Interactions between microsporidia and other members of the microbiome.

The microbiome is the collection of microbes that are associated with a host. Microsporidia are intracellular eukaryotic parasites that can infect most types of animals. In the last decade, there has been much progress to define the relationship between microsporidia and the microbiome. In this review, we cover an increasing number of reports suggesting that microsporidia are common components of the microbiome in both invertebrates and vertebrates. These microsporidia infections can range from mutualistic to pathogenic, causing several physiological phenotypes, including death. Infection with microsporidia often causes a disruption in the normal microbiome, with both increases and decreases of bacterial, fungal, viral, and protozoan species being observed. This impact on the microbiome can occur through upregulation and downregulation of innate immunity as well as morphological changes to tissues that impact interactions with these microbes. Other microbes, particularly bacteria, can inhibit microsporidia and have been exploited to control microsporidia infections. These bacteria can function through regulating immunity, secreting anti-microsporidia compounds, and, in engineered versions, expressing double-stranded RNA targeting microsporidia genes. We end this review by discussing potential future directions to further understand the complex interactions between microsporidia and the other members of the microbiome.

The ins and outs of host-microsporidia interactions during invasion, proliferation and exit.

Microsporidia are a large group of fungal-related obligate intracellular parasites. They are responsible for infections in humans as well as in agriculturally and environmentally important animals. Although microsporidia are abundant in nature, many of the molecular mechanisms employed during infection have remained enigmatic. In this review, we highlight recent work showing how microsporidia invade, proliferate and exit from host cells. During invasion, microsporidia use spore wall and polar tube proteins to interact with host receptors and adhere to the host cell surface. In turn, the host has multiple defence mechanisms to prevent and eliminate these infections. Microsporidia encode numerous transporters and steal host nutrients to facilitate proliferation within host cells. They also encode many secreted proteins which may modulate host metabolism and inhibit host cell defence mechanisms. Spores exit the host in a non-lytic manner that is dependent on host actin and endocytic recycling proteins. Together, this work provides a fuller picture of the mechanisms that these fascinating organisms use to infect their hosts.

Isolation and Identification of Nematode-Infecting Microsporidia.

Nematodes are naturally infected by the fungal-related pathogen microsporidia. These ubiquitous eukaryotic parasites are poorly understood, despite infecting most types of animals. Identifying novel species of microsporidia and studying them in an animal model can expedite our understanding of their infection biology and evolution. Nematodes present an excellent avenue for pursuing such work, as they are abundant in the environment and many species are easily culturable in the laboratory. The protocols presented here describe how to isolate bacterivorous nematodes from rotting substrates, screen them for microsporidia infection, and molecularly identify the nematode and microsporidia species. Additionally, we detail how to remove environmental contaminants and generate a spore preparation of microsporidia from infected samples. We also discuss potential pitfalls and provide suggestions on how to mitigate them. These protocols allow for the identification of novel microsporidia species, which can serve as an excellent starting point for genomic analysis, determination of host specificity, and infection characterization. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Gathering samples Support Protocol 1: Generating 10× and 40× Escherichia coli OP50 and seeding NGM plates Basic Protocol 2: Microsporidia screening, testing for Caenorhabditis elegans susceptibility, and sample freezing Basic Protocol 3: DNA extraction, PCR amplification, and sequencing to identify nematode and microsporidia species Basic Protocol 4: Removal of contaminating microbes and preparation of microsporidia spores Support Protocol 2: Bleach-synchronizing nematodes.

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection.

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.

An intestinally secreted host factor promotes microsporidia invasion of C. elegans.

Microsporidia are ubiquitous obligate intracellular pathogens of animals. These parasites often infect hosts through an oral route, but little is known about the function of host intestinal proteins that facilitate microsporidia invasion. To identify such factors necessary for infection by Nematocida parisii, a natural microsporidian pathogen of Caenorhabditis elegans, we performed a forward genetic screen to identify mutant animals that have a Fitness Advantage with Nematocida (Fawn). We isolated four fawn mutants that are resistant to Nematocida infection and contain mutations in T14E8.4, which we renamed aaim-1 (Antibacterial and Aids invasion by Microsporidia). Expression of AAIM-1 in the intestine of aaim-1 animals restores N. parisii infectivity and this rescue of infectivity is dependent upon AAIM-1 secretion. N. parisii spores in aaim-1 animals are improperly oriented in the intestinal lumen, leading to reduced levels of parasite invasion. Conversely, aaim-1 mutants display both increased colonization and susceptibility to the bacterial pathogen Pseudomonas aeruginosa and overexpression ofaaim-1 reduces P. aeruginosa colonization. Competitive fitness assays show that aaim-1 mutants are favored in the presence of N. parisii but disadvantaged on P. aeruginosa compared to wild-type animals. Together, this work demonstrates how microsporidia exploits a secreted protein to promote host invasion. Our results also suggest evolutionary trade-offs may exist to optimizing host defense against multiple classes of pathogens.

Integrative genomic analysis reveals mechanisms of immune evasion in P. falciparum malaria.

The mechanisms behind the ability of Plasmodium falciparum to evade host immune system are poorly understood and are a major roadblock in achieving malaria elimination. Here, we use integrative genomic profiling and a longitudinal pediatric cohort in Burkina Faso to demonstrate the role of post-transcriptional regulation in host immune response in malaria. We report a strong signature of miRNA expression differentiation associated with P. falciparum infection (127 out of 320 miRNAs, B-H FDR 5%) and parasitemia (72 miRNAs, B-H FDR 5%). Integrative miRNA-mRNA analysis implicates several infection-responsive miRNAs (e.g., miR-16-5p, miR-15a-5p and miR-181c-5p) promoting lymphocyte cell death. miRNA cis-eQTL analysis using whole-genome sequencing data identified 1,376 genetic variants associated with the expression of 34 miRNAs (B-H FDR 5%). We report a protective effect of rs114136945 minor allele on parasitemia mediated through miR-598-3p expression. These results highlight the impact of post-transcriptional regulation, immune cell death processes and host genetic regulatory control in malaria.

KLF4 Nuclear Export Requires ERK Activation and Initiates Exit from Naive Pluripotency.

Cooperative action of a transcription factor complex containing OCT4, SOX2, NANOG, and KLF4 maintains the naive pluripotent state; however, less is known about the mechanisms that disrupt this complex, initiating exit from pluripotency. We show that, as embryonic stem cells (ESCs) exit pluripotency, KLF4 protein is exported from the nucleus causing rapid decline in Nanog and Klf4 transcription; as a result, KLF4 is the first pluripotency transcription factor removed from transcription-associated complexes during differentiation. KLF4 nuclear export requires ERK activation, and phosphorylation of KLF4 by ERK initiates interaction of KLF4 with nuclear export factor XPO1, leading to KLF4 export. Mutation of the ERK phosphorylation site in KLF4 (S132) blocks KLF4 nuclear export, the decline in Nanog, Klf4, and Sox2 mRNA, and differentiation. These findings demonstrate that relocalization of KLF4 to the cytoplasm is a critical first step in exit from the naive pluripotent state and initiation of ESC differentiation.