scholarly journals Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay

2021 ◽  
Vol 12 ◽  
Author(s):  
Emma L. Farquharson ◽  
Ashlyn Lightbown ◽  
Elsi Pulkkinen ◽  
Téa Russell ◽  
Brenda Werner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Range ◽  
Infection Process ◽  
Bacterial Disease ◽  
Tail Fiber ◽  
Long Tail ◽  
Bacterial Surface ◽  
Specific Phage ◽  
Phage Tail ◽  
Tail Fibers

Phages have demonstrated significant potential as therapeutics in bacterial disease control and as diagnostics due to their targeted bacterial host range. Host range has typically been defined by plaque assays; an important technique for therapeutic development that relies on the ability of a phage to form a plaque upon a lawn of monoculture bacteria. Plaque assays cannot be used to evaluate a phage’s ability to recognize and adsorb to a bacterial strain of interest if the infection process is thwarted post-adsorption or is temporally delayed, and it cannot highlight which phages have the strongest adsorption characteristics. Other techniques, such as classic adsorption assays, are required to define a phage’s “adsorptive host range.” The issue shared amongst all adsorption assays, however, is that they rely on the use of a complete bacteriophage and thus inherently describe when all adsorption-specific machinery is working together to facilitate bacterial surface adsorption. These techniques cannot be used to examine individual interactions between a singular set of a phage’s adsorptive machinery (like long tail fibers, short tail fibers, tail spikes, etc.) and that protein’s targeted bacterial surface receptor. To address this gap in knowledge we have developed a high-throughput, filtration-based, bacterial binding assay that can evaluate the adsorptive capability of an individual set of a phage’s adsorption machinery. In this manuscript, we used a fusion protein comprised of an N-terminal bioluminescent tag translationally fused to T4’s long tail fiber binding tip (gp37) to evaluate and quantify gp37’s relative adsorptive strength against the Escherichia coli reference collection (ECOR) panel of 72 Escherichia coli isolates. Gp37 could adsorb to 61 of the 72 ECOR strains (85%) but coliphage T4 only formed plaques on 8 of the 72 strains (11%). Overlaying these two datasets, we were able to identify ECOR strains incompatible with T4 due to failed adsorption, and strains T4 can adsorb to but is thwarted in replication at a step post-adsorption. While this manuscript only demonstrates our assay’s ability to characterize adsorptive capabilities of phage tail fibers, our assay could feasibly be modified to evaluate other adsorption-specific phage proteins.

scholarly journals SodA Contributes to the Virulence of Avian PathogenicEscherichia coliO2 Strain E058 in Experimentally Infected Chickens

2019 ◽  
Vol 201 (6) ◽  
Author(s):  
Qingqing Gao ◽  
Le Xia ◽  
Xiaobo Wang ◽  
Zhengqin Ye ◽  
Jinbiao Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Virulence Factor ◽  
Infection Process ◽  
Strain Identification ◽  
Bacterial Disease ◽  
Wild Type ◽  
Content Type

ABSTRACTStrains of avian pathogenicEscherichia coli(APEC), the common pathogen of avian colibacillosis, encounter reactive oxygen species (ROS) during the infection process. Superoxide dismutases (SODs), acting as antioxidant factors, can protect against ROS-mediated host defenses. Our previous reports showed that thesodAgene (encoding a Mn-cofactor-containing SOD [MnSOD]) is highly expressed during the septicemic infection process of APEC.sodAhas been proven to be a virulence factor of certain pathogens, but its role in the pathogenicity of APEC has not been fully identified. In this study, we deleted thesodAgene from the virulent APEC O2 strain E058 and examined thein vitroandin vivophenotypes of the mutant. ThesodAmutant was more sensitive to hydrogen peroxide in terms of both its growth and viability than was the wild type. The ability to form a biofilm was weakened in thesodAmutant. ThesodAmutant was significantly more easily phagocytosed by chicken macrophages than was the wild-type strain. Chicken infection assays revealed significantly attenuated virulence of thesodAmutant compared with the wild type at 24 h postinfection. The virulence phenotype was restored by complementation of thesodAgene. Quantitative real-time reverse transcription-PCR revealed that the inactivation ofsodAreduced the expression of oxidative stress response geneskatE,perR, andosmCbut did not affect the expression ofsodBandsodC. Taken together, our studies indicate that SodA is important for oxidative resistance and virulence of APEC E058.IMPORTANCEAvian colibacillosis, caused by strains of avian pathogenicEscherichia coli, is a major bacterial disease of severe economic significance to the poultry industry worldwide. The virulence mechanisms of APEC are not completely understood. This study investigated the influence of an antioxidant protein, SodA, on the phenotype and pathogenicity of APEC O2 strain E058. This is the first report demonstrating that SodA plays an important role in protecting a specific APEC strain against hydrogen peroxide-induced oxidative stress and contributes to the virulence of this pathotype strain. Identification of this virulence factor will enhance our knowledge of APEC pathogenic mechanisms, which is crucial for designing successful strategies against associated infections and transmission.

scholarly journals Genetic definition of two functional elements in a bacteriophage T4 host-range "cassette".

Genetics ◽  
1989 ◽  
Vol 122 (3) ◽  
pp. 471-479
Author(s):  
M Snyder ◽  
W B Wood
Keyword(s):  
Host Range ◽  
Bacteriophage T4 ◽  
Temperature Sensitive ◽  
Tail Fiber ◽  
Neighboring Gene ◽  
Distal Half ◽  
Bacterial Surface ◽  
Fiber Assembly ◽  
Proximal Half ◽  
Carboxy Terminal

Abstract Gene 37 of T4 encodes the major subunit of the distal half of the tail fiber. The distal tip of the fiber, comprised of the carboxy-terminal ends of two molecules of gene 37 product (gp37), carries the principal determinant of the phage host range. The gp37 carboxyl termini recognize the bacterial surface during infection, and, in addition, include a site required for interaction with the product of gp38 during distal half-fiber assembly. In the absence of interaction with gp38, gp37 polypeptides do not dimerize. Eleven temperature-sensitive mutants with defects located near the promoter-distal end of gene 37 were tested at nonpermissive temperatures for production of an antigen that is diagnostic of distal half-fiber assembly. Six of the mutations prevent distal half-fiber assembly. The other five allow assembly of distal half fibers, which combine with proximal half fibers and attach to phage particles, but the resulting phage do not adsorb to bacteria. These two classes of mutations define two adjacent but separate genetic regions, corresponding to two different functional domains in gp37. These two regions and the neighboring gene 38 comprise a functional unit that can be considered as a host-range "cassette," with features that are strikingly similar to corresponding functional units in other unrelated as well as related phages.

scholarly journals Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis

Cell ◽  
2019 ◽  
Vol 179 (2) ◽  
pp. 459-469.e9 ◽  
Author(s):  
Kevin Yehl ◽  
Sébastien Lemire ◽  
Andrew C. Yang ◽  
Hiroki Ando ◽  
Mark Mimee ◽  
...  
Keyword(s):  
Host Range ◽  
Bacterial Resistance ◽  
Tail Fiber ◽  
Phage Tail

scholarly journals Mechanisms Responsible for a ΦX174 Mutant's Ability To Infect Escherichia coli by Phosphorylation

2010 ◽  
Vol 84 (9) ◽  
pp. 4860-4863 ◽  
Author(s):  
Jennifer Cox ◽  
Catherine Putonti
Keyword(s):  
Escherichia Coli ◽  
Host Range ◽  
Viral Entry ◽  
Bacterial Surface ◽  
E Coli ◽  
K 12 ◽  
Native Host ◽  
Virion Surface

ABSTRACT The ability for a virus to expand its host range is dependent upon a successful mode of viral entry. As such, the host range of the well-studied ΦX174 bacteriophage is dictated by the presence of a particular lipopolysaccharide (LPS) on the bacterial surface. The mutant ΦX174 strain JACS-K, unlike its ancestor, is capable of infecting both its native host Escherichia coli C and E. coli K-12, which does not have the necessary LPS. The conversion of an alanine to a very reactive threonine on its virion surface was found to be responsible for the strain's expanded host range.

Morphological examination of cell surface structures of enterotoxigenic strains of Escherichia coli

1984 ◽  
Vol 30 (4) ◽  
pp. 451-460 ◽  
Author(s):  
R. Chan ◽  
S. D. Acres ◽  
J. W. Costerton
Keyword(s):  
Escherichia Coli ◽  
Ruthenium Red ◽  
Surface Structures ◽  
Bacterial Disease ◽  
Morphological Examination ◽  
Bacterial Surface ◽  
Sectioned Material ◽  
Cell Surface Structures

The very fine sinuous K99 pili of enterotoxigenic strains of Escherichia coli can be visualized in shadowed and in negatively stained preparations, especially if the amorphous K30 glycocalyx is not produced, but these very delicate structures cannot be directly resolved in sectioned material. The K99 pili can, however, be thickened by the nonspecific accretion of K30 glycocalyx material, during its condensation as a result of dehydration, to the point where it can be resolved in sectioned material. This visualization is enhanced if the accreted and condensed glycocalyx is stained with ruthenium red. Alternatively and additionally, the K99 pilus can be thickened by the specific accretion of monoclonal antibodies so that it is made visible in sectioned material. The condensation of the hydrated K30 antigen glycocalyx of enterotoxigenic strains of Escherichia coli during dehydration can be prevented by stabilization using specific antibodies so that this capsular glycocalyx structure is identified in sectioned material and is seen in its correct distribution and dimensions. These methods allow the identification and visualization of bacterial surface structures, both in vitro and in vivo, and they provide a useful means of assessing the presence and distribution of these structures at all stages of the bacterial disease and a possible means of assessing their roles in the pathogenic process.

scholarly journals Engineering Phage Host-Range and Suppressing Bacterial Resistance Through Phage Tail Fiber Mutagenesis

2019 ◽  
Author(s):  
Kevin Yehl ◽  
Sébastien Lemire ◽  
Andrew C. Yang ◽  
Hiroki Ando ◽  
Mark Mimee ◽  
...  
Keyword(s):  
Host Range ◽  
Bacterial Resistance ◽  
Mouse Skin ◽  
Infection Model ◽  
Tail Fiber ◽  
List Type ◽  
Phage Tail ◽  
Genetically Engineer

SUMMARYThe rapid emergence of antibiotic-resistant infections is prompting increased interest in phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the successful development of phage therapies. Through natural evolution and structural modeling, we identified host-range determining regions (HRDR) in the T3 phage tail fiber protein and developed a high-throughput strategy to genetically engineer these regions through site-directed mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional diversity (>107 different members), while minimizing disruptions to the overall protein structure, resulting in synthetic “phagebodies”. We showed that mutating HRDRs yields phagebodies with altered host-ranges. Select phagebodies enable long-term suppression of bacterial growth by preventing the appearance of resistance in vitro and are functional in vivo using a mouse skin infection model. We anticipate this approach may facilitate the creation of next-generation antimicrobials that slow resistance development and could be extended to other viral scaffolds for a broad range of applications.HighlightsVastly diverse phagebody libraries containing 107 different members were created.Structure-informed engineering of viral tail fibers efficiently generated host-range alterations.Phagebodies prevented the development of bacterial resistance across long timescales in vitro and are functional in vivo.

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