Nichole A. Broderick

Drosophila Intestinal Response to Bacterial Infection: Activation of Host Defense and Stem Cell Proliferation

Although Drosophila systemic immunity is extensively studied, little is known about the flys intestine-specific responses to bacterial infection. Global gene expression analysis of Drosophila intestinal tissue to oral infection with the Gram-negative bacterium Erwinia carotovora revealed that immune responses in the gut are regulated by the Imd and JAK-STAT pathways, but not the Toll pathway. Ingestion of bacteria had a dramatic impact on the physiology of the gut that included modulation of stress response and increased stem cell proliferation and epithelial renewal. Our data suggest that gut homeostasis is maintained through a balance between cell damage due to the collateral effects of bacteria killing and epithelial repair by stem cell division. The Drosophila gut provides a powerful model to study the integration of stress and immunity with pathways associated with stem cell control, and this study should prove to be a useful resource for such further studies.

Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila

Gut homeostasis is controlled by both immune and developmental mechanisms, and its disruption can lead to inflammatory disorders or cancerous lesions of the intestine. While the impact of bacteria on the mucosal immune system is beginning to be precisely understood, little is known about the effects of bacteria on gut epithelium renewal. Here, we addressed how both infectious and indigenous bacteria modulate stem cell activity in Drosophila. We show that the increased epithelium renewal observed upon some bacterial infections is a consequence of the oxidative burst, a major defense of the Drosophila gut. Additionally, we provide evidence that the JAK-STAT (Janus kinase-signal transducers and activators of transcription) and JNK (c-Jun NH(2) terminal kinase) pathways are both required for bacteria-induced stem cell proliferation. Similarly, we demonstrate that indigenous gut microbiota activate the same, albeit reduced, program at basal levels. Altered control of gut microbiota in immune-deficient or aged flies correlates with increased epithelium renewal. Finally, we show that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection. Altogether, these results indicate that gut homeostasis is achieved by a complex interregulation of the immune response, gut microbiota, and stem cell activity.

Census of the bacterial community of the gypsy moth larval midgut by using culturing and culture-independent methods

ABSTRACT Little is known about bacteria associated with Lepidoptera, the large group of mostly phytophagous insects comprising the moths and butterflies. We inventoried the larval midgut bacteria of a polyphagous foliivore, the gypsy moth (Lymantria dispar L.), whose gut is highly alkaline, by using traditional culturing and culture-independent methods. We also examined the effects of diet on microbial composition. Analysis of individual third-instar larvae revealed a high degree of similarity of microbial composition among insects fed on the same diet. DNA sequence analysis indicated that most of the PCR-amplified 16S rRNA genes belong to the γ-Proteobacteria and low G+C gram-positive divisions and that the cultured members represented more than half of the phylotypes identified. Less frequently detected taxa included members of the α-Proteobacterium, Actinobacterium, and Cytophaga/Flexibacter/Bacteroides divisions. The 16S rRNA gene sequences from 7 of the 15 cultured organisms and 8 of the 9 sequences identified by PCR amplification diverged from previously reported bacterial sequences. The microbial composition of midguts differed substantially among larvae feeding on a sterilized artificial diet, aspen, larch, white oak, or willow. 16S rRNA analysis of cultured isolates indicated that an Enterococcus species and culture-independent analysis indicated that an Entbacter sp. were both present in all larvae, regardless of the feeding substrate; the sequences of these two phylotypes varied less than 1% among individual insects. These results provide the first comprehensive description of the microbial diversity of a lepidopteran midgut and demonstrate that the plant species in the diet influences the composition of the gut bacterial community.

Midgut bacteria required for Bacillus thuringiensis insecticidal activity

Bacillus thuringiensis is the most widely applied biological insecticide and is used to manage insects that affect forestry and agriculture and transmit human and animal pathogens. This ubiquitous spore-forming bacterium kills insect larvae largely through the action of insecticidal crystal proteins and is commonly deployed as a direct bacterial spray. Moreover, plants engineered with the cry genes encoding the B. thuringiensis crystal proteins are the most widely cultivated transgenic crops. For decades, the mechanism of insect killing has been assumed to be toxin-mediated lysis of the gut epithelial cells, which leads to starvation, or B. thuringiensis septicemia. Here, we report that B. thuringiensis does not kill larvae of the gypsy moth in the absence of indigenous midgut bacteria. Elimination of the gut microbial community by oral administration of antibiotics abolished B. thuringiensis insecticidal activity, and reestablishment of an Enterobacter sp. that normally resides in the midgut microbial community restored B. thuringiensis-mediated killing. Escherichia coli engineered to produce the B. thuringiensis insecticidal toxin killed gypsy moth larvae irrespective of the presence of other bacteria in the midgut. However, when the engineered E. coli was heat-killed and then fed to the larvae, the larvae did not die in the absence of the indigenous midgut bacteria. E. coli and the Enterobacter sp. achieved high populations in hemolymph, in contrast to B. thuringiensis, which appeared to die in hemolymph. Our results demonstrate that B. thuringiensis-induced mortality depends on enteric bacteria.

Gut homeostasis in a microbial world: insights from Drosophila melanogaster

Intestinal homeostasis is achieved, in part, by the integration of a complex set of mechanisms that eliminate pathogens and tolerate the indigenous microbiota. Drosophila melanogaster feeds on microorganism-enriched matter and therefore has developed efficient mechanisms to control ingested microorganisms. Regulatory mechanisms ensure an appropriate level of immune reactivity in the gut to accommodate the presence of beneficial and dietary microorganisms, while allowing effective immune responses to clear pathogens. Maintenance of D. melanogaster gut homeostasis also involves regeneration of the intestine to repair damage associated with infection. Entomopathogenic bacteria have developed common strategies to subvert these defence mechanisms and kill their host.

Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection

BackgroundGut homeostasis is central to whole organism health, and its disruption is associated with a broad range of pathologies. Following damage, complex physiological events are required in the gut to maintain proper homeostasis. Previously, we demonstrated that ingestion of a nonlethal pathogen, Erwinia carotovora carotovora 15, induces a massive increase in stem cell proliferation in the gut of Drosophila. However, the precise cellular events that occur following infection have not been quantitatively described, nor do we understand the interaction between multiple pathways that have been implicated in epithelium renewal.ResultsTo understand the process of infection and epithelium renewal in more detail, we performed a quantitative analysis of several cellular and morphological characteristics of the gut. We observed that the gut of adult Drosophila undergoes a dynamic remodeling in response to bacterial infection. This remodeling coordinates the synthesis of new enterocytes, their proper morphogenesis and the elimination of damaged cells through delamination and anoikis. We demonstrate that one signaling pathway, the epidermal growth factor receptor (EGFR) pathway, is key to controlling each of these steps through distinct functions in intestinal stem cells and enterocytes. The EGFR pathway is activated by the EGF ligands, Spitz, Keren and Vein, the latter being induced in the surrounding visceral muscles in part under the control of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Additionally, the EGFR pathway synergizes with the JAK/STAT pathway in stem cells to promote their proliferation. Finally, we show that the EGFR pathway contributes to gut morphogenesis through its activity in enterocytes and is required to properly coordinate the delamination and anoikis of damaged cells. This function of the EGFR pathway in enterocytes is key to maintaining homeostasis, as flies lacking EGFR are highly susceptible to infection.ConclusionsThis study demonstrates that restoration of normal gut morphology following bacterial infection is a more complex phenomenon than previously described. Maintenance of gut homeostasis requires the coordination of stem cell proliferation and differentiation, with the incorporation and morphogenesis of new cells and the expulsion of damaged enterocytes. We show that one signaling pathway, the EGFR pathway, is central to all these stages, and its activation at multiple steps could synchronize the complex cellular events leading to gut repair and homeostasis.

Gut-associated microbes of Drosophila melanogaster

There is growing interest in using Drosophila melanogaster to elucidate mechanisms that underlie the complex relationships between a host and its microbiota. In addition to the many genetic resources and tools Drosophila provides, its associated microbiota is relatively simple (1–30 taxa), in contrast to the complex diversity associated with vertebrates (> 500 taxa). These attributes highlight the potential of this system to dissect the complex cellular and molecular interactions that occur between a host and its microbiota. In this review, we summarize what is known regarding the composition of gut-associated microbes of Drosophila and their impact on host physiology. We also discuss these interactions in the context of their natural history and ecology and describe some recent insights into mechanisms by which Drosophila and its gut microbiota interact. “Workers with Drosophila have been considered fortunate in that they deal with the first multicellular invertebrate to be cultured monoxenically (Delcourt and Guyenot, 1910); the first to be handled axenically on a semisynthetic diet (Guyenot, 1917); and the first to be grown on a defined diet (Schultz et al., 1946). This list of advantages is somewhat embarrassing, since it implies an interest in nutrition that, in reality, was only secondary. The very first studies were concerned with the reduction of variability in genetic experiments (Delcourt and Guyenot, 1910) and standardization of the nutritional environment.” -James Sang, 1959 Ann NY Acad 1

Bloom of resident antibiotic-resistant bacteria in soil following manure fertilization

Significance The increasing prevalence of antibiotic-resistant bacteria is one of the most serious threats to public health in the 21st century. One route by which resistance genes enter the food system is through amendment of soils with manure from antibiotic-treated animals, which are considered a reservoir of such genes. Previous studies have associated application of pig manure with the dispersal of sulfonamide-resistance genes to soil bacteria. In this study, we found that dairy cow manure amendment enhanced the proliferation of resident antibiotic-resistant bacteria and genes encoding β-lactamases in soil even though the cows from which the manure was derived had not been treated with antibiotics. Our findings provide previously unidentified insight into the mechanism by which amendment with manure enriches antibiotic-resistant bacteria in soil. The increasing prevalence of antibiotic-resistant bacteria is a global threat to public health. Agricultural use of antibiotics is believed to contribute to the spread of antibiotic resistance, but the mechanisms by which many agricultural practices influence resistance remain obscure. Although manure from dairy farms is a common soil amendment in crop production, its impact on the soil microbiome and resistome is not known. To gain insight into this impact, we cultured bacteria from soil before and at 10 time points after application of manure from cows that had not received antibiotic treatment. Soil treated with manure contained a higher abundance of β-lactam–resistant bacteria than soil treated with inorganic fertilizer. Functional metagenomics identified β-lactam–resistance genes in treated and untreated soil, and indicated that the higher frequency of resistant bacteria in manure-amended soil was attributable to enrichment of resident soil bacteria that harbor β-lactamases. Quantitative PCR indicated that manure treatment enriched the blaCEP-04 gene, which is highly similar (96%) to a gene found previously in a Pseudomonas sp. Analysis of 16S rRNA genes indicated that the abundance of Pseudomonas spp. increased in manure-amended soil. Populations of other soil bacteria that commonly harbor β-lactamases, including Janthinobacterium sp. and Psychrobacter pulmonis, also increased in response to manure treatment. These results indicate that manure amendment induced a bloom of certain antibiotic-resistant bacteria in soil that was independent of antibiotic exposure of the cows from which the manure was derived. Our data illustrate the unintended consequences that can result from agricultural practices, and demonstrate the need for empirical analysis of the agroecosystem.

Microbiota-Induced Changes in Drosophila melanogaster Host Gene Expression and Gut Morphology

ABSTRACT To elucidate mechanisms underlying the complex relationships between a host and its microbiota, we used the genetically tractable model Drosophila melanogaster. Consistent with previous studies, the microbiota was simple in composition and diversity. However, analysis of single flies revealed high interfly variability that correlated with differences in feeding. To understand the effects of this simple and variable consortium, we compared the transcriptome of guts from conventionally reared flies to that for their axenically reared counterparts. Our analysis of two wild-type fly lines identified 121 up- and 31 downregulated genes. The majority of these genes were associated with immune responses, tissue homeostasis, gut physiology, and metabolism. By comparing the transcriptomes of young and old flies, we identified temporally responsive genes and showed that the overall impact of microbiota was greater in older flies. In addition, comparison of wild-type gene expression with that of an immune-deficient line revealed that 53% of upregulated genes exerted their effects through the immune deficiency (Imd) pathway. The genes included not only classic immune response genes but also those involved in signaling, gene expression, and metabolism, unveiling new and unexpected connections between immunity and other systems. Given these findings, we further characterized the effects of gut-associated microbes on gut morphology and epithelial architecture. The results showed that the microbiota affected gut morphology through their impacts on epithelial renewal rate, cellular spacing, and the composition of different cell types in the epithelium. Thus, while bacteria in the gut are highly variable, the influence of the microbiota at large has far-reaching effects on host physiology. IMPORTANCE The guts of animals are in constant association with microbes, and these interactions are understood to have important roles in animal development and physiology. Yet we know little about the mechanisms underlying the establishment and function of these associations. Here, we used the fruit fly to understand how the microbiota affects host function. Importantly, we found that the microbiota has far-reaching effects on host physiology, ranging from immunity to gut structure. Our results validate the notion that important insights on complex host-microbe relationships can be obtained from the use of a well-established and genetically tractable invertebrate model. The guts of animals are in constant association with microbes, and these interactions are understood to have important roles in animal development and physiology. Yet we know little about the mechanisms underlying the establishment and function of these associations. Here, we used the fruit fly to understand how the microbiota affects host function. Importantly, we found that the microbiota has far-reaching effects on host physiology, ranging from immunity to gut structure. Our results validate the notion that important insights on complex host-microbe relationships can be obtained from the use of a well-established and genetically tractable invertebrate model.

Contributions of gut bacteria to Bacillus thuringiensis-induced mortality vary across a range of Lepidoptera

BackgroundGut microbiota contribute to the health of their hosts, and alterations in the composition of this microbiota can lead to disease. Previously, we demonstrated that indigenous gut bacteria were required for the insecticidal toxin of Bacillus thuringiensis to kill the gypsy moth, Lymantria dispar. B. thuringiensis and its associated insecticidal toxins are commonly used for the control of lepidopteran pests. A variety of factors associated with the insect host, B. thuringiensis strain, and environment affect the wide range of susceptibilities among Lepidoptera, but the interaction of gut bacteria with these factors is not understood. To assess the contribution of gut bacteria to B. thuringiensis susceptibility across a range of Lepidoptera we examined larval mortality of six species in the presence and absence of their indigenous gut bacteria. We then assessed the effect of feeding an enteric bacterium isolated from L. dispar on larval mortality following ingestion of B. thuringiensis toxin.ResultsOral administration of antibiotics reduced larval mortality due to B. thuringiensis in five of six species tested. These included Vanessa cardui (L.), Manduca sexta (L.), Pieris rapae (L.) and Heliothis virescens (F.) treated with a formulation composed of B. thuringiensis cells and toxins (DiPel), and Lymantria dispar (L.) treated with a cell-free formulation of B. thuringiensis toxin (MVPII). Antibiotics eliminated populations of gut bacteria below detectable levels in each of the insects, with the exception of H. virescens, which did not have detectable gut bacteria prior to treatment. Oral administration of the Gram-negative Enterobacter sp. NAB3, an indigenous gut resident of L. dispar, restored larval mortality in all four of the species in which antibiotics both reduced susceptibility to B. thuringiensis and eliminated gut bacteria, but not in H. virescens. In contrast, ingestion of B. thuringiensis toxin (MVPII) following antibiotic treatment significantly increased mortality of Pectinophora gossypiella (Saunders), which was also the only species with detectable gut bacteria that lacked a Gram-negative component. Further, mortality of P. gossypiella larvae reared on diet amended with B. thuringiensis toxin and Enterobacter sp. NAB3 was generally faster than with B. thuringiensis toxin alone.ConclusionThis study demonstrates that in some larval species, indigenous gut bacteria contribute to B. thuringiensis susceptibility. Moreover, the contribution of enteric bacteria to host mortality suggests that perturbations caused by toxin feeding induce otherwise benign gut bacteria to exert pathogenic effects. The interaction between B. thuringiensis and the gut microbiota of Lepidoptera may provide a useful model with which to identify the factors involved in such transitions.