ABSTRACT: Persister cells are a sub-population of all bacterial cultures which exhibit a non-inheritable, multi-drug tolerance when subjected to lethal antibiotic challenge. These persisters arise as a result of metabolic dormancy, and can resume growth subsequent to antibiotic challenge, leading to recalcitrance of bacterial infections. Overproduction of DosP, an oxygen sensing protein with phosphodiesterase activity, increases bacterial persistence. Here we performed a microarray to determine the expression profile induced by DosP as a means to elucidate mechanisms of persister cell formation. dosP was overexpressed in Escherichia coli K-12 BW25113 and compared to the empty vector.
Project description:Persisters are a subpopulation of metabolically-dormant cells in biofilms that are resistant to antibiotics; hence, understanding persister cell formation is important for controlling bacterial infections. Previously we discerned that MqsR and MqsA of Escherichia coli are a toxin/antitoxin pair that influence persister cell production via their regulation of Hha, CspD, and HokA. Here, to gain more insights into the origin of persisters, we used protein engineering to increase the toxicity of toxin MqsR by reasoning it would be easier to understand the effect of this toxin if it were more toxic. We found that two mutations (K3N and N31Y) increase the toxicity four fold and increase persistence 73 fold compared to native MqsR by making the protein less labile. A whole transcriptome study revealed that the MqsR variant represses acid resistance genes (gadABCEWX and hdeABD), multidrug resistance genes (mdtEF), and osmotic resistance genes (osmEY). Corroborating these microarray results, deletion of rpoS as well as the genes that the master stress response regulator RpoS controls, gadB, gadX, mdtF, and osmY, increased persister formation dramatically to the extent that nearly the whole population became persistent. Therefore, the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS-based regulation of acid resistance, multidrug resistance, and osmotic resistance systems. For the whole-transcriptome study of BW25113 mqsR/pBS(Kan)-mqsR 2-1 versus BW25113 mqsR/pBS(Kan)-mqsR, planktonic cells were grown to a turbidity of 0.5 at 600 nm in LB medium with 1 mM IPTG at 37 °C, adjusted the turbidity to 1, and exposed to 20 μg/mL ampicillin with 1 mM IPTG for 1 h. Cells were isolated by centrifuging at 0°C, and RNALater® buffer (Ambion, Cat# AM7021) was added to stabilize RNA during the RNA preparation steps. Total RNA was isolated from cell pellets with Qiagen RNeasy mini Kit (Cat# 74104) using a bead beater. cDNA was synthesized and fragmented to obtain 50-200 base cDNA fragments. The fragmented cDNA was labelled with Biotin-ddUTP, and hybridization was performed at 45°C with 60 rpm for 16 hours. The probe array was washed, stained using Affymetrix Genechip Fluidics Station 450 and the software GenomeChipOperating Software (GCOS)”, and scanned using Affymetrix Genechip scanner GCS3000 7G system and the software GenomeChipOperating Software (GCOS). Data were processed with MAS 5.0 Expression Analysis Default Setting. Genes were identified as differentially expressed if the expression ratio was higher than the standard deviation: 4.0 fold (enriched and differentially degraded) cutoff for the DNA microarrays (standard deviation 1.3 fold), and if the p-value for comparing two chips was less than 0.05.
Project description:Toxin-antitoxin (TA) systems are ubiquitous throughout bacterial and archaeal genomes. TA systems consist of a stable toxin that inhibits growth and a labile antitoxin that prevents toxicity of the toxin. Here we made an artificial TA system (arT/arA) and performed a DNA microarray study for overproduction of the toxin. arT was overexpressed in Escherichia coli BW25113 and compared to the empty vector.
Project description:An antivirulence approach targets bacterial virulence rather than cell viability in the antibiotic approach that can readily lead to drug resistance. Opportunistic human pathogen Pseudomonas aeruginosa produces a variety of virulence factors, and biofilm cells of this bacterium are much more resistant to antibiotics than planktonic cells. To identify novel inorganic antivirulence compounds, the dual screenings of thirty-six metal ions were performed to identify that zinc ions and ZnO nanoparticle inhibited the pyocyanin production and biofilm formation in P. aeruginosa without affecting the growth of planktonic cells. Moreover, zinc ion and ZnO nanoparticle markedly reduced the production of 2-heptyl-3-hydroxy-4(1H)-quinolone and siderophore pyochelin, while increased the production of another sideropore pyoverdine and swarming motility. Further, zinc ion and ZnO nanoparticle clearly suppressed hemolytic activity in P. aeruginosa. Transcriptome analyses showed that ZnO nanoparticle induced zinc cation efflux pump czc operon, porin genes (oprD and opdT), and Pseudomonas type III repressor A ptrA, while repressed pyocyanin-related phz operon, which partially explains the phenotypic changes. Overall, ZnO nanoparticle is a potential candidate for use in an antivirulence approach against persistent P. aeruginosa infection. P. aeruginosa Genechip Genome Array (Affymetrix, P/N 900339) was used in order to study the cells after the addition of ZnO nanoparticles. DNA microarray analysis with one biological replicate was performed with an Affymetrix system. P. aeruginosa was inoculated in 25 ml of LB medium in 250 ml shaker flasks with overnight cultures (1 : 100 dilution). Cells were cultured for 5 h with shaking at 250 rpm with and without ZnO nanoparticles (1 mM). Before sample collection, RNase inhibitor (RNAlater, Ambion, TX, USA) was added, and the cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 s before centrifugation at 16,000 g for 2 min. The cell pellets were immediately frozen with dry ice and stored at –80°C. Total RNA was isolated using a Qiagen RNeasy mini Kit (Valencia, CA, USA).
Project description:Bacterial persister cells are phenotypic variants of regular cells that are tolerant to antibiotics. High-persister (hip) mutants of Mycobacterium tuberculosis produce 10- to 1,000-fold more persister cells than the wild type strain when challenged with various antibiotics. Comparison of gene expression pattern of the hip mutants may provide clues as to the genetic mechanisms underlying persister formation. Transcriptome analysis will provide information on what differentiates M. tuberculosis hip strains from regular strains, which will be useful in the development of anti-persister therapy for persister cell eradication. Twelve independent M. tuberculosis hip mutants and the wild type strain were grown to stationary phase in duplicate. To avoid analyzing the drug effect, total RNA was extracted from the cultures prior to the addition of antibiotic for hybridization to Affymetrix microarrays. Data analysis was performed by comparing the hip mutants to the wild type control.
Project description:Contamination with enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a worldwide problem but there is no effective therapy available for EHEC infection. Biofilm formation is closely related with EHEC infection and is one of the mechanisms of antimicrobial resistance. Antibiofilm screening of 560 plant secondary metabolites against EHEC shows that ginkgolic acids C15:1 and C17:1 at 5 μg/ml and Ginko biloba extract at 100 μg/ml significantly inhibited EHEC biofilm formation on the surface of polystyrene, nylon membrane, and glass. Importantly, the working concentration of ginkgolic acids and G. biloba extract did not affect bacterial growth and has been known to be non-toxic to human. Transcriptional analyses showed that ginkgolic acid C15:1 repressed curli genes and prophage genes in EHEC, which were corroborated by reduced fimbriae production and biofilm reduction in EHEC. Interestingly, ginkgolic acids and G. biloba extract did not inhibit the biofilm formation of commensal E. coli K-12 strain. The current study suggests that plant secondary metabolites are important resource of biofilm inhibitors, as well as other bioactive compounds. E. coli GeneChip Genome 2.0 Array (Affymetrix, P/N 900551, Santa Clara, USA) was used to study the differential gene expression of the E. coli O157:H7 cells after the treatment with ginkgolic acid C15:1 (0.005 mg/ml). Cells were inoculated into 25 ml LB medium in 250 mL shake flasks with a starting OD600 of 0.05, and cultured at 37oC for 8 h without shaking in the presence or absence of ginkgolic acid C15:1 (5 μg/ml). To prevent RNA degradation, RNase inhibitor (RNAlater, Ambion, TX, USA) was added, and the EHEC cells were collected by centrifugation at 10,000 rpm for 1 min. The cell pellets obtained were immediately frozen with dry ice and stored at -80°C. Total RNA was isolated using a Qiagen RNeasy mini Kit (Valencia, CA, USA).
Project description:MqsR/MqsA is a well-characterized toxin/antitoxin system with several regulatory roles. Deletion of mqsRA reduced growth with deoxycholate stress. Here we performed a microarray to determine the expression profile with and without mqsRA under deoxycholate stress to determine transcriptional regulation. Escherichia coli K-12 BW25113 with and without mqsRA was exposed to deoxycholate stress.
Project description:Quorum sensing controls the expression of multiple virulence factors. PA14 genes lasR and rhlR are necessary for quorum sensing via homoserine lactones. A PA14 lasR rhlR deficient mutant exhibits a reduced oxidative stress response. Here we conducted a microarray to determine oxidative stress response gene regulation mediated by the homoserine lactone quorum sensing circuits. A PA14 lasR rhlR deficient mutant was compared to the wild-type with and without H2O2 stress.
Project description:Honey has been widely used against bacterial infection for centuries. Previous studies suggested that honeys in high concentrations inhibited bacterial growth due to the presence of anti-microbial compounds, such as methylglyoxal, hydrogen peroxide, and peptides. In this study, we found that three honeys (acacia, clover, and polyfloral) in a low concentration as below as 0.5% (v/v) significantly suppress virulence and biofilm formation in enterohemorrhagic E. coli O157:H7 affecting the growth of planktonic cells while these honeys do not harm commensal E. coli K-12 biofilm formation. Transcriptome analyses show that honeys (0.5%) markedly repress quorum sensing genes (e.g., AI-2 import and indole biosynthesis), virulence genes (e.g., LEE genes), and curli genes (csgBAC). We found that glucose and fructose in honeys are key compounds to reduce the biofilm formation of E. coli O157:H7 via suppressing curli production, but not that of E. coli K-12. Additionally, we observed the temperature-dependent response of honeys and glucose on commensal E. coli K-12 biofilm formation; honey and glucose increase E. coli K-12 biofilm formation at 37°C, while they decrease E. coli K-12 biofilm formation at 26°C. These results suggest that honey can be a practical tool for reducing virulence and colonization of the pathogenic E. coli O157:H7, while honeys do not harm commensal E. coli community in the human. For the microarray experiments, E. coli O157:H7 EDL933 was inoculated in 250 ml of LB in 1000 ml flasks with overnight cultures that were diluted at 1:100. Cells were shaken with 10g of glass wool at 100 rpm and 37°C for 7 hrs. Cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 sec before centrifugation in 50 ml centrifuge tubes at 13,000 g for 2 min; cell pellets were frozen immediately with dry ice and stored -80°C. RNA was isolated using Qiagen RNeasy mini Kit (Valencia, CA, USA). To eliminate DNA contamination, Qiagen RNase-free DNase I was used to digest DNA. RNA quality was assessed by Agilent 2100 bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and quantity was determined by ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., DE, USA).
Project description:VS94 gene expression at different time-points in SAPI medium in absence and presence of AI-2 was studied. Autoinducer-2 (AI-2) is produced by many species of bacteria, including various commensal bacteria and is involved in inter-species communication. Since, pathogens encounter AI-2 once they enter the human gastro-intestinal tract; we studied the effects of presence of AI-2 on various phenotypes associated with infection and colonization of enterohemorrhagic Escherichia coli (EHEC) namely, chemotaxis, motility and attachment to HeLa cells. AI-2 attracted EHEC when observed in agarose plug assays and also increased EHEC motility by 1.44-fold. AI-2 also increased EHEC attachment to HeLa cells by 1.6-fold; hence, suggesting that exposure to AI-2 inside the gastro-intestinal tract can play an important role in EHEC colonization. We then investigated the global effects of AI-2 on EHEC gene expression using DNA microarrays at various time-points. We found that AI-2 controls virulence gene expression and several other groups of genes (flagellar genes, iron related genes, biofilm genes etc.) associated with virulence in a time-dependent manner. Hence, through these studies we have shown that AI-2 may be a key component in EHEC infection of human gastro-intestinal tract. Experiment Overall Design: Strain: VS94 (EHEC luxS mutant) Experiment Overall Design: Medium: SAPI Experiment Overall Design: Temperature: 37C Experiment Overall Design: Cell type: Suspension Experiment Overall Design: Chemical: 100 uM AI-2 Experiment Overall Design: Overnight cultures of VS94 were diluted in SAPI medium to a turbidity of 0.1. The cells were allowed to grow to a turbidity of 0.5 at 37 oC and 100 µM AI-2 was added to the culture. In the control flasks, no AI-2 was added. The cultures were then allowed to grow for further 3.5 hours, 4 hours, 4.5 hours and 5.5 hours before cell pellets were collected by centrifugation at the above mentioned time-points and stored at -80oC. Experiment Overall Design: Total RNA was isolated from isolated cell pellets and RNA quality was assessed using gel electrophoresis. E. coli Genome 2.0 arrays (Affymetrix, California) containing 10,208 probe sets for all 20,366 genes present in four strains of E. coli, including E. coli O157:H7, were used to profile changes in gene expression using RNA samples for each treatment. Hybridization was performed for 16 hours, and the total cell intensity was scaled automatically in the software to an average value of 500. The data were inspected for quality and analyzed according to the procedures described by the manufacturer (Affymetrix: Data Analysis Fundamentals) which include using premixed polyadenylated transcripts of the B. subtilis genes (lys, phe, thr, and dap) at different concentrations. Genes were identified as differentially expressed if the expression ratio (between AI-2 and control cells at different time-points) was greater than 1.5 (based on the standard deviation between values measuring relative changes in expression) (Ren et al. 2004) and the change in the P value was less than 0.05. The differentially expressed genes were annotated using gene ontology definitions available in the Affymetrix NetAffx Analysis Center (http://www.affymetrix.com/analysis/index.affx).