Rapid Microscopic Detection of Bacillus anthracis by Fluorescent Receptor Binding Proteins of Bacteriophages

Braun, Peter; Wolfschläger, Immanuel; Reetz, Leonie; Bachstein, Lilia; Jacinto, Ana Clara; Tocantins, Carolina; Poppe, Johannes; Grass, Gregor

doi:10.3390/microorganisms8060934

Cited by 13 publications

(44 citation statements)

References 59 publications

(103 reference statements)

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“…PCR primer oligonucleotides were designed using Geneious Prime software (Biomatters Limited, Auckland, New Zealand) from DNA sequences of phage ΦA1122 (accession #NC_004777), phage L-413C (accession #NC_004745), mCherry-pBAD or eGFP-pBAD ( Supplementary Table S2 ). Cloning of phage RBP genes was carried out as described previously [ 43 ]. For cloning of genes of fluorescent proteins and the putative RBP genes, the primers were designed in such a way that the recognition sites for SalI, EcoRI and BsrGI upstream and XhoI, PstI and BsiWI downstream of the fluorescence genes, which were inserted into the vector chassis pASG-IBA 105 (IBA GmbH, Göttingen, Germany), can be used for subsequent cloning of additional genes.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were harvested by centrifugation (7500× g , 10 min) and cell pellets stored at −20 °C until further use. Cell pellets were resuspended and processed as described previously [ 43 ]. Cell lysis was accomplished by mechanical means using a French Press (Emulsiflex-C3; Avestin Europe GmbH, Mannheim, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…RBP-reporter assays were carried out as described previously for B. anthracis with slight modifications [ 43 ]. Briefly, 500 µL of an overnight culture of Y. pestis or other Yersinia spp.…”

Section: Methodsmentioning

confidence: 99%

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Specific Detection of Yersinia pestis Based on Receptor Binding Proteins of Phages

Born¹,

Braun²,

Scholz³

et al. 2020

Pathogens

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The highly pathogenic bacterium Yersinia pestis is the causative agent of plague, a notorious infectious zoonotic disease. When transmitted from person to person as pneumonic plague via droplets, Y. pestis is highly contagious and in most cases is fatal if left untreated. Thus, when plague is suspected, rapid diagnosis is crucial, as a serious course of the infection is only averted by early antibiotic therapy. The bacterium is easy to cultivate, accessible and has a high potential for nefarious use such as bioterrorism. Highly specific, rapid and easy-to-use confirmatory diagnostic methods are required to reliably identify the pathogen independently from PCR-based methods or F1 antigen-based immunological detection. Yersinia pestis specific phages such as L-413C and ΦA1122 are already used for detection of Y. pestis in bacterial plaque or biosensor assays. Here, we made use of the host specificities conferred by phage receptor binding (or tail fiber/spike) proteins (RBP) for developing a specific, fast and simple fluorescence-microscopy-based detection method for Y. pestis. Genes of putative RBP of phages L-413C (gpH) and ΦA1122 (gp17) were fused with those of fluorescent proteins and recombinant receptor-reporter fusion proteins were produced heterologously in Escherichia coli. When first tested on attenuated Y. pestis strain EV76, RBP-reporters bound to the bacterial cell surface. This assay could be completed within a few minutes using live or formaldehyde-inactivated cells. Specificity tests using cultures of closely related Yersinia species and several inactivated fully virulent Y. pestis strains exhibited high specificities of the RBP-reporters against Y. pestis. The L-413C RBP proved to be especially specific, as it only detected Y. pestis at all temperatures tested, whereas the RBP of ΦA1122 also bound to Y. pseudotuberculosis strains at 37 °C (but not at 28, 20 or 6 °C). Finally, the Y. pestis-specific capsule, produced when grown at 37 °C, significantly reduced binding of phage ΦA1122 RBP, whereas the capsule only slightly diminished binding of L-413C RBP.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Specific Detection of Yersinia pestis Based on Receptor Binding Proteins of Phages

Born¹,

Braun²,

Scholz³

et al. 2020

Pathogens

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“…These phage RBP-based assays are already widely used as versatile tools for pathogen detection [ 9 , 10 ]. Target bacteria include biothreat agents that cause melioidosis ( Burkholderia pseudomallei ) [ 11 ], plague ( Yersinia pestis ) [ 12 ], or anthrax [ 13 ]. For B. anthracis phage Gamma, the GamR protein has been previously identified as the phage’s host cell receptor [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

Enzyme-Linked Phage Receptor Binding Protein Assays (ELPRA) Enable Identification of Bacillus anthracis Colonies

Braun¹,

Rupprich²,

Neif³

et al. 2021

Viruses

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Bacteriophage receptor binding proteins (RBPs) are employed by viruses to recognize specific surface structures on bacterial host cells. Recombinant RBPs have been utilized for detection of several pathogens, typically as fusions with reporter enzymes or fluorescent proteins. Identification of Bacillus anthracis, the etiological agent of anthrax, can be difficult because of the bacterium’s close relationship with other species of the Bacillus cereussensu lato group. Here, we facilitated the identification of B. anthracis using two implementations of enzyme-linked phage receptor binding protein assays (ELPRA). We developed a single-tube centrifugation assay simplifying the rapid analysis of suspect colonies. A second assay enables identification of suspect colonies from mixed overgrown solid (agar) media derived from the complex matrix soil. Thus, these tests identified vegetative cells of B. anthracis with little processing time and may support or confirm pathogen detection by molecular methods such as polymerase chain reaction.

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“…al. were able to predict a putative RBP (P28 + P29) encoded by phage AP50c based on genomic synteny and small identical stretches of protein sequence with the RBP (P24) polypeptide sequences from phage Wip1 [18]. Phages AP50c and Wip1 are similar phages belonging to the Tectiviridae family.…”

Section: Anthraxmentioning

confidence: 99%

The State of the Art in Biodefense Related Bacterial Pathogen Detection Using Bacteriophages: How It Started and How It’s Going

Sozhamannan

Hofmann

2020

Viruses

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Accurate pathogen detection and diagnosis is paramount in clinical success of treating patients. There are two general paradigms in pathogen detection: molecular and immuno-based, and phage-based detection is a third emerging paradigm due to its sensitivity and selectivity. Molecular detection methods look for genetic material specific for a given pathogen in a sample usually by polymerase chain reaction (PCR). Immuno-methods look at the pathogen components (antigens) by antibodies raised against that pathogen specific antigens. There are different variations and products based on these two paradigms with advantages and disadvantages. The third paradigm at least for bacterial pathogen detection entails bacteriophages specific for a given bacterium. Sensitivity and specificity are the two key parameters in any pathogen detection system. By their very nature, bacteriophages afford the best sensitivity for bacterial detection. Bacteria and bacteriophages form the predator-prey pair in the evolutionary arms race and has coevolved over time to acquire the exquisite specificity of the pair, in some instances at the strain level. This specificity has been exploited for diagnostic purposes of various pathogens of concern in clinical and other settings. Many recent reviews focus on phage-based detection and sensor technologies. In this review, we focus on a very special group of pathogens that are of concern in biodefense because of their potential misuse in bioterrorism and their extremely virulent nature and as such fall under the Centers for Disease and Prevention (CDC) Category A pathogen list. We describe the currently available phage methods that are based on the usual modalities of detection from culture, to molecular and immuno- and fluorescent methods. We further highlight the gaps and the needs for more modern technologies and sensors drawing from technologies existing for detection and surveillance of other pathogens of clinical relevance.

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Rapid Microscopic Detection of Bacillus anthracis by Fluorescent Receptor Binding Proteins of Bacteriophages

Cited by 13 publications

References 59 publications

Specific Detection of Yersinia pestis Based on Receptor Binding Proteins of Phages

Specific Detection of Yersinia pestis Based on Receptor Binding Proteins of Phages

Enzyme-Linked Phage Receptor Binding Protein Assays (ELPRA) Enable Identification of Bacillus anthracis Colonies

The State of the Art in Biodefense Related Bacterial Pathogen Detection Using Bacteriophages: How It Started and How It’s Going

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