A genetic screen in Drosophila reveals the role of fucosylation in host susceptibility to Candida infection.

Candida infections constitute a blind spot in global public health as very few new anti-fungal drugs are being developed. Genetic surveys of host susceptibilities to such infections using mammalian models have certain disadvantages in that obtaining results is time-consuming, owing to relatively long lifespans, and these results have low statistical resolution because sample sizes are usually small. Here, we report a targeted genetic screening of 5698 RNAi lines encompassing 4135 Drosophila genes with human homologues, several of which we identify as important for host survival after Candida albicans infection. These include genes in a variety of functional classes encompassing gene expression, intracellular signalling, metabolism and enzymatic regulation. Analysis of one of the screen hits, the infection-induced α-(1,3)-fucosylase FucTA, showed that N-glycan fucosylation has several targets among proteins involved in host defence, which provides multiple avenues of investigation for the mechanistic analysis of host survival to systemic C. albicans infection.

Correction: Bacterial recognition by PGRP-SA and downstream signalling by Toll/DIF sustain commensal gut bacteria in Drosophila.

[This corrects the article DOI: 10.1371/journal.pgen.1009992.].

Bacterial recognition by PGRP-SA and downstream signalling by Toll/DIF sustain commensal gut bacteria in Drosophila.

The gut sets the immune and metabolic parameters for the survival of commensal bacteria. We report that in Drosophila, deficiency in bacterial recognition upstream of Toll/NF-κB signalling resulted in reduced density and diversity of gut bacteria. Translational regulation factor 4E-BP, a transcriptional target of Toll/NF-κB, mediated this host-bacteriome interaction. In healthy flies, Toll activated 4E-BP, which enabled fat catabolism, which resulted in sustaining of the bacteriome. The presence of gut bacteria kept Toll signalling activity thus ensuring the feedback loop of their own preservation. When Toll activity was absent, TOR-mediated suppression of 4E-BP made fat resources inaccessible and this correlated with loss of intestinal bacterial density. This could be overcome by genetic or pharmacological inhibition of TOR, which restored bacterial density. Our results give insights into how an animal integrates immune sensing and metabolism to maintain indigenous bacteria in a healthy gut.

MicroRNAs That Contribute to Coordinating the Immune Response in Drosophila melanogaster.

Small noncoding RNAs called microRNAs (miRNAs) have emerged as post-transcriptional regulators of gene expression related to host defenses. Here, we have used Drosophila melanogaster to explore the contribution of individual or clusters of miRNAs in countering systemic Candida albicans infection. From a total of 72 tested, we identify 6 miRNA allelic mutant backgrounds that modulate the survival response to infection and the ability to control pathogen number. These mutants also exhibit dysregulation of the Toll pathway target transcripts Drosomycin (Drs) and Immune-Induced Molecule 1 (IM1). These are characteristics of defects in Toll signaling, and consistent with this, we demonstrate dependency for one of the miRNA mutants on the NF-κΒ homolog Dif. We also quantify changes in the miRNA expression profile over time in response to three pathogen types, and identify 13 mature miRNA forms affected by pathogens that stimulate Toll signaling. To complement this, we provide a genome-wide map of potential NF-κB sites in proximity to miRNA genes. Finally, we demonstrate that systemic C. albicans infection contributes to a reduction in the total amount of branch-chained amino acids, which is miRNA-regulated. Overall, our data reveal a new layer of miRNA complexity regulating the fly response to systemic fungal infection.

Wall teichoic acids of Staphylococcus aureus limit recognition by the drosophila peptidoglycan recognition protein-SA to promote pathogenicity.

The cell wall of gram-positive bacteria is a complex network of surface proteins, capsular polysaccharides and wall teichoic acids (WTA) covalently linked to Peptidoglycan (PG). The absence of WTA has been associated with a reduced pathogenicity of Staphylococcus aureus (S. aureus). Here, we assessed whether this was due to increased detection of PG, an important target of innate immune receptors. Antibiotic-mediated or genetic inhibition of WTA production in S. aureus led to increased binding of the non-lytic PG Recognition Protein-SA (PGRP-SA), and this was associated with a reduction in host susceptibility to infection. Moreover, PGRP-SD, another innate sensor required to control wild type S. aureus infection, became redundant. Our data imply that by using WTA to limit access of innate immune receptors to PG, under-detected bacteria are able to establish an infection and ultimately overwhelm the host. We propose that different PGRPs work in concert to counter this strategy.

Wild-type Drosophila melanogaster as an alternative model system for investigating the pathogenicity of Candida albicans.

Candida spp. are opportunistic pathogens in humans, and their systemic infections display upwards of 30% mortality in immunocompromised patients. Current mammalian model systems have certain disadvantages in that obtaining results is time consuming owing to the relatively long life spans and these results have low statistical resolution because sample sizes are usually small. We have therefore evaluated the potential of Drosophila melanogaster as an additional model system with which to dissect the host-pathogen interactions that occur during Candida albicans systemic infection. To do this, we monitored the survival of wild-type flies infected with various C. albicans clinical isolates that were previously ranked for murine virulence. From our lifetime data we computed two metrics of virulence for each isolate. These correlated significantly with murine survival, and were also used to group the isolates, and this grouping made relevant predictions regarding their murine virulence. Notably, differences in virulence were not predictably resolvable using immune-deficient spz(-/-) flies, suggesting that Toll signalling might actually be required to predictably differentiate virulence. Our analysis reveals wild-type D. melanogaster as a sensitive and relevant model system; one that offers immense genetic tractability (having an extensive RNA interference library that enables tissue-specific gene silencing), and that is easy to manipulate and culture. Undoubtedly, it will prove to be a valuable addition to the model systems currently used to study C. albicans infection.

Pathogen and host factors are needed to provoke a systemic host response to gastrointestinal infection of Drosophila larvae by Candida albicans.

Candida albicans systemic dissemination in immunocompromised patients is thought to develop from initial gastrointestinal (GI) colonisation. It is unclear what components of the innate immune system are necessary for preventing C. albicans dissemination from the GI tract, but studies in mice have indicated that both neutropenia and GI mucosal damage are crucial for allowing widespread invasive C. albicans disease. Mouse models, however, provide limited applicability to genome-wide screens for pathogen or host factors - factors that might influence systemic dissemination following GI colonisation. For this reason we developed a Drosophila model to study intestinal infection by Candida. We found that commensal flora aided host survival following GI infection. Candida provoked extensive JNK-mediated death of gut cells and induced antimicrobial peptide expression in the fat body. From the side of the host, nitric oxide and blood cells influenced systemic antimicrobial responses. The secretion of SAP4 and SAP6 (secreted aspartyl proteases) from Candida was also essential for activating systemic Toll-dependent immunity.

Toll-dependent antimicrobial responses in Drosophila larval fat body require Spätzle secreted by haemocytes.

In Drosophila, the humoral response characterised by the synthesis of antimicrobial peptides (AMPs) in the fat body (the equivalent of the mammalian liver) and the cellular response mediated by haemocytes (blood cells) engaged in phagocytosis represent two major reactions that counter pathogens. Although considerable analysis has permitted the elucidation of mechanisms pertaining to the two responses individually, the mechanism of their coordination has been unclear. To characterise the signals with which infection might be communicated between blood cells and fat body, we ablated circulating haemocytes and defined the parameters of AMP gene activation in larvae. We found that targeted ablation of blood cells influenced the levels of AMP gene expression in the fat body following both septic injury and oral infection. Expression of the AMP gene drosomycin (a Toll target) was blocked when expression of the Toll ligand Spätzle was knocked down in haemocytes. These results show that in larvae, integration of the two responses in a systemic reaction depend on the production of a cytokine (spz), a process that strongly parallels the mammalian immune response.

CYLD: a multifunctional deubiquitinase.

The nuclear factor-kappaB (NF-kappaB) and c-Jun NH2-terminal kinase (JNK) signaling pathways regulate diverse biological processes, including the immune and inflammatory response, cell growth, apoptosis, and tumour formation. Not surprisingly therefore defects to either pathway contributes to the progression of numerous human disorders. Enhancing our understanding of the mechanisms that control signaling through these pathways is therefore significant as it may enable development of specific treatments. In this regard, CYLD was recently identified as a negative regulator of NF-kappaB and JNK signaling. CYLD has a C-terminal catalytic domain characteristic of deubiquitinating enzymes, and this is essential for CYLD to remove ubiquitin from certain proteins that positively mediate signaling through the NF-kappaB and JNK pathways. Recent studies have revealed a requirement for CYLD in many different processes and have provided some insight into the underlying mechanisms.

Role of conserved intracellular motifs in Serrate signalling, cis-inhibition and endocytosis.

Notch is the receptor in a signalling pathway that operates in a diverse spectrum of developmental processes. Its ligands (e.g. Serrate) are transmembrane proteins whose signalling competence is regulated by the endocytosis-promoting E3 ubiquitin ligases, Mindbomb1 and Neuralized. The ligands also inhibit Notch present in the same cell (cis-inhibition). Here, we identify two conserved motifs in the intracellular domain of Serrate that are required for efficient endocytosis. The first, a dileucine motif, is dispensable for trans-activation and cis-inhibition despite the endocytic defect, demonstrating that signalling can be separated from bulk endocytosis. The second, a novel motif, is necessary for interactions with Mindbomb1/Neuralized and is strictly required for Serrate to trans-activate and internalise efficiently but not for it to inhibit Notch signalling. Cis-inhibition is compromised when an ER retention signal is added to Serrate, or when the levels of Neuralized are increased, and together these data indicate that cis-inhibitory interactions occur at the cell surface. The balance of ubiquitinated/unubiquitinated ligand will thus affect the signalling capacity of the cell at several levels.