scholarly journals Metabolomics Reveal Potential Natural Substrates of AcrB in Escherichia coli and Salmonella enterica Serovar Typhimurium

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Xuan Wang-Kan ◽  
Giovanny Rodríguez-Blanco ◽  
Andrew D. Southam ◽  
Catherine L. Winder ◽  
Warwick B. Dunn ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Drug Resistance ◽  
Salmonella Enterica ◽  
Efflux Pump ◽  
Metabolic Adaptation ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Chemically Modified ◽  
Serovar Typhimurium

ABSTRACT In the fight against antibiotic resistance, drugs that target resistance mechanisms in bacteria can be used to restore the therapeutic effectiveness of antibiotics. The multidrug resistance efflux complex AcrAB-TolC is the most clinically relevant efflux pump in Enterobacterales and is a target for drug discovery. Inhibition of the pump protein AcrB allows the intracellular accumulation of a wide variety of antibiotics, effectively restoring their therapeutic potency. To facilitate the development of AcrB efflux inhibitors, it is desirable to discover the native substrates of the pump, as these could be chemically modified to become inhibitors. We analyzed the native substrate profile of AcrB in Escherichia coli MG1655 and Salmonella enterica serovar Typhimurium SL1344 using an untargeted metabolomics approach. We analyzed the endo- and exometabolome of the wild-type strain and their respective AcrB loss-of-function mutants (AcrB D408A) to determine the metabolites that are native substrates of AcrB. Although there is 95% homology between the AcrB proteins of S. Typhimurium and E. coli, we observed mostly different metabolic responses in the exometabolomes of the S. Typhimurium and E. coli AcrB D408A mutants relative to those in the wild type, potentially indicating a differential metabolic adaptation to the same mutation in these two species. Additionally, we uncovered metabolite classes that could be involved in virulence of S. Typhimurium and a potential natural substrate of AcrB common to both species. IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a global threat to human health. The AcrB efflux pump confers inherent and evolved drug resistance to Enterobacterales, including Escherichia coli and Salmonella enterica serovar Typhimurium. We provide insights into the physiological role of AcrB: (i) we observe that loss of AcrB function in two highly related species, E. coli and S. Typhimurium, has different biological effects despite AcrB conferring drug resistance to the same groups of antibiotics in both species, and (ii) we identify potential natural substrates of AcrB, some of which are in metabolite classes implicated in the virulence of S. Typhimurium. Molecules that inhibit multidrug efflux potentiate the activity of old, licensed, and new antibiotics. The additional significance of our research is in providing data about the identity of potential natural substrates of AcrB in both species. Data on these will facilitate the discovery of, and/or could be chemically modified to become, new efflux inhibitors.

scholarly journals Effects of Norspermidine and Spermidine on Biofilm Formation by Potentially Pathogenic Escherichia coli and Salmonella enterica Wild-Type Strains

2015 ◽  
Vol 81 (6) ◽  
pp. 2226-2232 ◽  
Author(s):  
Live L. Nesse ◽  
Kristin Berg ◽  
Lene K. Vestby
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Biofilm Formation ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Serovar Typhimurium ◽  
And Control ◽  
Type Strains

ABSTRACTPolyamines are present in all living cells. In bacteria, polyamines are involved in a variety of functions, including biofilm formation, thus indicating that polyamines may have potential in the control of unwanted biofilm. In the present study, the effects of the polyamines norspermidine and spermidine on biofilms of 10 potentially pathogenic wild-type strains ofEscherichia coliserotype O103:H2,Salmonella entericasubsp.entericaserovar Typhimurium, andS. entericaserovar Agona were investigated. We found that exogenously supplied norspermidine and spermidine did not mediate disassembly of preformed biofilm of any of theE. coliandS. entericastrains. However, the polyamines did affect biofilm production. Interestingly, the two species reacted differently to the polyamines. Both polyamines reduced the amount of biofilm formed byE. colibut tended to increase biofilm formation byS. enterica. Whether the effects observed were due to the polyamines specifically targeting biofilm formation, being toxic for the cells, or maybe a combination of the two, is not known. However, there were no indications that the effect was mediated through binding to exopolysaccharides, as earlier suggested forE. coli. Our results indicate that norspermidine and spermidine do not have potential as inhibitors ofS. entericabiofilm. Furthermore, we found that the commercial polyamines used contributed to the higher pH of the test medium. Failure to acknowledge and control this important phenomenon may lead to misinterpretation of the results.

scholarly journals Members of the Conserved DedA Family Are Likely Membrane Transporters and Are Required for Drug Resistance in Escherichia coli

2013 ◽  
Vol 58 (2) ◽  
pp. 923-930 ◽  
Author(s):  
Sujeet Kumar ◽  
William T. Doerrler
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Drug Resistance ◽  
Public Health Problem ◽  
Membrane Transporters ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Acidic Amino Acids ◽  
Genes Encoding

ABSTRACTBacterial resistance to antibiotics and biocides is an increasing public health problem. Genes encoding integral membrane proteins belonging to the DedA family are present in most bacterial genomes, includingEscherichia coli. AnE. colistrain lacking partially redundant DedA family genesyqjAandyghB(strain BC202) displays temperature sensitivity and cell division defects. These phenotypes can be corrected by overexpression ofmdfA, an Na+-K+/H+antiporter of the major facilitator superfamily. We show that BC202 is hypersensitive to several biocides and cationic compounds that are known substrates of several multidrug resistance transporters, including MdfA, EmrE, and AcrB. The introduction of deletions of genes encoding these drug transporters into BC202 results in additional sensitivity. Expression of wild-typeyghBoryqjAcan restore drug resistance, but this is eliminated upon mutation of two membrane-embedded acidic amino acids (E39 or D51 in either protein). This dependence upon membrane-embedded acidic amino acids is a hallmark of proton-dependent antiporters. Overexpression ofmdfAin BC202 or artificially restoring proton motive force (PMF) restores wild-type resistance to substrates of MdfA as well as other drug resistance transporters such as EmrE and AcrAB. These results suggest that YqjA and YghB may be membrane transporters required for PMF-dependent drug efflux inE. coli.

scholarly journals The Acyl Homoserine Lactone Receptor, SdiA, of Escherichia coli and Salmonella enterica Serovar Typhimurium Does Not Respond to Indole

2012 ◽  
Vol 78 (15) ◽  
pp. 5424-5431 ◽  
Author(s):  
Anice Sabag-Daigle ◽  
Jitesh A. Soares ◽  
Jenée N. Smith ◽  
Mohamed E. Elmasry ◽  
Brian M. M. Ahmer
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Biofilm Formation ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Homoserine Lactone ◽  
Acyl Homoserine Lactone ◽  
Content Type ◽  
E Coli ◽  
Serovar Typhimurium ◽  
High Concentrations

ABSTRACTIn this study, we tested the hypothesis that the SdiA proteins ofEscherichia coliandSalmonella entericaserovar Typhimurium respond to indole. While indole was found to have effects on gene expression and biofilm formation, these effects were notsdiAdependent. However, high concentrations of indole did inhibitN-acyl-l-homoserine lactone (AHL) sensing by SdiA. We conclude that SdiA does not respond to indole but indole can inhibit SdiA activity inE. coliandSalmonella.

scholarly journals The ABC-Type Efflux Pump MacAB Protects Salmonella enterica serovar Typhimurium from Oxidative Stress

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Lydia M. Bogomolnaya ◽  
Katharine D. Andrews ◽  
Marissa Talamantes ◽  
Aimee Maple ◽  
Yury Ragoza ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Salmonella Enterica ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Drug Efflux Pump

ABSTRACTMultidrug efflux pumps are integral membrane proteins known to actively excrete antibiotics. The macrolide-specific pump MacAB, the only ABC-type drug efflux pump inSalmonella, has previously been linked to virulence in mice. The molecular mechanism of this link betweenmacABand infection is unclear. We demonstrate thatmacABplays a role in the detoxification of reactive oxygen species (ROS), compounds that salmonellae are exposed to at various stages of infection.macABis induced upon exposure to H2O2and is critical for survival ofSalmonella entericaserovar Typhimurium in the presence of peroxide. Furthermore, we determined thatmacABis required for intracellular replication inside J774.A1 murine macrophages but is not required for survival in ROS-deficient J774.D9 macrophages.macABmutants also had reduced survival in the intestine in the mouse colitis model, a model characterized by a strong neutrophilic intestinal infiltrate where bacteria may experience the cytotoxic actions of ROS. Using an Amplex red-coupled assay,macABmutants appear to be unable to induce protection against exogenous H2O2in vitro, in contrast to the isogenic wild type. In mixed cultures, the presence of the wild-type organism, or media preconditioned by the growth of the wild-type organism, was sufficient to rescue themacABmutant from peroxide-mediated killing. Our data indicate that the MacAB drug efflux pump has functions beyond resistance to antibiotics and plays a role in the protection ofSalmonellaagainst oxidative stress. Intriguingly, our data also suggest the presence of a soluble anti-H2O2compound secreted bySalmonellacells through a MacAB-dependent mechanism.IMPORTANCEThe ABC-type multidrug efflux pump MacAB is known to be required forSalmonella entericaserovar Typhimurium virulence after oral infection in mice, yet the function of this pump during infection is unknown. We show that this pump is necessary for colonization of niches in infected mice where salmonellae encounter oxidative stress during infection. MacAB is required for growth in cultured macrophages that produce reactive oxygen species (ROS) but is not needed in macrophages that do not generate ROS. In addition, we show that MacAB is required to resist peroxide-mediated killingin vitroand for the inactivation of peroxide in the media. Finally, wild-type organisms, or supernatant from wild-type organisms grown in the presence of peroxide, rescue the growth defect ofmacABmutants in H2O2. MacAB appears to participate in the excretion of a compound that induces protection against ROS-mediated killing, revealing a new role for this multidrug efflux pump.

scholarly journals Differential Regulation of Multiple Proteins of Escherichia coli and Salmonella enterica Serovar Typhimurium by the Transcriptional Regulator SlyA

2002 ◽  
Vol 184 (13) ◽  
pp. 3549-3559 ◽  
Author(s):  
Andrea Spory ◽  
Armin Bosserhoff ◽  
Christine von Rhein ◽  
Werner Goebel ◽  
Albrecht Ludwig
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Transcriptional Regulator ◽  
Host Cells ◽  
Wild Type ◽  
E Coli ◽  
Serovar Typhimurium

ABSTRACT SlyA is a transcriptional regulator of Escherichia coli, Salmonella enterica, and other bacteria belonging to the Enterobacteriaceae. The SlyA protein has been shown to be involved in the virulence of S. enterica serovar Typhimurium, but its role in E. coli is unclear. In this study, we employed the proteome technology to analyze the SlyA regulons of enteroinvasive E. coli (EIEC) and Salmonella serovar Typhimurium. In both cases, comparative analysis of the two-dimensional protein maps of a wild-type strain, a SlyA-overproducing derivative, and a corresponding slyA mutant revealed numerous proteins whose expression appeared to be either positively or negatively controlled by SlyA. Twenty of the putative SlyA-induced proteins and 13 of the putative SlyA-repressed proteins of the tested EIEC strain were identified by mass spectrometry. The former proteins included several molecular chaperones (GroEL, GroES, DnaK, GrpE, and CbpA), proteins involved in acid resistance (HdeA, HdeB, and GadA), the “starvation lipoprotein” (Slp), cytolysin ClyA (HlyE or SheA), and several enzymes involved in metabolic pathways, whereas most of the latter proteins proved to be biosynthetic enzymes. Consistently, the resistance of the EIEC slyA mutant to heat and acid stress was impaired compared to that of the wild-type strain. Furthermore, the implication of SlyA in the regulation of several of the identified E. coli proteins was confirmed at the level of transcription with lacZ fusions. Twenty-three of the Salmonella serovar Typhimurium proteins found to be affected by SlyA were also identified by mass spectrometry. With the exception of GroEL these differed from those identified in the EIEC strain and included proteins involved in various processes. The data suggest that gene regulation by SlyA might be crucial for intracellular survival and/or replication of both EIEC and Salmonella serovar Typhimurium in phagocytic host cells.

scholarly journals UDP-N-Acetylmuramic Acid l-Alanine Ligase (MurC) Inhibition in atolCMutant Escherichia coli Strain Leads to Cell Death

2014 ◽  
Vol 58 (10) ◽  
pp. 6165-6171 ◽  
Author(s):  
Vaishali Humnabadkar ◽  
K. R. Prabhakar ◽  
Ashwini Narayan ◽  
Sreevalli Sharma ◽  
Supreeth Guptha ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Coli Strain ◽  
Cellular Activity ◽  
Wild Type ◽  
Compound A ◽  
Content Type ◽  
E Coli ◽  
In The Wild ◽  
High Concentrations

ABSTRACTThe Mur ligases play an essential role in the biosynthesis of bacterial peptidoglycan and hence are attractive antibacterial targets. A screen of the AstraZeneca compound library led to the identification of compound A, a pyrazolopyrimidine, as a potent inhibitor ofEscherichia coliandPseudomonas aeruginosaMurC. However, cellular activity againstE. coliorP. aeruginosawas not observed. Compound A was active against efflux pump mutants of both strains. Experiments using anE. colitolCmutant revealed accumulation of the MurC substrate and a decrease in the level of product upon treatment with compound A,indicating inhibition of MurC enzyme in these cells. Such a modulation was not observed in theE. coliwild-type cells. Further, overexpression of MurC in theE. colitolCmutant led to an increase in the compound A MIC by ≥16-fold, establishing a correlation between MurC inhibition and cellular activity. In addition, estimation of the intracellular compound A level showed an accumulation of the compound over time in thetolCmutant strain. A significant compound A level was not detected in the wild-typeE. colistrain even upon treatment with high concentrations of the compound. Therefore, the lack of MIC and absence of MurC inhibition in wild-typeE. coliwere possibly due to suboptimal compound concentration as a consequence of a high efflux level and/or poor permeativity of compound A.

scholarly journals Evaluation of Near-Infrared Pasteurization in Controlling Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium, and Listeria monocytogenes in Ready-To-Eat Sliced Ham

2012 ◽  
Vol 78 (18) ◽  
pp. 6458-6465 ◽  
Author(s):  
Jae-Won Ha ◽  
Sang-Ryeol Ryu ◽  
Dong-Hyun Kang
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Salmonella Enterica ◽  
Near Infrared ◽  
Conventional Heating ◽  
Convective Heating ◽  
Content Type ◽  
E Coli ◽  
Texture Change ◽  
Serovar Typhimurium

ABSTRACTThis study was conducted to investigate the efficacy of near-infrared (NIR) heating to reduceSalmonella entericaserovar Typhimurium,Escherichia coliO157:H7, andListeria monocytogenesin ready-to-eat (RTE) sliced ham compared to conventional convective heating, and the effect of NIR heating on quality was determined by measuring the color and texture change. A cocktail of three pathogens was inoculated on the exposed or protected surfaces of ham slices, followed by NIR or conventional heating at 1.8 kW. NIR heating for 50 s achieved 4.1-, 4.19-, and 3.38-log reductions in surface-inoculatedS.Typhimurium,E. coliO157:H7, andL. monocytogenes, respectively, whereas convective heating needed 180 s to attain comparable reductions for each pathogen. There were no statistically significant (P> 0.05) differences in reduction between surface- and internally inoculated pathogens at the end of NIR treatment (50 s). However, when treated with conventional convective heating, significant (P< 0.05) differences were observed at the final stages of the treatment (150 and 180 s). Color values and texture parameters of NIR-treated (50-s treatment) ham slices were not significantly (P> 0.05) different from those of nontreated samples. These results suggest that NIR heating can be applied to control internalized pathogens as well as surface-adhering pathogens in RTE sliced meats without affecting product quality.

scholarly journals Occurrence of Antimicrobial-Resistant Escherichia coli and Salmonella enterica in the Beef Cattle Production and Processing Continuum

2014 ◽  
Vol 81 (2) ◽  
pp. 713-725 ◽  
Author(s):  
John W. Schmidt ◽  
Getahun E. Agga ◽  
Joseph M. Bosilevac ◽  
Dayna M. Brichta-Harhay ◽  
Steven D. Shackelford ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Generation Cephalosporin ◽  
Resistant Bacteria ◽  
Processing Plant ◽  
Cattle Production ◽  
Content Type ◽  
E Coli ◽  
Beef Processing ◽  
Tract Infections

ABSTRACTSpecific concerns have been raised that third-generation cephalosporin-resistant (3GCr)Escherichia coli, trimethoprim-sulfamethoxazole-resistant (COTr)E. coli, 3GCrSalmonella enterica, and nalidixic acid-resistant (NALr)S. entericamay be present in cattle production environments, persist through beef processing, and contaminate final products. The prevalences and concentrations of these organisms were determined in feces and hides (at feedlot and processing plant), pre-evisceration carcasses, and final carcasses from three lots of fed cattle (n= 184). The prevalences and concentrations were further determined for strip loins from 103 of the carcasses. 3GCrSalmonellawas detected on 7.6% of hides during processing and was not detected on the final carcasses or strip loins. NALrS. entericawas detected on only one hide. 3GCrE. coliand COTrE. coliwere detected on 100.0% of hides during processing. Concentrations of 3GCrE. coliand COTrE. colion hides were correlated with pre-evisceration carcass contamination. 3GCrE. coliand COTrE. coliwere each detected on only 0.5% of final carcasses and were not detected on strip loins. Five hundred and 42 isolates were screened for extraintestinal pathogenicE. coli(ExPEC) virulence-associated markers. Only two COTrE. coliisolates from hides were ExPEC, indicating that fed cattle products are not a significant source of ExPEC causing human urinary tract infections. The very low prevalences of these organisms on final carcasses and their absence on strip loins demonstrate that current sanitary dressing procedures and processing interventions are effective against antimicrobial-resistant bacteria.

scholarly journals Display of Recombinant Proteins on Bacterial Outer Membrane Vesicles by Using Protein Ligation

2018 ◽  
Vol 84 (8) ◽  
pp. e02567-17 ◽  
Author(s):  
H. Bart van den Berg van Saparoea ◽  
Diane Houben ◽  
Marien I. de Jonge ◽  
Wouter S. P. Jong ◽  
Joen Luirink
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Membrane Vesicles ◽  
Therapeutic Agents ◽  
Outer Membrane Vesicles ◽  
High Density ◽  
Content Type ◽  
Protein Ligation ◽  
Serovar Typhimurium

ABSTRACT The Escherichia coli virulence factor hemoglobin protease (Hbp) has been engineered into a surface display system that can be expressed to high density on live E. coli and Salmonella enterica serovar Typhimurium cells or derived outer membrane vesicles (OMVs). Multiple antigenic sequences can be genetically fused into the Hbp core structure for optimal exposure to the immune system. Although the Hbp display platform is relatively tolerant, increasing the number, size, and complexity of integrated sequences generally lowers the expression of the fused constructs and limits the density of display. This is due to the intricate mechanism of Hbp secretion across the outer membrane and the efficient quality control of translocation-incompetent chimeric Hbp molecules in the periplasm. To address this shortcoming, we explored the coupling of purified proteins to the Hbp carrier after its translocation across the outer membrane using the recently developed SpyTag/SpyCatcher protein ligation system. As expected, fusion of the small SpyTag to Hbp did not hamper display on OMVs. Subsequent addition of purified proteins fused to the SpyCatcher domain resulted in efficient covalent coupling to Hbp-SpyTag. Using in addition the orthogonal SnoopTag/SnoopCatcher system, multiple antigen modules could be coupled to Hbp in a sequential ligation strategy. Not only antigens proved suitable for Spy-mediated ligation but also nanobodies. Addition of this functionality to the platform might allow the targeting of live bacterial or OMV vaccines to certain tissues or immune cells to tailor immune responses.IMPORTANCE Outer membrane vesicles (OMVs) derived from Gram-negative bacteria attract increasing interest in the development of vaccines and therapeutic agents. We aim to construct a semisynthetic OMV platform for recombinant antigen presentation on OMVs derived from attenuated Salmonella enterica serovar Typhimurium cells displaying an adapted Escherichia coli autotransporter, Hbp, at the surface. Although this autotransporter accepts substantial modifications, its capacity with respect to the number, size, and structural complexity of the antigens genetically fused to the Hbp carrier is restricted. Here we describe the application of SpyCatcher/SpyTag protein ligation technology to enzymatically link antigens to Hbp present at high density in OMVs. Protein ligation was apparently unobstructed by the membrane environment and allowed a high surface density of coupled antigens, a property we have shown to be important for vaccine efficacy. The OMV coupling procedure appears versatile and robust, allowing fast production of experimental vaccines and therapeutic agents through a modular plug-and-display procedure.

Detection of Escherichia coli O157, Salmonella enterica Serovar Typhimurium, and Staphylococcal Enterotoxin B in a Single Sample Using Enzymatic Bio-Nanotransduction

2007 ◽  
Vol 70 (4) ◽  
pp. 841-850 ◽  
Author(s):  
JOSH R. BRANEN ◽  
MARTHA J. HASS ◽  
ERIN R. DOUTHIT ◽  
WUSI C. MAKI ◽  
A. LARRY BRANEN
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Staphylococcal Enterotoxin B ◽  
Escherichia Coli O157 ◽  
Dna Templates ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Biological Recognition ◽  
Enterotoxin B

Enzymatic bio-nanotransduction is a biological detection scheme based on the production of nucleic acid nano-signals (RNA) in response to specific biological recognition events. In this study, we applied an enzymatic bio-nanotransduction system to the detection of important food-related pathogens and a toxin. Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and staphylococcal enterotoxin B (SEB) were chosen because of the implications of these targets to food safety. Primary antibodies to each of the targets were used to functionalize magnetic beads and produce biological recognition elements (antibodies) conjugated to nano-signal–producing DNA templates. Immunomagnetic capture that was followed by in vitro transcription of DNA templates bound to target molecules produced RNA nano-signals specific for every target in the sample. Discrimination of RNA nano-signals with a standard enzyme-linked oligonucleotide fluorescence assay provided a correlation between nano-signal profiles and target concentrations. The estimated limit of detection was 2.4 × 103 CFU/ml for E. coli O157:H7, 1.9 × 104 CFU/ml for S. enterica serovar Typhimurium, and 0.11 ng/ml for SEB with multianalyte detection in buffer. Low levels of one target were also detected in the presence of interference from high levels of the other targets. Finally, targets were detected in milk, and detection was improved for E. coli O157 by heat treatment of the milk.

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