Front. Microbiol.

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“…In contrast, the INS1 is expressed exclusively in macrogamonts, indicating that these M16 metalloproteases have different biological roles. (27), while INS15 is expressed in a middleanterior compartment in sporozoites and merozoites (28). In contrast, INS5 is expressed at lower levels in sporozoites and increases to peak at 36-48 hpi (29).…”

Section: Discussionmentioning

confidence: 97%

“…INS5 is present in a punctate pattern in sporozoites and during merogony (29). Antibodies to these proteins have been shown to inhibit parasite invasion in vitro (27)(28)(29), suggesting they play some role in processing or maturation of substrates involved in host cell recognition. INS1 is a classic M16A protease containing a signal peptide and an active motif "HLIEH" in the N-terminal domain followed by three domains that lack this catalytic motif.…”

Section: Discussionmentioning

confidence: 99%

“…Ten of these contain the active site "HXXEH" indicating they might act as enzymes in parasite, although none of their substrates have been defined. Previous studies have shown that antibodies against INS20-19 (although originally annotated as two genes, later assemblies collapsed these into a single gene), INS15, or INS5 inhibit parasite invasion of host cells in vitro, suggesting they are involved processing substrates important for host cell recognition or entry (27)(28)(29). In the present study, we focused on INS1, which is present among all Cryptosporidium spp.. INS1 is a classic M16A family member with one active functional motif "HXXEH" followed by three inactive domains.…”

Section: Mainmentioning

confidence: 99%

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Insulinase like protease 1 contributes to macrogamont formation inCryptosporidium parvum

Xu¹,

Feng²,

Xiao³

et al. 2020

Preprint

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The apicomplexan parasite Cryptosporidium parvum contains an expanded family of 22 insulinase like proteases (INS), a feature that contrasts with their otherwise streamlined genome. Here we examined the function of INS1, which is most similar to the human insulinase protease that cleaves a variety of small peptide substrates. INS1 is a M16A clan member and contains a signal peptide, an N-terminal domain with the HxxEH active site, followed by three inactive domains. Unlike previously studied C. parvum INS proteins that are expressed in sporozoites and during merogony, INS1 was expressed exclusively in macrogamonts, where it was localized in small cytoplasmic vesicles. Although INS1 did not colocalize with the oocyst wall protein recognized by the antibody OW50, immune-electron microscopy indicated that INS1 resides in small vesicles in the secretory system. Notably, these small INS1 positive vesicles often subtend large vacuoles resembling wall forming bodies, which contain precursors for oocyst wall formation. Genetic deletion of INS1, or replacement with an active site mutant, resulted in lower formation of macrogamonts in vitro and reduced oocyst shedding in vivo. Our findings reveal that INS1 functions in formation or maturation of macrogamonts and that its loss results in attenuated virulence in immunocompromised mice.ImportanceCryptosporidiosis is a debilitating diarrheal disease in young children in developing countries. Absence of effective treatments or vaccines makes this infection very difficult to manage in susceptible populations. Although the oral dose of oocysts needed to cause infection is low, infected individuals shed very high numbers of oocysts, hence readily contaminating the environment. Our studies demonstrate that the protease INS1 is important for formation of female sexual stages and that in its absence, parasites produce fewer oocysts and are attenuated in immunocompromised mice. These findings suggest that mutants lacking INS1, or related proteases, may be useful for producing attenuated vaccines to induce immunity without causing disease.

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Cryptosporidium

Köster,

Dacal,

Dashti

et al. 2024

Molecular Medical Microbiology

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Characterization of INS-15, A Metalloprotease Potentially Involved in the Invasion of Cryptosporidium parvum

Cited by 20 publications

References 31 publications

Expression and Functional Studies of INS-5, an Insulinase-Like Protein in Cryptosporidium parvum

Expression and Functional Studies of INS-5, an Insulinase-Like Protein in Cryptosporidium parvum

Insulinase like protease 1 contributes to macrogamont formation inCryptosporidium parvum

Cryptosporidium

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