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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 contact. This parasite attacks the small intestine, and the main symptom is a watery diarrhea 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 analyzed 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 and other “intestinal” species lack 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.

Cryptosporidium parvum is a common cause of a zoonotic disease and a main cause of diarrhea in newborns. Effective drugs or vaccines are still lacking. Oocyst is the infective form of the parasite; after its ingestion, the oocyst excysts and releases four sporozoites into the host 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) that was not previously described. To investigate this phenomenon, we isolated and resolved EVs from the excystation medium by differential ultracentrifugation. Fluorescence flow cytometry and transmission electron microscopy (TEM) experiments identified two types of sporozoite-derived vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs). Nanoparticle tracking analysis (NTA) revealed mode diameter of 181 nm for LEVs and 105 nm for SEVs, respectively. Immunodetection experiments proved the presence of CpRom1 and the Golgi protein CpGRASP in LEVs, while immune-electron microscopy trials demonstrated the localization of CpRom1 on the LEVs surface. TEM and scanning electron microscopy (SEM) showed that LEVs were generated by means of the budding of the outer membrane of sporozoites; conversely, the origin of SEVs remained uncertain. Distinct protein compositions were observed between LEVs and SEVs as evidenced by their corresponding electrophoretic profiles. Indeed, a dedicated proteomic analysis identified 5 and 16 proteins unique for LEVs and SEVs, respectively. 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. and this last group included the immunodominant parasite antigen glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.

BACKGROUND: Several species of protozoa cause acute or chronic gastroenteritis in humans, worldwide. The burden of disease is particularly high among children living in developing areas of the world, where transmission is favored by lower hygienic standards and scarce availability of safe water. However, asymptomatic infection and polyparasitism are also commonly observed in poor settings. Here, we investigated the prevalence of intestinal protozoa in two small fishing villages, Porto Said (PS) and Santa Maria da Serra (SM), situated along the river Tietê in the State of São Paolo, Brazil. The villages lack basic public infrastructure and services, such as roads, public water supply, electricity and public health services. METHODS: Multiple fecal samples were collected from 88 individuals in PS and from 38 individuals in SM, who were asymptomatic at the time of sampling and had no recent history of diarrheal disease. To gain insights into potential transmission routes, 49 dog fecal samples (38 from PS and 11 from SM) and 28 river water samples were also collected. All samples were tested by microscopy and PCR was used to genotype Giardia duodenalis, Blastocystis sp., Dientamoeba fragilis and Cryptosporidium spp. RESULTS: By molecular methods, the most common human parasite was Blastocystis sp. (prevalence, 45% in PS and 71% in SM), followed by D. fragilis (13.6% in PS, and 18.4% in SM) and G. duodenalis (18.2% in PS and 7.9% in SM); Cryptosporidium spp. were not detected. Sequence analysis revealed large genetic variation among Blastocystis samples, with subtypes (STs) 1 and 3 being predominant, and with the notable absence of ST4. Among G. duodenalis samples, assemblages A and B were detected in humans, whereas assemblages A, C and D were found in dogs. Finally, all D. fragilis samples from humans were genotype 1. A single dog was found infected with Cryptosporidium canis. River water samples were negative for the investigated parasites. CONCLUSIONS: This study showed a high carriage of intestinal parasites in asymptomatic individuals from two poor Brazilian villages, and highlighted a large genetic variability of Blastocystis spp. and G. duodenalis.

Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli , suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.

Background Cryptosporidium parvum is a major cause of diarrhea in children and ruminants at the earliest stages of life. Maternal antibodies represent the main shield of neonate mammals for most of the infections. Two recombinant antigens (SA35 and SA40), portions of two C. parvum proteins, were tested for their ability to induce immune responses in adult mice and for protection on neonate BALB/c mice born from females immunised by mucosal delivery of both peptides. Methods Adult BALB/c mice were intraperitoneally immunised with SA35 and SA40, separately or mixed, and their immune response was characterised. Furthermore, BALB/c pregnant mice were immunised by mucosal delivery with an SA35/40 mix, before and during pregnancy. Soon after birth, their offspring were infected with two doses (1 × 10 5 and 5 × 10 3 ) of C. parvum oocysts and the parasitic burden was determined at 5 and 9 days post-infection. Results Intraperitoneal immunisation with SA35 and SA40 induced specific IgG and IgG1 in serum, specific IgA in the intestinal mucosa, increase of CD3+/CD4+ and CD30+ cells in splenocytes, which produced IFN-γ. Neonates born from immunised mice and infected with 1 × 10 5 oocysts showed a significant reduction of oocysts and intestinal forms (23 and 42%, respectively). A reduction of all parasitic forms (96%; P  < 0.05) was observed when neonates were infected with 5 × 10 3 oocysts. Conclusions SA35 and SA40 peptides induce specific humoral and cell-mediated immune responses to C. parvum in adult mice. Moreover, mucosal administration of the SA35/40 mix in pregnant mice reduces C. parvum burden in their litters.

In order to provide additional data on the prevalence and genetic diversity of Dientamoeba fragilis in human populations, we conducted a study in children from low-income communities in Sao Paulo State, Brazil. Fecal samples from daycare center attendees up to 6 years old (n=156) and staff members (n=18) were submitted to PCR and sequencing of D. fragilis as well as to microscopic examination for the presence of other intestinal parasites. All children assessed were asymptomatic and 10.3% (16/156) were positive for D. fragilis. No worker was found to be positive. An association between Dientamoeba and coinfection with other intestinal parasites was observed. Concerning the genetic diversity, 14 and only two isolates were genotype 1 and genotype 2, respectively. Our findings outline interesting aspects: (1) asymptomatic children as carriers of Dientamoeba in communities in which environmental conditions ensure parasite transmission and, (2) association between Dientamoeba infection in young children and coinfection with other enteric parasites, reinforcing its transmission via the fecal-oral route.

The flagellated protozoan Giardia duodenalis is a common gastrointestinal parasite of mammals, including humans. Molecular characterizations have shown the existence of eight genetic groups (or assemblages) in the G. duodenalis species complex. Human infections are caused by assemblages A and B, which infect other mammals as well. Whether transmission routes, animal reservoirs and associations with specific symptoms differ for assemblage A and assemblage B is not clear. Furthermore, the occurrence and clinical significance of mixed (A+B) infections is also poorly understood. To date, the majority of PCR assays has been developed to identify all G. duodenalis assemblages based on the use of primers that bind to conserved regions, yet a reliable identification of specific assemblages is better achieved by ad hoc methods. The aim of this work was to design simple PCR assays that, based on the use of assemblage-specific primers, produce diagnostic bands of different lengths for assemblage A and B. We first generated novel sequence information from assemblage B, identified homologous sequences in the assemblage A genome, and designed primers at six independent loci. Experiments performed on DNA extracted from axenic cultures showed that two of the six assays can detect the equivalent of a single cyst and are not negatively influenced by disproportions between DNA of each assemblage, at least up to a 9:1 ratio. Further experiments on DNAs extracted from feces showed that the two assays can detect both assemblages in single tube reactions with excellent reliability. Finally, the robustness of these assays was demonstrated by testing a large collection of human isolates previously typed by multi-locus genotyping.

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.

Two class 1 integrons, In-t1 and In-t2, were previously identified in IncFI plasmids of Salmonella enterica serotype Typhimurium. Molecular analysis revealed a close physical link between the two integrons. In-t1 is preceded by the transposase genes of Tn 21 , whereas In-t2 is located downstream the 3′-conserved segment (3′-CS) of In-t1, in a head-to-tail configuration. In-t1 shows a peculiar sequence downstream the 3′-CS, containing an extended version of the open reading frame known as ORF341 (referred to as ORF341E) and a novel trimethoprim resistance gene, designated dfrA18. Retrospective analysis provided evidence for In-t1 insertion within Tn 1935 , a Tn 21 -related transposon identified in IncFI plasmids circulating among epidemic clones of multidrug-resistant S. enterica during the 1970s. Structural comparison between Tn 21 derivatives from recent and ancestor IncFI plasmids showed that In-t2 has been conserved by these replicons. In-t1 belongs to a novel family of class 1 integrons containing the ORF341E sequence, and appears to have been acquired by IncFI plasmids after the assembly of Tn 1935 . In-t1 insertion occurred within the 5′-conserved segment (5′-CS) proximal region of the resident In-t2.

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 which rapidly attack the enterocytes. The membrane protein CpRom1 is a large Rhomboid protease which is expressed by sporozoites and recognized as antigen by the host immune system. We observed the release of CpRom1 with extracellular micro vesicles not previously described. To investigate this phenomenon, we isolated extracellular vesicles (EVs) from 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) that had different mean sizes (150 nm and 60 nm, respectively). CpRom1 as well as the Golgi protein CpGRASP were exclusively found on LEVs by immunodetection and localization of CpRom1 on LEVs surface was further demonstrated by immune-electron microscopy. The LEVs can be generated by budding of the outer membrane of sporozoites as demonstrated by TEM and scanning electron microscopy (SEM). Differently, the origin of SEVs remained uncertain. Furthermore, differences between LEVs and SEVs were also observed in protein composition as showed by the electrophoretic profiles of the types of vesicles. 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) have been already identified in the molecular cargo of extracellular vesicles. Noteworthy, we identified 12 proteins unique to Cryptosporidium spp and that had never been associated with extracellular vesicles. This last group included the immunodominant parasite antigen, glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.Competing Interest StatementThe authors have declared no competing interest.Footnotes* To correct the order of figures in the manuscript.