Glycan-based shaping of the microbiota during primate evolution
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Evaluation Summary:
30 million years ago the ancestors of Old World primates lost the ability to produce alpha-gal due to the fixation of several loss-of-function mutations in the GGTA1 gene. The evolutionary advantage of such loss remains elusive. Here, the authors provide additional insights into the pleiotropic role of ggta1 in shaping the gut microbiota, immune function, susceptibility to sepsis, and eventual fitness advantage.
(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 #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Genes encoding glycosyltransferases can be under relatively high selection pressure, likely due to the involvement of the glycans synthesized in host-microbe interactions. Here, we used mice as an experimental model system to investigate whether loss of α−1,3-galactosyltransferase gene ( GGTA1 ) function and Galα1-3Galβ1-4GlcNAcβ1-R (αGal) glycan expression affects host-microbiota interactions, as might have occurred during primate evolution. We found that Ggta1 deletion shaped the composition of the gut microbiota. This occurred via an immunoglobulin (Ig)-dependent mechanism, associated with targeting of αGal-expressing bacteria by IgA. Systemic infection with an Ig-shaped microbiota inoculum elicited a less severe form of sepsis compared to infection with non-Ig-shaped microbiota. This suggests that in the absence of host αGal, antibodies can shape the microbiota towards lower pathogenicity. Given the fitness cost imposed by bacterial sepsis, we infer that the observed reduction in microbiota pathogenicity upon Ggta1 deletion in mice may have contributed to increase the frequency of GGTA1 loss-of-function mutations in ancestral primates that gave rise to humans.
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Author Response:
Reviewer #3 (Public Review):
About 30 million years ago the ancestors of Old World primates lost the ability to produce the glycan a-gal due to the fixation of several loss-of-function mutations in the GGTA1 gene. The evolutionary advantage of such loss remains elusive. The current study builds upon previous work by the authors showing (i) that the presence of a-gal expressing bacteria in ggta1 deficient mice led to production of antibodies capable of clearance of malaria-causing plasmodia carrying a-gal (Yilmaz et al., 2014), and (ii) that ggta1 deficiency is associated with increased resistance to sepsis via the enhancement of IgG effector function (Sigh et al., 2021). Here they expand on these findings to show that ggta1 deletion in mice is associated with altered composition of the gut microbiome due to the …
Author Response:
Reviewer #3 (Public Review):
About 30 million years ago the ancestors of Old World primates lost the ability to produce the glycan a-gal due to the fixation of several loss-of-function mutations in the GGTA1 gene. The evolutionary advantage of such loss remains elusive. The current study builds upon previous work by the authors showing (i) that the presence of a-gal expressing bacteria in ggta1 deficient mice led to production of antibodies capable of clearance of malaria-causing plasmodia carrying a-gal (Yilmaz et al., 2014), and (ii) that ggta1 deficiency is associated with increased resistance to sepsis via the enhancement of IgG effector function (Sigh et al., 2021). Here they expand on these findings to show that ggta1 deletion in mice is associated with altered composition of the gut microbiome due to the action of IgA targeting of a-Gal expressing bacteria. In addition, they show that the absence of a-gal results in a microbiome that is less pathogenic (i.e., less likely to induce sepsis in their experimental model). Although some aspects of the work are not very novel (e.g., the fact that ggta1 is associated with a remodeled microbiome had already been shown in their previous publications) the work does provide additional insights into the pleiotropic role of ggta1 in immune function, susceptibility to sepsis, and eventual fitness advantage. The work is extremely well done and all conclusions are supported by solid data. Indeed, I felt that the authors were reading my mind every step of the way. Each time I questioned one of the conclusions the next paragraph would address that exact concern. There are, however, a few points that I think would deserve additional clarification.
1 - I was a little surprised that they found no difference in the microbiome of F2 mice between a-gal deficient and wild-type mice. Although I understand that this might be due to antibodies received by the mom, the fact that the divergence in only seen in F3 to F5 would also be compatible with drift and not necessarily a genotype-driven phenotype. Are the microbiome differences detected in F3-F5 overlapping to those observed at F0? If the original differences were controlled by host genetics - the hypothesis being tested - we would expect to see some convergent (at least at the level of specific taxa)
We agree essentially with the comment: “… would also be compatible with drift and not necessarily a genotype-driven phenotype” and have addressed this issue by adding the following statement in the Discussion section:
“On the basis of this observation alone (Figure 1), one cannot exclude the observed divergence in the microbiota bacterial population frequencies of wild type vs. Ggta1-deleted mice (Figure 1) from being a stochastic event. However, the observation that these changes occur via an Ig-dependent mechanism that differs in wild type vs. Ggta1- deleted mice (Figure 3) does support that loss of αGal contributes critically to shape the microbiota composition of Ggta1- deficient mice.”
We have previously shown that homogenization of the microbiota occurs between the littermates in the F2 generation (Singh et al., 2021). Having confirmed this finding in this manuscript (Figure 1C, Figure 3-figure supplement 7A-B), we find that the effect of the genotype and Ig is seen only from the F3 generation onwards (Figure 1D-F, Figure 3). Presumably, the inability of F1 Ggta1+/- mothers to produce anti-αGal antibodies accounts for the absence of overt shaping of the F2 microbiota. In these experiments, anti-αGal antibodies can only be generated from αGal-deficient F2 Ggta1-/- mice, being vertically transferred and shaping the microbiota from F3 Ggta1-/- mice onwards. We propose that the differences in the microbiota composition of the two F3 genotypes onwards are driven by a cumulative effect of maternal anti-αGal antibodies over the offspring microbiota composition.
2 - I was really surprised that ggta1 deficient mice lacking a functional adaptive immune system (Figure S8) were equally resistant to systemic infection with the cecal inoculum isolated from ggta1 deficient mice. In the previous work they show that the increases resistance to sepsis comes from increases effector function of IgG. If that is the case, how come mice not having an adaptive system (hence no IgG) are equally protected? Is the pathogenicity of the microbiome of ggta1 deficient mice that reduced? It seems unlikely. More generally, I would like to have seen a better discussion about how these new findings connect to their past work. In the context of increased resistance to sepsis what seems to be more important - the remodeling of the microbiome by IgA or the increased effector function of IgG?
The data reported in our manuscript does indeed support the conclusion that shaping of the microbiota composition of Ggta1-deficient mice is associated with an overall reduction of the microbiome pathogenicity. This finding is in keeping with host-microbe commensal interactions not being hard- wired but instead oscillating from pathogenic to symbiotic (Ayres, 2016; Vonaesch et al., 2018). Our findings suggest that the loss of Ggta1 function can modify the nature of host-microbiota interactions, through a mechanism whereby the absence of host αGal and the emergence of antibodies targeting this glycan in microbes, shapes and reduces the microbiome pathogenicity.
We have shown that loss of αGal can enhance resistance to bacterial sepsis via a mechanism that increases IgG effector function (Singh et al., 2021). This was demonstrated by systemically infecting Ggta1-deficient mice with a “non-shaped” microbiota inoculum, isolated from Ggta1-deficient mice lacking adaptive immunity (Rag2-/-Ggta1-/- mice). As discussed in the manuscript “the gut microbiota of Rag2-/-Ggta1-/- mice, lacking adaptive immunity, is highly enriched in pathobionts such as Proteobacteria, including Helicobacter (Singh et al., 2021)”. Under these experimental conditions, resistance to infection is IgG dependent, explaining why modulation of IgG effector function by αGal impacts on the outcome of sepsis.
In the current manuscript we describe another survival advantage against bacterial sepsis associated with Ggta1 deletion in mice. Namely, antibodies generated by Ggta1-deficient mice can shape and reduce the microbiota pathogenicity. This was demonstrated by infecting systemically Ggta1-deficient mice lacking adaptive immunity (Rag2-/-Ggta1-/- mice) with a “shaped- microbiota” inoculum isolated from Ggta1-deficient mice. While the mechanism underlying microbiota shaping is antibody-dependent, the effector mechanism conferring resistance against the shaped microbiota acts irrespectively of adaptive immunity, including IgG. This conclusion is supported by the observation that systemic infection by the shaped microbiota (isolated from Ggta1-deficient mice) failed to induce sepsis in Rag2-/-Ggta1-/- mice, which was not the case upon systemic infection with a non-shaped microbiota (isolated from Rag2-/-Ggta1-/- mice). We conclude that Ggta1 deletion in mice increases resistance to bacterial sepsis via two interrelated antibody-dependent mechanisms: i) Increased IgG effector function (Singh et al., 2021) and ii) Antibody shaping and reduction of microbiota pathogenicity (current manuscript). To what extent these two traits are related remains to be established.
It is possible that similarly to what was demonstrated for IgG (Singh et al., 2021), the absence of αGal from glycan structures in other Ig isotypes, including IgA, might modify their effector function. We do not yet know if this is the case, as in our manuscript, what we find is an altered antibody response targeting immunogenic bacteria in the microbiota of Ggta1-deficient mice. This is associated with modulation of the microbiota bacterial composition, i.e. antibody shaping of the microbiota, and with a reduction of the microbiome pathogenicity. The latter explains why the Ggta1-deficient mice do not rely on circulating antibodies to prevent the development of sepsis upon systemic infection by bacteria emanating for their own “shaped” microbiota.
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Reviewer #3 (Public Review):
About 30 million years ago the ancestors of Old World primates lost the ability to produce the glycan a-gal due to the fixation of several loss-of-function mutations in the GGTA1 gene. The evolutionary advantage of such loss remains elusive. The current study builds upon previous work by the authors showing (i) that the presence of a-gal expressing bacteria in ggta1 deficient mice led to production of antibodies capable of clearance of malaria-causing plasmodia carrying a-gal (Yilmaz et al., 2014), and (ii) that ggta1 deficiency is associated with increased resistance to sepsis via the enhancement of IgG effector function (Sigh et al., 2021). Here they expand on these findings to show that ggta1 deletion in mice is associated with altered composition of the gut microbiome due to the action of IgA targeting …
Reviewer #3 (Public Review):
About 30 million years ago the ancestors of Old World primates lost the ability to produce the glycan a-gal due to the fixation of several loss-of-function mutations in the GGTA1 gene. The evolutionary advantage of such loss remains elusive. The current study builds upon previous work by the authors showing (i) that the presence of a-gal expressing bacteria in ggta1 deficient mice led to production of antibodies capable of clearance of malaria-causing plasmodia carrying a-gal (Yilmaz et al., 2014), and (ii) that ggta1 deficiency is associated with increased resistance to sepsis via the enhancement of IgG effector function (Sigh et al., 2021). Here they expand on these findings to show that ggta1 deletion in mice is associated with altered composition of the gut microbiome due to the action of IgA targeting of a-Gal expressing bacteria. In addition, they show that the absence of a-gal results in a microbiome that is less pathogenic (i.e., less likely to induce sepsis in their experimental model). Although some aspects of the work are not very novel (e.g., the fact that ggta1 is associated with a remodeled microbiome had already been shown in their previous publications) the work does provide additional insights into the pleiotropic role of ggta1 in immune function, susceptibility to sepsis, and eventual fitness advantage. The work is extremely well done and all conclusions are supported by solid data. Indeed, I felt that the authors were reading my mind every step of the way. Each time I questioned one of the conclusions the next paragraph would address that exact concern. There are, however, a few points that I think would deserve additional clarification.
1 - I was a little surprised that they found no difference in the microbiome of F2 mice between a-gal deficient and wild-type mice. Although I understand that this might be due to antibodies received by the mom, the fact that the divergence in only seen in F3 to F5 would also be compatible with drift and not necessarily a genotype-driven phenotype. Are the microbiome differences detected in F3-F5 overlapping to those observed at F0? If the original differences were controlled by host genetics - the hypothesis being tested - we would expect to see some convergent (at least at the level of specific taxa)
2 - I was really surprised that ggta1 deficient mice lacking a functional adaptive immune system (Figure S8) were equally resistant to systemic infection with the cecal inoculum isolated from ggta1 deficient mice. In the previous work they show that the increases resistance to sepsis comes from increases effector function of IgG. If that is the case, how come mice not having an adaptive system (hence no IgG) are equally protected? Is the pathogenicity of the microbiome of ggta1 deficient mice that reduced? It seems unlikely. More generally, I would like to have seen a better discussion about how these new findings connect to their past work. In the context of increased resistance to sepsis what seems to be more important - the remodeling of the microbiome by IgA or the increased effector function of IgG?
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Reviewer #2 (Public Review):
The authors aimed to examine the impact of GGTA1 deletion on host-microbial interactions using a mouse model of a primate-specific mutation. This is a very informative model system that provided interesting insights into the consequences of aGal elimination from host glycoproteins, with subsequent 'release' of immune tolerance breaks and generation of antibody responses agains bacterial aGal epitopes.
The study is well executed and and the conclusions are well supported by the provided evidence. The findings are interesting for a broad audience of biologists.
The identity of IgA targeted bacteria in GGTA1 vs WT mice would be interesting to investigate in the future studies.
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Reviewer #1 (Public Review):
This work is a powerful example of thinking across silos. It combines much knowledge of innate and adaptive immunity, with primate evolution of certain antigens lost only in certain primate lineages and tests an important idea about host-mediated, antibody dependent shaping of gut microbiota using laboratory mice with different engineered genetic alterations. Gut microbiota are all the rage these days, but is often forgotten that these microbial communities represent formidable danger that is really too close (one epithelial layer away) for comfort. The authors demonstrate in laboratory mice, how antibodies against non-self sugar molecules present on bacteria can shape the microbiome. Claims and conclusions seem justified by the data presented.
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Evaluation Summary:
30 million years ago the ancestors of Old World primates lost the ability to produce alpha-gal due to the fixation of several loss-of-function mutations in the GGTA1 gene. The evolutionary advantage of such loss remains elusive. Here, the authors provide additional insights into the pleiotropic role of ggta1 in shaping the gut microbiota, immune function, susceptibility to sepsis, and eventual fitness advantage.
(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 #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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