Midgut immunity of Ixodes ricinus – What is behind the unstable midgut microbiome of the tick?

Kód výsledku v IS VaVaI

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Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Midgut immunity of Ixodes ricinus – What is behind the unstable midgut microbiome of the tick?

Popis výsledku v původním jazyce

Ticks are obligate hematophagous arthropods that transmit a wide range of pathogens to humans and animals. They also harbor a non-pathogenic microbiota, mainly in the ovaries and midgut. In our previous study on the midgut microbiome of Ixodes ricinus, we found a diverse but quantitatively poor bacterial microbiome and the absence of a core microbe. Experiments with artificial infection by capillary feeding with model bacteria from the environment (Gram-positive Micrococcus luteus and Gram-negative Pantoea sp.) showed a surprisingly rapid clearance of these microbes from the midgut of unfed females. In search of the molecules that might be responsible for this rapid defense effect, we performed RNAseq analysis of unfed I. ricinus midgut (Bioproject PRJNA685402), complemented by proteomic analysis. These high-throughput approaches led to the identification of potential antimicrobial molecules that hypothetically act as “sentinels&quot; against infections with environmental microbes. In addition to the recently characterized domesticated amidase effector2 (dae2), we also identified two different types of midgutspecific defensins: (i) early defensin, which is mainly present in unfed midgut, and (ii) late defensin, whose expression increases during blood feeding. These cationic peptides, approximately 4 kDa in size, have been commercially synthesized and exhibit a conserved pattern of three disulfide bridges. Antibacterial assays performed in microtiter plates in the concentration range of defensins from 250 μM to 8 nM showed their antibacterial potency against Gram-positive bacteria (Staphylococcus aureus or Staphylococcus epidermidis) with a minimum inhibitory concentration (MIC) of about 2 μM, while for M. luteus it was even at least two orders of magnitude lower in the nanomolar range. In contrast, no antimicrobial activity was exerted against Gram-negative bacteria. The role of the conserved disulfide bridges for defensins was also tested by reducing them with dithitreitol (DTT). This experiment revealedsurprising differences between the two defensins. While the antimicrobial activity of the reduced late defensin decreased significantly (~250-fold), DTT treatment had no effect on the activity of the early defensins, the MIC of which remained the same as the untreated peptide. Whether the activity of the defensins and other antimicrobial molecules such as Dae-2 is mainly due to the almost sterile environment of the tick midgut or whether other factors (such as epithelial immunity) play a role remains to be investigated.

Název v anglickém jazyce

Midgut immunity of Ixodes ricinus – What is behind the unstable midgut microbiome of the tick?

Popis výsledku anglicky

Ticks are obligate hematophagous arthropods that transmit a wide range of pathogens to humans and animals. They also harbor a non-pathogenic microbiota, mainly in the ovaries and midgut. In our previous study on the midgut microbiome of Ixodes ricinus, we found a diverse but quantitatively poor bacterial microbiome and the absence of a core microbe. Experiments with artificial infection by capillary feeding with model bacteria from the environment (Gram-positive Micrococcus luteus and Gram-negative Pantoea sp.) showed a surprisingly rapid clearance of these microbes from the midgut of unfed females. In search of the molecules that might be responsible for this rapid defense effect, we performed RNAseq analysis of unfed I. ricinus midgut (Bioproject PRJNA685402), complemented by proteomic analysis. These high-throughput approaches led to the identification of potential antimicrobial molecules that hypothetically act as “sentinels&quot; against infections with environmental microbes. In addition to the recently characterized domesticated amidase effector2 (dae2), we also identified two different types of midgutspecific defensins: (i) early defensin, which is mainly present in unfed midgut, and (ii) late defensin, whose expression increases during blood feeding. These cationic peptides, approximately 4 kDa in size, have been commercially synthesized and exhibit a conserved pattern of three disulfide bridges. Antibacterial assays performed in microtiter plates in the concentration range of defensins from 250 μM to 8 nM showed their antibacterial potency against Gram-positive bacteria (Staphylococcus aureus or Staphylococcus epidermidis) with a minimum inhibitory concentration (MIC) of about 2 μM, while for M. luteus it was even at least two orders of magnitude lower in the nanomolar range. In contrast, no antimicrobial activity was exerted against Gram-negative bacteria. The role of the conserved disulfide bridges for defensins was also tested by reducing them with dithitreitol (DTT). This experiment revealedsurprising differences between the two defensins. While the antimicrobial activity of the reduced late defensin decreased significantly (~250-fold), DTT treatment had no effect on the activity of the early defensins, the MIC of which remained the same as the untreated peptide. Whether the activity of the defensins and other antimicrobial molecules such as Dae-2 is mainly due to the almost sterile environment of the tick midgut or whether other factors (such as epithelial immunity) play a role remains to be investigated.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

40301 - Veterinary science

Projekt

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Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2024

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů