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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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Related in: MedlinePlus

Endpoint PCR analysis of esap1 and esap2 in adult squid tissues and juvenile light organ. The RT reactions for each tissue were performed using 1 µg of total RNA. PCRs were run for 35 cycles, and 10 μl of PCR mixture was loaded into each lane. The 40S ribosomal gene was used as a housekeeping gene control. A control reaction where no reverse transcriptase was added was run to assess genomic contamination. Results are representative of three independent extractions.
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fig4: Endpoint PCR analysis of esap1 and esap2 in adult squid tissues and juvenile light organ. The RT reactions for each tissue were performed using 1 µg of total RNA. PCRs were run for 35 cycles, and 10 μl of PCR mixture was loaded into each lane. The 40S ribosomal gene was used as a housekeeping gene control. A control reaction where no reverse transcriptase was added was run to assess genomic contamination. Results are representative of three independent extractions.

Mentions: Because transcripts encoding different alkaline phosphatase isoforms vary in tissue expression in other animals, we sought to determine whether the two esap transcripts are differentially expressed in host squid tissues. We performed endpoint semiquantitative reverse transcriptase PCR (RT-PCR) on cDNA amplified by poly(dT) primer from RNA extracted from tissues (Fig. 4). Our results revealed the presence of esap1 mRNA in guts and bacterium-containing central epithelial light organ cores (central cores) of adult animals, which can be dissected cleanly from adult light organs, and whole light organs of juvenile animals, which will contain both juvenile central core and gut. esap2 was highly expressed in other tissues, specifically, the skin, arm, and white body (hematopoietic organ). It had lower but detectable levels of expression in the tentacle and gut. Expression in the central core, gill, and mature circulating hemocytes, which are blood cells produced by the white body, was undetectable.


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)

Endpoint PCR analysis of esap1 and esap2 in adult squid tissues and juvenile light organ. The RT reactions for each tissue were performed using 1 µg of total RNA. PCRs were run for 35 cycles, and 10 μl of PCR mixture was loaded into each lane. The 40S ribosomal gene was used as a housekeeping gene control. A control reaction where no reverse transcriptase was added was run to assess genomic contamination. Results are representative of three independent extractions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Endpoint PCR analysis of esap1 and esap2 in adult squid tissues and juvenile light organ. The RT reactions for each tissue were performed using 1 µg of total RNA. PCRs were run for 35 cycles, and 10 μl of PCR mixture was loaded into each lane. The 40S ribosomal gene was used as a housekeeping gene control. A control reaction where no reverse transcriptase was added was run to assess genomic contamination. Results are representative of three independent extractions.
Mentions: Because transcripts encoding different alkaline phosphatase isoforms vary in tissue expression in other animals, we sought to determine whether the two esap transcripts are differentially expressed in host squid tissues. We performed endpoint semiquantitative reverse transcriptase PCR (RT-PCR) on cDNA amplified by poly(dT) primer from RNA extracted from tissues (Fig. 4). Our results revealed the presence of esap1 mRNA in guts and bacterium-containing central epithelial light organ cores (central cores) of adult animals, which can be dissected cleanly from adult light organs, and whole light organs of juvenile animals, which will contain both juvenile central core and gut. esap2 was highly expressed in other tissues, specifically, the skin, arm, and white body (hematopoietic organ). It had lower but detectable levels of expression in the tentacle and gut. Expression in the central core, gill, and mature circulating hemocytes, which are blood cells produced by the white body, was undetectable.

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