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Table of Contents
Year : 2015  |  Volume : 2  |  Issue : 2  |  Page : 68-71

Novel polymorphisms with in TLR4 exon1 sequences in visceral leishmaniasis and pulmonary tuberculosis patients

1 Assistant Professor, Faculty of Science, University of Princess Nora bint Abdul Rahman, Riyadh, Saudi Arabia
2 Professor, Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan

Date of Web Publication5-Jul-2017

Correspondence Address:
Hadeel Faisal Gad
Assistant Professor, Faculty of Science, University of Princess Nora bint Abdul Rahman, Riyadh
Saudi Arabia
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Source of Support: None, Conflict of Interest: None

DOI: 10.5530/ami.2015.3.4

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Introduction: Leishmania and Mycobacterium Tuberculosis share many similarities in their pathogenesis and both pathogens are macrophage parasites. The present study was carried out to determine the diversity of TLR4 gene in VL and pulmonary TB as innate immunity marker. Materials and Method: Confirmed VL patients, pulmonary TB patients and healthy individuals DNAs were analyzedfor TLR 4 exon1 mutationsafter stimulation with live Leishmania promastigotes and BCG. PCR based sequencing was done to determine the diversity of for toll-liked receptor 4 exon-1. Results: Three polymorphisms were detected for the first time in TLR4 exon1, TLR4-35C/T and TLR4-5C/T located in the transcription factor binding site, and TLR4+18C/T located in the coding region which resulted in the change from the amino-acid Threonine (polar) to Alanine (non-polar). Conclusion: The diversity in TLR4 suggests possible variation in the innate immune responses of the two patients groups.

Keywords: Polymorphisms, TLR4, Leishmaniasis & Tuberculosis

How to cite this article:
Gad HF, Mukhtar MM. Novel polymorphisms with in TLR4 exon1 sequences in visceral leishmaniasis and pulmonary tuberculosis patients. Acta Med Int 2015;2:68-71

How to cite this URL:
Gad HF, Mukhtar MM. Novel polymorphisms with in TLR4 exon1 sequences in visceral leishmaniasis and pulmonary tuberculosis patients. Acta Med Int [serial online] 2015 [cited 2022 Aug 10];2:68-71. Available from: https://www.actamedicainternational.com/text.asp?2015/2/2/68/209655

  Introduction Top

The recent discovery of Toll-liked receptors (TLRs) in cells of the innate immune system was a major advance in our understanding of the molecular mechanisms involved in host-parasite interaction. (TLRs) are the prototype for receptors that recognize pathogen-related structures and constitute a familyof at least 10 receptors that differentially recognizepathogen-associated molecular patterns (PAMP) through an extra cellular domain and initiate inflammatory signaling pathways throughan intracellular domain.

Previously published studies provided evidence for a role of TLR2, TLR3 and TLR4 in the response of NK cells and macrophages to Leishmania parasites.[1],[2],[3],[4] Recent work indicated that TLR4 contributes to the control of Leishmania parasite growth in both phases of the immune response.[4]

The initial recognition of mycobacterial components by the innate immune system through TLRs, and likely other PRR, contributes to triggering the host immune response.[5]

TLR2, TLR4 and, more recently, TLR1/TLR6 that heterodimerise with TLR2, have been implicated in the recognition of mycobacterial antigens.[6],[7]

This study was conducted to analyses Toll-liked receptor 4 gene of diversity among leishmaniasis and tuberculosis patients.

  Materials and Methods Top

Case Control Study Design

This study was conducted during the period from February 2011 to August 2012. Visceral leishmaniasis (VL) patients recruited for this study were mostly from the White Nile state and Gadarif state (Sudan), age range from 3 years to 55 years, males andfemales with symptoms duration from one to six months.

Ten (10) visceral leishmaniasis patients (diagnosed detection of agglutinating antibodies in patients' sera using DAT (Direct Agglutination Test), and confirmed by demonstration of the parasite in lymph node aspirate isolated and cultured in NNN biphasic media, at the Institute of Endemic Diseases) and ten (10) tuberculosis patients (diagnosed by demonstration of acid fast bacilli,Mycobacteriumtuberculosis in sputum samplesin Educational Khartoum Hospital laboratories) were randomly selected and referred to the study. Ten healthy controls were both Mantoux and DAT negative.

Preparation of Antigens for Stimulation


Leishmania parasites were isolated according to Evans (1989) using a modified biphasic NNN media (Novy-MacNeal- Nicolle medium)consisting of solid and liquid phases.

BCG preparation

Freeze-dried Bacillus Calmette-Guerin (BCG, manufactured by BB-NCIPD Ltd, Sofia, Bulgaria) tuberculosis vaccine was used as antigen for whole blood stimulation. The BCG was prepared in sterile environment by removing metal ring with a vial opener and adding 1 ml of diluent for BCG Vaccine (Saline) (BB-NCIPD Ltd, Sofia, Bulgaria). The prepared solution was kept in -20°C till used.

Whole Blood Stimulation

Dilution of the whole blood

One ml of heparinized venous blood from each patient from both groups and the healthy control was placed into a 15 ml centrifuge tubes (Corning, #25330, USA) and diluted by adding two ml of sterile RPMI-1640 medium (250 μl blood containing about 5x105 lymphocytes).


One ml/well of diluted whole blood were dispensed into a sterile 24-well flat-bottomed cell culture plates (Coaster # 3526, USA). Stimulation of whole blood was done by adding 4x106 parasite/well of live L. donovani, BCG in a concentration of 5x105 viable unit to another well and the third well remained as a non-stimulated negative control for each sample. The culture plates were incubated at 37°C with 5% CO2. After 48 hours, the cells were harvested by centrifugation at 1200 rpm for 10 min, kept in -80°C till used for DNA extraction and detection by PCR.

  Polymerase Chain Reaction Top

DNA Extraction

Genomic DNA (Deoxyribonuclic acid) was extracted from the harvested cells of the blood samples kept in -80°C after, using G-DEX™IIb GENOMIC DNA Extraction Kit (G-DEX IIb for blood, Genomic DNA Extraction Kit, iNtRON Biotechnology Inc., LotNo. 11910145, Korea). This procedure was done according to the manufacturer instructions. Three hundred (300) μl of whole blood was added to a 1.5 ml tube containing 900 μl RBC Lysis buffer Solution. After mixing and vortexing, it was incubated for 5 min at room temperature and centrifugated at 10,000 x g for 1 min. Supernatant was removed with remaining about 50 μl to re-suspend the pellet by vigorous vortexing. 300 μl of Cell Lysis Solution was added to the resuspended cells and incubated in 37°C for complete lysis of the cells. After chilling the homogeneous solution to room temperature, 100 μl of Protein PPT Buffer was added and vortexed vigorously at high speed for 20 seconds. The cell lysate was then centrifugated at 13,000 x g for 5 min and the precipitated proteins formed a tight white pellet. 300 μl of supernatant containing DNA was transferred into a new 1,5 ml tube and a 300 μl of 100% Isopropanol (2-propanol) was added and mixed. The solution was then centrifugated at 13,000 x g for 1 min, supernatant poured off and the tube was drained briefly on clean absorbent paper. 1 ml of 70% Ethanol was added, tube inverted many times for washing the DNA pellet, centrifugated at 13,000 x g for 1 min, supernatant carefully poured off and the tube was inverted and drained on the clean absorbent paper for air dry for 10 min. Then, 150 μl of DNA Rehydration Buffer was added to the tube to rehydrate the DNA and incubated at 4°C for overnight. Collected DNA concentration was measured using Spectrophotometer ND-1000 (Thermo Fisher Scientific, USA) and stored at -20°C.

Amplification of TLR4 Sequence

PCR reactions were performed in a PCR premix tubes (Maxime PCR PreMix (i-Taq), iNtRON co. ltd.) containing 2.5 U of Tag polymerase, 250 μM each deoxynucleotidetriophosphate, 1.5 mM MgCl2, 10 X buffer contain (10 mMTris HCL, 40 μM KCL) and gel loading dye. 1.5 μl of each primer was added (TLR4 exon1, [Table 1] and 5μl of DNA was added to a complete volume of 25 μl; The whole mixture was gently tapped to be evenly mixed. The reaction mixtures were subjected to 35 cycles of amplification (30 sec at 94°C, 30 sec at 72°C and 30 sec at 72°C) after an initial denaturation at 94°C for 5 min and final extension step at 74°C for 10 min.
Table 1: Sequence of the primers used for amplification of TLR4 exon1

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Agarose Gel Electrophoresis

Efficient amplification was confirmed by gel electrophoresis in 1.5% agarose gel in a flask (1.5 g agarose + 100ml 1X TBE buffer). The mixture was boiled till it completely dissolved. After cooling the gel down to 60-70°C, 4 μl ethidium bromide (0.5 mg/ml) was added. Then the 100ml of agarose was loaded without making air bubbles into the 100 ml gel cast and was left for 30 min for polymerization. The running buffer composed of 10 ml of 10X TBE buffer and 90 ml of distilled water was poured in a gel cast. Six μl PCR product of each sample and the controls were loaded into a well of agarose gel starting from lane 2 and 4 μl ladder was loaded into lane 1. Finally, the tank was plugged into power supply run at 60 Volt for 60 min until loading buffer has moved sufficiently into the gel. The gel was illuminated with a Gel-Documentation System to visualization and documentation the PCR results.

DNA Sequencing

PCR amplified region of TLR4 exon1 was commercially sequenced at Macrogene sequencing facility (Macrogen, Seoul, Korea).

Alignment of the Sequences of the PCR Amplified Fragments

VL, TB and healthy controls samples mentioned before were sequenced commercially. The generated sequences were compared with TLR4-exon 1 reference gene (Homo sapiens Toll-like receptor 4 gene, exon 1) using Bioedit Sequence program for DNA sequence comparisons.

Statistical Analysis

The sequencing results were analyzed using Bioedit software program and FASTA software.

  Results Top

Amplification of Toll-liked Receptor 4 Exon 1 Sequence by PCR

Amplification of Toll-liked receptor 4 exon 1gene of VL and TB patients and healthy controls' genomic DNA produced DNA fragments of 300 bp [Figure 1].
Figure 1: Amplification of the Toll-like receptor 4 exon 1gene in both patients and healthy controls' (labels from 1-10 respectivily: VL4, VL2, TB3, VL7, TB1, C3, C2, Positive control, Negative control, DNA ladder)

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Analysis of Sequences of the Amplified DNA Fragment

Alignment of the sequences of the PCR amplified fragments with human TLR4 sequences showed 3 single nucleotide polymorphisms (SNPs) which were not reported in the DNA database: TLR4 +18C/T, TLR4 -35C/T and TLR4 -5C/T. The major alleles' frequencies of the novel SNPs TLR4 -35C/T, TLR4 +18C/T and TLR4 -5C/T are 0.7, 0.9 and 0.8, respectively.

When AliBaba software was used to search for transcription factors that can bind at site of TLR4 -35C/T (four TB patients' samples) and TLR4 -5C/T (two VL patients' samples). The result showed that, in the presence of C allele in TLR4 -35C/T there was one transcription factor (Sp1) and I the presence of T allele was also one transcription factor (SRF). In the presence of allele C in TLR4 -5C/T there was one transcription factor (Nf-1), while in the presence of the allele T there were four transcription factors (CRBE, CPE bind,c-JUN and CRE-BP1) [Table 2].
Table 2: Transcription factors consensus binding sequence in the regions of TLR4-35C/T and TLR4-5C/T

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The TLR4 +18C/T (three VL patients' samples) polymorphism resulted in amino acid change from Threonine (T) (polar) into Alanine (A) (non-polar).

  Discussion Top

Visceral leishmaniasis and Pulmonary Tuberculosis are both endemic diseases in Sudan. Visceral leishmaniasis and pulmonary tuberculosis share several similarities both are caused by intracellular pathogens, both pathogens target macrophages and they stimulate similar immune responses.[8]

In this short study three polymorphisms in TLR4 exon1 were detected: TLR4+18C/T –found in three VL patents” samples - which is located in the coding region resulted in the change from amino-acid Threonine (polar)to Alanine (non-polar) which can lead to a change in the gene type [Figure 2]. The other polymorphisms found were TLR4- 5C/T (found in two VL patients samples) & TLR4-35C/T (found in four TB patients samples) which are located in the transcription factors binding i.e., functional polymorphisms as it can gain or lose transcription factors which would lead to a change in the gene regulation. Transcription factors found in the presence of C allele in TLR4-35C/T and TLR4- 5 were Sp1 and Nf 1 respectively. Transcription factors found in the presence of T allele in TLR4-35C/T was SRF and in TLR4-5C/T were CREB, CPE bind, c-Jun and BP1. As a conclusion, three snips polymorphisms were detected for the first time in the TLR4 in both VL and TB patients' samples although of the small number of the population's samples. Therefore, more investigation on the TLRs' polymorphisms role in the susceptibility to VL and TB diseases are required as more discoveries in the roll of TLRs would lead to the more understanding of the molecular mechanisms of host-parasite interaction.
Figure 2: Locations of Novel polymorphismswithin TLR4 exon1 sequences

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  References Top

Gad H and Muktar M M. In-vitro analysis of cytokines responses of visceral leishmaniasis and pulmonary tuberculosis patients to homologous and heterologous antigen stimulation. J Clin Cell Immunol 2014; 5:5.  Back to cited text no. 1
Becker R, Zilberstein S, Lesser V, and Goldman, C. V. Transition-independent decentralized Markov decision processes. In AAMAS 2003; 41–48.  Back to cited text no. 2
Flandin JF, Chano F, Descoteaux A. RNA interference reveals a role for TLR-2 and TLR3 in the recognition of Leishmaniadonovani promastigotes by interferon-gamma – primed macrophages. Eur J Immunol2006;36: 411 – 420.  Back to cited text no. 3
Kropf P, Freudenberg MA, Modolell M. “Toll like receptor 4 contributes to efficient control of infection with the protozoan parasite Leishmania major”.Infection andImmunity 2004; (72) 4:1920–1928.  Back to cited text no. 4
Kleinnijenhuis J, Marije O, Leo A, B. Joosten, Mihai GN and Reinout VC. Innate Immune Recognition of Mycobacterium tuberculosis. Clinical and Developmental Immunology 2011;(2011): Article ID 405310.  Back to cited text no. 5
Bulut Y, Faure E, Thomas L, Equils O andArditi M. Cooperation of Toll-like receptor 2 and 6 for cellular activation by soluble tuberculosis factor and Borreliaburgdorferiouter surface protein A lipoprotein: role of Toll-interacting protein and IL-1 receptor signaling molecules in Toll-like receptor 2 signaling, J. Immunol. 2001; 167:987–994.  Back to cited text no. 6
Hajjar AM, O'Mahony DS, Ozinsky A, Underhill DM, Aderem A, Klebanoff SJ and Wilson CB. Cutting edge: functional interactions between toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin, J. Immunol. 2004; 166 (2001) 15–19.  Back to cited text no. 7
El-Safi SH, Hamid N, Omer A, Abdel-Haleem A, Hammad A, Kareem HG and Boelaert M. Infection rates with Leishmaniadonovani and Mycobacterium tuberculosis in a village in eastern Sudan. Trop Med Int Health. 2004; 9(12): 1305 – 11.  Back to cited text no. 8


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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