Browsing by Subject "Escherichia coli"
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Item Open Access A noisy linear map underlies oscillations in cell size and gene expression in bacteria.(Nature, 2015-07-16) Tanouchi, Yu; Pai, Anand; Park, Heungwon; Huang, Shuqiang; Stamatov, Rumen; Buchler, Nicolas E; You, LingchongDuring bacterial growth, a cell approximately doubles in size before division, after which it splits into two daughter cells. This process is subjected to the inherent perturbations of cellular noise and thus requires regulation for cell-size homeostasis. The mechanisms underlying the control and dynamics of cell size remain poorly understood owing to the difficulty in sizing individual bacteria over long periods of time in a high-throughput manner. Here we measure and analyse long-term, single-cell growth and division across different Escherichia coli strains and growth conditions. We show that a subset of cells in a population exhibit transient oscillations in cell size with periods that stretch across several (more than ten) generations. Our analysis reveals that a simple law governing cell-size control-a noisy linear map-explains the origins of these cell-size oscillations across all strains. This noisy linear map implements a negative feedback on cell-size control: a cell with a larger initial size tends to divide earlier, whereas one with a smaller initial size tends to divide later. Combining simulations of cell growth and division with experimental data, we demonstrate that this noisy linear map generates transient oscillations, not just in cell size, but also in constitutive gene expression. Our work provides new insights into the dynamics of bacterial cell-size regulation with implications for the physiological processes involved.Item Open Access A prospective study of Escherichia coli bloodstream infection among adolescents and adults in northern Tanzania.(Transactions of the Royal Society of Tropical Medicine and Hygiene, 2020-05) Madut, Deng B; Rubach, Matthew P; Kalengo, Nathaniel; Carugati, Manuela; Maze, Michael J; Morrissey, Anne B; Mmbaga, Blandina T; Lwezaula, Bingileki F; Kilonzo, Kajiru G; Maro, Venance P; Crump, John ABackground
Characterization of the epidemiology of Escherichia coli bloodstream infection (BSI) in sub-Saharan Africa is lacking. We studied patients with E. coli BSI in northern Tanzania to describe host risk factors for infection and to describe the antimicrobial susceptibility of isolates.Methods
Within 24 h of admission, patients presenting with a fever at two hospitals in Moshi, Tanzania, were screened and enrolled. Cases were patients with at least one blood culture yielding E. coli and controls were those without E. coli isolated from any blood culture. Logistic regression was used to identify host risk factors for E. coli BSI.Results
We analyzed data from 33 cases and 1615 controls enrolled from 2007 through 2018. The median (IQR) age of cases was 47 (34-57) y and 24 (72.7%) were female. E. coli BSI was associated with (adjusted OR [aOR], 95% CI) increasing years of age (1.03, 1.01 to 1.05), female gender (2.20, 1.01 to 4.80), abdominal tenderness (2.24, 1.06 to 4.72) and urinary tract infection as a discharge diagnosis (3.71, 1.61 to 8.52). Of 31 isolates with antimicrobial susceptibility results, the prevalence of resistance was ampicillin 29 (93.6%), ceftriaxone three (9.7%), ciprofloxacin five (16.1%), gentamicin seven (22.6%) and trimethoprim-sulfamethoxazole 31 (100.0%).Conclusions
In Tanzania, host risk factors for E. coli BSI were similar to those reported in high-resource settings and resistance to key antimicrobials was common.Item Open Access Autochthonous ST405 NDM-5 producing Escherichia coli causing fatal sepsis in Northern Italy.(International journal of antimicrobial agents, 2020-05) Peri, Anna Maria; Piazza, Aurora; De Zan, Valentina; Carugati, Manuela; Muscatello, Antonio; Comandatore, Francesco; De Lorenzis, Elisa; Pluderi, Mauro; Arghittu, Milena; Cariani, Lisa; Cantù, Anna Paola; Bandi, Claudio; Cugno, Massimo; Gori, Andrea; Bandera, AlessandraItem Open Access Bacterial effector activates jasmonate signaling by directly targeting JAZ transcriptional repressors.(PLoS pathogens, 2013-10-31) Jiang, Shushu; Yao, Jian; Ma, Ka-Wai; Zhou, Huanbin; Song, Jikui; He, Sheng Yang; Ma, WenboGram-negative bacterial pathogens deliver a variety of virulence proteins through the type III secretion system (T3SS) directly into the host cytoplasm. These type III secreted effectors (T3SEs) play an essential role in bacterial infection, mainly by targeting host immunity. However, the molecular basis of their functionalities remains largely enigmatic. Here, we show that the Pseudomonas syringae T3SE HopZ1a, a member of the widely distributed YopJ effector family, directly interacts with jasmonate ZIM-domain (JAZ) proteins through the conserved Jas domain in plant hosts. JAZs are transcription repressors of jasmonate (JA)-responsive genes and major components of the jasmonate receptor complex. Upon interaction, JAZs can be acetylated by HopZ1a through a putative acetyltransferase activity. Importantly, P. syringae producing the wild-type, but not a catalytic mutant of HopZ1a, promotes the degradation of HopZ1-interacting JAZs and activates JA signaling during bacterial infection. Furthermore, HopZ1a could partially rescue the virulence defect of a P. syringae mutant that lacks the production of coronatine, a JA-mimicking phytotoxin produced by a few P. syringae strains. These results highlight a novel example by which a bacterial effector directly manipulates the core regulators of phytohormone signaling to facilitate infection. The targeting of JAZ repressors by both coronatine toxin and HopZ1 effector suggests that the JA receptor complex is potentially a major hub of host targets for bacterial pathogens.Item Open Access Biochemical and structural mechanisms of multidrug efflux pump transcription regulators, Neisseria gonorrhoeae MtrR and Escherichia coli MprA(2021) Beggs, Grace AnneAs bacterial resistance to multiple antibiotics continues to become a growing problem across the globe, the imperativeness for understanding mechanisms of antibiotic and multidrug resistance is increasingly apparent. Currently, the gram-negative bacteria Neisseria gonorrhoeae and Escherichia coli are considered urgent public health threats due to the rise in multidrug resistant strains. Mechanisms by which these bacteria become resistant to antibiotics include the overexpression of multidrug efflux systems. Prior to the introduction of antibiotics, the primary purpose of these multidrug efflux systems was to protect the bacteria from cytotoxins in the environment including innate host defense molecules or toxic molecules produced by the bacteria. Overtime, the bacteria have adapted these efflux systems to protect against clinically relevant antibiotics used to clear these bacterial infections. These multidrug efflux systems are energetically expensive to synthesize; thus, they are often tightly regulated at the transcription level by transcription repressors or activators. Many multidrug efflux systems are regulated by transcriptional regulators with proximal genes that specifically regulate a single multidrug efflux system. However, the expression of a few multidrug efflux systems is controlled by unique repressors that act as global regulators, which have a larger role in regulating complex virulence and stress response systems. Specifically, examples of multidrug efflux regulators in N. gonorrhoeae and E. coli that act as global regulators within their respective genomes include N. gonorrhoeae MtrR and E. coli MprA. Understanding the global regulatory activities of these two transcription regulators will broaden our understanding of the regulatory mechanisms that enable bacterial survival during host infection, the mechanisms that contribute to antibiotic resistance, as well as the fundamentals of bacterial transcription regulation. To provide novel insight into the global regulatory activities and function of N. gonorrhoeae MtrR, this dissertation expounds a series of original structural, biochemical, and in vivo studies identifying cytotoxin and DNA recognition mechanisms of MtrR. Previous work showed that MtrR represses directly the mtrCDE efflux transporter genes by binding an operator between the mtrR and mtrC genes; additionally, MtrR represses directly the rpoH oxidative stress response sigma factor. MtrR-mediated repression of the mtrCDE genes had been shown to be relieved upon exposure of gonococci to toxic hydrophobic agents and detergents (i. e. MtrR is “induced” by these toxic molecules). However, physiologically relevant innate host molecules recognized by MtrR had not been identified. In this work, we identify bile salts present at extra-urogenital gonococcal infection sites that MtrR directly binds, to result in derepression of the mtrCDE genes in vitro and in vivo. Furthermore, we use x-ray crystallography to solve structures of MtrR in its induced form and bound to the mtrCDE and rpoH operators. With these structures, we determined the structural mechanism of induction of MtrR. In addition, the MtrR-operator structures reveal a degenerate consensus sequence to which MtrR binds within the mtrCDE and rpoH operators. Mechanisms for cytotoxin and DNA recognition were confirmed by structure-guided site-directed mutagenesis studies and a combination of biochemical binding assays utilizing isothermal titration calorimetry (ITC) or fluorescence polarization (FP). Importantly, this structural and biochemical work also reveals the mechanisms by which common mutations in multidrug resistant strains of N. gonorrhoeae confer resistance. To elucidate the function of E. coli MprA and realize its potential as a drug target, this dissertation also includes research describing the ligand-binding mechanisms of MprA. MprA (formerly EmrR) represses directly the EmrAB efflux pump in E. coli. Previously published work identified MprA as the molecular target of a small molecule inhibitor (DU011) of the biosynthesis of an important virulence factor in E. coli, the polysaccharide capsule. This lead molecule has the potential for optimization for drug development and reveals a novel function of MprA as a regulator of polysaccharide capsule synthesis. We characterized the interaction between MprA and DU011 and compared this to the binding between MprA and other previously identified ligands including salicylate and 2,4-dinitrophenol (DNP) utilizing ITC assays. Through these studies, we revealed a novel binding mode for MprA and laid the groundwork for future structural studies and drug optimization. Collectively, this work provides important insight into the breadth of regulatory functions of N. gonorrhoeae MtrR and E. coli MprA, two key global regulators from highly prevalent multidrug resistant pathogens. Specifically, the original research presented here provides a biochemical evaluation of the bacterial stress response mechanisms controlled by MtrR and MprA and their contribution to antibiotic resistance. Indeed, the biochemical and structural characterization of these two regulators will inform future work to combat multidrug resistance.
Item Open Access Bloodstream infections in community hospitals in the 21st century: a multicenter cohort study.(PLoS One, 2014) Anderson, Deverick J; Moehring, Rebekah W; Sloane, Richard; Schmader, Kenneth E; Weber, David J; Fowler, Vance G; Smathers, Emily; Sexton, Daniel JBACKGROUND: While the majority of healthcare in the US is provided in community hospitals, the epidemiology and treatment of bloodstream infections in this setting is unknown. METHODS AND FINDINGS: We undertook this multicenter, retrospective cohort study to 1) describe the epidemiology of bloodstream infections (BSI) in a network of community hospitals and 2) determine risk factors for inappropriate therapy for bloodstream infections in community hospitals. 1,470 patients were identified as having a BSI in 9 community hospitals in the southeastern US from 2003 through 2006. The majority of BSIs were community-onset, healthcare associated (n = 823, 56%); 432 (29%) patients had community-acquired BSI, and 215 (15%) had hospital-onset, healthcare-associated BSI. BSIs due to multidrug-resistant pathogens occurred in 340 patients (23%). Overall, the three most common pathogens were S. aureus (n = 428, 28%), E. coli (n = 359, 24%), coagulase-negative Staphylococci (n = 148, 10%), though type of infecting organism varied by location of acquisition (e.g., community-acquired). Inappropriate empiric antimicrobial therapy was given to 542 (38%) patients. Proportions of inappropriate therapy varied by hospital (median = 33%, range 21-71%). Multivariate logistic regression identified the following factors independently associated with failure to receive appropriate empiric antimicrobial therapy: hospital where the patient received care (p<0.001), assistance with ≥3 ADLs (p = 0.005), Charlson score (p = 0.05), community-onset, healthcare-associated infection (p = 0.01), and hospital-onset, healthcare-associated infection (p = 0.02). Important interaction was observed between Charlson score and location of acquisition. CONCLUSIONS: Our large, multicenter study provides the most complete picture of BSIs in community hospitals in the US to date. The epidemiology of BSIs in community hospitals has changed: community-onset, healthcare-associated BSI is most common, S. aureus is the most common cause, and 1 of 3 patients with a BSI receives inappropriate empiric antimicrobial therapy. Our data suggest that appropriateness of empiric antimicrobial therapy is an important and needed performance metric for physicians and hospital stewardship programs in community hospitals.Item Open Access Contribution of urinary tract infection to the burden of febrile illnesses in young children in rural Kenya.(PLoS One, 2017) Masika, Wechuli Geoffrey; O'Meara, Wendy Prudhomme; Holland, Thomas L; Armstrong, JaniceINTRODUCTION: The clinical features of UTI in young children may not localize to the urinary tract and closely resemble other febrile illnesses. In malaria endemic areas, a child presenting with fever is often treated presumptively for malaria without investigation for UTI. Delayed or inadequate treatment of UTI increases the risk of bacteremia and renal scarring in young children and subsequently complications as hypertension and end stage renal disease in adulthood. METHODS: A cross-sectional study was carried out in a hospital in western Kenya. Inpatients and outpatients 2 months to five years with axillary temperature ≥37.5°C and no antibiotic use in the previous week were enrolled between September 2012 and April 2013. Urine dipstick tests, microscopy, and cultures were done and susceptibility patterns to commonly prescribed antibiotics established. UTI was defined as presence of pyuria (a positive urine dipstick or microscopy test) plus a positive urine culture. RESULTS: A total of 260 subjects were recruited; 45.8% were female and the median age was 25months (IQR: 13, 43.5). The overall prevalence of UTI was 11.9%. Inpatients had a higher prevalence compared to outpatients (17.9% v 7.8%, p = 0.027). UTI co-existed with malaria but the association was not significant (OR 0.80, p = 0.570). The most common organisms isolated were Escherichia coli (64.5%) and Staphylococcus aureus (12.9%) and were sensitive to ciproflaxin, cefuroxime, ceftriaxone, gentamycin and nitrofurantoin but largely resistant to more commonly used antibiotics such as ampicillin (0%), amoxicillin (16.7%), cotrimoxazole (16.7%) and amoxicillin-clavulinate (25%). CONCLUSION: Our study demonstrates UTI contributes significantly to the burden of febrile illness in young children and often co-exists with other infections. Multi-drug resistant organisms are common therefore choice of antimicrobial therapy should be based on local sensitivity pattern.Item Open Access Cyanoacrylate dressings: are they microbiologically impermeable?(The Journal of hospital infection, 2010-06) Rocos, B; Blom, AW; Bowker, KItem Open Access Differential coordination demands in Fe versus Mn water-soluble cationic metalloporphyrins translate into remarkably different aqueous redox chemistry and biology.(Inorganic chemistry, 2013-05-06) Tovmasyan, Artak; Weitner, Tin; Sheng, Huaxin; Lu, MiaoMiao; Rajic, Zrinka; Warner, David S; Spasojevic, Ivan; Reboucas, Julio S; Benov, Ludmil; Batinic-Haberle, InesThe different biological behavior of cationic Fe and Mn pyridylporphyrins in Escherichia coli and mouse studies prompted us to revisit and compare their chemistry. For that purpose, the series of ortho and meta isomers of Fe(III) meso-tetrakis-N-alkylpyridylporphyrins, alkyl being methyl to n-octyl, were synthesized and characterized by elemental analysis, UV/vis spectroscopy, mass spectrometry, lipophilicity, protonation equilibria of axial waters, metal-centered reduction potential, E(1/2) for M(III)P/M(II)P redox couple (M = Fe, Mn, P = porphyrin), kcat for the catalysis of O2(•-) dismutation, stability toward peroxide-driven porphyrin oxidative degradation (produced in the catalysis of ascorbate oxidation by MP), ability to affect growth of SOD-deficient E. coli, and toxicity to mice. Electron-deficiency of the metal site is modulated by the porphyrin ligand, which renders Fe(III) porphyrins ≥5 orders of magnitude more acidic than the analogous Mn(III) porphyrins, as revealed by the pKa1 of axially coordinated waters. The 5 log units difference in the acidity between the Mn and Fe sites in porphyrin translates into the predominance of tetracationic (OH)(H2O)FeP complexes relative to pentacationic (H2O)2MnP species at pH ∼7.8. This is additionally evidenced in large differences in the E(1/2) values of M(III)P/M(II)P redox couples. The presence of hydroxo ligand labilizes trans-axial water which results in higher reactivity of Fe relative to Mn center. The differences in the catalysis of O2(•-) dismutation (log kcat) between Fe and Mn porphyrins is modest, 2.5-5-fold, due to predominantly outer-sphere, with partial inner-sphere character of two reaction steps. However, the rate constant for the inner-sphere H2O2-based porphyrin oxidative degradation is 18-fold larger for (OH)(H2O)FeP than for (H2O)2MnP. The in vivo consequences of the differences between the Fe and Mn porphyrins were best demonstrated in SOD-deficient E. coli growth. On the basis of fairly similar log kcat(O2(•-)) values, a very similar effect on the growth of SOD-deficient E. coli was anticipated by both metalloporphyrins. Yet, while (H2O)2MnTE-2-PyP(5+) was fully efficacious at ≥20 μM, the Fe analogue (OH)(H2O)FeTE-2-PyP(4+) supported SOD-deficient E. coli growth at as much as 200-fold lower doses in the range of 0.1-1 μM. Moreover the pattern of SOD-deficient E. coli growth was different with Mn and Fe porphyrins. Such results suggested a different mode of action of these metalloporphyrins. Further exploration demonstrated that (1) 0.1 μM (OH)(H2O)FeTE-2-PyP(4+) provided similar growth stimulation as the 0.1 μM Fe salt, while the 20 μM Mn salt provides no protection to E. coli; and (2) 1 μM Fe porphyrin is fully degraded by 12 h in E. coli cytosol and growth medium, while Mn porphyrin is not. Stimulation of the aerobic growth of SOD-deficient E. coli by the Fe porphyrin is therefore due to iron acquisition. Our data suggest that in vivo, redox-driven degradation of Fe porphyrins resulting in Fe release plays a major role in their biological action. Possibly, iron reconstitutes enzymes bearing [4Fe-4S] clusters as active sites. Under the same experimental conditions, (OH)(H2O)FePs do not cause mouse arterial hypotension, whereas (H2O)2MnPs do, which greatly limits the application of Mn porphyrins in vivo.Item Open Access Differential inhibition of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin-mediated chemokine and gamma interferon production.(AIDS Res Hum Retroviruses, 2010-03) Geonnotti, Anthony R; Bilska, Miroslawa; Yuan, Xing; Ochsenbauer, Christina; Edmonds, Tara G; Kappes, John C; Liao, Hua-Xin; Haynes, Barton F; Montefiori, David CBacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.Item Open Access Differential Packaging of Outer Membrane Proteins of Enterotoxigenic Escherichia coli into Outer Membrane Vesicles under Oxidative Stress Conditions Reveals a Potential Mechanism for Vesicle Cargo Selectivity(2020) Orench-Rivera, NicholeOuter membrane vesicles (OMVs) are spherical structures that bud from the outer membrane (OM) of bacteria containing OM and periplasmic material. They are known to be produced by all bacteria studied to date and play important roles in inter-bacterial communication, bacterial-host interactions, toxin delivery, survival, nutrient acquisition, and biofilm development. The process of OMV production is known to be genetically regulated and selective cargo packaging into bacterial vesicles has been reported and implicated in many biological processes. While much is known about how cargo gets incorporated into vesicles in eukaryotic systems, the mechanism behind cargo selectivity in bacteria has remained largely unexplored. In this study we aimed to characterize preferential sorting trends in OMV packaging in Escherichia coli under oxidative stress, and investigate the mechanism behind selective sorting into OMVs. Proteomic analysis of outer membrane (OM) and OM vesicle fractions from enterotoxigenic E. coli (ETEC) revealed significant differences in protein abundance in the OMV and OM fractions for cultures shifted to oxidative stress conditions. Analysis of sequences of proteins preferentially packaged into OMVs showed that proteins with oxidizable residues were more packaged into OMVs in comparison with those retained in the membrane. In addition, the results indicated two distinct classes of OM-associated proteins were differentially packaged into OMVs as a function of peroxide treatment and we observed a slight increase in periplasmic content. Implementing a Bayesian hierarchical model, OM lipoproteins were determined to be preferentially exported during stress whereas integral OM proteins were preferentially retained in the cell. We first inquired whether this sorting was due to the need of the cell to discard or retain OM proteins and tested oxidative stress sensitivity of mutants of lipoproteins and integral proteins. We hypothesized that mutants of lipoproteins would not be more sensitive than integral proteins however both groups showed increased sensitivity. Therefore, this did not explain this preferential sorting. We next wondered if selectivity was dependent on gene expression. By mining gene expression databases and performing qRT-PCR we found the sorting to be independent of transcriptional regulation of the proteins upon oxidative stress. We were also able to validate these preferential sorting trends of lipoproteins vs integral proteins using randomly selected protein candidates from the different cargo classes. We also observed that a shift to oxidative stress conditions improved the fitness of bacteria to a secondary oxidative challenge, suggesting the differential sorting resulted in an OMV-mediated remodeling of the OM during stress. Together, our data showed that oxidative stress induced a differential sorting of proteins into OMVs and OM of E. coli and that OMV production might serve as a disposal mechanism for the cell to rid itself of oxidized proteins. Since our data revealed that the preferentially retained proteins were those known to have ties to other cell envelope components, a hypothetical functional and mechanistic basis for cargo selectivity was tested using OmpA as a model. A full-length and a truncated version of OmpA were used to test whether physical tethering to the cell is a determinant for protein retention in the OM. Quantifying OMV protein packaging of both OmpA constructs revealed a basic mechanism for cargo selectivity into OMVs. We show that the untethered version of OmpA was more likely to be exported than the tethered version and that this preferential selection was exacerbated under oxidative stress. The findings of this study provide insight into the dynamics of bacterial cargo selection and membrane remodeling during stress as well as propose and test a mechanism for cargo incorporation in E. coli.
Item Open Access Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis.(MBio, 2016-03-22) Young, Hayley E; Zhao, Jinshi; Barker, Jeffrey R; Guan, Ziqiang; Valdivia, Raphael H; Zhou, PeiConstitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, includingChlamydia trachomatis Although nearly all of the enzymes in the lipid A biosynthetic pathway are highly conserved across Gram-negative bacteria, the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN (UDP-DAGn) to form lipid X is carried out by two unrelated enzymes: LpxH in beta- and gammaproteobacteria and LpxI in alphaproteobacteria. The intracellular pathogenC. trachomatislacks an ortholog for either of these two enzymes, and yet, it synthesizes lipid A and exhibits conservation of genes encoding other lipid A enzymes. Employing a complementation screen against aC. trachomatisgenomic library using a conditional-lethallpxHmutantEscherichia colistrain, we have identified an open reading frame (Ct461, renamedlpxG) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function inE. coliand catalyzes the conversion of UDP-DAGn to lipid Xin vitro LpxG shows little sequence similarity to either LpxH or LpxI, highlighting LpxG as the founding member of a third class of UDP-DAGn hydrolases. Overexpression of LpxG results in toxic accumulation of lipid X and profoundly reduces the infectivity ofC. trachomatis, validating LpxG as the long-sought-after UDP-DAGn pyrophosphatase in this prominent human pathogen. The complementation approach presented here overcomes the lack of suitable genetic tools forC. trachomatisand should be broadly applicable for the functional characterization of other essentialC. trachomatisgenes.IMPORTANCEChlamydia trachomatisis a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of manyChlamydiagenes remain uncharacterized, including the essential gene encoding the UDP-DAGn pyrophosphatase activity for the biosynthesis of lipid A, the membrane anchor of lipooligosaccharide and the predominant lipid species of the outer leaflet of the bacterial outer membrane. We designed a complementation screen against theC. trachomatisgenomic library using a conditional-lethal mutant ofE. coliand identified the missing essential gene in the lipid A biosynthetic pathway, which we designatedlpxG We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activityin vitro Overexpression of LpxG inC. trachomatisleads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating thein vivofunction of LpxG and highlighting the importance of regulated lipid A biosynthesis inC. trachomatis.Item Open Access Drug design from the cryptic inhibitor envelope.(Nat Commun, 2016-02-25) Lee, Chul-Jin; Liang, Xiaofei; Wu, Qinglin; Najeeb, Javaria; Zhao, Jinshi; Gopalaswamy, Ramesh; Titecat, Marie; Sebbane, Florent; Lemaitre, Nadine; Toone, Eric J; Zhou, PeiConformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC--an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target--access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics.Item Open Access Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic Escherichia coli.(2011) Bateman, Stacey LynnEscherichia coli is a typical constituent of the enteric tract in many animals, including humans. However, specialized extraintestinal pathogenic E. colistrains (ExPEC) may transition from benign occupation of the enteric and vaginal tracts to sterile sites such as the urinary tract, bloodstream, and central nervous system. ExPEC isolates of urinary tract origin express type 1 pili as a critical virulence determinant mediating adherence to and invasion into urinary tract tissues. Type 1 pili expression is under epigenetic regulation by a family of site-specific recombinases, including FimX, which is encoded from a genomic islet called PAI-X for Pathogenicity Islet of FimX. A goal of this study was to determine the prevalence of the type 1 pili epigenetic regulator genes (fimB, fimE, fimX, ipuA, ipuB) and associated PAI-X genes (hyxR, hyxA, hyxB) present among an extended, diverse collection of pathogenic and commensal E. coli isolates. Using a new multiplex PCR, fimX and the additional PAI-X genes were found to be highly associated with ExPEC (83.2%) and more prevalent in ExPEC of lower urinary tract origin (87.5%) than upper urinary tract origin (73.6%) or human commensal isolates (20.6%; p < 0.05, all comparisons). Fim-like recombinase genes ipuA and ipuB also had a significant association with ExPEC compared to commensal isolates, but had a low overall prevalence (23.8% vs. 11.1%; p < 0.05). PAI-X also showed a strong positive correlation with the presence of virulence genes in the genomes of pathogenic isolates. Combined, our molecular epidemiology studies indicate PAI-X is highly associated with ExPEC isolates, and its high prevalence suggests a potential role in the ExPEC lifestyle. Further investigation into the regulation of PAI-X factors showed that FimX is also an epigenetic regulator of a LuxR-like response regulator HyxR, encoded on PAI-X. In multiple clinical ExPEC isolates, FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the inversion sites of the type 1 pili promoter and independent of integration host factor IHF. Additional studies into the role of HyxR during ExPEC pathogenesis uncovered that HyxR is involved in regulation of the nitrosative stress response. In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNI), primarily through derepression of hmpA, encoding a nitric oxide detoxifying flavohemoglobin. However, in the macrophage, HyxR expression produced large effects on intracellular survival in the presence and absence of RNI, and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX. ExPEC reside in the enteric tract as commensal reservoirs, but can transition to a pathogenic state by invading normally sterile niches, establishing infection, and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. This study demonstrates the functional versatility of the FimX recombinase and identifies novel epigenetic and transcriptional regulatory controls for ExPEC tolerance to RNI challenge and survival during intracellular macrophage infection. Further investigation of these pathways may shed light on the regulatory cues and programming that provoke the commensal to pathogen transition.Item Open Access Escherichia coli global gene expression in urine from women with urinary tract infection.(PLoS pathogens, 2010-11) Hagan, Erin C; Lloyd, Amanda L; Rasko, David A; Faerber, Gary J; Mobley, Harry LTMurine models of urinary tract infection (UTI) have provided substantial data identifying uropathogenic E. coli (UPEC) virulence factors and assessing their expression in vivo. However, it is unclear how gene expression in these animal models compares to UPEC gene expression during UTI in humans. To address this, we used a UPEC strain CFT073-specific microarray to measure global gene expression in eight E. coli isolates monitored directly from the urine of eight women presenting at a clinic with bacteriuria. The resulting gene expression profiles were compared to those of the same E. coli isolates cultured statically to exponential phase in pooled, sterilized human urine ex vivo. Known fitness factors, including iron acquisition and peptide transport systems, were highly expressed during human UTI and support a model in which UPEC replicates rapidly in vivo. While these findings were often consistent with previous data obtained from the murine UTI model, host-specific differences were observed. Most strikingly, expression of type 1 fimbrial genes, which are among the most highly expressed genes during murine experimental UTI and encode an essential virulence factor for this experimental model, was undetectable in six of the eight E. coli strains from women with UTI. Despite the lack of type 1 fimbrial expression in the urine samples, these E. coli isolates were generally capable of expressing type 1 fimbriae in vitro and highly upregulated fimA upon experimental murine infection. The findings presented here provide insight into the metabolic and pathogenic profile of UPEC in urine from women with UTI and represent the first transcriptome analysis for any pathogenic E. coli during a naturally occurring infection in humans.Item Open Access FtsZ at mid-cell is essential in Escherichia coli until the late stage of constriction.(Microbiology (Reading, England), 2022-06) Corbin Goodman, Lauren C; Erickson, Harold PThere has been recent debate as to the source of constriction force during cell division. FtsZ can generate a constriction force on tubular membranes in vitro, suggesting it may generate the constriction force in vivo. However, another study showed that mutants of FtsZ did not affect the rate of constriction, whereas mutants of the PG assembly did, suggesting that PG assembly may push the constriction from the outside. Supporting this model, two groups found that cells that have initiated constriction can complete septation while the Z ring is poisoned with the FtsZ targeting antibiotic PC190723. PC19 arrests treadmilling but leaves FtsZ in place. We sought to determine if a fully assembled Z ring is necessary during constriction. To do this, we used a temperature-sensitive FtsZ mutant, FtsZ84. FtsZ84 supports cell division at 30 °C, but it disassembles from the Z ring within 1 min upon a temperature jump to 42 °C. Following the temperature jump we found that cells in early constriction stop constricting. Cells that had progressed to the later stage of division finished constriction without a Z ring. These results show that in Escherichia coli, an assembled Z ring is essential for constriction except in the final stage, contradicting the simplest interpretation of previous studies using PC19.Item Open Access Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division.(Proceedings of the National Academy of Sciences of the United States of America, 2013-11-11) Kiro, Ruth; Molshanski-Mor, Shahar; Yosef, Ido; Milam, Sara L; Erickson, Harold P; Qimron, UdiBacteriophages take over host resources primarily via the activity of proteins expressed early in infection. One of these proteins, produced by the Escherichia coli phage T7, is gene product (Gp) 0.4. Here, we show that Gp0.4 is a direct inhibitor of the E. coli filamenting temperature-sensitive mutant Z division protein. A chemically synthesized Gp0.4 binds to purified filamenting temperature-sensitive mutant Z protein and directly inhibits its assembly in vitro. Consequently, expression of Gp0.4 in vivo is lethal to E. coli and results in bacteria that are morphologically elongated. We further show that this inhibition of cell division by Gp0.4 enhances the bacteriophage's competitive ability. This division inhibition is thus a fascinating example of a strategy in bacteriophages to maximize utilization of their hosts' cell resources.Item Open Access Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli.(PLoS One, 2015) Kulp, Adam J; Sun, Bo; Ai, Teresa; Manning, Andrew J; Orench-Rivera, Nichole; Schmid, Amy K; Kuehn, Meta JThe production of outer membrane vesicles by Gram-negative bacteria has been well documented; however, the mechanism behind the biogenesis of these vesicles remains unclear. Here a high-throughput experimental method and systems-scale analysis was conducted to determine vesiculation values for the whole genome knockout library of Escherichia coli mutant strains (Keio collection). The resultant dataset quantitatively recapitulates previously observed phenotypes and implicates nearly 150 new genes in the process of vesiculation. Gene functional and biochemical pathway analyses suggest that mutations that truncate outer membrane structures such as lipopolysaccharide and enterobacterial common antigen lead to hypervesiculation, whereas mutants in oxidative stress response pathways result in lower levels. This study expands and refines the current knowledge regarding the cellular pathways required for outer membrane vesiculation in E. coli.Item Open Access Heterogeneities in fullerene nanoparticle aggregates affecting reactivity, bioactivity, and transport.(ACS Nano, 2010-09-28) Chae, So-Ryong; Badireddy, Appala R; Farner Budarz, Jeffrey; Lin, Shihong; Xiao, Yao; Therezien, Mathieu; Wiesner, Mark RProperties of nanomaterial suspensions are typically summarized by average values for the purposes of characterizing these materials and interpreting experimental results. We show in this work that the heterogeneity in aqueous suspensions of fullerene C(60) aggregates (nC(60)) must be taken into account for the purposes of predicting nanomaterial transport, exposure, and biological activity. The production of reactive oxygen species (ROS), microbial inactivation, and the mobility of the aggregates of the nC(60) in a silicate porous medium all increased as suspensions were fractionated to enrich with smaller aggregates by progressive membrane filtration. These size-dependent differences are attributed to an increasing degree of hydroxylation of nC(60) aggregates with decreasing size. As the quantity and influence of these more reactive fractions may increase with time, experiments evaluating fullerene transport and toxicity end points must take into account the evolution and heterogeneity of fullerene suspensions.Item Open Access High-resolution crystal structures of Escherichia coli FtsZ bound to GDP and GTP.(Acta crystallographica. Section F, Structural biology communications, 2020-02-05) Schumacher, Maria A; Ohashi, Tomoo; Corbin, Lauren; Erickson, Harold PBacterial cytokinesis is mediated by the Z-ring, which is formed by the prokaryotic tubulin homolog FtsZ. Recent data indicate that the Z-ring is composed of small patches of FtsZ protofilaments that travel around the bacterial cell by treadmilling. Treadmilling involves a switch from a relaxed (R) state, favored for monomers, to a tense (T) conformation, which is favored upon association into filaments. The R conformation has been observed in numerous monomeric FtsZ crystal structures and the T conformation in Staphylococcus aureus FtsZ crystallized as assembled filaments. However, while Escherichia coli has served as a main model system for the study of the Z-ring and the associated divisome, a structure has not yet been reported for E. coli FtsZ. To address this gap, structures were determined of the E. coli FtsZ mutant FtsZ(L178E) with GDP and GTP bound to 1.35 and 1.40 Å resolution, respectively. The E. coli FtsZ(L178E) structures both crystallized as straight filaments with subunits in the R conformation. These high-resolution structures can be employed to facilitate experimental cell-division studies and their interpretation in E. coli.
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