Browsing by Subject "Escherichia coli"
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Item Accumulation of dihydrostreptomycin in Escherichia coli(1974) Andry, Kim Blanche ElaineItem Alkaline phosphatase formation in Escherichia coli K10(1968) Salanitro, Joseph PatrickItem Annual Precipitation and Discharge Drive Increases in Escherichia Coli Concentrations in an Urban Stream(Elsevier, 2022) Li, Rui; Filippelli, Gabriel; Wang, Lixin; Earth and Environmental Sciences, School of ScienceDetermining climate change influences on E. coli dynamics in urban aquatic systems and predicting future E. coli changes are important to regulate water quality. In this study, data from 6985 measurements of E. coli from 1999-2019 in the Indianapolis, Indiana (USA) urban waterway Pleasant Run were analyzed by Mann-Kendall and multiple linear regression to examine long term trends in E. coli concentrations and loads, and to project E. coli concentrations under future climate change scenarios. E. coli concentrations and loads monotonically increased over the last two decades, with E. coli concentrations increasing from 111 MPN/100 mL in 1999 to 911 MPN/100 mL in 2019. E. coli loads increased from 5×10 12 MPN/year to 90×10 12 MPN/year over the same period. E. coli showed peak concentration in summer, and significantly higher concentration in sites with Combined Sewer Outfalls relative to those without. Precipitation had both direct and indirect impacts on E. coli concentrations, meditated by stream discharge. Multiple linear regression results showed annual precipitation and discharge accounted for 60% of E. coli concentration variations. Based on the observed precipitation-discharge- E. coli concentration relationship, the projection results showed that, in the highest emission RCP 8.5 climate scenario, E. coli concentrations in 2020s, 2050s, and 2080s will be 1350 MPN/100mL, 1386 MPN/100mL, and 1443 MPN/100mL, respectively.Item Coordination logic of the sensing machinery in the transcriptional regulatory network of Escherichia coli(2007-10) Janga, Sarath Chandra; Salgado, Heladia; Martínez-Antonio, Agustino; Collado-Vides, JulioThe active and inactive state of transcription factors in growing cells is usually directed by allosteric physicochemical signals or metabolites, which are in turn either produced in the cell or obtained from the environment by the activity of the products of effector genes. To understand the regulatory dynamics and to improve our knowledge about how transcription factors (TFs) respond to endogenous and exogenous signals in the bacterial model, Escherichia coli, we previously proposed to classify TFs into external, internal and hybrid sensing classes depending on the source of their allosteric or equivalent metabolite. Here we analyze how a cell uses its topological structures in the context of sensing machinery and show that, while feed forward loops (FFLs) tightly integrate internal and external sensing TFs connecting TFs from different layers of the hierarchical transcriptional regulatory network (TRN), bifan motifs frequently connect TFs belonging to the same sensing class and could act as a bridge between TFs originating from the same level in the hierarchy. We observe that modules identified in the regulatory network of E. coli are heterogeneous in sensing context with a clear combination of internal and external sensing categories depending on the physiological role played by the module. We also note that propensity of two-component response regulators increases at promoters, as the number of TFs regulating a target operon increases. Finally we show that evolutionary families of TFs do not show a tendency to preserve their sensing abilities. Our results provide a detailed panorama of the topological structures of E. coli TRN and the way TFs they compose off, sense their surroundings by coordinating responses.Item Coordination of bacterial proteome with metabolism by cyclic AMP signalling(Springer Nature, 2013) You, Conghui; Okano, Hiroyuki; Hui, Sheng; Zhang, Zhongge; Kim, Minsu; Gunderson, Carl W.; Wang, Yi-Ping; Lenz, Peter; Yan, Dalai; Hwa, Terence; Microbiology and Immunology, School of MedicineThe cyclic AMP (cAMP)-dependent catabolite repression effect in Escherichia coli is among the most intensely studied regulatory processes in biology. However, the physiological function(s) of cAMP signalling and its molecular triggers remain elusive. Here we use a quantitative physiological approach to show that cAMP signalling tightly coordinates the expression of catabolic proteins with biosynthetic and ribosomal proteins, in accordance with the cellular metabolic needs during exponential growth. The expression of carbon catabolic genes increased linearly with decreasing growth rates upon limitation of carbon influx, but decreased linearly with decreasing growth rate upon limitation of nitrogen or sulphur influx. In contrast, the expression of biosynthetic genes showed the opposite linear growth-rate dependence as the catabolic genes. A coarse-grained mathematical model provides a quantitative framework for understanding and predicting gene expression responses to catabolic and anabolic limitations. A scheme of integral feedback control featuring the inhibition of cAMP signalling by metabolic precursors is proposed and validated. These results reveal a key physiological role of cAMP-dependent catabolite repression: to ensure that proteomic resources are spent on distinct metabolic sectors as needed in different nutrient environments. Our findings underscore the power of quantitative physiology in unravelling the underlying functions of complex molecular signalling networks.Item CpxA Phosphatase Inhibitor Activates CpxRA and Is a Potential Treatment for Uropathogenic Escherichia coli in a Murine Model of Infection(American Society for Microbiology, 2022-04-27) Fortney, Kate R.; Smith, Sara N.; van Rensburg, Julia J.; Brothwell, Julie A.; Gardner, Jessi J.; Katz, Barry P.; Ahsan, Nagib; Duerfeldt, Adam S.; Mobley, Harry L.T.; Spinola, Stanley M.; Microbiology and Immunology, School of MedicineCpxRA is an envelope stress response system that is highly conserved in the Enterobacteriaceae. CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR (CpxR-P), a transcription factor. In response to membrane stress, CpxR-P is produced and upregulates genes involved in membrane repair and downregulates genes that encode virulence factors that are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and in uropathogenic Escherichia coli (UPEC) are attenuated in murine models. We hypothesized that pharmacologic activation of CpxR could serve as an antimicrobial/antivirulence strategy and recently showed that 2,3,4,9-tetrahydro-1H-carbazol-1-amines activate the CpxRA system by inhibiting CpxA phosphatase activity. Here, we tested the ability of a series of three CpxRA-activating compounds with increasing potency to clear UPEC stain CFT073 in a murine urinary tract infection model. We show that these compounds are well tolerated and achieve sufficient levels to activate CpxR in the kidneys, bladder, and urine. Although the first two compounds were ineffective in promoting clearance of CFT073 in the murine model, the most potent derivative, compound 26, significantly reduced bacterial recovery in the urine and trended toward reducing bacterial recovery in the bladder and kidneys, with efficacy similar to ciprofloxacin. Treatment of CFT073 cultured in human urine with compound 26 fostered accumulation of CpxR-P and decreased the expression of proteins involved in siderophore biosynthesis and binding, heme degradation, and flagellar movement. These studies suggest that chemical activation of CpxRA may present a viable strategy for treating infections due to UPEC. IMPORTANCE: The increasing prevalence of urinary tract infections (UTIs) due to antibiotic-resistant uropathogenic Escherichia coli (UPEC) is a major public health concern. Bacteria contain proteins that sense their environment and have no human homologs and, thus, are attractive drug targets. CpxRA is a conserved sensing system whose function is to reduce stress in the bacterial cell membrane; activation of CpxRA reduces the expression of virulence determinants, which must cross the cell membrane to reach the bacterial surface. We previously identified a class of compounds that activate CpxRA. We show in a mouse UTI model that our most potent compound significantly reduced recovery of UPEC in the urine, trended toward reducing bacterial recovery in the bladder and kidneys, did not kill UPEC, and downregulated multiple proteins involved in UPEC virulence. Since these compounds do not act by a killing mechanism, they have potential to treat UTIs caused by antibiotic-resistant bacteria.Item Development and validation of a high-throughput cell-based screen to identify activators of a bacterial two-component signal transduction system(ACC, 2015-07) van Rensburg, Julia J.; Fortney, Kate R.; Chen, Lan; Krieger, Andrew J.; Lima, Bruno P.; Wolfe, Alan J.; Katz, Barry P.; Zhang, Zhong-Yin; Spinola, Stanley M.; Department of Microbiology and Immunology, IU School of MedicineCpxRA is a two-component signal transduction system (2CSTS) found in many drug-resistant Gram-negative bacteria. In response to periplasmic stress, CpxA autophosphorylates and donates a phosphoryl group to its cognate response regulator, CpxR. Phosphorylated CpxR (CpxR-P) upregulates genes involved in membrane repair and downregulates multiple genes that encode virulence factors, which are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and Haemophilus ducreyi are avirulent in mice and humans, respectively. Thus, the activation of CpxRA has high potential as a novel antimicrobial/antivirulence strategy. Using a series of Escherichia coli strains containing a CpxR-P-responsive lacZ reporter and deletions in genes encoding CpxRA system components, we developed and validated a novel cell-based high-throughput screen (HTS) for CpxRA activators. A screen of 36,000 compounds yielded one hit compound that increased reporter activity in wild-type cells. This is the first report of a compound that activates, rather than inhibits, a 2CSTS. The activity profile of the compound against CpxRA pathway mutants in the presence of glucose suggested that the compound inhibits CpxA phosphatase activity. We confirmed that the compound induced the accumulation of CpxR-P in treated cells. Although the hit compound contained a nitro group, a derivative lacking this group retained activity in serum and had lower cytotoxicity than that of the initial hit. This HTS is amenable for the screening of larger libraries to find compounds that activate CpxRA by other mechanisms, and it could be adapted to find activators of other two-component systems.Item Development of an Online 2D Ultrahigh-Pressure Nano-LC System for High-pH and Low-pH Reversed Phase Separation in Top-Down Proteomics(American Chemical Society, 2020-08-28) Wang, Zhe; Yu, Dahang; Cupp-Sutton, Kellye A.; Liu, Xiaowen; Smith, Kenneth; Wu, Si; Computer and Information Science, School of ScienceThe development of novel high-resolution separation techniques is crucial for advancing the complex sample analysis necessary for high-throughput top-down proteomics. Recently, our group developed an offline 2D high-pH RPLC/low-pH RPLC separation method and demonstrated good orthogonality between these two RPLC formats. Specifically, ultrahigh-pressure long capillary column RPLC separation has been applied as the second dimensional low-pH RPLC separation for the improvement of separation resolution. To further improve the throughput and sensitivity of the offline approach, we developed an online 2D ultrahigh-pressure nano-LC system for high-pH and low-pH RPLC separations in top-down proteomics. An online microtrap column with a dilution setup was used to collect eluted proteins from the first dimension high-pH separation and inject the fractions for ultrahigh-pressure long capillary column low-pH RPLC separation in the second dimension. This automatic platform enables the characterization of 1000+ intact proteoforms from 5 μg of intact E. coli cell lysate in 10 online-collected fractions. Here, we have demonstrated that our online 2D pH RP/RPLC system coupled with top-down proteomics holds the potential for deep proteome characterization of mass-limited samples because it allows the identification of hundreds of intact proteoforms from complex biological samples at low microgram sample amounts.Item Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence(MyJove Corporation, 2021-07-05) Warden, Cassandra; Simmons, Alan J.; Pasic, Lejla; Pitzer, Ashley; Davies, Sean S.; Layer, Justin H.; Mernaugh, Raymond L.; Kirabo, Annet; Medicine, School of MedicineIsolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.