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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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Inhibition of endogenous alkaline phosphatase in vivo. (A) Effect of 10 ng ml−1 levamisole on the luminescence of juvenile animals at 24 and 48 h of colonization. Bars, average relative light units per treatment (±standard error; n = 18 animals per treatment). *, inhibitor-treated animal significantly different from untreated animal (linear regression, t test on slopes, P < 0.05). (B) Effect of inhibitor on symbiont number (CFU) in the light organ of animals at 48 h of colonization. Average relative light units per treatment (±standard error; n = 18 animals per treatment). *, inhibitor-treated animal significantly different from untreated animal (t test on slopes with unequal variances, P < 0.05). (C) Effect of inhibitor on ES114 growth in culture measured at OD600. Black line, growth in LBS medium alone; gray line, growth in LBS medium supplemented with 10 ng ml−1 levamisole.
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fig7: Inhibition of endogenous alkaline phosphatase in vivo. (A) Effect of 10 ng ml−1 levamisole on the luminescence of juvenile animals at 24 and 48 h of colonization. Bars, average relative light units per treatment (±standard error; n = 18 animals per treatment). *, inhibitor-treated animal significantly different from untreated animal (linear regression, t test on slopes, P < 0.05). (B) Effect of inhibitor on symbiont number (CFU) in the light organ of animals at 48 h of colonization. Average relative light units per treatment (±standard error; n = 18 animals per treatment). *, inhibitor-treated animal significantly different from untreated animal (t test on slopes with unequal variances, P < 0.05). (C) Effect of inhibitor on ES114 growth in culture measured at OD600. Black line, growth in LBS medium alone; gray line, growth in LBS medium supplemented with 10 ng ml−1 levamisole.

Mentions: The patterns of regulation described above suggested that EsAPs are important to the symbiosis. To test this hypothesis, we assayed for the effects of the AP inhibitor levamisole on early stages of the association. We incubated hatchling animals for 48 h in the presence of V. fischeri ES114 alone or ES114 with 10 ng ml−1 levamisole and measured luminescence at 24 and 48 h and CFU/light organ at 48 h (Fig. 7A and 7B). At 24 h, the luminescence output of inhibitor-treated animals was ~75% of that of untreated animals (Fig. 7A). At 48 h, both luminescence and CFU/light organ were lower by more than 80% in treated animals (Fig. 7A and 7B). Per-cell luminescence of the bacteria was not affected by the inhibitor, nor was growth rate under culture conditions affected (Fig. 7C). These data suggest that active alkaline phosphatase is required to achieve and maintain a normal symbiosis.


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)

Inhibition of endogenous alkaline phosphatase in vivo. (A) Effect of 10 ng ml−1 levamisole on the luminescence of juvenile animals at 24 and 48 h of colonization. Bars, average relative light units per treatment (±standard error; n = 18 animals per treatment). *, inhibitor-treated animal significantly different from untreated animal (linear regression, t test on slopes, P < 0.05). (B) Effect of inhibitor on symbiont number (CFU) in the light organ of animals at 48 h of colonization. Average relative light units per treatment (±standard error; n = 18 animals per treatment). *, inhibitor-treated animal significantly different from untreated animal (t test on slopes with unequal variances, P < 0.05). (C) Effect of inhibitor on ES114 growth in culture measured at OD600. Black line, growth in LBS medium alone; gray line, growth in LBS medium supplemented with 10 ng ml−1 levamisole.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Inhibition of endogenous alkaline phosphatase in vivo. (A) Effect of 10 ng ml−1 levamisole on the luminescence of juvenile animals at 24 and 48 h of colonization. Bars, average relative light units per treatment (±standard error; n = 18 animals per treatment). *, inhibitor-treated animal significantly different from untreated animal (linear regression, t test on slopes, P < 0.05). (B) Effect of inhibitor on symbiont number (CFU) in the light organ of animals at 48 h of colonization. Average relative light units per treatment (±standard error; n = 18 animals per treatment). *, inhibitor-treated animal significantly different from untreated animal (t test on slopes with unequal variances, P < 0.05). (C) Effect of inhibitor on ES114 growth in culture measured at OD600. Black line, growth in LBS medium alone; gray line, growth in LBS medium supplemented with 10 ng ml−1 levamisole.
Mentions: The patterns of regulation described above suggested that EsAPs are important to the symbiosis. To test this hypothesis, we assayed for the effects of the AP inhibitor levamisole on early stages of the association. We incubated hatchling animals for 48 h in the presence of V. fischeri ES114 alone or ES114 with 10 ng ml−1 levamisole and measured luminescence at 24 and 48 h and CFU/light organ at 48 h (Fig. 7A and 7B). At 24 h, the luminescence output of inhibitor-treated animals was ~75% of that of untreated animals (Fig. 7A). At 48 h, both luminescence and CFU/light organ were lower by more than 80% in treated animals (Fig. 7A and 7B). Per-cell luminescence of the bacteria was not affected by the inhibitor, nor was growth rate under culture conditions affected (Fig. 7C). These data suggest that active alkaline phosphatase is required to achieve and maintain a normal symbiosis.

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