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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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Phylogenetic analysis of the amino acid sequences of EsAP1 and EsAP2. Sequences were chosen to provide broad representation across the animal kingdom (see Table S1 in the supplemental material). (A) Maximum likelihood inference phylogeny based on alkaline phosphatase sequences. Support values of >50 obtained after 100 bootstrap replicates are shown at corresponding nodes. (B) Bayesian inference phylogeny based on alkaline phosphatase sequences. Bayesian posterior probabilities of >0.9 are shown at corresponding nodes. Positions of E. scolopes sequences are shown in bold. Euprymna scolopes 1, EsAP1; Euprymna scolopes 2, EsAP2.
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fig3: Phylogenetic analysis of the amino acid sequences of EsAP1 and EsAP2. Sequences were chosen to provide broad representation across the animal kingdom (see Table S1 in the supplemental material). (A) Maximum likelihood inference phylogeny based on alkaline phosphatase sequences. Support values of >50 obtained after 100 bootstrap replicates are shown at corresponding nodes. (B) Bayesian inference phylogeny based on alkaline phosphatase sequences. Bayesian posterior probabilities of >0.9 are shown at corresponding nodes. Positions of E. scolopes sequences are shown in bold. Euprymna scolopes 1, EsAP1; Euprymna scolopes 2, EsAP2.

Mentions: An EST library created from juvenile E. scolopes light organ mRNA (22) had two partial alkaline phosphatase (AP) sequences. Rapid amplification of cDNA ends (RACE) of both sequences revealed that esap1 (open reading frame [ORF], 1,585 bp) and esap2 (ORF, 1,581 bp) are identical isoforms up to position 1386, where they differ, possibly as a result of alternative splicing. This variation in the 3′ end results in two isoforms that are 90% identical to one another in amino acid sequence, 44 to 53% identical to eukaryotes in the nonredundant database (BLASTp), and 24% identical to Escherichia coli (BLASTp). Analysis of the alignment of the EsAPs (Fig. 2) revealed a conserved signal sequence consisting of the first 21 amino acids, as well as conservation of the active-site residues I106 to T116, residues critical for metal binding (D61, T175, E333, D338, H342, D379, H380, H456), and essential N-glycosylation sites (N239 to T241 and N432 to D435) (23, 24). These two sequences have differences in the carboxy-terminal region of the protein, including different amino acids and gaps at alternative locations. In phylogenetic analyses, the EsAPs branch together and cluster with the other mollusk APs. Other closely related invertebrates form a group with the mollusks (Fig. 3) (see also Table S1 in the supplemental material).


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)

Phylogenetic analysis of the amino acid sequences of EsAP1 and EsAP2. Sequences were chosen to provide broad representation across the animal kingdom (see Table S1 in the supplemental material). (A) Maximum likelihood inference phylogeny based on alkaline phosphatase sequences. Support values of >50 obtained after 100 bootstrap replicates are shown at corresponding nodes. (B) Bayesian inference phylogeny based on alkaline phosphatase sequences. Bayesian posterior probabilities of >0.9 are shown at corresponding nodes. Positions of E. scolopes sequences are shown in bold. Euprymna scolopes 1, EsAP1; Euprymna scolopes 2, EsAP2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Phylogenetic analysis of the amino acid sequences of EsAP1 and EsAP2. Sequences were chosen to provide broad representation across the animal kingdom (see Table S1 in the supplemental material). (A) Maximum likelihood inference phylogeny based on alkaline phosphatase sequences. Support values of >50 obtained after 100 bootstrap replicates are shown at corresponding nodes. (B) Bayesian inference phylogeny based on alkaline phosphatase sequences. Bayesian posterior probabilities of >0.9 are shown at corresponding nodes. Positions of E. scolopes sequences are shown in bold. Euprymna scolopes 1, EsAP1; Euprymna scolopes 2, EsAP2.
Mentions: An EST library created from juvenile E. scolopes light organ mRNA (22) had two partial alkaline phosphatase (AP) sequences. Rapid amplification of cDNA ends (RACE) of both sequences revealed that esap1 (open reading frame [ORF], 1,585 bp) and esap2 (ORF, 1,581 bp) are identical isoforms up to position 1386, where they differ, possibly as a result of alternative splicing. This variation in the 3′ end results in two isoforms that are 90% identical to one another in amino acid sequence, 44 to 53% identical to eukaryotes in the nonredundant database (BLASTp), and 24% identical to Escherichia coli (BLASTp). Analysis of the alignment of the EsAPs (Fig. 2) revealed a conserved signal sequence consisting of the first 21 amino acids, as well as conservation of the active-site residues I106 to T116, residues critical for metal binding (D61, T175, E333, D338, H342, D379, H380, H456), and essential N-glycosylation sites (N239 to T241 and N432 to D435) (23, 24). These two sequences have differences in the carboxy-terminal region of the protein, including different amino acids and gaps at alternative locations. In phylogenetic analyses, the EsAPs branch together and cluster with the other mollusk APs. Other closely related invertebrates form a group with the mollusks (Fig. 3) (see also Table S1 in the supplemental material).

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