Publications by authors named "Yineng Wu"

21 Publications

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Paratransgenic manipulation of a tsetse microRNA alters the physiological homeostasis of the fly's midgut environment.

PLoS Pathog 2021 Jun 9;17(6):e1009475. Epub 2021 Jun 9.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, United States of America.

Tsetse flies are vectors of parasitic African trypanosomes, the etiological agents of human and animal African trypanosomoses. Current disease control methods include fly-repelling pesticides, fly trapping, and chemotherapeutic treatment of infected people and animals. Inhibiting tsetse's ability to transmit trypanosomes by strengthening the fly's natural barriers can serve as an alternative approach to reduce disease. The peritrophic matrix (PM) is a chitinous and proteinaceous barrier that lines the insect midgut and serves as a protective barrier that inhibits infection with pathogens. African trypanosomes must cross tsetse's PM in order to establish an infection in the fly, and PM structural integrity negatively correlates with trypanosome infection outcomes. Bloodstream form trypanosomes shed variant surface glycoproteins (VSG) into tsetse's gut lumen early during the infection establishment, and free VSG molecules are internalized by the fly's PM-producing cardia. This process results in a reduction in the expression of a tsetse microRNA (miR275) and a sequential molecular cascade that compromises PM integrity. miRNAs are small non-coding RNAs that are critical in regulating many physiological processes. In the present study, we investigated the role(s) of tsetse miR275 by developing a paratransgenic expression system that employs tsetse's facultative bacterial endosymbiont, Sodalis glossinidius, to express tandem antagomir-275 repeats (or miR275 sponges). This system induces a constitutive, 40% reduction in miR275 transcript abundance in the fly's midgut and results in obstructed blood digestion (gut weights increased by 52%), a significant increase (p-value < 0.0001) in fly survival following infection with an entomopathogenic bacteria, and a 78% increase in trypanosome infection prevalence. RNA sequencing of cardia and midgut tissues from paratransgenic tsetse confirmed that miR275 regulates processes related to the expression of PM-associated proteins and digestive enzymes as well as genes that encode abundant secretory proteins. Our study demonstrates that paratransgenesis can be employed to study microRNA regulated pathways in arthropods that house symbiotic bacteria.
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http://dx.doi.org/10.1371/journal.ppat.1009475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8216540PMC
June 2021

Mutualist-Provisioned Resources Impact Vector Competency.

mBio 2019 06 4;10(3). Epub 2019 Jun 4.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Yale University, New Haven, Connecticut, USA

Many symbionts supplement their host's diet with essential nutrients. However, whether these nutrients also enhance parasitism is unknown. In this study, we investigated whether folate (vitamin B) production by the tsetse fly ( spp.) essential mutualist, , aids auxotrophic African trypanosomes in completing their life cycle within this obligate vector. We show that the expression of folate biosynthesis genes changes with the progression of trypanosome infection within tsetse. The disruption of folate production caused a reduction in the percentage of flies that housed midgut (MG) trypanosome infections. However, decreased folate did not prevent MG trypanosomes from migrating to and establishing an infection in the fly's salivary glands, thus suggesting that nutrient requirements vary throughout the trypanosome life cycle. We further substantiated that trypanosomes rely on symbiont-generated folate by feeding this vitamin to , which exhibits low trypanosome vector competency and houses incapable of producing folate. Folate-supplemented flies were significantly more susceptible to trypanosome infection, further demonstrating that this vitamin facilitates parasite infection establishment. Our cumulative results provide evidence that provides a key metabolite (folate) that is "hijacked" by trypanosomes to enhance their infectivity, thus indirectly impacting tsetse species vector competency. Parasite dependence on symbiont-derived micronutrients, which likely also occurs in other arthropod vectors, represents a relationship that may be exploited to reduce disease transmission. Parasites elicit several physiological changes in their host to enhance transmission. Little is known about the functional association between parasitism and microbiota-provisioned resources typically dedicated to animal hosts and how these goods may be rerouted to optimize parasite development. This study is the first to identify a specific symbiont-generated metabolite that impacts insect vector competence by facilitating parasite establishment and, thus, eventual transmission. Specifically, we demonstrate that the tsetse fly obligate mutualist provisions folate (vitamin B) that pathogenic African trypanosomes exploit in an effort to successfully establish an infection in the vector's MG. This process is essential for the parasite to complete its life cycle and be transmitted to a new vertebrate host. Disrupting metabolic contributions provided by the microbiota of arthropod disease vectors may fuel future innovative control strategies while also offering minimal nontarget effects.
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http://dx.doi.org/10.1128/mBio.00018-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6550517PMC
June 2019

Colonization of the tsetse fly midgut with commensal Kosakonia cowanii Zambiae inhibits trypanosome infection establishment.

PLoS Pathog 2019 02 28;15(2):e1007470. Epub 2019 Feb 28.

Yale School of Public Health, Department of Epidemiology of Microbial Diseases, New Haven, Connecticut, United States of America.

Tsetse flies (Glossina spp.) vector pathogenic trypanosomes (Trypanosoma spp.) in sub-Saharan Africa. These parasites cause human and animal African trypanosomiases, which are debilitating diseases that inflict an enormous socio-economic burden on inhabitants of endemic regions. Current disease control strategies rely primarily on treating infected animals and reducing tsetse population densities. However, relevant programs are costly, labor intensive and difficult to sustain. As such, novel strategies aimed at reducing tsetse vector competence require development. Herein we investigated whether Kosakonia cowanii Zambiae (Kco_Z), which confers Anopheles gambiae with resistance to Plasmodium, is able to colonize tsetse and induce a trypanosome refractory phenotype in the fly. Kco_Z established stable infections in tsetse's gut and exhibited no adverse effect on the fly's survival. Flies with established Kco_Z infections in their gut were significantly more refractory to infection with two distinct trypanosome species (T. congolense, 6% infection; T. brucei, 32% infection) than were age-matched flies that did not house the exogenous bacterium (T. congolense, 36% infected; T. brucei, 70% infected). Additionally, 52% of Kco_Z colonized tsetse survived infection with entomopathogenic Serratia marcescens, compared with only 9% of their wild-type counterparts. These parasite and pathogen refractory phenotypes result from the fact that Kco_Z acidifies tsetse's midgut environment, which inhibits trypanosome and Serratia growth and thus infection establishment. Finally, we determined that Kco_Z infection does not impact the fecundity of male or female tsetse, nor the ability of male flies to compete with their wild-type counterparts for mates. We propose that Kco_Z could be used as one component of an integrated strategy aimed at reducing the ability of tsetse to transmit pathogenic trypanosomes.
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http://dx.doi.org/10.1371/journal.ppat.1007470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6394900PMC
February 2019

A fine-tuned vector-parasite dialogue in tsetse's cardia determines peritrophic matrix integrity and trypanosome transmission success.

PLoS Pathog 2018 04 3;14(4):e1006972. Epub 2018 Apr 3.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, United States of America.

Arthropod vectors have multiple physical and immunological barriers that impede the development and transmission of parasites to new vertebrate hosts. These barriers include the peritrophic matrix (PM), a chitinous barrier that separates the blood bolus from the midgut epithelia and modulates vector-pathogens interactions. In tsetse flies, a sleeve-like PM is continuously produced by the cardia organ located at the fore- and midgut junction. African trypanosomes, Trypanosoma brucei, must bypass the PM twice; first to colonize the midgut and secondly to reach the salivary glands (SG), to complete their transmission cycle in tsetse. However, not all flies with midgut infections develop mammalian transmissible SG infections-the reasons for which are unclear. Here, we used transcriptomics, microscopy and functional genomics analyses to understand the factors that regulate parasite migration from midgut to SG. In flies with midgut infections only, parasites fail to cross the PM as they are eliminated from the cardia by reactive oxygen intermediates (ROIs)-albeit at the expense of collateral cytotoxic damage to the cardia. In flies with midgut and SG infections, expression of genes encoding components of the PM is reduced in the cardia, and structural integrity of the PM barrier is compromised. Under these circumstances trypanosomes traverse through the newly secreted and compromised PM. The process of PM attrition that enables the parasites to re-enter into the midgut lumen is apparently mediated by components of the parasites residing in the cardia. Thus, a fine-tuned dialogue between tsetse and trypanosomes at the cardia determines the outcome of PM integrity and trypanosome transmission success.
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http://dx.doi.org/10.1371/journal.ppat.1006972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5898766PMC
April 2018

Tsetse fly (Glossina pallidipes) midgut responses to Trypanosoma brucei challenge.

Parasit Vectors 2017 Dec 19;10(1):614. Epub 2017 Dec 19.

Department of Biochemistry, Biotechnology Research Institute, Kenya Agricultural and Livestock Research Organization, P.O. Box 362, Kikuyu, Kenya.

Background: Tsetse flies (Glossina spp.) are the prominent vector of African trypanosome parasites (Trypanosoma spp.) in sub-Saharan Africa, and Glossina pallidipes is the most widely distributed species in Kenya. This species displays strong resistance to infection by parasites, which are typically eliminated in the midgut shortly after acquisition from the mammalian host. Although extensive molecular information on immunity for the related species Glossina morsitans morsitans exists, similar information is scarce for G. pallidipes.

Methods: To determine temporal transcriptional responses of G. pallidipes to Trypanosoma brucei brucei challenge, we conducted Illumina based RNA-seq on midgut organ and carcass from teneral females G. pallidipes at 24 and 48 h post-challenge (hpc) with T. b. brucei relative to their respective controls that received normal blood meals (without the parasite). We used a suite of bioinformatics tools to determine differentially expressed and enriched transcripts between and among tissues, and to identify expanded transcripts in G. pallidipes relative to their orthologs G. m. morsitans.

Results: Midgut transcripts induced at 24 hpc encoded proteins were associated with lipid remodelling, proteolysis, collagen metabolism, apoptosis, and cell growth. Midgut transcripts induced at 48 hpc encoded proteins linked to embryonic growth and development, serine endopeptidases and proteosomal degradation of the target protein, mRNA translation and neuronal development. Temporal expression of immune responsive transcripts at 48 relative to 24 hpc was pronounced, indicative of a gradual induction of host immune responses the following challenge. We also searched for G. m. morsitans orthologous groups that may have experienced expansions in the G. pallidipes genome. We identified ten expanded groups in G. pallidipes with putative immunity-related functions, which may play a role in the higher refractoriness exhibited by this species.

Conclusions: There appears to be a lack of strong immune responses elicited by gut epithelia of teneral adults. This in combination with a compromised peritrophic matrix at this stage during the initial phase of T. b. brucei challenge may facilitate the increased parasite infection establishment noted in teneral flies relative to older adults. Although teneral flies are more susceptible than older adults, the majority of tenerals are still able to eliminate parasite infections. Hence, robust responses elicited at a later time point, such as 72 hpc, may clear parasite infections from the majority of flies. The expanded G. m. morsitans orthologous groups in G. pallidipes may also be functionally associated with the enhanced refractoriness to trypanosome infections reported in G. pallidipes relative to G. m. morsitans.
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http://dx.doi.org/10.1186/s13071-017-2569-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738168PMC
December 2017

Molecular characterization of tsetse's proboscis and its response to Trypanosoma congolense infection.

PLoS Negl Trop Dis 2017 Nov 20;11(11):e0006057. Epub 2017 Nov 20.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, United States of America.

Tsetse flies (Glossina spp.) transmit parasitic African trypanosomes (Trypanosoma spp.), including Trypanosoma congolense, which causes animal African trypanosomiasis (AAT). AAT detrimentally affects agricultural activities in sub-Saharan Africa and has negative impacts on the livelihood and nutrient availability for the affected communities. After tsetse ingests an infectious blood meal, T. congolense sequentially colonizes the fly's gut and proboscis (PB) organs before being transmitted to new mammalian hosts during subsequent feedings. Despite the importance of PB in blood feeding and disease transmission, little is known about its molecular composition, function and response to trypanosome infection. To bridge this gap, we used RNA-seq analysis to determine its molecular characteristics and responses to trypanosome infection. By comparing the PB transcriptome to whole head and midgut transcriptomes, we identified 668 PB-enriched transcripts that encoded proteins associated with muscle tissue, organ development, chemosensation and chitin-cuticle structure development. Moreover, transcripts encoding putative mechanoreceptors that monitor blood flow during tsetse feeding and interact with trypanosomes were also expressed in the PB. Microscopic analysis of the PB revealed cellular structures associated with muscles and cells. Infection with T. congolense resulted in increased and decreased expression of 38 and 88 transcripts, respectively. Twelve of these differentially expressed transcripts were PB-enriched. Among the transcripts induced upon infection were those encoding putative proteins associated with cell division function(s), suggesting enhanced tissue renewal, while those suppressed were associated with metabolic processes, extracellular matrix and ATP-binding as well as immunity. These results suggest that PB is a muscular organ with chemosensory and mechanosensory capabilities. The mechanoreceptors may be point of PB-trypanosomes interactions. T. congolense infection resulted in reduced metabolic and immune capacity of the PB. The molecular knowledge on the composition and putative functions of PB forms the foundation to identify new targets to disrupt tsetse's ability to feed and parasite transmission.
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http://dx.doi.org/10.1371/journal.pntd.0006057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695773PMC
November 2017

Symbiont-induced odorant binding proteins mediate insect host hematopoiesis.

Elife 2017 01 12;6. Epub 2017 Jan 12.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, United States.

Symbiotic bacteria assist in maintaining homeostasis of the animal immune system. However, the molecular mechanisms that underlie symbiont-mediated host immunity are largely unknown. Tsetse flies ( spp.) house maternally transmitted symbionts that regulate the development and function of their host's immune system. Herein we demonstrate that the obligate mutualist, , up-regulates expression of in the gut of intrauterine tsetse larvae. This process is necessary and sufficient to induce systemic expression of the hematopoietic RUNX transcription factor and the subsequent production of crystal cells, which actuate the melanotic immune response in adult tsetse. Larval indigenous microbiota, which is acquired from the environment, regulates an orthologous hematopoietic pathway in their host. These findings provide insight into the molecular mechanisms that underlie enteric symbiont-stimulated systemic immune system development, and indicate that these processes are evolutionarily conserved despite the divergent nature of host-symbiont interactions in these model systems.
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http://dx.doi.org/10.7554/eLife.19535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5231409PMC
January 2017

The peritrophic matrix mediates differential infection outcomes in the tsetse fly gut following challenge with commensal, pathogenic, and parasitic microbes.

J Immunol 2014 Jul 9;193(2):773-82. Epub 2014 Jun 9.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06520.

The insect gut is lined by a protective, chitinous peritrophic matrix (PM) that separates immunoreactive epithelial cells from microbes present within the luminal contents. Tsetse flies (Glossina spp.) imbibe vertebrate blood exclusively and can be exposed to foreign microorganisms during the feeding process. We used RNA interference-based reverse genetics to inhibit the production of a structurally robust PM and then observed how this procedure impacted infection outcomes after per os challenge with exogenous bacteria (Enterobacter sp. and Serratia marcescens strain Db11) and parasitic African trypanosomes. Enterobacter and Serratia proliferation was impeded in tsetse that lacked an intact PM because these flies expressed the antimicrobial peptide gene, attacin, earlier in the infection process than did their counterparts that housed a fully developed PM. After challenge with trypanosomes, attacin expression was latent in tsetse that lacked an intact PM, and these flies were thus highly susceptible to parasite infection. Our results suggest that immunodeficiency signaling pathway effectors, as opposed to reactive oxygen intermediates, serve as the first line of defense in tsetse's gut after the ingestion of exogenous microorganisms. Furthermore, tsetse's PM is not a physical impediment to infection establishment, but instead serves as a barrier that regulates the fly's ability to immunologically detect and respond to the presence of these microbes. Collectively, our findings indicate that effective insect antimicrobial responses depend largely upon the coordination of multiple host and microbe-specific developmental factors.
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http://dx.doi.org/10.4049/jimmunol.1400163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4107339PMC
July 2014

Analysis of multiple tsetse fly populations in Uganda reveals limited diversity and species-specific gut microbiota.

Appl Environ Microbiol 2014 Jul 9;80(14):4301-12. Epub 2014 May 9.

Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut, USA Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Yale University, New Haven, Connecticut, USA.

The invertebrate microbiome contributes to multiple aspects of host physiology, including nutrient supplementation and immune maturation processes. We identified and compared gut microbial abundance and diversity in natural tsetse flies from Uganda using five genetically distinct populations of Glossina fuscipes fuscipes and multiple tsetse species (Glossina morsitans morsitans, G. f. fuscipes, and Glossina pallidipes) that occur in sympatry in one location. We used multiple approaches, including deep sequencing of the V4 hypervariable region of the 16S rRNA gene, 16S rRNA gene clone libraries, and bacterium-specific quantitative PCR (qPCR), to investigate the levels and patterns of gut microbial diversity from a total of 151 individuals. Our results show extremely limited diversity in field flies of different tsetse species. The obligate endosymbiont Wigglesworthia dominated all samples (>99%), but we also observed wide prevalence of low-density Sodalis (tsetse's commensal endosymbiont) infections (<0.05%). There were also several individuals (22%) with high Sodalis density, which also carried coinfections with Serratia. Albeit in low density, we noted differences in microbiota composition among the genetically distinct G. f. fuscipes flies and between different sympatric species. Interestingly, Wigglesworthia density varied in different species (10(4) to 10(6) normalized genomes), with G. f. fuscipes having the highest levels. We describe the factors that may be responsible for the reduced diversity of tsetse's gut microbiota compared to those of other insects. Additionally, we discuss the implications of Wigglesworthia and Sodalis density variations as they relate to trypanosome transmission dynamics and vector competence variations associated with different tsetse species.
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http://dx.doi.org/10.1128/AEM.00079-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068677PMC
July 2014

Trypanosome infection establishment in the tsetse fly gut is influenced by microbiome-regulated host immune barriers.

PLoS Pathog 2013 18;9(4):e1003318. Epub 2013 Apr 18.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, United States of America.

Tsetse flies (Glossina spp.) vector pathogenic African trypanosomes, which cause sleeping sickness in humans and nagana in domesticated animals. Additionally, tsetse harbors 3 maternally transmitted endosymbiotic bacteria that modulate their host's physiology. Tsetse is highly resistant to infection with trypanosomes, and this phenotype depends on multiple physiological factors at the time of challenge. These factors include host age, density of maternally-derived trypanolytic effector molecules present in the gut, and symbiont status during development. In this study, we investigated the molecular mechanisms that result in tsetse's resistance to trypanosomes. We found that following parasite challenge, young susceptible tsetse present a highly attenuated immune response. In contrast, mature refractory flies express higher levels of genes associated with humoral (attacin and pgrp-lb) and epithelial (inducible nitric oxide synthase and dual oxidase) immunity. Additionally, we discovered that tsetse must harbor its endogenous microbiome during intrauterine larval development in order to present a parasite refractory phenotype during adulthood. Interestingly, mature aposymbiotic flies (Gmm(Apo)) present a strong immune response earlier in the infection process than do WT flies that harbor symbiotic bacteria throughout their entire lifecycle. However, this early response fails to confer significant resistance to trypanosomes. Gmm(Apo) adults present a structurally compromised peritrophic matrix (PM), which lines the fly midgut and serves as a physical barrier that separates luminal contents from immune responsive epithelial cells. We propose that the early immune response we observe in Gmm(Apo) flies following parasite challenge results from the premature exposure of gut epithelia to parasite-derived immunogens in the absence of a robust PM. Thus, tsetse's PM appears to regulate the timing of host immune induction following parasite challenge. Our results document a novel finding, which is the existence of a positive correlation between tsetse's larval microbiome and the integrity of the emerging adult PM gut immune barrier.
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http://dx.doi.org/10.1371/journal.ppat.1003318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3630092PMC
January 2014

Wolbachia association with the tsetse fly, Glossina fuscipes fuscipes, reveals high levels of genetic diversity and complex evolutionary dynamics.

BMC Evol Biol 2013 Feb 5;13:31. Epub 2013 Feb 5.

Department of Ecology and Evolutionary Biology, Yale University, 21 Sachem St, New Haven, CT, USA.

Background: Wolbachia pipientis, a diverse group of α-proteobacteria, can alter arthropod host reproduction and confer a reproductive advantage to Wolbachia-infected females (cytoplasmic incompatibility (CI)). This advantage can alter host population genetics because Wolbachia-infected females produce more offspring with their own mitochondrial DNA (mtDNA) haplotypes than uninfected females. Thus, these host haplotypes become common or fixed (selective sweep). Although simulations suggest that for a CI-mediated sweep to occur, there must be a transient phase with repeated initial infections of multiple individual hosts by different Wolbachia strains, this has not been observed empirically. Wolbachia has been found in the tsetse fly, Glossina fuscipes fuscipes, but it is not limited to a single host haplotype, suggesting that CI did not impact its population structure. However, host population genetic differentiation could have been generated if multiple Wolbachia strains interacted in some populations. Here, we investigated Wolbachia genetic variation in G. f. fuscipes populations of known host genetic composition in Uganda. We tested for the presence of multiple Wolbachia strains using Multi-Locus Sequence Typing (MLST) and for an association between geographic region and host mtDNA haplotype using Wolbachia DNA sequence from a variable locus, groEL (heat shock protein 60).

Results: MLST demonstrated that some G. f. fuscipes carry Wolbachia strains from two lineages. GroEL revealed high levels of sequence diversity within and between individuals (Haplotype diversity = 0.945). We found Wolbachia associated with 26 host mtDNA haplotypes, an unprecedented result. We observed a geographical association of one Wolbachia lineage with southern host mtDNA haplotypes, but it was non-significant (p = 0.16). Though most Wolbachia-infected host haplotypes were those found in the contact region between host mtDNA groups, this association was non-significant (p = 0.17).

Conclusions: High Wolbachia sequence diversity and the association of Wolbachia with multiple host haplotypes suggest that different Wolbachia strains infected G. f. fuscipes multiple times independently. We suggest that these observations reflect a transient phase in Wolbachia evolution that is influenced by the long gestation and low reproductive output of tsetse. Although G. f. fuscipes is superinfected with Wolbachia, our data does not support that bidirectional CI has influenced host genetic diversity in Uganda.
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http://dx.doi.org/10.1186/1471-2148-13-31DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3574847PMC
February 2013

OmpA-mediated biofilm formation is essential for the commensal bacterium Sodalis glossinidius to colonize the tsetse fly gut.

Appl Environ Microbiol 2012 Nov 31;78(21):7760-8. Epub 2012 Aug 31.

Yale School of Public Health, Department of Epidemiology of Microbial Diseases, New Haven, Connecticut, USA.

Many bacteria successfully colonize animals by forming protective biofilms. Molecular processes that underlie the formation and function of biofilms in pathogenic bacteria are well characterized. In contrast, the relationship between biofilms and host colonization by symbiotic bacteria is less well understood. Tsetse flies (Glossina spp.) house 3 maternally transmitted symbionts, one of which is a commensal (Sodalis glossinidius) found in several host tissues, including the gut. We determined that Sodalis forms biofilms in the tsetse gut and that this process is influenced by the Sodalis outer membrane protein A (OmpA). Mutant Sodalis strains that do not produce OmpA (Sodalis ΔOmpA mutants) fail to form biofilms in vitro and are unable to colonize the tsetse gut unless endogenous symbiotic bacteria are present. Our data indicate that in the absence of biofilms, Sodalis ΔOmpA mutant cells are exposed to and eliminated by tsetse's innate immune system, suggesting that biofilms help Sodalis evade the host immune system. Tsetse is the sole vector of pathogenic African trypanosomes, which also reside in the fly gut. Acquiring a better understanding of the dynamics that promote Sodalis colonization of the tsetse gut may enhance the development of novel disease control strategies.
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http://dx.doi.org/10.1128/AEM.01858-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485708PMC
November 2012

Intercommunity effects on microbiome and GpSGHV density regulation in tsetse flies.

J Invertebr Pathol 2013 Mar 19;112 Suppl:S32-9. Epub 2012 Apr 19.

Division of Epidemiology of Microbial Disease, Yale School of Public Health, Yale University, New Haven, CT, USA.

Tsetse flies have a highly regulated and defined microbial fauna made of 3 bacterial symbionts (obligate Wigglesworthia glossinidia, commensal Sodalis glossinidius and parasitic Wolbachia pipientis) in addition to a DNA virus (Glossina pallidipes Salivary gland Hypertrophy Virus, GpSGHV). It has been possible to rear flies in the absence of either Wigglesworthia or in totally aposymbiotic state by dietary supplementation of tsetse's bloodmeal. In the absence of Wigglesworthia, tsetse females are sterile, and adult progeny are immune compromised. The functional contributions for Sodalist are less known, while Wolbachia cause reproductive manupulations known as cytoplasmic incompatibility (CI). High GpSGHV virus titers result in reduced fecundity and lifespan, and have compromised efforts to colonize flies in the insectary for large rearing purposes. Here we investigated the within community effects on the density regulation of the individual microbiome partners in tsetse lines with different symbiotic compositions. We show that absence of Wigglesworthia results in loss of Sodalis in subsequent generations possibly due to nutritional dependancies between the symbiotic partners. While an initial decrease in Wolbachia and GpSGHV levels are also noted in the absence of Wigglesworthia, these infections eventually reach homeostatic levels indicating adaptations to the new host immune environment or nutritional ecology. Absence of all bacterial symbionts also results in an initial reduction of viral titers, which recover in the second generation. Our findings suggest that in addition to the host immune system, interdependencies between symbiotic partners result in a highly tuned density regulation for tsetse's microbiome.
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http://dx.doi.org/10.1016/j.jip.2012.03.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772524PMC
March 2013

Transcript expression analysis of putative Trypanosoma brucei GPI-anchored surface proteins during development in the tsetse and mammalian hosts.

PLoS Negl Trop Dis 2012 19;6(6):e1708. Epub 2012 Jun 19.

Division of Epidemiology of Microbial Diseases, Yale School of Public Health, Yale University, New Haven, Connecticut, USA.

Human African Trypanosomiasis is a devastating disease caused by the parasite Trypanosoma brucei. Trypanosomes live extracellularly in both the tsetse fly and the mammal. Trypanosome surface proteins can directly interact with the host environment, allowing parasites to effectively establish and maintain infections. Glycosylphosphatidylinositol (GPI) anchoring is a common posttranslational modification associated with eukaryotic surface proteins. In T. brucei, three GPI-anchored major surface proteins have been identified: variant surface glycoproteins (VSGs), procyclic acidic repetitive protein (PARP or procyclins), and brucei alanine rich proteins (BARP). The objective of this study was to select genes encoding predicted GPI-anchored proteins with unknown function(s) from the T. brucei genome and characterize the expression profile of a subset during cyclical development in the tsetse and mammalian hosts. An initial in silico screen of putative T. brucei proteins by Big PI algorithm identified 163 predicted GPI-anchored proteins, 106 of which had no known functions. Application of a second GPI-anchor prediction algorithm (FragAnchor), signal peptide and trans-membrane domain prediction software resulted in the identification of 25 putative hypothetical proteins. Eighty-one gene products with hypothetical functions were analyzed for stage-regulated expression using semi-quantitative RT-PCR. The expression of most of these genes were found to be upregulated in trypanosomes infecting tsetse salivary gland and proventriculus tissues, and 38% were specifically expressed only by parasites infecting salivary gland tissues. Transcripts for all of the genes specifically expressed in salivary glands were also detected in mammalian infective metacyclic trypomastigotes, suggesting a possible role for these putative proteins in invasion and/or establishment processes in the mammalian host. These results represent the first large-scale report of the differential expression of unknown genes encoding predicted T. brucei surface proteins during the complete developmental cycle. This knowledge may form the foundation for the development of future novel transmission blocking strategies against metacyclic parasites.
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http://dx.doi.org/10.1371/journal.pntd.0001708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378594PMC
September 2012

Implications of microfauna-host interactions for trypanosome transmission dynamics in Glossina fuscipes fuscipes in Uganda.

Appl Environ Microbiol 2012 Jul 27;78(13):4627-37. Epub 2012 Apr 27.

Yale University School of Public Health, New Haven, Connecticut, USA.

Tsetse flies (Diptera: Glossinidae) are vectors for African trypanosomes (Euglenozoa: kinetoplastida), protozoan parasites that cause African trypanosomiasis in humans (HAT) and nagana in livestock. In addition to trypanosomes, two symbiotic bacteria (Wigglesworthia glossinidia and Sodalis glossinidius) and two parasitic microbes, Wolbachia and a salivary gland hypertrophy virus (SGHV), have been described in tsetse. Here we determined the prevalence of and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in Glossina fuscipes fuscipes in Uganda over a large geographical scale spanning the range of host genetic and spatial diversity. Using a multivariate analysis approach, we uncovered complex coinfection dynamics between the pathogens and statistically significant associations between host genetic groups and pathogen prevalence. It is important to note that these coinfection dynamics and associations with the host were not apparent by univariate analysis. These associations between host genotype and pathogen are particularly evident for Wolbachia and SGHV where host groups are inversely correlated for Wolbachia and SGHV prevalence. On the other hand, trypanosome infection prevalence is more complex and covaries with the presence of the other two pathogens, highlighting the importance of examining multiple pathogens simultaneously before making generalizations about infection and spatial patterns. It is imperative to note that these novel findings would have been missed if we had employed the standard univariate analysis used in previous studies. Our results are discussed in the context of disease epidemiology and vector control.
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http://dx.doi.org/10.1128/AEM.00806-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370491PMC
July 2012

Transcriptome analysis of reproductive tissue and intrauterine developmental stages of the tsetse fly (Glossina morsitans morsitans).

BMC Genomics 2010 Mar 9;11:160. Epub 2010 Mar 9.

Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT, USA.

Background: Tsetse flies, vectors of African trypanosomes, undergo viviparous reproduction (the deposition of live offspring). This reproductive strategy results in a large maternal investment and the deposition of a small number of progeny during a female's lifespan. The reproductive biology of tsetse has been studied on a physiological level; however the molecular analysis of tsetse reproduction requires deeper investigation. To build a foundation from which to base molecular studies of tsetse reproduction, a cDNA library was generated from female tsetse (Glossina morsitans morsitans) reproductive tissues and the intrauterine developmental stages. 3438 expressed sequence tags were sequenced and analyzed.

Results: Analysis of a nonredundant catalogue of 1391 contigs resulted in 520 predicted proteins. 475 of these proteins were full length. We predict that 412 of these represent cytoplasmic proteins while 57 are secreted. Comparison of these proteins with other tissue specific tsetse cDNA libraries (salivary gland, fat body/milk gland, and midgut) identified 51 that are unique to the reproductive/immature cDNA library. 11 unique proteins were homologous to uncharacterized putative proteins within the NR database suggesting the identification of novel genes associated with reproductive functions in other insects (hypothetical conserved). The analysis also yielded seven putative proteins without significant homology to sequences present in the public database (unknown genes). These proteins may represent unique functions associated with tsetse's viviparous reproductive cycle. RT-PCR analysis of hypothetical conserved and unknown contigs was performed to determine basic tissue and stage specificity of the expression of these genes.

Conclusion: This paper identifies 51 putative proteins specific to a tsetse reproductive/immature EST library. 11 of these proteins correspond to hypothetical conserved genes and 7 proteins are tsetse specific.
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http://dx.doi.org/10.1186/1471-2164-11-160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2846916PMC
March 2010

Interactions between mutualist Wigglesworthia and tsetse peptidoglycan recognition protein (PGRP-LB) influence trypanosome transmission.

Proc Natl Acad Sci U S A 2009 Jul 8;106(29):12133-8. Epub 2009 Jul 8.

Department of Epidemiology of Microbial Diseases, Yale School of Public Heath, New Haven, CT 06520, USA.

Tsetse flies, the sole vectors of African trypanosomes, have coevolved with mutualistic endosymbiont Wigglesworthia glossinidiae. Elimination of Wigglesworthia renders tsetse sterile and increases their trypanosome infection susceptibility. We show that a tsetse peptidoglycan recognition protein (PGRP-LB) is crucial for symbiotic tolerance and trypanosome infection processes. Tsetse pgrp-lb is expressed in the Wigglesworthia-harboring organ (bacteriome) in the midgut, and its level of expression correlates with symbiont numbers. Adult tsetse cured of Wigglesworthia infections have significantly lower pgrp-lb levels than corresponding normal adults. RNA interference (RNAi)-mediated depletion of pgrp-lb results in the activation of the immune deficiency (IMD) signaling pathway and leads to the synthesis of antimicrobial peptides (AMPs), which decrease Wigglesworthia density. Depletion of pgrp-lb also increases the host's susceptibility to trypanosome infections. Finally, parasitized adults have significantly lower pgrp-lb levels than flies, which have successfully eliminated trypanosome infections. When both PGRP-LB and IMD immunity pathway functions are blocked, flies become unusually susceptible to parasitism. Based on the presence of conserved amidase domains, tsetse PGRP-LB may scavenge the peptidoglycan (PGN) released by Wigglesworthia and prevent the activation of symbiont-damaging host immune responses. In addition, tsetse PGRP-LB may have an anti-protozoal activity that confers parasite resistance. The symbiotic adaptations and the limited exposure of tsetse to foreign microbes may have led to the considerable differences in pgrp-lb expression and regulation noted in tsetse from that of closely related Drosophila. A dynamic interplay between Wigglesworthia and host immunity apparently is influential in tsetse's ability to transmit trypanosomes.
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http://dx.doi.org/10.1073/pnas.0901226106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2715537PMC
July 2009

An insect symbiosis is influenced by bacterium-specific polymorphisms in outer-membrane protein A.

Proc Natl Acad Sci U S A 2008 Sep 24;105(39):15088-93. Epub 2008 Sep 24.

Department of Epidemiology and Public Health, Section of Vector Biology, Yale University School of Medicine, LEPH 606, 60 College Street, New Haven, CT 06520, USA.

Beneficial bacterial symbioses are ubiquitous in nature. However, the functional and molecular basis of host tolerance to resident symbiotic microbes, in contrast to resistance to closely related bacteria that are recognized as foreign, remain largely unknown. We used the tsetse fly (Glossina morsitans), which depends on symbiotic flora for fecundity and has limited exposure to foreign microbes, to investigate the tolerance phenomenon exhibited during symbiosis. We examined the potential role of bacterium-specific polymorphisms present in the major bacterial surface protein, outer-membrane protein A (OmpA), on host infection outcomes. Tsetse were successfully superinfected with their mutualistic facultative symbiont, Sodalis glossinidius, whereas infections with Escherichia coli K12 were lethal. In contrast, tsetse were resistant to an E. coli OmpA mutant strain, whereas recombinant Sodalis expressing E. coli OmpA became pathogenic. Profiling of tsetse immunity-related gene expression incriminated peptidoglycan recognition protein (pgrp)-lb as a determinant of the infection outcomes we observed. RNAi-induced knockdown of tsetse pgrp-lb significantly reduced host mortality after infection with otherwise lethal E. coli K12. Our results show that polymorphisms in the exposed loop domains of OmpA represent a microbial adaptation that mediates host tolerance of endogenous symbiotic bacteria.
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http://dx.doi.org/10.1073/pnas.0805666105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567497PMC
September 2008

The obligate mutualist Wigglesworthia glossinidia influences reproduction, digestion, and immunity processes of its host, the tsetse fly.

Appl Environ Microbiol 2008 Oct 8;74(19):5965-74. Epub 2008 Aug 8.

Yale School of Public Heath, Department of Epidemiology of Microbial Diseases, New Haven, CT 06520, USA.

Tsetse flies (Diptera: Glossinidae) are vectors for trypanosome parasites, the agents of the deadly sleeping sickness disease in Africa. Tsetse also harbor two maternally transmitted enteric mutualist endosymbionts: the primary intracellular obligate Wigglesworthia glossinidia and the secondary commensal Sodalis glossinidius. Both endosymbionts are transmitted to the intrauterine progeny through the milk gland secretions of the viviparous female. We administered various antibiotics either continuously by per os supplementation of the host blood meal diet or discretely by hemocoelic injections into fertile females in an effort to selectively eliminate the symbionts to study their individual functions. A symbiont-specific PCR amplification assay and fluorescence in situ hybridization analysis were used to evaluate symbiont infection outcomes. Tetracycline and rifampin treatments eliminated all tsetse symbionts but reduced the fecundity of the treated females. Ampicillin treatments did not affect the intracellular Wigglesworthia localized in the bacteriome organ and retained female fecundity. The resulting progeny of ampicillin-treated females, however, lacked Wigglesworthia but still harbored the commensal Sodalis. Our results confirm the presence of two physiologically distinct Wigglesworthia populations: the bacteriome-localized Wigglesworthia involved with nutritional symbiosis and free-living Wigglesworthia in the milk gland organ responsible for maternal transmission to the progeny. We evaluated the reproductive fitness, longevity, digestion, and vectorial competence of flies that were devoid of Wigglesworthia. The absence of Wigglesworthia completely abolished the fertility of females but not that of males. Both the male and female Wigglesworthia-free adult progeny displayed longevity costs and were significantly compromised in their blood meal digestion ability. Finally, while the vectorial competence of the young newly hatched adults without Wigglesworthia was comparable to that of their wild-type counterparts, older flies displayed higher susceptibility to trypanosome infections, indicating a role for the mutualistic symbiosis in host immunobiology. The ability to rear adult tsetse that lack the obligate Wigglesworthia endosymbionts will now enable functional investigations into this ancient symbiosis.
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http://dx.doi.org/10.1128/AEM.00741-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2565960PMC
October 2008

Molecular aspects of transferrin expression in the tsetse fly (Glossina morsitans morsitans).

J Insect Physiol 2007 Jul 1;53(7):715-23. Epub 2007 Apr 1.

Department of Epidemiology and Public Health, Yale University School of Medicine, 60 College Street, 606 LEPH, New Haven, CT 06510, USA.

Iron is an essential element for metabolic processes intrinsic to life, and yet the properties that make iron a necessity also make it potentially deleterious. To avoid harm, iron homeostasis is achieved via proteins involved in transport and storage of iron, one of which is transferrin. We describe the temporal and spatial aspects of transferrin (GmmTsf) expression and its transcriptional regulation in tsetse where both the male and female are strictly hematophagous. Using Northern, Western and immunohistochemical analysis, we show that GmmTsf is abundant in the hemolymph and is expressed in the adult developmental stages of male and female insects. It is preferentially expressed in the female milk gland tubules and its expression appears to be cyclical and possibly regulated in synchrony with the oogenic and/or larvigenic cycle. Although no mRNA is detected, GmmTsf protein is present in the immature stages of development, apparently being transported into the intrauterine larva from the mother via the milk gland ducts. Transferrin is also detected in the vitellogenic ovary and the adult male testes, further supporting its classification as a vitellogenic protein. Similar to reports in other insects, transferrin mRNA levels increase upon bacterial challenge in tsetse suggesting that transferrin may play an additional role in immunity. Although transferrin expression is induced following bacterial challenge, it is significantly reduced in tsetse carrying midgut trypanosome infections. Analysis of tsetse that have cured the parasite challenge shows normal levels of GmmTsf. This observation suggests that the parasite in competing for the availability of limited dietary iron may manipulate host gene expression.
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http://dx.doi.org/10.1016/j.jinsphys.2007.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2065764PMC
July 2007
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