A structural view of the antibiotic degradation enzyme NDM-1 from a superbug

Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1 (NDM-1) are a type of newly discovered antibiotic-resistant bacteria. The rapid pandemic spread of NDM-1 bacteria worldwide (spreading to India, Pakistan, Europe, America, and Chinese Taiwan) in less than 2 months characterizes these microbes as a potentially major global health problem. The drug resistance of NDM-1 bacteria is largely due to plasmids containing the blaNDM-1 gene shuttling through bacterial populations. The NDM-1 enzyme encoded by the blaNDM-1 gene hydrolyzes β-lactam antibiotics, allowing the bacteria to escape the action of antibiotics. Although the biological functions and structural features of NDM-1 have been proposed according to results from functional and structural investigation of its homologues, the precise molecular characteristics and mechanism of action of NDM-1 have not been clarified. Here, we report the three-dimensional structure of NDM-1 with two catalytic zinc ions in its active site. Biological and mass spectroscopy results revealed that D-captopril can effectively inhibit the enzymatic activity of NDM-1 by binding to its active site with high binding affinity. The unique features concerning the primary sequence and structural conformation of the active site distinguish NDM-1 from other reported metallo-β-lactamases (MBLs) and implicate its role in wide spectrum drug resistance. We also discuss the molecular mechanism of NDM-1 action and its essential role in the pandemic of drug-resistant NDM-1 bacteria. Our results will provide helpful information for future drug discovery targeting drug resistance caused by NDM-1 and related metallo-β-lactamases.     

Refined models of New Delhi metallo-beta-lactamase-1 with inhibitors: an QM/MM modeling study

New Delhi metallo-beta-lactamase 1 (NDM-1) has been identified as a potential target for the treatment of multi-drug resistance bacterial infections. We used molecular docking, normal MD, SIE, QM/MM MD simulations, QM/MM GBSA binding free energy, and QM/MM GBSA alanine-scanning mutagenesis techniques to investigate interactions of the NDM-1 with 11 inhibitors (Tigecycline, BAL30072, D-captopril, Penicillin G, Ampicillin, Carbenicillin, Cephalexin, Cefaclor, Nitrocefin, Meropenem, and Imipenem). From our normal MD and QM/MM simulations, the correlation coefficients between the predicted binding free energies and experimental values are .88 and .93, respectively. Then simulations, which combined QM/MM/GBSA and alanine-scanning mutagenesis techniques, were performed and our results show that two residues (Lys211 and His250) have the strongest impact on the binding affinities of the 11 NDM-1/inhibitors. Therefore, our approach theoretically suggests that the two residues (Lys211 and His250) are responsible for the selectivity of NDM-1 associated inhibitors.     

Insights from modeling the 3D structure of New Delhi metallo-β-lactamse and its binding interactions with antibiotic drugs

New Delhi metallo-beta-lactamase (NDM-1) is an enzyme that makes bacteria resistant to a broad range of beta-lactam antibiotic drugs. This is because it can inactivate most beta-lactam antibiotic drugs by hydrolyzing them. For in-depth understanding of the hydrolysis mechanism, the three-dimensional structure of NDM-1 was developed. With such a structural frame, two enzyme-ligand complexes were derived by respectively docking Imipenem and Meropenem (two typical beta-lactam antibiotic drugs) to the NDM-1 receptor. It was revealed from the NDM-1/Imipenem complex that the antibiotic drug was hydrolyzed while sitting in a binding pocket of NDM-1 formed by nine residues. And for the case of NDM-1/Meropenem complex, the antibiotic drug was hydrolyzed in a binding pocket formed by twelve residues. All these constituent residues of the two binding pockets were explicitly defined and graphically labeled. It is anticipated that the findings reported here may provide useful insights for developing new antibiotic drugs to overcome the resistance problem.     

泛耐药细菌NDM-1基困Taqman PCR快速检测方法的建立

产NDM-1(New Delhi Metallo-β-lactamase 1,Ⅰ型新德里金属β-内酰胺酶)细菌是新近报道的一种泛耐药细菌,由于对绝大多数常用抗生素均耐药,又被称为超级细菌.目的:建立一种可快速检测泛耐药细菌NDM-1基因的Taqman探针实时荧光定量PCR法.方法:根据NDM-1基因序列,设计引物和Ta...     

Studies on New Delhi Metallo-Beta-Lactamse-1 producing Acinetobacter baumannii isolated from donor swab in a tertiary eye care centre, India and structural analysis of its antibiotic binding interactions

Gram-negative bacilli, Enterobacteriaceae and Non-fermentors with resistance to carbapenems and metallo beta-lactams are the major cause of concern in clinical problems in current human healthcare. The most highly emerging dreadful Metallo Beta-lactamses is New Delhi metallo-beta-lactamase (blaNDM-1) which confers resistance to carbapenems; susceptible only to colistin and, less consistently to tigecycline, leading to no therapeutic options. In the present study, we demonstrate the effects of cephalosporins and carbepenems on biofilm producing A. baumanii clinical isolate and also to infer the probable inhibitory binding mode through molecular docking studies. The result of MIC on Biofilm producing A. baumanii and the docking analysis results were found to be concordant. Moreover, we also found cephalosporins and carbepenem groups to interact with 162-166 region of blaNDM-1, which is unique for NDM-1 and also documented to be a potential drug targeting region.     

Proteomic Analysis of Clinical Isolate of Stenotrophomonas maltophilia with bla(NDM-1,)bla(L1) and bla(L2) β-Lactamase Genes under Imipenem Treatment

The co-occurrence of L1 and AmpR-L2 with bla(NDM-1) gene with an upstream 250-bp promoter was detected in a clinical isolate of Stenotrophomonas maltophilia DCPS-01, which was resistant to all β-lactams and sensitive only to colistin and fluoroquinolones. To investigate expression of resistance genes and the molecular mechanisms of bacteria resistance to carbapenems, proteomic profiles of the isolate was passaged with and without the drug by using 2D-PAGE. The results showed that 33 genes exhibiting a ≥3-fold change were identified as candidates that may help S. maltophilia survive drug selection. Strikingly, L1 was expressed more highly in cells grown with imipenem, and the abundant NDM-1 further increased, while very little L2 was detected even following induction. Specific activities for β-lactamase revealed that L2 remained at constitutive low levels (10.6 U/mg), while L1 and NDM-1 showed clear activity (69.8 U/mg). Our data support that imipenem could specifically and reversibly induce L1 and NDM-1, which together played key roles in drug resistance in DCPS-01. Although NDM-1 mediated resistance to carbapenems has been found in very few cases, to our knowledge, this is the first proteomics research of S. maltophilia with NDM-1, giving very broad-spectrum antibiotic resistance profiles.     

Molecular basis of NDM-1, a new antibiotic resistance determinant

The New Delhi Metallo-β-lactamase (NDM-1) was first reported in 2009 in a Swedish patient. A recent study reported that Klebsiella pneumonia NDM-1 positive strain or Escherichia coli NDM-1 positive strain was highly resistant to all antibiotics tested except tigecycline and colistin. These can no longer be relied on to treat infections and therefore, NDM-1 now becomes potentially a major global health threat.In this study, we performed modeling studies to obtain its 3D structure and NDM-1/antibiotics complex. It revealed that the hydrolytic mechanisms are highly conserved. In addition, the detailed analysis indicates that the more flexible and hydrophobic loop1, together with the evolution of more positive-charged loop2 leads to NDM-1 positive strain more potent and extensive in antibiotics resistance compared with other MBLs. Furthermore, through biological experiments, we revealed the molecular basis for antibiotics catalysis of NDM-1 on the enzymatic level. We found that NDM-1 enzyme was highly potent to degrade carbapenem antibiotics, while mostly susceptible to tigecycline, which had the ability to slow down the hydrolysis velocity of meropenem by NDM-1. Meanwhile, the mutagenesis experiments, including D124A, C208A, K211A and K211E, which displayed down-regulation on meropenem catalysis, proved the accuracy of our model.At present, there are no effective antibiotics against NDM-1 positive pathogen. Our study will provide clues to investigate the molecular basis of extended antibiotics resistance of NDM-1 and then accelerate the search for new antibiotics against NDM-1 positive strain in clinical studies.     

泛耐药基因NDM-1在大肠杆菌中的克隆表达及耐药性检测

目的:构建bla(NDM-1)基因重组质粒,表达新德里金属β内酰胺酶1(NDM-1),并检测携带bla(NDM-1)基因重组质粒的大肠杆菌的耐药状况。方法:PCR扩增编码NDM-1的基因bla(NDM-1),构建表达载体pGEX4T-1-NDM-1,并转化至大肠杆菌,转化子经PCR后测序,以确认构建和转化成功;用Western印迹验证重组蛋白的表达;用药敏纸片法检测含重组质粒pGEX4T-1-NDM-1的大肠杆菌的耐药谱;用E-test法测定其最低抑菌浓度(MIC)。结果:PCR及测序结果显示载体构建和转化成功;含重组质粒pGEX4T-1-NDM-1的大肠杆菌在37℃时,经1 mmol/LIPTG诱导5 h后,SDS-PAGE可见目的条带;除对替加环素和粘菌素敏感外,该重组子对多种碳青霉烯类抗生素耐药,E-test检测其对亚胺培南的MIC为64μg/mL。结论:构建了含泛耐药基因bla(NDM-1)的重组质粒,转入大肠杆菌后表达了融合蛋白,并对多种碳青霉烯类抗生素耐药。为进一步研究bla(NDM-1)基因和蛋白的功能奠定了基础。     

Molecular and computational approaches to understand resistance of New Delhi metallo β-lactamase variants (NDM-1, NDM-4, NDM-5, NDM-6, NDM-7)-producing strains against carbapenems

The discovery of NDM-1 and its variants has caused the emergence of antibiotic resistance in the community and hospital setting, causing major concern for health care across the globe. New Delhi Metallo-β-lactamase is known to hydrolyse almost all β-lactam antibiotics. Studies have shown the hydrolytic activates of NDM-1 and some of its variants, however a comparative study of these NDM variants has not been explored in detail. Hence, we proposed to check their catalytic activity by performing a comparative study between NDM-1 and its variants. The study was initiated to clone NDM variants (NDM-1, NDM-4, NDM-5, NDM-6 and NDM-7) followed by overexpression of the recombinant proteins to check their hydrolytic properties against β-lactam antibiotics. The minimum inhibitory concentration of carbapenems antibiotics for bla_NDM-5 clone was found four fold increased, whereas no change was observed in the clones having other variants. The hydrolytic activity of carbapenem with NDM-5 variant was found to be augmented as per the kinetics parameter where Km was decreased and kcat, kcat/Km values increased as compared to the NDM-1. Molecular docking studies were employed to identify the variations in the binding ability among all NDM variants with imipenem or meropenem. Simulation studies at 100?ns showed a good stability of NDM-5 with imipenem and meropenem as compared to NDM-1. CD spectroscopy data revealed significant changes in the secondary structure of NDM variants. We conclude that NDM-5 showed higher hydrolytic activity as compared to other variants. This study provides a comparative analysis of the severity of NDM producing strains.     

New Delhi Metallo-β-Lactamase 1(NDM-1), the Dominant Carbapenemase Detected in Carbapenem-Resistant Enterobacter cloacae from Henan Province,China

The emergence of New Delhi metallo-β-lactamase 1 (NDM-1) has become established as a major public health threat and represents a new challenge in the treatment of infectious diseases. In this study, we report a high incidence and endemic spread of NDM-1-producing carbapenem-resistant Enterobacter cloacae isolates in Henan province, China. Eight (72.7%) out of eleven non-duplicated carbapenem-resistant E. cloacae isolates collected between June 2011 and May 2013 were identified as NDM-1 positive. The bla _NDM-1 gene surrounded by an entire ISAba125 element and a bleomycin resistance gene ble _MBL in these isolates were carried by diverse conjugatable plasmids (IncA/C, IncN, IncHI2 and untypeable) ranging from ~55 to ~360 kb. Molecular epidemiology analysis revealed that three NDM-1-producing E. cloacae belonged to the same multilocus sequence type (ST), ST120, two of which were classified as extensively drug-resistant (XDR) isolates susceptible only to tigecycline and colistin. The two XDR ST120 E. cloacae isolates co-harbored bla _NDM-1, armA and fosA3 genes and could transfer resistance to carbapenems, fosfomycin and aminoglycosides simultaneously via a conjugation experiment. Our study demonstrated NDM-1 was the most prevalent metallo-β-lactamase (MBL) among carbapenem-resistant E.cloacae isolates and identified a potential endemic clone of ST120 in Henan province. These findings highlight the need for enhanced efforts to monitor the further spread of NDM-1 and XDR ST120 E. cloacae in this region.     

Identification of New Delhi metallo-β-lactamase gene (NDM-1) from a clinical isolate of Acinetobacter junii in China

New Delhi metallo-β-lactamase-1 (NDM-1) is a novel type of metallo-β-lactamase (MBL) responsible for bacterial resistance to β-lactam antibiotics. Acinetobacter junii was previously shown to possess a MBL phenotype; however, the genes responsible for this phenotype were not identified. In this study, we reported the identification of NDM-1 gene in a clinical isolate of A. junii from a child patient in China, which was resistant to all β-lactams except aztreonam but sensitive to aminoglycosides and quinolones. The cloned NDM-1 gene contained an open reading frame of 813 bp and had a nucleotide sequence 99.9% identical (812/813) to reported NDM-1 genes carried by Acinetobacter baumannii , Enterococcus faecium , Escherichia coli , and Klebsiella pneumoniae . Recombinant NDM-1 protein was successfully expressed in E.?coli BL21, and antibiotic sensitivities of the NDM-1-producing E.?coli were largely similar to the A.?junii 1454 isolate. The findings of this study raise attention to the emergence and spread of NDM-1-carrying bacteria in China.     

Copy Number Change of the NDM-1 Sequence in a Multidrug-Resistant Klebsiella pneumoniae Clinical Isolate

The genetic features of the antimicrobial resistance of a multidrug resistant Klebsiella pneumoniae strain harboring bla _NDM-1 were investigated to increase our understanding of the evolution of NDM-1. The strain, KPX, came from a Taiwanese patient with a hospitalization history in New Delhi. Complete DNA sequencing was performed; and the genes responsible for antimicrobial resistance were systematically examined and isolated by library screening. KPX harbored two resistance plasmids, pKPX-1 and pKPX-2, which are 250-kb and 141-kb in size, respectively, with bla _NDM-1 present on pKPX-1. The plasmid pKPX-1 contained genes associated with the IncR and IncF groups, while pKPX-2 belonged to the IncF family. Each plasmid carried multiple antimicrobial resistance genetic determinants. The gene responsible for resistance to carbapenems was found on pKPX-1 and that for resistance to aztreonam was found on pKPX-2. To our surprise, we discovered that bla _NDM-1 exists on pKPX-1 as multiple copies in the form of tandem repeats. Amplification of bla _NDM-1 was found to occur by duplication of an 8.6-kb unit, with the copy number of the repeat varying from colony to colony. This repeat sequence is identical to that of the pNDM-MAR except for two base substitutions. The copy number of bla _NDM-1 of colonies under different conditions was assessed by Southern blotting and quantitative PCR. The bla _NDM-1 sequence was maintained in the presence of the antimicrobial selection; however, removal of antimicrobial selection led to the emergence of susceptible bacterial populations with a reduced copy number or even the complete loss of the bla _NDM-1 sequence. The dynamic nature of the NDM-1 sequence provides a strong argument for judicious use of the broad-spectrum antimicrobials in order to reduce the development and spread of antimicrobial resistance among pathogens.     

Biochemical Characteristics of New Delhi Metallo-β-Lactamase-1 Show Unexpected Difference to Other MBLs

New Delhi metallo-β-lactamase (NDM-1) is a new metallo-β-lactamase (MBL) that has recently emerged as a global threat because it confers bacteria with resistance to almost all clinically used β-lactam antibiotics. To determine the molecular basis of this threat, NDM-1 was purified from Escherichia coli TransB (DE3) carrying cloned blaNDM-1 gene by an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme was stable even in extremely alkaline buffer (pH 11) and reached its highest activity at a low temperature (15°C), which was different from other MBLs. The 50% inhibition concentration of EDTA against NDM-1 was 412 nM, which showed that NDM-1 was more susceptible to EDTA than other MBLs. The effects of zinc on NDM-1 differed between cephem and carbapenem complexes, but inhibition at high Zn²⁺ concentration was observed for all of tested β-lactam compounds.     

Caenorhabditis elegans mom-4 is required for the activation of the p38 MAPK signaling pathway in the response to Pseudomonas aeruginosa infection

The p38 mitogen-activated protein kinase (MAPK) plays an evolutionarily conserved role in the cellular response to microbial infection and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. In the Caenorhabditis elegans, the p38 MAPK (also called PMK-1) signaling pathway has been shown to be required in its resistance to bacterial infection. However, how different upstream MAP2Ks and MAP3Ks specifically contribute to the activation of PMK-1 in response to bacterial infection still is not clearly understood. By using double-stranded RNA-mediated interference (RNAi) and genetic mutants of C. elegans, we demonstrate that C. elegans MOM-4, a mammalian TAK1 homolog, is required for the resistance of C. elegans to a P. aeruginosa infection. We have also found that the MKK-4 of C. elegans is required for P. aeruginosa resistance, but not through the regulation of DLK-1. In summary, our results indicate that different upstream MAPKKKs or MAPKKs regulate the activation of PMK-1 in response to P. aeruginosa.     

In silico based unraveling of New Delhi metallo-β-lactamase (NDM-1) inhibitors from natural compounds: a molecular docking and molecular dynamics simulation study

Abstract

The development of pathogenic microbial resistance toward antibiotics has become a global clinical concern. New Delhi metallo-β-lactmase-1 (NDM-1) and its variants have recently drawn immense attention for its biological ability to catalyze the hydrolysis of almost all of β-lactam antibiotics including the Carbapenems which are generally considered as the last-resort antibiotics. Also, the horizontal gene transfer is expediting the rapid spread of NDM-1 in bacteria. In the wake of this serious antibiotic resistance problem it becomes imperative to find inhibitors which can render the present antibiotics functional and useful. In the present study, we have used Molecular docking and Molecular Dynamics (MD) simulation approach to find out suitable inhibitors against NDM-1 from an array of different natural compounds. We have screened unique natural compounds from ZINC database and also a set of standard antibiotics and inhibitors. Based upon the highest binding affinity demonstrated by docking with NDM-1, the best binding antibiotic Meropenem and the top five natural compounds, viz., Withaferin A, Beta-Sitosterol, Aristolochic acid, Diosgenin and Guggulsterone E were selected and subjected to MD simulations study. The docked NDM-1 complex with withaferin A, beta-sitosterol and diosgenin were found to be more stable as compared to the one with meropenem throughout the MD simulation process with the relative RMSD and RMSF in acceptable range. In conclusion, these compounds can be readily tested in vitro and in vivo to fully establish and confirm their inhibition potentiality and can also serve as lead molecules for the development of future functional inhibitors.

Communicated by Ramaswamy H. Sarma     

3D-QSAR and molecular recognition of Klebsiella pneumoniae NDM-1 inhibitors

New Delhi metallo-β-lactamase-1 (NDM-1) as a target for the development of anti-superbug agents, plays an important role in the resistance of β-lactam antibiotics and has received worldwide attention. Sulfhydryl propionic acid derivatives can effectively inhibit the catalytic activity of NDM-1, but the quantitative structure–activity relationship (QSAR) and inhibitor-target recognition mechanism both remain unclear. In this work, CoMFA and CoMSIA models of sulfhydryl propionic acid inhibitors with high predictive ability were obtained, from which the effect of different substituents on the inhibitory activity against NDM-1 were revealed at the molecular level. Then, two 120-nanosecond comparative molecular dynamics (MD) simulations for NDM-1 enzyme and NDM-1-inhibitor complex systems were performed to study the recognition and inhibition mechanism of sulfhydryl propionic acid derivatives. The binding of inhibitors alters the entire H-bond network of the NDM-1 system accompanied by the formation of strong interactions with I35, W93, H120, H122, D124, H189 and H250, further weakens the recognition of NDM-1 by its endogenic substrates. Finally, the results of free energy landscape and conformation cluster analyses show that NDM-1 underwent a significant conformational change after the association with sulfhydryl propionic acid inhibitors. Our findings can provide theoretical support and help for anti-superbug agents design based on the structures of NDM-1 and sulfhydryl propionic acid derivatives.     

Programmed cell death may act as a surveillance mechanism to safeguard male gametophyte development in Arabidopsis

Programmed cell death (PCD) plays an important role in plant growth and development as well as in stress responses. During male gametophyte development, it has been proposed that PCD may act as a cellular surveillance mechanism to ensure successful progression of male gametogenesis, and this suicide protective machinery is repressed under favorable growth conditions. However, the regulatory mechanism of male gametophyte-specific PCD remains unknown. Here, we report the use of a TdT-mediated dUTP nick-end labeling-based strategy for genetic screening of Arabidopsis mutants that present PCD phenotype during male gametophyte development. By using this approach, we identified 12 mutants, designated as pcd in male gametogenesis (pig). pig mutants are defective at various stages of male gametophyte development, among which nine pig mutants show a microspore-specific PCD phenotype occurring mainly around pollen mitosis I or the bicellular stage. The PIG1 gene was identified by map-based cloning, and was found to be identical to ATAXIA TELANGIECTASIA MUTATED (ATM), a highly conserved gene in eukaryotes and a key regulator of the DNA damage response. Our results suggest that PCD may act as a general mechanism to safeguard the entire process of male gametophyte development.     

Higher Isolation of NDM-1 Producing Acinetobacter baumannii from the Sewage of the Hospitals in Beijing

Multidrug resistant microbes present in the environment are a potential public health risk. In this study, we investigate the presence of New Delhi metallo-β-lactamase 1 (NDM-1) producing bacteria in the 99 water samples in Beijing City, including river water, treated drinking water, raw water samples from the pools and sewage from 4 comprehensive hospitals. For the bla _NDM-1 positive isolate, antimicrobial susceptibility testing was further analyzed, and Pulsed Field Gel Electrophoresis (PFGE) was performed to determine the genetic relationship among the NDM-1 producing isolates from sewage and human, as well as the clinical strains without NDM-1. The results indicate that there was a higher isolation of NDM-1 producing Acinetobacter baumannii from the sewage of the hospitals, while no NDM-1 producing isolates were recovered from samples obtained from the river, drinking, or fishpond water. Surprisingly, these isolates were markedly different from the clinical isolates in drug resistance and pulsed field gel electrophoresis profiles, suggesting different evolutionary relationships. Our results showed that the hospital sewage may be one of the diffusion reservoirs of NDM-1 producing bacteria.     

Characterization of purified New Delhi metallo-β-lactamase-1

New Delhi metallo-β-lactmase-1 (NDM-1) has recently emerged as a global threat because of its ability to confer resistance to almost all clinically used β-lactam antibiotics, its presence within an easily transmissible plasmid bearing a number of other antibiotic resistance determinants, its carriage in a variety of enterobacteria, and its presence in both nosocomial and community-acquired infections. To improve our understanding of the molecular basis of this threat, NDM-1 was purified and characterized. Recombinant NDM-1 bearing its native leader sequence was expressed in Escherichia coli BL21 cells. The major processed form found to be released into culture media contains a 35-residue truncation at the N-terminus. This form of NDM-1 is monomeric and can be purified with 1.8 or 1.0 equiv of zinc ion, depending on the experimental conditions. Treatment of dizinc NDM-1 with EDTA results in complete removal of both zinc ions, but the relatively weaker chelator PAR chelates only 1 equiv of zinc ion from folded protein but 1.9 equiv of zinc ion from denatured protein, indicating different affinities for each metal binding site. UV-vis spectroscopy of the dicobalt metalloform along with molecular dynamics simulations of the dizinc metallo form indicates that the dinuclear metal cluster at the active site of NDM-1 is similar in structure to other class B1 metallo-β-lactamases. Supplementation of excess zinc ions to monozinc NDM-1 has differential effects on enzyme activity with respect to three different classes of β-lactam substrates tested, penems, cephems, and carbapenems, and likely reflects dissimilar contributions of the second equivalent of metal ion to the catalysis of the hydrolysis of these substrates. Fits to these concentration dependencies are used to approximate the K(d) value of the more weakly bound zinc ion (2 μM). NDM-1 achieved maximal activity with all substrates tested when supplemented with approximately 10 μM ZnSO(4), displaying k(cat)/K(M) values ranging from 1.4 × 10(6) to 2.0 × 10(7) M(-1) s(-1), and a slight preference for cephem substrates. This work provides a foundation for an improved understanding of the molecular basis of NDM-1-mediated antibiotic resistance and should allow more quantitative studies to develop targeted therapeutics.     

Human pathogenic fungus Trichophytonschoenleinii activates the NLRP3 inflammasome

The fungus Trichophyton schoenleinii (T. schoenleinii) is the causative agent of Trichophytosis and Tinea favosa of the scalp in certain regions of Eurasia and Africa. Human innate immune system plays an important role in combating with various pathogens including fungi. The inflammasome is one of the most critical arms of host innate immunity, which is a protein complex controlling maturation of IL-1β. To clarify whether T. schoenleinii is able to activate the inflammasome, we analyzed human monocytic cell line THP-1 for IL-1β production upon infection with T. schoenleinii strain isolated from Tinea favosa patients, and rapid IL-1β secretion from THP-1 cells was observed. Moreover, applying competitive inhibitors and gene specific silencing with shRNA, we found that T. schoenleinii induced IL-1β secretion, ASC pyroptosome formation as well as caspase-1 activation were all dependent on NLRP3. Cathepsin B activity, ROS production and K⁺ efflux were required for the inflammasome activation by T. schoenleinii. Our data thus reveal that the NLRP3 inflammasome plays an important role in host defense against T. schoenleinii, and suggest that manipulating NLRP3 signaling can be a novel approach for control of diseases caused by T. schoenleinii infection.