The gut commensal fungus, Candida parapsilosis, promotes high fat-diet induced obesity in mice

Gut fungi is known to play many important roles in human health regulations. Herein, we investigate the anti-obesity efficacy of the antifungal antibiotics (amphotericin B, fluconazole and 5-fluorocytosine) in the high fat diet-fed (HFD) mice. Supplementation of amphotericin B or fluconazole in water can effectively inhibit obesity and its related disorders, whereas 5-fluorocytosine exhibit little effects. The gut fungus Candida parapsilosis is identified as a key commensal fungus related to the diet-induced obesity by the culture-dependent method and the inoculation assay with C. parapsilosis in the fungi-free mice. In addition, the increase of free fatty acids in the gut due to the production of fungal lipases from C. parapsilosis is confirmed as one mechanism by which C. parapsilosis promotes obesity. The current study demonstrates the gut C. parapsilosis as a causal fungus for the development of diet-induced obesity in mice and highlights the therapeutic strategy targeting the gut fungi.

The authors Sun et al. have investigated the role of anti-fungal antibiotics such as amphotericin B, fluconazole and 5-fluorocytosinegut and fungal "mycobiome" microbiome in high-fat induced obesity in mice. To do so, they have supplemented Candida parapsilosis to see the casual role and further, the number of Candida parapsilosis have reduced with antifungals with in-turn reduced obesity in mice. They found the role of Candida parapsilosis spp., a gut commensal fungi in induction of obesity. The manuscript is well-written and clearly discussed. That said, information on dosing strategy, high-fat diet and clinical replication may be correlated with caution, due to supplementation of either fungi or antifungals. The authors have greater potential to promote and provide clinical significance by comparing human/mice samples as they have collected both.
Comments: 1. It is a surprising not see a trace of Candida and Nagashina genera in SD. It is specific to obesity or high-fat diet supplied to the mice. There is no information about the HFD composition? 2. C. parapsilosis was supplemented for 40 days at 2 x 10^7 cfu/ml, how did the author select the dosing strategy? Can this be replicated in humans? 3. Have the authors noticed any adverse events after supplementing fungi in mice, there is no information.
Minor comments: It will be wonderful opportunity to show: 1. Number of reads bacterial versus fungal in mice across the groups. 2. Also, the authors have collected fecal and cecal samples, it will be good to show microbial comparisons of sample type across the groups. 3. Further, the authors have human fecal samples, beneficial to to microbial comparisons between human/mice samples? 4. Number of mice per cage could have impacted due to coprophagous nature of mice?
Reviewer #2 (Remarks to the Author): The manuscript entitled "Identification of the gut commensal Candida parapsilosis as a causative fungus for the development of high fat-diet induced obesity in mice" by Hongwei Liu et al. demonstrates the role of the C. parapsilosis produced lipase in HFD-obesity. The manuscript is particularly interesting, well written and the conclusion are strongly supported by results. Experimental design is well organized, methods are comprehensive and sufficient to demonstrate the working hypothesis. One major concern is related with Figure 3D: there are 6W of CP enrichment with depletion of other fungi by Amphotericin B in the drinking water followed by 40 days of CP and HFD. What is the meaning of CP administration during the first 6 weeks if as been demonstrated that CD is susceptible to Ampho? What is the effect of CP enrichment followed by SD? Are mice gaining more weight than CP free mice?
Minor: Figure 2C there is a substantia variability in SD diet and SD+Ampho. are plasma triglyceride more abundant when Ampho is administered to SD feed mice? SD + Ampho is missing in Figure 2K Overall the manuscript is really well done and extremely interesting, a revision is strongly encouraged Reviewer #3 (Remarks to the Author): The manuscript in the title: Identification of the gut commensal Candida parapsilosis as a causative fungus for the development of high fat-diet induced obesity in mice is very interesting to be accepted for publication in Journal of Communictions Biology. The experiments were well organized, performed with appropriate methods and data were clearly presented.
I would only recommend the following changes: -This study demonstrates the gut Candida parapsilosis as a causal fungus for the development of diet-induced obesity in mice and you highlights the therapeutic strategy targeting the gut fungi. How do you want to use antifugal for the therapeutic strategy in patients?
-Please show the prevalence or incidence of fungal infection or fungal including Candida parapsilosis in the gut of obese patients in Introduction part.
-The increase of free fatty acids (FFA) in the gut because of the production of fungal lipases from C. parapsilosis was confirmed as one mechanism by which C. parapsilosis promotes obesity. What kind of the other mechanisms that C. parapsilosis induce obesity? -In result: two enriched yeasts including Candida parapsilosis and Naganishia globosa were identified in the feces of the HFD-fed mice. Why are you only interested in C. parapsilosis to test in mice?
- Figure 4. Please discuss the microbiome result in discussion part and also show the link between bacterial microbiota and mycobiota in your work.
-Please discuss the effects of the wild type and the lipase mutant strain C. parapsilosis on inflammation in HFD-induced obese mice in the discussion part.
-Please discuss the effect of plasma LPS in HFD-induced obese mice in the discussion part.

Answers to Reviewer 1
Reviewer #1 (Remarks to the Author): The authors Sun et al. have investigated the role of anti-fungal antibiotics such as amphotericin B, fluconazole and 5-fluorocytosinegut and fungal "mycobiome" microbiome in high-fat induced obesity in mice. To do so, they have supplemented Candida parapsilosis to see the casual role and further, the number of Candida parapsilosis have reduced with antifungals with in-turn reduced obesity in mice. They found the role of Candida parapsilosis spp., a gut commensal fungi in induction of obesity. The manuscript is well-written and clearly discussed. That said, information on dosing strategy, high-fat diet and clinical replication may be correlated with caution, due to supplementation of either fungi or antifungals. The authors have greater potential to promote and provide clinical significance by comparing human/mice samples as they have collected both.
Comments: 1. It is a surprising not see a trace of Candida and Nagashina genera in SD. It is specific to obesity or high-fat diet supplied to the mice. There is no information about the HFD composition? Answers: Heisel showed that mouse fecal fungal microbiomes are not solely structured by diet, including Candida and Nagashina genera, these fungi may obtained by a variety of environmental factors 1 . The low abundance in the mice given SD may make it difficult to culture under lab condition. We have added the HFD composition in part of Methods. Page25, lines 446-447.
2. C. parapsilosis was supplemented for 40 days at 2 x 10^7 cfu/ml, how did the author select the dosing strategy? Can this be replicated in humans? Answers: We refer to the dosage of gavage C. parapsilosis in the literature 2 . Whether this can be repeated in humans requires more clinical and experimental data to support.
3. Have the authors noticed any adverse events after supplementing fungi in mice, there is no information. Answers: We have not observed any side effects after supplementing fungi in mice, including active state and behavioristics, however, the adverse events of supplementing fungi in mice need be study systematically in further study.
Minor comments: It will be wonderful opportunity to show: 1. Number of reads bacterial versus fungal in mice across the groups. Answers: We tended to show influence of C. parapsilosis supplementation on gut bacterial community in the antifungal-preteated mice in Figure 4. To make it clear, the title of Figure 4 is changed to "Changes of gut bacterial community in the live C. parpsilosis-treated and antifungal pretreated HFD mice." Due to pretreatment with amphotericin B, the number of fungal reads in the vehicle group should be trace. Thus, the number of reads bacterial versus fungal in mice can not be obtained in this work.
2. Also, the authors have collected fecal and cecal samples, it will be good to show microbial comparisons of sample type across the groups. Answers: We only collected cecal contents of the the C. parapsilosis -treated mice in current study, the microbial composition can not be compared.

Answers to Reviewer 2
Reviewer #2 (Remarks to the Author): The manuscript entitled "Identification of the gut commensal Candida parapsilosis as a causative fungus for the development of high fat-diet induced obesity in mice" by Hongwei Liu et al. demonstrates the role of the C. parapsilosis produced lipase in HFD-obesity. The manuscript is particularly interesting, well written and the conclusion are strongly supported by results. Experimental design is well organized, methods are comprehensive and sufficient to demonstrate the working hypothesis.
One major concern is related with Figure 3D: there are 6W of CP enrichment with depletion of other fungi by Amphotericin B in the drinking water followed by 40 days of CP and HFD. What is the meaning of CP administration during the first 6 weeks if as been demonstrated that CP is susceptible to Ampho? Answers: As described in the scheme of Figure 3D, the HFD-fed mice were first treated with supplementation of amphotericin B in drinking water for six weeks to eliminate original intestinal fungus and then were orally given live C. parapsilosis or PBS to observe the effect of C. parapsilosis on obesity.
What is the effect of CP enrichment followed by SD? Are mice gaining more weight than CP free mice? Answers: The oversupply of triglyceride in the high-fat feed are important for the development of obesity. Therefore, we did not evaluate the effect of CP enrichment followed by SD.
Minor: Figure 2C there is a substantia variability in SD diet and SD+Ampho. are plasma triglyceride more abundant when Ampho is administered to SD feed mice? Answers: We are sorry for this mistake. The indicaiton of significance should be SD vers HFD groups. There is no significant difference in plasma triglycerides between SD and SD+Ampho groups. SD + Ampho is missing in Figure 2K. Answers: We have added SD+Ampho groups in Figure 2K Overall the manuscript is really well done and extremely interesting, a revision is strongly encouraged

Answers to Reviewer 3
Reviewer #3 (Remarks to the Author): The manuscript in the title: Identification of the gut commensal Candida parapsilosis as a causative fungus for the development of high fat-diet induced obesity in mice is very interesting to be accepted for publication in Journal of Communictions Biology. The experiments were well organized, performed with appropriate methods and data were clearly presented. I would only recommend the following changes: -This study demonstrates the gut Candida parapsilosis as a causal fungus for the development of diet-induced obesity in mice and you highlights the therapeutic strategy targeting the gut fungi. How do you want to use antifugal for the therapeutic strategy in patients? Answers: The antifungal agents without gut absorption can be expected for treatment of obesity patients after deep and extensive studies in preclinical and clinical investigation. But we do not recommend antifungal treatment for patients, because the extensive use of antifungal drugs may lead to the emergence of drug-resistant strains. We believe that the best treatment is to reestablish the balance of the intestinal microbiota.
-Please show the prevalence or incidence of fungal infection or fungal including Candida parapsilosis in the gut of obese patients in Introduction part. Answers: We have added the prevalence or incidence of fungal infection or fungal including Candida parapsilosis in the gut of obese patients in Introduction part. Page 4, lines 67-70. Recently, Tahliyah's study showed the genera Thermomyces and Saccharomyces most strongly associate with metabolic disturbance and weight gain. And Borges reported a mycobiota shift towards obesity, the increased yeast in obese human individuals, and more filamentous fungi in the eutrophic human individuals.
-The increase of free fatty acids (FFA) in the gut because of the production of fungal lipases from C. parapsilosis was confirmed as one mechanism by which C. parapsilosis promotes obesity. What kind of the other mechanisms that C. parapsilosis induce obesity? Answers: We have added a brief discussion about the other mechanisms that C. parapsilosis induce obesity. Page 23, lines 393-399: Several study showed that Candida can produces ligands for pattern recognition receptors (PRRs), including beta-glucans, chitin, mannans, beta-(1,2)-linked oligomannosides, and fungal nucleic acids, which stimulate innate immune responses. And Candida produces proinflammatory small molecular compounds like alcohol and prostaglandin. Excessive fat accumulation is also associated with a low grade systemic and chronic inflammatory condition. Therefore, these may also be the cause of obesity caused by C. parapsilosis.
-In result: two enriched yeasts including Candida parapsilosis and Naganishia globosa were identified in the feces of the HFD-fed mice. Why are you only interested in C. parapsilosis to test in mice? Answers: In the fecal samples of the chow diet-fed mice, amphotericin B-treated HFD-fed mice, and fluconazole-treated HFD-fed mice, no fungi were successfully cultured. Different from that of amphotericin B-treated and fluconazole-treated mice, C. parapsilosis were obtained in the 5-fluorocytosine-treated HFD-fed mice that showed similar obesity-related disorders as that of HFD-fed mice. Based on above evidence, we hypothesize that the enrichment of C. parapsilosis is closely associated with the occurrence and development of diet-induced obisity.
- Figure 4. Please discuss the microbiome result in discussion part and also show the link between bacterial microbiota and mycobiota in your work. Answers: Added in lines 400-413 on page 23-24. There are complex interactions between the intestinal microorganisms that colonize the human body. This interaction is determined by many factors, including the host's physiology, immune status, and nutritional competition. Study showed antibiotic treatment resulted in the overgrowth of a commensal fungal. Another study showed Enterobacteriaceae are essential for the modulation of colitis severity by fungi. To reveal the influence of C. parapsilosis on gut microbiota, high-throughput sequencing (HiSeq) of the V3-V4 region of 16S rRNA genes from the caecum contents was conducted. A significant increase in the phylum of Proteobacteria and Actinobacteria and the family of Desulfovibrionaceae was found in the gut microbiota of live C. parapsilosis group by analysis with linear discriminant analysis (LDA) effect size (LEFSe) method, accompanying with the decrease of the phylum of Firmicutes and the family of Lachnospiracea. The result reflected C. parapsilosis enrichment affects the composition of intestinal bacteria. This may also one important mechanism that C. parapsilosis induce obesity.
-Please discuss the effects of the wild type and the lipase mutant strain C. parapsilosis on inflammation in HFD-induced obese mice in the discussion part. Answers: Added in line 387-393 on page 22-23. In the state of infection, deletion of lipase increased the inflammatory response of the host to C. parapsilosis. In this investigation, the similar increase in the levels of TNF-α, IL-1β, IL-6, and IL-8 was observed in the lipase-negative mutant C. parapsilosis-treated mice and the wild strain-treated mice, as compared with those of amphotericin B-treated HFD mice ( Supplementary Fig. 5), which indicates less influence of gut fungi-secreted lipases on the host immune.
-Please discuss the effect of plasma LPS in HFD-induced obese mice in the discussion part. Answers: Added in line 323-329 on page 20. The involvement of the gut microbiota in host health and pathogenesis of obesity has been demonstrated. Several studies showed that Families in the phylum Proteobacteria, such as Enterobacteriaceae and Desulfovibrionaceae that cover species producing LPS, are enriched in obese humans and rodents. LPS, the major component of the outer membrane of Gram-negative bacteria that induces inflammation after entering the circulation, has been regarded as the major cause for the initiation of obesity.