Male and female Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal e... more Male and female Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal egress from the red blood cells by rupturing the two surrounding membranes, the parasitophorous vacuole and the red blood cell membranes. Proteins of the so‐called osmiophilic bodies, (OBs), secretory organelles resident in the cytoplasm, are important players in this process. Once gametes emerge, the female is ready to be fertilized while the male develops into motile flagellar gametes. Here, we describe the function(s) of PBANKA_1115200, which we named Gamete Egress Protein (GEP), a protein specific to malaria parasites. GEP is restricted to gametocytes, expressed in gametocytes of both genders and partly localizes to the OBs. A mutant lacking the protein shows aberrant rupture of the two surrounding membranes, while OBs discharge is delayed but not aborted. Moreover, we identified a second function of GEP during exflagellation since the axonemes of the male flagellar gametes were not motile. Genetic crossing experiments reveal that both genders are unable to establish infections in mosquitoes and thus the lack of GEP leads to a complete block in Plasmodium transmission from mice to mosquitoes. The combination of our results reveals essential and pleiotropic functions of GEP in Plasmodium gametogenesis.
Malaria long-term elimination depends on parasite transmission control. Plasmodium sexual stage m... more Malaria long-term elimination depends on parasite transmission control. Plasmodium sexual stage maturation in the mosquito, including egress from the host erythrocyte, is one of the prime targets for transmission-blocking interventions. This work aims to identify candidate molecules potentially involved in gamete emergence from the host erythrocyte, as novel transmission blocking targets. We analyzed by quantitative mass spectrometry the proteins released/secreted by purified Plasmodium falciparum gametocytes upon induction of gametogenesis. The proteome obtained showed a good overlap (74%) with the one previously characterized in similar conditions from gametocytes of the rodent malaria parasite P. berghei. Four candidates were selected based on comparative analysis of their abundance values in released vs total gametocyte proteome. We also characterized the P. falciparum orthologue of the microgamete surface protein (MiGS), a marker of male gametocyte secretory vesicles in murine ...
Research article Clinical and epidemiological correlates of antibody response to human papillomav... more Research article Clinical and epidemiological correlates of antibody response to human papillomaviruses (HPVs) as measured by a novel ELISA based on denatured recombinant HPV16 late (L) and early (E) antigens
Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and prote... more Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and protein composition. These dynamic assemblies are involved in several biological processes, including infection by intracellular pathogens. This work provides a comprehensive analysis of the composition of human erythrocyte membrane microdomains. Based on their floating properties, we also categorized the microdomain-associated proteins into clusters. Interestingly, erythrocyte microdomains include the vast majority of the proteins known to be involved in invasion by the malaria parasite Plasmodium falciparum. We show here that the Ecto-ADP-ribosyltransferase 4 (ART4) and Aquaporin 1 (AQP1), found within one specific cluster, containing the essential host determinant CD55, are recruited to the site of parasite entry and then internalized to the newly formed parasitophorous vacuole membrane. By generating null erythroid cell lines, we showed that one of these proteins, ART4, plays a role in P....
Hypertension affects 25% of the world's population and is considered a risk factor for cardiovasc... more Hypertension affects 25% of the world's population and is considered a risk factor for cardiovascular disorders and other diseases. The aim of this study was to examine the evidence regarding the acute effect of exercise on blood pressure (BP) using meta-analytic measures. Sixty-five studies were compared using effect sizes (ES), and heterogeneity and Z tests to determine whether the ES were different from zero. The mean corrected global ES for exercise conditions were-0.56 (-4.80 mmHg) for systolic BP (sBP) and-0.44 (-3.19 mmHg) for diastolic BP (dBP; z ≠ 0 for all; p < 0.05). The reduction in BP was significant regardless of the participant's initial BP level, gender, physical activity level, antihypertensive drug intake, type of BP measurement, time of day in which the BP was measured, type of exercise performed, and exercise training program (p < 0.05 for all). ANOVA tests revealed that BP reductions were greater if participants were males, not receiving antihypertensive medication, physically active, and if the exercise performed was jogging. A significant inverse correlation was found between age and BP ES, body mass index (BMI) and sBP ES, duration of the exercise's session and sBP ES, and between the number of sets performed in the resistance exercise program and sBP ES (p < 0.05). Regardless of the characteristics of the participants and exercise, there was a reduction in BP in the hours following an exercise session. However, the hypotensive effect was greater when the exercise was performed as a preventive strategy in those physically active and without antihypertensive medication.
Chlamydia trachomatis (Ct) and human papillomavirus (HPV) are the most common sexually transmitte... more Chlamydia trachomatis (Ct) and human papillomavirus (HPV) are the most common sexually transmitted pathogens. Whereas it is well known that infection with oncogenic HPV genotypes increases the risk of cervical cancer (CC), the implication of Ct in the pathogenesis of CC is still controversial. Hence, to investigate the possible implication of Ct infection alone, or with concomitant HPV infection, in the severity of cervical lesions, we conducted a study in 164 Caucasian HIV-negative women with abnormal Pap. Genomic HPV and Ct DNA were detected in 97 (59%) and 16 (10%) women respectively, and 15 (9%) of women were infected by both. Of the HPV positive samples, 89 (79%) were HR-HPV types or probable HR types and HPV16 was the most represented genotype. Interestingly, it was observed that co-infection was more frequent than HPV infection alone in women with high grade lesions.
By a differential quantitative proteomic approach, we identified proteins released by Plasmodium ... more By a differential quantitative proteomic approach, we identified proteins released by Plasmodium gametocytes from a transgenic line defective in male gametogenesis and its parental line, when activated to form gametes. Gender specificity of gametocyte egress-secretome was defined by a comparative analysis of proteins quantified in the two lines. Candidate proteins selected for validation confirmed the predicted gender-specificity and showed that these secreted proteins localize to different types of vesicles, suggesting that emergence from the host cell involves a tightly coordinated secretory apparatus. Graphical Abstract Highlights Quantitative analysis of Plasmodium sexual stage egress secretome. Activated gametocytes release gender-related proteins. Gametocyte egress process involves different types of vesicles. Plasmodium, the malaria parasite, undergoes a complex life cycle alternating between a vertebrate host and a mosquito vector of the genus Anopheles. In red blood cells of the vertebrate host, Plasmodium multiplies asexually or differentiates into gamete precursors, the male and female gametocytes, responsible for parasite transmission. Sexual stage maturation occurs in the midgut of the mosquito vector, where male and female gametes egress from the host erythrocytes to fuse and form a zygote. Gamete egress entails the successive rupture of two membranes surrounding the parasite, the parasitophorous vacuole membrane and the erythrocyte plasma membrane. In this study, we used the rodent model parasite Plasmodium berghei to design a label-free quantitative proteomic approach aimed at identifying gender-related proteins differentially released/secreted by purified mature gametocytes when activated to form gametes. We compared the abundance of molecules secreted by wild type gametocytes of both genders with that of a transgenic line defective in male gamete maturation and egress. This enabled us to provide a comprehensive data set of egress-related molecules and their gender specificity. Using specific antibodies, we validated eleven candidate molecules, predicted as either gender-specific or common to both male and female gametocytes. All of them localize to punctuate, vesicle-like structures that relocate to cell periphery upon activation, but only three of them localize to the gametocyte-specific secretory vesicles named osmiophilic bodies. Our results confirm that the egress process involves a tightly coordinated secretory apparatus that includes different types of vesicles and may put the basis for functional studies aimed at designing novel transmission-blocking molecules.
Male and female Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal e... more Male and female Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal egress from the red blood cells by rupturing the two surrounding membranes, the parasitophorous vacuole and the red blood cell membranes. Proteins of the so‐called osmiophilic bodies, (OBs), secretory organelles resident in the cytoplasm, are important players in this process. Once gametes emerge, the female is ready to be fertilized while the male develops into motile flagellar gametes. Here, we describe the function(s) of PBANKA_1115200, which we named Gamete Egress Protein (GEP), a protein specific to malaria parasites. GEP is restricted to gametocytes, expressed in gametocytes of both genders and partly localizes to the OBs. A mutant lacking the protein shows aberrant rupture of the two surrounding membranes, while OBs discharge is delayed but not aborted. Moreover, we identified a second function of GEP during exflagellation since the axonemes of the male flagellar gametes were not motile. Genetic crossing experiments reveal that both genders are unable to establish infections in mosquitoes and thus the lack of GEP leads to a complete block in Plasmodium transmission from mice to mosquitoes. The combination of our results reveals essential and pleiotropic functions of GEP in Plasmodium gametogenesis.
An enzyme-linked immunosorbent assay (ELISA) based on the recombinant Toscana virus nucleoprotein... more An enzyme-linked immunosorbent assay (ELISA) based on the recombinant Toscana virus nucleoprotein (rN) has been developed. Its sensitivity and specificity for the detection of virus-specific immunoglobulins G and M in human sera were similar to those of the ELISA that is based on an antigen extracted from infected mouse brain and that is routinely used for serodiagnosis.
Molecular & cellular proteomics : MCP, Aug 10, 2017
Membrane microdomains that include lipid rafts, are involved in key physiological and pathologica... more Membrane microdomains that include lipid rafts, are involved in key physiological and pathological processes and participate in the entry of endocellular pathogens. These assemblies, enriched in cholesterol and sphingolipids, form highly dynamic, liquid-ordered phases that can be separated from the bulk membranes thanks to their resistance to solubilization by non-ionic detergents. To characterize complexity and dynamics of detergent-resistant membranes of sexual stages of the rodent malaria parasite Plasmodium berghei, here we propose an integrated study of raft components based on proteomics, lipid analysis and bioinformatics. This analysis revealed unexpected heterogeneity and unexplored pathways associated with these specialized assemblies. Protein-protein relationships and protein-lipid co-occurrence were described through multi-component networks. The proposed approach can be widely applied to virtually every cell type in different contexts and perturbations, under physiologic...
The M RNA segment of Toscana (TOS) phlebovirus was cloned and the complete nucleotide sequence de... more The M RNA segment of Toscana (TOS) phlebovirus was cloned and the complete nucleotide sequence determined. The M RNA segment is 4215 nucleotides in length, and it contains a single major open reading frame (ORF) in the viral-complementary sequence, between nucleotides 18 and 4034, which can encode for a polyprotein of 1339 amino acids (Mr 149 kDa). The viral segment is expressed via a unique mRNA containing 10-14 non-templated nucleotides at the 5' end and it is truncated at the 3' end by about 140 nucleotides in a purine-rich region. In M predicted amino acid sequences, several hydrophobic regions have been identified. They could function as a signal sequence or a transmembrane region for the different proteins. Comparison of the deduced amino acid sequence of M precursor product revealed 38, 36, and 25% identity and 58, 56, and 47% similarity with those of Rift Valley fever (RVF), Punta Toro (PT) and Uukuniemi (UUK) viruses, respectively. Residues conserved among the proteins are mainly located at the COOH-portion of the precursor, while the major divergence is in the NSm coding regions. Based on sequence comparison and similarity of hydropathic pattern of TOS M segment with other phleboviruses the N-termini of TOS GN and Gc glycoproteins were placed at residues 297 and 936 of the precursor.
The nucleotide sequence of the Toscana (TOS) virus M RNA segment contains a single major open rea... more The nucleotide sequence of the Toscana (TOS) virus M RNA segment contains a single major open reading frame in the viral-complementary sequence, which can encode a polyprotein of 1339 amino acids. To map the TOS M segment product(s), different regions of the putative M polypeptide were expressed as glutathione S-transferase fusion proteins, which were purified and inoculated into mice to produce hyperimmune sera. By Western blot analysis, a protein of approximately 30 kDa and two glycoproteins, G1 and G2, with the same molecular mass (approximately 65 kDa) were identified in TOS virus-infected cells. The 30 kDa protein, which reacted with antibodies raised to the NH2-terminal, was found to be a non-structural protein (designated NSm). By immunoprecipitation analysis of TOS virus-infected cell lysates, both treated or untreated with tunicamycin, the relative positions of glycoproteins G1 and G2 were determined. The gene order, with respect to the genomic M RNA, was found to be 3'...
Human papillomaviruses (HPVs) are the primary etiological agents of cervical cancer and are also ... more Human papillomaviruses (HPVs) are the primary etiological agents of cervical cancer and are also involved in the development of other tumours (skin, head and neck). Serological survey of the HPV infections is important to better elucidate their natural history and to disclose antigen determinants useful for vaccine development. At present, the analysis of the HPV-specific antibodies has not diagnostic value for the viral infections, and new approaches are needed to correlate the antibody response to the disease outcome. The aim of this study is to develop a novel ELISA, based on five denatured recombinant HPV16 proteins, to be used for detection HPV-specific antibodies. The HPV16 L1, L2, E4, E6 and E7 genes were cloned in a prokaryotic expression vector and expressed as histidine-tagged proteins. These proteins, in a denatured form, were used in ELISA as coating antigens. Human sera were collected from women with abnormal PAP smear enrolled during an ongoing multicenter HPV-Pathogen...
Here we report a novel strategy for the induction of CD8 + T cell adaptive immune response agains... more Here we report a novel strategy for the induction of CD8 + T cell adaptive immune response against viral and tumor antigens. This approach relies on high levels of incorporation in HIV-1 VLPs of a mutant of HIV-1 Nef (Nef mut) which can act as anchoring element for foreign proteins. By in vitro assay, we found that VLPassociated Nef mut is efficiently cross-presented by antigen presenting cells. Inoculation in mice of VLPs incorporating the HPV-16 E7 protein fused to Nef mut led to an anti-E7 CD8 + T cell response much stronger than that elicited by E7 recombinant protein inoculated with incomplete Freund's adjuvant and correlating with well-detectable anti-E7 CTL activity. Most relevantly, mice immunized with Nef mut-E7 VLPs developed a protective immune response against tumors induced by E7 expressing tumor cells. These results make Nef mut VLPs a promising candidate for new vaccine strategies focused on the induction of CD8 + T cell immunity.
The arthropod-borne Toscana virus has been associated with acute neurological disease in humans. ... more The arthropod-borne Toscana virus has been associated with acute neurological disease in humans. In this study, the viral envelope glycoproteins were expressed in soluble form in a baculovirus system. The recombinant sGN and sGC proteins were used as viral antigens in a Western blot assay to analyze the specific immune response in sera from patients with recognized virus-associated aseptic meningitis. The anti-glycoprotein and the anti-nucleoprotein N IgG responses were compared by an immunoassay based on the recombinant proteins. In this system, all the sera showed a high reactivity to the N protein, but they differed in the response to the glycoproteins.
Persistent infection with high risk genotypes of human papillomavirus (HPV) is the cause of cervi... more Persistent infection with high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, one of most common cancer among woman worldwide, and represents an important risk factor associated with other anogenital and oropharyngeal cancers in men and women. Here, we designed a therapeutic vaccine based on integrase defective lentiviral vector (IDLV) to deliver a mutated nononcogenic form of HPV16 E7 protein, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer, fused to calreticulin (CRT), a protein able to enhance major histocompatibility complex class I antigen presentation (IDLV-CRT/E7). Vaccination with IDLV-CRT/E7 induced a potent and persistent E7-specific T cell response up to 1 year after a single immunization. Importantly, a single immunization with IDLV-CRT/E7 was able to prevent growth of E7-expressing TC-1 tumor cells and to eradicate established tumors in mice. The strong therapeutic effect induced by the IDLV-based vaccine in this preclinical model suggests that this strategy may be further exploited as a safe and attractive anticancer immunotherapeutic vaccine in humans.
Background: At present, seroreactivity is not a valuable parameter for diagnosis of Human Papillo... more Background: At present, seroreactivity is not a valuable parameter for diagnosis of Human Papillomavirus (HPV) infection but, it is potentially valuable as marker of viral exposure in elucidating the natural history of this infection. More data are needed to asses the clinical relevance of serological response to HPV. Objectives: The objective was to assess the clinical and epidemiological correlates of HPVseroreactivity in a cohort of HIV-negative and HIV-positive women. Methods: Seroreactivity of 96 women, evaluated in an ELISA test based on denatured HPV16 late (L) and early (E) antigens, was correlated with their clinical and epidemiological data previously collected for a multi-centre Italian study, HPV-PathogenISS study. Results: No significant correlation was found between HPV DNA detection and seroreactivity. Women, current smokers showed significantly less seroreactivity to L antigens as compared with the non-smokers. HIV-positive women showed significantly less (66.7%) antibody response as compared with HIV-negative women (89.3%), with particularly impaired response to L antigens. Women, HIV-positive and current smokers, showed by far the lowest seroprevalence (33.3%) as compared to 75.9% among all other women (OR = 0.158; 95%CI 0.036-0.695, p = 0.014; Fisher's exact test). Importantly, this association did not loose its significance when controlled for
Male and female Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal e... more Male and female Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal egress from the red blood cells by rupturing the two surrounding membranes, the parasitophorous vacuole and the red blood cell membranes. Proteins of the so‐called osmiophilic bodies, (OBs), secretory organelles resident in the cytoplasm, are important players in this process. Once gametes emerge, the female is ready to be fertilized while the male develops into motile flagellar gametes. Here, we describe the function(s) of PBANKA_1115200, which we named Gamete Egress Protein (GEP), a protein specific to malaria parasites. GEP is restricted to gametocytes, expressed in gametocytes of both genders and partly localizes to the OBs. A mutant lacking the protein shows aberrant rupture of the two surrounding membranes, while OBs discharge is delayed but not aborted. Moreover, we identified a second function of GEP during exflagellation since the axonemes of the male flagellar gametes were not motile. Genetic crossing experiments reveal that both genders are unable to establish infections in mosquitoes and thus the lack of GEP leads to a complete block in Plasmodium transmission from mice to mosquitoes. The combination of our results reveals essential and pleiotropic functions of GEP in Plasmodium gametogenesis.
Malaria long-term elimination depends on parasite transmission control. Plasmodium sexual stage m... more Malaria long-term elimination depends on parasite transmission control. Plasmodium sexual stage maturation in the mosquito, including egress from the host erythrocyte, is one of the prime targets for transmission-blocking interventions. This work aims to identify candidate molecules potentially involved in gamete emergence from the host erythrocyte, as novel transmission blocking targets. We analyzed by quantitative mass spectrometry the proteins released/secreted by purified Plasmodium falciparum gametocytes upon induction of gametogenesis. The proteome obtained showed a good overlap (74%) with the one previously characterized in similar conditions from gametocytes of the rodent malaria parasite P. berghei. Four candidates were selected based on comparative analysis of their abundance values in released vs total gametocyte proteome. We also characterized the P. falciparum orthologue of the microgamete surface protein (MiGS), a marker of male gametocyte secretory vesicles in murine ...
Research article Clinical and epidemiological correlates of antibody response to human papillomav... more Research article Clinical and epidemiological correlates of antibody response to human papillomaviruses (HPVs) as measured by a novel ELISA based on denatured recombinant HPV16 late (L) and early (E) antigens
Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and prote... more Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and protein composition. These dynamic assemblies are involved in several biological processes, including infection by intracellular pathogens. This work provides a comprehensive analysis of the composition of human erythrocyte membrane microdomains. Based on their floating properties, we also categorized the microdomain-associated proteins into clusters. Interestingly, erythrocyte microdomains include the vast majority of the proteins known to be involved in invasion by the malaria parasite Plasmodium falciparum. We show here that the Ecto-ADP-ribosyltransferase 4 (ART4) and Aquaporin 1 (AQP1), found within one specific cluster, containing the essential host determinant CD55, are recruited to the site of parasite entry and then internalized to the newly formed parasitophorous vacuole membrane. By generating null erythroid cell lines, we showed that one of these proteins, ART4, plays a role in P....
Hypertension affects 25% of the world's population and is considered a risk factor for cardiovasc... more Hypertension affects 25% of the world's population and is considered a risk factor for cardiovascular disorders and other diseases. The aim of this study was to examine the evidence regarding the acute effect of exercise on blood pressure (BP) using meta-analytic measures. Sixty-five studies were compared using effect sizes (ES), and heterogeneity and Z tests to determine whether the ES were different from zero. The mean corrected global ES for exercise conditions were-0.56 (-4.80 mmHg) for systolic BP (sBP) and-0.44 (-3.19 mmHg) for diastolic BP (dBP; z ≠ 0 for all; p < 0.05). The reduction in BP was significant regardless of the participant's initial BP level, gender, physical activity level, antihypertensive drug intake, type of BP measurement, time of day in which the BP was measured, type of exercise performed, and exercise training program (p < 0.05 for all). ANOVA tests revealed that BP reductions were greater if participants were males, not receiving antihypertensive medication, physically active, and if the exercise performed was jogging. A significant inverse correlation was found between age and BP ES, body mass index (BMI) and sBP ES, duration of the exercise's session and sBP ES, and between the number of sets performed in the resistance exercise program and sBP ES (p < 0.05). Regardless of the characteristics of the participants and exercise, there was a reduction in BP in the hours following an exercise session. However, the hypotensive effect was greater when the exercise was performed as a preventive strategy in those physically active and without antihypertensive medication.
Chlamydia trachomatis (Ct) and human papillomavirus (HPV) are the most common sexually transmitte... more Chlamydia trachomatis (Ct) and human papillomavirus (HPV) are the most common sexually transmitted pathogens. Whereas it is well known that infection with oncogenic HPV genotypes increases the risk of cervical cancer (CC), the implication of Ct in the pathogenesis of CC is still controversial. Hence, to investigate the possible implication of Ct infection alone, or with concomitant HPV infection, in the severity of cervical lesions, we conducted a study in 164 Caucasian HIV-negative women with abnormal Pap. Genomic HPV and Ct DNA were detected in 97 (59%) and 16 (10%) women respectively, and 15 (9%) of women were infected by both. Of the HPV positive samples, 89 (79%) were HR-HPV types or probable HR types and HPV16 was the most represented genotype. Interestingly, it was observed that co-infection was more frequent than HPV infection alone in women with high grade lesions.
By a differential quantitative proteomic approach, we identified proteins released by Plasmodium ... more By a differential quantitative proteomic approach, we identified proteins released by Plasmodium gametocytes from a transgenic line defective in male gametogenesis and its parental line, when activated to form gametes. Gender specificity of gametocyte egress-secretome was defined by a comparative analysis of proteins quantified in the two lines. Candidate proteins selected for validation confirmed the predicted gender-specificity and showed that these secreted proteins localize to different types of vesicles, suggesting that emergence from the host cell involves a tightly coordinated secretory apparatus. Graphical Abstract Highlights Quantitative analysis of Plasmodium sexual stage egress secretome. Activated gametocytes release gender-related proteins. Gametocyte egress process involves different types of vesicles. Plasmodium, the malaria parasite, undergoes a complex life cycle alternating between a vertebrate host and a mosquito vector of the genus Anopheles. In red blood cells of the vertebrate host, Plasmodium multiplies asexually or differentiates into gamete precursors, the male and female gametocytes, responsible for parasite transmission. Sexual stage maturation occurs in the midgut of the mosquito vector, where male and female gametes egress from the host erythrocytes to fuse and form a zygote. Gamete egress entails the successive rupture of two membranes surrounding the parasite, the parasitophorous vacuole membrane and the erythrocyte plasma membrane. In this study, we used the rodent model parasite Plasmodium berghei to design a label-free quantitative proteomic approach aimed at identifying gender-related proteins differentially released/secreted by purified mature gametocytes when activated to form gametes. We compared the abundance of molecules secreted by wild type gametocytes of both genders with that of a transgenic line defective in male gamete maturation and egress. This enabled us to provide a comprehensive data set of egress-related molecules and their gender specificity. Using specific antibodies, we validated eleven candidate molecules, predicted as either gender-specific or common to both male and female gametocytes. All of them localize to punctuate, vesicle-like structures that relocate to cell periphery upon activation, but only three of them localize to the gametocyte-specific secretory vesicles named osmiophilic bodies. Our results confirm that the egress process involves a tightly coordinated secretory apparatus that includes different types of vesicles and may put the basis for functional studies aimed at designing novel transmission-blocking molecules.
Male and female Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal e... more Male and female Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal egress from the red blood cells by rupturing the two surrounding membranes, the parasitophorous vacuole and the red blood cell membranes. Proteins of the so‐called osmiophilic bodies, (OBs), secretory organelles resident in the cytoplasm, are important players in this process. Once gametes emerge, the female is ready to be fertilized while the male develops into motile flagellar gametes. Here, we describe the function(s) of PBANKA_1115200, which we named Gamete Egress Protein (GEP), a protein specific to malaria parasites. GEP is restricted to gametocytes, expressed in gametocytes of both genders and partly localizes to the OBs. A mutant lacking the protein shows aberrant rupture of the two surrounding membranes, while OBs discharge is delayed but not aborted. Moreover, we identified a second function of GEP during exflagellation since the axonemes of the male flagellar gametes were not motile. Genetic crossing experiments reveal that both genders are unable to establish infections in mosquitoes and thus the lack of GEP leads to a complete block in Plasmodium transmission from mice to mosquitoes. The combination of our results reveals essential and pleiotropic functions of GEP in Plasmodium gametogenesis.
An enzyme-linked immunosorbent assay (ELISA) based on the recombinant Toscana virus nucleoprotein... more An enzyme-linked immunosorbent assay (ELISA) based on the recombinant Toscana virus nucleoprotein (rN) has been developed. Its sensitivity and specificity for the detection of virus-specific immunoglobulins G and M in human sera were similar to those of the ELISA that is based on an antigen extracted from infected mouse brain and that is routinely used for serodiagnosis.
Molecular & cellular proteomics : MCP, Aug 10, 2017
Membrane microdomains that include lipid rafts, are involved in key physiological and pathologica... more Membrane microdomains that include lipid rafts, are involved in key physiological and pathological processes and participate in the entry of endocellular pathogens. These assemblies, enriched in cholesterol and sphingolipids, form highly dynamic, liquid-ordered phases that can be separated from the bulk membranes thanks to their resistance to solubilization by non-ionic detergents. To characterize complexity and dynamics of detergent-resistant membranes of sexual stages of the rodent malaria parasite Plasmodium berghei, here we propose an integrated study of raft components based on proteomics, lipid analysis and bioinformatics. This analysis revealed unexpected heterogeneity and unexplored pathways associated with these specialized assemblies. Protein-protein relationships and protein-lipid co-occurrence were described through multi-component networks. The proposed approach can be widely applied to virtually every cell type in different contexts and perturbations, under physiologic...
The M RNA segment of Toscana (TOS) phlebovirus was cloned and the complete nucleotide sequence de... more The M RNA segment of Toscana (TOS) phlebovirus was cloned and the complete nucleotide sequence determined. The M RNA segment is 4215 nucleotides in length, and it contains a single major open reading frame (ORF) in the viral-complementary sequence, between nucleotides 18 and 4034, which can encode for a polyprotein of 1339 amino acids (Mr 149 kDa). The viral segment is expressed via a unique mRNA containing 10-14 non-templated nucleotides at the 5' end and it is truncated at the 3' end by about 140 nucleotides in a purine-rich region. In M predicted amino acid sequences, several hydrophobic regions have been identified. They could function as a signal sequence or a transmembrane region for the different proteins. Comparison of the deduced amino acid sequence of M precursor product revealed 38, 36, and 25% identity and 58, 56, and 47% similarity with those of Rift Valley fever (RVF), Punta Toro (PT) and Uukuniemi (UUK) viruses, respectively. Residues conserved among the proteins are mainly located at the COOH-portion of the precursor, while the major divergence is in the NSm coding regions. Based on sequence comparison and similarity of hydropathic pattern of TOS M segment with other phleboviruses the N-termini of TOS GN and Gc glycoproteins were placed at residues 297 and 936 of the precursor.
The nucleotide sequence of the Toscana (TOS) virus M RNA segment contains a single major open rea... more The nucleotide sequence of the Toscana (TOS) virus M RNA segment contains a single major open reading frame in the viral-complementary sequence, which can encode a polyprotein of 1339 amino acids. To map the TOS M segment product(s), different regions of the putative M polypeptide were expressed as glutathione S-transferase fusion proteins, which were purified and inoculated into mice to produce hyperimmune sera. By Western blot analysis, a protein of approximately 30 kDa and two glycoproteins, G1 and G2, with the same molecular mass (approximately 65 kDa) were identified in TOS virus-infected cells. The 30 kDa protein, which reacted with antibodies raised to the NH2-terminal, was found to be a non-structural protein (designated NSm). By immunoprecipitation analysis of TOS virus-infected cell lysates, both treated or untreated with tunicamycin, the relative positions of glycoproteins G1 and G2 were determined. The gene order, with respect to the genomic M RNA, was found to be 3'...
Human papillomaviruses (HPVs) are the primary etiological agents of cervical cancer and are also ... more Human papillomaviruses (HPVs) are the primary etiological agents of cervical cancer and are also involved in the development of other tumours (skin, head and neck). Serological survey of the HPV infections is important to better elucidate their natural history and to disclose antigen determinants useful for vaccine development. At present, the analysis of the HPV-specific antibodies has not diagnostic value for the viral infections, and new approaches are needed to correlate the antibody response to the disease outcome. The aim of this study is to develop a novel ELISA, based on five denatured recombinant HPV16 proteins, to be used for detection HPV-specific antibodies. The HPV16 L1, L2, E4, E6 and E7 genes were cloned in a prokaryotic expression vector and expressed as histidine-tagged proteins. These proteins, in a denatured form, were used in ELISA as coating antigens. Human sera were collected from women with abnormal PAP smear enrolled during an ongoing multicenter HPV-Pathogen...
Here we report a novel strategy for the induction of CD8 + T cell adaptive immune response agains... more Here we report a novel strategy for the induction of CD8 + T cell adaptive immune response against viral and tumor antigens. This approach relies on high levels of incorporation in HIV-1 VLPs of a mutant of HIV-1 Nef (Nef mut) which can act as anchoring element for foreign proteins. By in vitro assay, we found that VLPassociated Nef mut is efficiently cross-presented by antigen presenting cells. Inoculation in mice of VLPs incorporating the HPV-16 E7 protein fused to Nef mut led to an anti-E7 CD8 + T cell response much stronger than that elicited by E7 recombinant protein inoculated with incomplete Freund's adjuvant and correlating with well-detectable anti-E7 CTL activity. Most relevantly, mice immunized with Nef mut-E7 VLPs developed a protective immune response against tumors induced by E7 expressing tumor cells. These results make Nef mut VLPs a promising candidate for new vaccine strategies focused on the induction of CD8 + T cell immunity.
The arthropod-borne Toscana virus has been associated with acute neurological disease in humans. ... more The arthropod-borne Toscana virus has been associated with acute neurological disease in humans. In this study, the viral envelope glycoproteins were expressed in soluble form in a baculovirus system. The recombinant sGN and sGC proteins were used as viral antigens in a Western blot assay to analyze the specific immune response in sera from patients with recognized virus-associated aseptic meningitis. The anti-glycoprotein and the anti-nucleoprotein N IgG responses were compared by an immunoassay based on the recombinant proteins. In this system, all the sera showed a high reactivity to the N protein, but they differed in the response to the glycoproteins.
Persistent infection with high risk genotypes of human papillomavirus (HPV) is the cause of cervi... more Persistent infection with high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, one of most common cancer among woman worldwide, and represents an important risk factor associated with other anogenital and oropharyngeal cancers in men and women. Here, we designed a therapeutic vaccine based on integrase defective lentiviral vector (IDLV) to deliver a mutated nononcogenic form of HPV16 E7 protein, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer, fused to calreticulin (CRT), a protein able to enhance major histocompatibility complex class I antigen presentation (IDLV-CRT/E7). Vaccination with IDLV-CRT/E7 induced a potent and persistent E7-specific T cell response up to 1 year after a single immunization. Importantly, a single immunization with IDLV-CRT/E7 was able to prevent growth of E7-expressing TC-1 tumor cells and to eradicate established tumors in mice. The strong therapeutic effect induced by the IDLV-based vaccine in this preclinical model suggests that this strategy may be further exploited as a safe and attractive anticancer immunotherapeutic vaccine in humans.
Background: At present, seroreactivity is not a valuable parameter for diagnosis of Human Papillo... more Background: At present, seroreactivity is not a valuable parameter for diagnosis of Human Papillomavirus (HPV) infection but, it is potentially valuable as marker of viral exposure in elucidating the natural history of this infection. More data are needed to asses the clinical relevance of serological response to HPV. Objectives: The objective was to assess the clinical and epidemiological correlates of HPVseroreactivity in a cohort of HIV-negative and HIV-positive women. Methods: Seroreactivity of 96 women, evaluated in an ELISA test based on denatured HPV16 late (L) and early (E) antigens, was correlated with their clinical and epidemiological data previously collected for a multi-centre Italian study, HPV-PathogenISS study. Results: No significant correlation was found between HPV DNA detection and seroreactivity. Women, current smokers showed significantly less seroreactivity to L antigens as compared with the non-smokers. HIV-positive women showed significantly less (66.7%) antibody response as compared with HIV-negative women (89.3%), with particularly impaired response to L antigens. Women, HIV-positive and current smokers, showed by far the lowest seroprevalence (33.3%) as compared to 75.9% among all other women (OR = 0.158; 95%CI 0.036-0.695, p = 0.014; Fisher's exact test). Importantly, this association did not loose its significance when controlled for
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Papers by Stefania Mochi