ABSTRACT
Os sistemas toxina-antitoxinas (TA) compreendem um conjunto de genes que são amplamente difundidos em procariotos. No cromossomo, os sistemas podem estar envolvidos na indução de morte celular em resposta a condições estressantes, indução de persistência, formação de biofilme, colonização de novos nichos, manutenção da mobilidade bacteriana e virulência de bactérias patogênicas. Em E. coli K12, 36 sistemas TA foram descritos, dos quais o do tipo II é o mais abundante e estudado. Dentre as oito toxinas pesquisadas nesse trabalho, o gene da toxina HipA está presente em 76 das 100 cepas de ExPEC estudadas. Apesar da abundância de hipA em ExPEC e em diversos genomas bacterianos, a participação dos sistemas hipA/B na indução da persistência ainda não é clara. Portanto, o sistema hipA/B de duas cepas ExPEC isoladas de infecção sanguínea foi deletado, e estas foram avaliadas quando a indução da persistência bacteriana na presença de antibióticos, formação de biofilme, resistência ao soro e sobrevivência em macrófagos. O sistema TA hipA/B não influenciou no fenótipo de resistência ao soro humano e na sobrevivência intracelular em macrófagos, no entanto, participou da indução da persistência por ciprofloxacino em um isolado (EC182); e da formação de biofilme em superfície de vidro do isolado (EC273)
Toxin-antitoxin (TA) systems comprise a set of genes that are widespread in prokaryotes. On the chromosome, the systems may be involved in the induction of cell death in response to stressful conditions, persistence induction, biofilm formation, colonization of new niches, maintenance of bacterial mobility and virulence. In E. coli K12, 36 TA systems have been described, of which type II is the most abundant. Among the eight toxins searched in this work, hipA is present in 76 bacteria of the 100 ExPEC strains studied. Despite the abundance of hipA in ExPEC and in several bacterial genomes, the participation of hipA/B modules in the persistence is still unclear. Therefore, hipA/B system of two ExPEC strains isolated from blood infection was deleted and consequently evaluated in bacterial persistence induced by antibiotics, serum resistance and macrophage survival. Despite the fact that, the TA hipA/B system did not influence the phenotype of resistance to human serum and intracellular survival in macrophages. Herein, we described that hipA/B was important for persistence induction in one isolate (EC182); and may participate in the biofilm formation on the glass surface in the other studied strain (EC273)
Subject(s)
Toxin-Antitoxin Systems , Biofilms , Extraintestinal Pathogenic Escherichia coli/classification , Anti-Bacterial Agents/adverse effectsABSTRACT
TA (Toxin-Antitoxin) systems are widely spread in chromosomes and plasmids of bacteria and archaea. These systems consist of two co-expression genes, encoding stable toxin and sensitive antitoxin, respectively. The toxicity of toxins usually inhibits bacterial growth and antitoxins can neutralize the toxins. Interaction between them would regulate the growth state of bacteria precisely. According to the composition of TA and nature of antitoxin, six types of TA have been found. The role of these TA systems in bacteria has been a hot research topic in recent years. Now, the research status on functions of bacterial TA is reviewed.
ABSTRACT
We discussed the function of four pairs of genes in the toxin-antitoxin system of Mycobacterium tuberculosis,providing theoretical foundation and scientific basis for studying the transmission mechanism of Mycobacterium tuberculosis.Four pairs of genes which belong to VapBC family,including four VapC genes (Rv1720c,Rv2103c,Rv2494,Rv3408) and four VapB genes (Rv1721c,Rv2104c,Rv2493,Rv3407) were chosen.We constructed a serial of arabinose-induced hybrid plasmid system in Escherichia coli and a serial of acetamide-induced hybrid plasmid system in Mycobacterium smegmatis respectively,in order to observe the potential inhibition effect of VapC and the release inhibition of homologous VapB.Results showed that only one toxin gene(Rv2103c) showed the function of bacteriostasis in both E.coli and M.smegmatis and the homologous antitoxin gene(Rv2104c) could release the inhibition of growth.We built the inducible systems of VapBC family in both E.coli and M.smegmatis respectively and found only a pair of toxin and antitoxin genes(Rv2103c,Rv2104c) had the function of inhibition and release for the growth of bacteria.And two pairs of toxin genes(Rv1720c,Rv2494) did not have the function of inhibition for the growth of both E.coli and M.smegmatis.Whereas,another toxin gene VapC47(Rv3408) also did not have the bacteriostastic activity,only this result was not consistent with the existing literature.We speculated that the reason for this kind of difference may be the different inducible systems we used.Cause the other three results were consistent with all existing literature and the doubtful result also appeared in other reports,so our protocol could be confirmed as reliable,and we would use it to build inducible systems and make further functional identification of certain toxin and antitoxin genes that we are interested in.
ABSTRACT
We investigate the different expression of toxin gene mazF3,6,9 and antitoxin gene mazE3,6,9 in the drug-resistance Mycobacterium tuberculosis,we used quantitative real-time polymerase chin reaction method to detect the expression level of toxin gene mazF3,6,9 and antitoxin gene mazE3,6,9 in M.tuberculosis (20 mono-resistance strains,20 multidrug resistance strains and standard strain H37Rv).The differences of gene expression levels between groups were analyzed by oneway ANOVA.Contrasting with control group,toxin genes mazF6,9 were up-regulated expression levels both in mono-resistance (11.1519±22.31721;8.4306±17.97897) and multidrug resistance (4.6016±1.29018;6.9627±6.92948),had statistical significance (P＜0.01),mazF3 expression levels had statistical significance neither in mono-resistance nor in multidrug resistance (P＞0.05);antitoxin genes mazE3 was in down-expression level,and had statistical significance both in mono-resistance (0.3606±0.12527) and multidrug resistance (0.2016±0.16542) (P＜0.01),mazE6 had no statistical significance (P＞0.05)either in mono-resistance or multi drug resistance,mazE9 only in multidrug resistance(0.3989±0.37679) was in downexpression level,and has statistical significance (P＜0.001).The toxin gene mazF6,9 and antitoxin gene mazE3,9 may participate in the drug-resistance formation of M.tuberculosis.
ABSTRACT
Objective To determine the function of toxic protein VapC in toxin/antitoxin system of Leptospira species and the cytotoxicity to host cells of the toxic protein.Methods Using genomic DNA of pathogenic L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai as the template,several PCRs were performed to amplify entire vapB,vapC and vapBC genes.Subsequently,the prokaryotic expression systems of vapB,vapC and vapBC genes were constructed.Expression of the target recombinant proteins rVapB and rVapC was detected by SDS-PAGE and the expressed rVapB and rVapC were extracted by NiNTA affinity chromatography.Activity of rVapB and rVapC to lyse the DNAs or RNAs from L.interrogans strain Lai and THP-1 cells were then determined.The changes of transcription and expression of vapB and vapC genes of L.interrogans strain Lai before and after infection of THP-1 cells were detected by real-time fluorescent quantitative RT-PCR and Western blot assay.The eukaryotic expression vectors of the vapB and vapC genes were generated for transfection of host cells and CCK-8 agent was used to detect the effect of leptospiral VapB and VapC proteins on activity of host cells.Results The nucleotide and putative amino acid sequences of the cloned vapB and vapC genes were completely identical with the reported corresponding genes.The constructed prokaryotic expression systems could express rVapB and rVapC,respectively.rVapC displayed RNase avtivity but did not lyse DNA.When L.interrogans strain Lai infected THP-1 cells,the transcription and expression of vapB and vapC genes were upregulated and partial VapC protein was secreted from the leptospiral cells.The mass mortality was observed in HEK293 human renal tubular epithelial ceils containing the vapC gene through transfection.Conclusion VapC protein of L.interrogans strain Lai is a RNase and is secreted during infection of host cells with obvious cytotoxicity.