TY - JOUR
T1 - Insulinase-like protease 1 contributes to macrogamont formation in cryptosporidium parvum
AU - Xu, Rui
AU - Feng, Yaoyu
AU - Xiao, Lihua
AU - David Sibley, L.
N1 - Publisher Copyright:
© 2021 Xu et al.
PY - 2021/3/1
Y1 - 2021/3/1
N2 - 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. par-vum 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. Ourfindings 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 immunocompro-mised 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.
AB - 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. par-vum 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. Ourfindings 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 immunocompro-mised 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.
KW - Active-site mutant
KW - CRISPR/Cas9
KW - Cryptosporidiosis
KW - Oocyst shedding
KW - Pathogenesis
KW - Sexual development
UR - http://www.scopus.com/inward/record.url?scp=85102112145&partnerID=8YFLogxK
U2 - 10.1128/mBio.03405-20
DO - 10.1128/mBio.03405-20
M3 - Article
C2 - 33688009
AN - SCOPUS:85102112145
SN - 2161-2129
VL - 12
SP - 1
EP - 16
JO - mBio
JF - mBio
IS - 2
M1 - e03405-20
ER -