The International Journal of Mycobacteriology

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 9  |  Issue : 4  |  Page : 380--390

Association of disease severity with toll-like receptor polymorphisms in multidrug-resistant tuberculosis patients


Soedarsono Soedarsono1, Muhammad Amin2, Katsushi Tokunaga3, Rika Yuliwulandari4, Desak Nyoman Surya Suameitria Dewi5, Ni Made Mertaniasih6,  
1 Department of Pulmonology and Respiratory Medicine; Laboratory of Tuberculosis, Institute of Tropical Disease, Airlangga University, Surabaya, Indonesia
2 Department of Pulmonology and Respiratory Medicine, Airlangga University, Surabaya, Indonesia
3 Department of Human Genetic, Graduate School of Medicine, Tokyo University, Tokyo, Japan
4 Department of Pharmacology, Faculty of Medicine, Yarsi University, Surabaya, Indonesia
5 Department of Microbiology, Faculty of Medicine, Universitas Ciputra, Surabaya, Indonesia
6 Department of Clinical Microbiology, Faculty of Medicine, Airlangga University, Surabaya, Indonesia

Correspondence Address:
Soedarsono Soedarsono
Jl. Mayjen Prof. Dr. Moestopo No. 47 Surabaya 60131
Indonesia

Abstract

Background: The disease severity in pulmonary Multidrug-resistant tuberculosis (MDR-TB) varies from mild to severe, which is determined by host and pathogen virulence factors. The difference of symptoms felt by TB patients were interesting to investigate in discovering whether its the human immune response or bacteria's virulence gene that plays the role. The aim of this research was to analyze association between disease severity degree of pulmonary MDR-TB patients with Single nucleotide polymorphisms (SNPs) found in toll-like receptors (TLRs) gene. Method: Blood samples were obtained from pulmonary MDR-TB patients in Dr. Soetomo Hospital, Surabaya, Indonesia. Polymerase chain reaction (PCR) multiplex and target SNPs were analyze using DigiTag2 assay. The variant of esxA gene was determined using PCR and sequencing. Severity degree was determined by chest X-ray, the lesions were scored according to their severity, score of =2.5 ranking as mild, 2.5–6 as moderate and =6 as severe. Association level between SNP in TLRs gene degree of pulmonary MDR-TB was analyzed using Chi-square test. Bonferroni correction for multiple comparison was used to anticipate genotyping error. Results: A total of 22 MDR-TB patients were classified into severe degree group, while 16 patients were moderate/mild degree. SNPs in encoding gene of TLRs were mostly found in intron, specifically in TLR-1, TLR-2, and TLR-6. HWE P value in rs5743572 was 0.841; in rs3804100 was 0.0176; and in rs5743808 was 0.562. Association analysis between SNP in TLRs genes and degree of disease revealed significant association in rs5743572, SNP of TLR-1, with P < 0.05; odds ratio [OR] = 11.67 (95% confidence interval [CI]: 3.94–34.52); rs3804100, SNP of TLR-2 had P < 0.05; OR = 37.59 (95% CI: 9.30–151.88); and rs5743808, the SNP of TLR-6 had P < 0.05; OR = 31.5 (95% CI: 8.60–115.34). Conclusions: We concluded that SNPs in TLR-1, TLR-2, and TLR-6 of pulmonary MDR-TB patients was found to have an association with disease severity. TLRs polymorphism had significant association was present in TLR-1 rs5743572 in intron, TLR-2 rs3804100 in exon, and TLR-6 rs5743808 in exon and among MDR-TB isolates from patients with pulmonary MDR-TB of severe and moderate/mild degree.



How to cite this article:
Soedarsono S, Amin M, Tokunaga K, Yuliwulandari R, Suameitria Dewi DN, Mertaniasih NM. Association of disease severity with toll-like receptor polymorphisms in multidrug-resistant tuberculosis patients.Int J Mycobacteriol 2020;9:380-390


How to cite this URL:
Soedarsono S, Amin M, Tokunaga K, Yuliwulandari R, Suameitria Dewi DN, Mertaniasih NM. Association of disease severity with toll-like receptor polymorphisms in multidrug-resistant tuberculosis patients. Int J Mycobacteriol [serial online] 2020 [cited 2021 Mar 7 ];9:380-390
Available from: https://www.ijmyco.org/text.asp?2020/9/4/380/303448


Full Text



 Introduction



Multidrug-resistant tuberculosis (MDR-TB) occurs when Mycobacterium tuberculosis (M. tuberculosis) is resistant to both potent first-line anti–TB drugs, namely, rifampicin (R) and isoniazid (H). Indonesia is one of 27 countries with high MDR-TB prevalence and is ranked ninth among countries with a high MDR-TB burden.[1] According to the Global TB Report in 2015, 1812 MDR-TB cases were reported in Indonesia, but only 1284 patients (71%) were included in the MDR-TB treatment program.[2] The WHO revealed that MDR-TB causes a high mortality rate, has a higher virulence and is more infectious than non-MDR TB.[3]

One of the reasons for the low enrollment rates of the treatment program was that some patients with MDR-TB only felt that their symptoms of disease were mild; thus, patients felt that they needed no treatment because the period of treatment itself can take a long time (18–24 months). The difference in symptoms experienced by TB patients was investigated to discover whether the human immune response or bacterial virulence genes play a role.

Several in vitro studies on the fitness cost of drug-resistant M. tuberculosis showed that mutant bacteria were fit; however, a different study discovered contradictory findings (i.e., that the mutant bacteria were less fit).[4] The study revealed that a lower fitness caused a reduction in the transmission power and destructive effect of the bacteria on host tissue. The previous research also showed no difference between the transmission power of anti-TB-sensitive and anti-TB-resistant strains.[5] Other molecular epidemiology studies have also shown contradictory results.[6],[7]

The causes or range of severity that exists in pulmonary MDR-TB in patients has yet to be explained. One of the suspected causes is that the mechanism of Toll-like receptors (TLRs) of each individual is different. Examining the mechanisms of innate immunity induced by pathogen invasion in the host conducted through TLRs is important. TLR stimulation is initiated by a signaling cascade that recruits many proteins, such as MyD88, as a common adapter involving the recruitment of IRAK and TRAF-6, leading to activation of MAPK and nuclear translocation of activated activator protein 1 (AP-1) and nuclear factor-kB (NF-kB).[8] The signaling cascade activates NF-kB, which induces the secretion of proinflammatory cytokines.[8] Several of the released proinflammatory cytokines will determine the manifestation of disease, mainly caused by the process of tissue destruction.

Single nucleotide polymorphisms (SNPs) in TLRs, such as TLR-1, TLR-2, TLR-3, TLR-4, TLR-6, TLR-8, and TLR-9, are thought to play a role in the recognition of M. tuberculosis.[9] Different manifestations of infectious disease usually cannot be explained by knowing the phenotypic risk factors alone. TLR gene polymorphisms may explain varying natural immune responses to M. tuberculosis that cause differences in disease manifestation.[10] Different manifestations of TB disease severity reflect a balance between pathogens and host immune responses.[11]

The severity degree of MDR-TB in patients could be determined by examining the profile of pulmonary radiography or chest X-ray. The pulmonary radiographic profile provides information on the severity of the patients' pulmonary TB (PTB). Ralph et al.[12] also reported that the assessment of the thoracic radiographic profile using numerical scoring might describe the severity of PTB disease.

In addition, we also examined whether changes in the virulence genes, such as the esxA gene, which releases ESAT-6, the essential protein affecting virulence, could affect the range of severity. Previous studies have reported that aside from mutations in genes involved in anti-TB drug resistance, the influence of agents on different symptoms can be determined by discovering whether there is a sequence mutation in potent virulence genes, such as ESAT-6.[13],[14] The esxA gene is considered a major pathogenic determinant and is known to play a crucial role in M. tuberculosis invasion and modulate macrophage activation to reduce virulence.[15],[16]

Previous studies only focused on investigating the relationship between TLR polymorphism and infection progress, investigating the effect of SNPs on the susceptibility and severity of PTB in the Asian Indian population, and these studies only reviewed numerous other studies regarding the SNPs in TLR receptors and susceptibility to infectious disease.[17],[18],[19]

However, in this research, we aimed to analyze TLR polymorphisms by detecting TLR1-TLR12 using a digital assay method, and we also analyzed Indonesian population samples, especially groups of mild, moderate, and severe pulmonary MDR-TB patients in Indonesia. Moreover, we sought to detect the variation in the esxA gene by polymerase chain reaction (PCR) and sequencing between the mild-moderate pulmonary MDR-TB group and severe pulmonary MDR-TB group objectively, based on the severity of the effect on lung parenchyma in their chest X-rays.

 Method



Study design, setting, and facility

This study had an observational analytic cross-sectional design. A total of 182 blood samples and clinical isolates were collected from Dr Soetomo Hospital from the end of 2013 until 2015. Samples were taken from pulmonary MDR-TB patients confirmed to have received or who were receiving MDR treatment and who were registered with demographic, clinical and radiological data at the MDR-TB outpatient clinic, Dr. Soetomo Hospital, with M. tuberculosis isolates identified as causative pathogens stored at the Center for Health Laboratory Surabaya. Data were collected after receiving approval from the IRB/ethics committee on health research of Dr. Soetomo Hospital (number 347/Panke. KKE/VI/2015).

Inclusion and exclusion criteria

Patients with pulmonary MDR-TB were confirmed as adults in terms of age based on Indonesian law, which was >21-year-old, who were willing to participate in this study with written consent. The exclusion criteria were patients with pulmonary MDR-TB with comorbid conditions such as diabetes, kidney failure, liver failure, bronchial asthma, chronic obstructive pulmonary disease and HIV, as well as MDR-TB patients who were being treated with corticosteroids. From 182 samples, 38 samples met the requirements.

Data collection and sample collection

The severity degree on chest X-ray was determined using a scoring method adapted from Yan et al.[20] Based on this scoring system, each lung was divided into three areas by drawing two horizontal lines at the carina level and lower level of the pulmonary vein, thus forming six areas. Each of these areas was assessed for severity scores based on the size of the lesions and cavities.

The final score from the severity of lung disease was acquired from the sum of each of the six lung area scores, and the details are shown in [Table 1]. The definition of moderate/mild and severe groups used in this study was according to the total score in [Table 2].{Table 1}{Table 2}

All isolated MDR-TB bacteria samples were stored in the Center for Health Laboratory Surabaya. Blood samples were also isolated from the infected patients. For further analysis, MDR-TB patients of moderate and mild degree were grouped into one moderate/mild group; thus, the combined group analyzed comprised two groups. The groups of severe and moderate/mild degree were then associated with SNPs found in TLR gene nucleotide sequences in patients with pulmonary MDR-TB, and the presence of nucleotide sequence variants of the esxA gene isolated from M. tuberculosis derived from pulmonary MDR-TB patient specimens was determined.

TLR-encoding genes were obtained from blood samples of pulmonary MDR-TB patients. PCR multiplex and target SNPs were genotyped using a DigiTag2 assay, which was adapted from Kobayashi et al.[21]

DigiTag2 procedure

DNA from blood samples was extracted with a QIAampTM DNA Blood Midi Kit (Qiagen GmbH, Max Volmer Straβe 4 Hilden, Germany). The DigiTag2 assay was conducted in the Human Genetic Laboratory, Tokyo University, Japan. The DigiTag2 assay can detect 96 or 32 SNPs with high accuracy. This research used 2 sets of 32-plex SNP typing arrays to detect the DNA samples. The procedure of this method was initiated by a multiplex PCR process.

The mixture solution consisted of 0.5 μl Multiplex PCR primer, ×5 multiplex PCR buffer, deoxynucleoside triphosphate (dNTPs), MgCl2, enzyme Kapa 2G Fast HS, dH2O, and 10 ng/μl genomic DNA. Amplification was performed using a Biometra Tgradient machine. The next step was encoding the reaction, which was performed by mixing the multiplex product with master mix that consisted of Q/C mix (50 nM), Taq DNA ligase buffer, dH2O, and Taq DNA ligase. The mixture was incubated at 95°C for 5 min and 58°C for 15 min before being cooled to 10°C using a Biometra Tgradient.

The third step was labeling the product from the previous action. The 6.0 μl ligation product was mixed into 6 μl labeling master mix consisting of Code mix (0.5 μM), primer mix (25 μM), 5x Buffer, 10 mM dNTPs, MgCl2 (25 mM), KAPA 2G Fast HS (5 U/μl) and dH2O. The mixtures were incubated at 95°C for 1 min followed by 25 cycles at 95°C for 15 s, 55°C for 2 min and 72°C for 5 s using a Biometra Tragradient. The last step was hybridization. This research used a hybrid control (3.75 fmol Alexa 555-labeled D1_100 and Alexa 647 labeled D1_100) with a hybrid solution containing 0.5 x standard sodium citrate (SSC), 0.1% sodium dodecyl sulfate (SDS), 15% formamide, and 1 mM ethylenediaminetetraacetic acid as the mixture.

Afterward, 8.0 μl from the hybridization mixture was applied to each area in the DNA microarray. The incubation process took 1 h at 37°C in a hybridization oven (Thermostat plus, Eppendorf). Later, the microarray was washed for 3 min using liquid buffer consisting of 0.1 × SSC and 0.1% SDS and was then washed again for 1 min in distilled water and dried through centrifugation for 1 min at 900 × g. The fluorescence image was analyzed using GENEPIX 400B and GENEPIX Pro 4.1 (Axon Instruments, Union City, CA).

Severity evaluation method

The association of the TLR gene and disease degree in the severe and moderate/mild MDR-TB groups was analyzed at both the allelic and genotypic levels. At the genotypic level, the analysis was performed using 3 models: Codominant, A-dominant, and A-recessive. In the codominant model, both alleles were assumed to have the same role. In the A-dominant model, one allele (for example, “A”) was assumed to have a more dominant role than the second allele (for example, “B”). In the A-recessive model, one allele (for example, “A”) was assumed to have a more recessive role than the second allele (for example, “B”).

Statistical calculations

The degree of association between SNPs in the TLR gene of pulmonary MDR-TB was analyzed statistically using the Chi-square test at a 5% (P = 0.05) significance level with Bonferroni correction to anticipate genotyping error. The P value was multiplied by the total number of SNPs analyzed (n = 30). The Hardy–Weinberg equilibrium (HWE) value is a model and theorem that is used for the analysis allele and genotype frequency and has also become a standard for analyzing large SNP databases used in genetic studies. The HWE measure of the genetic/allelic continuity of a specific population is useful in determining how specific mutations occur and their source.[22] The HWE value of each SNP as a standard for genetic analysis that was used in this research is shown in [Table 3].[21]{Table 3}

DNA isolation, polymerase chain reaction, and sequencing of the esxA gene

Variant of the esxA gene from MDR-TB isolates was determined using PCR at the Institute of Tropical Disease, Surabaya, Indonesia. DNA isolation for the esxA examination was conducted using a QiAmp DNA Kit (QIAGEN). The PCR solution consisted of the KAPA2G Fast Ready Mix PCR Kit as PCR Master Mix, a pair of LIC-ESAT-6 primers (R: 5'-GAG GAG AAG CCC GGT TGC CCT TTC GCT ATT CTA CG-3' and F: 5'-GAC GAC GAC AAG ATG ACA GAG CAG CAG TGG AAT-3'), nuclease-free water, and the DNA template.

The PCR product was sent to 1st Base using an ABI PRISM 3730 × l Genetic Analyzer. The sequence results were analyzed using GENETYX Ver. 10. The PCR product was sent to 1st Base, Singapore for sequencing. Sequencing of the esxA gene was performed using an ABI PRISM 3730 × l Genetic Analyzer with 96 capillaries developed by Applied Biosystems and a Big Dye ® Terminator v3.1 Cycle Sequencing Kit.[23]

 Results



Background information of patients

Thirty-eight patients with pulmonary MDR-TB (21 men and 17 women) were analyzed. The ages of the patients examined were between 21 and 64 years. Most of the patient isolates were resistant to rifampicin, isoniazid, and ethambutol (RHE) and the isolates of as many as 18 patients (47.37%) and 11 patients (28.95%) were resistant to rifampicin, isoniazid, ethambutol, and streptomycin (RHES). Eight patient isolates (21.05%) were resistant to rifampicin and isoniazid (RH), and one patient isolate (2.63%) was resistant to rifampicin, isoniazid and streptomycin (RHS).

Degree of severity

By means of scoring abnormalities found on chest X-rays, 22 of the 38 patients (57.9%) had severe pulmonary MDR-TB with a mean score of 7.61, followed by 10 patients (26.3%) with a moderate MDR-TB with a mean score of 4.50, and the lowest number, 6 patients (15.8%), with mild MDR-TB and a mean score of 1.58. The lowest score for the mild degree of severity was 1.0, while the highest score was 1.5, with a mean of 1.58. The lowest score for the moderate severity group was 3, and the highest score was 5.5, with a mean of 4.5. For the severe degree of severity, the lowest score was 6, while the highest score was 11, with a mean of 7.61 (data not shown).

The severe degree group comprised approximately 22 patients (57.9%) with a score greater than 6 points, and the moderate/mild degree group comprised 16 patients (42.1%) with a score between 0 and 5 [Table 4].{Table 4}

The severe and moderate/mild severity degree groups were then correlated with SNPs found in TLR gene nucleotide sequences in patients with pulmonary MDR-TB, and the presence of nucleotide sequence variants of the esxA gene isolated from M. tuberculosis derived from pulmonary MDR-TB patient specimens was determined.

The profile of lung damage observed on a chest X-ray in patients with pulmonary MDR-TB was based on lung destruction, either bilateral or unilateral, and whether cavities were present or absent. In the current study, among pulmonary MDR-TB patients with severe disease, 18 patients (81.82%) had bilateral lung damage and 4 patients (18.18%) had unilateral lung damage. Ten patients (45.45%) with severe disease had a lung cavity, and the remaining 12 patients (54.55%) had no lung cavities. In the group of patients with pulmonary MDR-TB disease of moderate/mild severity degree, 6 patients (37.5%) had bilateral lung damage, 10 patients (62.5%) had unilateral lung damage, 4 patients (25%) had a cavity, and 12 patients (75%) had no cavities [Table 5].{Table 5}

Single nucleotide polymorphisms distribution in the nucleotide sequence of the toll-like receptors protein signaling gene in pulmonary multidrug-resistant tuberculosis patients

We found SNPs with specific alleles in the analysis of the nucleotide sequence of protein signaling genes TLR-1 through TLR-9, of the blood cells of all patients with pulmonary MDR-TB of severe and moderate/mild severity degree (TLR-5 was not evaluated further due to loss of quality control).

In the TLR-1 signaling gene sequence, SNPs were apparent in the intron. The SNP located in the exon was rs4833095, while the SNPs in the intron were rs5743599, rs5743572, rs5743567, rs5743564, and rs2101521. In the TLR-1 gene, in the exon, we found SNP rs4833095 with genotype C/C (40.9%) among pulmonary MDR-TB patients of a severe degree and genotype T/C (50%) among pulmonary MDR-TB patients of a moderate/mild degree. In SNP rs5743599, we found allele G/G (40.91%) and A/G (40.91%) among patients of a severe degree and allele A/G (62.5%) in patients of a moderate/mild degree.

In the TLR-2 gene sequence, we found two SNPs, one in the intron and the other in exon. In each group of pulmonary MDR-TB patients, the highest SNP percentages were found with the same genotype, either in the intron or exon. In the exon, SNP rs3804100 with A/A (62.5%) was found in moderate/mild MDR-TB patients, while in severe MDR-TB patients, A/A (45.46%) was found. In the TLR-3 gene sequence, 12 SNPs were all in the intron. In all SNPs with the highest percentage, the genotypes were found to be similar in both severe and moderate/mild MDR-TB patients.

In the TLR-4 gene sequence, all SNPs were found in the intron. In rs1098375 and rs1153688, the same alleles were found with the highest percentage in MDR-TB patients of both severe degree and moderate/mild degree. The SNP profile in the TLR-6 signaling gene sequence revealed a SNP in the exon and one in the intron. The allele with the highest percentage in all SNPs was found to be the same in severe and moderate/mild MDR-TB patients, with SNP rs5743808 in the exon having the T/T genotype in 87.5% of the moderate/mild degree group and in 68.18% of the severe degree group.

In the TLR-7 signaling gene sequence, it was apparent that all SNPs were found in the intron, including rs5935436, rs1634323, rs179016, and rs179012. All SNPs showed the same alleles with the highest percentage between pulmonary MDR-TB patients of both the severe degree and moderate/mild degree. In the TLR-8 gene sequence, we found only SNP rs3764880 in the intron, and there was no difference in the genotype with the highest percentage between the severe degree and moderate/mild degree groups. The SNP profile in the TLR-9 gene sequence only showed SNP rs352139 in the intron, for which the G/G genotype (50%) among MDR-TB patients of a severe degree and the A/G genotype (56.25%) among MDR-TB patients of a moderate/mild patients were found.

Association between single nucleotide polymorphisms in the toll-like receptors genes of pulmonary multidrug-resistant tuberculosis patients and the degree of disease

The association between SNPs in the TLR gene and the degree of pulmonary MDR-TB severity was analyzed statistically using the Chi-square test at a 5% (P = 0.05) significance level. The Bonferroni correction for multiple comparisons was used to anticipate a genotyping error. The P value was multiplied by the total SNP number analyzed (n = 30).

TheHWE P value in rs5743572 was 0.841; in rs3804100, the P value was 0.0176; and in rs5743808 the P value was 0.562. Only SNPs with HWE > 0.0016 were included in the analysis. The odds ratio (OR) with 95% confidence interval (CI) is also displayed in [Table 6]. Association analysis between SNPs in the TLR genes and the degree of disease severity revealed a significant association with rs5743572 in TLR-1 (P < 0.05, OR = 11.67, 95% CI: 3.94–34.52); rs3804100 in TLR-2 (P < 0.05, OR = 37.59, 95% CI: 9.30–151.88); and rs5743808 in TLR-6 (P < 0.05, OR = 31.5, 95% CI: 8.60–115.34) [Table 6].{Table 6}

Toll-like receptors structure model analysis

Analysis of the TLR-1 protein structure model (Swiss model program) showed the presence of SNP rs5743572 (in intron) T/T, T/C, and C/C, resulting in two variants of protein amino acids Asparagine (Asn) 248 and Serine (Ser) 248 in the tertiary structure of protein TLR-1, which was the protein domain on which the interaction between the receptor ligand and epitope of M. tuberculosis occurred.

The conformation of the TLR-1 receptor protein structure of both proteins was not altered, but several amino acids showed changes, namely, Asparagine and Serine at position 248 [Figure 1];a and [Figure 1]b. The TLR-2 protein structure model was apparent in exon SNP rs3804100 A/A, A/G, as well as G/G in the third base position that did not alter any amino acids. In the TLR-2 protein structure model, no change was observed in the conformational structure of either the protein or amino acid composition.{Figure 1}

The TLR-6 protein structure model was present in exon SNP rs5743808 T/C, C/C and T/T, with two protein variants, isoleucine (Ile) 120 and threonine (Thr) 120. The conformation of the TLR-6 receptor protein structure of both proteins was not altered, but several amino acids were changed, namely, isoleucine and threonine at position 120 [Figure 1];c and [Figure 1]d.

Sequence analysis of the esxA gene

The sequence of the esxA gene from clinical isolate samples was analyzed in the open reading frame (ORF) region with nucleotide base length 289 by PCR, and a positive result was detected at 351 bp [Figure 2]. Analysis of the nucleotide sequence in the esxA gene from the M. tuberculosis isolate was derived from specimens from pulmonary MDR-TB patients with severe and moderate/mild disease. No apparent difference was found in the nucleotide structure compared to the esxA gene nucleotide sequence from the virulent wild type strain of M. tuberculosis H37Rv [Figure 3].{Figure 2}{Figure 3}

Analysis of epitope protein prediction in the esxA protein using the Genetyc Ver 10 program demonstrated that the esxA protein had 4 T-cell epitopes; ASAIQS, VTSIHS, TKLAAA and VTGMFA. The amino acid sequence of the epitopes was in position 4–9, 11–16, 26–31 and 79–84, respectively.

 Discussion



A study by Zahirifard et al.[24] on the chest X-ray profile of pulmonary MDR-TB showed that there are often extensive and destructive abnormal patterns present. The damage visible on chest X-ray mostly involved both lungs (bilateral). The existence of multiple cavities, infiltrative lesions, nodular and pleural effusion are the main features of pulmonary MDR-TB.

In this study, among all patients with pulmonary MDR-TB, 63.16% of patients with pulmonary MDR-TB showed bilateral pulmonary damage, while the remaining 36.84% showed unilateral pulmonary damage. Lesions that presented as cavities on chest X-ray were found in 36.84% of all patients with pulmonary MDR-TB, while the remaining 63.16% did not indicate the presence of cavities. A severe degree of disease was based on the scoring system of Yan et al.[20] and this severe disease was most prevalent among patients with pulmonary MDR-TB (57.9%).

In the pathogenesis of pulmonary MDR-TB, there was the possibility of an extensive pathological destruction process in the lesion site; thus, the pathological damage process continued to progress along with the formation of cavities, either single or multiple. This process could also be associated with continuous active multiplication of MDR-TB pathogens due to anti-TB drug resistance.

In sequence analysis of the protein signaling genes TLR-1, 2, 3, 4, 6, 7, 8, and 9 of blood cells in pulmonary MDR-TB patients, SNP alleles were found at the intron position in all TLR protein signaling genes. SNPs in the exon position were only present in TLR-1, TLR-2 and TLR-6 protein signaling genes. There were differences in the SNP frequency between pulmonary MDR-TB of a severe degree and of a moderate/mild degree. SNPs in the intron position of the TLR-1 gene in pulmonary MDR-TB patients of a severe degree showed protein differences compared to those in pulmonary MDR-TB patients of a moderate/mild degree. Moreover, TLR polymorphism in healthy controls is shown in Table 3. The data included in this manuscript are from a previous publication.[21]

TLR-1 is a type I transmembrane protein that has 786 amino acids with a 581-amino acid leucine-rich extracellular domain, a 23-amino acid transmembrane domain (amino acids 582–604) and a 181-amino acid cytoplasmic signaling domain. Human TLR-1 plays a particularly important role in host defense against M. tuberculosis. To date, studies on TLR polymorphisms, including TLR-1, have tended to be more associated with the onset of TB sensitivity. Various studies have mostly shown that TLR-1 rs4833095 and TLR-1 rs5743618 are significantly associated with TB incidence.[13] However, thus far, no studies have been conducted relating TLR-1 to the severity of TB patients' suffering. In this study, a strong association was proven to be present between TLR-1 rs 5743572 in the intron position and the severity of pulmonary MDR-TB disease in patients of a severe degree and moderate/mild degree of disease.

The TLR-2 polymorphism associated with the sensitivity of individuals acquiring TB has previously been studied and has been extensively shown.[25] However, no study has demonstrated an association with TB disease severity in the lungs. A study by Thuong et al. reported a strong correlation between SNP T597C TLR-2 gene and the development of PTB and meningitis TB, which indicated that the TLR-2 gene had an effect on the dissemination of M. tuberculosis.[26]

Some studies summarized by Texereau et al. reported that TLR-2 polymorphism was related to the severity of infectious diseases such as Streptococcus aureus, Streptococcus pneumoniae and M. tuberculosis.[27] However, another study showed that TLR-2 and TLR-4 did not play important roles in Chagas disease.[28] Further study is required to confirm this association. In this study, there was no difference found in the TLR-2 protein in pulmonary MDR-TB patients of severe and moderate/mild degree, but in terms of the SNP frequency, a significant difference was found between the two groups of pulmonary MDR-TB patients examined.

A study by Thuong et al. and the current study, although using different parameters in assessing the severity of the TB disease process, showed the same outcome. Thuong's study measured the presence of TB dissemination (extrapulmonary),[26] while this current study measured damage severity in the lungs. Both studies resulted in a significant association between certain SNPs on TLR-2 and the severity of the disease.

TLR-6 had only one exon and comprised 796 amino acid polypeptides. TLR-6 was expressed in the spleen and peripheral white blood cells and was a coreceptor for TLR-2. TLR-6 was activated through MyD88 and TRAF6, resulting in NF-kB activation, cytokine secretion, and the inflammatory response. In this study, TLR-6 rs5743808 in the exon position had been shown to have a strong association with the severity of pulmonary MDR-TB in patients with both severe and moderate/mild disease. The results of this study were different from,[13] in which the TLR-6 allele rs5743810 was reported to be associated with TB. The association indicated that TLR-6 was not a sensitivity factor for TB emergence, but rather, it was protective in nature for most ethnicities. This difference was understandable, since SNPs found in the respective studies were also different.

In humans, gene structure is composed mainly of exons and introns. SNPs located in exons might cause changes in amino acids that are composed of proteins and result in alterations of protein functional structure. Although not directly determined by amino acid sequences, SNPs in the intron position play a role in the process of mRNA splicing. Changes in SNPs might result in alterations of the protein concentration produced.[29]

In the study of SNPs among M. tuberculosis isolated from patients with pulmonary MDR-TB of a severe degree or moderate/mild degree of disease, no SNPs or mutations were found in the sequence of the ORF gene region along 289 nucleotide bases. The ORF is part of a nucleotide sequence potentially encoding proteins. Although not directly involved in encoding proteins, both regions played a role in the activation of the protein transcription process.

However, although it could be stated that there was no difference in the structure of the functional protein ESAT-6, it was still possible that different SNPs were present in downstream or upstream gene regions located outside the esxA gene ORF, which played a role in ESAT-6 protein expression and regulation of protein production, resulting in different concentrations of the ESAT-6 protein product. On the evaluation of the M. tuberculosis genome complex, there had never been a study performed on the presence of SNPs both in upstream and downstream regions, which were noncoding regions that might function as regulators and could determine protein expression or expression concentration.

A previous study involving the sequencing of 24 M. tuberculosis antigens from 16 clinical strains revealed that there was no variation in the esxA gene sequence.[30] Another study showed that the genetic diversity of esxA, esxH and fbpB genes among 88 clinical isolates reported the same finding, namely, that there was no SNP found at esxA.[31]

The ORF of the esxA gene encoding the ESAT-6 protein from the isolate population of patients of a severe or moderate/mild degree of pulmonary MDR-TB was similar or identical to the sequence of the ORF from the wild-type virulence M. tuberculosis H37Rv. Therefore, the functional structure of the protein epitope of immunodominant and pathogenesis determinants that induced disease was the same as those of the virulence strains.

The ORF of the gene encoding ESAT-6 protein signaling is a conserved region. In this study, the sequence of the esxA ORF was extracted from the MDR-TB strain of patients with similar chronic disease conditions, possibly derived from the same microenvironment supporting the formation of stable amino acid structures or conserved nucleotide sequences. A study by Mertaniasih et al. found similar results in which no SNP was found in the nucleotide sequence of the esxA genes of M. tuberculosis among clinical isolates from the sputum of TB patients sensitive to first-line anti-TB drugs. The esxA nucleotide sequence was examined along with base 351. Prediction of the T-cell epitopes found four epitopes that were also synonymous with the current findings. The strains identified from isolates of patients with pulmonary MDR-TB possibly originated from the same geographic area.[32]

The results of this study and reports from other authors suggested the possibility that the ESAT-6 protein structure was stable. This finding could be attributed to the role of TB pathogenesis that is focused on the inflammatory reaction of inducing M. tuberculosis virulence agent secretion, including ESAT-6.[32]

Based on the esxA gene from the MDR-TB isolate, which is the most virulent gene of M. tuberculosis, there were no changes found in the arrangement of the nucleotide esxA gene compared to the wild-type M. tuberculosis. Previous studies have shown that the esxA gene has the potential to be a gene target for the M. tuberculosis complex.[11]

However, the authors did not check the sequence of the esxA gene in the MDR-TB sample. The results of this research confirmed that the sequence of the esxA gene is still conserved even in the MDR-TB sample and emphasized that the virulence gene in the M. tuberculosis mutant can work well. New in this study is the finding that shows the relationship between disease severity and polymorphisms in TLR-1, 2, and 6.

A limitation of this study was that the sequencing analysis had not been carried out outside of the ORF region, such as in the downstream or upstream regions. In addition, there were several virulence genes, such Ag85 and CFP-10, that were not measured in this study. It could be beneficial if other virulence genes besides ESAT-6 in MDR-TB patients could be analyzed by whole genome sequencing; these other genes form the region of the regulator gene that can be evaluated in patients with MDR-TB and other forms of TB. Moreover, studies of whole genome sequencing in regions of TLR genes could resolve the controversy over the role of some TLRs in TB patients.

Based on the results of this research, it should be understood that in MDR-TB patients with disease of mild severity, even if the symptoms are not severe, the bacteria are not less virulent. Physicians are advised to remain cautious when managing pulmonary MDR-TB patients who have been bacteriologically confirmed despite being of a mild severity. Physicians need to give comprehensive education to patients; thus, they will be more likely to agree to receive the full medication regimen, which will prevent transmission to other people.

 Conclusion



TLR polymorphism in pulmonary MDR-TB patients with a significant association with severe and moderate/mild disease was present as TLR-1 rs5743572 in the intron, TLR-2 rs3804100 in the exon, and TLR-6 rs5743808 in the exon. There was no difference in the functional structure of the ESAT-6 protein among the isolates of MDR M. tuberculosis, and no variation was found in the esxA gene.

Acknowledgment

We would like to thank the Chief of Dr. Soetomo Hospital, Surabaya, Indonesia, the Chairman of the Institute of Tropical Disease, Airlangga University, Surabaya, Indonesia and the Dean of the Faculty of Medicine, Airlangga University, Surabaya, Indonesia. In addition, members of the Laboratory of Human Genetics, Graduate School of Medicine, Tokyo University, Japan; members of the Center for Health Laboratory, Surabaya, Indonesia; and the Institute of Tropical Diseases Universitas Airlangga crew, especially Agnes Dwi Sis Perwitasari, Amd. K, S. Si, Mochamad Amin, S. Si., M. Si. and Much Zaenal Fanani for helping with the data analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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