MiRNA profile of THP-1 macrophage infected with different MTB strains
ABSTRACT: We examined the microRNA profiles of THP-1 macrophages upon the MTB infection of (1) Beijing/W and non-Beijing/W clinical strains, and (2) susceptible and multidrug-resistant (MDR-) MTB strains. THP-1 cells were induced differentiation into a macrophage phenotype. Then cells were infected with three MDR (INHR, RIFR) Beijing/W, three sensitive (INHS, RIFS) Beijing/W, three MDR(INHR, RIFR) non-Beijing/W, and three sensitive (INHS, RIFS) non-Beijing/W strains. Total RNA were extracted and transfered into cDNA for miRNA profile analysis. Non-infected cells were used as control.
Project description:BACKGROUND:China is a country with high burden of tuberculosis (TB), especially drug-resistant TB (DR-TB), which is still a serious health problem in Yunnan Province. Mycobacterium tuberculosis (MTB) is the pathogenic microorganism of TB. The epidemiological characteristics of MTB strains in local areas need to be described. METHODS:A total of 430 clinical MTB isolates were collected from Yunnan Province and genotyped through the method of 24-locus mycobacterial interspersed repetitive unit-variable number tandem DNA repeats (MIRU-VNTR). RESULTS:The genotypes of the 24 loci showed abundantly genetic diversity, and allelic diversity index (h) of these loci varied from 0.012 to 0.817. Among the 430 strains, 30 clusters and 370 unique genotypes were identified. Beijing family was the predominant lineage (70.47%) in Yunnan MTB strains, and the other lineages contained T family (5.81%), MANU2 (0.70%), LAM (3.26%), CAS (0.23%), New-1 (8.37%), and some unknown clades (11.16%). A total of 74 TB strains were identified as drug resistance through drug susceptibility testing (DST), including 38 multidrug-resistant TB (MDR-TB) and 36 single-drug-resistant TB (SDR-TB). The frequency of MDR-TB strains was significantly higher in Beijing family (10.89%) than that in non-Beijing family (3.94%, P = 0.032). CONCLUSIONS:Although MTB strains showed high genetic diversity in Yunnan, China, the Beijing family was still the dominant strain. A high frequency of MDR-TB strains was recorded in the Beijing family.
Project description:The role of microRNAs in association with Mycobacterium tuberculosis (MTB) infection and the immunology regulated by microRNAs upon MTB infection have not been fully unravelled. We examined the microRNA profiles of THP-1 macrophages upon the MTB infection of Beijing/W and non-Beijing/W clinical strains. We also studied the microRNA profiles of the host macrophages by microarray in a small cohort with active MTB disease, latent infection (LTBI), and from healthy controls.The results revealed that 14 microRNAs differentiated infections of Beijing/W from non-Beijing/W strains (P<0.05). A unique signature of 11 microRNAs in human macrophages was identified to differentiate active MTB disease from LTBI and healthy controls. Pathway analyses of these differentially expressed miRNAs suggest that the immune-regulatory interactions involving TGF-? signalling pathway take part in the dysregulation of critical TB processes in the macrophages, resulting in active expression of both cell communication and signalling transduction systems.We showed for the first time that the Beijing/W TB strains repressed a number of miRNAs expressions which may reflect their virulence characteristics in altering the host response. The unique signatures of 11 microRNAs may deserve further evaluation as candidates for biomarkers in the diagnosis of MTB and Beijing/W infections.
Project description:Background:Levofloxacin (LVX) and Moxifloxacin (MXF) are the cornerstones for treatment of multidrug-resistant tuberculosis (MDR-TB). China is one of the highest MDR- and fluoroquinolones (FQ)-resistant TB burdens countries. DNA gyrase encoded by gyr genes is the main target of FQ in Mycobacterium tuberculosis (MTB). The prevalence and molecular characterization of LVX- and MXF-resistant MTB strains from southern China were examined in this study. Methods:Drug susceptibility testing (DST) of 400 MTB clinical isolates was evaluated by proportion method on Löwenstein-Jensen (LJ) medium against ten drugs. The sequencing of entire gyrA and gyrB genes and multiplex PCR were performed to distinguish the prevalence of mutant types in Beijing and non-Beijing genotypes. Results:Three hundred and twenty-one out of four hundred (80.25%) drug-resistant isolates (resistant > one drug) were categorized as 83/321 (25.80%) MDR, 174/321 (54.20%) pre-XDR and 64/321 (19.93%) XDR-MTB. Overall, 303/400 (75.75%) LVX- and 292/400 (73.00%) MXF-resistant (R) MTB strains were identified. Two hundred seventy-one out of three hundred and three (89.43%) resistant strains carried mutations in gyrA and 91/303 (30.03%) in gyrB. Interestingly, 18 novel mutations were detected in gyrA and gyrB genes. Mutations at (A90, D94) and (T500, G510, G512) frequently existed in QRDR(s) of gyrA and gyrB respectively in 286/400 (71.50%) LVXRMXFR strains. The novel mutations in- and out-side the QRDR of gyrA (L105R, A126E, M127K, D151T, V165A) and gyrB (D461H, N499S, G520A) increased the sensitivity and consistency of genotypic tests. Notably, 25 LVXRMXFR strains were found with unknown resistance mechanisms. Conclusions:Mutations in QRDR(s) were concomitantly associated with Beijing and non-Beijing genotypes. The prevalence of resistance and cross-resistance between LVX and MXF in MTB isolates from southern China was immensely higher than other countries. Our valuable findings provide the substantial implications to improve the reliability of genotypic diagnostic tests relying on potential resistance conferring mutations in entire gyr genes.
Project description:Tuberculosis is a significant global health threat, with one-third of the world's population infected with its causative agent Mycobacterium tuberculosis (Mtb). The emergence of multidrug-resistant (MDR) Mtb that is resistant to the frontline anti-tubercular drugs rifampicin and isoniazid forces treatment with toxic second-line drugs. Currently, ~4% of new and ~21% of previously treated tuberculosis cases are either rifampicin-drug-resistant or MDR Mtb infections1. The specific molecular host-pathogen interactions mediating the rapid worldwide spread of MDR Mtb strains remain poorly understood. W-Beijing Mtb strains are highly prevalent throughout the world and associated with increased drug resistance2. In the early 1990s, closely related MDR W-Beijing Mtb strains (W strains) were identified in large institutional outbreaks in New York City and caused high mortality rates3. The production of interleukin-1? (IL-1?) by macrophages coincides with the shift towards aerobic glycolysis, a metabolic process that mediates protection against drug-susceptible Mtb4. Here, using a collection of MDR W-Mtb strains, we demonstrate that the overexpression of Mtb cell wall lipids, phthiocerol dimycocerosates, bypasses the interleukin 1 receptor, type I (IL-1R1) signalling pathway, instead driving the induction of interferon-? (IFN-?) to reprogram macrophage metabolism. Importantly, Mtb carrying a drug resistance-conferring single nucleotide polymorphism in rpoB (H445Y)5 can modulate host macrophage metabolic reprogramming. These findings transform our mechanistic understanding of how emerging MDR Mtb strains may acquire drug resistance single nucleotide polymorphisms, thereby altering Mtb surface lipid expression and modulating host macrophage metabolic reprogramming.
Project description:Tuberculosis incidence among aborigines is significantly higher than for Han Chinese in Taiwan, but the extent to which Mycobacterium tuberculosis (MTB) strain characteristics contribute to this difference is not well understood. MTB isolates from aborigines and Han Chinese living in eastern and southern Taiwan, the major regions of aborigines, were analyzed by spoligotyping and 24-loci MIRU-VNTR. In eastern Taiwan, 60% of aboriginal patients were ?20 years old, significantly younger than the non-aboriginal patients there; aborigines were more likely to have clustered MTB isolates than Han Chinese (odds ratio (OR)?=?5.98, p<0.0001). MTB lineages with high clustering were EAI (54.9%) among southern people, and Beijing (62.5%) and Haarlem (52.9%) among eastern aborigines. Resistance to first-line drugs and multidrug resistance (MDR) were significantly higher among eastern aborigines (?15%) than in any other geographic and ethnic group (p<0.05); MDR was detected in 5 of 28 eastern aboriginal patients ?20 years old. Among patients from the eastern region, clustered strains (p?=?0.01) and aboriginal ethnicity (p?=?0.04) were independent risk factors for MDR. The lifestyles of aborigines in eastern Taiwan may explain why the percentage of infected aborigines is much higher than for their Han Chinese counterparts. The significantly higher percentage of the MDR-MTB strains in the aboriginal population warrants close attention to control policy and vaccination strategy.
Project description:Prothionamide (PTH) has been widely used in the treatment of tuberculosis (TB), especially multidrug resistant tuberculosis (MDR-TB), while data regarding prevalence of resistance-causing mutation is limited. In this study, we aimed to investigate the molecular characteristics of PTH-resistant MTB isolates, and also analyzed the risk factors for PTH resistance among Mycobacterium tuberculosis (MTB) isolates in southern China. A total of 282 MTB isolates were enrolled in from Guangzhou Chest Hospital. Among these isolates, 46 (16.3%) were resistant to PTH. Statistical analysis revealed that PTH resistance was more likely to be associated with resistance to levofloxacin (LFX; OR: 2.18, 95% CI: 1.02-4.63; P = 0.04). Of the 46 PTH-resistant MTB isolates, 37 (80.4%) isolates harbored 19 different mutation types, including 10 (21.7%) isolates with double nucleotide substitutions and 27 (58.7%) with single nucleotide substitution. The mutations in ethA (51.4%, 19/37) were most frequently observed among PTH-resistant isolates, followed by 16 (43.2%) in the promoter of inhA and 6 (16.2%) in inhA. In addition, no significant difference was found in the distribution of isolates with different mutation types between Beijing and non-Beijing genotypes (P > 0.05). In conclusion, our data demonstrate that high diversity of genetic mutations conferring PTH resistance is identified among MTB isolates from southern China. Mutations in inhA, ethA, mshA, and ndh genes confer increased resistance of MTB to PTH. Ancient Beijing genotype strains have higher proportion of drug resistance compared with modern Beijing strains. In addition, PTH resistance is more likely to be observed in the LFX-resistant MTB isolates.
Project description:Tuberculosis caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) strains is a growing problem in many countries. The availability of the complete nucleotide sequences of several MTB genomes allows to use the comparative genomics as a tool to study the relationships of strains and differences in their evolutionary history including acquisition of drug-resistance. In our work, we sequenced three genomes of Russian MTB strains of different phenotypes--drug susceptible, MDR and XDR. Of them, MDR and XDR strains were collected in Tomsk (Siberia, Russia) during the local TB outbreak in 1998-1999 and belonged to rare KQ and KY families in accordance with IS6110 typing, which are considered endemic for Russia. Based on phylogenetic analysis, our isolates belonged to different genetic families, Beijing, Ural and LAM, which made the direct comparison of their genomes impossible. For this reason we performed their comparison in the broader context of all M. tuberculosis genomes available in GenBank. The list of unique individual non-synonymous SNPs for each sequenced isolate was formed by comparison with all SNPs detected within the same phylogenetic group. For further functional analysis, all proteins with unique SNPs were ascribed to 20 different functional classes based on Clusters of Orthologous Groups (COG). We have confirmed drug resistant status of our isolates that harbored almost all known drug-resistance associated mutations. Unique SNPs of an XDR isolate CTRI-4(XDR), belonging to a Beijing family were compared in more detail with SNPs of additional 14 Russian XDR strains of the same family. Only type specific mutations in genes of repair, replication and recombination system (COG category L) were found common within this group. Probably the other unique SNPs discovered in CTRI-4(XDR) may have an important role in adaptation of this microorganism to its surrounding and in escape from antituberculosis drugs treatment.
Project description:It is unclear to what extent the host-responses elicited by Beijing versus non-Beijing strains of Mycobacterium tuberculosis (MTB) contribute to the predominance of modern Beijing strains in Taiwan and some other Asian countries. The purpose of this study was to compare the expression profiles of virulence-related genes in human monocyte-derived macrophages infected in vitro with Beijing (ancient and modern strains) and non-Beijing strains (EAI strains) of MTB that are epidemic in Taiwan. We found that modern Beijing strains induced lower levels of pro-inflammatory cytokines, whereas EAI strains induced higher levels. Notably, the most prevalent modern Beijing sub-lineage, possessing intact RD150 and RD142 chromosomal regions, induced very low levels of pro-inflammatory cytokines, especially interleukin-1?. Moreover, in an intracellular growth assay, the survival of the same modern Beijing strain in human monocyte-derived macrophages was significantly higher than that of an ancient Beijing strain and an EAI strain. Taken together, these results may explain why modern Beijing strains of MTB predominate in Taiwan.
Project description:Background Molecular epidemiological studies of Mycobacterium tuberculosis (MTB) are the core of current research to find out the association of the M. tuberculosis genotypes with its outbreak and transmission. The high prevalence of the Beijing genotype strain among multidrug resistance (MDR) TB has already been reported in various studies around India. The overall objective of this study was to detect the prevalence of Beijing genotype strains of MDR M. tuberculosis and their association with the clinical characteristics of TB patients. Methods In this study 381?M. tuberculosis clinical isolates were obtained from sputum samples from 2008 to 2014. The multiplex-PCR and Spoligotyping (n?=?131) methods were used to investigate the prevalence of the Beijing genotype strain by targeting the Rv2820 gene and their association with drug resistance and clinical characteristics of TB patients. The drug susceptibility testing of first-line anti-TB drugs was performed by using the proportion method and MGIT960. A collection of isolates having Beijing and non-Beijing strains were also characterized to see if Beijing genotype strains had a higher rate of mutations at codons 516, 526 and 531 of the 81-bp region of the rpoB gene, codon 315 of the katG gene, and codon 306 of the embB gene. Results The sensitivities and specificities of multiplex-PCR assay compared to that of standard Spoligotyping was detected to be 100%. Further, we observe that the multi drug-resistance was significantly associated with Beijing genotype strains (p?=?0.03) and a strong correlation between Beijing genotype strains and specific resistance mutations at the katG315, rpoB531, and embB306 codons (p?=?<?0.0001, <?0.0001 & 0.0014 respectively) was also found. Conclusions This rapid, simple, and cost-effective multiplex PCR assay can effectively be used for monitoring the prevalence of Beijing genotype strains in low resource settings. Findings of this study may provide a scientific basis for the development of new diagnostic tools for detection and effective management of DR-TB in countries with a higher incidence rate of Beijing genotype strains.
Project description:Infants are vulnerable to disseminated forms of tuberculosis and suffer disproportionately high morbidity and mortality, but the reasons for this are unknown. We hypothesized that since alveolar macrophages (AMs) are critical in the uptake and containment of Mycobacterium tuberculosis (Mtb) in the lung, their function may be impaired in early life. We developed a method of obtaining AMs during rigid bronchoscopy of healthy infants with suspected airway abnormality. RNAseq analysis of Mtb-stimulated AMs from 4 infants and 4 adults was performed.