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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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Localization of EsAP activity in intact juvenile light organs. Images of fluorescence in 48-h nonsymbiotic and 48-h symbiotic light organs. (A and B) Detection of EsAP activity using ELF-97 substrate. The inset in panel A outlines the area of the light organ depicted in the images. (C and D) Localization of V. fischeri producing red fluorescent protein (RFP). Arrows indicate where the AP activity and bacteria colocalize. (E and F) Light organs incubated in ELF-97 substrate with the AP inhibitor levamisole (10 ng ml−1) as a control. Angle brackets indicate the region where AP activity is present in the absence of inhibitor. Bar, 200 µm; aa, anterior appendage; ac, antechamber; d, ducts; dc, deep crypts.
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fig8: Localization of EsAP activity in intact juvenile light organs. Images of fluorescence in 48-h nonsymbiotic and 48-h symbiotic light organs. (A and B) Detection of EsAP activity using ELF-97 substrate. The inset in panel A outlines the area of the light organ depicted in the images. (C and D) Localization of V. fischeri producing red fluorescent protein (RFP). Arrows indicate where the AP activity and bacteria colocalize. (E and F) Light organs incubated in ELF-97 substrate with the AP inhibitor levamisole (10 ng ml−1) as a control. Angle brackets indicate the region where AP activity is present in the absence of inhibitor. Bar, 200 µm; aa, anterior appendage; ac, antechamber; d, ducts; dc, deep crypts.

Mentions: Because the results showed that EsAP activity is high in total light organ extracts when symbiont density is high, we sought to determine the specific location of EsAP activity. Fixed light organs from 48-h symbiotic and nonsymbiotic animals were incubated with a substrate, ELF-97, which has been used in the study of alkaline phosphatase activity (26) and which produces a fluorescent precipitate when dephosphorylated. Nonsymbiotic and symbiotic juvenile light organs had intense signal in the duct and antechamber, although these regions appeared brighter in symbiotic animals than in nonsymbiotic animals (Fig. 8A and 8B). However, fluorescence in the crypts was detected only in symbiotic animals (Fig. 8A to 8D); as the bacteria had no detectable AP activity (see Materials and Methods), the substrate fluorescence signal was host associated. Light organs were also incubated (i) in the presence of substrate containing the AP inhibitor levamisole to confirm that the observed signal was specific to AP (Fig. 8E and 8F) or (ii) in detection buffer alone to ensure that the solution itself did not induce autofluorescence of light organ tissues (data not shown). Levamisole treatment abrogated the fluorescence signal of ELF-97, confirming that the signal was associated with AP activity.


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)

Localization of EsAP activity in intact juvenile light organs. Images of fluorescence in 48-h nonsymbiotic and 48-h symbiotic light organs. (A and B) Detection of EsAP activity using ELF-97 substrate. The inset in panel A outlines the area of the light organ depicted in the images. (C and D) Localization of V. fischeri producing red fluorescent protein (RFP). Arrows indicate where the AP activity and bacteria colocalize. (E and F) Light organs incubated in ELF-97 substrate with the AP inhibitor levamisole (10 ng ml−1) as a control. Angle brackets indicate the region where AP activity is present in the absence of inhibitor. Bar, 200 µm; aa, anterior appendage; ac, antechamber; d, ducts; dc, deep crypts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3569863&req=5

fig8: Localization of EsAP activity in intact juvenile light organs. Images of fluorescence in 48-h nonsymbiotic and 48-h symbiotic light organs. (A and B) Detection of EsAP activity using ELF-97 substrate. The inset in panel A outlines the area of the light organ depicted in the images. (C and D) Localization of V. fischeri producing red fluorescent protein (RFP). Arrows indicate where the AP activity and bacteria colocalize. (E and F) Light organs incubated in ELF-97 substrate with the AP inhibitor levamisole (10 ng ml−1) as a control. Angle brackets indicate the region where AP activity is present in the absence of inhibitor. Bar, 200 µm; aa, anterior appendage; ac, antechamber; d, ducts; dc, deep crypts.
Mentions: Because the results showed that EsAP activity is high in total light organ extracts when symbiont density is high, we sought to determine the specific location of EsAP activity. Fixed light organs from 48-h symbiotic and nonsymbiotic animals were incubated with a substrate, ELF-97, which has been used in the study of alkaline phosphatase activity (26) and which produces a fluorescent precipitate when dephosphorylated. Nonsymbiotic and symbiotic juvenile light organs had intense signal in the duct and antechamber, although these regions appeared brighter in symbiotic animals than in nonsymbiotic animals (Fig. 8A and 8B). However, fluorescence in the crypts was detected only in symbiotic animals (Fig. 8A to 8D); as the bacteria had no detectable AP activity (see Materials and Methods), the substrate fluorescence signal was host associated. Light organs were also incubated (i) in the presence of substrate containing the AP inhibitor levamisole to confirm that the observed signal was specific to AP (Fig. 8E and 8F) or (ii) in detection buffer alone to ensure that the solution itself did not induce autofluorescence of light organ tissues (data not shown). Levamisole treatment abrogated the fluorescence signal of ELF-97, confirming that the signal was associated with AP activity.

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