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Autophagy and phagocytosis are cellular immune mechanisms for internalization and elimination of intracellular and extracellular pathogens. Some pathogens have evolved the ability to inhibit or manipulate these processes, raising the prospect of adaptive reciprocal co-evolution by the host. We performed population genetic analyses on phagocytosis and autophagy genes in Drosophila melanogaster and D. simulans to test for molecular evolutionary signatures of immune adaptation. We found that phagocytosis and autophagy genes as a whole exhibited an elevated level of haplotype homozygosity in both species. In addition, we detected signatures of recent selection, notably in the Atg14 and Ykt6 genes in D. melanogaster and a pattern of elevated sequence divergence in the genderblind (gb) gene on the D. simulans lineage. These results suggest that the evolution of the host cellular immune system as a whole may be shaped by a dynamic conflict between Drosophila and its pathogens even without pervasive evidence of strong adaptive evolution at the individual gene level.

Background Host sexual dimorphism is being increasingly recognized to generate strong differences in the outcome of infectious disease, but the mechanisms underlying immunological differences between males and females remain poorly characterized. Here, we used Drosophila melanogaster to assess and dissect sexual dimorphism in the innate response to systemic bacterial infection. Results We demonstrated sexual dimorphism in susceptibility to infection by a broad spectrum of Gram-positive and Gram-negative bacteria. We found that both virgin and mated females are more susceptible than mated males to most, but not all, infections. We investigated in more detail the lower resistance of females to infection with Providencia rettgeri, a Gram-negative bacterium that naturally infects D. melanogaster. We found that females have a higher number of phagocytes than males and that ablation of hemocytes does not eliminate the dimorphism in resistance to P. rettgeri, so the observed dimorphism does not stem from differences in the cellular response. The Imd pathway is critical for the production of antimicrobial peptides in response to Gram-negative bacteria, but mutants for Imd signaling continued to exhibit dimorphism even though both sexes showed strongly reduced resistance. Instead, we found that the Toll pathway is responsible for the dimorphism in resistance. The Toll pathway is dimorphic in genome-wide constitutive gene expression and in induced response to infection. Toll signaling is dimorphic in both constitutive signaling and in induced activation in response to P. rettgeri infection. The dimorphism in pathway activation can be specifically attributed to Persephone-mediated immune stimulation, by which the Toll pathway is triggered in response to pathogen-derived virulence factors. We additionally found that, in absence of Toll signaling, males become more susceptible than females to the Gram-positive Enterococcus faecalis. This reversal in susceptibility between male and female Toll pathway mutants compared to wildtype hosts highlights the key role of the Toll pathway in D. melanogaster sexual dimorphism in resistance to infection. Conclusion Altogether, our data demonstrate that Toll pathway activity differs between male and female D. melanogaster in response to bacterial infection, thus identifying innate immune signaling as a determinant of sexual immune dimorphism. Electronic supplementary material The online version of this article (doi:10.1186/s12915-017-0466-3) contains supplementary material, which is available to authorized users.

Organismal fitness depends on the ability of gene networks to function robustly in the face of environmental and genetic perturbations. Understanding the mechanisms of this stability is one of the key aims of modern systems biology. Dissecting the basis of robustness to mutation has proven a particular challenge, with most experimental models relying on artificial DNA sequence variants engineered in the laboratory. In this work, we hypothesized that negative regulatory feedback could stabilize gene expression against the disruptions that arise from natural genetic variation. We screened yeast transcription factors for feedback and used the results to establish ROX1 ( R epressor of hyp OX ia) as a model system for the study of feedback in circuit behaviors and its impact across genetically heterogeneous populations. Mutagenesis experiments revealed the mechanism of Rox1 as a direct transcriptional repressor at its own gene, enabling a regulatory program of rapid induction during environmental change that reached a plateau of moderate steady-state expression. Additionally, in a given environmental condition, Rox1 levels varied widely across genetically distinct strains; the ROX1 feedback loop regulated this variation, in that the range of expression levels across genetic backgrounds showed greater spread in ROX1 feedback mutants than among strains with the ROX1 feedback loop intact. Our findings indicate that the ROX1 feedback circuit is tuned to respond to perturbations arising from natural genetic variation in addition to its role in induction behavior. We suggest that regulatory feedback may be an important element of the network architectures that confer mutational robustness across biology.

Host responses to infection encompass many processes in addition to activation of the immune system, including metabolic adaptations, stress responses, tissue repair, and other reactions. The response to bacterial infection in Drosophila melanogaster has been classically described in studies that focused on the immune response elicited by a small set of largely avirulent microbes. Thus, we have surprisingly limited knowledge of responses to infection that are outside the canonical immune response, of how the response to pathogenic infection differs from that to avirulent bacteria, or even of how generic the response to various microbes is and what regulates that core response. In this study, we addressed these questions by profiling the D. melanogaster transcriptomic response to 10 bacteria that span the spectrum of virulence. We found that each bacterium triggers a unique transcriptional response, with distinct genes making up to one third of the response elicited by highly virulent bacteria. We also identified a core set of 252 genes that are differentially expressed in response to the majority of bacteria tested. Among these, we determined that the transcription factor CrebA is a novel regulator of infection tolerance. Knock-down of CrebA significantly increased mortality from microbial infection without any concomitant change in bacterial number. Upon infection, CrebA is upregulated by both the Toll and Imd pathways in the fat body, where it is required to induce the expression of secretory pathway genes. Loss of CrebA during infection triggered endoplasmic reticulum (ER) stress and activated the unfolded protein response (UPR), which contributed to infection-induced mortality. Altogether, our study reveals essential features of the response to bacterial infection and elucidates the function of a novel regulator of infection tolerance., Author summary How does an organism survive infection? How generic or specific is the host response to diverse pathogens? To address these questions, we infected fruit flies with 10 different bacteria that vary in their ability to kill flies and measured changes in global gene expression. In general, we found that the host response is highly specific to individual bacteria. However, we also discovered a set of genes that changed expression in response to the majority of bacteria tested. Among these genes, we determined that the transcription factor CrebA is a novel regulator of the host response to infection. We found that upon infection, the immune system induces the expression of CrebA. CrebA-deficient flies are more likely to die from infection despite carrying the same number of bacteria as wildtype flies. CrebA is expressed in the fat body, an organ analogous to the mammalian liver and adipose tissues, where it regulates the transcription of multiple secretory pathway genes. Loss of CrebA during infection triggers endoplasmic reticulum (ER) stress (a type of cellular stress), which is sufficient to sensitize flies to infection. These results suggest that the immune system can modulate host physiology to prevent the deleterious effect of infection-associated cellular stress.

Gene expression varies widely between individuals of a population, and regulatory change can underlie phenotypes of evolutionary and biomedical relevance. A key question in the field is how DNA sequence variants impact gene expression, with most mechanistic studies to date focused on the effects of genetic change on regulatory regions upstream of protein-coding sequence. By contrast, the role of RNA 3′-end processing in regulatory variation remains largely unknown, owing in part to the challenge of identifying functional elements in 3′ untranslated regions. In this work, we conducted a genomic survey of transcript ends in lymphoblastoid cells from genetically distinct human individuals. Our analysis mapped the cis-regulatory architecture of 3′ gene ends, finding that transcript end positions did not fall randomly in untranslated regions, but rather preferentially flanked the locations of 3′ regulatory elements, including miRNA sites. The usage of these transcript length forms and motifs varied across human individuals, and polymorphisms in polyadenylation signals and other 3′ motifs were significant predictors of expression levels of the genes in which they lay. Independent single-gene experiments confirmed the effects of polyadenylation variants on steady-state expression of their respective genes, and validated the regulatory function of 3′ cis-regulatory sequence elements that mediated expression of these distinct RNA length forms. Focusing on the immune regulator IRF5, we established the effect of natural variation in RNA 3′-end processing on regulatory response to antigen stimulation. Our results underscore the importance of two mechanisms at play in the genetics of 3′-end variation: the usage of distinct 3′-end processing signals and the effects of 3′ sequence elements that determine transcript fate. Our findings suggest that the strategy of integrating observed 3′-end positions with inferred 3′ regulatory motifs will prove to be a critical tool in continued efforts to interpret human genome variation., Author Summary Messenger RNAs carry the instructions necessary to synthesize proteins that do work for the cell. Extending beyond the protein-coding sequence of a given mRNA is an additional stretch of sequence, harboring signals that govern how much protein is made and how long the mRNA remains in the cell before it is broken down. The incorporation of this end region into mature mRNA is itself subject to change; for the vast majority of human genes, how and why cells use different mRNA ends remains largely unknown. In this work, we surveyed mRNA ends from ∼10,000 genes in immune cells from genetically distinct human individuals. We found that mRNA end positions were not randomly distributed, but rather preferentially flanked the locations of regulatory signals that govern mRNA fate. The usage of these mRNA length forms and regulatory elements varied across individuals and could be dissected molecularly. Our results uncover key mechanisms and regulatory effects of transcript end processing, particularly as these are perturbed by genetic differences between humans.