G-Quadruplex DNAzyme-Substrated CRISPR/Cas12 Assay for Label-Free Detection of Single-Celled Parasitic Infection.

Early diagnosis of parasitic diseases can dramatically alleviate medical, economic, and social burdens. Herein, we report a sensitive and label-free assay for diagnosing single-celled parasitic infections using G-quadruplex (G4) DNAzyme as a reporter for CRISPR/Cas12. The substitution of a fluorescent DNA reporter with G4 DNAzyme increased the sensitivity for detecting Leishmania donovani (L. donovani) by 5 times and obviated the need for using chemically labeled DNA probes. The G4 DNAzyme-substrated CRISPR/Cas12 (GsubCas12) assay yielded a limit of detection of 3.1 parasites in the detection of cultured L. donovani and was further applied to analyze L. donovani in infected mice. The results showed that the GsubCas12 assay could positively detect L. donovani in spleen samples from infected mice about 2 weeks after low-dose inoculation, nearly 2 weeks earlier than that of parasitological analysis. GsubCas12 assay is promising as a diagnostic tool for parasitic infection in resource-limited regions.

In Leishmania major, the Homolog of the Oncogene PES1 May Play a Critical Role in Parasite Infectivity.

Leishmaniasis is a neglected tropical disease caused by Leishmania spp. The improvement of existing treatments and the discovery of new drugs remain ones of the major goals in control and eradication of this disease. From the parasite genome, we have identified the homologue of the human oncogene PES1 in Leishmania major (LmjPES). It has been demonstrated that PES1 is involved in several processes such as ribosome biogenesis, cell proliferation and genetic transcription. Our phylogenetic studies showed that LmjPES encodes a highly conserved protein containing three main domains: PES N-terminus (shared with proteins involved in ribosomal biogenesis), BRCT (found in proteins related to DNA repair processes) and MAEBL-type domain (C-terminus, related to erythrocyte invasion in apicomplexan). This gene showed its highest expression level in metacyclic promastigotes, the infective forms; by fluorescence microscopy assay, we demonstrated the nuclear localization of LmjPES protein. After generating mutant parasites overexpressing LmjPES, we observed that these clones displayed a dramatic increase in the ratio of cell infection within macrophages. Furthermore, BALB/c mice infected with these transgenic parasites exhibited higher footpad inflammation compared to those inoculated with non-overexpressing parasites.

Gene-Specific Sex Effects on Susceptibility to Infectious Diseases.

Inflammation is an integral part of defense against most infectious diseases. These pathogen-induced immune responses are in very many instances strongly influenced by host's sex. As a consequence, sexual dimorphisms were observed in susceptibility to many infectious diseases. They are pathogen dose-dependent, and their outcomes depend on pathogen and even on its species or subspecies. Sex may differentially affect pathology of various organs and its influence is modified by interaction of host's hormonal status and genotype: sex chromosomes X and Y, as well as autosomal genes. In this Mini Review we summarize the major influences of sex in human infections and subsequently focus on 22 autosomal genes/loci that modify in a sex-dependent way the response to infectious diseases in mouse models. These genes have been observed to influence susceptibility to viruses, bacteria, parasites, fungi and worms. Some sex-dependent genes/loci affect susceptibility only in females or only in males, affect both sexes, but have stronger effect in one sex; still other genes were shown to affect the disease in both sexes, but with opposite direction of effect in females and males. The understanding of mechanisms of sex-dependent differences in the course of infectious diseases may be relevant for their personalized management.

How much epigenetics and quantitative trait loci (QTL) mapping tell us about parasitism maintenance and resistance/susceptibility to hosts.

Interactions between the environment, parasites, vectors, and/or intermediate hosts are complex and involve several factors that define the success or failure of an infection. Among these interactions that can affect infections by a parasite, it is possible to highlight the genetic and epigenetic mechanisms in hosts and parasites. The interaction between genetics, epigenetics, infection, and the host's internal and external environment is decisive and dictates the outcome of a parasitic infection and the resistance, susceptibility, and transmission of this parasite. Epigenetic changes become important mediators in the regulation of gene expression, allowing the evasion of the parasite to immune host barriers, its transmission to new hosts, and the end of its development cycle. Epigenetics is a new frontier in the understanding of the interaction mechanisms between parasite and host that, along with information from the gene regions associated with complex phenotypic variations, the Quantitative Trait Loci, brings new possibilities to investigate more modern and efficient approaches to the treatment, control, and eradication of parasitic diseases. In this brief review, a general overview of the use of epigenetic information and mapping of Quantitative Trait Loci was summarized, both in genes of parasites and hosts, for understanding the mechanisms of resistance and/or susceptibility in parasitic relationships; also, the main search platforms were quantitatively compared, aiming to facilitate access data produced over a period of twenty years.

The Autophagy Machinery in Human-Parasitic Protists; Diverse Functions for Universally Conserved Proteins.

Autophagy is a eukaryotic cellular machinery that is able to degrade large intracellular components, including organelles, and plays a pivotal role in cellular homeostasis. Target materials are enclosed by a double membrane vesicle called autophagosome, whose formation is coordinated by autophagy-related proteins (ATGs). Studies of yeast and Metazoa have identified approximately 40 ATGs. Genome projects for unicellular eukaryotes revealed that some ATGs are conserved in all eukaryotic supergroups but others have arisen or were lost during evolution in some specific lineages. In spite of an apparent reduction in the ATG molecular machinery found in parasitic protists, it has become clear that ATGs play an important role in stage differentiation or organelle maintenance, sometimes with an original function that is unrelated to canonical degradative autophagy. In this review, we aim to briefly summarize the current state of knowledge in parasitic protists, in the light of the latest important findings from more canonical model organisms. Determining the roles of ATGs and the diversity of their functions in various lineages is an important challenge for understanding the evolutionary background of autophagy.

The potential role of necroptosis in clinical diseases (Review).

As an important type of programmed cell death in addition to apoptosis, necroptosis occurs in a variety of pathophysiological processes, including infections, liver diseases, kidney injury, neurodegenerative diseases, cardiovascular diseases, and human tumors. It can be triggered by a variety of factors, such as tumor necrosis factor receptor and Toll­like receptor families, intracellular DNA and RNA sensors, and interferon, and is mainly mediated by receptor­interacting protein kinase 1 (RIP1), RIP3, and mixed lineage kinase domain­like protein. A better understanding of the mechanism of necroptosis may be useful in the development of novel drugs for necroptosis­related diseases. In this review, the focus is on the molecular mechanisms of necroptosis, exploring the role of necroptosis in different pathologies, discussing their potential as a novel therapeutic target for disease therapy, and providing suggestions for further study in this area.

Aquatic Exposure to Abscisic Acid Transstadially Enhances Anopheles stephensi Resistance to Malaria Parasite Infection.

The ancient stress signaling molecule abscisic acid (ABA) is ubiquitous in animals and plants but is perhaps most well-known from its early discovery as a plant hormone. ABA can be released into water by plants and is found in nectar, but is also present in mammalian blood, three key contexts for mosquito biology. We previously established that addition of ABA to Anopheles stephensi larval rearing water altered immature development and life history traits of females derived from treated larvae, while addition of ABA to an infected bloodmeal increased resistance of adult female A. stephensi to human malaria parasite infection. Here we sought to determine whether larval treatment with ABA could similarly impact resistance to parasite infection in females derived from treated larvae and, if so, whether resistance could be extended to another parasite species. We examined nutrient levels and gene expression to demonstrate that ABA can transstadially alter resistance to a rodent malaria parasite with hallmarks of previously observed mechanisms of resistance following provision of ABA in blood to A. stephensi.

The Role of MicroRNAs in Parasitology.

MicroRNAs (miRNAs), as epigenetic regulators, are small non-coding RNAs regulating gene expression in eukaryotes at the post-transcriptional level to control biological functions. MicroRNAs play a role in development, physiology, infection, immunity and the complex life cycles of parasites. Also, parasite infection can alter host miRNA expression that might result in either parasite clearance or infection. Over the past 20 years, thousands of miRNAs have been identified in the nematode Caenorhabditis elegans and other parasites. Thus, miRNA pathways are potential targets for the diagnostic and therapeutic control of parasitic diseases. Here, we review the current status and potential functions of miRNAs related to protozoans, helminths, and arthropods.

CCR5 and CCR5Δ32 in bacterial and parasitic infections: Thinking chemokine receptors outside the HIV box.

The CCR5 molecule was reported in 1996 as the main HIV-1 co-receptor. In that same year, the CCR5Δ32 genetic variant was described as a strong protective factor against HIV-1 infection. These findings led to extensive research regarding the CCR5, culminating in critical scientific advances, such as the development of CCR5 inhibitors for the treatment of HIV infection. Recently, the research landscape surrounding CCR5 has begun to change. Different research groups have realized that, since CCR5 has such important effects in the chemokine system, it could also affect other different physiological systems. Therefore, the effect of reduced CCR5 expression due to the presence of the CCR5Δ32 variant began to be further studied. Several studies have investigated the role of CCR5 and the impacts of CCR5Δ32 on autoimmune and inflammatory diseases, various types of cancer, and viral diseases. However, the role of CCR5 in diseases caused by bacteria and parasites is still poorly understood. Therefore, the aim of this article is to review the role of CCR5 and the effects of CCR5Δ32 on bacterial (brucellosis, osteomyelitis, pneumonia, tuberculosis and infection by Chlamydia trachomatis) and parasitic infections (toxoplasmosis, leishmaniasis, Chagas disease and schistosomiasis). Basic information about each of these infections was also addressed. The neglected role of CCR5 in fungal disease and emerging studies regarding the action of CCR5 on regulatory T cells are briefly covered in this review. Considering the "renaissance of CCR5 research," this article is useful for updating researchers who develop studies involving CCR5 and CCR5Δ32 in different infectious diseases.

Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba.

Pseudosuccinea columella snails transmit the trematode Fasciola hepatica, but in Cuba, six naturally occurring populations successfully resist parasite infection. Here, we present an updated distribution of P. columella in Cuba; 68 positive sites with the earliest records more abundant in west-central Cuba and with east-central populations generally corresponding to the newest samples. No records were found farther east. The IPA site reported 10.5% prevalence of F. hepatica-infected snails. Population genetics, studied through microsatellites, showed low allelic and multilocus genotypic richness (MLGT), mainly in susceptible populations, strong deviations from panmixia and high self-fertilization rates. Susceptible individuals were grouped in one major cluster containing the majority of MLGT, and two independent clusters grouped the MLGT of resistant individuals from western and central populations, respectively. From these, we propose that several introductions of P. columella occurred in Cuba, primarily in the west, with the early arrivals deriving on the resistant populations. A more recent introduction of susceptible P. columella carrying MLGT T and Y may have occurred, where the latter spread quickly through the island and possibly increase the risk of parasite transmission in Cuba since all snails naturally infected with F. hepatica were carriers of the MLGT Y. Interestingly, even though resistant populations are highly diverse and are likely the oldest within Cuba, they are only found in six localities characterized by soft (total hardness, TH = 6.3 ± 1.03°d) and slightly acidic (pH = 6.2 ± 0.12) waters with low richness in snail species (3.2 ± 1.02). This tendency was also observed in a two-year follow-up ecological study that was conducted on a farm where both phenotypes occurred in sympatry; colonization events by resistant over susceptible snails coincided with a reduction in the pH and TH of the water. A comparison of life traits in susceptible and resistant isolates reared at two different pH/TH conditions (5.9/4°d or 7.8/14°d) showed that low pH/TH negatively affects P. columella, irrespective of the phenotype. However, evidence of higher tolerance (higher survival, life expectancy, egg viability) to such conditions was observed in resistant isolates. Finally, we speculate that the limited distribution of resistant populations might be related to a better exploitation of sites that are less suitable to snails (thus, with lower competition), rather than to a differential ecological restriction to specific environmental conditions from susceptible P. columella.

Analysis of genome-wide SNPs based on 2b-RAD sequencing of pooled samples reveals signature of selection in different populations of Haemonchus contortus.

The parasitic nematode Haemonchus contortus is one of the world's most important parasites of small ruminants that causes significant economic losses to the livestock sector. The population structure and selection in its various strains are poorly understood. No study so far compared its different populations using genome-wide data. Here, we focused on different geographic populations of H. contours from China (Tibet, TB; Hubei, HB; Inner Mongolia, IM; Sichuan, SC), UK and Australia (AS), using genome-wide population-genomic approaches, to explore genetic diversity, population structure and selection. We first performed next-generation high-throughput 2b RAD pool sequencing using Illumina technology, and identified single-nucleotide polymorphisms (SNPs) in all the strains. We identified 75,187 SNPs for TB, 82,271 for HB, 82,420 for IM, 79,803 for SC, 83,504 for AS and 78,747 for UK strain. The SNPs revealed low-nucleotide diversity (pi= 0.0092-0.0133) within each strain, and a significant differentiation level (average Fst = 0.34264) among them. Chinese populations TB and SC, along with the UK strain, were more divergent populations. Chinese populations IM and HB showed affinities to the Australian strain. We then analysed signature of selection and detected 44 (UK) and 03 (AS) private selective sweeps containing 49 and 05 genes, respectively. Finally, we performed the functional annotation of selective sweeps and proposed biological significance to signature of selection. Our data suggest that 2b-RAD pool sequencing can be used to assess the signature of selection in H. contortus.

Non-Viral Parasites Associated with Carcinogenesis.

Cancer is a proficient evader of the immune system and is responsible for a high number of deaths annually. Most of these cancer cases are associated with genetic mutations, viruses, radiations or other carcinogenic substances like tobacco smoke. However, a significant number of cases arise as a result of infection by certain parasitic organisms other than viruses. This review tries to explore various less studied mechanisms by which these parasites induce cancer and lead to its progression. The changes brought by organisms in the genetic makeup are enumerated along with the effects of various protein products synthesised by these organisms.

Parasitic Protozoa: Unusual Roles for G-Quadruplexes in Early-Diverging Eukaryotes.

Guanine-quadruplex (G4) motifs, at both the DNA and RNA levels, have assumed an important place in our understanding of the biology of eukaryotes, bacteria and viruses. However, it is generally little known that their very first description, as well as the foundational work on G4s, was performed on protozoans: unicellular life forms that are often parasitic. In this review, we provide a historical perspective on the discovery of G4s, intertwined with their biological significance across the protozoan kingdom. This is a history in three parts: first, a period of discovery including the first characterisation of a G4 motif at the DNA level in ciliates (environmental protozoa); second, a period less dense in publications concerning protozoa, during which DNA G4s were discovered in both humans and viruses; and third, a period of renewed interest in protozoa, including more mechanistic work in ciliates but also in pathogenic protozoa. This last period has opened an exciting prospect of finding new anti-parasitic drugs to interfere with parasite biology, thus adding new compounds to the therapeutic arsenal.

Migrants and allergy: a new view of the atopic march.

Summary: Atopy is the result of the influence of environmental factors on genetically predisposed individuals. Migration flows represent an interesting model to study the possible reciprocal roles of genes and environment. In this review the following issues influencing the development of allergic sensitization and/or atopic disorders in migrants will be rooted out: 1) ethnicity, genetic polymorphisms and risk of atopy; 2) double faceted effects of parasitic infestations; 3) biodiversity loss and industrial progress. Moreover, an extensive revision of the literature about the relationship between the migratory status and allergy development is provided.

Analysis of Predicted Host-Parasite Interactomes Reveals Commonalities and Specificities Related to Parasitic Lifestyle and Tissues Tropism.

The study of molecular host-parasite interactions is essential to understand parasitic infection and adaptation within the host system. As well, prevention and treatment of infectious diseases require a clear understanding of the molecular crosstalk between parasites and their hosts. Yet, large-scale experimental identification of host-parasite molecular interactions remains challenging, and the use of computational predictions becomes then necessary. Here, we propose a computational integrative approach to predict host-parasite protein-protein interaction (PPI) networks resulting from the human infection by 15 different eukaryotic parasites. We used an orthology-based approach to transfer high-confidence intraspecies interactions obtained from the STRING database to the corresponding interspecies homolog protein pairs in the host-parasite system. Our approach uses either the parasites predicted secretome and membrane proteins, or only the secretome, depending on whether they are uni- or multi-cellular, respectively, to reduce the number of false predictions. Moreover, the host proteome is filtered for proteins expressed in selected cellular localizations and tissues supporting the parasite growth. We evaluated the inferred interactions by analyzing the enriched biological processes and pathways in the predicted networks and their association with known parasitic invasion and evasion mechanisms. The resulting PPI networks were compared across parasites to identify common mechanisms that may define a global pathogenic hallmark. We also provided a study case focusing on a closer examination of the human-S. mansoni predicted interactome, detecting central proteins that have relevant roles in the human-S. mansoni network, and identifying tissue-specific interactions with key roles in the life cycle of the parasite. The predicted PPI networks can be visualized and downloaded at http://orthohpi.jensenlab.org.

Progress and challenges in aminoacyl-tRNA synthetase-based therapeutics.

Aminoacyl-tRNA synthetases (ARSs) are universal enzymes that catalyze the attachment of amino acids to the 3' ends of their cognate tRNAs. The resulting aminoacylated tRNAs are escorted to the ribosome where they enter protein synthesis. By specifically matching amino acids to defined anticodon sequences in tRNAs, ARSs are essential to the physical interpretation of the genetic code. In addition to their canonical role in protein synthesis, ARSs are also involved in RNA splicing, transcriptional regulation, translation, and other aspects of cellular homeostasis. Likewise, aminoacylated tRNAs serve as amino acid donors for biosynthetic processes distinct from protein synthesis, including lipid modification and antibiotic biosynthesis. Thanks to the wealth of details on ARS structures and functions and the growing appreciation of their additional roles regulating cellular homeostasis, opportunities for the development of clinically useful ARS inhibitors are emerging to manage microbial and parasite infections. Exploitation of these opportunities has been stimulated by the discovery of new inhibitor frameworks, the use of semi-synthetic approaches combining chemistry and genome engineering, and more powerful techniques for identifying leads from the screening of large chemical libraries. Here, we review the inhibition of ARSs by small molecules, including the various families of natural products, as well as inhibitors developed by either rational design or high-throughput screening as antibiotics and anti-parasitic therapeutics.

The role of myeloid-derived suppressor cells in chronic infectious diseases and the current methodology available for their study.

An effective pathogen has the ability to evade the immune response. The strategies used to achieve this may be based on the direct action of virulence factors or on the induction of host factors. Myeloid-derived suppressor cells (MDSCs) are immune cells with an incredible ability to suppress the inflammatory response, which makes them excellent targets to be exploited by pathogenic bacteria, viruses, or parasites. In this review, we describe the origin and suppressive mechanisms of MDSCs, as well as their role in chronic bacterial, viral, and parasitic infections, where their expansion seems to be essential in the chronicity of the disease. We also analyze the disadvantages of current MDSC depletion strategies and the different in vitro generation methods, which can be useful tools for the deeper study of these cells in the context of microbial infections.

Gene expression profiles alteration after infection of virus, bacteria, and parasite in the Olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is one of economically valuable fish species in the East Asia. In comparison with its economic importance, available genomic information of the olive flounder is very limited. The mass mortality caused by variety of pathogens (virus, bacteria and parasites) is main problem in aquaculture industry, including in olive flounder culture. In this study, we carried out transcriptome analysis using the olive flounder gill tissues after infection of three types of pathogens (Virus; Viral hemorrhagic septicemia virus, Bacteria; Streptococcus parauberis, and Parasite; Miamiensis avidus), respectively. As a result, we identified total 12,415 differentially expressed genes (DEG) from viral infection, 1,754 from bacterial infection, and 795 from parasite infection, respectively. To investigate the effects of pathogenic infection on immune response, we analyzed Gene ontology (GO) enrichment analysis with DEGs and sorted immune-related GO terms per three pathogen groups. Especially, we verified various GO terms, and genes in these terms showed down-regulated expression pattern. In addition, we identified 67 common genes (10 up-regulated and 57 down-regulated) present in three pathogen infection groups. Our goals are to provide plenty of genomic knowledge about olive flounder transcripts for further research and report genes, which were changed in their expression after specific pathogen infection.

The effect of parasite infection on the recombination rate of the mosquito Aedes aegypti.

Sexual reproduction and meiotic recombination generate new genetic combinations and may thereby help an individual infected by a parasite to protect its offspring from being infected. While this idea is often used to understand the evolutionary forces underlying the maintenance of sex and recombination, it also suggests that infected individuals should increase plastically their rate of recombination. We tested the latter idea with the mosquito Aedes aegypti and asked whether females infected by the microsporidian Vavraia culicis were more likely to have recombinant offspring than uninfected females. To measure the rate of recombination over a chromosome we analysed combinations of microsatellites on chromosome 3 in infected and uninfected females, in the (uninfected) males they copulated with and in their offspring. As predicted, the infected females were more likely to have recombinant offspring than the uninfected ones. These results show the ability of a female to diversify her offspring in response to parasitic infection by plastically increasing her recombination rate.