Dietary restriction ameliorates TBI-induced phenotypes in Drosophila melanogaster.

Traumatic brain injury (TBI) affects millions annually and is associated with long-term health decline. TBI also shares molecular and cellular hallmarks with neurodegenerative diseases (NDs), typically increasing in prevalence with age, and is a major risk factor for developing neurodegeneration later in life. While our understanding of genes and pathways that underlie neurotoxicity in specific NDs has advanced, we still lack a complete understanding of early molecular and physiological changes that drive neurodegeneration, particularly as an individual ages following a TBI. Recently Drosophila has been introduced as a model organism for studying closed-head TBI. In this paper, we deliver a TBI to flies early in adult life, and then measure molecular and physiological phenotypes at short-, mid-, and long-term timepoints following the injury. We aim to identify the timing of changes that contribute to neurodegeneration. Here we confirm prior work demonstrating a TBI-induced decline in lifespan, and present evidence of a progressive decline in locomotor function, robust acute and modest chronic neuroinflammation, and a late-onset increase in protein aggregation. We also present evidence of metabolic dysfunction, in the form of starvation sensitivity and decreased lipids, that persists beyond the immediate injury response, but does not differ long-term. An intervention of dietary restriction (DR) partially ameliorates some TBI-induced phenotypes, including lifespan and locomotor function, though it does not alter the pattern of starvation sensitivity of injured flies. In the future, molecular pathways identified as altered following TBI-particularly in the short-, or mid-term-could present potential therapeutic targets.

A Drosophila model of Fragile X syndrome exhibits defects in phagocytosis by innate immune cells.

Fragile X syndrome, the most common known monogenic cause of autism, results from the loss of FMR1, a conserved, ubiquitously expressed RNA-binding protein. Recent evidence suggests that Fragile X syndrome and other types of autism are associated with immune system defects. We found that Drosophila melanogaster Fmr1 mutants exhibit increased sensitivity to bacterial infection and decreased phagocytosis of bacteria by systemic immune cells. Using tissue-specific RNAi-mediated knockdown, we showed that Fmr1 plays a cell-autonomous role in the phagocytosis of bacteria. Fmr1 mutants also exhibit delays in two processes that require phagocytosis by glial cells, the immune cells in the brain: neuronal clearance after injury in adults and the development of the mushroom body, a brain structure required for learning and memory. Delayed neuronal clearance is associated with reduced recruitment of activated glia to the site of injury. These results suggest a previously unrecognized role for Fmr1 in regulating the activation of phagocytic immune cells both in the body and the brain.

period-Regulated Feeding Behavior and TOR Signaling Modulate Survival of Infection.

Most metazoans undergo dynamic, circadian-regulated changes in behavior and physiology. Currently, it is unknown how circadian-regulated behavior impacts immunity against infection. Two broad categories of defense against bacterial infection are resistance, control of microbial growth, and tolerance, control of the pathogenic effects of infection. Our study of behaviorally arrhythmic Drosophila circadian period mutants identified a novel link between nutrient intake and tolerance of infection with B. cepacia, a bacterial pathogen of rising importance in hospital-acquired infections. We found that infection tolerance in wild-type animals is stimulated by acute exposure to dietary glucose and amino acids. Glucose-stimulated tolerance was induced by feeding or direct injection; injections revealed a narrow window for glucose-stimulated tolerance. In contrast, amino acids stimulated tolerance only when ingested. We investigated the role of a known amino-acid-sensing pathway, the TOR (Target of Rapamycin) pathway, in immunity. TORC1 is circadian regulated and inhibition of TORC1 decreased resistance, as in vertebrates. Surprisingly, inhibition of the less well-characterized TOR complex 2 (TORC2) dramatically increased survival, through both resistance and tolerance mechanisms. This work suggests that dietary intake on the day of infection by B. cepacia can make a significant difference in long-term survival. We further demonstrate that TOR signaling mediates both resistance and tolerance of infection and identify TORC2 as a novel potential therapeutic target for increasing survival of infection.

Message in a biota: gut microbes signal to the circadian clock.

Circadian rhythm involves diurnal oscillations in biological processes. In this issue of Cell Host & Microbe, Leone et al. (2015) show that the gut microbiota influences the circadian clock and undergoes circadian oscillations. Microbiota-produced metabolites change with host diet and may affect circadian rhythm, highlighting functional links between diet and physiology.

Rational manipulation of mRNA folding free energy allows rheostat control of pneumolysin production by Streptococcus pneumoniae.

The contribution of specific factors to bacterial virulence is generally investigated through creation of genetic "knockouts" that are then compared to wild-type strains or complemented mutants. This paradigm is useful to understand the effect of presence vs. absence of a specific gene product but cannot account for concentration-dependent effects, such as may occur with some bacterial toxins. In order to assess threshold and dose-response effects of virulence factors, robust systems for tunable expression are required. Recent evidence suggests that the folding free energy (ΔG) of the 5' end of mRNA transcripts can have a significant effect on translation efficiency and overall protein abundance. Here we demonstrate that rational alteration of 5' mRNA folding free energy by introduction of synonymous mutations allows for predictable changes in pneumolysin (PLY) expression by Streptococcus pneumoniae without the need for chemical inducers or heterologous promoters. We created a panel of isogenic S. pneumoniae strains, differing only in synonymous (silent) mutations at the 5' end of the PLY mRNA that are predicted to alter ΔG. Such manipulation allows rheostat-like control of PLY production and alters the cytotoxicity of whole S. pneumoniae on primary and immortalized human cells. These studies provide proof-of-principle for further investigation of mRNA ΔG manipulation as a tool in studies of bacterial pathogenesis.

The circadian clock protein timeless regulates phagocytosis of bacteria in Drosophila.

Survival of bacterial infection is the result of complex host-pathogen interactions. An often-overlooked aspect of these interactions is the circadian state of the host. Previously, we demonstrated that Drosophila mutants lacking the circadian regulatory proteins Timeless (Tim) and Period (Per) are sensitive to infection by S. pneumoniae. Sensitivity to infection can be mediated either by changes in resistance (control of microbial load) or tolerance (endurance of the pathogenic effects of infection). Here we show that Tim regulates resistance against both S. pneumoniae and S. marcescens. We set out to characterize and identify the underlying mechanism of resistance that is circadian-regulated. Using S. pneumoniae, we found that resistance oscillates daily in adult wild-type flies and that these oscillations are absent in Tim mutants. Drosophila have at least three main resistance mechanisms to kill high levels of bacteria in their hemolymph: melanization, antimicrobial peptides, and phagocytosis. We found that melanization is not circadian-regulated. We further found that basal levels of AMP gene expression exhibit time-of-day oscillations but that these are Tim-independent; moreover, infection-induced AMP gene expression is not circadian-regulated. We then show that phagocytosis is circadian-regulated. Wild-type flies exhibit up-regulated phagocytic activity at night; Tim mutants have normal phagocytic activity during the day but lack this night-time peak. Tim appears to regulate an upstream event in phagocytosis, such as bacterial recognition or activation of phagocytic hemocytes. Interestingly, inhibition of phagocytosis in wild type flies results in survival kinetics similar to Tim mutants after infection with S. pneumoniae. Taken together, these results suggest that loss of circadian oscillation of a specific immune function (phagocytosis) can have significant effects on long-term survival of infection.

Genomic and Experimental Evidence Suggests that Verrucomicrobium spinosum Interacts with Eukaryotes.

Our knowledge of pathogens and symbionts is heavily biased toward phyla containing species that are straightforward to isolate in pure culture. Novel bacterial phyla are often represented by a handful of strains, and the number of species interacting with eukaryotes is likely underestimated. Identification of predicted pathogenesis and symbiosis determinants such as the Type III Secretion System (T3SS) in the genomes of "free-living" bacteria suggests that these microbes participate in uncharacterized interactions with eukaryotes. Our study aimed to test this hypothesis on Verrucomicrobium spinosum (phylum Verrucomicrobia) and to begin characterization of its predicted T3SS. We showed the putative T3SS structural genes to be transcriptionally active, and that expression of predicted effector proteins was toxic to yeast in an established functional screen. Our results suggest that the predicted T3SS genes of V. spinosum could encode a functional T3SS, although further work is needed to determine whether V. spinosum produces a T3SS injectisome that delivers the predicted effectors. In the absence of a known eukaryotic host, we made use of invertebrate infection models. The injection or feeding of V. spinosum to Drosophila melanogaster and Caenorhabditis elegans, respectively, was shown to result in increased mortality rates relative to controls, a phenomenon exaggerated in C. elegans mutants hypersensitive to pathogen infection. This finding, although not conclusively demonstrating pathogenesis, suggests that V. spinosum is capable of pathogenic activity toward an invertebrate host. Symbiotic interactions with a natural host provide an alternative explanation for the results seen in the invertebrate models. Further work is needed to determine whether V. spinosum can establish and maintain interactions with eukaryotic species found in its natural habitat, and whether the predicted T3SS is directly involved in pathogenic or symbiotic activity.

Confronting physiology: how do infected flies die?

Fruit fly immunology is on the verge of an exciting new path. The fruit fly has served as a strong model for innate immune responses; the field is now expanding to use the fruit fly to study pathogenesis. We argue here that, to understand pathogenesis in the fly, we need to understand pathology - and to understand pathology, we need to confront physiology with molecular tools. When flies are infected with a pathogen, they get sick. We group the events following infection into three categories: innate immune responses (defence mechanisms by which the fly attempts to kill or neutralize the microbe, some of which can themselves cause harm to the fly); microbial virulence (mechanisms by which the microbe evades the immune response); and host pathology (physiologies adversely affected by either the immune response or microbial virulence). We divide this review into sections mirroring these categories. The molecular study of infection in the fruit fly has focused on the first category, has begun to explore the second, and has yet to tap the full potential of the fly regarding the third.

Drosophila eiger mutants are sensitive to extracellular pathogens.

We showed previously that eiger, the Drosophila tumor necrosis factor homolog, contributes to the pathology induced by infection with Salmonella typhimurium. We were curious whether eiger is always detrimental in the context of infection or if it plays a role in fighting some types of microbes. We challenged wild-type and eiger mutant flies with a collection of facultative intracellular and extracellular pathogens, including a fungus and Gram-positive and Gram-negative bacteria. The response of eiger mutants divided these microbes into two groups: eiger mutants are immunocompromised with respect to extracellular pathogens but show no change or reduced sensitivity to facultative intracellular pathogens. Hence, eiger helps fight infections but also can cause pathology. We propose that eiger activates the cellular immune response of the fly to aid clearance of extracellular pathogens. Intracellular pathogens, which can already defeat professional phagocytes, are unaffected by eiger.