protozoan parasite
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Phytomonas serpens is a protozoan parasite that alternates its life cycle between two hosts: an invertebrate vector and the tomato fruit. This phytoflagellate is able to synthesize proteins displaying similarity to the cysteine peptidase named cruzipain, an important virulence factor from Trypanosoma cruzi, the etiologic agent of Chagas disease. Herein, the growth of P. serpens in complex medium (BHI) supplemented with natural tomato extract (NTE) resulted in the increased expression of cysteine peptidases, as verified by the hydrolysis of the fluorogenic substrate Z-Phe-Arg-AMC and by gelatin-SDS-PAGE. Phytoflagellates showed no changes in morphology, morphometry and viability, but the proliferation was slightly reduced when cultivated in the presence of NTE. The enhanced proteolytic activity was accompanied by a significant increase in the expression of cruzipain-like molecules, as verified by flow cytometry using anti-cruzipain antibodies. In parallel, parasites incubated under chemically defined conditions (PBS supplemented with glucose) and added of different concentration of NTE revealed an augmentation in the production of cruzipain-like molecules in a typically dose-dependent way. Similarly, P. serpens recovered from the infection of mature tomatoes showed an increase in the expression of molecules homologous to cruzipain; however, cells showed a smaller size compared to parasites grown in BHI medium. Furthermore, phytoflagellates incubated with dissected salivary glands from Oncopeltus fasciatus or recovered from the hemolymph of infected insects also showed a strong enhance in the expression of cruzipain-like molecules that is more relevant in the hemolymph. Collectively, our results showed that cysteine peptidases displaying similarities to cruzipain are more expressed during the life cycle of the phytoflagellate P. serpens both in the invertebrate and plant hosts.

Cryptosporidium parvum is a zoonotic, protozoan parasite that causes potentially life-threatening diarrhea in the host and can be transmitted via the fecal-oral route. C. parvum can infect cattle and may be detected in their feces using a variety of tests. We compared the level of agreement, ease of procedure, and cost among PCR, lateral flow immunoassay, fluorescent antibody, and Kinyoun acid-fast stain direct smear tests. Over the course of 9 mo, 74 calf fecal samples were submitted and tested for C. parvum using all 4 tests. A Fleiss kappa value of 0.813 was obtained, indicating an excellent level of agreement among tests. Overall, the best test based on cost and ease of procedure was the Kinyoun acid-fast stain direct smear.

Background Globally distributed with variable prevalence depending on geography, toxoplasmosis is a zoonosis caused by an obligate intracellular protozoan parasite, Toxoplasma gondii. This disease is usually benign but poses a risk for immunocompromised people and for newborns of mothers with a primary infection during pregnancy because of the risk of congenital toxoplasmosis (CT). CT can cause severe damage to fetuses-newborns. To our knowledge, no study has been conducted in sub-Saharan Africa on toxoplasmosis seroprevalence, seroconversion and CT in a large longitudinal cohort and furthermore, no observation has been made of potential relationships with malaria. Methods We performed a retrospective toxoplasmosis serological study using available samples from a large cohort of 1,037 pregnant women who were enrolled in a malaria follow-up during the 2008–2010 period in a rural area in Benin. We also used some existing data to investigate potential relationships between the maternal toxoplasmosis serological status and recorded malaria infections. Results Toxoplasmosis seroprevalence, seroconversion and CT rates were 52.6%, 3.4% and 0.2%, respectively, reflecting the population situation of toxoplasmosis, without targeted medical intervention. The education level influences the toxoplasmosis serological status of women, with women with little or no formal education have greater immunity than others. Surprisingly, toxoplasmosis seropositive pregnant women tended to present lower malaria infection during pregnancy (number) or at delivery (presence) and to have lower IgG levels to Plasmodium falciparum Apical Membrane Antigen 1, compared to toxoplasmosis seronegative women. Conclusions The high toxoplasmosis seroprevalence indicates that prevention against this parasite remains important to deploy and must be accessible and understandable to and for all individuals (educated and non-educated). A potential protective role against malaria conferred by a preexisting toxoplasmosis infection needs to be explored more precisely to examine the environmental, parasitic and/or immune aspects.

Toxoplasma gondii is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause foetal damage and abortion in both animals and humans. Sphingolipids have indispensable functions as signaling molecules and are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Ceramide is the precursor for all sphingolipids, and here we report the identification, localisation and analyses of the Toxoplasma ceramide synthases Tg CerS1 and Tg CerS2 and, using a conditional gene regulation approach, establish their roles in pathogenicity and parasite fitness. Interestingly, we observed that whilst Tg CerS1 was a fully functional orthologue of the yeast Lag1p capable of catalysing the conversion of sphinganine to ceramide, in contrast Tg CerS2 was catalytically inactive. Furthermore, genomic deletion of Tg CerS1 using CRISPR/Cas-9 led to viable but slow growing parasites indicating its importance but not indispensability. In contrast, genomic knock out of Tg CerS2 was only accessible utilising the rapamycin-inducible Cre recombinase system. Surprisingly, the results demonstrated that this ‘pseudo’ ceramide synthase, Tg CerS2, has an even greater role in parasite fitness than its catalytically active orthologue (Tg CerS1). Phylogenetic analyses indicated that, as in humans and plants, the ceramide synthase isoforms found in Toxoplasma and other Apicomplexa arose through gene duplication. However, in the Apicomplexa the duplicated copy subsequently evolved into a non-functional ‘pseudo’ ceramide synthase. This arrangement is unique to the Apicomplexa and further illustrates the unusual biology that characterize these protozoan parasites, a feature that could potentially be exploited in the development of new antiprotozoals.

Understanding the frequency, spatiotemporal dynamics and impacts of parasite coinfections is fundamental to developing control measures and predicting disease impacts. The European turtle dove (Streptopelia turtur) is one of Europe’s most threatened bird species. High prevalence of infection by the protozoan parasite Trichomonas gallinae has previously been identified, but the role of this and other coinfecting parasites in turtle dove declines remains unclear. Using a high-throughput sequencing approach, we identified seven strains of T. gallinae, including two novel strains, from ITS1/5.8S/ITS2 ribosomal sequences in turtle doves on breeding and wintering grounds, with further intra-strain variation and four novel sub-types revealed by the iron-hydrogenase gene. High spatiotemporal turnover was observed in T. gallinae strain composition, and infection was prevalent in all populations (89–100%). Coinfection by multiple Trichomonas strains was rarer than expected (1% observed compared to 38.6% expected), suggesting either within-host competition, or high mortality of coinfected individuals. In contrast, coinfection by multiple haemosporidians was common (43%), as was coinfection by haemosporidians and T. gallinae (90%), with positive associations between strains of T. gallinae and Leucocytozoon suggesting a mechanism such as parasite-induced immune modulation. We found no evidence for negative associations between coinfections and host body condition. We suggest that longitudinal studies involving the recapture and investigation of infection status of individuals over their lifespan are crucial to understand the epidemiology of coinfections in natural populations.

Chagas disease (CD) is a vector-borne parasitosis, caused by the protozoan parasite Trypanosoma cruzi, that affects millions of people worldwide. Although endemic in South America, CD is emerging throughout the world due to climate change and increased immigratory flux of infected people to non-endemic regions. Containing of the diffusion of CD is challenged by the asymptomatic nature of the disease in early infection stages and by the lack of a rapid and effective diagnostic test. With the aim of designing new serodiagnostic molecules to be implemented in a microarray-based diagnostic set-up for early screening of CD, herein, we report the recombinant production of the extracellular domain of a surface membrane antigen from T. cruzi (TcSMP) and confirm its ability to detect plasma antibodies from infected patients. Moreover, we describe its high-resolution (1.62 Å) crystal structure, to which in silico epitope predictions were applied in order to locate the most immunoreactive regions of TcSMP in order to guide the design of epitopes that may be used as an alternative to the full-length antigen for CD diagnosis. Two putative, linear epitopes, belonging to the same immunogenic region, were synthesized as free peptides, and their immunological properties were tested in vitro. Although both peptides were shown to adopt a structural conformation that allowed their recognition by polyclonal antibodies raised against the recombinant protein, they were not serodiagnostic for T. cruzi infections. Nevertheless, they represent good starting points for further iterative structure-based (re)design cycles.

Trypanosoma cruzi, the protozoan parasite causative agent of Chagas disease, affects about seven million people worldwide, representing a major global public health concern with relevant socioeconomic consequences, particularly in developing countries. In this review, we discuss the multiple roles of galectins, a family of β-galactoside-binding proteins, in modulating both T. cruzi infection and immunoregulation. Specifically, we focus on galectin-driven circuits that link parasite invasion and inflammation and reprogram innate and adaptive immune responses. Understanding the dynamics of galectins and their β-galactoside-specific ligands during the pathogenesis of T. cruzi infection and elucidating their roles in immunoregulation, inflammation, and tissue damage offer new rational opportunities for treating this devastating neglected disease.

Malaria is a life-threatening infectious disease caused by a protozoan parasite of the genus Plasmodium. It is transmitted through the bites of infected female Anopheles mosquitoes. The global burden is estimated to be around 219 million cases in 87 countries. Natural compounds have been used primarily in the traditional medicine for thousands of years. For the treatment of malaria, natural products were used until the development of synthetic drugs, and most of the currently available anti-malarial drugs have been derived based on the compounds from these traditional medicinal plants. The current chapter tries to briefly indicate the emerging resistance against anti-malarial drugs and to discuss the recent research on natural products that have been evaluated for anti-malarial activity. Rigorous evaluation of the efficacy and safety of traditional medicines is required along with identification of active constituents in order to develop new drugs with novel mechanisms of action.

Trypanosoma cruzi is a flagellated protozoan parasite that causes Chagas disease, which represents a serious health problem in the Americas. Nucleoside diphosphate kinases (NDPKs) are key enzymes that are implicated in cellular energy management. TcNDPK1 is the canonical isoform in the T. cruzi parasite. TcNDPK1 has a cytosolic, perinuclear and nuclear distribution. It is also found in non-membrane-bound filaments adjacent to the nucleus. In the present work, X-ray diffraction and in vivo studies of TcNDPK1 are described. The structure reveals a novel, multi-hexameric, left-handed helical oligomer structure. The results of directed mutagenesis studies led to the conclusion that the microscopic TcNDPK1 granules observed in vivo in T. cruzi parasites are made up by the association of TcNDPK1 oligomers. In the absence of experimental data, analysis of the interactions in the X-ray structure of the TcNDPK1 oligomer suggests the probable assembly and disassembly steps: dimerization, assembly of the hexamer as a trimer of dimers, hexamer association to generate the left-handed helical oligomer structure and finally oligomer association in a parallel manner to form the microscopic TcNDPK1 filaments that are observed in vivo in T. cruzi parasites. Oligomer disassembly takes place on the binding of substrate in the active site of TcNDPK1, leading to dissociation of the hexamers. This study constitutes the first report of such a protein arrangement, which has never previously been seen for any protein or NDPK. Further studies are needed to determine its physiological role. However, it may suggest a paradigm for protein storage reflecting the complex mechanism of action of TcNDPK1.