Immunogenic and Antioxidant Effects of a Pathogen-Associated Prenyl Pyrophosphate in Anopheles gambiae

Despite efficient vector transmission, Plasmodium parasites suffer great bottlenecks during their developmental stages within Anopheles mosquitoes. The outcome depends on a complex three-way interaction between host, parasite and gut bacteria. Although considerable progress has been made recently in deciphering Anopheles effector responses, little is currently known regarding the underlying microbial immune elicitors. An interesting candidate in this sense is the pathogen-derived prenyl pyrophosphate and designated phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), found in Plasmodium and most eubacteria but not in higher eukaryotes. HMBPP is the most potent stimulant known of human V gamma 9V delta 2 T cells, a unique lymphocyte subset that expands during several infections including malaria. In this study, we show that V(Y)9V delta 2 T cells proliferate when stimulated with supernatants from intraerythrocytic stages of Plasmodium falciparum cultures, suggesting that biologically relevant doses of phosphoantigens are excreted by the parasite. Next, we used Anopheles gambiae to investigate the immune-and redox-stimulating effects of HMBPP. We demonstrate a potent activation in vitro of all but one of the signaling pathways earlier implicated in the human V(Y)9V delta 2 T cell response, as p38, JNK and PI3K/Akt but not ERK were activated in the A. gambiae 4a3B cell line. Additionally, both HMBPP and the downstream endogenous metabolite isopentenyl pyrophosphate displayed antioxidant effects by promoting cellular tolerance to hydrogen peroxide challenge. When provided in the mosquito blood meal, HMBPP induced temporal changes in the expression of several immune genes. In contrast to meso-diaminopimelic acid containing peptidoglycan, HMBPP induced expression of dual oxidase and nitric oxide synthase, two key determinants of Plasmodium infection. Furthermore, temporal fluctuations in midgut bacterial numbers were observed. The multifaceted effects observed in this study indicates that HMBPP is an important elicitor in common for both Plasmodium and gut bacteria in the mosquito.AuthorCount:7;</p

Immunogenic and Antioxidant Effects of a Pathogen-Associated Prenyl Pyrophosphate in Anopheles gambiae

Despite efficient vector transmission, Plasmodium parasites suffer great bottlenecks during their developmental stages within Anopheles mosquitoes. The outcome depends on a complex three-way interaction between host, parasite and gut bacteria. Although considerable progress has been made recently in deciphering Anopheles effector responses, little is currently known regarding the underlying microbial immune elicitors. An interesting candidate in this sense is the pathogen-derived prenyl pyrophosphate and designated phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), found in Plasmodium and most eubacteria but not in higher eukaryotes. HMBPP is the most potent stimulant known of human Vγ9Vδ2 T cells, a unique lymphocyte subset that expands during several infections including malaria. In this study, we show that Vγ9Vδ2 T cells proliferate when stimulated with supernatants from intraerythrocytic stages of Plasmodium falciparum cultures, suggesting that biologically relevant doses of phosphoantigens are excreted by the parasite. Next, we used Anopheles gambiae to investigate the immune- and redox- stimulating effects of HMBPP. We demonstrate a potent activation in vitro of all but one of the signaling pathways earlier implicated in the human Vγ9Vδ2 T cell response, as p38, JNK and PI3K/Akt but not ERK were activated in the A. gambiae 4a3B cell line. Additionally, both HMBPP and the downstream endogenous metabolite isopentenyl pyrophosphate displayed antioxidant effects by promoting cellular tolerance to hydrogen peroxide challenge. When provided in the mosquito blood meal, HMBPP induced temporal changes in the expression of several immune genes. In contrast to meso-diaminopimelic acid containing peptidoglycan, HMBPP induced expression of dual oxidase and nitric oxide synthase, two key determinants of Plasmodium infection. Furthermore, temporal fluctuations in midgut bacterial numbers were observed. The multifaceted effects observed in this study indicates that HMBPP is an important elicitor in common for both Plasmodium and gut bacteria in the mosquito

Immunogenic and Antioxidant Effects of a Pathogen-Associated Prenyl Pyrophosphate in Anopheles gambiae

Despite efficient vector transmission, Plasmodium parasites suffer great bottlenecks during their developmental stages within Anopheles mosquitoes. The outcome depends on a complex three-way interaction between host, parasite and gut bacteria. Although considerable progress has been made recently in deciphering Anopheles effector responses, little is currently known regarding the underlying microbial immune elicitors. An interesting candidate in this sense is the pathogen-derived prenyl pyrophosphate and designated phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), found in Plasmodium and most eubacteria but not in higher eukaryotes. HMBPP is the most potent stimulant known of human Vγ9Vδ2 T cells, a unique lymphocyte subset that expands during several infections including malaria. In this study, we show that Vγ9Vδ2 T cells proliferate when stimulated with supernatants from intraerythrocytic stages of Plasmodium falciparum cultures, suggesting that biologically relevant doses of phosphoantigens are excreted by the parasite. Next, we used Anopheles gambiae to investigate the immune- and redox- stimulating effects of HMBPP. We demonstrate a potent activation in vitro of all but one of the signaling pathways earlier implicated in the human Vγ9Vδ2 T cell response, as p38, JNK and PI3K/Akt but not ERK were activated in the A. gambiae 4a3B cell line. Additionally, both HMBPP and the downstream endogenous metabolite isopentenyl pyrophosphate displayed antioxidant effects by promoting cellular tolerance to hydrogen peroxide challenge. When provided in the mosquito blood meal, HMBPP induced temporal changes in the expression of several immune genes. In contrast to meso-diaminopimelic acid containing peptidoglycan, HMBPP induced expression of dual oxidase and nitric oxide synthase, two key determinants of Plasmodium infection. Furthermore, temporal fluctuations in midgut bacterial numbers were observed. The multifaceted effects observed in this study indicates that HMBPP is an important elicitor in common for both Plasmodium and gut bacteria in the mosquito

Draft Genome Sequences of Elizabethkingia anophelis Strains R26T and Ag1 from the Midgut of the Malaria Mosquito Anopheles gambiae

Elizabethkingia anophelis is a species in the family Flavobacteriaceae. It is a dominant resident in the mosquito gut and also a human pathogen. We present the draft genome sequences of two strains of E. anophelis, R26T and Ag1, which were isolated from the midgut of the malaria mosquito Anopheles gambiae

Prenyl pyrophosphate stimulation increased cellular tolerance to H₂O₂-induced oxidative stress.

<p><i>A. gambiae</i> 4a3B cells were seeded in 96-well plates. (<b>A</b>) HMBPP or IPP (10 µM) were added at indicated time points prior to oxidative insult (n = 3). (<b>B</b>) Cells were preincubated for 3 h with HMBPP at different concentrations (n = 6-8). At 30 min before H₂O₂ addition, cells were loaded with (<b>A</b>) CellROX Deep Red Reagent or (<b>B</b>) DCFDA for oxidative stress detection. Cells were washed and challenged with H₂O₂ for 2 h. Bars represent mean relative fluorescence ± SE, asterisks denote significant differences (*P < 0.05, **P < 0.01, *** P < 0.001) compared to H₂O₂ control.</p

Expression in midgut and carcass and abundance of midgut bacteria relative to control feed.

<p>(<b>A</b>–<b>B</b>) At 3 and 6 hpi, 5-10 midguts and corresponding carcasses per sample were dissected and subjected to qRT-PCR analysis of DEF1, DUOX and NOS. (<b>C</b>–<b>D</b>) Effects of HMBPP on relative midgut bacterial 16S were quantified using qPCR. (<b>C</b>) At 24 hpi, bacterial gDNA was extracted from midguts (n = 4). (<b>D</b>) Midgut cDNA from 3 and 6 hpi (n = 3). Bars represent mean ± SE (*<i>P</i> < 0.05, ** <i>P</i> < 0.01).</p

HMBPP activates phosphorylation of JNK, p38 and FOXO in <i>A. gambiae</i> 4a3B cells.

<p>(<b>A</b>–<b>D</b>) Mean fold changes ± SE relative to control stimulation and representative western blot images of target phosphoproteins. Membranes where stripped and reprobed for GAPDH to normalize relative band intensities. Asterisks represent significant differences (*<i>P</i> < 0.05, *** <i>P</i> < 0.001) from at least three independent experiments.</p

Insights from the Genome Annotation of <i>Elizabethkingia anophelis</i> from the Malaria Vector <i>Anopheles gambiae</i>

<div><p><i>Elizabethkingia anophelis</i> is a dominant bacterial species in the gut ecosystem of the malaria vector mosquito <i>Anopheles gambiae</i>. We recently sequenced the genomes of two strains of <i>E. anophelis</i>, R26^T and Ag1, isolated from different strains of <i>A. gambiae</i>. The two bacterial strains are identical with a few exceptions. Phylogenetically, <i>Elizabethkingia</i> is closer to <i>Chryseobacterium</i> and <i>Riemerella</i> than to <i>Flavobacterium.</i> In line with other Bacteroidetes known to utilize various polymers in their ecological niches, the <i>E. anophelis</i> genome contains numerous TonB dependent transporters with various substrate specificities. In addition, several genes belonging to the polysaccharide utilization system and the glycoside hydrolase family were identified that could potentially be of benefit for the mosquito carbohydrate metabolism. In agreement with previous reports of broad antibiotic resistance in <i>E. anophelis</i>, a large number of genes encoding efflux pumps and β-lactamases are present in the genome. The component genes of resistance-nodulation-division type efflux pumps were found to be syntenic and conserved in different taxa of Bacteroidetes. The bacterium also displays hemolytic activity and encodes several hemolysins that may participate in the digestion of erythrocytes in the mosquito gut. At the same time, the OxyR regulon and antioxidant genes could provide defense against the oxidative stress that is associated with blood digestion. The genome annotation and comparative genomic analysis revealed functional characteristics associated with the symbiotic relationship with the mosquito host.</p></div

Temporal expression of selected immune genes relative to control blood meal as determined by qRT-PCR.

<p>(<b>A</b>–<b>E</b>) Bars represent mean ± SE from at least three (HMBPP) or two (PGN) independent experiments. Whole bodies were used from 5–10 fully engorged females. Asterisks (*<i>P</i> < 0.05, **<i>P</i> < 0.01) denote significantly different expression compared to control feed.</p

Summary of the functional subcategories of resistance genes.

<p>Average identity reflects the mean identity of genes within the subcategory compared to R26^T. <i>E. m</i>., <i>Elizabethkingia meningoseptica</i>; <i>C. g.</i>, <i>Chryseobacterium gleum</i>; <i>F. b., Flavobacterium branchiophilum</i>.</p