Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

Epithelial cells line the surfaces of the body, and are on the front lines of defense against microbial infection. Like many other metazoans, the nematode C. elegans lacks known professional immune cells and relies heavily on defense mediated by epithelial cells. New results indicate that epithelial defense in C. elegans can be triggered through detection of pathogen-induced perturbation of core physiology within host cells and through autophagic defense against intracellular and extracellular pathogens. Recent studies have also illuminated a diverse array of pathogenic attack strategies used against C. elegans. These findings are providing insight into the underpinnings of host/pathogen interactions in a simple animal host that can inform studies of infectious diseases in humans.

Defense against intracellular infection has been extensively studied in vertebrate hosts, but less is known about invertebrate hosts; specifically, the transcription factors that induce defense against intracellular intestinal infection in the model nematode Caenorhabditis elegans remain understudied. Two different types of intracellular pathogens that naturally infect the C. elegans intestine are the Orsay virus, which is an RNA virus, and microsporidia, which comprise a phylum of fungal pathogens. Despite their molecular differences, these pathogens induce a common host transcriptional response called the intracellular pathogen response (IPR). Here we show that zip-1 is an IPR regulator that functions downstream of all known IPR-activating and regulatory pathways. zip-1 encodes a putative bZIP transcription factor, and we show that zip-1 controls induction of a subset of genes upon IPR activation. ZIP-1 protein is expressed in the nuclei of intestinal cells, and is at least partially required in the intestine to upregulate IPR gene expression. Importantly, zip-1 promotes resistance to infection by the Orsay virus and by microsporidia in intestinal cells. Altogether, our results indicate that zip-1 represents a central hub for triggers of the IPR, and that this transcription factor has a protective function against intracellular pathogen infection in C. elegans.

Fungal infections, widespread throughout the world, affect a broad range of life forms, including agriculturally relevant plants, humans, and insects. In defending against fungal infections, the fruit fly Drosophila melanogaster employs the Toll pathway to induce a large number of immune peptides. Some have been investigated, such as the antimicrobial peptides (AMPs) and Bomanins (Boms); many, however, remain uncharacterized. Here, we examine the role in innate immunity of two related peptides, Daisho1 and Daisho2 (formerly IM4 and IM14, respectively), found in hemolymph following Toll pathway activation. By generating a CRISPR/Cas9 knockout of both genes, daisho, we find that the Daisho peptides are required for defense against a subset of filamentous fungi, including Fusarium oxysporum, but not other Toll-inducible pathogens, such as Enterococcus faecalis and Candida glabrata. Analysis of null alleles and transgenes revealed that the two daisho genes are each required for defense, although their functions partially overlap. Generating and assaying a genomic epitope-tagged Daisho2 construct, we detected interaction in vitro of Daisho2 peptide in hemolymph with the hyphae of F. oxysporum. Together, these results identify the Daisho peptides as a new class of innate immune effectors with humoral activity against a select set of filamentous fungi.

The fruit fly Drosophila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. Here we describe a novel Drosophila antifungal peptide gene that we name Baramicin A. We show that BaraA encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, we show that flies lacking BaraA are viable but susceptible to the entomopathogenic fungus Beauveria bassiana. Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA mutant males but not females display an erect wing phenotype upon infection. Here, we characterize a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to the Drosophila antimicrobial response.