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

ABSTRACT: 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.

SUBMITTER: Weiss BL

PROVIDER: S-EPMC4107339 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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

Weiss Brian L BL Savage Amy F AF Griffith Bridget C BC Wu Yineng Y Aksoy Serap S

Journal of immunology (Baltimore, Md. : 1950) 20140609 2

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 ...[more]

PMID: 24913976

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Project description:Tsetse flies are the principal insect vectors of African trypanosomes-sleeping sickness in humans and Nagana in cattle. One of the tsetse fly species, Glossina morsitans morsitans, is host to the parasite, Trypanosoma brucei, a major cause of African trypanosomiasis. Precise details of the life cycle have yet to be established, but the parasite life cycle involves crossing the insect peritrophic matrix (PM). The PM consists of the polysaccharide chitin, several hundred proteins, and both glycosamino- and galactosaminoglycan (GAG) polysaccharides. Owing to the technical challenges of detecting small amounts of GAG polysaccharides, their conclusive identification and composition have not been possible until now. Following removal of PMs from the insects and the application of heparinases (bacterial lyase enzymes that are specific for heparan sulphate (HS) GAG polysaccharides), dot blots with a HS-specific antibody showed heparan sulphate proteoglycans (HSPGs) to be present, consistent with Glossina morsitans morsitans genome analysis, as well as the likely expression of the HSPGs syndecan and perlecan. Exhaustive HS digestion with heparinases, fluorescent labeling of the resulting disaccharides with BODIPY fluorophore, and separation by strong anion exchange chromatography then demonstrated the presence of HS for the first time and provided the disaccharide composition. There were no significant differences in the type of disaccharide species present between genders or between ages (24 vs. 48 h post emergence), although the HS from female flies was more heavily sulphated overall. Significant differences, which may relate to differences in infection between genders or ages, were evident, however, in overall levels of 2-O-sulphation between sexes and, for females, between 24 and 48 h post-emergence, implying a change in expression or activity for the 2-O-sulphotransferase enzyme. The presence of significant quantities of disaccharides containing the monosaccharide GlcNAc6S contrasts with previous findings in Drosophila melanogaster and suggests subtle differences in HS fine structure between species of the Diptera.

Project description: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.

Project description:BackgroundTsetse flies (genus Glossina) are large blood-sucking dipteran flies that are important as vectors of human and animal trypanosomiasis in sub-Saharan Africa. Tsetse anatomy has been well described, including detailed accounts of the functional anatomy of the proboscis for piercing host skin and sucking up blood. The proboscis also serves as the developmental site for the infective metacyclic stages of several species of pathogenic livestock trypanosomes that are inoculated into the host with fly saliva. To understand the physical environment in which these trypanosomes develop, we have re-examined the microarchitecture of the tsetse proboscis.ResultsWe examined proboscises from male and female flies of Glossina pallidipes using light microscopy and scanning electron microscopy (SEM). Each proboscis was removed from the fly head and either examined intact or dissected into the three constituent components: Labrum, labium and hypopharynx. Our light and SEM images reaffirm earlier observations that the tsetse proboscis is a formidably armed weapon, well-adapted for piercing skin, and provide comparative data for G. pallidipes. In addition, the images reveal that the hypopharynx, the narrow tube that delivers saliva to the wound site, ends in a remarkably ornate and complex structure with around ten finger-like projections, each adorned with sucker-like protrusions, contradicting previous descriptions that show a simple, bevelled end like a hypodermic needle. The function of the finger-like projections is speculative; they appear to be flexible and may serve to protect the hypopharynx from influx of blood or microorganisms, or control the flow of saliva. Proboscises were examined after colonisation by Trypanosoma congolense savannah. Consistent with the idea that colonisation commences in the region nearest the foregut, the highest densities of trypanosomes were found in the region of the labrum proximal to the bulb, although high densities were also found in other regions of the labrum. Trypanosomes were visible through the thin wall of the hypopharynx by both light microscopy and SEM.ConclusionsWe highlight the remarkable architecture of the tsetse proboscis, in particular the intricate structure of the distal end of the hypopharynx. Further work is needed to elucidate the function of this intriguing structure.

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

Publications

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

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