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

Ni, Ni; Jia, Ruilian; Guo, Yaqiong; Li, Na; Wu, Haizhen; Feng, Yaoyu; Xiao, Lihua

doi:10.3389/fmicb.2020.00719

Cited by 7 publications

(16 citation statements)

References 29 publications

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“…In contrast, INS5 is expressed at lower levels in sporozoites and increases to peak at 36-48 hpi (29). 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.…”

Section: Discussionmentioning

confidence: 99%

“…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%

“…Of the 22 M16 metalloproteases in encoded the C. parvum genome, 18 of them belong to the M16A family and several of these have previously been studied including INS5, INS15, and INS20-19. INS15 and INS20-19 contain signal peptides suggesting they are in the secretory pathway, while INS5 does not have a predicted signal peptide (27–29). INS20-19 is expressed in sporozoites and localized to an apical compartment (27), while INS15 is expressed in a middle-anterior compartment in sporozoites and merozoites (28).…”

Section: Discussionmentioning

confidence: 99%

“…INS20-19 is expressed in sporozoites and localized to an apical compartment (27), while INS15 is expressed in a middle-anterior 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). INS5 is present in a punctate pattern in sporozoites and during merogony (29).…”

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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Mainmentioning

confidence: 99%

See 3 more Smart Citations

Insulinase like protease 1 contributes to macrogamont formation inCryptosporidium parvum

Xu¹,

Feng²,

Xiao³

et al. 2020

Preprint

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Insulinase-like Protease 1 Contributes to Macrogamont Formation in Cryptosporidium parvum

Feng

Xiao

et al. 2021

mBio

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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 an 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 were often in close proximity to large OW50-positive 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 the formation or maturation of macrogamonts and that its loss results in attenuated virulence in immunocompromised mice. IMPORTANCE Cryptosporidiosis is a debilitating diarrheal disease in young children in developing countries. The 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, 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, are useful for further characterizing the pathway that leads to macrogamont maturation and oocyst wall formation.

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Metallopeptidases as Key Virulence Attributes of Clinically Relevant Protozoa: New Discoveries, Perspectives, and Frontiers of Knowledge

Rigo

Cardoso

Galego

et al. 2023

CPPS

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This article provides a comprehensive review of several subclasses of metallo-type peptidases expressed by the main clinically relevant protozoa, including Plasmodium spp., Toxoplasma gondii, Cryptosporidium spp., Leishmania spp., Trypanosoma spp., Entamoeba histolytica, Giardia duodenalis, and Trichomonas vaginalis. These species comprise a diverse group of unicellular eukaryotic microorganisms responsible for widespread and severe human infections. Metallopeptidases, defined as hydrolases with activity mediated by divalent metal cation, play important roles in the induction and maintenance of parasitic infections. In this context, metallopeptidases can be considered veritable virulence factors in protozoa with direct/indirect participation in several key pathophysiological processes, including adherence, invasion, evasion, excystation, central metabolism, nutrition, growth, proliferation, and differentiation. Indeed, metallopeptidases have become an important and valid target to search for new compounds with chemotherapeutic purposes. The present review aims to gather updates regarding metallopeptidase subclasses, exploring their participation in protozoa virulence as well as investigating the similarity of peptidase sequences through bioinformatics techniques in order to discover clusters of greater relevance for the development of new broad antiparasitic molecules.

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

Cited by 7 publications

References 29 publications

Insulinase like protease 1 contributes to macrogamont formation inCryptosporidium parvum

Insulinase like protease 1 contributes to macrogamont formation inCryptosporidium parvum

Insulinase-like Protease 1 Contributes to Macrogamont Formation in Cryptosporidium parvum

Metallopeptidases as Key Virulence Attributes of Clinically Relevant Protozoa: New Discoveries, Perspectives, and Frontiers of Knowledge

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