Summary Bacterial multidrug tolerance is largely responsible for the inability of antibiotics to eradicate infections and is caused by a small population of dormant bacteria called persisters. HipA is a critical Escherichia coli persistence factor that is normally neutralized by HipB, a transcription repressor, which also regulates hipBA expression. Here we report multiple structures of HipA and a HipA-HipB-DNA complex. HipA has a eukaryotic Ser/Thr kinase-like fold and can phosphorylate the translation factor, EF-Tu, suggesting a persistence mechanism via cell stasis. The HipA-HipB-DNA structure reveals the HipB-operator binding mechanism, ~70° DNA bending and unexpected HipA-DNA contacts. Dimeric HipB interacts with two HipA molecules to inhibit its kinase activity through sequestration and conformational inactivation. Combined, these studies suggest mechanisms for HipA-mediated persistence and its neutralization by HipB.
Bacterial populations produce a small number of persister cells that exhibit multidrug tolerance. Persister cells are largely responsible for the antibiotic recalcitrance of biofilm infections. The mechanism of persister cell formation largely remains unknown due to the challenges in identifying persister genes. We screened an ordered comprehensive library of 3,985 Escherichia coli knockout strains to identify mutants with altered antibiotic tolerance. Stationary-state cultures in 96-well plates were exposed to ofloxacin at a concentration which allows only tolerant persister cells to survive. The persister cell level of each culture was determined. A total of 150 mutants with decreased persistence were identified in the initial screen, and subsequent validation confirmed that neither the growth rate nor the ofloxacin MIC was affected for 10 of them. The genes affected in these strains were dnaJ and dnaK (chaperones), apaH (diadenosine tetraphosphatase), surA (peptidyl-prolyl cis-trans isomerase), fis and hns (global regulators), hnr (response regulator of RpoS), dksA (transcriptional regulator of rRNA transcription), ygfA (5-formyl-tetrahydrofolate cyclo-ligase), and yigB (flavin mononucleotide [FMN] phosphatase). The prominent presence of global regulators among these strains pointed to the likely redundancy of persister cell formation mechanisms: the elimination of a regulator controlling several redundant persister genes would be expected to produce a phenotype. This observation is consistent with previous findings for a possible role of redundant genes such as toxin/antitoxin modules in persister cell formation. ygfA and yigB were of special interest. The mammalian homolog of YgfA (methenyltetrahydrofolate synthetase) catalyzes the conversion of 5-formyl-tetrahydrofolate (THF) into the rapidly degraded 5,10-methenyl-THF, depleting the folate pool. The YigB protein is a phosphatase of FMN which would deplete the pool of this cofactor. Stochastic overexpression of these genes could lead to dormancy and, hence, tolerance by depleting the folate and FMN pools, respectively. Consistent with this scenario, the overexpression of both genes produced increased tolerance to ofloxacin.Persister cells are multidrug-tolerant cells that contribute to the antibiotic recalcitrance of biofilm infections (12,27,53). Biofilms are implicated in many bacterial infections, including those associated with indwelling devices, dental disease, endocarditis, and cystic fibrosis (16,18,23,38,46). Understanding the the mechanism of persister cell formation/maintenance is likely to lead to new effective therapies for the treatment of biofilm infections. However, research in this area has been hindered by the difficulties in identifying persister genes.Persisters are phenotypic variants of the wild type that are tolerant to killing by antibiotics (8, 27). They are survivor cells that make up a small part of the population: 10 Ϫ6 to 10 Ϫ4 in exponentially growing cultures and ϳ10 Ϫ2 in stationary phase (33). A targeted search for mutants ...
Multidrug tolerance is largely responsible for chronic infections and caused by a small population of dormant cells called persisters. Selection for survival in the presence of antibiotics produced the first genetic link to multidrug tolerance: a mutant in the Escherichia coli hipA locus. HipA encodes a serine-protein kinase, the multidrug tolerance activity of which is neutralized by binding to the transcriptional regulator HipB and hipBA promoter. The physiological role of HipA in multidrug tolerance, however, has been unclear. Here we show that wild-type HipA contributes to persister formation and that high-persister hipA mutants cause multidrug tolerance in urinary tract infections. Perplexingly, high-persister mutations map to the N-subdomain-1 of HipA far from its active site. Structures of higher-order HipA-HipB-promoter complexes reveal HipA forms dimers in these assemblies via N-subdomain-1 interactions that occlude their active sites. High-persistence mutations, therefore, diminish HipA-HipA dimerization, thereby unleashing HipA to effect multidrug tolerance. Thus, our studies reveal the mechanistic basis of heritable, clinically relevant antibiotic tolerance.
Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon - background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence.
Examining global effects of toxins on gene expression profiles is proving to be a powerful method for toxicity assessment and for investigating mechanisms of toxicity. This study demonstrated the application of prokaryotic real-time gene expression profiling in Escherichia coli for toxicity assessment of environmental pollutants in water samples, by use of a cell-array library of 93 E. coli K12 strains with transcriptional green fluorescent protein (GFP) fusions covering most known stress response genes. The high-temporal-resolution gene expression data, for the first time, revealed complex and time-dependent transcriptional activities of various stress-associated genes in response to mercury and mitomycin (MMC) exposure and allowed for gene clustering analysis based on temporal response patterns. Compound-specific and distinctive gene expression profiles were obtained for MMC and mercury at different concentrations. MMC (genotoxin) induced not only the SOS response, which regulates DNA damage and repair, but also many other stress genes associated with drug resistance/sensitivity and chemical detoxification. A number of genes belonging to the P-type ATPase family and the MerR family were identified to be related to mercury resistance, among which zntA was found to be up-regulated at an increasing level as the mercury concentration increased. A mechanism-based evaluation of toxins based on real-time gene expression profiles promises, to be an efficient and informative method for toxicity assessment in environmental samples.
Europe is being severely challenged by the ageing of the population, and although for well over a decade now is looking for strategies to effectively increase the labour force participation of older workers and reduce the rates of early retirement and labour market exit, the unemployment amongst older people remains particularly high. The design and realization of age-friendly living and working environments is a huge challenge that we have just only started to address as the number of older citizens who are and want to continue being active members of society and live independently is constantly increasing. This paper introduces the SmartWork project, which aims at building a worker-centric Artificial Intelligence system for work ability sustainability, integrating unobtrusive sensing
Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lonbackground. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence.
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