Investigation of TLR2 and TLR4 Polymorphisms and Sepsis Susceptibility: Computational and Experimental Approaches

Abstract

Toll-like receptors (TLR) play an eminent role in the regulation of immune responses to invading pathogens during sepsis. TLR genetic variants might influence individual susceptibility to developing sepsis. The current study aimed to investigate the association of genetic polymorphisms of the TLR2 and TLR4 with the risk of developing sepsis with both a pilot study and in silico tools. Different in silico tools were used to predict the impact of our SNPs on protein structure, stability, and function. Furthermore, in our prospective study, all patients matching the inclusion criteria in the intensive care units (ICU) were included and followed up, and DNA samples were genotyped using real-time polymerase chain reaction (RT-PCR) technology. There was a significant association between TLR2 Arg753Gln polymorphisms and sepsis under the over-dominant model (p = 0.043). In contrast, we did not find a significant difference with the TLR4 Asp299Gly polymorphism with sepsis. However, there was a significant association between TLR4 Asp299Gly polymorphisms and Acinetobacter baumannii infection which is quite a virulent organism in ICU (p = 0.001) and post-surgical cohorts (p = 0.033). Our results conclude that the TLR2 genotype may be a risk factor for sepsis in adult patients.

Keywords: TLR; infection; polymorphism; sepsis; septic shock.

Figure 2

Functional Analysis of TLR2 and TLR4 proteins. (A) Subcellular localization of the TLR2 protein. The gradient of green color indicates the degree of confidence (genecards.org) with (compartments.jensenlab.org) as the source of the image. (B) Predicted network of protein–protein interactions of the TLR2 protein. Proteins are represented by nodes, while predicted associations are represented by edges that could be drawn with 7 colored lines that indicate different types of evidence. Redline fusion evidence, Light blue line—database evidence. Green line—neighborhood evidence. Blue line—co-occurrence evidence. Purple line—experimental evidence. Black line—coexpression evidence. Yellow line—text mining evidence. HMGB1: High mobility group protein B1, TIRAP: Toll/interleukin-1 receptor domain-containing adapter protein, VCAN: Versican core protein, HSPD1: 60 kDa heat shock protein, LY96: Lymphocyte antigen 96, CD14: Monocyte differentiation antigen CD14, TOLLIP: Toll-interacting protein, IRAK1: Interleukin-1 receptor-associated kinase 1, CLEC7A: C-type lectin domain family 7 member A, HSP90B1: Endoplasmin. STRING analysis (version 11.5). (C) TLR2 Gene Coexpression matrix. Predict association between protein functions, Color intensity shows the confidence level in the association between protein functions. TLR2 shows coexpression with CD14, CLEC7A, and LY96 with scores of 0.611, 0.281, and 0.130, respectively (https://string-db.org (accessed on 29 August 2021)). (D) Subcellular localization of TLR4 protein (genecards.org) with (compartments.jensenlab.org) as the source of the image. (compartments.jensenlab.org). (E) 1B predicted a network of protein–protein interactions of the TLR4 protein. TICAM1: TIR domain-containing adapter molecule 1, TICAM2: TIR domain-containing adapter molecule 2, TRAF6: TNF receptor-associated factor 6, LY86: Lymphocyte antigen 86. STRING analysis (version 11.5). (F) TLR4 Gene Coexpression matrix. TLR4 shows coexpression with LY86, CD14, and LY96 with scores of 0.301, 0.264, and 0.176, respectively (https://string-db.org (accessed on 29 August 2021)).

Figure 3

Functional and structural consequences of SNPs (A). Predicting the impact of rs5743708 on TLR2 function—the score ranged from benign (0) to damaging (1) (B). Table showing the transcripts of rs5743708, allele (transcript allele), consequence type, amino acid fate, codons, and PolyPhen score. R: Arginine, Q: Glutamine (ensemble.org). (C) Predicting the impact of rs4986790 on TLR4 function the score ranges from benign (0) to damaging (1) (D). Table showing the transcripts of rs4986790, allele (transcript allele), consequence type, amino acid fate, codons, and PolyPhen score. D: Aspartate, V: valine, G: Glycine (ensemble.org).

Figure 1

Scheme illustrating the outline of the study plan.

Figure 4

Evolutionary conservation analysis of TLR2 by Consurf.

Figure 5

Evolutionary conservation analysis of TLR4 by Consurf.

Figure 6

HOPE illustration of mutation structural impacts (A). Project HOPE illustration of the structural replacement of Aspartic acid with Glycine at position 299 in the TLR4 protein (colored with grey). The side chain of the wild type is colored in green while Glycine only has a hydrogen atom in its side chain (B). Project HOPE illustration of the structural replacement of Arginine with Glutamine at position 753 in the TLR2 protein (colored with grey). The side chain of the wild type is colored green while the side chain of the mutant type is colored red.