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Studying Inherited Immunity in a <em>Caenorhabditis elegans</em> 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 ...

University of Toronto

Evolution of microsporidia: An extremely successful group of eukaryotic intracellular parasites.

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.

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

Pathogens are abundant and drive evolution of host immunity. Whilst immune memory is classically associated with adaptive immunity, studies in diverse species now show that priming of innate immune defences can also protect against secondary infection. Remarkably, priming may also be passed on to progeny to enhance pathogen resistance and promote survival in future generations. Phenotypic changes that occur independent of DNA sequence underlie both 'within-generation' priming and 'multigenerational' priming. However, the molecular mechanisms responsible for these phenomena are still poorly understood. Caenorhabditis elegans is a simple and genetically tractable model organism that has enabled key advances in immunity and environmental epigenetics. Using both natural and human pathogens, researchers have uncovered numerous examples of innate immune priming in this animal. Viral infection models have provided key evidence for a conserved antiviral RNA silencing mechanism that is inherited in progeny. Bacterial infection models have explored mechanisms of within-generation and multigenerational priming that span chromatin modification and transcriptional changes, small RNA pathways, maternal provisioning and pathogen avoidance strategies. Together, these studies are providing novel insight into the immune reactivity of the genome and have important consequences for our understanding of health and evolution. In this review, we present the current evidence for learned protection against pathogens in C. elegans, discuss the significance and limitations of these findings and highlight important avenues of future investigation.

Remembering your enemies: mechanisms of within-generation and multigenerational immune priming in Caenorhabditis elegans.

Parental infection can result in the production of offspring with enhanced immunity phenotypes. Critically, the mechanisms underlying inherited immunity are poorly understood. Here, we show that  infected with the intracellular microsporidian parasite  produce progeny that are resistant to microsporidia infection. We determine the kinetics of the response and show that intergenerational immunity prevents host-cell invasion by  and enhances survival to the bacterial pathogen  We demonstrate that immunity is induced by the parental transcriptional response to infection, which can be mimicked through maternal somatic depletion of PALS-22 and the retinoblastoma protein ortholog, LIN-35. We find that other biotic and abiotic stresses (viral infection and cadmium exposure) that induce a similar transcriptional response as microsporidia also induce immunity in progeny. Together, our results reveal how a parental transcriptional signal can be induced by distinct stimuli and protect offspring against multiple classes of pathogens.

A parental transcriptional response to microsporidia infection induces inherited immunity in offspring.

Aaron Reinke studies microsporidian evolution and how microsporidia interact with their hosts. In this mSphere of Influence article, he reflects on how the papers "A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells" (K. J. Roux, D. I. Kim, M. Raida, and B. Burke, J Cell Biol 196:801-810, 2012, https://doi.org/10.1083/jcb.201112098) and "Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging" (H.-W. Rhee, P. Zou, N. D. Udeshi, J. D. Martell, et al., Science 339:1328-1331, 2013, https://doi.org/10.1126/science.1230593) impacted his thinking on how to determine where proteins from intracellular pathogens are located within host cells.

mSphere of Influence: Where the Pathogen Proteins Are.

Microsporidia are a large group of fungus-related obligate intracellular parasites. Though many microsporidia species have been identified over the past 160 years, depiction of the full diversity of this phylum is lacking. To systematically describe the characteristics of these parasites, we created a database of 1,440 species and their attributes, including the hosts they infect and spore characteristics. We find that microsporidia have been reported to infect 16 metazoan and 4 protozoan phyla, with smaller phyla being underrepresented. Most species are reported to infect only a single host, but those that are generalists are also more likely to infect a broader set of host tissues. Strikingly, polar tubes are threefold longer in species that infect tissues besides the intestine, suggesting that polar tube length is a determinant of tissue specificity. Phylogenetic analysis revealed four clades which each contain microsporidia that infect hosts from all major habitats. Although related species are more likely to infect similar hosts, we observe examples of changes in host specificity and convergent evolution. Taken together, our results show that microsporidia display vast diversity in their morphology and the hosts they infect, illustrating the flexibility of these parasites to evolve new traits.  Microsporidia are a large group of parasites that cause death and disease in humans and many agriculturally important animal species. To fully understand the diverse properties of these parasites, we curated species reports from the last 160 years. Using these data, we describe when and where microsporidia were identified and what types of animals and host tissues these parasites infect. Microsporidia infect hosts using a conserved apparatus known as the polar tube. We observe that the length of this tube is correlated with the tissues that are being infected, suggesting that the polar tube controls where within the animals that the parasite infects. Finally, we show that microsporidia species often exist in multiple environments and are flexible in their ability to evolve new traits. Our study provides insight into the ecology and evolution of microsporidia and provides a useful resource to further understand these fascinating parasites.

Generation of a Microsporidia Species Attribute Database and Analysis of the Extensive Ecological and Phenotypic Diversity of Microsporidia.

The Caenorhabditis elegans genome encodes nineteen functional Argonaute proteins that use 22G-RNAs, 26G-RNAs, miRNAs or piRNAs to regulate target transcripts. Only one Argonaute is essential under normal laboratory conditions: CSR-1. While CSR-1 has been studied widely, nearly all studies have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR-1a. Using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG into the long (CSR-1a) and short (CSR-1b) isoforms, we found that CSR-1a is expressed during spermatogenesis and in several somatic tissues, including the intestine. CSR-1b is expressed constitutively in the germline. small RNA sequencing of CSR-1 complexes shows that they interact with partly overlapping sets of 22G-RNAs. Phenotypic analyses reveal that the essential functions of csr-1 described in the literature coincide with CSR-1b, while CSR-1a plays tissue specific functions. During spermatogenesis, CSR-1a integrates into an sRNA regulatory network including ALG-3, ALG-4 and WAGO-10 that is necessary for fertility at 25°C. In the intestine, CSR-1a silences immunity and pathogen-responsive genes, and its loss results in improved survival from the pathogen Pseudomonas aeruginosa. Our findings functionally distinguish the CSR-1 isoforms and highlight the importance of studying each AGO isoform independently.

Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility.

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 , a natural microsporidian pathogen of , we performed a forward genetic screen to identify mutant animals that have a Fitness Advantage with  (Fawn). We isolated four  mutants that are resistant to  infection and contain mutations in , which we renamed  (Antibacterial and Aids invasion by Microsporidia). Expression of AAIM-1 in the intestine of  animals restores  infectivity and this rescue of infectivity is dependent upon AAIM-1 secretion.  spores in  animals are improperly oriented in the intestinal lumen, leading to reduced levels of parasite invasion. Conversely,  mutants display both increased colonization and susceptibility to the bacterial pathogen  and overexpression of reduces  colonization. Competitive fitness assays show that  mutants are favored in the presence of  but disadvantaged on  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.

Aaron W. Reinke

Department of Molecular Genetics

University of Toronto

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection