Dataset Information

Inhibition of P2X Receptors Protects Human Monocytes against Damage by Leukotoxin from Aggregatibacter actinomycetemcomitans and ?-Hemolysin from Escherichia coli.

ABSTRACT: ?-Hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans are important virulence factors in ascending urinary tract infections and aggressive periodontitis, respectively. The extracellular signaling molecule ATP is released immediately after insertion of the toxins into plasma membranes and, via P2X receptors, is essential for the erythrocyte damage inflicted by these toxins. Moreover, ATP signaling is required for the ensuing recognition and phagocytosis of damaged erythrocytes by the monocytic cell line THP-1. Here, we investigate how these toxins affect THP-1 monocyte function. We demonstrate that both toxins trigger early ATP release and a following increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in THP-1 monocytes. The HlyA- and LtxA-induced [Ca²⁺]_i response is diminished by the P2 receptor antagonist in a pattern that fits the functional P2 receptor expression in these cells. Both toxins are capable of lysing THP-1 cells, with LtxA being more aggressive. Either desensitization or blockage of P2X₁, P2X₄, or P2X₇ receptors markedly reduces toxin-induced cytolysis. This pattern is paralleled in freshly isolated human monocytes from healthy volunteers. Interestingly, only a minor fraction of the toxin-damaged THP-1 monocytes eventually lyse. P2X₇ receptor inhibition generally prevents cell damage, except from a distinct cell shrinkage that prevails in response to the toxins. Moreover, we find that preexposure to HlyA preserves the capacity of THP-1 monocytes to phagocytose damaged erythrocytes and may induce readiness to discriminate between damaged and healthy erythrocytes. These findings suggest a new pharmacological target for protecting monocytes during exposure to pore-forming cytolysins during infection or injury.

SUBMITTER: Fagerberg SK

PROVIDER: S-EPMC5067745 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Inhibition of P2X Receptors Protects Human Monocytes against Damage by Leukotoxin from Aggregatibacter actinomycetemcomitans and α-Hemolysin from Escherichia coli.

Fagerberg Steen K SK Jakobsen Martin R MR Skals Marianne M Praetorius Helle A HA

Infection and immunity 20161017 11

α-Hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans are important virulence factors in ascending urinary tract infections and aggressive periodontitis, respectively. The extracellular signaling molecule ATP is released immediately after insertion of the toxins into plasma membranes and, via P2X receptors, is essential for the erythrocyte damage inflicted by these toxins. Moreover, ATP signaling is required for the ensuing recognition and ph ...[more]

PMID: 27528275

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Project description:Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previous in vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2) some caution should be used when interpreting the effect attributed to targeted gene mutations when seen in a competitive in vivo environment.

Dataset Information

Inhibition of P2X Receptors Protects Human Monocytes against Damage by Leukotoxin from Aggregatibacter actinomycetemcomitans and ?-Hemolysin from Escherichia coli.

Publications

Inhibition of P2X Receptors Protects Human Monocytes against Damage by Leukotoxin from Aggregatibacter actinomycetemcomitans and α-Hemolysin from Escherichia coli.

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