Biology subjects

Bertuccini, L.

Publications and source records attributed to Bertuccini, L..

3 recordsLinked to original sources

bioRxiv2025

Exploring the Repertoire of Rhomboid Proteases in Cryptosporidium parvum Parasite: Phylogenesis, Structural motifs and Cellular Localization in Sporozoite Cells

Cryptosporidium parvum is an apicomplexan parasite and an important pathogen of mammals and humans, which can be infected by zoonotic transmission or directly by human-to-human contacts. This parasite attacks the small intestine, and the main symptom is a watery diarrhoea that can be particularly severe in newborns and deadly in immunodeficient subjects. Rhomboids are ubiquitous proteases embedded in cell membranes that act by cleaving other membrane proteins in or near their transmembrane domains. Apicomplexan rhomboids play an important role in approaching and invading the host cell. This study analysed the phylogenetic origin, the structural motifs and the subcellular localization of C. parvum rhomboids. Altogether, C. parvum possesses three rhomboids, namely CpRom1, CpRom2 and CpRom3. The similarity search in Cryptosporidium genus revealed that C. parvum as well as other "intestinal" species lacks a PARL-like rhomboid whereas this type of mitochondrial rhomboid was present in "gastric" species like Cryptosporidium muris and Cryptosporidium andersoni. At the genome level this was revealed by a precise excision of the PARL-like gene in intestinal species whereas the rest of chromosomal synteny was well conserved among the Cryptosporidium species. The analysis of the structural domains revealed that C. parvum rhomboids can be classified as mixed secretases and the comparison with orthologs from Toxoplasma gondii and Plasmodium falciparum showed that C. parvum rhomboids can be distinguished in two separate clusters based on similarities at the level of the catalytic sites. The three rhomboids were expressed simultaneously in the invasive stage of sporozoite, but each of them had a different spatial distribution. Indeed, CpRom1 had a dual localization: this rhomboid was internal at the apical complex, and it was also accumulated at the posterior pole of the sporozoite. Otherwise, CpRom2 was prevalently contained in the apical complex, and a point of accumulation was on the surface of the apical end. Differently from CpRom1 and CpRom2, CpRom3 is distributed along the entire surface of sporozoites. Finally, we listed 10 membrane proteins as candidate substrates for the C. parvum rhomboids based on the similarities with some proven substrates of apicomplexan rhomboids and the copresence in subcellular structures with the three rhomboids.

microbiology↗

bioRxiv2023

Unveiling Cryptosporidium parvum Sporozoite-Derived Extracellular Vesicles: Profiling, Origin, and Protein Composition

Cryptosporidium parvum is a common cause of a zoonotic disease and a main cause of diarrhea in newborns around the world. Effective drugs or vaccines are still lacking. Oocyst is the infective form of the parasite; after the ingestion the oocyst excysts and releases four sporozoites into the intestine that rapidly attack the enterocytes. The membrane protein CpRom1 is a large rhomboid protease that is expressed by sporozoites and recognized as antigen by the host immune system. In this study, we observed the release of CpRom1 with extracellular vesicles (EVs) not previously described. To investigate this phenomenon, we isolated and resolved EVs from the excystation medium by differential ultracentrifugation. By fluorescence flow cytometry and transmission electron microscopy (TEM), we identified two types of sporozoite-derived vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs), having different a mean size of 150 nm and 60 nm, respectively. Immunodetection experiments proved the occurrence of CpRom1 and the Golgi protein CpGRASP in LEVs, while immune-electron microscopy experiments demonstrated localization of CpRom1 on LEVs surface. TEM and scanning electron microscopy (SEM) showed the generation of LEVs by budding of the outer membrane of sporozoites; conversely, the origin of SEVs remained uncertain. Differences between LEVs and SEVs were observed for protein composition as proved by the corresponding electrophoretic profiles. Indeed, a dedicated proteomic analysis identified 5 proteins unique to LEVs composition and 16 proteins unique to SEVs. Overall, 60 proteins were identified in the proteome of both types of vesicles and most of these proteins (48 in number) were already identified in the molecular cargo of extracellular vesicles from other organisms. Noteworthy, we identified 12 proteins unique to Cryptosporidium spp. that had never been associated with EVs. This last group included the immunodominant parasite antigen glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.

microbiology↗

bioRxiv2022

Functional characterization of the thrombospondin-related paralogous proteins rhoptry discharge factor 1 and 2 unveils phenotypic plasticity in Toxoplasma gondii rhoptry exocytosis

To gain access to the intracellular cytoplasmic niche essential for their growth and replication, apicomplexan parasites such as Toxoplasma gondii rely on the timely secretion of two types of apical organelles named micronemes and rhoptries. Rhoptry proteins are key to host cell invasion and remodelling, however, the molecular mechanisms underlying the thight control of rhoptry discharge are poorly understood. Here, we report the identification and functional characterization of two novel T. gondii thrombospondin-related proteins implicated in rhoptry exocytosis. The two proteins, already annotated as MIC15 and MIC14, were renamed rhoptry discharge factor 1 (RDF1) and rhoptry discharge factor 2 (RDF2) and found to be exclusive of the Coccidia class of apicomplexan parasites. Furthermore, they were shown to have a paralogous relationship and share a C-terminal transmembrane domain followed by a short cytoplasmic tail. Immunofluorescence analysis of T. gondii tachyzoites revealed that RDF1 presents a diffuse cytoplasmic localization not reminiscent of any know subcellular compartment, whereas RDF2 was not detected. Using a conditional knockout approach, we demonstrated that RDF1 loss caused a marked growth defect. The lack of the protein did not affect parasite gliding motility, host cell attachment, replication and egress, whereas invasion was dramatically reduced. Notably, while RDF1 depletion did not result in altered microneme exocytosis, rhoptry discharge was found to be heavily impaired. Interestingly, rhoptry secretion was partially reversed by spontaneous upregulation of the RDF2 gene in knockdown parasites grown under constant RDF1 repression. Collectively, our results identify RDF1 and RDF2 as additional key players in the pathway controlling rhoptry discharge. Furthermore, this study unveils a new example of compensatory mechanism contributing to phenotypic plasticity in T. gondii.

microbiology↗