Molecular studies on microbes and Toll like receptors signaling pathway in obesity associated inflammation

Abstract

Obesity is a complicated disease that affects equally populations in industrial and developing countries. Europe and USA currently have the highest prevalence of obesity (60%) which accounts for 2.8 million of all global fatalities. It is predicted that adult obesity to increase up to 78% by 2030. In recent years, an upward trend in obesity prevalence is even observed in Pakistan placing it at 145th position as per Global observatory report after an overall obesity increase from 5.1 to 8.60% in 2022, where 11.3% are females and 6% males. Obesity being a multifactorial metabolic disorder, it involves interplay of multiple risk factors including dietary components, environmental, immunological and genetic attributes along with most recent addition of gut microbial diversity. Present study was designed to decipher the role of TLR-2 and TLR-4 associated polymorphisms to understand the genetic susceptibility. Also, possible dysbiosis of gut microbiota, dietary choices, socio demographic and lifestyle habits as risk for obesity in local Pakistani population. The present study was conducted on total 687 obese and healthy controls, in which various predisposing risk factors for obesity were assessed after recording data in designed questionnaire. Among various demographic factors, marital status [p <0.01, OR= 2.529(1.683-3.802)], area of residence [p= 0.001, OR=2.090(1.370-3.189)], lower and middle socioeconomic strata [p=0.002, OR= 2.900(1.461-5.757)] and [p=0.011, OR=2.250(1.207-4.193)] respectively were posing risk for obesity. Upon regression analysis, increasing portion of meals [p=0.006, OR= 46.45 (3.05-705.45)], snacks [p <0.01, OR= 0.002 (0.01-0.05)] and fruits/vegetable [p<0.01, OR=26.54(5.72-123.06)] were positively associated as well as showing increased risk for obesity. The regular beef consumption at least 1-2 times/week [p=0.016, OR=3.60 (1.26-10.25)] had three fold odds for obesity. Detection of genetic variations was done by using polymerase chain reaction and Single Stranded Conformational Polymerase followed by Sanger sequencing. By using Bio edit software four novel variants in TLR-2 gene (153688371 T >C, 153702295 T >C, 153703504 T >C and 153705074 C >A) and 7 variants in TLR-4 gene (117707870 G>A, 117708080 A>G, 117708777 C>G, 117708780 G>A, 117713024 A>G, 117715449 T>A and 117715853 G>C) were detected. The genotypic and allelic frequencies have also shown positive association with obesity susceptibility. xviii Different in silico analysis were performed to predict effects of detected genetic variations on the mRNA structure and gene regulation by miRNAs. Mutation taster showed that the 4 detected TLR-2 variants changed polyA signal and the splice sites. miRBase predicted creation of novel binding site for hsa-miR-4523 in variant 153688371 T >C coding region. Moreover, Visual gene graphic predicted that the variants 153688371 T >C and 153702295 T >C were increasing mRNA stability while variant 153703504 T >C and 153705074 C >A decreased mRNA stability. In the 7 detected TLR-4 gene variants, in four variants there was loss of splice site and other were predicted a change in TLR-4 protein. Most of genetic variation either led to loss or dislocation of miRNA binding sites; variant 117707870 G>A (hsa-miR-576-5p and hsa-miR-122-3p), 117708780 G>A (hsa-miR-215-3p, hsa-miR 130b-3p) and 117715449 T>A (hsa-miR-1246). Also, for detected TLR-4 polymorphisms in variants 117707870 G>A, 117713024 A>G and 117715449 T>A, decreased stability of mRNA was predicted. However, variants 117708080 A>G, 117708777 C>G and 117715853 G>C enhanced stability of predicted mRNA. The variant 153703504 T >C of TLR-2 and 117708780 G>A TLR-4 both upon in-silico were affecting the LRR region, which are ligand binding site of the TLRs. Also, TLR-4’s variant 117713024 A>G disrupted the disulphide bond formation and hence might affect the ligand binding. The variant in TLR-2 encoding the cytoplasmic domain was also detected, which can affect the downstream signaling and pro-inflammatory cytokines formation. All the TLR-2 and TLR 4 gene variants` after mutational and in-silico analysis found change in mRNAs and some were annotated to have alteration in their protein. Most of these genetic variations had strong association for their involvement in obesity associated inflammation. The culturable fungi was isolated using standard microbiological techniques from fecal samples of the obese and control study individuals. From all samples, different Candida species were identified which were Candida kefyr, Teunomyces krusei and Candida albican. C.kefyr was most prevalent with increased abundance in obese gut. Moreover, increased diversity was present in obese gut with additional species including Candida glabrata, Candida dubliensis and Candida parapsilosis. The percentage positivity of many virulence factors was high among isolates from obese group where majorly expressed virulence factors were; biofilm formation 95.2% and phospholipase 84.6% with high resistance to Fluconazole 31.7%. The pathogenic potential in Candida (C.kefyr, C. albicans and T. krusei) isolated from obese individuals was very potent, which can be trigger for low grade inflammation by these opportunistic pathogens in the obesity. xix The gut microbial diversity was checked by both culture dependent approaches using microbiological standard techniques and the culture independent approach using 16SrRNA based sequencing. Among the culturable bacterial gut colonizers, change in bacterial abundance and diversity was observed with increase in Klebsiella pneumonia (16.1%), Pseudomonas aeruginosa 64 (19.5), Shigella species (6.4%) and Acinetobacter (1.5%). The change in diversity was seen with Proteus vulgaris (0.9%) and Streptococcus specie (19.0%) that were present in obese group only. Based on 16SrRNA sequencing relative abundance of Firmicutes (obese=42.31% and controls=6.59%) was increased and Phylum Bacteroidota (obese= 40.53% and control= 90.95%) was decreased among obese subjects. Moreover, the Phylum Proteobacteria (11.2%), Verrucomicrobiota (1.31%), Cyanobacteria (0.4%), Desulfobacterota (0.17%) and Patescibacteria (0.01%) were detected only in obese samples showing increased diversity in obese faecal samples with dysbiosis in case of obesity. According to alpha and beta diversity analysis, the change in bacterial gut diversity was significantly associated with the obesity. Phylum Proteobacteria population was significantly increased in the obese group. Moreover, the member of Proteobacteria namely E.coli and Enterobacter’s presence was associated with detection of lower levels of LPS (23.8EU/mL) in obese serum samples only, which were indicative of endotoxemia. The detection of LPS and Proteobacteria point toward possible role of secreted LPS by gut microbiota for TLR-mediated inflammation in obesity. Also the gut bacterial diversity had intricate and complex network of interactions, which need to be further evaluate to understand their role in digestion and energy harvesting in case of obesity. There was dysbiosis in gut bacterial diversity with more Gram negative in core microbiome and change in culturable gut microflora with Gram negatives too with detection of LPS in serum of obese individuals’ points towards triggering of TLRs for generation of low grade inflammation in obesity. All the risk factors were further studied for their association with the each another as multifactorial risk for the obesity. The detected single nucleotide polymorphisms of TLR-2 and TLR-4 were common in females. Interestingly age 30 years for men and above 40 for women was risk in individuals carry polymorphisms in study population. There was association of detected polymorphisms with demographic risk factors like lower to middle socioeconomic strata, secondary level and urbanization with obesity. Moreover, three variants 117708080 A>G, 117707870 G>A and 117715853 G>C were significantly xx associated with both culturable dysbiosis as well as dietary choices (beef, poultry and fruits/vegetable) and lifestyle (no physical exercise) changes. Moreover, dysbiosis of uncultured bacteria was associated with gender, beef intake and marital status. These analysis showed there was strong association of gut microbial dysbiosis (culturable and unculturable) with the demographics, dietary habits and lifestyle choices linking TLRs with obesity. Overall, it can be concluded that all the risk factors were associated to each other including Toll like receptor SNPs, dietary habits, demographics, lifestyle, fungal and bacterial diversity change and increased LPS with the obesity. All these were posing risk for sustaining obesity and may have been triggered by low grade inflammation in the obesity. At the end it can be inferred that there is need of comprehensive study including genetics, demographics and dietary habits to control the increasing trends of obesity around the globe and in various regions of Pakistan. Based on this work, it is recommended that there is need for nationwide awareness campaign to reduce prevalence of obesity in Pakistan for better weight management and healthy life style to combat this pandemic.