The Lyme disease agent co-opts adiponectin receptor-mediated signaling in its arthropod vector

Article activity feed

1. 

   Version published to 10.7554/elife.72568 on eLife

   Nov 16, 2021
2. 

   eLife

   Oct 7, 2021

   Author Response:

   Reviewer #1 (Public Review):

   In this manuscript, the authors present evidence that mouse blood meals containing Lyme disease spirochetes induce upregulation and activation of an adiponectin receptor (ISARL) in the midguts of Ixodes scapularis ticks. Activation of the receptor initiates transcriptional changes, not seen with blood meals from uninfected mice, that give rise to metabolic alterations in the midguts required for replication of spirochetes. Using RNA-seq, they trace these critical alterations to genes encoding enzymes for synthesis of phospholipids. Although mouse adiponectin induced transcriptional changes in engorged tick midguts related to glucose and energy metabolism, it did not influence B. burgdorferi colonization. The authors conclude by presenting evidence that tick complement C1q-like protein …

   Author Response:

   Reviewer #1 (Public Review):

   In this manuscript, the authors present evidence that mouse blood meals containing Lyme disease spirochetes induce upregulation and activation of an adiponectin receptor (ISARL) in the midguts of Ixodes scapularis ticks. Activation of the receptor initiates transcriptional changes, not seen with blood meals from uninfected mice, that give rise to metabolic alterations in the midguts required for replication of spirochetes. Using RNA-seq, they trace these critical alterations to genes encoding enzymes for synthesis of phospholipids. Although mouse adiponectin induced transcriptional changes in engorged tick midguts related to glucose and energy metabolism, it did not influence B. burgdorferi colonization. The authors conclude by presenting evidence that tick complement C1q-like protein (C1QL3), also upregulated in response to a blood meal containing spirochetes, is an ISARL ligand and that knockdown of C1QL3 impairs spirochete colonization.

   This work extends our understanding of the complex and intimate physiologic interactions between Borrelia burgdorferi and Ixodes ticks that sustain the spirochete's enzootic cycle. It builds upon prior work by others showing that feeding ticks provide spirochetes with glycerol, an alternative carbohydrate energy source and essential building block for phospholipid biosynthesis. It also appears relevant to previously published work showing that B. burgdorferi can extract lipids from the membranes of eukaryotic cells to which they are attached.

   The strength of the study is that it uses state-of-the-art genetic, bioinformatic, and transcriptional approaches to garner novel insights into the unique transcriptional/metabolic changes that occur in ticks when they ingest blood from B. burgdorferi¬-infected mice. It enhances the now well-established, but still far from well understood, viewpoint that ticks are not mere biologic syringes for injection of spirochetes. And it demonstrates, probably more than any preceding study, the extent to which tick midgut interactions with Lyme disease spirochetes re-configure metabolic responses/adaptations to the blood meal. Viewed from these contexts, the major outcomes of this study - identification of ISARL as a midgut metabolic regulator and a tick derived ISARL ligand - are groundbreaking. On the other hand, the main conclusions of the paper, though consistent with the data, are less than definitive. The authors can only infer that spirochetes take up phospholipids produced within the tick midgut following ISARL stimulation, and they stop short of showing that C1QL3-ISARL interactions mediate the transcriptional/metabolic changes involving phospholipid biosynthesis attributed to activation of ISARL during an infection blood meal.

   We agree that the studies on glycerol and lipid uptake of Borrelia from the host supports our findings. We have added all the suggestions to the Discussion section of our manuscript.

   Reviewer #2 (Public Review):

   The authors searched for human and murine Adiponectin and Adiponectin receptors homologous sequences in the I. scapularis NCBI database. They found one homologous sequence for Adiponectin receptor 1 and 2, called I. scapularis Adiponectin receptor-like (ISARL) and none for Adiponectin. ISARL showed 71% homology with AdipoR1 and 2 human and murine, 384 amino acids long, and 87% homology with the D. melanogaster ortholog.

   Then the authors, characterized ISARL functionally in the tripartite interaction between vector (I. scapularis, deer tick), mammal (mice) and Lyme disease spirochete (B. burgdorferi, bacteria). They used an elegant paradigm by which they intervened the interaction of B. burgdorferi with its vector I. scapularis by injecting siRNAs or adiponectin in the nymphal tick guts to silence or activate ISARL and other proteins of interest. They observed that the blood meal from mice infected with B. burgdorferi increased the expression of ISARL in the tick guts and by silencing ISARL they were able to reduce the colonization of the bacteria in the tick gut without affecting the feeding habits. The silencing of ISARL, however, did not prevent or reduced the ability of the spirochete to infect mice after being biten by the tick.

   The authors then, screened for genes related to B. burgdorferi on the tick guts by comparing the RNAseq profile of tick guts when fed from uninfected and infected mice and ISARL were silenced. The comparisons shown in figure 3 were clean and follow a logical line of reasoning. On one hand, the comparison between silenced and non-silenced fed blood meal from uninfected mice showed 17 differentially expressed gene, one of those was the silenced ISARL. On the other hand, the comparison between silenced and non-silenced from infected bloodmeal showed 35 differentially expressed genes, from which one was the silenced ISARL. None of the two sets, showed genes in common except from ISARL. The GO analysis showed that several metabolic pathways were modified by B. burgdorferi. From those the authors chose 18 genes that were robustly represented and confirmed their expression using qPCR 17 of them passed the analysis and four of them changed significantly. They chose Phosphatidylserine synthase 1 (PTDSS1) as a paradigm to silence because it is involved in the synthesis of phospholipids (PE and thus PC) and B. burgdorferi lack the machinery to synthesize them. The silencing of PTDSS1 effectively reduced the content of the spirochete in the tick guts without affecting its feeding behavior and moreover, silencing of PSD another enzyme downstream PTDSS1 also involved in PE synthesis induced the same effect. This was an elegant demonstration that the pathway involved downstream ISARL was the phospholipid synthesis pathway.

   Because ISARL resembles AdipoR1 and 2 and bloodmeal may contain its natural ligand adiponectin, the authors investigated the influence of Adiponectin on B. burgdorferi effects on Tick guts. They injected adiponectin or they fed bloodmeal from mice wild type and Adiponectin KO and found in both cases that Adiponectin presence decreases the expression of G6P1 and 2 (2 isomers found in ticks) just as it does in mammalian systems, but the injection of Adiponectin only reduced the expression of two of the three phosphoenolpyruvate carboxykinase PEPCKs found downstream in ticks glycolytic and gluconeogenic pathways (PEPCK2 and 3 but not with 1). On the contrary, Bloodmeal does not reduce any of them. But what it is more important Adiponectin and glucose metabolism does not have any effect in the infectivity or colonization of B. burgdorferi.

   Because ISARL respond to ligands, the authors searched for one in the database in the tick using the C1Q motif of human Adiponectin than interacts with the receptor. They found a match of 181 aa (pre-protein) and 157 aa, the protein mature (without the signal peptide). The proposed ligand, called C1QL3, was increased in expression when the tick was fed bloodmeal of infected mice (as it was ISARL), and when silenced feeding behavior did not changed but the content of B. burgdorferi in the tick gut decreased. They demonstrated in a heterologous system (human HEK cells expressing ISARL) that recombinant C1QL3 interacted with ISARL by immunocytochemistry and pull-down assay.

   After silencing C1QL3, ISARL expression was decreased and the bloodmeal with infected mice lost the ability to increase the receptor expression level but the tick's gut.

   We are grateful for the comments to improve our manuscript. We have carefully considered each point and addressed them all. Please see below for point-to-point response.

   Critique:

   Overall, the authors have done an impeccable job in the demonstration of the pathways involving ISARL in the tripartite interaction of mammalian-insect-bacteria system. However, the medical relevance of the interaction portrayed in the present manuscript, although very interesting from the biological-evolutive, point of view remains to be demonstrated and it is an opportunity that was not taken in the discussion. In the discussion the authors just described the systems in the three species which is usually done in the introduction, instead they repeated all the conclusions drawn in results and minus a couple of paragraphs results a waste of space give such a good scientific work made with the results section. I would suggest the authors to concentrate in areas where their work would be elevated challenging the reader with new ideas. For instance, there is literature about the role of Adiponectin receptors in lipids metabolism and uptake that was not mentioned. ADIPOR1 is expressed in the retina and retinal pigment epithelial cells. Mutant of the Adipor1 gene in these cells results in the inability to take up and retain the essential fatty acid family member docosahexaenoic acid (DHA, 22:6,n-3). Therefore, phospholipids in those cells display a selective shortage in DHA, not in arachidonic acid. In addition, the elongation products 32:6 ,n-3 and 34:6,n-3 are depleted. A consequence is photoreceptor cell death and retinal degeneration( Rice DS, Calandria JM, Gordon WC, et al. Adiponectin receptor 1 conserves docosahexaenoic acid and promotes photoreceptor cell survival. Nat Commun. 2015;6:6228-6242.).

   ADIPOR1 recently has been shown critical for retinal degenerative diseases for axample a single amino acid mutation of Adipor1 occurs in different forms of retinitis pigmentosa. Also polymorphisms of this receptor have been found in age-related macular degeneration (AMD). AdipoR1 has an adiponectin independent role. This was demonstrated by the fact that adiponectin KO do not change DHA and do not result in retinal degeneration. Therefore it seems that is a regulatory switch of DHA uptake, retention, conservation, and elongation in retinal cells, to sustain photoreceptor cell integrity.

   There is also literature regarding PEs and survival pathways involving ceramides, route that was not taken by the authors. It would be specially interesting to analyze this aspect from the point of view of the therapy against Lyme disease. Other aspects would be from the point of view of control of reproduction of B. burgdorferi in tick's population and strategies to control the disease.

   Thank you so much for the suggestion. We have deleted the repetitive paragraphs, such as RNA-seq portion, and added a more meaningful discussion that reflects a combination of the reviewers’ comments.

   Therapeutics for Lyme disease are a complicated topic, as there is both early and late stage disease, and the pathogenesis is likely different. We agree that it is important to consider this issue and have added comments in the revised manuscript (Page 19-20, Line 564-592).

   Reviewer #3 (Public Review):

   Following up on an initial observation that the genome of the black-legged tick encodes an adiponectin-like receptor (ISARL), but lacks an obvious cognate adiponectin homolog, Tang et. al uncover the interesting finding that ISARL is important for colonization of the tick by the Lyme disease agent, Borrelia burgdorferi. Spurred by compelling data that silencing of the ISARL gene significantly attenuates tick acquisition of B. burgdorferi from infected mice, the authors link ISARL production to the differential expression of tick genes involved in metabolism. They show that ISARL mediates regulation of tick phospholipid metabolic pathways and that this phenotype is unique to bloodmeals taken from B. burgdorferi infected mice. Data are presented that support the contention that tick metabolic pathways linked to phosphatidylserine synthase I are critical to spirochete colonization. To investigate potential ligands for ISARL, the authors first examine mammalian adiponectin using knock-out mice. They show that adiponectin regulates glucose metabolism pathways in an ISARL-dependent manner, but has no impact on B. burgdorferi colonization. Instead, a homology search of the Ixodes genome using the C1q globular domain of adiponectin as a query led to the identification of tick C1q-like protein 3 (C1QL3). The authors show that tick C1QL3 regulates ISARL expression and like ISARL is critical for B. burgdorferi colonization. The authors conclude that B. burgdorferi influences tick C1QL3 expression through an undefined mechanism, leading to increased ISARL-mediated signaling effects on metabolic pathways that aid B. burgdorferi colonization of the tick.

   Strengths:

   This is a well written and carefully designed study that lays the foundation for asking many new questions about the complex interplay between the Lyme disease spirochete, its tick vector, and its vertebrate hosts. I agree with the authors that these findings are also likely relevant to other important arthropod-borne pathogens.

   The extensive use of an in vivo system that relies on tick acquisition from the blood of infected mice is a significant strength of the study. By silencing a series of genes in the tick the authors develop a convincing case for the mechanistic relationship of ISARL to B. burgdorferi colonization.

   Thank you for appreciating the significance of this work. We agree with the comments and have addressed all of them. Please see below for point-to-point response.

   Weaknesses:

   1. Potential mechanisms of B. burgdorferi influence on C1QL3 expression are not addressed. While outside the scope of the current work, the manuscript would be improved by some consideration of this issue in the discussion.

   Thank you for your suggestion. We have added this consideration in the revised manuscript (Page 19, Line 559-560).

   1. Adiponectin and C1QL3 are shown to be ISARL ligands that cause differential regulation of tick metabolic pathways. B. burgdorferi infection does not alter adiponectin concentrations in the blood of mice (Fig. 3H). Presumably tick C1QL3 competes with mammalian adiponectin for ISARL-binding, but this is not addressed. Similarly, the homology of murine or human C1QL3 (i.e. CTRP13) is not shown and its potential relevance, along with other C1Q/TNF-related proteins are not discussed in the context of ISARL, but are instead discussed in their known host associated roles only. An improved discussion of how adiponectin, CTRP13, and other C1Q/TNF-related vertebrate proteins may act (or not act) in the tick C1QL3/ISARL pathway should be provided.

   We agree that it is important to discuss if, or how, C1QL3 homologs in mammals may influence tick C1QL3/ISARL pathway. We have added the discussion in the revised manuscript (Page 19, Line 560-562).

   1. The authors conclude that mammalian adiponectin/ISARL-mediated glucose metabolism changes have no impact on B. burgdorferi colonization. However, the authors report a significant difference in engorgement weights between the GFP controls and G6p1/2 knock downs. Furthermore, a majority of the samples evaluated for G6P1 exhibited flaB/actin ratios near zero, indicating low colonization, including for the GFP control. The authors should clarify how these factors potentially influenced the claim that glucose metabolism changes (particularly G6p1) did not cause statistically significant differences in B. burgdorferi acquisition.

   Thank you for this comment. It is interesting that significantly increased engorgement weights were observed after silencing tick G6p genes. We hypothesized that the low glucose level probably makes ticks “hungrier”, which could lead to more feeding. However, this doesn’t result in a greater Borrelia burden in tick gut. We set two segments in the Y-axis and flaB/actin ratios for clarification.

   Read the original source
3. 

   eLife

   Oct 7, 2021

   Evaluation Summary:

   This article identifies a new metabolic pathway in ticks that Borrelia burgdorferi, the agent of Lyme disease, requires for survival. The authors show that the adiponectin receptor (ISARL) is upregulated after a blood meal and find that the tick complement C1q-like protein (C1QL3) is an ISARL ligand whose knockdown impairs spirochete colonization.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

   Read the original source
4. 

   eLife

   Oct 7, 2021

   Reviewer #1 (Public Review):

   In this manuscript, the authors present evidence that mouse blood meals containing Lyme disease spirochetes induce upregulation and activation of an adiponectin receptor (ISARL) in the midguts of Ixodes scapularis ticks. Activation of the receptor initiates transcriptional changes, not seen with blood meals from uninfected mice, that give rise to metabolic alterations in the midguts required for replication of spirochetes. Using RNA-seq, they trace these critical alterations to genes encoding enzymes for synthesis of phospholipids. Although mouse adiponectin induced transcriptional changes in engorged tick midguts related to glucose and energy metabolism, it did not influence B. burgdorferi colonization. The authors conclude by presenting evidence that tick complement C1q-like protein (C1QL3), also …

   Reviewer #1 (Public Review):

   In this manuscript, the authors present evidence that mouse blood meals containing Lyme disease spirochetes induce upregulation and activation of an adiponectin receptor (ISARL) in the midguts of Ixodes scapularis ticks. Activation of the receptor initiates transcriptional changes, not seen with blood meals from uninfected mice, that give rise to metabolic alterations in the midguts required for replication of spirochetes. Using RNA-seq, they trace these critical alterations to genes encoding enzymes for synthesis of phospholipids. Although mouse adiponectin induced transcriptional changes in engorged tick midguts related to glucose and energy metabolism, it did not influence B. burgdorferi colonization. The authors conclude by presenting evidence that tick complement C1q-like protein (C1QL3), also upregulated in response to a blood meal containing spirochetes, is an ISARL ligand and that knockdown of C1QL3 impairs spirochete colonization.

   This work extends our understanding of the complex and intimate physiologic interactions between Borrelia burgdorferi and Ixodes ticks that sustain the spirochete's enzootic cycle. It builds upon prior work by others showing that feeding ticks provide spirochetes with glycerol, an alternative carbohydrate energy source and essential building block for phospholipid biosynthesis. It also appears relevant to previously published work showing that B. burgdorferi can extract lipids from the membranes of eukaryotic cells to which they are attached.

   The strength of the study is that it uses state-of-the-art genetic, bioinformatic, and transcriptional approaches to garner novel insights into the unique transcriptional/metabolic changes that occur in ticks when they ingest blood from B. burgdorferi¬-infected mice. It enhances the now well-established, but still far from well understood, viewpoint that ticks are not mere biologic syringes for injection of spirochetes. And it demonstrates, probably more than any preceding study, the extent to which tick midgut interactions with Lyme disease spirochetes re-configure metabolic responses/adaptations to the blood meal. Viewed from these contexts, the major outcomes of this study - identification of ISARL as a midgut metabolic regulator and a tick derived ISARL ligand - are groundbreaking. On the other hand, the main conclusions of the paper, though consistent with the data, are less than definitive. The authors can only infer that spirochetes take up phospholipids produced within the tick midgut following ISARL stimulation, and they stop short of showing that C1QL3-ISARL interactions mediate the transcriptional/metabolic changes involving phospholipid biosynthesis attributed to activation of ISARL during an infection blood meal.

   Read the original source
5. 

   eLife

   Oct 7, 2021

   Reviewer #2 (Public Review):

   The authors searched for human and murine Adiponectin and Adiponectin receptors homologous sequences in the I. scapularis NCBI database. They found one homologous sequence for Adiponectin receptor 1 and 2, called I. scapularis Adiponectin receptor-like (ISARL) and none for Adiponectin. ISARL showed 71% homology with AdipoR1 and 2 human and murine, 384 amino acids long, and 87% homology with the D. melanogaster ortholog.

   Then the authors, characterized ISARL functionally in the tripartite interaction between vector (I. scapularis, deer tick), mammal (mice) and Lyme disease spirochete (B. burgdorferi, bacteria). They used an elegant paradigm by which they intervened the interaction of B. burgdorferi with its vector I. scapularis by injecting siRNAs or adiponectin in the nymphal tick guts to silence or activate …

   Reviewer #2 (Public Review):

   The authors searched for human and murine Adiponectin and Adiponectin receptors homologous sequences in the I. scapularis NCBI database. They found one homologous sequence for Adiponectin receptor 1 and 2, called I. scapularis Adiponectin receptor-like (ISARL) and none for Adiponectin. ISARL showed 71% homology with AdipoR1 and 2 human and murine, 384 amino acids long, and 87% homology with the D. melanogaster ortholog.

   Then the authors, characterized ISARL functionally in the tripartite interaction between vector (I. scapularis, deer tick), mammal (mice) and Lyme disease spirochete (B. burgdorferi, bacteria). They used an elegant paradigm by which they intervened the interaction of B. burgdorferi with its vector I. scapularis by injecting siRNAs or adiponectin in the nymphal tick guts to silence or activate ISARL and other proteins of interest. They observed that the blood meal from mice infected with B. burgdorferi increased the expression of ISARL in the tick guts and by silencing ISARL they were able to reduce the colonization of the bacteria in the tick gut without affecting the feeding habits. The silencing of ISARL, however, did not prevent or reduced the ability of the spirochete to infect mice after being biten by the tick.

   The authors then, screened for genes related to B. burgdorferi on the tick guts by comparing the RNAseq profile of tick guts when fed from uninfected and infected mice and ISARL were silenced. The comparisons shown in figure 3 were clean and follow a logical line of reasoning. On one hand, the comparison between silenced and non-silenced fed blood meal from uninfected mice showed 17 differentially expressed gene, one of those was the silenced ISARL. On the other hand, the comparison between silenced and non-silenced from infected bloodmeal showed 35 differentially expressed genes, from which one was the silenced ISARL. None of the two sets, showed genes in common except from ISARL. The GO analysis showed that several metabolic pathways were modified by B. burgdorferi. From those the authors chose 18 genes that were robustly represented and confirmed their expression using qPCR 17 of them passed the analysis and four of them changed significantly. They chose Phosphatidylserine synthase 1 (PTDSS1) as a paradigm to silence because it is involved in the synthesis of phospholipids (PE and thus PC) and B. burgdorferi lack the machinery to synthesize them. The silencing of PTDSS1 effectively reduced the content of the spirochete in the tick guts without affecting its feeding behavior and moreover, silencing of PSD another enzyme downstream PTDSS1 also involved in PE synthesis induced the same effect. This was an elegant demonstration that the pathway involved downstream ISARL was the phospholipid synthesis pathway.

   Because ISARL resembles AdipoR1 and 2 and bloodmeal may contain its natural ligand adiponectin, the authors investigated the influence of Adiponectin on B. burgdorferi effects on Tick guts. They injected adiponectin or they fed bloodmeal from mice wild type and Adiponectin KO and found in both cases that Adiponectin presence decreases the expression of G6P1 and 2 (2 isomers found in ticks) just as it does in mammalian systems, but the injection of Adiponectin only reduced the expression of two of the three phosphoenolpyruvate carboxykinase PEPCKs found downstream in ticks glycolytic and gluconeogenic pathways (PEPCK2 and 3 but not with 1). On the contrary, Bloodmeal does not reduce any of them. But what it is more important Adiponectin and glucose metabolism does not have any effect in the infectivity or colonization of B. burgdorferi.

   Because ISARL respond to ligands, the authors searched for one in the database in the tick using the C1Q motif of human Adiponectin than interacts with the receptor. They found a match of 181 aa (pre-protein) and 157 aa, the protein mature (without the signal peptide). The proposed ligand, called C1QL3, was increased in expression when the tick was fed bloodmeal of infected mice (as it was ISARL), and when silenced feeding behavior did not changed but the content of B. burgdorferi in the tick gut decreased. They demonstrated in a heterologous system (human HEK cells expressing ISARL) that recombinant C1QL3 interacted with ISARL by immunocytochemistry and pull-down assay.

   After silencing C1QL3, ISARL expression was decreased and the bloodmeal with infected mice lost the ability to increase the receptor expression level but the tick's gut.

   Critique:

   Overall, the authors have done an impeccable job in the demonstration of the pathways involving ISARL in the tripartite interaction of mammalian-insect-bacteria system. However, the medical relevance of the interaction portrayed in the present manuscript, although very interesting from the biological-evolutive, point of view remains to be demonstrated and it is an opportunity that was not taken in the discussion. In the discussion the authors just described the systems in the three species which is usually done in the introduction, instead they repeated all the conclusions drawn in results and minus a couple of paragraphs results a waste of space give such a good scientific work made with the results section. I would suggest the authors to concentrate in areas where their work would be elevated challenging the reader with new ideas. For instance, there is literature about the role of Adiponectin receptors in lipids metabolism and uptake that was not mentioned. ADIPOR1 is expressed in the retina and retinal pigment epithelial cells. Mutant of the Adipor1 gene in these cells results in the inability to take up and retain the essential fatty acid family member docosahexaenoic acid (DHA, 22:6,n-3). Therefore, phospholipids in those cells display a selective shortage in DHA, not in arachidonic acid. In addition, the elongation products 32:6 ,n-3 and 34:6,n-3 are depleted. A consequence is photoreceptor cell death and retinal degeneration( Rice DS, Calandria JM, Gordon WC, et al. Adiponectin receptor 1 conserves docosahexaenoic acid and promotes photoreceptor cell survival. Nat Commun. 2015;6:6228-6242.).

   ADIPOR1 recently has been shown critical for retinal degenerative diseases for axample a single amino acid mutation of Adipor1 occurs in different forms of retinitis pigmentosa. Also polymorphisms of this receptor have been found in age-related macular degeneration (AMD). AdipoR1 has an adiponectin independent role. This was demonstrated by the fact that adiponectin KO do not change DHA and do not result in retinal degeneration. Therefore it seems that is a regulatory switch of DHA uptake, retention, conservation, and elongation in retinal cells, to sustain photoreceptor cell integrity.

   There is also literature regarding PEs and survival pathways involving ceramides, route that was not taken by the authors. It would be specially interesting to analyze this aspect from the point of view of the therapy against Lyme disease. Other aspects would be from the point of view of control of reproduction of B. burgdorferi in tick's population and strategies to control the disease.

   Read the original source
6. 

   eLife

   Oct 7, 2021

   Reviewer #3 (Public Review):

   Following up on an initial observation that the genome of the black-legged tick encodes an adiponectin-like receptor (ISARL), but lacks an obvious cognate adiponectin homolog, Tang et. al uncover the interesting finding that ISARL is important for colonization of the tick by the Lyme disease agent, Borrelia burgdorferi. Spurred by compelling data that silencing of the ISARL gene significantly attenuates tick acquisition of B. burgdorferi from infected mice, the authors link ISARL production to the differential expression of tick genes involved in metabolism. They show that ISARL mediates regulation of tick phospholipid metabolic pathways and that this phenotype is unique to bloodmeals taken from B. burgdorferi infected mice. Data are presented that support the contention that tick metabolic pathways linked …

   Reviewer #3 (Public Review):

   Following up on an initial observation that the genome of the black-legged tick encodes an adiponectin-like receptor (ISARL), but lacks an obvious cognate adiponectin homolog, Tang et. al uncover the interesting finding that ISARL is important for colonization of the tick by the Lyme disease agent, Borrelia burgdorferi. Spurred by compelling data that silencing of the ISARL gene significantly attenuates tick acquisition of B. burgdorferi from infected mice, the authors link ISARL production to the differential expression of tick genes involved in metabolism. They show that ISARL mediates regulation of tick phospholipid metabolic pathways and that this phenotype is unique to bloodmeals taken from B. burgdorferi infected mice. Data are presented that support the contention that tick metabolic pathways linked to phosphatidylserine synthase I are critical to spirochete colonization. To investigate potential ligands for ISARL, the authors first examine mammalian adiponectin using knock-out mice. They show that adiponectin regulates glucose metabolism pathways in an ISARL-dependent manner, but has no impact on B. burgdorferi colonization. Instead, a homology search of the Ixodes genome using the C1q globular domain of adiponectin as a query led to the identification of tick C1q-like protein 3 (C1QL3). The authors show that tick C1QL3 regulates ISARL expression and like ISARL is critical for B. burgdorferi colonization. The authors conclude that B. burgdorferi influences tick C1QL3 expression through an undefined mechanism, leading to increased ISARL-mediated signaling effects on metabolic pathways that aid B. burgdorferi colonization of the tick.

   Strengths:

   This is a well written and carefully designed study that lays the foundation for asking many new questions about the complex interplay between the Lyme disease spirochete, its tick vector, and its vertebrate hosts. I agree with the authors that these findings are also likely relevant to other important arthropod-borne pathogens.

   The extensive use of an in vivo system that relies on tick acquisition from the blood of infected mice is a significant strength of the study. By silencing a series of genes in the tick the authors develop a convincing case for the mechanistic relationship of ISARL to B. burgdorferi colonization.

   Weaknesses:

   1. Potential mechanisms of B. burgdorferi influence on C1QL3 expression are not addressed. While outside the scope of the current work, the manuscript would be improved by some consideration of this issue in the discussion.

   2. Adiponectin and C1QL3 are shown to be ISARL ligands that cause differential regulation of tick metabolic pathways. B. burgdorferi infection does not alter adiponectin concentrations in the blood of mice (Fig. 3H). Presumably tick C1QL3 competes with mammalian adiponectin for ISARL-binding, but this is not addressed. Similarly, the homology of murine or human C1QL3 (i.e. CTRP13) is not shown and its potential relevance, along with other C1Q/TNF-related proteins are not discussed in the context of ISARL, but are instead discussed in their known host associated roles only. An improved discussion of how adiponectin, CTRP13, and other C1Q/TNF-related vertebrate proteins may act (or not act) in the tick C1QL3/ISARL pathway should be provided.

   3. The authors conclude that mammalian adiponectin/ISARL-mediated glucose metabolism changes have no impact on B. burgdorferi colonization. However, the authors report a significant difference in engorgement weights between the GFP controls and G6p1/2 knock downs. Furthermore, a majority of the samples evaluated for G6P1 exhibited flaB/actin ratios near zero, indicating low colonization, including for the GFP control. The authors should clarify how these factors potentially influenced the claim that glucose metabolism changes (particularly G6p1) did not cause statistically significant differences in B. burgdorferi acquisition.

   Read the original source
7. 

   Version published to 10.1101/2021.09.13.460039v1 on bioRxiv

   Sep 15, 2021