Oxidation of dCTP contributes to antibiotic lethality in stationary-phase mycobacteria.

Growing evidence shows that generation of reactive oxygen species (ROS) derived from antibiotic-induced metabolic perturbation contribute to antibiotic lethality. However, our knowledge of the mechanisms by which antibiotic-induced oxidative stress actually kills cells remains elusive. Here, we show that oxidation of dCTP underlies ROS-mediated antibiotic lethality via induction of DNA double-strand breaks (DSBs). Deletion of mazG-encoded 5-OH-dCTP-specific pyrophosphohydrolase potentiates antibiotic killing of stationary-phase mycobacteria, but did not affect antibiotic efficacy in exponentially growing cultures. Critically, the effect of mazG deletion on potentiating antibiotic killing is associated with antibiotic-induced ROS and accumulation of 5-OH-dCTP. Independent lines of evidence presented here indicate that the increased level of DSBs observed in the ΔmazG mutant is a dead-end event accounting for enhanced antibiotic killing. Moreover, we provided genetic evidence that 5-OH-dCTP is incorporated into genomic DNA via error-prone DNA polymerase DnaE2 and repair of 5-OH-dC lesions via the endonuclease Nth leads to the generation of lethal DSBs. This work provides a mechanistic view of ROS-mediated antibiotic lethality in stationary phase and may have broad implications not only with respect to antibiotic lethality but also to the mechanism of stress-induced mutagenesis in bacteria.

Mycobacterial MazG safeguards genetic stability via housecleaning of 5-OH-dCTP.

Generation of reactive oxygen species and reactive nitrogen species in phagocytes is an important innate immune response mechanism to eliminate microbial pathogens. It is known that deoxynucleotides (dNTPs), the precursor nucleotides to DNA synthesis, are one group of the significant targets for these oxidants and incorporation of oxidized dNTPs into genomic DNA may cause mutations and even cell death. Here we show that the mycobacterial dNTP pyrophosphohydrolase MazG safeguards the bacilli genome by degrading 5-OH-dCTP, thereby, preventing it from incorporation into DNA. Deletion of the (d)NTP pyrophosphohydrolase-encoding mazG in mycobacteria leads to a mutator phenotype both under oxidative stress and in the stationary phase of growth, resulting in increased CG to TA mutations. Biochemical analyses demonstrate that mycobacterial MazG can efficiently hydrolyze 5-OH-dCTP, an oxidized nucleotide that induces CG to TA mutation upon incorporation by polymerase. Moreover, chemical genetic analyses show that direct incorporation of 5-OH-dCTP into mazG-null mutant strain of Mycobacterium smegmatis (Msm) leads to a dose-dependent mutagenesis phenotype, indicating that 5-OH-dCTP is a natural substrate of mycobacterial MazG. Furthermore, deletion of mazG in Mycobacterium tuberculosis (Mtb) leads to reduced survival in activated macrophages and in the spleen of infected mice. This study not only characterizes the mycobacterial MazG as a novel pyrimidine-specific housecleaning enzyme that prevents CG to TA mutation by degrading 5-OH-dCTP but also reveals a genome-safeguarding mechanism for survival of Mtb in vivo.

[Sequence homology analysis on lactate dehydrogenase (LDH) of Plasmodium vivax Anhui isolates].

DNA in dried blood spots of 39 vivax malaria patients (2009-2010) from Anhui (Bengbu urban district and counties of Wuhe, Huaiyuan, Mengcheng and Lixin) was extracted. The Plasmodium vivax LDH (PvLDH) gene was amplified, cloned and sequenced. The sequences were subjected to NCBI Blast program. The results showed that the targeted DNA fragment size was 951 bp without difference among the 39 samples (accession No. GU078391), and was more than 99% homologous to the PvLDH sequences in other strains from GenBank. There was only one different amino acid in the protein sequences between the isolates from Anhui and EJEU60134 or MIA061251 strains.

Housecleaning of pyrimidine nucleotide pool coordinates metabolic adaptation of nongrowing Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis (Mtb) to adopt a slowly growing or nongrowing state within the host plays a critical role for the bacilli to persist in the face of a prolonged multidrug therapy, establish latency and sustain chronic infection. In our previous study, we revealed that genome maintenance via MazG-mediated elimination of oxidized dCTP contributes to the antibiotic tolerance of nongrowing Mtb. Here, we provide evidence that housecleaning of pyrimidine nucleotide pool via MazG coordinates metabolic adaptation of Mtb to nongrowing state. We found that the ΔmazG mutant fails to maintain a nongrowing and metabolic quiescence state under dormancy models in vitro. To investigate bacterial metabolic changes during infection, we employed RNA-seq to compare the global transcriptional response of wild-type Mtb and the ΔmazG mutant after infection of macrophages. Pathway enrichment analyses of the differentially regulated genes indicate that the deletion of mazG in Mtb not only results in DNA instability, but also perturbs pyrimidine metabolism, iron and carbon source uptake, catabolism of propionate and TCA cycle. Moreover, these transcriptional signatures reflect anticipatory metabolism and regulatory activities observed during cell cycle re-entry in the ΔmazG mutant. Taken together, these results provide evidence that pyrimidine metabolism is a metabolic checkpoint during mycobacterial adaptation to nongrowing state.

Cloning identification and functional analysis of human IL-17A promoter.

OBJECTIVE: To conduct the cloning identification and characterization of the sequence of human IL-17A promoter so as to analyze the regulatory mechanism of the gene expression of IL-17. METHODS: First of all, the potential promoter region of IL-17A was found by means of the bioinformatics methods. Then, it was cloned into the reporter vector with PCR technique. Finally, the activity of the test promoter was determined by dual luciferase reporter system. RESULTS: Two transcriptional start points of the upper region, 600 bp and 1000 bp, of IL-17A were obtained by PCR clone and proved to have certain activities by dual luciferase reporter system. Also, they could be activated by IL-17A activator STAT3, which could start the expression of the reported gene. CONCLUSIONS: Clone established the regulatory region of human IL-17A promoter, which provided bases to the subsequent function research.

[Effects of veratryl alcohol and tween 80 on ligninase production and its roles in decolorization of azo dyes by white-rot basidiomycete PM2].

Basidiomycete PM2, a lignin-degrading white rot fungus, produces lgnin peroxidase (Lip) and manganese peroxidase (Mnp) in nutrient nitrogen limited liquid cultures. This fungus was selected for its ability to decolorize azo group of dyes. In order to improve production of the peroxidases and rapid dye decolorizing activity by basidiomycete PM2, the addition of veratryl alcohol or Tween 80 to nutrient nitrogen limited liquid cultures were tested. It was found to have a large stimulatory effect on Mnp activities and decolorization rate of azo dyes. A maximum Mnp activities of 254.2 u/L with veratryl alcohol and 192.2 u/L with Tween 80 were achieved respectively. These values were about 3.4-fold and 2.5-fold higher than that obtained in the control cultures (without alcohol or Tween 80), whereas the levels of Lip activity detected were very low (about 12 u/L)in all the cultures. In further experiments using three kinds of azo dyes of congo red, orange G and orange IV, enzyme activities and dye decolorization were investigated in the above-mentioned cultures. The results showed that Mnp activities and decolorization were notably higher than those obtained in the control cultures in the presence of azo dyes. Cultures supplemented with Tween 80 were more adequate for dye decolorization. The rates of the decolorization with Tween 80 of congo red (95.4%), orange G (98.5%) and orange IV (54.4%) after 24 hours of dye incubation were higher than that supplemented with veratryl alcohol. According to the results, Mnp activities secreted by basidiomycete PM2 play an essential role in the process of dye decolorization. Tween 80 was the main factor affecting the decolorization. The analysis of structure of the three kinds of azo dyes indicats that the extent of decolorization is affected by the dye molecular structure. The types and quantity of the substituted groups on the aromatic ring of azo dyes have effect on the percentage of biological decolorization.