Construction and Modeling of a Coculture Microplate for Real-Time Measurement of Microbial Interactions.

The dynamic structures of microbial communities emerge from the complex network of interactions between their constituent microorganisms. Quantitative measurements of these interactions are important for understanding and engineering ecosystem structure. Here, we present the development and application of the BioMe plate, a redesigned microplate device in which pairs of wells are separated by porous membranes. BioMe facilitates the measurement of dynamic microbial interactions and integrates easily with standard laboratory equipment. We first applied BioMe to recapitulate recently characterized, natural symbiotic interactions between bacteria isolated from the Drosophila melanogaster gut microbiome. Specifically, the BioMe plate allowed us to observe the benefit provided by two Lactobacillus strains to an Acetobacter strain. We next explored the use of BioMe to gain quantitative insight into the engineered obligate syntrophic interaction between a pair of Escherichia coli amino acid auxotrophs. We integrated experimental observations with a mechanistic computational model to quantify key parameters associated with this syntrophic interaction, including metabolite secretion and diffusion rates. This model also allowed us to explain the slow growth observed for auxotrophs growing in adjacent wells by demonstrating that, under the relevant range of parameters, local exchange between auxotrophs is essential for efficient growth. The BioMe plate provides a scalable and flexible approach for the study of dynamic microbial interactions. IMPORTANCE Microbial communities participate in many essential processes from biogeochemical cycles to the maintenance of human health. The structure and functions of these communities are dynamic properties that depend on poorly understood interactions among different species. Unraveling these interactions is therefore a crucial step toward understanding natural microbiota and engineering artificial ones. Microbial interactions have been difficult to measure directly, largely due to limitations of existing methods to disentangle the contribution of different organisms in mixed cocultures. To overcome these limitations, we developed the BioMe plate, a custom microplate-based device that enables direct measurement of microbial interactions, by detecting the abundance of segregated populations of microbes that can exchange small molecules through a membrane. We demonstrated the possible application of the BioMe plate for studying both natural and artificial consortia. BioMe is a scalable and accessible platform that can be used to broadly characterize microbial interactions mediated by diffusible molecules.

Intracellular Density of Wolbachia Is Mediated by Host Autophagy and the Bacterial Cytoplasmic Incompatibility Gene cifB in a Cell Type-Dependent Manner in Drosophila melanogaster.

Autophagy is an intracellular degradation pathway involved in innate immunity. Pathogenic bacteria have evolved several mechanisms to escape degradation or exploit autophagy to acquire host nutrients. In the case of endosymbionts, which often have commensal or mutualistic interactions with the host, autophagy is not well characterized. We utilized tissue-specific autophagy mutants to determine if Wolbachia, a vertically transmitted obligate endosymbiont of Drosophila melanogaster, is regulated by autophagy in somatic and germ line cell types. Our analysis revealed core autophagy proteins Atg1 and Atg8 and a selective autophagy-specific protein Ref(2)p negatively regulate Wolbachia in the hub, a male gonad somatic cell type. Furthermore, we determined that the Wolbachia effector protein, CifB, modulates autophagy-Wolbachia interactions, identifying a new host-related pathway which these bacterial proteins interact with. In the female germ line, the cell type necessary for inheritance of Wolbachia through vertical transmission, we discovered that bulk autophagy mediated by Atg1 and Atg8 positively regulates Wolbachia density, whereas Ref(2)p had no effect. Global metabolomics of fly ovaries deficient in germ line autophagy revealed reduced lipid and carbon metabolism, implicating metabolites from these pathways as positive regulators of Wolbachia Our work provides further understanding of how autophagy affects bacteria in a cell type-dependent manner.IMPORTANCE Autophagy is a eukaryotic intracellular degradation pathway which can act as an innate immune response to eliminate pathogens. Conversely, pathogens can evolve proteins which modulate the autophagy pathway to subvert degradation and establish an infection. Wolbachia, a vertically transmitted obligate endosymbiont which infects up to 40% of insect species, is negatively regulated by autophagy in whole animals, but the specific molecular mechanism and tissue which govern this interaction remain unknown. Our studies use cell type-specific autophagy mutants to reveal that Wolbachia is negatively regulated by selective autophagy in the soma, while nonselective autophagy positively regulates Wolbachia in the female germ line. These data provide evidence that cell type can drive different basal autophagy programs which modulate intracellular microbes differently. Additionally, we identified that the Wolbachia effector CifB acts in the selective autophagy pathway to aid in intracellular bacterial survival, providing a new function for CifB beyond its previously identified role in reproductive manipulation.

Wolbachia and Sirtuin-4 interaction is associated with alterations in host glucose metabolism and bacterial titer.

Wolbachia is an intracellular bacterial symbiont of arthropods notorious for inducing many reproductive manipulations that foster its dissemination. Wolbachia affects many aspects of host biology, including metabolism, longevity and physiology, being described as a nutrient provisioning or metabolic parasite, depending on the host-microbe association. Sirtuins (SIRTs) are a family of NAD+-dependent post-translational regulatory enzymes known to affect many of the same processes altered by Wolbachia, including aging and metabolism, among others. Despite a clear overlap in control of host-derived pathways and physiology, no work has demonstrated a link between these two regulators. We used genetically tractable Drosophila melanogaster to explore the role of sirtuins in shaping signaling pathways in the context of a host-symbiont model. By using transcriptional profiling and metabolic assays in the context of genetic knockouts/over-expressions, we examined the effect of several Wolbachia strains on host sirtuin expression across distinct tissues and timepoints. We also quantified the downstream effects of the sirtuin x Wolbachia interaction on host glucose metabolism, and in turn, how it impacted Wolbachia titer. Our results indicate that the presence of Wolbachia is associated with (1) reduced sirt-4 expression in a strain-specific manner, and (2) alterations in host glutamate dehydrogenase expression and ATP levels, key components of glucose metabolism. We detected high glucose levels in Wolbachia-infected flies, which further increased when sirt-4 was over-expressed. However, under sirt-4 knockout, flies displayed a hypoglycemic state not rescued to normal levels in the presence of Wolbachia. Finally, whole body sirt-4 over-expression resulted in reduced Wolbachia ovarian titer. Our results expand knowledge of Wolbachia-host associations in the context of a yet unexplored class of host post-translational regulatory enzymes with implications for conserved host signaling pathways and bacterial titer, factors known to impact host biology and the symbiont's ability to spread through populations.

Author Correction: Large Enriched Fragment Targeted Sequencing (LEFT-SEQ) Applied to Capture of Wolbachia Genomes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Large Enriched Fragment Targeted Sequencing (LEFT-SEQ) Applied to Capture of Wolbachia Genomes.

Symbiosis is a major force of evolutionary change, influencing virtually all aspects of biology, from population ecology and evolution to genomics and molecular/biochemical mechanisms of development and reproduction. A remarkable example is Wolbachia endobacteria, present in some parasitic nematodes and many arthropod species. Acquisition of genomic data from diverse Wolbachia clades will aid in the elucidation of the different symbiotic mechanisms(s). However, challenges of de novo assembly of Wolbachia genomes include the presence in the sample of host DNA: nematode/vertebrate or insect. We designed biotinylated probes to capture large fragments of Wolbachia DNA for sequencing using PacBio technology (LEFT-SEQ: Large Enriched Fragment Targeted Sequencing). LEFT-SEQ was used to capture and sequence four Wolbachia genomes: the filarial nematode Brugia malayi, wBm, (21-fold enrichment), Drosophila mauritiana flies (2 isolates), wMau (11-fold enrichment), and Aedes albopictus mosquitoes, wAlbB (200-fold enrichment). LEFT-SEQ resulted in complete genomes for wBm and for wMau. For wBm, 18 single-nucleotide polymorphisms (SNPs), relative to the wBm reference, were identified and confirmed by PCR. A limit of LEFT-SEQ is illustrated by the wAlbB genome, characterized by a very high level of insertion sequences elements (ISs) and DNA repeats, for which only a 20-contig draft assembly was achieved.

Dual Insect specific virus infection limits Arbovirus replication in Aedes mosquito cells.

Aedes mosquitoes are vectors for many pathogenic viruses. Cell culture systems facilitate the investigation of virus growth in the mosquito vector. We found Zika virus (ZIKV) growth to be consistent in A. albopictus cells but hypervariable in A. aegypti cell lines. As a potential explanation of this variability, we tested the hypothesis that our cells harbored opportunistic viruses. We screened Aedes cell lines for the presence of insect specific viruses (ISVs), Cell-fusing agent virus (CFAV) and Phasi charoen-like virus (PCLV). PCLV was present in the ZIKV-growth-variable A. aegypti cell lines but absent in A. albopictus lines, suggesting that these ISVs may interfere with ZIKV growth. In support of this hypothesis, PCLV infection of CFAV-positive A. albopictus cells inhibited the growth of ZIKV, dengue virus and La Crosse virus. These data suggest ISV infection of cell lines can impact arbovirus growth leading to significant changes in cell permissivity to arbovirus infection.

Polar cell fate stimulates Wolbachia intracellular growth.

Bacteria are crucial partners in the development and evolution of vertebrates and invertebrates. A large fraction of insects harbor Wolbachia, bacterial endosymbionts that manipulate host reproduction to favor their spreading. Because they are maternally inherited, Wolbachia are under selective pressure to reach the female germline and infect the offspring. However, Wolbachia infection is not limited to the germline. Somatic cell types, including stem cell niches, have higher Wolbachia loads compared with the surrounding tissue. Here, we show a novel Wolbachia tropism to polar cells (PCs), specialized somatic cells in the Drosophila ovary. During oogenesis, all stages of PC development are easily visualized, facilitating the investigation of the kinetics of Wolbachia intracellular growth. Wolbachia accumulation is triggered by particular events of PC morphogenesis, including differentiation from progenitors and between stages 8 and 9 of oogenesis. Moreover, induction of ectopic PC fate is sufficient to promote Wolbachia accumulation. We found that Wolbachia PC tropism is evolutionarily conserved across most Drosophila species, but not in Culex mosquitos. These findings highlight the coordination of endosymbiont tropism with host development and cell differentiation.

Group B Wolbachia Strain-Dependent Inhibition of Arboviruses.

Mosquito-borne viruses, including Zika virus (ZIKV) and dengue virus (DENV), are global threats that continue to infect millions annually. Historically, efforts to combat the spread of these diseases have sought to eradicate the mosquito population. This has had limited success. Recent efforts to combat the spread of these diseases have targeted the mosquito population and the mosquito's ability to transmit viruses by altering the mosquito's microbiome. The introduction of particular strains of Wolbachia bacteria into mosquitos suppresses viral growth and blocks disease transmission. This novel strategy is being tested worldwide to reduce DENV and has early indications of success. The Wolbachia genus comprised divergent strains that are divided in major phylogenetic clades termed supergroups. All Wolbachia field trials currently utilize supergroup A Wolbachia in Aedes aegypti mosquitos to limit virus transmission. Here we discuss our studies of Wolbachia strains not yet used in virus control strategies but that show strong potential to reduce ZIKV replication. These strains are important opportunities in the search for novel tools to reduce the levels of mosquito-borne viruses and provide additional models for mechanistic studies.

The Gut Commensal Microbiome of Drosophila melanogaster Is Modified by the Endosymbiont Wolbachia.

Endosymbiotic Wolbachia bacteria and the gut microbiome have independently been shown to affect several aspects of insect biology, including reproduction, development, life span, stem cell activity, and resistance to human pathogens, in insect vectors. This work shows that Wolbachia bacteria, which reside mainly in the fly germline, affect the microbial species present in the fly gut in a lab-reared strain. Drosophila melanogaster hosts two main genera of commensal bacteria-Acetobacter and Lactobacillus. Wolbachia-infected flies have significantly reduced titers of Acetobacter. Sampling of the microbiome of axenic flies fed with equal proportions of both bacteria shows that the presence of Wolbachia bacteria is a significant determinant of the composition of the microbiome throughout fly development. However, this effect is host genotype dependent. To investigate the mechanism of microbiome modulation, the effect of Wolbachia bacteria on Imd and reactive oxygen species pathways, the main regulators of immune response in the fly gut, was measured. The presence of Wolbachia bacteria does not induce significant changes in the expression of the genes for the effector molecules in either pathway. Furthermore, microbiome modulation is not due to direct interaction between Wolbachia bacteria and gut microbes. Confocal analysis shows that Wolbachia bacteria are absent from the gut lumen. These results indicate that the mechanistic basis of the modulation of composition of the microbiome by Wolbachia bacteria is more complex than a direct bacterial interaction or the effect of Wolbachia bacteria on fly immunity. The findings reported here highlight the importance of considering the composition of the gut microbiome and host genetic background during Wolbachia-induced phenotypic studies and when formulating microbe-based disease vector control strategies. IMPORTANCEWolbachia bacteria are intracellular bacteria present in the microbiome of a large fraction of insects and parasitic nematodes. They can block mosquitos' ability to transmit several infectious disease-causing pathogens, including Zika, dengue, chikungunya, and West Nile viruses and malaria parasites. Certain extracellular bacteria present in the gut lumen of these insects can also block pathogen transmission. However, our understanding of interactions between Wolbachia and gut bacteria and how they influence each other is limited. Here we show that the presence of Wolbachia strain wMel changes the composition of gut commensal bacteria in the fruit fly. Our findings implicate interactions between bacterial species as a key factor in determining the overall composition of the microbiome and thus reveal new paradigms to consider in the development of disease control strategies.

Variable Inhibition of Zika Virus Replication by Different Wolbachia Strains in Mosquito Cell Cultures.

Mosquito-borne arboviruses are a major source of human disease. One strategy to reduce arbovirus disease is to reduce the mosquito's ability to transmit virus. Mosquito infection with the bacterial endosymbiont Wolbachia pipientis wMel is a novel strategy to reduce Aedes mosquito competency for flavivirus infection. However, experiments investigating cyclic environmental temperatures have shown a reduction in maternal transmission of wMel, potentially weakening the integration of this strain into a mosquito population relative to that of other Wolbachia strains. Consequently, it is important to investigate additional Wolbachia strains. All Zika virus (ZIKV) suppression studies are limited to the wMel Wolbachia strain. Here we show ZIKV inhibition by two different Wolbachia strains: wAlbB (isolated from Aedes albopictus mosquitoes) and wStri (isolated from the planthopper Laodelphax striatellus) in mosquito cells. Wolbachia strain wStri inhibited ZIKV most effectively. Single-cycle infection experiments showed that ZIKV RNA replication and nonstructural protein 5 translation were reduced below the limits of detection in wStri-containing cells, demonstrating early inhibition of virus replication. ZIKV replication was rescued when Wolbachia was inhibited with a bacteriostatic antibiotic. We observed a partial rescue of ZIKV growth when Wolbachia-infected cells were supplemented with cholesterol-lipid concentrate, suggesting competition for nutrients as one of the possible mechanisms of Wolbachia inhibition of ZIKV. Our data show that wAlbB and wStri infection causes inhibition of ZIKV, making them attractive candidates for further in vitro mechanistic and in vivo studies and future vector-centered approaches to limit ZIKV infection and spread.IMPORTANCE Zika virus (ZIKV) has swiftly spread throughout most of the Western Hemisphere. This is due in large part to its replication in and spread by a mosquito vector host. There is an urgent need for approaches that limit ZIKV replication in mosquitoes. One exciting approach for this is to use a bacterial endosymbiont called Wolbachia that can populate mosquito cells and inhibit ZIKV replication. Here we show that two different strains of Wolbachia, wAlbB and wStri, are effective at repressing ZIKV in mosquito cell lines. Repression of virus growth is through the inhibition of an early stage of infection and requires actively replicating Wolbachia Our findings further the understanding of Wolbachia viral inhibition and provide novel tools that can be used in an effort to limit ZIKV replication in the mosquito vector, thereby interrupting the transmission and spread of the virus.

Extreme divergence of Wolbachia tropism for the stem-cell-niche in the Drosophila testis.

Microbial tropism, the infection of specific cells and tissues by a microorganism, is a fundamental aspect of host-microbe interactions. The intracellular bacteria Wolbachia have a peculiar tropism for the stem cell niches in the Drosophila ovary, the microenvironments that support the cells producing the eggs. The molecular underpinnings of Wolbachia stem cell niche tropism are unknown. We have previously shown that the patterns of tropism in the ovary show a high degree of conservation across the Wolbachia lineage, with closely related Wolbachia strains usually displaying the same pattern of stem cell niche tropism. It has also been shown that tropism to these structures in the ovary facilitates both vertical and horizontal transmission, providing a strong selective pressure towards evolutionary conservation of tropism. Here we show great disparity in the evolutionary conservation and underlying mechanisms of stem cell niche tropism between male and female gonads. In contrast to females, niche tropism in the male testis is not pervasive, present in only 45% of niches analyzed. The patterns of niche tropism in the testis are not evolutionarily maintained across the Wolbachia lineage, unlike what was shown in the females. Furthermore, hub tropism does not correlate with cytoplasmic incompatibility, a Wolbachia-driven phenotype imprinted during spermatogenesis. Towards identifying the molecular mechanism of hub tropism, we performed hybrid analyses of Wolbachia strains in non-native hosts. These results indicate that both Wolbachia and host derived factors play a role in the targeting of the stem cell niche in the testis. Surprisingly, even closely related Wolbachia strains in Drosophila melanogaster, derived from a single ancestor only 8,000 years ago, have significantly different tropisms to the hub, highlighting that stem cell niche tropism is rapidly diverging in males. These findings provide a powerful system to investigate the mechanisms and evolution of microbial tissue tropism.

Evolutionarily conserved Wolbachia-encoded factors control pattern of stem-cell niche tropism in Drosophila ovaries and favor infection.

Wolbachia are intracellular bacteria that infect invertebrates at pandemic levels, including insect vectors of devastating infectious diseases. Although Wolbachia are providing novel strategies for the control of several human pathogens, the processes underlying Wolbachia's successful propagation within and across species remain elusive. Wolbachia are mainly vertically transmitted; however, there is also evidence of extensive horizontal transmission. Here, we provide several lines of evidence supporting Wolbachia's targeting of ovarian stem cell niches--referred to as "niche tropism"--as a previously overlooked strategy for Wolbachia thriving in nature. Niche tropism is pervasive in Wolbachia infecting the Drosophila genus, and different patterns of niche tropism are evolutionarily conserved. Phylogenetic analysis, confirmed by hybrid introgression and transinfection experiments, demonstrates that bacterial factors are the major determinants of differential patterns of niche tropism. Furthermore, bacterial load is increased in germ-line cells passing through infected niches, supporting previous suggestions of a contribution of Wolbachia from stem-cell niches toward vertical transmission. These results support the role of stem-cell niches as a key component for the spreading of Wolbachia in the Drosophila genus and provide mechanistic insights into this unique tissue tropism.

Wolbachia enhance Drosophila stem cell proliferation and target the germline stem cell niche.

Wolbachia are widespread maternally transmitted intracellular bacteria that infect most insect species and are able to alter the reproduction of innumerous hosts. The cellular bases of these alterations remain largely unknown. Here, we report that Drosophila mauritiana infected with a native Wolbachia wMau strain produces about four times more eggs than the noninfected counterpart. Wolbachia infection leads to an increase in the mitotic activity of germline stem cells (GSCs), as well as a decrease in programmed cell death in the germarium. Our results suggest that up-regulation of GSC division is mediated by a tropism of Wolbachia for the GSC niche, the cellular microenvironment that supports GSCs.

Drosophila brain tumor metastases express both neuronal and glial cell type markers.

Loss of either lgl or brat gene activity in Drosophila larvae causes neoplastic brain tumors. Fragments of tumorous brains from either mutant transplanted into adult hosts over-proliferate, and kill their hosts within 2 weeks. We developed an in vivo assay for the metastatic potential of tumor cells by quantifying micrometastasis formation within the ovarioles of adult hosts after transplantation and determined that specific metastatic properties of lgl and brat tumor cells are different. We detected micrometastases in 15.8% of ovarioles from wild type host females 12 days after transplanting lgl tumor cells into their abdominal cavities. This frequency increased significantly with increased proliferation time. We detected micrometastases in 15% of ovarioles from wild type host females 10 days after transplanting brat tumor cells into their abdominal cavities. By contrast, this frequency did not change significantly with increased proliferation time. We found that nearly all lgl micrometastases co-express the neuronal cell marker, ELAV, and the glial cell marker, REPO. These markers are not co-expressed in normal brain cells nor in tumorous brain cells. This indicates deregulated gene expression in these metastatic cells. By contrast, most of the brat micrometastases expressed neither marker. While mutations in both lgl and brat cause neoplastic brain tumors, our results reveal that metastatic cells arising from these tumors have quite different properties. These data may have important implications for the treatment of tumor metastasis.

Somatic stem cell niche tropism in Wolbachia.

Wolbachia are intracellular bacteria found in the reproductive tissue of all major groups of arthropods. They are transmitted vertically from the female hosts to their offspring, in a pattern analogous to mitochondria inheritance. But Wolbachia phylogeny does not parallel that of the host, indicating that horizontal infectious transmission must also occur. Insect parasitoids are considered the most likely vectors, but the mechanism for horizontal transfer is largely unknown. Here we show that newly introduced Wolbachia cross several tissues and infect the germline of the adult Drosophila melanogaster female. Through investigation of bacterial migration patterns during the course of infection, we found that Wolbachia reach the germline through the somatic stem cell niche in the D. melanogaster germarium. In addition, our data suggest that Wolbachia are highly abundant in the somatic stem cell niche of long-term infected hosts, implying that this location may also contribute to efficient vertical transmission. This is, to our knowledge, the first report of an intracellular parasite displaying tropism for a stem cell niche.

Wolbachia utilizes host microtubules and Dynein for anterior localization in the Drosophila oocyte.

To investigate the role of the host cytoskeleton in the maternal transmission of the endoparasitic bacteria Wolbachia, we have characterized their distribution in the female germ line of Drosophila melanogaster. In the germarium, Wolbachia are distributed to all germ cells of the cyst, establishing an early infection in the cell destined to become the oocyte. During mid-oogenesis, Wolbachia exhibit a distinct concentration between the anterior cortex and the nucleus in the oocyte, where many bacteria appear to contact the nuclear envelope. Following programmed rearrangement of the microtubule network, Wolbachia dissociate from this anterior position and become dispersed throughout the oocyte. This localization pattern is distinct from mitochondria and all known axis determinants. Manipulation of microtubules and cytoplasmic Dynein and Dynactin, but not Kinesin-1, disrupts anterior bacterial localization in the oocyte. In live egg chambers, Wolbachia exhibit movement in nurse cells but not in the oocyte, suggesting that the bacteria are anchored by host factors. In addition, we identify mid-oogenesis as a period in the life cycle of Wolbachia in which bacterial replication occurs. Total bacterial counts show that Wolbachia increase at a significantly higher rate in the oocyte than in the average nurse cell, and that normal Wolbachia levels in the oocyte depend on microtubules. These findings demonstrate that Wolbachia utilize the host microtubule network and associated proteins for their subcellular localization in the Drosophila oocyte. These interactions may also play a role in bacterial motility and replication, ultimately leading to the bacteria's efficient maternal transmission.