Genomic analysis of two phlebotomine sand fly vectors of Leishmania from the New and Old World.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.

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.

The Phlebotomus papatasi systemic transcriptional response to trypanosomatid-contaminated blood does not differ from the non-infected blood meal.

BACKGROUND: Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that affects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand fly. Novel strategies for disease control require a better understanding of the key step for transmission, namely the establishment of infection inside the fly. METHODS: The aim of this work was to identify sand fly systemic transcriptomic signatures associated with Leishmania infection. We used next generation sequencing to describe the transcriptome of whole Phlebotomus papatasi sand flies when fed with blood alone (control) or with blood containing one of three trypanosomatids: Leishmania major, L. donovani and Herpetomonas muscarum, the latter being a parasite not transmitted to humans. RESULTS: Of the trypanosomatids studied, only L. major was able to successfully establish an infection in the host P. papatasi. However, the transcriptional signatures observed after each parasite-contaminated blood meal were not specific to success or failure of a specific infection and they did not differ from each other. The transcriptional signatures were also indistinguishable after a non-contaminated blood meal. CONCLUSIONS: The results imply that sand flies perceive Leishmania as just one feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment. Alternatively, Leishmania could suppress host responses. These results will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect.

HYD3, a conidial hydrophobin of the fungal entomopathogen Metarhizium acridum induces the immunity of its specialist host locust.

Conidial hydrophobins in fungal pathogens of plants, insects, and humans are required for fungal attachment and are associated with high virulence. They are believed to contribute to the pathogenesis of infection by preventing immune recognition. Here, we refute this generalisation offering a more nuanced analysis. We show that MacHYD3, a hydrophobin located on the conidial surface of the specialist entomopathogenic fungus Metarhizium acridum (narrow host range, kills only locusts and grasshoppers), activates specifically the humoral and cellular immunity of its own host insect, Locusta migratoria manilensis (Meyen) but not that of other non-host insects. When topically applied to the cuticle, purified MacHYD3 improved the resistance of locusts to both specialist and generalist fungal pathogens (wide host range) but had no effect on the fungal resistance of other insects, including Spodoptera frugiperda and Galleria mellonella. Hydrophobins extracted from the generalist fungal pathogens M. anisopliae and Beauveria bassiana had no effect on the resistance of locusts to fungal infection. Thus, the host locust has evolved to recognize the conidial hydrophobin of its specialist fungal pathogen, whereas conidial hydrophobins from generalist fungi are able to evade recognition. Our results distinguish the immunogenic potential of conidial hydrophobins between specialist and generalist fungi.

Beyond Host Defense: Deregulation of Drosophila Immunity and Age-Dependent Neurodegeneration.

Age-dependent neurodegenerative disorders are a set of diseases that affect millions of individuals worldwide. Apart from a small subset that are the result of well-defined inherited autosomal dominant gene mutations (e.g., those encoding the ß-amyloid precursor protein and presenilins), our understanding of the genetic network that underscores their pathology, remains scarce. Genome-wide association studies (GWAS) especially in Alzheimer's disease patients and research in Parkinson's disease have implicated inflammation and the innate immune response as risk factors. However, even if GWAS etiology points toward innate immunity, untangling cause, and consequence is a challenging task. Specifically, it is not clear whether predisposition to de-regulated immunity causes an inadequate response to protein aggregation (such as amyloid or α-synuclein) or is the direct cause of this aggregation. Given the evolutionary conservation of the innate immune response in Drosophila and humans, unraveling whether hyperactive immune response in glia have a protective or pathological role in the brain could be a potential strategy in combating age-related neurological diseases.

From pathogen to a commensal: modification of the Microbacterium nematophilum-Caenorhabditis elegans interaction during chronic infection by the absence of host insulin signalling.

The nematode worm Caenorhabditis elegans depends on microbes in decaying vegetation as its food source. To survive in an environment rich in opportunistic pathogens, Celegans has evolved an epithelial defence system where surface-exposed tissues such as epidermis, pharynx, intestine, vulva and hindgut have the capacity of eliciting appropriate immune defences to acute gut infection. However, it is unclear how the worm responds to chronic intestinal infections. To this end, we have surveyed Celegans mutants that are involved in inflammation, immunity and longevity to find their phenotypes during chronic infection. Worms that grew in a monoculture of the natural pathogen Microbacterium nematophilum (CBX102 strain) had a reduced lifespan and vigour. This was independent of intestinal colonisation as both CBX102 and the derived avirulent strain UV336 were early persistent colonisers. In contrast, the long-lived daf-2 mutant was resistant to chronic infection, showing reduced colonisation and higher vigour. In fact, UV336 interaction with daf-2 resulted in a host lifespan extension beyond OP50, the Escherichia coli strain used for laboratory Celegans culture. Longevity and vigour of daf-2 mutants growing on CBX102 was dependent on the FOXO orthologue DAF-16. Our results indicate that the interaction between host genotype and strain-specific bacteria determines longevity and health for C. elegans.

Tools for the Genetic Manipulation of Herpetomonas muscarum.

Trypanosomatid parasites are causative agents of important human and animal diseases such as sleeping sickness and leishmaniasis. Most trypanosomatids are transmitted to their mammalian hosts by insects, often belonging to Diptera (or true flies). With resistance to both vector-targeted pesticides and trypanocidal drugs being reported, there is a need for novel transmission blocking strategies to be developed. Studies using the blood-feeding vectors themselves are not broadly accessible, as such, new model systems are being developed to unpick insect-trypanosmatids interactions. One such case is the interactions between the model dipteran Drosophila melanogaster and its natural trypanosomatid Herpetomonas muscarum Our previous work has found that much of the transcriptomic changes triggered in H. muscarum after ingestion by Drosophila reflect what is known for disease-causing trypanosomatids. Here we describe a set of tools to genetically manipulate the parasite and therefore create a truly tractable insect-parasite interaction system from both sides of this association. These include transgenic fluorescently tagged parasites to follow infection dynamics in the fly gut as well as iterations of plasmids that can be used for generating knock-in and knock-out strains. The tools presented in this short report will facilitate further characterization of trypanosomatid establishment in a model dipteran.

Transcriptional and genomic parallels between the monoxenous parasite Herpetomonas muscarum and Leishmania.

Trypanosomatid parasites are causative agents of important human and animal diseases such as sleeping sickness and leishmaniasis. Most trypanosomatids are transmitted to their mammalian hosts by insects, often belonging to Diptera (or true flies). These are called dixenous trypanosomatids since they infect two different hosts, in contrast to those that infect just insects (monoxenous). However, it is still unclear whether dixenous and monoxenous trypanosomatids interact similarly with their insect host, as fly-monoxenous trypanosomatid interaction systems are rarely reported and under-studied-despite being common in nature. Here we present the genome of monoxenous trypanosomatid Herpetomonas muscarum and discuss its transcriptome during in vitro culture and during infection of its natural insect host Drosophila melanogaster. The H. muscarum genome is broadly syntenic with that of human parasite Leishmania major. We also found strong similarities between the H. muscarum transcriptome during fruit fly infection, and those of Leishmania during sand fly infections. Overall this suggests Drosophila-Herpetomonas is a suitable model for less accessible insect-trypanosomatid host-parasite systems such as sand fly-Leishmania.

Accessibility to Peptidoglycan Is Important for the Recognition of Gram-Positive Bacteria in Drosophila.

In Drosophila, it is thought that peptidoglycan recognition proteins (PGRPs) SA and LC structurally discriminate between bacterial peptidoglycans with lysine (Lys) or diaminopimelic (DAP) acid, respectively, thus inducing differential antimicrobial transcription response. Here, we find that accessibility to PG at the cell wall plays a central role in immunity to infection. When wall teichoic acids (WTAs) are genetically removed from S. aureus (Lys type) and Bacillus subtilis (DAP type), thus increasing accessibility, the binding of both PGRPs to either bacterium is increased. PGRP-SA and -LC double mutant flies are more susceptible to infection with both WTA-less bacteria. In addition, WTA-less bacteria grow better in PGRP-SA/-LC double mutant flies. Finally, infection with WTA-less bacteria abolishes any differential activation of downstream antimicrobial transcription. Our results indicate that accessibility to cell wall PG is a major factor in PGRP-mediated immunity and may be the cause for discrimination between classes of pathogens.

Intestinal NF-κB and STAT signalling is important for uptake and clearance in a Drosophila-Herpetomonas interaction model.

Dipteran insects transmit serious diseases to humans, often in the form of trypanosomatid parasites. To accelerate research in more difficult contexts of dipteran-parasite relationships, we studied the interaction of the model dipteran Drosophila melanogaster and its natural trypanosomatid Herpetomonas muscarum. Parasite infection reduced fecundity but not lifespan in NF-κB/Relish-deficient flies. Gene expression analysis implicated the two NF-κB pathways Toll and Imd as well as STAT signalling. Tissue specific knock-down of key components of these pathways in enterocytes (ECs) and intestinal stem cells (ISCs) influenced initial numbers, infection dynamics and time of clearance. Herpetomonas triggered STAT activation and proliferation of ISCs. Loss of Relish suppressed ISCs, resulting in increased parasite numbers and delayed clearance. Conversely, overexpression of Relish increased ISCs and reduced uptake. Finally, loss of Toll signalling decreased EC numbers and enabled parasite persistence. This network of signalling may represent a general mechanism with which dipteran respond to trypanosomatids.

Convergence of longevity and immunity: lessons from animal models.

An increasing amount of data implicate immunity-mostly innate immunity-in the ageing process; both during healthy ageing as well as in neurodegenerative diseases. Despite the aetiology however, the underlying mechanisms are poorly understood. Here we review what we know from model organisms (worms, flies and mice) on the possible mechanistic details that connect immunity and ageing. These links provide evidence that inter-tissue communication (especially the interaction between gut and brain), hormonal control mechanisms and intestinal microbiota determine immune system activity and thus influence lifespan.

A Host-Pathogen Interaction Screen Identifies ada2 as a Mediator of Candida glabrata Defenses Against Reactive Oxygen Species.

Candida glabrata (C. glabrata) forms part of the normal human gut microbiota but can cause life-threatening invasive infections in immune-compromised individuals. C. glabrata displays high resistance to common azole antifungals, which necessitates new treatments. In this investigation, we identified five C. glabrata deletion mutants (∆ada2, ∆bas1, ∆hir3, ∆ino2 and ∆met31) from a library of 196 transcription factor mutants that were unable to grow and activate an immune response in Drosophila larvae. This highlighted the importance of these transcription factors in C. glabrata infectivity. Further ex vivo investigation into these mutants revealed the requirement of C. glabrata ADA2 for oxidative stress tolerance. We confirmed this observation in vivo whereby growth of the C. glabrata Δada2 strain was permitted only in flies with suppressed production of reactive oxygen species (ROS). Conversely, overexpression of ADA2 promoted C. glabrata replication in infected wild type larvae resulting in larval killing. We propose that ADA2 orchestrates the response of C. glabrata against ROS-mediated immune defenses during infection. With the need to find alternative antifungal treatment for C. glabrata infections, genes required for survival in the host environment, such as ADA2, provide promising potential targets.

Functional analysis of the C. elegans cyld-1 gene reveals extensive similarity with its human homolog.

The human cylindromatosis tumor suppressor (HsCyld) has attracted extensive attention due to its association with the development of multiple types of cancer. HsCyld encodes a deubiquitinating enzyme (HsCYLD) with a broad range of functions that include the regulation of several cell growth, differentiation and death pathways. HsCyld is an evolutionarily conserved gene. Homologs of HsCyld have been identified in simple model organisms such as Drosophila melanogaster and Caenorhabditis elegans (C. elegans) which offer extensive possibilities for functional analyses. In the present report we have investigated and compared the functional properties of HsCYLD and its C. elegans homolog (CeCYLD). As expected from the mammalian CYLD expression pattern, the CeCyld promoter is active in multiple tissues with certain gastrointestinal epithelia and neuronal cells showing the most prominent activity. CeCYLD is a functional deubiquitinating enzyme with similar specificity to HsCYLD towards K63- and M1-linked polyubiquiting chains. CeCYLD was capable of suppressing the TRAF2-mediated activation of NF-kappaB and AP1 similarly to HsCYLD. Finally, CeCYLD could suppress the induction of TNF-dependent gene expression in mammalian cells similarly to HsCYLD. Our results demonstrate extensively overlapping functions between the HsCYLD and CeCYLD, which establish the C. elegans protein as a valuable model for the elucidation of the complex activity of the human tumor suppressor protein.

Immunity: Insect Immune Memory Goes Viral.

Adaptive memory in insect immunity has been controversial. In this issue, Andino and co-workers propose that acquisition of viral sequences in the host genome gives rise to anti-sense, anti-viral piRNAs. Such sequences can be regarded as both a genomic archive of past infections and as an armour of potential heritable memory.

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.

NF-κB Immunity in the Brain Determines Fly Lifespan in Healthy Aging and Age-Related Neurodegeneration.

During aging, innate immunity progresses to a chronically active state. However, what distinguishes those that "age well" from those developing age-related neurological conditions is unclear. We used Drosophila to explore the cost of immunity in the aging brain. We show that mutations in intracellular negative regulators of the IMD/NF-κB pathway predisposed flies to toxic levels of antimicrobial peptides, resulting in early locomotor defects, extensive neurodegeneration, and reduced lifespan. These phenotypes were rescued when immunity was suppressed in glia. In healthy flies, suppressing immunity in glial cells resulted in increased adipokinetic hormonal signaling with high nutrient levels in later life and an extension of active lifespan. Thus, when levels of IMD/NF-κB deviate from normal, two mechanisms are at play: lower levels derepress an immune-endocrine axis, which mobilizes nutrients, leading to lifespan extension, whereas higher levels increase antimicrobial peptides, causing neurodegeneration. Immunity in the fly brain is therefore a key lifespan determinant.