Studies On The Changes Of Gut Microbiota Due To Insulin Resistance Provoked By Staphylococcus Aureus ELtaS Protein And The Mechanism Of Glucose Metabolism Disorder Induced By Staphylococcus Aureus β-hemolysin

Glucose metabolism disorders include impaired fasting glucose,reduced glucose tolerance and diabetes mellitus.Diabetes mellitus,caused by glucose metabolism disorders,is one of the three major chronic non-communicable diseases that threaten human health.It can cause acute and chronic complications,damage to a variety of organs and tissues in the body and seriously endangering human health.Staphylococcus aureus（S.aureus）is a Gram-positive conditionally pathogenic bacterium that is widely colonized in the human body.Studies have found that obese,diabetic and insulin-resistant patients are more likely to colonize S.aureus than healthy individuals,suggesting that S.aureus may be involved in host glucose metabolism disorders.Based on the previous work,to explore the relationship between S.aureus and the development of host glucose metabolism disorders by studying the relationship between extracellular proteins of S.aureus and host glucose metabolism disorders.The details are as follows.Chapter 1:The relationship between insulin resistance and changes in gut microbiota was investigated using an animal model of insulin resistance(e Lta S^transmice),with the following main studies:S.aureus LTA synthase（Lta S）is a transmembrane protein in which the protease Sps B recognises and cleaves the Ala²¹⁵Leu²¹⁶Ala²¹⁷ fragment of Lta S to generate and release the water-soluble Lta S extracellular structural domain（e Lta S）.Our previous studies confirmed that e Lta S proteins are insulin-binding proteins that trigger insulin resistance by blocking the interaction of insulin with its receptor,and constructed an animal model of insulin resistance(e Lta S transgenic mice,e Lta S^trans,Nature Microbiolog 2018;3:622-631.).Insulin resistance is a key component in the development of type 2 diabetes,and studies have confirmed that multiple factors can cause insulin resistance,including inflammation,alterations in lipid metabolism and changes in the gut microbiota.The gut microbiota mediates the development of related metabolic diseases by regulating the body’s ability to take in and store energy and lipid metabolism.The composition of the gut microbiota is significantly altered in patients with insulin resistance,metabolic syndrome and type 2 diabetes.Most of the models currently used to study insulin resistence are non-single factor induced,but the insulin resistance in e Lta S^trans mice developed by our group is mediated by e Lta S binding to insulin and inhibiting its signalling pathway,which is an ideal single factor induced model to study the effect of insulin resistance on the body.In this section,we use e Lta S transgenic mice as a model to explore the relationship between insulin resistance and altered gut microbiota,with the following main studies:1.Glucose tolerance testing in transgenic mice.Glucose tolerance was tested in e Lta S^trans mice and wild-type（WT）mice respectively.The results showed that e Lta S^transmice showed impaired glucose tolerance compared to WT mice.2.Mouse faecal samples were subjected to macrogenomic and 16S r DNA sequencing and correlation analysis.To investigate the effect of insulin resistance on gut microbiota,high-throughput sequencing of the faecal flora of mice was performed;the results showed significant differences in composition and function of gut microbiota were observed between e Lta S^trans mice and WT mice（P=0.028）.Compared with WT mice,the diversity of gut microbiota in e Lta S^trans mice increased evidently（P<0.0001）,moreover the relative abundance of Phylum Firmicutes in e Lta S^trans mice significantly increased（P<0.0001）.However,the relative abundance of Phylum Bacteroides and Phylum Verrucomicrobia decreased visibly（P=0.042,P=0.033）.We found that the relative abundance of Akkermansia muciniphila and Parabacterides distasonis related to metabolic diseases to decreased significantly in e Lta S^trans mice（P=0.033,P=0.013）.The gut microbiota of e Lta S^trans mice was clearly different from that of WT mice in carbohydrate metabolism,lipid metabolism,biosynthesis of other secondary metabolites,metabolism of other amino acids,energy production and transformation.3.The"dirty cage"sharing experiment explored the relationship between the altered intestinal flora and glucose tolerance.The faeces from e Lta S^trans mice were transplanted into WT mice and glucose tolerance was measured at different time points after transplantation.Glucose tolerance was measured at different time points after transplantation.The glucose tolerance of WT mice was impaired at 7th,8th and 9th week after faecal transplantation in e Lta S^trans mice,and recovered at 1 week after cessation of faecal transplantation.It is suggested that the change of gut microbiota will further promote the impairment of glucose tolerance.The results of this chapter show that insulin resistance induced by Staphylococcus aureus e Lta S can lead to significant changes in the composition and function of the intestinal flora of mice,while the intestinal flora of insulin-resistant mice can further induce abnormal glucose tolerance in the body,suggesting that the altered intestinal flora caused by insulin resistance can further lead to abnormal glucose metabolism in the host.Chapter Ⅱ:Preliminary study on the glucose metabolism disorder induced by Staphylococcus aureusβ-hemolysin.β-hemolysin（Hlb）is the only"non-perforating toxin"of S.aureus that has sphingolipase activity and affects the fluidity and fragility of cell membranes by degrading sphingolipids in the cell membrane.Ongoing research in our group has found that Hlb can affect the activation of the cell surface receptor tyrosine kinase（RTK）.Two key receptors involved in host glucose metabolism（Insulin Receptor（IR）and Insulin-like Growth Factory 1 Receptor（IGF-1R））belong to the RTK family and are two important transmembrane proteins that play an important role in host glucose metabolism.They play an important role in host glucose metabolism.Therefore,in this section,we investigated whether Hlb proteins could influence the phosphorylation of IGF-1R and IR and whether they could further influence host glucose metabolism.The details are as follows.1.Hlb promotes phosphorylation of IGF-1R.We found that Hlb protein did not affect IR phosphorylation,but promoted IGF-1R phosphorylation,which depended on Hlb’s sphingolipase activity and dose dependent effect.Preliminary results showed that Hlb protein promoted IGF-1 expression but did not bind to the IGF-1R extracellular alpha chain.2.Hlb causes disruption of glucose metabolism in mice.We constructed transgenic mice that induce systemic expression of Hlb protein(Hlb^trans mice).Compared with wild-type mice,Hlb^trans mice showed reduced fasting glucose levels and increased serum levels of IGF-1,but no significant differences in insulin levels,random glucose and glucose tolerance.The results of this chapter suggest that Hlb proteins may activate IGF-1R by up-regulating the expression of IGF1,which is dependent on its sphingomyelinase activity and participates in host glucose metabolism.In our subsequent studies,we will explore the molecular mechanisms underlying the upregulation of IGF-1 expression by Hlb proteins and their role in host glucose metabolism and other pathological biological processes.In summary,we found that the e Lta S protein of S.aureus induced abnormal glucose tolerance by inhibiting insulin function,which further led to significant changes in the intestinal flora of mice,and that the above-mentioned altered gut microbiota could further induce disorders of glucose metabolism in the organism.In addition,we found that the Hlb protein of S.aureus could promote the expression of IGF-1 and thus activate IGF-1R,thus participating in the regulation of host glucose metabolism.Our results suggest that S.aureus can interact with the host through its extracellular proteins and directly or indirectly participate in the glucose metabolism of the organism.In our subsequent studies,we will further investigate the molecular mechanisms underlying the regulation of host glucose metabolism by different functional molecules of S.aureus,and elucidate in detail the association between S.aureus and the development of host glucose metabolism disorders,so as to promote the study of the long-term harmful effects of S.aureus on the host.