Determinación de anticuerpos para péptidos derivados de la proteína ROP18 en individuos con toxoplasmosis ocular

Introducción: La importancia de la proteína ROP 18 de Toxoplasma gondii surge en su sobreexpresión de esta proteína en la invasión a la célula hospedera, la cual varía de acuerdo con el linaje clonal. No se conoce si existen diferencias en el reconocimiento por anticuerpos específicos contra esta proteína en la toxoplasmosis ocular. Objetivo: Establecer la presencia de anticuerpos por medio de la técnica ELISA en los individuos con toxoplasmosis ocular para los péptidos derivados de la proteína ROP18 de cada uno de los linajes clónales (I, II, y III) de Toxoplasma gondiiPregradoLicenciado en Biología y Educación Ambienta

Molecular cloning and characterization of NcROP2Fam-1, a member of the ROP2 family of rhoptry proteins in Neospora caninum that is targeted by antibodies neutralizing host cell invasion in vitro

Recent publications demonstrated that a fragment of a Neospora caninum ROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein, N. caninum ROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of ‘cysts' produced in vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasion in vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccine

The effects of development and virulence on gene expression in the protozoan parasite Neospora caninum

Neospora caninum is an obligate intracellular protozoan parasite which causes abortion in cattle and neuromuscular disease in dogs. Neosporosis causes substantial economic losses in both dairy and beef cattle industries worldwide. This thesis explores changes in genes and proteins expression associated with development and virulence of N. caninum in order to gain more biological information in this parasite. To achieve the global quantification of genes and proteins expression under different phenotypic conditions, transcriptomics and proteomics approaches are used to investigate these phenomenons. Chapter 2 examines the differential protein expression during tachyzoites-bradyzoites stage conversion using a label-free proteomics approach and finds that the differential protein expression of most apical secretory proteins decrease in abundance in bradyzoites stage. In addition, most of proteins associated with parasite motility were also reduced in abundance in bradyzoites. This indicates that proteins associated with host cell adhesion, invasion and gliding motility are down-regulated in the quiescent cyst-forming stage. Chapter 3 compares the transcriptomic profiles of low- and high-virulence N. caninum using RNA-Seq and finds that protein phosphatase 2C (PP2C) was preferentially expressed in the high-virulence strain, while a group of SRS family proteins was preferentially expressed in low-virulence conditions. This finding suggests that PP2C might play a role in virulence, while a group of SRS proteins might be associated with to limiting N. caninum virulence. Chapter 4 compares protein expression in low- and high-virulence N. caninum strains at three different time points using a label-free quantitative proteomic approach and finds that Rop24 was preferentially expressed in high-virulence N. caninum at all time points. Proteins associated with host cell attachment such as SAG3, TgSRS35A and cathepsin C2 also showed preferentially expression throughout the time course. This finding indicates that Rop24 might be associated with the N. caninum virulence, while the discovery of proteins associated with host cell attachment in low-virulence conditions might suggest that such strains are more efficient in invading host cells than high-virulence strains. Overall, this thesis identifies genes and proteins such as the rhoptry associated proteins PP2C and Rop24, which might be associated with virulence in neosporosis. It also shows that many proteins involved in host cell adhesion, invasion and parasite movement decrease in abundance in the cyst-forming bradyzoite stage. These discoveries enhance our understanding of parasite biology, providing a basis for future research into novel ways to prevent and control the disease by inhibiting parasite survival and transmission

Immunostimulatory efficacy and protective potential of putative TgERK7 protein in mice experimentally infected by Toxoplasma gondii

© 2020 The Author(s) The extracellular signal-regulated kinases (ERKs) serve as important determinants of cellular signal transduction pathways, and hence may play important roles during infections. Previous work suggested that putative ERK7 of Toxoplasma gondii is required for efficient intracellular replication of the parasite. However, the antigenic and immunostimulatory properties of TgERK7 protein remain unknown. The objective of this study was to produce a recombinant TgERK7 protein in vitro and to evaluate its effect on the induction of humoral and T cell-mediated immune responses against T. gondii infection in BALB/c mice. Immunization using TgERK7 mixed with Freund's adjuvants significantly increased the ratio of CD3e+CD4+ T/CD3e+CD8a+ T lymphocytes in spleen and elevated serum cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-12p70, IL-23, MCP-1, and TNF-α) in immunized mice compared to control mice. On the contrary, immunization did not induce high levels of serum IgG antibodies. Five predicted peptides of TgERK7 were synthesized and conjugated with KLH and used to analyze the antibody specificity in the sera of immunized mice. We detected a progressive increase in the antibody level only against TgERK7 peptide A (DEVDKHVLRKYD). Antibody raised against this peptide significantly decreased intracellular proliferation of T. gondii in vitro, suggesting that peptide A can potentially induce a protective antibody response. We also showed that immunization improved the survival rate of mice challenged with a virulent strain and significantly reduced the parasite cyst burden within the brains of chronically infected mice. Our data show that TgERK7-based immunization induced TgERK7 peptide A-specific immune responses that can impart protective immunity against T. gondii infection. The therapeutic potential of targeting ERK7 signaling pathway for future toxoplasmosis treatment is warranted

A comparison of host-parasite interactions between Toxoplasma gondii and Neospora caninum

Toxoplasma gondii and Neospora caninum are closely related obligatory intracellular apicomplexan parasites that invade and multiply in almost all mammalian host cells. They cause disease in immunocompromised animals, abortion in the intermediates host and great economic losses to the farming industry. However, there are several biological differences between these parasites, including host range, zoonotic capacity, transmission, virulence and definitive host. What causes these biological differences is not well understood. To fully understand these biological differences, the host-parasite interactions of these parasites have been investigated in this study using several different approaches at the molecular level. Due to the importance of protein-protein interactions (PPIs) and multiple protein complexes (MPCs) in host-parasite interactions, blue native polyacrylamide gel electrophoresis (BN-PAGE) combined with liquid chromatography tandem mass spectrometry (LC MS/MS) was used to study T. gondii and N. caninum tachyzoites. Several interesting complexes were identified in N. caninum tachyzoites and include mitochondrial complexes, proteasome, glideosome and moving junction molecules that play an important role in the physiology and invasion of host cells. In addition, in T. gondii the microneme MIC1/6 complex was found migrated and/or co-associated with the important surface antigen glycoprotein SAG1, which is critical in the initial interaction with host surface peptidoglycan. In order to understand the direct interactions between parasite secretory proteins such as dense granule proteins (GRA2 and GRA7) and host cell proteins; a pull-down assay has been used to elucidate the binding partners of expressed recombinant GRA2 and GRA7 in both parasites within the host cell lysate. Several methods were applied to purify the recombinant GRA proteins such as affinity chromatography using nickel or cobalt, salting-out, denaturing buffer using urea and reverse phase HPLC. TgGRA2 was successfully purified by HPLC and attempts have been made to study its role in host-parasite interactions using a pull-down assay. Since these parasites secrete an array of secretory proteins, including kinases, to manipulate host cell responses; phosphopeptide enrichment, combined with LC MS/MS has been used to study the global response in the host signalling pathway through protein phosphorylation and signal transduction in response to infection with T. gondii and N. caninum. Three important differences were identified; about one-third of the phosphoproteomes of the host cell in response to infection by T. gondii and N. caninum was different. Approximately 21 % of the phospho-motifs were found differentially enriched between host cells infected with T. gondii compared to N. caninum infection and finally the pathway analysis showed that a few pathways were differentially enriched between infections with these parasites, such as glycolysis/gluconeogenesis and mTOR signalling pathway in infection with T. gondii Abstract vi but not with N. caninum. The differences in the host cell phosphoproteome indicated that these parasites interact with the host cell differently. As a means of understanding the broader host response to infection with these parasites at the systems biology level, integrated data analyses were performed on quantitative data from the transcriptome, proteome and phosphoproteome of host cells infected with the two parasites. Data analyses showed that host cells produce more proteins in response to infection with T. gondii than with N. caninum after 36 hours post infection (p.i.). In addition, data analysis showed that T. gondii inhibits apoptosis and acute inflammatory responses more when compared to N. caninum. Overall, the results presented in this thesis have provided new insights into the biological differences between T. gondii and N. caninum. Several interesting differences in host-parasite interactions at both the qualitative and quantitative levels were identified. These interactions are related to the virulence and transmission strategy of the parasites and so are potentially associated with the biological differences between these parasites

A comparison of host-parasite interactions between Toxoplasma gondii and Neospora caninum

Toxoplasma gondii and Neospora caninum are closely related obligatory intracellular apicomplexan parasites that invade and multiply in almost all mammalian host cells. They cause disease in immunocompromised animals, abortion in the intermediates host and great economic losses to the farming industry. However, there are several biological differences between these parasites, including host range, zoonotic capacity, transmission, virulence and definitive host. What causes these biological differences is not well understood. To fully understand these biological differences, the host-parasite interactions of these parasites have been investigated in this study using several different approaches at the molecular level. Due to the importance of protein-protein interactions (PPIs) and multiple protein complexes (MPCs) in host-parasite interactions, blue native polyacrylamide gel electrophoresis (BN-PAGE) combined with liquid chromatography tandem mass spectrometry (LC MS/MS) was used to study T. gondii and N. caninum tachyzoites. Several interesting complexes were identified in N. caninum tachyzoites and include mitochondrial complexes, proteasome, glideosome and moving junction molecules that play an important role in the physiology and invasion of host cells. In addition, in T. gondii the microneme MIC1/6 complex was found migrated and/or co-associated with the important surface antigen glycoprotein SAG1, which is critical in the initial interaction with host surface peptidoglycan. In order to understand the direct interactions between parasite secretory proteins such as dense granule proteins (GRA2 and GRA7) and host cell proteins; a pull-down assay has been used to elucidate the binding partners of expressed recombinant GRA2 and GRA7 in both parasites within the host cell lysate. Several methods were applied to purify the recombinant GRA proteins such as affinity chromatography using nickel or cobalt, salting-out, denaturing buffer using urea and reverse phase HPLC. TgGRA2 was successfully purified by HPLC and attempts have been made to study its role in host-parasite interactions using a pull-down assay. Since these parasites secrete an array of secretory proteins, including kinases, to manipulate host cell responses; phosphopeptide enrichment, combined with LC MS/MS has been used to study the global response in the host signalling pathway through protein phosphorylation and signal transduction in response to infection with T. gondii and N. caninum. Three important differences were identified; about one-third of the phosphoproteomes of the host cell in response to infection by T. gondii and N. caninum was different. Approximately 21 % of the phospho-motifs were found differentially enriched between host cells infected with T. gondii compared to N. caninum infection and finally the pathway analysis showed that a few pathways were differentially enriched between infections with these parasites, such as glycolysis/gluconeogenesis and mTOR signalling pathway in infection with T. gondii Abstract vi but not with N. caninum. The differences in the host cell phosphoproteome indicated that these parasites interact with the host cell differently. As a means of understanding the broader host response to infection with these parasites at the systems biology level, integrated data analyses were performed on quantitative data from the transcriptome, proteome and phosphoproteome of host cells infected with the two parasites. Data analyses showed that host cells produce more proteins in response to infection with T. gondii than with N. caninum after 36 hours post infection (p.i.). In addition, data analysis showed that T. gondii inhibits apoptosis and acute inflammatory responses more when compared to N. caninum. Overall, the results presented in this thesis have provided new insights into the biological differences between T. gondii and N. caninum. Several interesting differences in host-parasite interactions at both the qualitative and quantitative levels were identified. These interactions are related to the virulence and transmission strategy of the parasites and so are potentially associated with the biological differences between these parasites

A comparison of host-parasite interactions between Toxoplasma gondii and Neospora caninum

Toxoplasma gondii and Neospora caninum are closely related obligatory intracellular apicomplexan parasites that invade and multiply in almost all mammalian host cells. They cause disease in immunocompromised animals, abortion in the intermediates host and great economic losses to the farming industry. However, there are several biological differences between these parasites, including host range, zoonotic capacity, transmission, virulence and definitive host. What causes these biological differences is not well understood. To fully understand these biological differences, the host-parasite interactions of these parasites have been investigated in this study using several different approaches at the molecular level. Due to the importance of protein-protein interactions (PPIs) and multiple protein complexes (MPCs) in host-parasite interactions, blue native polyacrylamide gel electrophoresis (BN-PAGE) combined with liquid chromatography tandem mass spectrometry (LC MS/MS) was used to study T. gondii and N. caninum tachyzoites. Several interesting complexes were identified in N. caninum tachyzoites and include mitochondrial complexes, proteasome, glideosome and moving junction molecules that play an important role in the physiology and invasion of host cells. In addition, in T. gondii the microneme MIC1/6 complex was found migrated and/or co-associated with the important surface antigen glycoprotein SAG1, which is critical in the initial interaction with host surface peptidoglycan. In order to understand the direct interactions between parasite secretory proteins such as dense granule proteins (GRA2 and GRA7) and host cell proteins; a pull-down assay has been used to elucidate the binding partners of expressed recombinant GRA2 and GRA7 in both parasites within the host cell lysate. Several methods were applied to purify the recombinant GRA proteins such as affinity chromatography using nickel or cobalt, salting-out, denaturing buffer using urea and reverse phase HPLC. TgGRA2 was successfully purified by HPLC and attempts have been made to study its role in host-parasite interactions using a pull-down assay. Since these parasites secrete an array of secretory proteins, including kinases, to manipulate host cell responses; phosphopeptide enrichment, combined with LC MS/MS has been used to study the global response in the host signalling pathway through protein phosphorylation and signal transduction in response to infection with T. gondii and N. caninum. Three important differences were identified; about one-third of the phosphoproteomes of the host cell in response to infection by T. gondii and N. caninum was different. Approximately 21 % of the phospho-motifs were found differentially enriched between host cells infected with T. gondii compared to N. caninum infection and finally the pathway analysis showed that a few pathways were differentially enriched between infections with these parasites, such as glycolysis/gluconeogenesis and mTOR signalling pathway in infection with T. gondii Abstract vi but not with N. caninum. The differences in the host cell phosphoproteome indicated that these parasites interact with the host cell differently. As a means of understanding the broader host response to infection with these parasites at the systems biology level, integrated data analyses were performed on quantitative data from the transcriptome, proteome and phosphoproteome of host cells infected with the two parasites. Data analyses showed that host cells produce more proteins in response to infection with T. gondii than with N. caninum after 36 hours post infection (p.i.). In addition, data analysis showed that T. gondii inhibits apoptosis and acute inflammatory responses more when compared to N. caninum. Overall, the results presented in this thesis have provided new insights into the biological differences between T. gondii and N. caninum. Several interesting differences in host-parasite interactions at both the qualitative and quantitative levels were identified. These interactions are related to the virulence and transmission strategy of the parasites and so are potentially associated with the biological differences between these parasites

Utilizing interspecies comparisons between Toxoplasma gondii and Hammondia hammondi to elucidate mechanisms driving life stage development, life cycle flexibility, and pathogenesis

Multi-host life cycles of eukaryotic parasites are fundamentally important for their success because they are linked to the way that these parasites replicate, disseminate, transmit, and cause disease in each host. As parasites of this nature progress through their life cycle, they transition between different developmental stages that function to promote survival and transmission in a specific host environment. The transitions between these life stages are especially important for the parasites Toxoplasma gondii and Hammondia hammondi. T. gondii and H. hammondi are closely related Apicomplexans that share a large majority of their genes in near perfect synteny. Despite this genomic similarity, these parasites have stark differences in their life cycle flexibility and virulence. To begin to understand the mechanisms for these differences, we have compared the developmental programs of these parasite species in head-to-head experiments and identified critical differences in the timing of stage conversion and response to stress. We were able to exploit this knowledge to produce the first ever transgenic H. hammondi line. We also used the developmental differences between T. gondii and H. hammondi and thorough transcriptional comparisons to identify new factors driving life stage conversion in T. gondii. Specifically, we identified the gene Regulator of Cystogenesis 1 (ROCY1), which encodes a zinc finger CCCH motif containing protein that plays a critical role in in vitro tissue cyst formation and is required for tissue cyst formation in vivo. Our data also suggest that ROCY1 is regulated, at least in part, by a T. gondii transcription factor that serves as a regulator of differentiation, Bradyzoite-Formation Deficient 1 (BFD1). Together, our findings begin to elucidate the regulatory networks driving stage conversion in T. gondii and H. hammondi

Designing a multiepitope vaccine against toxoplasma gondii

Toxoplasmosis is a significant, life-threatening disease with medical, veterinary, and economic importance caused by Toxoplasma gondii. T. gondii infects about 25-30% of human population globally. There have been noteworthy efforts to control and limit the disease incidence. At present, the only approved vaccine for use in veterinary is attenuated tachyzoites of strain S48 that control congenital infection of ewe known as Toxovax, which has significantly reduced the rate of abortion in sheep. However, the vaccine is expensive and has the probability of changing into a pathogenic form thus, it is inappropriate for human use. At the moment, there is no effective vaccine for preventing the formation of chronic tissue cysts in an infected host. Therefore, this research aims to design a multiepitope vaccine against T. gondii using in silico method to predict and analyze B-cell and T-cell epitopes of ROP2, MIC3, and GRA7. The result showed that the selected epitopes were antigenic, non-allergen, non-toxic, and non-human homology which makes them appropriate for the construction of vaccines. The secondary and tertiary structure, as well as the physiochemical properties of the vaccine construct, were determined and validation experiments such as molecular docking and immune simulation were conducted. This validation process predicted that the candidate vaccine is stable and soluble in the biological environment. In conclusion, in silico method can be used to design a good vaccine for T. gondii