scholarly journals CovR and VicRKX Regulate Transcription of the Collagen Binding Protein Cnm ofStreptococcus mutans

2018 ◽  
Vol 200 (23) ◽  
Author(s):  
Lívia Araújo Alves ◽  
Tridib Ganguly ◽  
Renata O. Mattos-Graner ◽  
Jessica Kajfasz ◽  
Erika N. Harth-Chu ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Bacterial Infections ◽  
Regulatory Region ◽  
Core Gene ◽  
Negative Regulator ◽  
Mobility Shift ◽  
Content Type ◽  
Intracellular Invasion ◽  
Systemic Infections

ABSTRACTCnm is a surface-associated protein present in a subset ofStreptococcus mutansstrains that mediates binding to extracellular matrices, intracellular invasion, and virulence. Here, we showed thatcnmtranscription is controlled by the global regulators CovR and VicRKX.In silicoanalysis identified multiple putative CovR- and VicR-binding motifs in the regulatory region ofcnmas well as in the downstream genepgfS, which is associated with the posttranslational modification of Cnm. Electrophoretic mobility shift assays revealed that CovR and VicR specifically and independently bind to thecnmandpgfSpromoter regions. Quantitative real-time PCR and Western blot analyses of ΔcovRand ΔvicKstrains as well as of a strain overexpressingvicRKXrevealed that CovR functions as a positive regulator ofcnm, whereas VicRKX acts as a negative regulator. In agreement with the role of VicRKX as a repressor, the ΔvicKstrain showed enhanced binding to collagen and laminin and higher intracellular invasion rates. Overexpression ofvicRKXwas associated with decreased rates of intracellular invasion but did not affect collagen or lamin binding activities, suggesting that this system controls additional genes involved in binding to these extracellular matrix proteins. As expected, based on the role of CovR incnmregulation, the ΔcovRstrain showed decreased intracellular invasion rates, but, unexpectedly collagen and laminin binding activities were increased in this mutant strain. Collectively, the results presented here expand the repertoire of virulence-related genes regulated by CovR and VicRKX to include the core genepgfSand the noncore genecnm.IMPORTANCEStreptococcus mutansis a major pathogen associated with dental caries and also implicated in systemic infections, in particular, infective endocarditis. The Cnm adhesin ofS. mutansis an important virulence factor associated with systemic infections and caries severity. Despite its role in virulence, the regulatory mechanisms governingcnmexpression are poorly understood. Here, we describe the identification of two independent regulatory systems controlling the transcription ofcnmand the downstreampgfS-pgfM1-pgfE-pgfM2operon. A better understanding of the mechanisms controlling expression of virulence factors like Cnm can facilitate the development of new strategies to treat bacterial infections.

scholarly journals Transcription of the Streptococcus pyogenes Hyaluronic Acid Capsule Biosynthesis Operon Is Regulated by Previously Unknown Upstream Elements

2014 ◽  
Vol 82 (12) ◽  
pp. 5293-5307 ◽  
Author(s):  
Marina Falaleeva ◽  
Oliwia W. Zurek ◽  
Robert L. Watkins ◽  
Robert W. Reed ◽  
Hadeel Ali ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Streptococcus Pyogenes ◽  
Regulatory Region ◽  
Regulatory System ◽  
Invasive Disease ◽  
Negative Regulator ◽  
Mobility Shift ◽  
Putative Promoter ◽  
Transcriptional Terminator ◽  
Content Type

ABSTRACTThe important human pathogenStreptococcus pyogenes(group AStreptococcus[GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that thehasABCoperon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription ofhasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion ofhasSor of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through,hasAtranscription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence.

scholarly journals Interleukin-7 Produced by Intestinal Epithelial Cells in Response to Citrobacter rodentium Infection Plays a Major Role in Innate Immunity against This Pathogen

2015 ◽  
Vol 83 (8) ◽  
pp. 3213-3223 ◽  
Author(s):  
Wei Zhang ◽  
Jiang-Yuan Du ◽  
Qing Yu ◽  
Jun-O Jin
Keyword(s):  
Epithelial Cells ◽  
Protective Immunity ◽  
Bacterial Infections ◽  
Intestinal Epithelial Cells ◽  
Intestinal Damage ◽  
Intestinal Epithelial ◽  
Citrobacter Rodentium ◽  
Content Type ◽  
Interleukin 7

Interleukin-7 (IL-7) engages multiple mechanisms to overcome chronic viral infections, but the role of IL-7 in bacterial infections, especially enteric bacterial infections, remains unclear. Here we characterized the previously unexplored role of IL-7 in the innate immune response to the attaching and effacing bacteriumCitrobacter rodentium.C. rodentiuminfection induced IL-7 production from intestinal epithelial cells (IECs). IL-7 production from IECs in response toC. rodentiumwas dependent on gamma interferon (IFN-γ)-producing NK1.1+cells and IL-12. Treatment with anti-IL-7Rα antibody duringC. rodentiuminfection resulted in a higher bacterial burden, enhanced intestinal damage, and greater weight loss and mortality than observed with the control IgG treatment. IEC-produced IL-7 was only essential for protective immunity againstC. rodentiumduring the first 6 days after infection. An impaired bacterial clearance upon IL-7Rα blockade was associated with a significant decrease in macrophage accumulation and activation in the colon. Moreover,C. rodentium-induced expansion and activation of intestinal CD4+lymphoid tissue inducer (LTi) cells was completely abrogated by IL-7Rα blockade. Collectively, these data demonstrate that IL-7 is produced by IECs in response toC. rodentiuminfection and plays a critical role in the protective immunity against this intestinal attaching and effacing bacterium.

scholarly journals The Novel Streptococcal Transcriptional Regulator XtgS Negatively Regulates Bacterial Virulence and Directly Represses PseP Transcription

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Guangjin Liu ◽  
Tingting Gao ◽  
Xiaojun Zhong ◽  
Jiale Ma ◽  
Yumin Zhang ◽  
...  
Keyword(s):  
Strong Association ◽  
Challenge Test ◽  
Transcriptional Regulator ◽  
Transcriptional Regulators ◽  
Negative Regulator ◽  
Broad Host Range ◽  
Bacterial Survival ◽  
Mobility Shift ◽  
Upstream Gene ◽  
Content Type

ABSTRACT Streptococcus agalactiae (group B streptococcus [GBS]) has received continuous attention for its involvement in invasive infections and its broad host range. Transcriptional regulators have an important impact on bacterial adaptation to various environments. Research on transcriptional regulators will shed new light on GBS pathogenesis. In this study, we identified a novel XRE-family transcriptional regulator encoded on the GBS genome, designated XtgS. Our data demonstrate that XtgS inactivation significantly increases bacterial survival in host blood and animal challenge test, suggesting that it is a negative regulator of GBS pathogenicity. Further transcriptomic analysis and quantitative reverse transcription-PCR (qRT-PCR) mainly indicated that XtgS significantly repressed transcription of its upstream gene pseP. Based on electrophoretic mobility shift and lacZ fusion assays, we found that an XtgS homodimer directly binds a palindromic sequence in the pseP promoter region. Meanwhile, the PseP and XtgS combination naturally coexists in diverse Streptococcus genomes and has a strong association with sequence type, serotype diversification and host adaptation of GBS. Therefore, this study reveals that XtgS functions as a transcriptional regulator that negatively affects GBS virulence and directly represses PseP expression, and it provides new insights into the relationships between transcriptional regulator and genome evolution.

scholarly journals Role of Toll Interleukin-1 Receptor (IL-1R) 8, a Negative Regulator of IL-1R/Toll-Like Receptor Signaling, in Resistance to Acute Pseudomonas aeruginosa Lung Infection

2011 ◽  
Vol 80 (1) ◽  
pp. 100-109 ◽  
Author(s):  
Tania Véliz Rodriguez ◽  
Federica Moalli ◽  
Nadia Polentarutti ◽  
Moira Paroni ◽  
Eduardo Bonavita ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Interleukin 1 ◽  
Bacterial Load ◽  
Lung Infection ◽  
Negative Regulator ◽  
Current Evidence ◽  
Toll Like Receptor ◽  
Content Type ◽  
Deficient Mice

ABSTRACTToll interleukin-1 receptor (IL-1R) 8 (TIR8), also known as single Ig IL-1 receptor (IL-R)-related molecule, or SIGIRR, is a member of the IL-1R-like family, primarily expressed by epithelial cells. Current evidence suggests that TIR8 plays a nonredundant role as a negative regulatorin vivounder different inflammatory conditions that are dependent on IL-R and Toll-like receptor (TLR) activation. In the present study, we examined the role of TIR8 in innate resistance to acute lung infections caused byPseudomonas aeruginosa, a Gram-negative pathogen responsible for life-threatening infections in immunocompromised individuals and cystic fibrosis patients. We show that Tir8 deficiency in mice was associated with increased susceptibility to acuteP. aeruginosainfection, in terms of mortality and bacterial load, and to exacerbated local and systemic production of proinflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], IL-1β, and IL-6) and chemokines (CXCL1, CXCL2, and CCL2). It has been reported that host defense againstP. aeruginosaacute lung infection can be improved by blocking IL-1 since exaggerated IL-1β production may be harmful for the host in this infection. In agreement with these data, IL-1RI deficiency rescues the phenotype observed in Tir8-deficient mice: in Tir8−/−IL-1RI−/−double knockout mice we observed higher survival rates, enhanced bacterial clearance, and reduced levels of local and systemic cytokine and chemokine levels than in Tir8-deficient mice. These results suggest that TIR8 has a nonredundant effect in modulating the inflammation caused byP. aeruginosa, in particular, by negatively regulating IL-1RI signaling, which plays a major role in the pathogenesis of this infectious disease.

scholarly journals Silencing the Honey Bee (Apis mellifera) Naked Cuticle Gene (nkd) Improves Host Immune Function and Reduces Nosema ceranae Infections

2016 ◽  
Vol 82 (22) ◽  
pp. 6779-6787 ◽  
Author(s):  
Wenfeng Li ◽  
Jay D. Evans ◽  
Qiang Huang ◽  
Cristina Rodríguez-García ◽  
Jie Liu ◽  
...  
Keyword(s):  
Apis Mellifera ◽  
Immune Function ◽  
Honey Bee ◽  
Honey Bees ◽  
Nosema Ceranae ◽  
Negative Regulator ◽  
Mrna Levels ◽  
Content Type ◽  
Bee Disease

ABSTRACTNosema ceranaeis a new and emerging microsporidian parasite of European honey bees,Apis mellifera, that has been implicated in colony losses worldwide. RNA interference (RNAi), a posttranscriptional gene silencing mechanism, has emerged as a potent and specific strategy for controlling infections of parasites and pathogens in honey bees. While previous studies have focused on the silencing of parasite/pathogen virulence factors, we explore here the possibility of silencing a host factor as a mechanism for reducing parasite load. Specifically, we used an RNAi strategy to reduce the expression of a honey bee gene,naked cuticle(nkd), which is a negative regulator of host immune function. Our studies found thatnkdmRNA levels in adult bees were upregulated byN. ceranaeinfection (and thus, the parasite may use this mechanism to suppress host immune function) and that ingestion of double-stranded RNA (dsRNA) specific tonkdefficiently silenced its expression. Furthermore, we found that RNAi-mediated knockdown ofnkdtranscripts inNosema-infected bees resulted in upregulation of the expression of several immune genes (Abaecin,Apidaecin,Defensin-1, andPGRP-S2), reduction ofNosemaspore loads, and extension of honey bee life span. The results of our studies clearly indicate that silencing the hostnkdgene can activate honey bee immune responses, suppress the reproduction ofN. ceranae, and improve the overall health of honey bees. This study represents a novel host-derived therapeutic for honey bee disease treatment that merits further exploration.IMPORTANCEGiven the critical role of honey bees in the pollination of agricultural crops, it is urgent to develop strategies to prevent the colony decline induced by the infection of parasites/pathogens. Targeting parasites and pathogens directly by RNAi has been proven to be useful for controlling infections in honey bees, but little is known about the disease impacts of RNAi silencing of host factors. Here, we demonstrate that knocking down the honey bee immune repressor-encodingnkdgene can suppress the reproduction ofN. ceranaeand improve the overall health of honey bees, which highlights the potential role of host-derived and RNAi-based therapeutics in controlling the infections in honey bees. The information obtained from this study will have positive implications for honey bee disease management practices.

scholarly journals Role of yoaE Gene Regulated by CpxR in the Survival of Salmonella enterica Serovar Enteritidis in Antibacterial Egg White

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Xiaozhen Huang ◽  
Mengjun Hu ◽  
Xiujuan Zhou ◽  
Yanhong Liu ◽  
Chunlei Shi ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Salmonella Enterica ◽  
Promoter Region ◽  
Antibacterial Properties ◽  
Egg White ◽  
Global Public Health ◽  
Mobility Shift ◽  
Content Type ◽  
Egg Products

ABSTRACT The survival ability of Salmonella enterica serovar Enteritidis in antibacterial egg white is an important factor leading to Salmonella outbreaks through eggs and egg products. In this study, the role of the gene yoaE, encoding an inner membrane protein, in the survival of Salmonella Enteritidis in egg white, and its transcriptional regulation by CpxR were investigated. Quantitative reverse transcription-PCR (RT-qPCR) results showed that the yoaE gene expression was upregulated 35-fold after exposure to egg white for 4 h compared to that in M9FeS medium, and the deletion of yoaE (ΔyoaE) dramatically decreased the survival rate of bacteria in egg white to less than 1% of the wild type (WT) and the complementary strain at both 37 and 20°C, indicating that yoaE was essential for bacteria to survive in egg white. Furthermore, the ΔyoaE strain was sensitive to a 3-kDa ultrafiltration matrix of egg white because of its high pH and antimicrobial peptide components. Putative conserved binding sites for the envelope stress response regulator CpxR were found in the yoaE promoter region. In vivo, the RT-qPCR assay results showed that the upregulation of yoaE in a ΔcpxR strain in egg white was 1/5 that of the WT. In vitro, results from DNase I footprinting and electrophoretic mobility shift assays further demonstrated that CpxR could directly bind to the yoaE promoter region, and a specific CpxR binding sequence was identified. In conclusion, it was shown for the first time that CpxR positively regulated the transcription of yoaE, which was indispensable for survival of Salmonella Enteritidis in egg white. IMPORTANCE Salmonella enterica serovar Enteritidis is the predominant Salmonella serotype that causes human salmonellosis mainly through contaminated chicken eggs or egg products and has been a global public health threat. The spread and frequent outbreaks of this serotype through eggs correlate significantly with its exceptional survival in eggs, despite the antibacterial properties of egg white. Research on the survival mechanisms of S. Enteritidis in egg white will help develop effective strategies to control the contamination of eggs by this Salmonella serotype and help further elucidate the complex antibacterial mechanisms of egg white. This study revealed the importance of yoaE, a gene with unknown function, on the survival of S. Enteritidis in egg white, as well as its transcriptional regulation by CpxR. Our work provides the basis to reveal the mechanisms of survival of S. Enteritidis in egg white and the specific function of the yoaE gene.

scholarly journals The Histone-Like Nucleoid Structuring Protein (H-NS) Is a Negative Regulator of the Lateral Flagellar System in the Deep-Sea Bacterium Shewanella piezotolerans WP3

2016 ◽  
Vol 82 (8) ◽  
pp. 2388-2398 ◽  
Author(s):  
Huahua Jian ◽  
Guanpeng Xu ◽  
Yingbao Gai ◽  
Jun Xu ◽  
Xiang Xiao
Keyword(s):  
Deep Sea ◽  
Negative Regulator ◽  
Mobility Shift ◽  
Swarming Motility ◽  
Content Type ◽  
Cold Environments ◽  
Expression Of Genes ◽  
Gel Mobility Shift ◽  
Pressure Stress ◽  
Flagellar Genes

ABSTRACTAlthough the histone-like nucleoid structuring protein (H-NS) is well known for its involvement in the adaptation of mesophilic bacteria, such asEscherichia coli, to cold environments and high-pressure stress, an understanding of the role of H-NS in the cold-adapted benthic microorganisms that live in the deep-sea ecosystem, which covers approximately 60% of the earth's surface, is still lacking. In this study, we characterized the function of H-NS inShewanella piezotoleransWP3, which was isolated from West Pacific sediment at a depth of 1,914 m. Anhnsgene deletion mutant (WP3Δhns) was constructed, and comparative whole-genome microarray analysis was performed. H-NS had a significant influence (fold change, >2) on the expression of a variety of WP3 genes (274 and 280 genes were upregulated and downregulated, respectively), particularly genes related to energy production and conversion. Notably, WP3Δhnsexhibited higher expression levels of lateral flagellar genes than WP3 and showed enhanced swarming motility and lateral flagellar production compared to those of WP3. The DNA gel mobility shift experiment showed that H-NS bound specifically to the promoter of lateral flagellar genes. Moreover, the high-affinity binding sequences of H-NS were identified by DNase I protection footprinting, and the results support the “binding and spreading” model for H-NS functioning. To our knowledge, this is the first attempt to characterize the function of the universal regulator H-NS in a deep-sea bacterium. Our data revealed that H-NS has a novel function as a repressor of the expression of genes related to the energy-consuming secondary flagellar system and to swarming motility.

scholarly journals Regulation ofperRExpression by Iron and PerR in Campylobacter jejuni

2011 ◽  
Vol 193 (22) ◽  
pp. 6171-6178 ◽  
Author(s):  
Minkyeong Kim ◽  
Sunyoung Hwang ◽  
Sangryeol Ryu ◽  
Byeonghwa Jeon
Keyword(s):  
Oxidative Stress ◽  
Campylobacter Jejuni ◽  
Regulatory Region ◽  
Transcriptional Level ◽  
Mobility Shift ◽  
Content Type ◽  
Dnase I Footprinting ◽  
Transcriptional Changes ◽  
Electrophoretic Mobility Shift Assays ◽  
Binding Sequence

Campylobacter jejuniis a leading food-borne pathogen causing gastroenteritis in humans. Although OxyR is a widespread oxidative stress regulator in many Gram-negative bacteria,C. jejunilacks OxyR and instead possesses the metalloregulator PerR. Despite the important role played by PerR in oxidative stress defense, little is known about the factors influencingperRexpression inC. jejuni. In this study, aperRpromoter-lacZfusion assay demonstrated that iron significantly reduced the level ofperRtranscription, whereas other metal ions, such as copper, cobalt, manganese, and zinc, did not affectperRtranscription. Notably, aperRmutation substantially increased the level ofperRtranscription and intranscomplementation restored the transcriptional changes, suggestingperRis transcriptionally autoregulated inC. jejuni. In theperRmutant, iron did not repressperRtranscription, indicating the iron dependence ofperRexpression results fromperRautoregulation. Electrophoretic mobility shift assays showed that PerR binds to theperRpromoter, and DNase I footprinting assays identified a PerR binding site overlapping the −35 region of the twoperRpromoters, further supportingperRautoregulation at the transcriptional level. Alignment of the PerR binding sequence in theperRpromoter with the regulatory region of other PerR regulon genes ofC. jejunirevealed a 16-bp consensus PerR binding sequence, which shares high similarities to theBacillus subtilisPerR box. The results of this study demonstrated that PerR directly interacts with theperRpromoter and regulatesperRtranscription and thatperRautoregulation is responsible for the repression ofperRtranscription by iron inC. jejuni.

scholarly journals Phenylacetic Acid Catabolism and Its Transcriptional Regulation in Corynebacterium glutamicum

2012 ◽  
Vol 78 (16) ◽  
pp. 5796-5804 ◽  
Author(s):  
Xi Chen ◽  
Thomas A. Kohl ◽  
Christian Rückert ◽  
Dmitry A. Rodionov ◽  
Ling-Hao Li ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Phenylacetic Acid ◽  
Specific Binding ◽  
Genomic Analysis ◽  
Negative Regulator ◽  
Comparative Genomic ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Content Type ◽  
Transcription Regulator

ABSTRACTThe industrially important organismCorynebacterium glutamicumhas been characterized in recent years for its robust ability to assimilate aromatic compounds. In this study,C. glutamicumstrain AS 1.542 was investigated for its ability to catabolize phenylacetic acid (PAA). Thepaagenes were identified; they are organized as a continuouspaagene cluster. The type strain ofC. glutamicum, ATCC 13032, is not able to catabolize PAA, but the recombinant strain ATCC 13032/pEC-K18mob2::paagained the ability to grow on PAA. ThepaaRgene, encoding a TetR family transcription regulator, was studied in detail. Disruption ofpaaRin strain AS 1.542 resulted in transcriptional increases of allpaagenes. Transcription start sites and putative promoter regions were determined. An imperfect palindromic motif (5′-ACTNACCGNNCGNNCGGTNAGT-3′; 22 bp) was identified in the upstream regions ofpaagenes. Electrophoretic mobility shift assays (EMSA) demonstrated specific binding of PaaR to this motif, and phenylacetyl coenzyme A (PA-CoA) blocked binding. It was concluded that PaaR is the negative regulator of PAA degradation and that PA-CoA is the PaaR effector. In addition, GlxR binding sites were found, and binding to GlxR was confirmed. Therefore, PAA catabolism inC. glutamicumis regulated by the pathway-specific repressor PaaR, and also likely by the global transcription regulator GlxR. By comparative genomic analysis, we reconstructed orthologous PaaR regulons in 57 species, including species ofActinobacteria,Proteobacteria, andFlavobacteria, that carry PAA utilization genes and operate by conserved binding motifs, suggesting that PaaR-like regulation might commonly exist in these bacteria.

scholarly journals Mutations in theembC-embAIntergenic Region Contribute to Mycobacterium tuberculosis Resistance to Ethambutol

2014 ◽  
Vol 58 (11) ◽  
pp. 6837-6843 ◽  
Author(s):  
Zhenling Cui ◽  
Yuanyuan Li ◽  
Song Cheng ◽  
Hua Yang ◽  
Junmei Lu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Transcriptional Activity ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Mobility Shift ◽  
Content Type ◽  
Clinical Strains ◽  
Gel Mobility Shift

ABSTRACTThe rapid increase inMycobacterium tuberculosisresistance to ethambutol (EMB) threatens the diagnosis and treatment of tuberculosis (TB). We investigated the role of mutations in theembC-embAintergenic region (IGR) in EMB-resistant clinical strains from east China. A total of 767M. tuberculosisclinical strains were collected and analyzed for their drug susceptibility to EMB using the MGIT 960 system and MIC assay, and theembC-embAIGRs of these strains were sequenced. The transcriptional activity of theembC-embAIGR mutations was examined by reporter gene assays in recombinantMycobacterium smegmatisstrains, and the effect of IGR mutations on its binding to EmbR, a transcription regulator ofembAB, was analyzed by gel mobility shift assays. Correlation coefficient analysis showed that theembC-embAIGR mutation is associated with EMB resistance. The clinical strains carrying IGR mutations had a much higher level ofembAandembBmRNA as well as higher MICs to EMB. IGR mutations had higher transcriptional activity when transformed intoM. smegmatisstrains. Mutated IGRs bound to EmbR with much higher affinity than wild-type fragments. The sensitivity of molecular drug susceptibility testing (DST) with IGR mutations as an additional marker increased from 65.5% to 73.5%. Mutations of theembC-embAIGR enhance the binding of EmbR to the promoter region ofembABand increase the expression ofembAB, thus contributing to EMB resistance. Therefore, identification of IGR mutations as markers of EMB resistance could increase the sensitivity of molecular DST.

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