Publications by authors named "Kaixia Mi"

24 Publications

  • Page 1 of 1

The pentapeptide-repeat protein, MfpA, interacts with mycobacterial DNA gyrase as a DNA T-segment mimic.

Proc Natl Acad Sci U S A 2021 Mar;118(11)

Department of Biological Chemistry, John Innes Centre, NR4 7UH Norwich, United Kingdom;

DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that MfpA (MsMfpA) inhibits negative supercoiling by gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA-MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins.
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http://dx.doi.org/10.1073/pnas.2016705118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980463PMC
March 2021

The plasmid-borne quinolone resistance protein QnrB, a novel DnaA-binding protein, increases the bacterial mutation rate by triggering DNA replication stress.

Mol Microbiol 2019 06 27;111(6):1529-1543. Epub 2019 Mar 27.

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.

Bacterial antibiotic resistance, a global health threat, is caused by plasmid transfer or genetic mutations. Quinolones are important antibiotics, partially because they are fully synthetic and resistance genes are unlikely to exist in nature; nonetheless, quinolone resistance proteins have been identified. The mechanism by which plasmid-borne quinolone resistance proteins promotes the selection of quinolone-resistant mutants is unclear. Here, we show that QnrB increases the bacterial mutation rate. Transcriptomic and genome sequencing analyses showed that QnrB promoted gene abundance near the origin of replication (oriC). In addition, the QnrB expression level correlated with the replication origin to terminus (oriC/ter) ratio, indicating QnrB-induced DNA replication stress. Our results also show that QnrB is a DnaA-binding protein that may act as an activator of DNA replication initiation. Interaction of QnrB with DnaA promoted the formation of the DnaA-oriC open complex, which leads to DNA replication over-initiation. Our data indicate that plasmid-borne QnrB increases bacterial mutation rates and that genetic changes can alleviate the fitness cost imposed by transmitted plasmids. Derivative mutations may impair antibiotic efficacy and threaten the value of antibiotic treatments. Enhanced understanding of how bacteria adapt to the antibiotic environment will lead to new therapeutic strategies for antibiotic-resistant infections.
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http://dx.doi.org/10.1111/mmi.14235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6617969PMC
June 2019

Mycobacterium tuberculosis universal stress protein Rv2623 interacts with the putative ATP binding cassette (ABC) transporter Rv1747 to regulate mycobacterial growth.

PLoS Pathog 2017 Jul 28;13(7):e1006515. Epub 2017 Jul 28.

Department of Medicine, Albert Einstein College of Medicine & Montefiore Medical Center, Bronx, New York, United States of America.

We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (ΔRv2623) displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs), while the ΔRv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ΔRv2623 is hypervirulent, while ΔRv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ΔRv2623 and ΔRv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv1747 might function as a transporter of PIMs. Because these glycolipids are major mycobacterial cell envelope components that can impact on the immune response, our findings raise the possibility that Rv2623 may regulate bacterial growth, virulence, and entry into persistence, at least in part, by modulating the levels of bacillary PIM expression, perhaps through negatively regulating the Rv1747-dependent export of the immunomodulatory PIMs to alter host-pathogen interaction, thereby influencing the fate of M. tuberculosis in vivo.
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http://dx.doi.org/10.1371/journal.ppat.1006515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5549992PMC
July 2017

Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain.

Structure 2017 08 14;25(8):1286-1294.e4. Epub 2017 Jul 14.

College of Life Sciences, Nankai University, Tianjin 300071, China; National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100101, China; Laboratory of Structural Biology, School of Medicine, Tsinghua University, Beijing 100084, China. Electronic address:

Protein kinases play central roles in the survival of Mycobacterium tuberculosis within host. Here we report the individual high-resolution crystal structures of the sensor domain (in both monomer and dimer forms) and the kinase domain of PknI, a transmembrane protein member of the serine/threonine protein kinases (STPKs) family. PknI is the first STPK identified whose sensor domain exists in a monomer-dimer equilibrium. Inspection of the two structures of the sensor domain (PknI_SD) revealed conformational changes upon dimerization, with an arm region of critical importance for dimer formation identified. Rapamycin-induced dimerization of unphosphorylated fusions of PknI juxtamembrane and the kinase domain, intended to mimic the dimerization effect presumably imposed by PknI_SD, was observed to be able to activate auto-phosphorylation activity of the kinase domain. In vivo experiments using an M. bovis model suggested PknI functions as a dimer in the regulation of M. tuberculosis growth.
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http://dx.doi.org/10.1016/j.str.2017.06.010DOI Listing
August 2017

Universal stress protein Rv2624c alters abundance of arginine and enhances intracellular survival by ATP binding in mycobacteria.

Sci Rep 2016 10 20;6:35462. Epub 2016 Oct 20.

Shanghai Municipal Center for Disease Control &Prevention, Shanghai 200336, China.

The universal stress protein family is a family of stress-induced proteins. Universal stress proteins affect latency and antibiotic resistance in mycobacteria. Here, we showed that Mycobacterium smegmatis overexpressing M. tuberculosis universal stress protein Rv2624c exhibits increased survival in human monocyte THP-1 cells. Transcriptome analysis suggested that Rv2624c affects histidine metabolism, and arginine and proline metabolism. LC-MS/MS analysis showed that Rv2624c affects the abundance of arginine, a modulator of both mycobacteria and infected THP-1 cells. Biochemical analysis showed that Rv2624c is a nucleotide-binding universal stress protein, and an Rv2624c mutant incapable of binding ATP abrogated the growth advantage in THP-1 cells. Rv2624c may therefore modulate metabolic pathways in an ATP-dependent manner, changing the abundance of arginine and thus increasing survival in THP-1 cells.
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http://dx.doi.org/10.1038/srep35462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5071874PMC
October 2016

Differential roles of the hemerythrin-like proteins of Mycobacterium smegmatis in hydrogen peroxide and erythromycin susceptibility.

Sci Rep 2015 Nov 26;5:16130. Epub 2015 Nov 26.

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, CAS, Beijing 100101, China.

Hemerythrin-like proteins are oxygen-carrying non-heme di-iron binding proteins and their functions have effect on oxidation-reduction regulation and antibiotic resistance. Recent studies using bioinformatic analyses suggest that multiple hemerythrin-like protein coding sequences might have been acquired by lateral gene transfer and the number of hemerythrin-like proteins varies amongst different species. Mycobacterium smegmatis contains three hemerythrin-like proteins, MSMEG_3312, MSMEG_2415 and MSMEG_6212. In this study, we have systematically analyzed all three hemerythrin-like proteins in M. smegmatis and our results identified and characterized two functional classes: MSMEG_2415 plays an important role in H2O2 susceptibility, and MSMEG_3312 and MSMEG_6212 are associated with erythromycin susceptibility. Phylogenetic analysis indicated that these three proteins have different evolutionary origins, possibly explaining their different physiological functions. Here, combined with biological and phylogenetic analyses, our results provide new insights into the evolutionary divergence of the hemerythrin-like proteins in M. smegmatis.
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http://dx.doi.org/10.1038/srep16130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4660385PMC
November 2015

HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions.

Nat Struct Mol Biol 2015 Nov 12;22(11):906-13. Epub 2015 Oct 12.

Ministry of Education Key Laboratory of Protein Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.

Adverse cellular conditions often lead to nonproductive translational stalling and arrest of ribosomes on mRNAs. Here, we used fast kinetics and cryo-EM to characterize Escherichia coli HflX, a GTPase with unknown function. Our data reveal that HflX is a heat shock-induced ribosome-splitting factor capable of dissociating vacant as well as mRNA-associated ribosomes with deacylated tRNA in the peptidyl site. Structural data demonstrate that the N-terminal effector domain of HflX binds to the peptidyl transferase center in a strikingly similar manner as that of the class I release factors and induces dramatic conformational changes in central intersubunit bridges, thus promoting subunit dissociation. Accordingly, loss of HflX results in an increase in stalled ribosomes upon heat shock. These results suggest a primary role of HflX in rescuing translationally arrested ribosomes under stress conditions.
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http://dx.doi.org/10.1038/nsmb.3103DOI Listing
November 2015

Functional Characterization of Sirtuin-like Protein in Mycobacterium smegmatis.

J Proteome Res 2015 Nov 29;14(11):4441-9. Epub 2015 Sep 29.

MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University , Beijing 100084, China.

Nicotinamide adenine dinucleotide (NAD)-dependent deacetylases (sirtuins) are well conserved from prokaryotes to eukaryotes. Functions and regulations of mammalian sirtuins have been extensively studied and indicate that sirtuins play an important role in regulation of biological processes, whereas functions of mycobacterial sirtuins were less explored. To examine functions of the sirtuin-like protein in mycobacteria, a Mycobacterium smegmatis sirtuin, MSMEG_5175, was overexpressed in a M. smegmatis strain mc(2)155 to generate an MSMEG_5175-overexpression strain (mc(2)155-MS5175) in the present study. The physiological aspects of mc(2)155-MS5175 strain were characterized showing that they had a lower intracellular NAD level and a higher resistance to isoniazid (INH) as compared to mc(2)155 containing empty pMV261 plasmid (mc(2)155-pMV261). Quantitative proteomic analysis was carried out to determine differentially expressed proteins between mc(2)155-pMV261 and mc(2)155-MS5175. Among 3032 identified proteins, overexpression of MSMEG_5175 results in up-regulation of 34 proteins and down-regulation of 72 proteins, which involve in diverse cellular processes including metabolic activation, transcription and translation, antioxidant, and DNA repair. Down-regulation of catalase peroxidase (KatG) expression in both mRNA and protein levels were observed in mc(2)155-MS5175 strain, suggesting that a decrease in cellular NAD content and down-regulation of KatG expression contribute to the higher resistance to INH in mc(2)155-MS5175. Using a combination of immunoprecipitation and proteomic analysis, we found that acetylation in 27 proteins was decreased in mc(2)155-MS5175 as compared to those in mc(2)155-pMV261, suggesting that these proteins including the beta prime subunit of RNA polymerase (rpoC), ribosomal proteins, and metabolic enzymes were substrates of MSMEG_5175. Acetylation changes in rpoC may affect its function and cause changes in global gene transcription. Taken together, these results suggest that MSMEG_5175 regulates diverse cellular processes resulting in an increase in INH resistance in mycobacteria, and provide a useful resource to further biological exploration into functions of protein acetylation in mycobacteria.
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http://dx.doi.org/10.1021/acs.jproteome.5b00359DOI Listing
November 2015

Distinct Responses of Mycobacterium smegmatis to Exposure to Low and High Levels of Hydrogen Peroxide.

PLoS One 2015 30;10(7):e0134595. Epub 2015 Jul 30.

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, CAS, Beijing, 100101, China; Beijing Key Laboratory of Microbial Drug Resistance and Resistome, Beijing 100101, Beijing, China.

Hydrogen peroxide (H2O2) is a natural oxidant produced by aerobic organisms and gives rise to oxidative damage, including DNA mutations, protein inactivation and lipid damage. The genus Mycobacterium utilizes redox sensors and H2O2 scavenging enzymes for the detoxification of H2O2. To date, the precise response to oxidative stress has not been fully elucidated. Here, we compared the effects of different levels of H2O2 on transcription in M. smegmatis using RNA-sequencing. A 0.2 mM H2O2 treatment had little effect on the growth and viability of M. smegmatis whereas 7 mM H2O2 was lethal. Analysis of global transcription showed that 0.2 mM H2O2 induced relatively few changes in gene expression, whereas a large proportion of the mycobacterial genome was found to be differentially expressed after treatment with 7 mM H2O2. Genes differentially expressed following treatment with 0.2 mM H2O2 included those coding for proteins involved in glycolysis-gluconeogenesis and fatty acid metabolism pathways, and expression of most genes encoding ribosomal proteins was lower following treatment with 7 mM H2O2. Our analysis shows that M. smegmatis utilizes the sigma factor MSMEG_5214 in response to 0.2 mM H2O2, and the RpoE1 sigma factors MSMEG_0573 and MSMEG_0574 in response to 7 mM H2O2. In addition, different transcriptional regulators responded to different levels of H2O2: MSMEG_1919 was induced by 0.2 mM H2O2, while high-level induction of DevR occurred in response to 7 mM H2O2. We detected the induction of different detoxifying enzymes, including genes encoding KatG, AhpD, TrxB and Trx, at different levels of H2O2 and the detoxifying enzymes were expressed at different levels of H2O2. In conclusion, our study reveals the changes in transcription that are induced in response to different levels of H2O2 in M. smegmatis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134595PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4520597PMC
May 2016

Proteomic Analysis of Drug-Resistant Mycobacteria: Co-Evolution of Copper and INH Resistance.

PLoS One 2015 2;10(6):e0127788. Epub 2015 Jun 2.

MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing, China.

Tuberculosis, caused by the pathogen Mycobacterium tuberculosis, is a worldwide public health threat. Mycobacterium tuberculosis is capable of resisting various stresses in host cells, including high levels of ROS and copper ions. To better understand the resistance mechanisms of mycobacteria to copper, we generated a copper-resistant strain of Mycobacterium smegmatis, mc2155-Cu from the selection of copper sulfate treated-bacteria. The mc2155-Cu strain has a 5-fold higher resistance to copper sulfate and a 2-fold higher resistance to isoniazid (INH) than its parental strain mc2155, respectively. Quantitative proteomics was carried out to find differentially expressed proteins between mc2155 and mc2155-Cu. Among 345 differentially expressed proteins, copper-translocating P-type ATPase was up-regulated, while all other ABC transporters were down-regulated in mc2155-Cu, suggesting copper-translocating P-type ATPase plays a crucial role in copper resistance. Results also indicated that the down-regulation of metabolic enzymes and decreases in cellular NAD, FAD, mycothiol, and glutamine levels in mc2155-Cu were responsible for its slowing growth rate as compared to mc2155. Down-regulation of KatG2 expression in both protein and mRNA levels indicates the co-evolution of copper and INH resistance in copper resistance bacteria, and provides new evidence to understanding of the molecular mechanisms of survival of mycobacteria under stress conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127788PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4452738PMC
April 2016

[A hemerythrin-like protein MSMEG_3312 influences erythromycin resistance in mycobacteria].

Wei Sheng Wu Xue Bao 2014 Nov;54(11):1279-88

Objective: Reactive oxygen species are natural products of metabolism in aerobic organisms, which lead to oxidative damage, such as DNA mutation, protein inactivation and drug resistance. MSMEG_3312 was predicted as a hemerythrin-like protein, which can carry oxygen and reversibly bind to oxygen, thus it might play important roles in the process of oxygen metabolism. In this study, we explored the role of MSMEG_3312 in drug resistance.

Methods: On the basis of bioinformatics, we identified the conserved sequence of HHE domain in MSMEG_3312 and it was predicted to have typical α-helix at secondary structure. To explore potential functions of MSMEG_3312, we constructed the msmeg_3312 knockout strain and compare the susceptibility to various drugs to its parent strain, mc2155. In addition, we also measured the promoter response when treatment of erythromycin.

Results: Genetic results showed that MSMEG_3312 is not necessary for M. smegmatis growth at 7H9 rich medium. The msmeg_3312 knockout strain showed increased erythromycin resistance. Moreover, the drug resistance is only limited to erythromycin which its mechanism of action is by binding to the 50S subunit of the bacteria ribosomal complex and then inhibit protein synthesis. However, there were no different MICs of other antibiotics, targets for protein synthesis inhibition, but not 50S subunit, such as tetracyclines, aminoglycosides and chloramphenicol. Moreover, we also showed that the promoter of msmeg_3312 responses to erythromycin.

Conclusions: Hemerythin-like protein MSMEG_3312 is involved in erythromycin resistance.
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November 2014

Quantitative proteomics reveals novel insights into isoniazid susceptibility in mycobacteria mediated by a universal stress protein.

J Proteome Res 2015 Mar 18;14(3):1445-54. Epub 2015 Feb 18.

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, CAS , Beijing 100101, China.

Tuberculosis (TB) is caused by the ancient pathogen, Mycobacterium tuberculosis, and is one of the most serious infectious diseases in the world. Isoniazid (INH) is an important first-line drug for the treatment of active and latent TB. INH resistance is an increasing problem in the treatment of TB. Phenotypic resistance to INH, however, is poorly understood. In this study, we constructed a strain of Mycobacterium bovis BCG that overexpresses the latency-related universal stress protein (USP), BCG_2013, and designated this strain BCG-2013. BCG_2013 overexpression increased susceptibility to INH compared with that of the wild-type strain, BCG-pMV261. Quantitative proteomic analysis revealed that BCG_2013 overexpression resulted in the upregulation of 50 proteins and the downregulation of 26 proteins among the 1500 proteins identified. Upregulation of catalase-peroxidase KatG expression in BCG-2013 was observed and confirmed by qPCR, whereas expression of other INH resistance-related proteins did not change. In addition, differential expression of the mycobacterial persistence regulator MprA and its regulatory proteins was observed. BCG_2013 and katG mRNA levels increased in a Wayne dormancy model, whereas MprA mRNA levels decreased. Taken together, our results suggest that the increase in KatG levels induced by increased BCG_2013 levels underlies the phenotypic susceptibility of mycobacteria to INH.
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http://dx.doi.org/10.1021/pr5011058DOI Listing
March 2015

A bacterial hemerythrin-like protein MsmHr inhibits the SigF-dependent hydrogen peroxide response in mycobacteria.

Front Microbiol 2014 15;5:800. Epub 2015 Jan 15.

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences Beijing, China ; Beijing Key Laboratory of Microbial Drug Resistance and Resistome Beijing, China.

Hydrogen peroxide (H2O2) is one of a variety of reactive oxygen species (ROS) produced by aerobic organisms. Host production of toxic H2O2 in response to pathogen infection is an important classical innate defense mechanism against invading microbes. Understanding the mechanisms by which pathogens, in response to oxidative stress, mediate defense against toxic ROS, can reveal anti-microbial targets and shed light on pathogenic mechanisms. In this study, we provide evidence that a Mycobacterium smegmatis hemerythrin-like protein MSMEG_2415, designated MsmHr, is a H2O2-modulated repressor of the SigF-mediated response to H2O2. Circular dichroism and spectrophotometric analysis of MsmHr revealed properties characteristic of a typical hemerythrin-like protein. An msmHr knockout strain of M. smegmatis mc(2)155 (ΔmsmHr) was more resistant to H2O2 than its parental strain, and overexpression of MsmHr increased mycobacterial susceptibility to H2O2. Mutagenesis studies revealed that the hemerythrin domain of MsmHr is required for the regulation of the H2O2 response observed in the overexpression study. We show that MsmHr inhibits the expression of SigF (MSMEG_1804), an alternative sigma factor that plays an important role in bacterial oxidative stress responses, including those elicited by H2O2, thus providing a mechanistic link between ΔmsmHr and its enhanced resistance to H2O2. Together, these results strongly suggest that MsmHr is involved in the response of mycobacteria to H2O2 by negatively regulating a sigma factor, a function not previously described for hemerythrins.
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http://dx.doi.org/10.3389/fmicb.2014.00800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4295536PMC
February 2015

[Quantitative proteomics analysis of ClpS-mediated rifampicin resistance in Mycobacterium].

Sheng Wu Gong Cheng Xue Bao 2014 Jul;30(7):1115-27

Adaptor protein ClpS is an essential regulator of prokaryotic ATP-dependent protease ClpAP, which delivers certain protein substrates with specific amino acid sequences to ClpAP for degradation. However, ClpS also functions as the inhibitor of the ClpAP-mediated protein degradation for other proteins. Here, we constructed the clpS-overexpression Mycobacterium smegmatis strain, and showed for the first time that overexpression of ClpS increased the resistance of M. smegmatis to rifampicin that is one of most widely used antibiotic drugs in treatment of tuberculosis. Using quantitative proteomic technology, we systematically analyzed effects of ClpS overexpression on changes in M. smegmatis proteome, and proposed that the increased rifampicin resistance was caused by ClpS-regulated drug sedimentation and drug metabolism. Our results indicate that the changes in degradation related proteins enhanced drug resistance and quantitative proteomic analysis is an important tool for understanding molecular mechanisms responsible for bacteria drug resistance.
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July 2014

The gain of hydrogen peroxide resistance benefits growth fitness in mycobacteria under stress.

Protein Cell 2014 Mar;5(3):182-5

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, CAS, Beijing, 100101, China.

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http://dx.doi.org/10.1007/s13238-014-0024-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967060PMC
March 2014

Draft Genome Sequence of mc251, a Highly Hydrogen Peroxide-Resistant Mycobacterium smegmatis Mutant Strain.

Genome Announc 2014 Feb 20;2(1). Epub 2014 Feb 20.

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, CAS, Beijing, China.

Here, we report the draft genome sequence of the Mycobacterium tuberculosis-like Mycobacterium smegmatis mutant strain, mc(2)51, compared to that of wild-type strain M. smegmatis mc(2)155. The draft genome sequence comprises 6,823,739 bp, revealing 6,569 coding DNA sequences (CDSs) and 50 RNA genes (4 rRNA genes and 46 tRNA genes).
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http://dx.doi.org/10.1128/genomeA.00092-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931361PMC
February 2014

[Sigma factor F regulates Mycobacterium smegmatis hydrogen peroxide resistance].

Wei Sheng Wu Xue Bao 2012 Nov;52(11):1352-9

Norman Bethune Medical College, Jilin University, Changchun 130021, China.

Objective: A sigma factor is an important component of RNA polymerase complex and is essential for initiation of RNA synthesis. The sigma factors fall into 2 categories: primary sigma factor is essential for bacterial growth and the alterative sigma factor is activated under different environmental conditions. Sigma F (SigF) is one of the sigma factors of Mycobacterium tuberculosis, affecting its virulence and pathogenesis. In contrast, the ortholog of the non-virulent, fast growing strain Mycobacterium smegmatis has been suggested without similar physiology roles. Here, we studied the functions of M. smegmatis SigF.

Methods: sigF knockout Mycobacterium smegmatis strain was constructed by specialized transduction. The wild type, knockout and complementary stains were challenged by oxidative stress and antibiotics.

Results: The knockout sigF stain was susceptible oxidative stress, compared to wild type. Furthermore, there was no defect in resistance to antibiotics including isoniazid between the knockout sigF strain and wild type strain. In addition, SigF is required for carotenoid pigment production in M. smegmatis.

Conclusion: Our data suggested that SigF is important to detoxify the reactive oxygen species, probably through photo-oxidative stress response pathway, which is independent on the pathway that is required for the isoniazid activation.
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November 2012

Mycobacterium fluoroquinolone resistance protein B, a novel small GTPase, is involved in the regulation of DNA gyrase and drug resistance.

Nucleic Acids Res 2013 Feb 28;41(4):2370-81. Epub 2012 Dec 28.

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, CAS, Beijing, 100101, China.

DNA gyrase plays a vital role in resolving DNA topological problems and is the target of antibiotics such as fluoroquinolones. Mycobacterium fluoroquinolone resistance protein A (MfpA) from Mycobacterium smegmatis is a newly identified DNA gyrase inhibitor that is believed to confer intrinsic resistance to fluoroquinolones. However, MfpA does not prevent drug-induced inhibition of DNA gyrase in vitro, implying the involvement of other as yet unknown factors. Here, we have identified a new factor, named Mycobacterium fluoroquinolone resistance protein B (MfpB), which is involved in the protection of DNA gyrase against drugs both in vivo and in vitro. Genetic results suggest that MfpB is necessary for MfpA protection of DNA gyrase against drugs in vivo; an mfpB knockout mutant showed greater susceptibility to ciprofloxacin than the wild-type, whereas a strain overexpressing MfpA and MfpB showed higher loss of susceptibility. Further biochemical characterization indicated that MfpB is a small GTPase and its GTP bound form interacts directly with MfpA and influences its interaction with DNA gyrase. Mutations in MfpB that decrease its GTPase activity disrupt its protective efficacy. Our studies suggest that MfpB, a small GTPase, is required for MfpA-conferred protection of DNA gyrase.
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http://dx.doi.org/10.1093/nar/gks1351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575795PMC
February 2013

Mycobacterium tuberculosis universal stress protein Rv2623 regulates bacillary growth by ATP-Binding: requirement for establishing chronic persistent infection.

PLoS Pathog 2009 May 29;5(5):e1000460. Epub 2009 May 29.

Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA.

Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.
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http://dx.doi.org/10.1371/journal.ppat.1000460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682197PMC
May 2009

Deletion of the Mycobacterium tuberculosis resuscitation-promoting factor Rv1009 gene results in delayed reactivation from chronic tuberculosis.

Infect Immun 2006 May;74(5):2985-95

Division of Infectious Diseases, Department of Medicine, Montefiore Medical Center and Albert Einstein College of Medicine, Forchheimer Building, Room 406, 1300 Morris Park Ave., Bronx, NY 10461, USA.

Approximately one-third of the human population is latently infected with Mycobacterium tuberculosis, comprising a critical reservoir for disease reactivation. Despite the importance of latency in maintaining M. tuberculosis in the human population, little is known about the mycobacterial factors that regulate persistence and reactivation. Previous in vitro studies have implicated a family of five related M. tuberculosis proteins, called resuscitation promoting factors (Rpfs), in regulating mycobacterial growth. We studied the in vivo role of M. tuberculosis rpf genes in an established mouse model of M. tuberculosis persistence and reactivation. After an aerosol infection with the M. tuberculosis Erdman wild type (Erdman) or single-deletion rpf mutants to establish chronic infections in mice, reactivation was induced by administration of the nitric oxide (NO) synthase inhibitor aminoguanidine. Of the five rpf deletion mutants tested, one (deltaRv1009) exhibited a delayed reactivation phenotype, manifested by delayed postreactivation growth kinetics and prolonged median survival times among infected animals. Immunophenotypic analysis suggested differences in pulmonary B-cell responses between Erdman- and deltaRv1009-infected mice at advanced stages of reactivation. Analysis of rpf gene expression in the lungs of Erdman-infected mice revealed that relative expression of four of the five rpf-like genes was diminished at late times following reactivation, when bacterial numbers had increased substantially, suggesting that rpf gene expression may be regulated in a growth phase-dependent manner. To our knowledge, deltaRv1009 is the first M. tuberculosis mutant to have a specific defect in reactivation without accompanying growth defects in vitro or during acute infection in vivo.
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http://dx.doi.org/10.1128/IAI.74.5.2985-2995.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1459759PMC
May 2006

Multiple interactions of rad23 suggest a mechanism for ubiquitylated substrate delivery important in proteolysis.

Mol Biol Cell 2004 Jul 30;15(7):3357-65. Epub 2004 Apr 30.

Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245, USA.

The mechanism underlying the delivery of ubiquitylated substrates to the proteasome is poorly understood. Rad23 is a putative adaptor molecule for this process because it interacts with ubiquitin chains through its ubiquitin-associated motifs (UBA) and with the proteasome through a ubiquitin-like element (UBL). Here, we demonstrate that the UBL motif of Rad23 also binds Ufd2, an E4 enzyme essential for ubiquitin chain assembly onto its substrates. Mutations in the UBL of Rad23 alter its interactions with Ufd2 and the proteasome, and impair its function in the UFD proteolytic pathway. Furthermore, Ufd2 and the proteasome subunit Rpn1 compete for the binding of Rad23, suggesting that Rad23 forms separate complexes with them. Importantly, we also find that the ability of other UBL/UBA proteins to associate with Ufd2 correlates with their differential involvement in the UFD pathway, suggesting that UBL-mediated interactions may contribute to the substrate specificity of these adaptors. We propose that the UBL motif, a protein-protein interaction module, may be used to facilitate coupling between substrate ubiquitylation and delivery, and to ensure the orderly handoff of the substrate from the ubiquitylation machinery to the proteasome.
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http://dx.doi.org/10.1091/mbc.e03-11-0835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC452589PMC
July 2004