Browsing by Subject "Escherichia coli"
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Item Allosteric Determinants of Guanine Nucleotide Binding Proteins and Methods to Crystallize the Cytosolic Domains of Adenylyl Cyclase(2004-05-04) Hatley, Mark Edward; Gilman, Alfred G.The cytosolic domains of mammalian adenylyl cyclases, termed C1 and C2, are responsible for catalytic activity and most regulatory properties. Crystal structures of the soluble catalytic core of adenylyl cyclase bound to activators Gsa and forskolin were previously determined. However, structural information regarding low activity (non-Gsa or forskolin bound) states of the enzyme is lacking. Genetic and biochemical methods were utilized to overcome the low affinity of the cytosolic domains in the absence of activators. A genetic screen in Saccharomyces cerevisiae identified mutations that activate mammalian adenylyl cyclase in the absence of Gsa. The increased affinity of the K1014N-C2 mutant protein for the C1 domain in the absence of Gsa was exploited to isolate a complex containing C1 and C2 in the absence of Gsa. Unfortunately, this complex crystallized but failed to diffract due to heterogeneity. Intein-mediated protein ligation and expression of a C1-C2 fusion protein in adenylyl cyclase deficient Escherichia coli were explored to circumvent the low affinity of the domains. However, the yields of products were insufficient for crystallization. Members of the G protein superfamily contain nucleotide-dependent switches that dictate the specificity of their interactions with binding partners. Using a new sequence-based method termed statistical coupling analysis (SCA), I identified the allosteric core of these proteins - the network of amino acid residues that couples the domains responsible for nucleotide binding and protein-protein interactions. One-third of the 38 residues identified by SCA were mutated in the G protein Gsa, and the interactions of GTPgamma S- and GDP-bound mutant proteins were tested with both adenylyl cyclase (preferential binding to GTP-Gsa) and the G protein beta gamma subunit complex (preferential binding to GDP-Gsa). A two-state allosteric model predicts that mutation of residues that control the equilibrium between GDP- and GTP-bound conformations of the protein will cause the ratio of affinities of these species for adenylyl cyclase and beta gamma to vary in a reciprocal fashion. Observed results were consistent with this prediction. The network of residues identified by the SCA appears to comprise a core allosteric mechanism conferring nucleotide-dependent switching; the specific features of different G protein family members are built upon this core.Item Changes in the Gut Metabolic Landscape Drive Inflammation-Associated Dysbiosis and Host Responses(2020-12-01T06:00:00.000Z) Hughes, Elizabeth Rose; Sperandio, Vanessa; Pfeiffer, Julie K.; Alto, Neal; Winter, Sebastian E.Intestinal inflammation is frequently associated with alterations in composition of gut microbial communities, termed dysbiosis. Inflammation-associated dysbiosis is characterized by an expansion of facultative anaerobic bacteria in the Proteobacteria phylum, such as Escherichia coli. A dysbiotic microbiota has been linked to increased disease severity in the context of inflammatory bowel disease. However, the mechanisms responsible for inflammation-associated dysbiosis and its impact on disease are incompletely understood. Utilizing bioinformatic analyses of gut microbiota composition and mechanistic studies with Escherichia coli as a model organism in murine models, we uncovered two metabolic pathways that are unique to intestinal inflammation and responsible for changes in microbiota composition. Aerobic respiration coupled with formate oxidation, and utilization of molecular hydrogen fuel expansion of Escherichia coli populations during intestinal inflammation. The impact of oxygen leakage into the gut lumen on obligate anaerobic bacterial metabolism was additionally investigated. In vitro metabolite measurements and use of bacterial genetics indicated formate production increased in Bacteroides exposed to low oxygen levels. Formate measurements and exogenous delivery of formate in mice suggested that intestinal formate levels increase during inflammation and may exacerbate disease. However, further study is required. In conclusion, we identified key changes that occur during non-infectious inflammation in the gut metabolic landscape, illustrating the importance of understanding bacterial metabolism in order to understand host-microbiota interactions.Item Characterizing the Role of the E. Coli Cytochrome Oxidase AppBCX During Intestinal Inflammation(August 2021) Chanin, Rachael Beth; Sperandio, Vanessa; Winter, Sebastian E.; Pfeiffer, Julie K.; Moreland, JessicaDuring non-infectious colitis, oral antibiotic treatment, and enteric infection, changes in colonocyte metabolism allow for increased oxygen availability in the gut lumen, supporting the growth of facultative anaerobic bacteria primarily from the family Enterobacteriaceae. Additionally, recruitment of inflammatory cells, especially neutrophils, has also been shown to influence local oxygen levels. The oxidative burst mounted by neutrophils consumes oxygen, which in turn creates a hyper-hypoxic microenvironment. These two seemingly contradictory findings remain to be reconciled. The findings described here delineate an additional mechanism that helps explain these seemingly contradictory observations on oxygen availability and bacterial respiratory processes during non-infectious colitis. The picture emerging from our work is that reactive oxygen species (ROS) generated by the NADPH oxidase 1 (NOX1) at the epithelial interface serve as a local source of oxygen. Specifically, H2O2 deriving from epithelial NOX1, is detoxified via bacterial catalases to molecular oxygen. Facultative anaerobic bacteria can then respire this pool of oxygen. In this work, we have used commensal strains of E. coli as representative members of Enterobacteriaceae, a family of facultative anaerobic bacteria that is observed to outgrow during episodes of inflammation. In particular, we have studied the physiological function of the cytochrome bd oxidase, AppBCX. Using both chemical and genetic models of non-infectious colitis we have shown that it allows E. coli to utilize low levels of oxygen early in inflammation. Additionally, we have characterized the regulation of this enzyme in vivo highlighting the delicate balance between growth and survival during oxidative and nitrosative stress.Item Dysbiosis-Associated Changes in Host Metabolism Produce Lactate to Support Enterobacterial Expansion During Inflammation(2019-04-04) Gillis, Caroline Catherine; Hendrixson, David R.; Winter, Sebastian E.; Sperandio, Vanessa; Burstein, EzraThe lumen of the gastrointestinal tract is heavily colonized by microbes, termed the gut microbiota. Under normal conditions, fermentative anaerobes constitute the majority of the gut microbiota. However, during inflammation there is a change in the nutritional environment of the gut that enables the outgrowth of facultative aerobic Enterobacteriaceae through respiratory metabolism. Salmonella enterica serovar Typhimurium (S. Tm) is a pathogenic member of the Enterobacteriaceae family that benefits from inflammation. We found that S. Tm uses lactate as a nutrient during infection, which maximizes colonization of the gut. During S. Tm infection, a profound change in the microbial community of the gut occurs. In particular, butyrate-producing Clostridia species are depleted. Butyrate is the preferred substrate for β-oxidation by intestinal epithelial cells (IEC). In the absence of butyrate, IEC perform a fermentative metabolism that produces lactate as a waste product. Lactate is then used in conjunction with oxygen as a terminal electron acceptor to support growth of S. Tm in the murine gut lumen. We next investigated the regulation of lactate utilization in S. Tm. We found that the lactate utilization genes (lldPRD), were inducible by electron acceptors and L-lactate. The transcriptional response to L-lactate was coordinated by the regulatory protein LldR, which maximized colonization of the murine gut. Under anaerobic conditions, lldPRD expression was repressed by the two-component system ArcAB. Commensal members of the Enterobacteriaceae family also expand during non-infectious colitis. We investigated whether lactate was also produced during non-infectious colitis and if commensal Enterobacteriaceae could use this nutrient. Butyrate was depleted and lactate was abundant in a murine model of colitis. Metagenomic sequencing demonstrated that lactate dehydrogenase genes were more abundant in the microbiome of inflamed mice than control mice. We next began to characterize putative lactate dehydrogenases in E. coli. We identified several putative lactate dehydrogenases, however, their role in E. coli fitness requires further study. In conclusion, we identified an important host-derived nutrient that promotes S. Tm fitness during infection and may serve as a nutrient for commensal Enterobacteriaceae during non-infectious colitis. This illustrates the importance of nutrient acquisition for Enterobacteriaceae during inflammatory colonization of the gut.Item Engineered E. Coli That Detect and Respond to Gut Inflammation Through Nitric Oxide Sensing(2014-07-25) Archer, Eric Jeffry; Tu, Benjamin; Süel, Gürol M.; Mangelsdorf, David J.; Hooper, Lora V.; Gardner, Kevin H.Within the last several years, advances in synthetic biology have allowed for the development of re-programmed microorganisms that perform useful tasks in areas like fuel production, bioremediation, and medicine. Several engineered microorganisms are in pre-clinical development for the treatment of human diseases, but may face critical limitations that decrease their utility in medicine due to adverse events like sepsis, caused by the introduction of bacteria within patients. Here I describe the design, construction, and characterization of a synthetic genetic network that is intended for use by E. coli within lumen of the intestine, which is presumed to be a safer location than other tissues, such as blood, for the introduction of engineered microbes. The synthetic gene regulatory circuit described here regulates gene expression through the activation of a permanent DNA switch in response to nitric oxide produced by inducible nitric oxide synthase. The detection of nitric oxide initiates the expression of a DNA recombinase, causing the permanent genetic rearrangement of a short DNA segment containing a gene promoter, allowing for the regulation of output gene expression upon nitric oxide sensing. Here I demonstrate that E. coli containing this synthetic genetic circuit respond to nitric oxide as designed from both chemical nitric oxide donors and from injured mouse intestinal explants. This synthetic genetic circuit could be optimized for clinical use by allowing E. coli to reliably detect and treat inflammation in patients with inflammatory bowel disease, but the circuit described herein now serves as the proof-of-concept for both bacterial sensing of mammalian inflammation and for the use of DNA recombinases to translate transient environmental signals into permanent responses in engineered bacteria.Item Exploiting Evolutionary Tradeoffs to Fight Evolution of Antibiotic Resistance(2019-08-02) Tamer, Yusuf Talha; Reynolds, Kimberly A.; Rosen, Michael K.; Koh, Andrew Y.; Toprak, ErdalEvolution of antibiotic resistance is a growing public health problem around the world, and the identification of novel antimicrobials is no longer a viable approach to tackling this problem. To design smart technologies that take the evolutionary dimension into account, it is essential to understand the evolutionary process underlying the development of resistance. In nature, a single organism cannot be the most fit under all possible conditions, implying that bacterial populations that evolve resistance to antimicrobials should be less fit under other conditions. In this thesis, we report two examples of two tradeoffs in antibiotic-resistant bacterial populations. First, by studying biophysical and biochemical properties of the dihydrofolate reductase (DHFR) enzyme in Escherichia coli, we found that some mutations that conferred resistance to trimethoprim, a DHFR inhibitor, decreased drug affinity while substantially increasing substrate affinity. In addition, many of the epistatic interactions between such mutations were due to changes in the catalytic activities of DHFR mutants, rather than changes in trimethoprim affinity. We found that the high-order epistasis in catalytic power of DHFR (kcat and Km) created a rugged fitness landscape under trimethoprim selection. Taken together, these data provide a concrete illustration of how epistatic coupling at the level of biochemical parameters can give rise to complex fitness landscapes, suggesting new strategies for developing mutant specific inhibitors. In the second part of the thesis, we report that E. coli cells that evolved resistance against aminoglycosides pleiotropically evolved hypersensitivity against non-aminoglycoside antibiotics. A point mutation in a the potassium channel called TrkH decreases antibiotic efflux by altering the bacterial membrane potential. To mimic this phenotype, we designed and successfully used peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to silence efflux genes. Specifically, by silencing the acrA gene, we transiently induced antibiotic hypersensitivity in E. coli. This sequence-specific perturbation decreased the minimum lethal dose of several antibiotics. Moreover, this approach enables combination therapies using several pairs of antagonistic drugs with non-overlapping resistance mechanisms.Item The Functional Characterization of the Quorum Sensing E. Coli Regulators B and C in EHEC(2005-12-19) Clarke, Marcie B.; Sperandio, VanessaEnterohemorrhagic E. coli (EHEC) is the causative agent of hemorrhagic colitis. During an infection, EHEC can sense and respond to environmental cues, including the cell density of the intestinal normal flora (through the floral-derived AI-3 signal) and the epinephrine/norepinephrine produced naturally by the host. This cell-to-cell signaling may aid in colonization and disease by allowing EHEC to up-regulate its flagella and motility genes to swim closer to the intestinal epithelium. Previously, Sperandio et al. (2002) have shown that the quorum sensing E. coli regulators B and C (QseB&C), a two-component system in EHEC, are responsible for the regulation of the master regulator of flagella and motility genes, flhDC, in response to cell-to-cell signaling [1]. Here, we show that QseC, the membrane-bound sensor kinase, can autophosphorylate itself in response to AI-3 or epinephrine/norepinephrine and then transfer this phosphate to the response regulator, QseB. The autophosphorylation of QseC is not affected by the addition of autoinducer-2 or intestinal hormones, including gastrin, galanin, and secretin. Additionally, autophosphorylation can be antagonized upon the addition of phentolamine, an a-adrenergic receptor antagonist. Given that enterocytes harbor a-adrenergic receptors, it would be consistent for a microbial adrenergic sensor (QseC) to mostly resemble (in an orthologous and not a homologous fashion) an a- and not a ᭡drenergic receptor. Taken together, these results suggest that QseC may be a microbial adrenergic receptor conserved amongst different bacterial and fungal species. After QseC has autophosphorylated and transferred its phosphate to QseB, QseB acts as a transcription factor to activate the expression of flhDC, the master regulator of flagella and motility genes. Nested deletion analyses of the flhDC promoter suggest that QseB may bind to three promoter regions, to either repress or activate transcription. Further transcriptional studies suggest that phosphorylated QseB autoregulates its own transcription in a similar manner. These analyses have identified a QseB consensus binding sequence, which was utilized in an in silico search to identify novel potential targets of QseB. Through the use of both biochemistry and genetics, a comprehensive model of the QseB&C signaling cascade was generated.Item Testing Isogenicity of Recurrent UTI in Postmenopausal Women(2017-01-17) Wong, Daniel; Sarir, Saloomeh; Dao, Ryan; Thomas, Collin; Zimmern, Philippe E.PURPOSE: Due to increase in antibiotic allergies and resistance, the care of older women with recurrent urinary tract infections (UTIs) can be extremely challenging. Antibiotic regimens assume that infections are due to a single genetically identical species or isogen. The aim of this study was to use the classic method of phage typing to test whether the UTI pathogen in a patient is isogenic. Our hypothesis posits that infection may be due to the existence of a complex ecology of simultaneous infection by multiple same-species strains. METHODS: Mid-stream urine samples were taken from postmenopausal women with history of documented recurrent UTIs. Standard urine culture confirmed the presence of Escherichia coli bacterial strain. Urine sample was spread on LB agar plate and incubated for 24hrs at 37 degrees Celsius. 50 separate colonies were picked from the incubated plate and were treated in a patch assay with novel UTI targeting phages from the Rajagopal/Thomas Lab. Transilluminated images where taken with Biorad Image Lab equipment; then sensitivity to each phage was rated. Urine sample from patient 9 was used because our Lab had the most phages specific to lysing it. Phage clearings from the patch were rated on a scale of 0-3 based upon prevailing phage-typing metrics. (Ward et al. year) RESULTS: Differences to phage sensitivity across the 50 colonies numbered 9-1 to 9-50 were noted. Colonies 9-8, 9-44, and 9-46 were notably more resistant to a set of phages that was effective on all the other colonies. DISCUSSION: These findings in one representative older woman with recurrent UTI caused by Escherichia coli may have clinical significance particularly if differential phage sensitivity correlates with virulence, biofilm production, and antibiotic sensitivity variations. Pathogen findings in our urine samples suggest that infection may be an ecology of related, but nonidentical bacteria. CONCLUSION: The observed differences in phage sensitivity suggest there are multiple related, but non isogenic, Escherichia coli in the same bladder, a mechanism possibly contributing to antibiotic resistance and thus leading to UTI recurrence. Next step is to test each colony clone for its antibiotic sensitivity profile to determine if this novel observation could be clinically relevant.Item [UT Southwestern Medical Center News](2009-03-09) Shear, Kristen HollandItem [UT Southwestern Medical Center News](2010-05-11) Shear, Kristen HollandItem [UT Southwestern Medical Center News](2006-06-26) Despres, CliffItem [UT Southwestern Medical Center News](2012-03-01) Wormser, Deborah