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Modulation of symbiont lipid A signaling by host alkaline phosphatases in the squid-vibrio symbiosis.

Rader BA, Kremer N, Apicella MA, Goldman WE, McFall-Ngai MJ - MBio (2012)

Bottom Line: With constant presentation of MAMPs by the normal microbiota, mechanisms to tolerate their effects have developed.The results of this contribution provide evidence that host alkaline phosphatases (APs) dephosphorylate and inactivate the symbiont MAMP lipid A.Not only may these activities serve to "tame" the MAMPs, but also the resulting products may themselves be important signals in persistent mutualisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA.

ABSTRACT

Unlabelled: The synergistic activity of Vibrio fischeri lipid A and the peptidoglycan monomer (tracheal cytotoxin [TCT]) induces apoptosis in the superficial cells of the juvenile Euprymna scolopes light organ during the onset of the squid-vibrio symbiosis. Once the association is established in the epithelium-lined crypts of the light organ, the host degrades the symbiont's constitutively produced TCT by the amidase activity of a peptidoglycan recognition protein (E. scolopes peptidoglycan recognition protein 2 [EsPGRP2]). In the present study, we explored the role of alkaline phosphatases in transforming the lipid A of the symbiont into a form that changes its signaling properties to host tissues. We obtained full-length open reading frames for two E. scolopes alkaline phosphatase (EsAP) mRNAs (esap1 and esap2); transcript levels suggested that the dominant light organ isoform is EsAP1. Levels of total EsAP activity increased with symbiosis, but only after the lipid A-dependent morphogenetic induction at 12 h, and were regulated over the day-night cycle. Inhibition of total EsAP activity impaired normal colonization and persistence by the symbiont. EsAP activity localized to the internal regions of the symbiotic juvenile light organ, including the lumina of the crypt spaces where the symbiont resides. These data provide evidence that EsAPs work in concert with EsPGRPs to change the signaling properties of bacterial products and thereby promote persistent colonization by the mutualistic symbiont.

Importance: The potential for microbe-associated molecular patterns (MAMPs) to compromise host-tissue health is reflected in the often-used nomenclature for these molecules: lipopolysaccharide (LPS) is also called "endotoxin" and the peptidoglycan monomer is also called "tracheal cytotoxin" (TCT). With constant presentation of MAMPs by the normal microbiota, mechanisms to tolerate their effects have developed. The results of this contribution provide evidence that host alkaline phosphatases (APs) dephosphorylate and inactivate the symbiont MAMP lipid A. As such, APs work in synergy with a peptidoglycan recognition protein, which inactivates symbiont-exported TCT, to alter the symbiont MAMPs and promote persistence of the partnership. Not only may these activities serve to "tame" the MAMPs, but also the resulting products may themselves be important signals in persistent mutualisms. The finding of lipid A modification by APs in an invertebrate mutualism provides evidence that this specific strategy for dealing with symbiotic partners is conserved across the animal kingdom.

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Effect of CIAP treatment of lipid A on its ability to induce juvenile light organ apoptosis. (A) Fluorescence micrographs of light organs that had been incubated in filter-sterilized artificial seawater alone (non-sym) or with ES114 (sym), non-sym animal light organs incubated in FSASW plus lipid A, or non-sym animal light organs incubated in FSASW plus CIAP-treated lipid A for 18 h and stained with acridine orange (green) to visualize pycnotic nuclei, which appear as regions of punctate staining across the diffusely stained field of epithelial cells (e.g., arrow). Bars, 150 or 200 µm. (B) The number of pycnotic nuclei per anterior appendage of the juvenile light organ (n = 12 per treatment). Bars, average (±standard deviation); *, treatments that are significantly different from other treatments but not each other (one-way analysis of variance with post hoc pairwise comparisons, P < 0.001). aa, anterior appendage, the region used for enumeration of pycnotic nuclei.
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fig9: Effect of CIAP treatment of lipid A on its ability to induce juvenile light organ apoptosis. (A) Fluorescence micrographs of light organs that had been incubated in filter-sterilized artificial seawater alone (non-sym) or with ES114 (sym), non-sym animal light organs incubated in FSASW plus lipid A, or non-sym animal light organs incubated in FSASW plus CIAP-treated lipid A for 18 h and stained with acridine orange (green) to visualize pycnotic nuclei, which appear as regions of punctate staining across the diffusely stained field of epithelial cells (e.g., arrow). Bars, 150 or 200 µm. (B) The number of pycnotic nuclei per anterior appendage of the juvenile light organ (n = 12 per treatment). Bars, average (±standard deviation); *, treatments that are significantly different from other treatments but not each other (one-way analysis of variance with post hoc pairwise comparisons, P < 0.001). aa, anterior appendage, the region used for enumeration of pycnotic nuclei.

Mentions: To determine whether alkaline phosphatase dephosphorylation can render V. fischeri lipid A nonreactive, we took advantage of the previous finding that V. fischeri lipid A is required for entry into early-stage apoptosis (chromatin condensation) of the superficial epithelial cells of the juvenile light organ (6). We incubated hatchling animals for 18 h in filter-sterilized artificial seawater with V. fischeri lipid A, lipid A that had been treated with calf intestinal alkaline phosphatase (CIAP), V. fischeri ES114 as a positive control for chromatin condensation, or seawater alone as a negative control. Whereas lipid A caused early-stage apoptosis at levels indistinguishable from those of symbiont-colonized juveniles, animals exposed to CIAP-treated lipid A had levels indistinguishable from those of animals exposed to seawater alone (Fig. 9).


Modulation of symbiont lipid A signaling by host alkaline phosphatases in the squid-vibrio symbiosis.

Rader BA, Kremer N, Apicella MA, Goldman WE, McFall-Ngai MJ - MBio (2012)

Effect of CIAP treatment of lipid A on its ability to induce juvenile light organ apoptosis. (A) Fluorescence micrographs of light organs that had been incubated in filter-sterilized artificial seawater alone (non-sym) or with ES114 (sym), non-sym animal light organs incubated in FSASW plus lipid A, or non-sym animal light organs incubated in FSASW plus CIAP-treated lipid A for 18 h and stained with acridine orange (green) to visualize pycnotic nuclei, which appear as regions of punctate staining across the diffusely stained field of epithelial cells (e.g., arrow). Bars, 150 or 200 µm. (B) The number of pycnotic nuclei per anterior appendage of the juvenile light organ (n = 12 per treatment). Bars, average (±standard deviation); *, treatments that are significantly different from other treatments but not each other (one-way analysis of variance with post hoc pairwise comparisons, P < 0.001). aa, anterior appendage, the region used for enumeration of pycnotic nuclei.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig9: Effect of CIAP treatment of lipid A on its ability to induce juvenile light organ apoptosis. (A) Fluorescence micrographs of light organs that had been incubated in filter-sterilized artificial seawater alone (non-sym) or with ES114 (sym), non-sym animal light organs incubated in FSASW plus lipid A, or non-sym animal light organs incubated in FSASW plus CIAP-treated lipid A for 18 h and stained with acridine orange (green) to visualize pycnotic nuclei, which appear as regions of punctate staining across the diffusely stained field of epithelial cells (e.g., arrow). Bars, 150 or 200 µm. (B) The number of pycnotic nuclei per anterior appendage of the juvenile light organ (n = 12 per treatment). Bars, average (±standard deviation); *, treatments that are significantly different from other treatments but not each other (one-way analysis of variance with post hoc pairwise comparisons, P < 0.001). aa, anterior appendage, the region used for enumeration of pycnotic nuclei.
Mentions: To determine whether alkaline phosphatase dephosphorylation can render V. fischeri lipid A nonreactive, we took advantage of the previous finding that V. fischeri lipid A is required for entry into early-stage apoptosis (chromatin condensation) of the superficial epithelial cells of the juvenile light organ (6). We incubated hatchling animals for 18 h in filter-sterilized artificial seawater with V. fischeri lipid A, lipid A that had been treated with calf intestinal alkaline phosphatase (CIAP), V. fischeri ES114 as a positive control for chromatin condensation, or seawater alone as a negative control. Whereas lipid A caused early-stage apoptosis at levels indistinguishable from those of symbiont-colonized juveniles, animals exposed to CIAP-treated lipid A had levels indistinguishable from those of animals exposed to seawater alone (Fig. 9).

Bottom Line: With constant presentation of MAMPs by the normal microbiota, mechanisms to tolerate their effects have developed.The results of this contribution provide evidence that host alkaline phosphatases (APs) dephosphorylate and inactivate the symbiont MAMP lipid A.Not only may these activities serve to "tame" the MAMPs, but also the resulting products may themselves be important signals in persistent mutualisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA.

ABSTRACT

Unlabelled: The synergistic activity of Vibrio fischeri lipid A and the peptidoglycan monomer (tracheal cytotoxin [TCT]) induces apoptosis in the superficial cells of the juvenile Euprymna scolopes light organ during the onset of the squid-vibrio symbiosis. Once the association is established in the epithelium-lined crypts of the light organ, the host degrades the symbiont's constitutively produced TCT by the amidase activity of a peptidoglycan recognition protein (E. scolopes peptidoglycan recognition protein 2 [EsPGRP2]). In the present study, we explored the role of alkaline phosphatases in transforming the lipid A of the symbiont into a form that changes its signaling properties to host tissues. We obtained full-length open reading frames for two E. scolopes alkaline phosphatase (EsAP) mRNAs (esap1 and esap2); transcript levels suggested that the dominant light organ isoform is EsAP1. Levels of total EsAP activity increased with symbiosis, but only after the lipid A-dependent morphogenetic induction at 12 h, and were regulated over the day-night cycle. Inhibition of total EsAP activity impaired normal colonization and persistence by the symbiont. EsAP activity localized to the internal regions of the symbiotic juvenile light organ, including the lumina of the crypt spaces where the symbiont resides. These data provide evidence that EsAPs work in concert with EsPGRPs to change the signaling properties of bacterial products and thereby promote persistent colonization by the mutualistic symbiont.

Importance: The potential for microbe-associated molecular patterns (MAMPs) to compromise host-tissue health is reflected in the often-used nomenclature for these molecules: lipopolysaccharide (LPS) is also called "endotoxin" and the peptidoglycan monomer is also called "tracheal cytotoxin" (TCT). With constant presentation of MAMPs by the normal microbiota, mechanisms to tolerate their effects have developed. The results of this contribution provide evidence that host alkaline phosphatases (APs) dephosphorylate and inactivate the symbiont MAMP lipid A. As such, APs work in synergy with a peptidoglycan recognition protein, which inactivates symbiont-exported TCT, to alter the symbiont MAMPs and promote persistence of the partnership. Not only may these activities serve to "tame" the MAMPs, but also the resulting products may themselves be important signals in persistent mutualisms. The finding of lipid A modification by APs in an invertebrate mutualism provides evidence that this specific strategy for dealing with symbiotic partners is conserved across the animal kingdom.

Show MeSH
Related in: MedlinePlus