Intestinal lysozyme1 deficiency alters microbiota composition and impacts host metabolism through the emergence of NAD+-secreting ASTB Qing110 bacteria

ABSTRACT The intestine plays a pivotal role in nutrient absorption and host defense against pathogens, orchestrated in part by antimicrobial peptides secreted by Paneth cells. Among these peptides, lysozyme has multifaceted functions beyond its bactericidal activity. Here, we uncover the intricate relationship between intestinal lysozyme, the gut microbiota, and host metabolism. Lysozyme deficiency in mice led to altered body weight, energy expenditure, and substrate utilization, particularly on a high-fat diet. Interestingly, these metabolic benefits were linked to changes in the gut microbiota composition. Cohousing experiments revealed that the metabolic effects of lysozyme deficiency were microbiota-dependent. 16S rDNA sequencing highlighted differences in microbial communities, with ASTB_g (OTU60) highly enriched in lysozyme knockout mice. Subsequently, a novel bacterium, ASTB Qing110, corresponding to ASTB_g (OTU60), was isolated. Metabolomic analysis revealed that ASTB Qing110 secreted high levels of NAD+, potentially influencing host metabolism. This study sheds light on the complex interplay between intestinal lysozyme, the gut microbiota, and host metabolism, uncovering the potential role of ASTB Qing110 as a key player in modulating metabolic outcomes. IMPORTANCE The impact of intestinal lumen lysozyme on intestinal health is complex, arising from its multifaceted interactions with the gut microbiota. Lysozyme can both mitigate and worsen certain health conditions, varying with different scenarios. This underscores the necessity of identifying the specific bacterial responses elicited by lysozyme and understanding their molecular foundations. Our research reveals that a deficiency in intestinal lysozyme1 may offer protection against diet-induced obesity by altering bacterial populations. We discovered a strain of bacterium, ASTB Qing110, which secretes NAD+ and is predominantly found in lyz1-deficient mice. Qing110 demonstrates positive effects in both C. elegans and mouse models of ataxia telangiectasia. This study sheds light on the intricate role of lysozyme in influencing intestinal health.

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Reviewer #1 (Comments for the Author): The manuscript by Zhang et al described the microbiota features associated with the metabolic alterations in lysozyme deficiency and identified a previously unknown gut bacteria strain, ASTB Qing110, enriched in the microbiota of lysozymedeficient mice.The authors also characterized the genomic and metabolic features of ASTB Qing110 and found that ASTB Qing110 was capable of producing NAD+.The authors then showed evidence that ASTB Qing110 could ameliorate disease conditions correlating with NAD+ production by the bacteria.The work identifies a novel metabolically-important gut microbe and provides key insights into its physiological function.However, the reviewer thinks there are a few questions to be addressed.
Major concerns: 1) Do NAD+ levels differ in the intestine or feces between WT and Lyz1-/-mice?2) How does the abundance of ASTB Qing110 change in response to HFD feeding? 3) In mice and worms treated with ASTB Qing110, what is the abundance of ASTB Qing110 achieved by the treatment?Does ASTB Qing110 treatment impact other components of the gut microbiota?
Minor concern: 1) As the Lyz1-/-mouse model used is a whole-body knockout, it is not appropriate to state "Intestinal Lysozyme Deficiency" in the title or the author should provide evidence that other body parts are not impacted.
2) The authors start with characterizing metabolism and HFD-induced obesity in Lyz1-/-mice and identify ASTB Qing110 as a significantly enriched gut microbe.I would suggest including the role of NAD+ in obesity and metabolic disorders in the discussion.

Response Letter
We'd like to express our gratitude to the reviewer for their comments and suggestions.We have carried out substantial new experimentation addressing all of the questions and concerns.These additional data have strengthened and clarified the revised manuscript.We have also compiled a point-by-point response to the reviewer.The corresponding changes are in yellow in main text.
Comments for the Author: The manuscript by Zhang et al described the microbiota features associated with the metabolic alterations in lysozyme deficiency and identified a previously unknown gut bacteria strain, ASTB Qing110, enriched in the microbiota of lysozyme-deficient mice.The authors also characterized the genomic and metabolic features of ASTB Qing110 and found that ASTB Qing110 was capable of producing NAD + .The authors then showed evidence that ASTB Qing110 could ameliorate disease conditions correlating with NAD + production by the bacteria.The work identifies a novel metabolically-important gut microbe and provides key insights into its physiological function.However, the reviewer thinks there are a few questions to be addressed.
Response: We thank the Reviewer's kind comments on our manuscript and appreciate the helpful suggestions.3) In mice and worms treated with ASTB Qing110, what is the abundance of ASTB Qing110 achieved by the treatment?Does ASTB Qing110 treatment impact other components of the gut microbiota?Response: We are grateful for the Reviewer's feedback.We assessed the relative abundance of Qing110 in WT mice treated with Qing110.The results showed the average relative abundance of Qing110 in Qing110-treated mice was 1.8% (RL-Fig.3a).To explore Qing110's impact on the gut microbiota's other components, we initially administered antibiotics (ABX) orally to WT mice for one week.This was followed by a three-month daily oral administration of Qing110.Post-treatment, we analyzed the microbial communities in both vehicle and Qing110-treated mice using 16S ribosomal RNA gene sequencing.A Principal Component Analysis (PCA) based on Bray-Curtis distances highlighted distinct taxonomic composition differences between the control and Qing110-treated mice (RL-Fig.3b).In a detailed comparison of the relative abundance, significant differences were identified for Erysipelotrichaceae which family Qing110 belongs to.Other families with lower abundance also have significant differences, such as UCG-010, Atopobiaceae, Peptococcaceae, Saccharimonadaceae (RL-Fig.3c-d (Zhang et al., Cell Res, 2019).
2) The authors start with characterizing metabolism and HFD-induced obesity in Lyz1 −/− mice and identify ASTB Qing110 as a significantly enriched gut microbe.I would suggest including the role of NAD + in obesity and metabolic disorders in the discussion.(Stromsdorfer et al., Cell Rep, 2016).These studies collectively provide compelling evidence that targeting NAD + metabolism can be an effective strategy against metabolic diseases.We have included the information in our revised manuscript (Discussion).
Major concerns: 1) Do NAD + levels differ in the intestine or feces between WT and Lyz1 −/− mice?Response: We express our gratitude to the reviewer for their insightful comment.In response, we conducted an evaluation of the NAD + content in the intestines of both WT and Lyz1 −/− mice.Our findings revealed that Lyz1 −/− mice exhibited elevated levels of NAD + level in the small intestinal epithelium compared to their WT counterparts, as depicted in RL-Fig.1a.Furthermore, upon administering a high-fat diet, Lyz1 −/− mice continued to demonstrate higher NAD + levels in the small intestinal epithelium relative to WT mice, as shown in RL-Fig.1b.This additional data has been duly incorporated into our revised manuscript, specifically in Fig S6.RL-Fig.1 NAD + levels in the small intestinal epithelium in WT and Lyz1 −/− mice on SFD(a) and HFD(b).The NAD + content was quantified using liquid chromatography-mass spectrometry (LC-MS).Mean and s.e.m. are plotted, with each dot representing one individual animal.2) How does the abundance of ASTB Qing110 change in response to HFD feeding?Response: We appreciate the Reviewer for the comment.Qing110 plays a pivotal role in the metabolic advantages seen in Lyz1 −/− mice.We assessed the abundance of ASTB Qing110 in Lyz1 −/− mice during a high-fat diet regimen.Our findings reveal a significant increase in Qing110 levels on day 14, with no notable changes on days 7 and 21.This suggests that Qing110's abundance remains stable throughout high-fat feeding, as depicted in RL-Fig.2a.Further exploring the impact of a high-fat diet (HFD) on Qing110 colonization, we orally administered Qing110 to wild-type (WT) mice on an HFD.The results showed that the average abundance of Qing110 was 4.065% (RL-Fig.2b),indicating that HFD does not impede Qing110 colonization.These results have been incorporated into our revised manuscript (see Fig 3).RL-Fig.2The abundance of ASTB Qing110 in response to HFD feeding.(a) Real-time PCR analysis of Qing110 in fecal samples from Lyz1 −/− mice on SFD and HFD.The relative abundance of Qing110 was quantified by normalizing to total bacteria, and the relative abundance of Qing110 on HFD was normalized to the relative abundance of Qing110 on SFD.(b) Real-time PCR analysis of Qing110 in fecal samples from WT mice treated with PBS or Qing110 for 1 month.The relative abundance of Qing110 was quantified by normalizing to total bacteria.Mean and s.e.m. are plotted, with each dot representing one individual animal.
).Overall, oral administration of Qing110 significantly altered the composition of gut microbiota in mice, with Erysipelotrichaceae family being the most significantly increased.We have included the information in our revised manuscript (Fig S3).RL-Fig.3The effect of oral administration of Qing110 on the gut microbiota of mice.(a) Real-time PCR analysis of Qing110 in fecal samples from vehicle or Qing110 treated mice.The relative abundance of Qing110 was quantified by normalizing to total bacteria.(b) PCA analysis of fecal microbiota based on the relative abundance of bacterial OTUs.(c) Relative abundance on family level in the feces of control and Qing110-treated WT mice.(d) Relative abundance and statistical description of different families.Mean and s.e.m. are plotted, with each dot representing one individual animal.Minor concern: 1) As the Lyz1 −/− mouse model used is a whole-body knockout, it is not appropriate to state " Intestinal Lysozyme Deficiency " in the title or the author should provide evidence that other body parts are not impacted.Response: We are thankful for the Reviewer's comment.In the mouse genome, there are two lysozyme genes: lysozyme M (lysozyme 2) and lysozyme P (lysozyme 1).Lysozyme M is expressed in myeloid cells, while Lyz1, also known as lysozyme P, is expressed in intestinal Paneth cells (Cross M et al, Proc Natl Acad Sci U S A, 1988; Klockars M and EF., J Histochem Cytochem, 1974).Paneth cells, located at the base of intestinal crypts, secrete a significant amount of Lyz1 into the intestinal lumen.Lyz1, being the primary lysozyme in the gut lumen, is also known as intestinal lysozyme.Our laboratory created Lyz1 knockout mice and verified lysozyme expression through various methods.Immunohistochemical and immunofluorescence staining on ileum paraffin sections from Lyz1 −/− mice showed an absence of lysozyme in their Paneth cells (RL-Fig.4A and RL-Fig.4B).Immunoblotting experiments further confirmed the lack of lysozyme expression in the crypts of Lyz1 −/− mice (RL-Fig.4C).Additionally, we conducted whole tissue immunofluorescence staining of the small intestine, using antibodies to label lysozyme and defensin, and FITC-labeled lectin to label mucin.In WT mice, lysozyme colocalized with mucin, but was absent in Lyz1 −/− mice (RL-Fig.4D).Procryptdin, however, colocalized with mucin in both WT and Lyz1 −/− mice (RL-Fig.4E),indicating that lysozyme absence does not impact Paneth cell exocytosis.Furthermore, Lyz2 mRNA levels in myeloid and Paneth cells of Lyz1−/− and WT mice were not significantly different (RL-Fig.5)(Zhang et al., Cell Res, 2019).Following the Reviewer's suggestion, we have revised the manuscript title to "Intestinal Lysozyme1 Deficiency" for greater clarity and to prevent any potential misunderstandings.RL-Fig.4Lyz1 −/− mice lack lysozyme in Paneth cells and intestinal lumen.(A) IHC staining of lysozyme in Paneth cells of the ileum from WT and Lyz1 −/− mice.(B) Immunostaining and confocal imaging of lysozyme in paraffin sections of ileum from WT and Lyz1 −/− mice.Green represents lysozyme, and blue represents cell nucleus.Scale bar=10 µm.(C) Immunoblotting analysis of lysozyme in the crypts of WT and Lyz1 −/− mice.(D-E) Immunostaining and confocal imaging of lysozyme, procryptdin and mucin in paraffin sections of small intestine from WT and Lyz1 −/− mice.Red represents (D) lysozyme or (E) procryptdin, while green represents mucin.Scale bar=20 µm.RL-Fig.5The relative levels of Lyz1 (a) and Lyz2 (b) mRNA in Paneth cells and bone marrow cells Response: We are grateful for the Reviewer's suggestion to enrich the discussion in our article.NAD + is central to metabolism, serving as a co-enzyme in redox reactions and a crucial co-factor or substrate for NAD + -dependent enzymes.It plays a critical role in a myriad of biological processes.Alterations in metabolic status, such as those induced by a high-fat diet, can lead to decreased NAD + levels, subsequently reducing the activity of NAD + -dependent cellular processes.Counteracting this decrease, the supplementation of NAD + precursors like NR and NMN has been shown to protect against obesity induced by a high-fat diet in rodent models (Canto et al., Cell Metab, 2012; Yoshino et al., Cell Metab, 2011).Furthermore, studies have demonstrated that inhibiting NAD + consuming enzymes can also offer protection against high-fat diet-induced obesity.Mice with Parp1 or Cd38 knockout, or those treated with PARP or CD38 inhibitors, exhibit elevated NAD + levels.These mice not only show resistance to obesity but also have enhanced metabolic rates during high-fat diets and aging (Bai et al., Cell Metab, 2011; Barbosa et al., FASEB J, 2007; Camacho-Pereira et al., Cell Metab, 2016; Tarrago et al., Cell Metab, 2018).Nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in NAD + biosynthesis, is notably affected by high-fat diets, leading to reduced NAD + biosynthesis (Yoshino et al., Cell Metab, 2011).Mice with an adipocyte-specific deletion of NAMPT exhibit lower NAD + levels in their fat tissues, suffer from multi-organ insulin resistance, and have impaired adipose tissue function.Remarkably, these issues can be ameliorated with NMN supplementation