Free-living amoebae and other neglected protistan pathogens: Health emergency signals?

Protistan parasites have an undisputed global health impact. However, outside of a few key exceptions, e.g. the agent of malaria, most of these infectious agents are neglected as important health threats. The Symposium entitled "Free-living amoebae and neglected pathogenic protozoa: health emergency signals?" held at the European Congress of Protistology in Rome, July 2019, brought together researchers addressing scientific and clinical questions about some of these fascinating organisms. Topics presented included the molecular basis of pathogenicity in Acanthamoeba; genomics of Naegleria fowleri; and epidemiology of poorly diagnosed enteric protistan species, including Giardia, Cryptosporidium, Blastocystis, Dientamoeba. The Symposium aim was to excite the audience about the opportunities and challenges of research in these underexplored organisms and to underline the public health implications of currently under-appreciated protistan infections. The major take home message is that any knowledge that we gain about these organisms will allow us to better address them, in terms of monitoring and treatment, as sources of future health emergencies.

Structural Basis for the Antibiotic Resistance of Eukaryotic Isoleucyl-tRNA Synthetase.

Pathogenic aminoacyl-tRNA synthetases (ARSs) are attractive targets for anti-infective agents because their catalytic active sites are different from those of human ARSs. Mupirocin is a topical antibiotic that specifically inhibits bacterial isoleucy-ltRNA synthetase (IleRS), resulting in a block to protein synthesis. Previous studies on Thermus thermophilus IleRS indicated that mupirocin-resistance of eukaryotic IleRS is primarily due to differences in two amino acids, His581 and Leu583, in the active site. However, without a eukaryotic IleRS structure, the structural basis for mupirocin-resistance of eukaryotic IleRS remains elusive. Herein, we determined the crystal structure of Candida albicans IleRS complexed with Ile-AMP at 2.9 Å resolution. The largest difference between eukaryotic and prokaryotic IleRS enzymes is closure of the active site pocket by Phe55 in the HIGH loop; Arg410 in the CP core loop; and the second Lys in the KMSKR loop. The Ile-AMP product is lodged in a closed active site, which may restrict its release and thereby enhance catalytic efficiency. The compact active site also prevents the optimal positioning of the 9-hydroxynonanoic acid of mupirocin and plays a critical role in resistance of eukaryotic IleRS to anti-infective agents.

Chromatin architecture and virulence-related gene expression in eukaryotic microbial pathogens.

A fundamental question in biology is to understand how appropriate transcriptional regulation and dense packaging of the genetic material within the eukaryotic nucleus are achieved. The exquisite gene expression control and other metabolic processes of DNA require a highly complex, multilayered, three-dimensional architecture of the chromatin and its specific compartmentalization within the nucleus. Some of these architectural and sub-nuclear positioning mechanisms have been extensively co-opted by eukaryotic pathogens to keep fine expression control and expansion of virulence-related gene families in Plasmodium falciparum, Trypanosoma brucei and Candida glabrata. For example non-linear interactions between distant cis-acting regions and the formation of chromatin loops are required for appropriate regulation of the expression of virulence-related multi-gene families encoding cell surface proteins. These gene families are located near the chromosome ends and tethered to the nuclear periphery. Consequently, only one or very few genes of the family are expressed at a time. These genes are involved in antigenic variation in parasites and the generation of subpopulations of cells with diverse antigenic proteins at the surface in some pathogenic fungi, making them highly efficient pathogens.

The 14th International Workshops on Opportunistic Protists (IWOP 14).

The 14th International Workshops on Opportunistic Protists (IWOP-14) was held August 10-12, 2017 in Cincinnati, OH, USA. The IWOP meetings focus on opportunistic protists (OIs); for example, free-living amoebae, Pneumocystis spp., Cryptosporidium spp., Toxoplasma, the Microsporidia, and kinetoplastid flagellates. The highlights of Pneumocystis spp. research included the reports of primary homothallism for mating; a potential requirement for sexual replication in its life cycle; a new antigen on the surface of small asci; roles for CLRs, Dectin-1, and Mincle in host responses; and identification of MSG families and mechanisms used for surface variation. Studies of Cryptosporidia spp. included comparative genomics, a new cryopreservation method; the role of mucin in attachment and invasion, and epidemiological surveys illustrating species diversity in animals. One of the five identified proteins in the polar tube of Microsporidia, PTP4, was shown to play a role in host infection. Zebrafish were used as a low cost vertebrate animal model for an evaluation of potential anti-toxoplasma drugs. Folk medicine compounds with anti-toxoplasma activity were presented, and reports on the chronic toxoplasma infection provided evidence for increased tractability for the study of this difficult life cycle stage. Escape from the parasitophorus vacuole and cell cycle regulation were the topics of the study in the acute phase.

Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples.

OBJECTIVE: Efficient and easy-to-use DNA extraction and purification methods are critical in implementing PCR-based diagnosis of pathogens. In order to optimize the routine clinical laboratory diagnosis of eukaryotic enteric pathogens, we compare, via quantitative PCR cycle threshold (Ct) values, the efficiency of two DNA extraction kits: the semi-automated EZ1® (Qiagen) and the manual QIAamp® DNA Stool Mini Kit (Qiagen), on six protozoa: Blastocystis spp., Cryptosporidium parvum/hominis, Cyclospora cayetanensis, Dientamoeba fragilis, Giardia intestinalis and Cystoisospora belli and one microsporidia: Enterocytozoon bieneusi. RESULTS: Whereas EZ1® (Qiagen) and QIAamp® DNA Stool Mini Kit (Qiagen) yielded similar performances for the detection of Cryptosporidium spp. and D. fragilis, significant lower Ct values (p < 0.002) pointed out a better performance of EZ1® on the five remaining pathogens. DNA extraction using the semi-automated EZ1® procedure was faster and as efficient as the manual procedure in the seven eukaryotic enteric pathogens tested. This procedure is suitable for DNA extraction from stools in both clinical laboratory diagnosis and epidemiological study settings.

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists.

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii.

Reshuffling transcriptional circuits: how microorganisms adapt to colonize the human body.

Several hundred taxa of microorganisms-including bacteria, archaea and eukaryotes-inhabit the human body. What did it take for these species to become stable residents of humans? Recent reports illustrate how evolutionary changes in transcriptional circuits played a pivotal role in the adaptation of single-celled eukaryotes to colonize mammals.

Pathogenic eukaryotes in gut microbiota of western lowland gorillas as revealed by molecular survey.

Although gorillas regarded as the largest extant species of primates and have a close phylogenetic relationship with humans, eukaryotic communities have not been previously studied in these populations. Herein, 35 eukaryotic primer sets targeting the 18S rRNA gene, internal transcribed spacer gene and other specific genes were used firstly to explore the eukaryotes in a fecal sample from a wild western lowland gorilla (Gorilla gorilla gorilla). Then specific real-time PCRs were achieved in additional 48 fecal samples from 21 individual gorillas to investigate the presence of human eukaryotic pathogens. In total, 1,572 clones were obtained and sequenced from the 15 cloning libraries, resulting in the retrieval of 87 eukaryotic species, including 52 fungi, 10 protozoa, 4 nematodes and 21 plant species, of which 52, 5, 2 and 21 species, respectively, have never before been described in gorillas. We also reported the occurrence of pathogenic fungi and parasites (i.e. Oesophagostomum bifurcum (86%), Necator americanus (43%), Candida tropicalis (81%) and other pathogenic fungi were identified). In conclusion, molecular techniques using multiple primer sets may offer an effective tool to study complex eukaryotic communities and to identify potential pathogens in the gastrointestinal tracts of primates.

Utilization of zebrafish for intravital study of eukaryotic pathogen-host interactions.

Unique imaging tools and practical advantages have made zebrafish a popular model to investigate in vivo host-pathogen interactions. These studies have uncovered details of the mechanisms involved in several human infections. Until recently, studies using this versatile host were limited to viral and prokaryotic pathogens. Eukaryotic pathogens are a diverse group with a major impact on the human and fish populations. The relationships of eukaryote pathogens with their hosts are complex and many aspects remain obscure. The small and transparent zebrafish, with its conserved immune system and amenability to genetic manipulation, make it an exciting model for quantitative study of the core strategies of eukaryotic pathogens and their hosts. The only thing to do now is realize its potential for advancement of biomedical and aquaculture research.

Water-related parasitic diseases in China.

Water-related parasitic diseases are directly dependent on water bodies for their spread or as a habitat for indispensable intermediate or final hosts. Along with socioeconomic development and improvement of sanitation, overall prevalence is declining in the China. However, the heterogeneity in economic development and the inequity of access to public services result in considerable burden due to parasitic diseases in certain areas and populations across the country. In this review, we demonstrated three aspects of ten major water-related parasitic diseases, i.e., the biology and pathogenicity, epidemiology and recent advances in research in China. General measures for diseases control and special control strategies are summarized.

A prepared mind--Ernest Edward Tyzzer's legacy of research into avian diseases.

Ernest Edward Tyzzer (1875-1965) was a physician, specializing at first (1902-1916) in cancer research and then from 1916 as a parasitologist. He was born of English parents in Wakefield, Massachusetts, where he lived all his life. Educated in Wakefield public schools, Brown University (Ph.B., A.M., Hon. Sc.D.), and Harvard University (M.D.), he established during his 40-yr career (1902-1942) an international reputation in oncology, pathology, virology, bacteriology, parasitology, and taxonomic zoology in relation to human and veterinary medicine. His contributions to knowledge of avian diseases were outstanding and wide-ranging. Seminal work included: new descriptions of tumors in chickens; the first record of Cryptosporidium in birds; studies on the biology, morphology, in vitro culture, and epizootiology of the blackhead (histomonosis) parasite and its reclassification under a new genus Histomonas; descriptions of eight new taxa of amebae and flagellates in chickens, turkeys, and ruffed grouse; descriptions of seven new species of Eimeria in chickens, turkeys, pheasants, and quail as well as studies on their biology, immunogenicity, virulence, and epizootiology; a description of the trematode Collyriclum in English sparrows; the first record of mycosis in ruffed grouse; the recognition of birds as a source of equine encephalomyelitis infections of humans; the first American record of infectious sinusitis in turkeys and discovery of a curative treatment; and studies of Newcastle disease and avian influenza during the war research program of the 1940s. Application of Tyzzer's histomonosis research to farm practice saved the Massachusetts turkey industry from extinction in the 1920s and significantly influenced the recovery of turkey farming nationally.

Characterization of the protozoan parasite Marteilia refringens infecting the dwarf oyster Ostrea stentina in Tunisia.

Marteilia refringens is a protozoan parasite recognized as a significant pathogen of the European flat oyster Ostrea edulis. The life cycle of this species is still poorly known, although there is evidence of the need for intermediate host(s). In the present study, we have used molecular approaches to identify this parasite in samples of the dwarf oyster Ostrea stentina after reports of massive mortality along the Tunisian coasts. In 2009 we evaluated the status of O. stentina from Monastir and checked if there was an infection with M. refringens, using polymerase chain reaction assays. Of the 103 tested O. stentina, 85 were PCR-positive using a Marteilia genus-specific assay. Additional assays were subsequently carried out on some samples collected in 2010 in Monastir and processed for histology, transmission electron microscopy and complementary molecular analyses. PCR was carried out to amplify the IGS and ITS regions. Histological and transmission electron microscopy analyses allowed us to confirm the presence of this parasite in the digestive gland tissue of O. stentina and to characterize it at the ultrastructural level. This is the first record of the occurrence of M. refringens in the oyster O. stentina along the Tunisian coasts.

Targeting UDP-galactopyranose mutases from eukaryotic human pathogens.

UDP-Galactopyranose mutase (UGM) is a unique flavin-dependent enzyme that catalyzes the conversion of UDP-galactopyranose(UDP-Galp) to UDP-galactofuranose (UDP-Galf). The product of this reaction is the precursor to Galf, a major component of the cell wall and of cell surface glycoproteins and glycolipids in many eukaryotic and prokaryotic human pathogens. The function of UGM is important in the virulence of fungi, parasites, and bacteria. Its role in virulence and its absence in humans suggest that UGM is an ideal drug target. Significant structural and mechanistic information has been accumulated on the prokaryotic UGMs; however, in the past few years the research interest has shifted to UGMs from eukaryotic human pathogens such as fungi and protozoan parasites. It has become clear that UGMs from prokaryotic and eukaryotic organisms have different structural and mechanistic features. The amino acid sequence identity between these two classes of enzymes is low, resulting in differences in oligomeric states, substrate binding, active site flexibility, and interaction with redox partners. However, the unique role of the flavin cofactor in catalysis is conserved among this enzyme family. In this review, recent findings on eukaryotic UGMs are discussed and presented in comparison with prokaryotic UGMs.

How to invade, replicate, and escape from host organisms. A challenge in defining virulence factors for parasites.

During millions of years, parasites have been adapting to different environments and hosts. During this time, they have acquired different molecules and peculiar structures, some absent in other living organisms, in order to successfully invade hosts, resist immune attack, and proliferate in the hosts. Nowadays, several genome sequences and a multitude of new information have been generated for many human and animal parasites, opening new possibilities for understanding in detail how they interact with the host and cause disease. Investigations of these molecules and the associated structures, together with their functional roles, are now emerging, providing key advances in understanding pathology that could be used for developing robust strategies to selectively target the parasites without damaging the host.

Sink or swim: lipid rafts in parasite pathogenesis.

Lipid rafts, sterol- and sphingolipid-rich membrane microdomains, have been extensively studied in mammalian cells. Recently, lipid rafts have been shown to control virulence in a variety of parasites including Entamoeba histolytica, Giardia intestinalis, Leishmania spp., Plasmodium spp., Toxoplasma gondii, and Trypanosoma spp. Parasite rafts regulate adhesion to host and invasion, and parasite adhesion molecules often localize to rafts. Parasite rafts also control vesicle trafficking, motility, and cell signaling. Parasites disrupt host cell rafts; the dysregulation of host membrane function facilitates the establishment of infection and evasion of the host immune system. Discerning the mechanism by which lipid rafts regulate parasite pathogenesis is essential to our understanding of virulence. Such insight may guide the development of new drugs for disease management.

Infection prevalence and phylogenetic analysis of Perkinsus olseni in Ruditapes philippinarum from East China.

The prevalence of Perkinsus sp. infection in Manila clam Ruditapes philippinarum was investigated in the coastal areas of east China. Thirteen groups of clams were collected from 5 sites: Dandong and Qingdao Bays (Yellow Sea), Weifang Bay (Bohai Sea), and Ningbo and Fuzhou Bays (East China Sea). The clams were tested for perkinsosis infection using Ray's fluid thioglycollate medium culture assay. Perkinsus sp. was found in samples from all 5 sites from May 2008 to May 2009. Infection prevalence ranged from 43.75 to 95.83%, and was significantly higher in October than in May. The only 3 uninfected groups of clams were collected from Weifang Bay, the site farthest from the ocean. There was no difference in the prevalence of infection among the remaining 4 sites. The conserved internal transcribed spacer regions of the ribosomal RNA gene complex in each of the Perkinsus sp. isolates were amplified by PCR. The resulting amplicons were sequenced and phylogenetically analyzed. All the Perkinsus isolates were identified as Perkinsus olseni.