scholarly journals Stable neutralization of virulent bacteria using temperate phage in the mammalian gut

2019 ◽  
Author(s):  
Bryan B. Hsu ◽  
Jeffrey C. Way ◽  
Pamela A. Silver
Keyword(s):  
Temperate Phage ◽  
Cascading Effects ◽  
E Coli ◽  
Bacterial Action ◽  
Exogenous Genes ◽  
Selection For ◽  
Mouse Model Of Infection ◽  
Bacterial Concentrations ◽  
New Framework ◽  
Lambdoid Phages

ABSTRACTElimination or alteration of select members of the gut microbiota is key to therapeutic efficacy. However, the complexity of these microbial inhabitants makes it challenging to precisely target bacteria without unexpected cascading effects. Here, we use bacteriophage to deliver exogenous genes to specific bacteria by genomic integration of temperate phage for long-lasting modification. As a real-world therapeutic test, we engineered λ phage to transcriptionally-repress shigatoxin by using genetic hybrids between λ and other lambdoid phages to overcome resistance encoded by the virulent prophage derived from enterohemorrhagic E. coli. We show that a single dose of engineered phage propagates throughout the bacterial community and reduces shigatoxin production in an enteric mouse model of infection without markedly affecting bacterial concentrations. We thus minimize the selection for resistance by relying on anti-virulence and not anti-bacterial action. Our work reveals a new framework for transferring functions to bacteria within their native environment.

scholarly journals Stable Neutralization of a Virulence Factor in Bacteria Using Temperate Phage in the Mammalian Gut

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Bryan B. Hsu ◽  
Jeffrey C. Way ◽  
Pamela A. Silver
Keyword(s):  
Virulence Factor ◽  
Shiga Toxin ◽  
Bacterial Infections ◽  
Bacterial Genome ◽  
Toxin Production ◽  
Temperate Phage ◽  
Bacterial Function ◽  
Mouse Model Of Infection ◽  
New Framework

ABSTRACT Elimination or alteration of select members of the gut microbiota is key to therapeutic efficacy. However, the complexity of these microbial inhabitants makes it challenging to precisely target bacteria. Here, we deliver exogenous genes to specific bacteria by genomic integration of temperate phage for long-lasting modification. As a real-world therapeutic test, we engineered λ phage to transcriptionally repress Shiga toxin by using genetic hybrids between λ and other lambdoid phages to overcome resistance encoded by the virulence-expressing prophage. We show that a single dose of engineered phage propagates throughout the bacterial community and reduces Shiga toxin production in an enteric mouse model of infection without markedly affecting bacterial concentrations. Our work reveals a new framework for transferring functions to bacteria within their native environment. IMPORTANCE With the increasing frequency of antibiotic resistance, it is critical to explore new therapeutic strategies for treating bacterial infections. Here, we use a temperate phage, i.e., one that integrates itself into the bacterial genome, to neutralize the expression of a virulence factor by modifying bacterial function at the genetic level. We show that Shiga toxin production can be significantly reduced in vitro and in the mammalian gut. Alternative to traditional applications of phage therapy that rely on killing bacteria, our genetics-based antivirulence approach introduces a new framework for treating bacterial infections.

scholarly journals Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

2014 ◽  
Vol 81 (1) ◽  
pp. 130-138 ◽  
Author(s):  
James Kirby ◽  
Minobu Nishimoto ◽  
Ruthie W. N. Chow ◽  
Edward E. K. Baidoo ◽  
George Wang ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Terpene Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Pathway Expression ◽  
Terpene Synthesis ◽  
Selection For

ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

scholarly journals Development and Characterization of a Fluorescent-Bacteriophage Assay for Detection of Escherichia coli O157:H7

1999 ◽  
Vol 65 (4) ◽  
pp. 1397-1404 ◽  
Author(s):  
Lawrence Goodridge ◽  
Jinru Chen ◽  
Mansel Griffiths
Keyword(s):  
Escherichia Coli ◽  
Flow Cytometry ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Lower Detection ◽  
Unreliable Method ◽  
Direct Epifluorescent Filter Technique ◽  
Bacterial Concentrations

ABSTRACT In this paper we describe evaluation and characterization of a novel assay that combines immunomagnetic separation and a fluorescently stained bacteriophage for detection of Escherichia coliO157:H7 in broth. When it was combined with flow cytometry, the fluorescent-bacteriophage assay (FBA) was capable of detecting 104 cells/ml. A modified direct epifluorescent-filter technique (DEFT) was employed in an attempt to estimate bacterial concentrations. Using regression analysis, we calculated that the lower detection limit was between 102 and 103cells/ml; however, the modified DEFT was found to be an unreliable method for determining bacterial concentrations. The results of this study show that the FBA, when combined with flow cytometry, is a sensitive technique for presumptive detection of E. coliO157:H7 in broth cultures.

Molecular Cloning of the gyrA andgyrB Genes of Bacteroides fragilis Encoding DNA Gyrase

1999 ◽  
Vol 43 (10) ◽  
pp. 2423-2429 ◽  
Author(s):  
Yoshikuni Onodera ◽  
Kenichi Sato
Keyword(s):  
Hot Spot ◽  
Bacteroides Fragilis ◽  
Dna Gyrase ◽  
E Coli ◽  
Important Target ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Gyrase A ◽  
Selection For

ABSTRACT The genes encoding the DNA gyrase A and B subunits ofBacteroides fragilis were cloned and sequenced. ThegyrA and gyrB genes code for proteins of 845 and 653 amino acids, respectively. These proteins were expressed inEscherichia coli, and the combination of GyrA and GyrB exhibited ATP-dependent supercoiling activity. To analyze the role of DNA gyrase in quinolone resistance of B. fragilis, we isolated mutant strains by stepwise selection for resistance to increasing concentrations of levofloxacin. We analyzed the resistant mutants and showed that Ser-82 of GyrA, equivalent to resistance hot spot Ser-83 of GyrA in E. coli, was in each case replaced with Phe. These results suggest that DNA gyrase is an important target for quinolones in B. fragilis.

scholarly journals Origin and Evolution of the AmpC β-Lactamases of Citrobacter freundii

2002 ◽  
Vol 46 (5) ◽  
pp. 1190-1198 ◽  
Author(s):  
Miriam Barlow ◽  
Barry G. Hall
Keyword(s):  
Escherichia Coli ◽  
Clinical Isolates ◽  
Citrobacter Freundii ◽  
Clinical Use ◽  
Origin And Evolution ◽  
E Coli ◽  
Selection For

ABSTRACT To determine whether the widespread clinical use of β-lactams has been selective for Citrobacter freundii-derived alleles of plasmid ampC genes, we generated a Bayesian consensus phylogeny of the published ampC sequences and compared the MICs of 16 β-lactam antibiotics for Escherichia coli strains containing cloned copies of the C. freundii ampC alleles. We found that for the majority of compounds investigated, there has been essentially no increase in β-lactam resistance conferred by those alleles. We also found that ampC alleles from the chromosomes of two β-lactam-sensitive C. freundii strains isolated in the 1920s, before the clinical use of antibiotics, were as effective at providing β-lactam resistance in E. coli as were the plasmid-borne alleles from β-lactam-resistant clinical isolates. These results suggest that selection for increased resistance to β-lactam antibiotics has not been a significant force directing the evolution of the C. freundii ampC alleles found in β-lactam-resistant clinical isolates.

scholarly journals Facilitated Oligomerization of Mycobacterial GroEL: Evidence for Phosphorylation-Mediated Oligomerization

2009 ◽  
Vol 191 (21) ◽  
pp. 6525-6538 ◽  
Author(s):  
C. M. Santosh Kumar ◽  
Garima Khare ◽  
C. V. Srikanth ◽  
Anil K. Tyagi ◽  
Abhijit A. Sardesai ◽  
...  
Keyword(s):  
Molecular Chaperones ◽  
Domain Swapping ◽  
Central Cavity ◽  
E Coli ◽  
Double Ring ◽  
Single Ring ◽  
Genes Encoding ◽  
Selection For ◽  
Oligomeric Forms ◽  
State Form

ABSTRACT The distinctive feature of the GroES-GroEL chaperonin system in mediating protein folding lies in its ability to exist in a tetradecameric state, form a central cavity, and encapsulate the substrate via the GroES lid. However, recombinant GroELs of Mycobacterium tuberculosis are unable to act as effective molecular chaperones when expressed in Escherichia coli. We demonstrate here that the inability of M. tuberculosis GroEL1 to act as a functional chaperone in E. coli can be alleviated by facilitated oligomerization. The results of directed evolution involving random DNA shuffling of the genes encoding M. tuberculosis GroEL homologues followed by selection for functional entities suggested that the loss of chaperoning ability of the recombinant mycobacterial GroEL1 and GroEL2 in E. coli might be due to their inability to form canonical tetradecamers. This was confirmed by the results of domain-swapping experiments that generated M. tuberculosis-E. coli chimeras bearing mutually exchanged equatorial domains, which revealed that E. coli GroEL loses its chaperonin activity due to alteration of its oligomerization capabilities and vice versa for M. tuberculosis GroEL1. Furthermore, studying the oligomerization status of native GroEL1 from cell lysates of M. tuberculosis revealed that it exists in multiple oligomeric forms, including single-ring and double-ring variants. Immunochemical and mass spectrometric studies of the native M. tuberculosis GroEL1 revealed that the tetradecameric form is phosphorylated on serine-393, while the heptameric form is not, indicating that the switch between the single- and double-ring variants is mediated by phosphorylation.

An E. coli-yeast shuttle cosmid with positive selection for inserted fragments

1985 ◽  
Vol 10 (1) ◽  
pp. 29-33 ◽  
Author(s):  
Manda E. Gent ◽  
Patrick Crowley ◽  
J. Richard Ludwig ◽  
Rashida Anwar ◽  
David A. Sugden ◽  
...  
Keyword(s):  
Positive Selection ◽  
E Coli ◽  
Selection For

scholarly journals Isolation and characterization of a novel bacteriophage, Kapi1, capable of O-antigen modification in commensal Escherichia coli.

2021 ◽  
Author(s):  
Kat Pick ◽  
Tracy Lyn Raivio
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Shigella Flexneri ◽  
Model System ◽  
Temperate Phage ◽  
Phage Displays ◽  
E Coli ◽  
O Antigen ◽  
Isolation And Characterization

In this study, we describe the isolation and characterization of novel bacteriophage Kapi1 (vB_EcoP_Kapi1) isolated from a strain of commensal Escherichia coli inhabiting the gastrointestinal tract of healthy mice. We show that Kapi1 is a temperate phage integrated into tRNA argW of strain MP1 and describe its genome annotation and structure. Kapi1 shows limited homology to other characterized prophages but is most similar to the phages of Shigella flexneri, and clusters taxonomically with P22-like phages. Investigation of the lifestyle of Kapi1 shows that this phage displays unstable lysogeny and influences the growth of its host. The receptor for Kapi1 is the lipopolysaccharide O-antigen, and we further show that Kapi1 alters the structure of its hosts O-antigen in multiple ways. We hope to use MP1 and Kapi1 as a model system to explore molecular mechanisms of mammalian colonization by E. coli and ask what the role(s) of prophages in this context might be.

scholarly journals Bactericidal activity of starch films containing albumin, collagen and eggshell membrane

2013 ◽  
Vol 3 (1) ◽  
pp. 50
Author(s):  
Jéssica F. da Silva ◽  
Renan Luiz Romano Gon ◽  
Angela Kwiatkowski ◽  
Regiane Da Silva
Keyword(s):  
Nitric Oxide ◽  
Bactericidal Activity ◽  
Low Cost ◽  
Field Research ◽  
Eggshell Membrane ◽  
Bactericidal Action ◽  
E Coli ◽  
Bacterial Action ◽  
Starch Films ◽  
Membrane Collagen

<p>The need for materials resistant to bacterial action has led to increases in biomaterials field research in food and pharmaceutical industry, particularly with regard to obtaining low cost materials. In this study, starch films easily obtained were used. The application is favorable to their application as biomaterials and studying the bactericidal activity against <em>Salmonella typhimurium</em>, <em>Escherichia coli</em> and <em>Staphylococcus aureus</em>. The results showed that the films were capable to release about 6 mmols of nitric oxide (NO) per gram of film and that they had potential antibacterial action against <em>S. typhimurium</em> and <em>S. aureus</em>, showing no significant results against <em>E. coli</em>. These results indicate that nitric oxide has bactericidal action and the starch films containing eggshell membrane, collagen and albumin can be used as packaging materials in contact with food or compounds which must be preserved from the bacterial action.</p><p>&nbsp;</p><p>DOI: http://dx.doi.org/10.14685/rebrapa.v3i1.92</p>

scholarly journals Evidence of evolutionary selection for co-translational folding

2017 ◽  
Author(s):  
William M. Jacobs ◽  
Eugene I. Shakhnovich
Keyword(s):  
Self Assembly ◽  
E Coli ◽  
Evolutionary Selection ◽  
Fitness Effects ◽  
Open Questions ◽  
Genome Wide ◽  
A Genome ◽  
Evolutionarily Conserved ◽  
Domain Boundaries ◽  
Selection For

Recent experiments and simulations have demonstrated that proteins can fold on the ribosome. However, the extent and generality of fitness effects resulting from co-translational folding remain open questions. Here we report a genome-wide analysis that uncovers evidence of evolutionary selection for co-translational folding. We describe a robust statistical approach to identify loci within genes that are both significantly enriched in slowly translated codons and evolutionarily conserved. Surprisingly, we find that domain boundaries can explain only a small fraction of these conserved loci. Instead, we propose that regions enriched in slowly translated codons are associated with co-translational folding intermediates, which may be smaller than a single domain. We show that the intermediates predicted by a native-centric model of co-translational folding account for the majority of these loci across more than 500 E. coli proteins. By making a direct connection to protein folding, this analysis provides strong evidence that many synonymous substitutions have been selected to optimize translation rates at specific locations within genes. More generally, our results indicate that kinetics, and not just thermodynamics, can significantly alter the efficiency of self-assembly in a biological context.

Sign in / Sign up

Export Citation Format

Share Document