Mechanisms of adaptation and evolution in Toxoplasma gondii.

Toxoplasma has high host flexibility, infecting all nucleated cells of mammals and birds. This implies that during its infective process the parasite must constantly adapt to different environmental situations, which in turn leads to modifications in its metabolism, regulation of gene transcription, translation of mRNAs and stage specific factors. There are conserved pathways that support these adaptations, which we aim to elucidate in this review. We begin by exploring the widespread epigenetic mechanisms and transcription regulators, continue with the supportive role of Heat Shock Proteins (Hsp), the translation regulation, stress granules, and finish with the emergence of contingency genes in highly variable genomic domains, such as subtelomeres. Within epigenetics, the discovery of a new histone variant of the H2B family (H2B.Z), contributing to T. gondii virulence and differentiation, but also gene expression regulation and its association with the metabolic state of the parasite, is highlighted. Associated with the regulation of gene expression are transcription factors (TFs). An overview of the main findings on TF and development is presented. We also emphasize the role of Hsp90 and Tgj1 in T. gondii metabolic fitness and the regulation of protein translation. Translation regulation is also highlighted as a mechanism for adaptation to conditions encountered by the parasite as well as stress granules containing mRNA and proteins generated in the extracellular tachyzoite. Another important aspect in evolution and adaptability are the subtelomeres because of their high variability and gene duplication rate. Toxoplasma possess multigene families of membrane proteins and contingency genes that are associated with different metabolic stresses. Among them parasite differentiation and environmental stresses stand out, including those that lead tachyzoite to bradyzoite conversion. Finally, we are interested in positioning protozoa as valuable evolution models, focusing on research related to the Extended Evolutionary Synthesis, based on models recently generated, such as extracellular adaptation and ex vivo cyst recrudescence.

Triclabendazole and clofazimine reduce replication and spermine uptake in vitro in Toxoplasma gondii.

Toxoplasmosis is a worldwide zoonosis caused by the protozoan parasite Toxoplasma gondii. Although this infection is generally asymptomatic in immunocompetent individuals, it can cause serious clinical manifestations in newborns with congenital infection or in immunocompromised patients. As current treatments are not always well tolerated, there is an urgent need to find new drugs against human toxoplasmosis. Drug repurposing has gained considerable momentum in the last decade and is a particularly attractive approach for the search of therapeutic alternatives to treat rare and neglected diseases. Thus, in this study, we investigated the antiproliferative effect of several repurposed drugs. Of these, clofazimine and triclabendazole displayed a higher selectivity against T. gondii, affecting its replication. Furthermore, both compounds inhibited spermine incorporation into the parasite, which is necessary for the formation of other polyamines. The data reported here indicate that clofazimine and triclabendazole could be used for the treatment of human toxoplasmosis and confirms that drug repurposing is an excellent strategy to find new therapeutic targets of intervention.

Prolonged survival of venereal Tritrichomonas foetus parasite in the gastrointestinal tract, bovine fecal extract, and water.

IMPORTANCE: Nowadays, the routine herd diagnosis is usually performed exclusively on bulls, as they remain permanently infected, and prevention and control of Tritrichomonas foetus transmission are based on identifying infected animals and culling practices. The existence of other forms of transmission and the possible role of pseudocysts or cyst-like structures as resistant forms requires rethinking the current management and control of this parasitic disease in the future in some livestock regions of the world.

Dicodon-based measures for modeling gene expression.

MOTIVATION: Codon usage preference patterns have been associated with modulation of translation efficiency, protein folding, and mRNA decay. However, new studies support that codon pair usage has also a remarkable effect at the gene expression level. Here, we expand the concept of CAI to answer if codon pair usage patterns can be understood in terms of codon usage bias, or if they offer new information regarding coding translation efficiency. RESULTS: Through the implementation of a weighting strategy to consider the dicodon contributions, we observe that the dicodon-based measure has greater correlations with gene expression level than CAI. Interestingly, we have noted that dicodons associated with a low value of adaptiveness are related to dicodons which mediate strong translational inhibition in yeast. We have also noticed that some codon-pairs have a smaller dicodon contribution than estimated by the product of the respective codon contributions. AVAILABILITY AND IMPLEMENTATION: Scripts, implemented in Python, are freely available for download at https://zenodo.org/record/7738276#.ZBIDBtLMIdU.

Proteomics Applications in Toxoplasma gondii: Unveiling the Host-Parasite Interactions and Therapeutic Target Discovery.

Toxoplasma gondii, a protozoan parasite with the ability to infect various warm-blooded vertebrates, including humans, is the causative agent of toxoplasmosis. This infection poses significant risks, leading to severe complications in immunocompromised individuals and potentially affecting the fetus through congenital transmission. A comprehensive understanding of the intricate molecular interactions between T. gondii and its host is pivotal for the development of effective therapeutic strategies. This review emphasizes the crucial role of proteomics in T. gondii research, with a specific focus on host-parasite interactions, post-translational modifications (PTMs), PTM crosstalk, and ongoing efforts in drug discovery. Additionally, we provide an overview of recent advancements in proteomics techniques, encompassing interactome sample preparation methods such as BioID (BirA*-mediated proximity-dependent biotin identification), APEX (ascorbate peroxidase-mediated proximity labeling), and Y2H (yeast two hybrid), as well as various proteomics approaches, including single-cell analysis, DIA (data-independent acquisition), targeted, top-down, and plasma proteomics. Furthermore, we discuss bioinformatics and the integration of proteomics with other omics technologies, highlighting its potential in unraveling the intricate mechanisms of T. gondii pathogenesis and identifying novel therapeutic targets.

Single cell gene expression profiling of nasal ciliated cells reveals distinctive biological processes related to epigenetic mechanisms in patients with severe COVID-19.

OBJECTIVE: To explore the molecular processes associated with cellular regulatory programs in patients with COVID-19, including gene activation or repression mediated by epigenetic mechanisms. We hypothesized that a comprehensive gene expression profiling of nasopharyngeal epithelial cells might expand our understanding of the pathogenic mechanisms of severe COVID-19. METHODS: We used single-cell RNA sequencing (scRNAseq) profiling of ciliated cells (n = 12,725) from healthy controls (SARS-CoV-2 negative n = 13) and patients with mild/moderate (n = 13) and severe (n = 14) COVID-19. ScRNAseq data at the patient level were used to perform gene set and pathway enrichment analyses. We prioritized candidate miRNA-target interactions and epigenetic mechanisms. RESULTS: We found that mild/moderate COVID-19 compared to healthy controls had upregulation of gene expression signatures associated with mitochondrial function, misfolded proteins, and membrane permeability. In addition, we found that compared to mild/moderate disease, severe COVID-19 had downregulation of epigenetic mechanisms, including DNA and histone H3K4 methylation and chromatin remodelling regulation. Furthermore, we found 11-ranked miRNAs that may explain miRNA-dependent regulation of histone methylation, some of which share seed sequences with SARS-CoV-2 miRNAs. CONCLUSION: Our results may provide novel insights into the epigenetic mechanisms mediating the clinical course of SARS-CoV-2 infection.

In-depth comparative analysis of Tritrichomonas foetus transcriptomics reveals novel genes linked with adaptation to feline host.

Tritrichomonas foetus is a flagellated parasite able to infect cattle, cats, and pigs. Despite its prevalence, feline tritrichomonosis has received markedly less attention than venereal infection, and little information about the molecular mechanisms that participate in feline host infection is available. Through a bioinformatics approach, we integrated public transcriptomic data for three T. foetus isolates and explored the differences at transcript level with a focus on pathogenesis and adaptation processes, particularly for the feline isolate. Our analysis revealed higher abundance levels of predicted virulence factors, such as proteases and surface antigens. Additionally, by a comparative and expression analysis of T. foetus genes, we proposed putative virulence factors that could be involved in feline infection. Finally, we identified a great proportion of predicted transcription factors of the MYB protein family and, by a promoter analysis, we revealed that MYB-related proteins could participate in the regulation of gene transcription in T. foetus. In conclusion, this integrated approach is a valuable resource for future studies of host-pathogen interactions and identifying new gene targets for improved feline tritrichomonosis diagnosis and treatment.

Evaluation of Toxoplasma gondii recombinant antigens for early diagnosis of congenital toxoplasmosis.

The performance of Toxoplasma rGra8, rMic1, and the chimeric rGra4-Gra7 antigens for early congenital toxoplasmosis (CT) diagnosis was evaluated. Sera from CT patients showed high IgG reactivity to rMic1, rGra8, and rGra4-Gra7. The seroreactivity of samples from uninfected infants was lost within 2 months of age.

Exploring protein myristoylation in Toxoplasma gondii.

Toxoplasma gondii is an important human and veterinary pathogen and the causative agent of toxoplasmosis, a potentially severe disease especially in immunocompromised or congenitally infected humans. Current therapeutic compounds are not well-tolerated, present increasing resistance, limited efficacy and require long periods of treatment. On this context, searching for new therapeutic targets is crucial to drug discovery. In this sense, recent works suggest that N-myristoyltransferase (NMT), the enzyme responsible for protein myristoylation that is essential in some parasites, could be the target of new anti-parasitic compounds. However, up to date there is no information on NMT and the extent of this modification in T. gondii. In this work, we decided to explore T. gondii genome in search of elements related with the N-myristoylation process. By a bioinformatics approach it was possible to identify a putative T. gondii NMT (TgNMT). This enzyme that is homologous to other parasitic NMTs, presents activity in vitro, is expressed in both intra- and extracellular parasites and interacts with predicted TgNMT substrates. Additionally, NMT activity seems to be important for the lytic cycle of Toxoplasma gondii. In parallel, an in silico myristoylome predicts 157 proteins to be affected by this modification. Myristoylated proteins would be affecting several metabolic functions with some of them being critical for the life cycle of this parasite. Together, these data indicate that TgNMT could be an interesting target of intervention for the treatment of toxoplasmosis.

Gene target discovery with network analysis in Toxoplasma gondii.

Infectious diseases are of great relevance for global health, but needed drugs and vaccines have not been developed yet or are not effective in many cases. In fact, traditional scientific approaches with intense focus on individual genes or proteins have not been successful in providing new treatments. Hence, innovations in technology and computational methods provide new tools to further understand complex biological systems such as pathogen biology. In this paper, we apply a gene regulatory network approach to analyze transcriptomic data of the parasite Toxoplasma gondii. By means of an optimization procedure, the phenotypic transitions between the stages associated with the life cycle of T. gondii were embedded into the dynamics of a gene regulatory network. Thus, through this methodology we were able to reconstruct a gene regulatory network able to emulate the life cycle of the pathogen. The community network analysis has revealed that nodes of the network can be organized in seven communities which allow us to assign putative functions to 338 previously uncharacterized genes, 25 of which are predicted as new pathogenic factors. Furthermore, we identified a small gene circuit that drives a series of phenotypic transitions that characterize the life cycle of this pathogen. These new findings can contribute to the understanding of parasite pathogenesis.

Prediction of cell position using single-cell transcriptomic data: an iterative procedure.

Single-cell sequencing reveals cellular heterogeneity but not cell localization. However, by combining single-cell transcriptomic data with a reference atlas of a small set of genes, it would be possible to predict the position of individual cells and reconstruct the spatial expression profile of thousands of genes reported in the single-cell study. With the purpose of developing new algorithms, the Dialogue for Reverse Engineering Assessments and Methods (DREAM) consortium organized a crowd-sourced competition known as DREAM Single Cell Transcriptomics Challenge (SCTC). Within this context, we describe here our proposed procedures for adequate reference genes selection, and an iterative procedure to predict spatial expression profile of other genes.

A comprehensive analysis of direct and photosensitized attenuation of Toxoplasma gondii tachyzoites.

In the present work, we have evaluated the effect of three different types of radiation: UVC (254±5nm), UVA (365±20nm) and visible (420±20nm) on different morphological and biological functions of Toxoplasma gondii tachyzoites. Briefly, UVC and UVA showed an inhibitory effect on parasite invasion in a dose-dependent manner. UVC showed the strongest effect inducing both structural damage (antigens) and functional inhibition (i.e., invasion and replication). On its own, visible light induces a quite distinctive and selective pattern of parasite-attenuation. This type of incident radiation inhibits the replication of the parasite affecting neither the capability of invasion/attachment nor the native structure of proteins (antigens) on parasites. Such effects are a consequence of photosensitized processes where phenol red might act as the active photosensitizer. The potential uses of the methodologies investigated herein are discussed.

Identification of new palmitoylated proteins in Toxoplasma gondii.

Protein palmitoylation has been shown to be an important post-translational modification in eukaryotic cells. This modification alters the localization and/or the function of the targeted protein. In recent years, protein palmitoylation has risen in importance in apicomplexan parasites as well. In Toxoplasma gondii, some proteins have been reported to be modified by palmitate. With the development of new techniques that allow the isolation of palmitoylated proteins, this significant post-translational modification has begun to be studied in more detail in T. gondii. Here we describe the palmitoylome of the tachyzoite stage of T. gondii using a combination of the acyl-biotin exchange chemistry method and mass spectrometry analysis. We identified 401 proteins found in multiple cellular compartments, with a wide range of functions that vary from metabolic processes, gliding and host-cell invasion to even regulation of transcription and translation. Besides, we found that more rhoptry proteins than the ones already described for Toxoplasma are palmitoylated, suggesting an important role for this modification in the invasion mechanism of the host-cell. This study documents that protein palmitoylation is a common modification in T. gondii that could have an impact on different cellular processes.

Wavelets-based clustering of air quality monitoring sites.

This paper aims at providing a variance/covariance profile of a set of 36 monitoring stations measuring ozone (O3) and nitrogen dioxide (NO2) hourly concentrations, collected over the period 2005-2013, in Portugal mainland. The resulting individual profiles are embedded in a wavelet decomposition-based clustering algorithm in order to identify groups of stations exhibiting similar profiles. The results of the cluster analysis identify three groups of stations, namely urban, suburban/urban/rural, and a third group containing all but one rural stations. The results clearly indicate a geographical pattern among urban stations, distinguishing those located in Lisbon area from those located in Oporto/North. Furthermore, for urban stations, intra-diurnal and daily time scales exhibit the highest variance. This is due to the more relevant chemical activity occurring in high NO2 emissions areas which are responsible for high variability on daily profiles. These chemical processes also explain the reason for NO2 and O3 being highly negatively cross-correlated in suburban and urban sites as compared with rural stations. Finally, the clustering analysis also identifies sites which need revision concerning classification according to environment/influence type.

Overview of object oriented data analysis.

Object oriented data analysis is the statistical analysis of populations of complex objects. In the special case of functional data analysis, these data objects are curves, where a variety of Euclidean approaches, such as principal components analysis, have been very successful. Challenges in modern medical image analysis motivate the statistical analysis of populations of more complex data objects that are elements of mildly non-Euclidean spaces, such as lie groups and symmetric spaces, or of strongly non-Euclidean spaces, such as spaces of tree-structured data objects. These new contexts for object oriented data analysis create several potentially large new interfaces between mathematics and statistics. The notion of object oriented data analysis also impacts data analysis, through providing a framework for discussion of the many choices needed in many modern complex data analyses, especially in interdisciplinary contexts.

Rabbit antibodies against Toxoplasma Hsp20 are able to reduce parasite invasion and gliding motility in Toxoplasma gondii and parasite invasion in Neospora caninum.

Toxoplasma gondii Hsp20 is a pellicle-associated functional chaperone whose biological role is still unknown. Hsp20 is present in different apicomplexan parasites, showing a high degree of conservation across the phylum, with Neospora caninum Hsp20 presenting an 82% identity to that of T. gondii. Hence rabbit anti-T. gondii Hsp20 serum was able to recognize the N. caninum counterpart. Interestingly, both N. caninum and T. gondii Hsp20 localized to the inner membrane complex and to the plasma membrane. Incubation of T. gondii and N. caninum tachyzoites with an anti-TgHsp20 serum reduced parasite invasion at rates of 57.23% and 54.7%, respectively. This anti-serum also reduced T. gondii gliding 48.7%. Together, all this data support a role for Hsp20 in parasite invasion and gliding motility.

Breast carcinomas fulfill the Warburg hypothesis and provide metabolic markers of cancer prognosis.

The aim of this study was to investigate selected proteomic markers of the metabolic phenotype of breast carcinomas as prognostic markers of cancer progression. For this purpose, a series of 101 breast carcinomas and 13 uninvolved breast samples were examined for quantitative differences in protein expression of mitochondrial and glycolytic markers. The beta-subunit of the mitochondrial H(+)-ATP synthase (beta-F1-ATPase) and heat shock protein 60 (Hsp60), and the glycolytic glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase were identified by immunological techniques. Correlations of the expression level of the protein markers and of the ratios derived from them were established with the clinicopathological information of the tumors and the follow-up data of the patients. The metabolic proteome of breast cancer specimens revealed a pronounced shift towards an enhanced glycolytic phenotype concurrent with a profound alteration on the mitochondrial beta-F1-ATPase/Hsp60 ratio when compared with normal samples. Discriminant analysis using markers of the metabolic signature as predictor variables revealed a classification sensitivity of approximately 97%. Kaplan-Meier survival analysis showed that several of the proteomic variables significantly correlated with overall and disease-free survival of the patients. The expression level of beta-F1-ATPase per se allowed the identification of a subgroup of breast cancer patients with significantly worse prognosis. Multivariate Cox regression analysis indicated that tumor expression of beta-F1-ATPase is a significant marker independent from clinical variables to assess the prognosis of the patients. We conclude that the alteration of the mitochondrial and glycolytic proteomes is a hallmark feature of breast cancer further providing relevant markers to aid in the prognosis of breast cancer patients.

Parent phenotype and age dependence, on rat glioma tumor rejection.

BACKGROUND: The brain, despite the blood-brain barrier, does not escape to the highly variable host rejection response mediated by a very strong and complex immune reaction when rat glioma cells are transplanted into the adult animal. METHODS: Crosses were performed among parents that are able or enable to reject a well-known brain tumor cell line (C6). Newborn animals were also challenged with rat glioma cells both in the brain and the side flanks. RESULTS: The percentage of susceptibility or resistance to develop a lethal glioma can be estimated knowing the parental phenotypes. When both parents had rejected an induced tumor, 63% of the progeny will also reject it. Similarly, if both parents died as a consequence of the tumor, 70% of the progeny would also be unable to reject the challenge of glioma C6 cells. Newborn animals do not have a mature immune system and they tolerate transplanted cells much better than adults. We found no rejection to glioma C6, at both brain and side flank sites, in 1-day-old neonatal Wistar rats. Tumors were beginning to be eliminated if the cells are inoculated at day 3 from birth on the flanks, and at 1 week from birth on the brain. CONCLUSIONS: There is a genetic component conferring susceptibility or resistance to the lethal effect of tumor development and progression depending on the parental phenotype of the adult rats. Neonatal rats represent a much more reliable model than adults to study experimental therapies against gliomas.

The bioenergetic signature of lung adenocarcinomas is a molecular marker of cancer diagnosis and prognosis.

The aim of this study was to investigate the mitochondrial bioenergetic signature of lung adenocarcinomas as a prognostic marker of cancer progression. For this purpose, a series of 90 lung adenocarcinomas and 10 uninvolved lung samples were examined for quantitative differences in protein expression using two-dimensional polyacrylamide gel electrophoresis. The beta subunit of the mitochondrial H(+)-ATP synthase (beta-F1-ATPase) and heat shock protein 60 (Hsp 60), and the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used to define the bioenergetic cellular (BEC) index, were identified using mass spectrometry and specific antibodies. Correlations of the expression level of the protein markers and of the BEC index were established with the clinicopathological information of the tumors and the follow-up data of the patients. The expression of beta-F1-ATPase is significantly reduced in lung adenocarcinomas in the absence of significant changes in the expression of Hsp 60 and of a major GAPDH isoform. Cross-validation analysis using the beta-F1-ATPase/Hsp 60 ratio and GAPDH expression as predictor variables revealed a classification sensitivity of 97.3%. The beta-F1-ATPase/Hsp 60 ratio is significantly higher in well differentiated and bronchioloalveolar tumors than in moderate or poorly differentiated and in bronchial-derived tumors. The BEC index of T1 tumors was significantly higher than that of T2 tumors. Likewise, stage IA tumors had a higher BEC index than stage IB tumors. Kaplan-Meier survival analysis using the BEC index as predictor of survival revealed that within tumors of the same size or stage I or with no lymph node metastasis (N0) the patients bearing 'low' BEC index tumors had a significant worse prognosis. We conclude that the bioenergetic signature of lung adenocarcinomas is altered, further providing a relevant marker for the diagnosis and classification of lung adenocarcinomas, and for the prognosis of lung cancer patients.