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Hu X.,CAS Institute of Microbiology | Li X.,CAS Institute of Microbiology | Huang L.,CAS Institute of Microbiology | Chan J.,Yeshiva University | And 4 more authors.
Journal of Proteome Research | Year: 2015

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. © 2015 American Chemical Society. Source

Li X.,CAS Institute of Microbiology | Tao J.,CAS Institute of Microbiology | Hu X.,CAS Institute of Microbiology | Chan J.,Yeshiva University | And 3 more authors.
Frontiers in Microbiology | Year: 2014

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 mc2 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. © 2014 Li, Tao, Hu, Chan, Xiao and Mi. Source

Zhang G.,CAS Institute of Microbiology | Wang C.,CAS Institute of Microbiology | Sui Z.,CAS Institute of Microbiology | Feng J.,CAS Institute of Microbiology | Feng J.,Beijing Key Laboratory of Microbial Drug Resistance and Resistome
Journal of Antimicrobial Chemotherapy | Year: 2015

Objectives: Fluoroquinolone resistance in Streptococcus pneumoniae typically arises through specific site mutations, but dynamic variation of mutations in the resistance evolution and interaction among these mutations have not been clearly demonstrated. The objectives of this study were to investigate the dynamics of allele frequency in populations evolved under fluoroquinolone pressure and pervasive interactions among mutations present in the evolutionary trajectories. Methods: Thirty-three evolved populationswere obtained by serial passages in the presence of antibiotic pressure and these populations were sequenced by using the Paired-End Illumina method. Mutants that occurred in the evolutionary trajectories were constructed by transforming the parental strain with PCR fragments containing corresponding mutations. Results: The number of target mutations increased progressively, consistent with phenotypic adaptation to moxifloxacin and levofloxacin. However, more mutations are required for high-level resistance to moxifloxacin than levofloxacin. Pervasive interactions, including positive epistasis between mutations, play a role in the evolutionary trajectories of resistance to the two drugs. Two mutations (R447C and P454S) in gyrB were identified to confer 2-fold increases in resistance to moxifloxacin and levofloxacin based on the background of the double mutant S81F/S79F in parC. Moreover, the dynamics of allele frequency in evolved populations was revealed and found to be directly correlated with the resistance levels of evolved populations. Clonal interference among alleles of mutations contributed to the molecular dynamics of resistance evolution. Conclusions: Our results provide novel insights into the evolutionary trajectories of resistance to fluoroquinolones and may serve as a theoretical basis for predicting resistance development and provide references for the clinical use of these drugs. © The Author 2015. Source

Zhou W.,CAS Institute of Microbiology | Yao K.,Capital Medical University | Zhang G.,CAS Institute of Microbiology | Yang Y.,Capital Medical University | And 4 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2014

Objectives The objective of this study was to identify the mechanism responsible for the horizontal transfer of transposon Tn2010 in Streptococcus pneumoniae, and the genomic alterations introduced by the transfer process. Methods Tn2010 was identified using PCR in 15 clinical isolates of S. pneumoniae with erythromycin resistance. S. pneumoniae and Enterococcus faecalis isolates were used as recipient cells in mating and transformation experiments to test the conjugative transferability and transformability of Tn2010. Whole-genome sequencing was used to assess the effects of the Tn2010 transfer on recipient genomes. The biological cost of the horizontal acquisition of Tn2010 and additional genomic changes was investigated by growth competition experiments. Results Tn2010 was transformed at a frequency of 3 × 10-7 transformants per cfu, whereas no transconjugants were detected using S. pneumoniae or E. faecalis as recipient cells. Genome analysis showed that many other recombinations were scattered throughout the genome of the transformants in addition to transposon Tn2010. The transformants demonstrated a negligible fitness cost compared with the wild-type strain. Conclusions Tn2010 tended to be transferred by transformation rather than conjugation in S. pneumoniae, and the spread of Tn2010 could have a profound effect on the evolution of the genome. The acquisition of Tn2010 with negligible fitness cost may facilitate spread of the transposon. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. Source

Zhu Y.,CAS Institute of Microbiology | Zhu Y.,Beijing Key Laboratory of Microbial Drug Resistance and Resistome | Yi Y.,The 306th Hospital of Peoples Liberation Army | Liu F.,CAS Institute of Microbiology | And 12 more authors.
Microbiological Research | Year: 2014

The class 1 integron is an important driver of the nosocomial dissemination of multidrug-resistant (MDR) bacteria, such as Acinetobacters. In this study, we characterized the gene cassette arrays of class 1 integrons in Acinetobacter baumannii, where the detailed structure of these integrons for 38 clinical strains was analyzed. The results showed that there are three types of gene cassette arrays that are carried by different class 1 integrons, among them the aac(6')-IId-catB8-aadA1 array was the most prevalent. For detailed analysis of the integron structure, whole genome sequencing was carried out on strain AB16, and it was found that a single integron on its chromosome has a partial Tn21 transposon in its 5' flanking region and two complete copies of the insertion element IS. 26 in both the 5' and 3' flanking regions, indicating that the integron could be acquired by horizontal gene transfer. Furthermore, there is one resistance island AbaR22, one bla gene containing a transposon, four intrinsic resistant genes and one efflux pump that together confer six types of antibiotic resistance. © 2014 Elsevier GmbH. Source

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